ARCHNES
MASSACHUSETTS
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The Influence of Mucins on
Bacterial Communities
JUL 06 2015
LIBRARIES
by
Julia Yin-Ting Co
B.S. Microbiology, Immunology, and Molecular Genetics
University of California, Los Angeles, 2010
Submitted to the Microbiology Graduate Program in partial fulfillment of the requirements for
the degree of Doctor of Philosophy at the Massachusetts Institute of Technology
June 2015
0 2015 Julia Co. All Rights Reserved.
The author hereby grants to MIT permission to reproduce and to distribute publicly paper and
electronic copies of this thesis document in whole or in part in any medium now known or
hereafter created.
Auth or .............................................................
Signature redacted
Julia Co
Microbiology Graduate Program
May 22, 2015
C ertified by ........................................................
Signature redacted
Katharina Ribbeck
Assistant Professor of Biological Engineering
Thesis Supervisor
Accepted by................................
Signature redacted
Michael Laub
Professor of Biology
Director Microbiology Graduate Program
The Influence of Mucins on Bacterial Communities
by
Julia Yin-Ting Co
Submitted to the Microbiology Graduate Program in partial fulfillment of the requirements for
the degree of Doctor of Philosophy at the Massachusetts Institute of Technology
ABSTRACT
Mucus is the hydrogel layer that coats all wet epithelia in the body. By supporting
commensal microbes and preventing pathogenic invasion, mucus maintains host-microbe
homeostasis. Mucin polymers, the primary gel-forming component of mucus, are an important
mediator of mucus-microbe interactions. In this thesis, I demonstrate that mucins impact
bacterial communities in their physical structure as well as microbe-microbe and microbe-host
dynamics.
In Chapter 2, 1 study the ability of mucin surface coatings to suppress bacterial surface
attachment, the first step in biofilm formation, for Streptococcuspneumoniae and
Staphylococcus aureus. Mucin-bound glycans were identified as a critical structural component
of mucin coatings that are necessary for bacterial repulsion.
In Chapter 3, 1 investigate how mucins impact established Pseudomonasaeruginosa
biofilms. The data reveal that mucins cause disassembly and structural rearrangement in P.
aeruginosabiofilms in a mucin concentration and flow rate dependent manner.
In Appendix A, I show evidence for the involvement of the bacterial flagella in mucinmediated biofilm disruption. Deletion of flagellar capfliD or flagellar stators motABCD results in
biofilms that are resistant to mucin-mediated dissociation.
In Appendix B, I examine how mucins affect dual-species bacterial communities. I show
that mucins promote S. aureus survival during co-culture with P. aeruginosaand also suppress
the anti-staphylococcal effects of P. aeruginosapyocyanin.
In Appendix C, I explore the impacts of mucins on P. aeruginosaquorum sensing, an
important pathogenic determinant in P. aeruginosainfections. I found that mucins suppress the
expression of P. aeruginosaLas and Rhl quorum sensing genes as well as downstream virulence
factors.
In Appendix D, I assess how mucins modulate P. aeruginosa-epitheliuminteractions.
The data show that mucins hinder the ability of P. aeruginosato attach to epithelial cells in vitro.
Additionally, mucins suppressed P. aeruginosa-associatedepithelial cytotoxicity in a mucin
concentration dependent manner.
Together, this thesis demonstrates that mucins modulate microbial communities in their
behavior and interactions. Understanding how mucus and mucins impact microbes provides
insight to host-microbe relationships, as well as for the development of novel bacteria-regulating
strategies.
Thesis Supervisor: Katharina Ribbeck
Title: Assistant Professor of Biological Engineering
3
4
Acknowledgements
First, I would like to thank my thesis advisor Prof. Katharina Ribbeck, whose infectious
enthusiasm for mucus brought me to this exciting field of research. Through my graduate studies,
Katharina's guidance has helped me grow into a more thorough and thoughtful scientist. I would
also like to thank Ribbeck lab members Alona, Andrew, Brad, Erica, Kate, Leon, Nicole B.,
Nicole K., Nikki, Tahoura, Thomas, Tu, and Wes for all of the cake and coffee, for the
intellectual/experimental support and collaboration, and above all for their friendship. To Nicole
B. and Thomas, thank you for your scientific guidance and for being driving forces in my
research projects. Thank you to Nicole B., Nicole K., and Wes for their contributions in'editing
this thesis. To Nicole K., thank you for being "lab mom" and for paving the way for me and all
future Ribbeck lab graduate students. A huge thank you to Denise, our administrative miracle
worker, for always going above and beyond to ensure smooth sailing for the Ribbeck lab.
I would like to thank all of my thesis committee members: Alan Grossman, James Fox,
Michael Laub, Stephen Lory (Harvard Medical School) and Katherine Lemon (Forsyth Institute).
I am thankful to Stephen Lory for sharing his expertise and resources for multiple facets of my P.
aeruginosaprojects. I am thankful to Katherine Lemon for inspiring me to explore microbial
interspecies interactions, and for helpful discussion. I am thankful to all committee members for
their time, guidance, and support.
I am very grateful for the support and comradery of fellow Microbiology Graduate
Program students, in both my class and others. I am constantly inspired by their drive, intellect,
and scientific prowess. I would also like to thank Bonnielee Whang, who keeps the program
running strong.
Last, but certainly not least, I am thankful for the continuous support from my mom, my
dad, and my brother Dennis. I am truly grateful for the intellectual, scientific, and emotional
guidance that my parents provided. I am thankful to Chris for his unwavering encouragement,
late-night rides home from lab, 3D printed custom laboratory equipment, and everything in
between.
5
6
Table of Contents
Abstract..........................................................................................................................................
3
Acknow ledgem nents .......................................................................................................................
5
Table of Contents ..........................................................................................................................
7
List of Tables ...............................................................................................................................
10
List of Figures..............................................................................................................................
11
Chapter 1: Introduction .............................................................................................................
13
Biofilm s......................................................................................................................................
13
Introduction to biofilm s ............................................................................................
Challenges in treating biofilm infections.............................................................................
.... 13
15
M ucus and M ucins.....................................................................................................................
16
Physiology of m ucus...............................................................................................................
16
Structure and regulation of m ucins ......................................................................................
17
M ucin-M icrobe Interactions ...................................................................................................
20
Types of relationships between m ucins and m icrobes........................................................
20
System s for investigating m ucin-m icrobe interactions........................................................
22
Thesis Sum m ary.........................................................................................................................
24
References..................................................................................................................................
25
Chapter 2: Mucin-bound glycans contribute to bacterial repulsion by mucin coatings...... 35
Abstract ......................................................................................................................................
35
Introduction................................................................................................................................
36
M aterials and M ethods...............................................................................................................
37
Results ........................................................................................................................................
40
Mucin coatings reduce surface attachment of S. pneumoniae and S. aureus, but not
P. aeruginosa..........................................................................................................................
40
Mucin-bound glycans contribute to repulsion of S. pneumoniae and S. aureus..................41
Structural analysis of m ucin coatings ................................................................................
Discussion ..................................................................................................................................
7
43
44
Chapter 2: Mucin-bound glycans contribute to bacterial repulsion by mucin coatings
(continued)
Acknow ledgem ents....................................................................................................................
45
References..................................................................................................................................
45
Chapter 3: Mucins trigger disassembly of Pseudomonas aeruginosa biofilms..................
49
Abstract ......................................................................................................................................
49
Introduction................................................................................................................................
50
M aterials and M ethods...............................................................................................................
50
Results .........................................................................................................................................
52
Mucins cause P. aeruginosabiofilm disassembly and structural rearrangement ............... 52
M ucins are not toxic to P. aeruginosa ...............................................................................
54
Biofilm disassembly and structural rearrangement are mucin concentration dependent ....... 56
Mucin treatment flow rate affects the degree of biofilm disassembly .................
57
M ucin specificity of biofilm disassem bly...............................................................................
58
D iscussion ...................................................................................................................................
59
Acknow ledgem ents....................................................................................................................
61
References..................................................................................................................................
61
Appendix A: Mucin-mediated P. aeruginosa biofilm disassembly is flagella dependent..... 65
Introduction.................................................................................................................................65
M aterials and M ethods...............................................................................................................
65
Results ........................................................................................................................................
66
Mucin-mediated biofilm disassembly involves the P. aeruginosaflagellar capfliD.......... 66
Role of flagellar m otility in m ucin-m ediated biofilm disassem bly ....................................
67
D iscussion..................................................................................................................................
68
References..................................................................................................................................
69
8
Appendix B: Mucins protect Staphylococcus aureus against Pseudomonas aeruginosa
secreted toxin pyocyanin ............................................................................................................
71
Introduction................................................................................................................................
71
M aterials and M ethods...............................................................................................................
72
Results ........................................................................................................................................
74
M ucins protect S. aureus againstP. aeruginosakilling ....................................................
74
M ucins protect S. aureus against pyocyanin toxicity ..........................................................
75
Mucins bind pyocyanin...........................................................................................................
76
D iscussion ..................................................................................................................................
77
References..................................................................................................................................
79
Appendix C: Mucins suppress P. aeruginosa quorum sensing and virulence genes ......
83
Introduction................................................................................................................................
83
M aterials and M ethods...............................................................................................................
83
Results ........................................................................................................................................
84
Mucins suppress P. aeruginosaLas and Rhi quorum sensing genes ..................
84
M ucins suppress quorum sensing-regulated virulence genes .................................................
85
D iscussion..................................................................................................................................
85
References..................................................................................................................................
86
Appendix D: Mucins reduce P. aeruginosa epithelial attachment and cytotoxicity .......... 89
Introduction................................................................................................................................
89
M aterials and M ethods...............................................................................................................
89
Results........................................................................................................................................
91
M ucins reduce P. aeruginosaepithelial attachm ent ............................................................
91
M ucins protect epithelial cells against P. aeruginosacytotoxicity ....................................
92
D iscussion..................................................................................................................................
93
References..................................................................................................................................
93
Chapter 4: D iscussion .................................................................................................................
97
References.................................................................................................................................100
9
List of Tables
Chapter 1: Introduction
Table 1: M ucin genes and their tissue distributions ...............................................................
10
18
List of Figures
Chapter 1: Introduction
Figure 1. Stages of the biofilm cycle .....................................................................................
14
Figure 2. Schem atic of secreted mucins .................................................................................
19
Figure 3. Mucins regulate bacterial attachment to the epithelia ............................................
21
Figure 4 Mucins suppress biofilm formation and maintain bacteria in their planktonic state... 22
Figure 5 Schematic of mucin systems studied in this thesis...................................................24
Chapter 2: Mucin-bound glycans contribute to bacterial repulsion by mucin coatings
Figure 1. M ucin coatings are homogeneous ..........................................................................
40
Figure 2. Mucin coatings reduce bacterial attachment .........................................................
41
Figure 3. Apo-mucin coatings are homogeneous .................................................................
42
Figure 4. Quantification of bacterial attachment to apo-mucin coatings................................
42
Figure 5. Mucin glycans contribute structural properties to mucin coatings.........................
43
Chapter 3: Mucins trigger disassembly and structural rearrangement in Pseudomonas
aeruginosabiofilms
Figure 1. Mucins trigger disassembly and structural disassembly ........................................
53
Figure 2. Mucins cause cellular alignment in P. aeruginosabiofilms ...................................
54
Figure 3. Mucins are neither bacteriostatic nor bactericidal toward PAOI -GFP ..................
55
Figure 4. Mucins impact P. aeruginosabiofilms in a concentration-dependent manner .....
55
Figure 5. Biofilms can continue development after mucin treatments .................................
56
Figure 6. Mucin treatment flow rate impacts P.aeruginosabiofilm disassembly ................
57
Figure 7. P.aeruginosabiofilm treatments with viscous polymer solutions .........................
59
Appendix A: Mucin-mediated P. aeruginosa biofilm disassembly is flagella dependent
Figure 1. P. aeruginosafliDand motABCD are required for mucin-associated biofilm
d isrup tio n ...................................................................................................................................
67
Figure 2. P. aeruginosaswimm ing motility ..........................................................................
68
11
Appendix B: Mucins protect Staphylococcus aureus against Pseudomonas aeruginosa
secreted toxin pyocyanin
Figure 1. P. aeruginosa-S. aureus co-culture growth............................................................
75
Figure 2. M ucins suppress pyocyanin toxicity .....................................................................
75
Figure 3. M ucins restrict pyocyanin diffusion ......................................................................
77
Appendix C: Mucins suppress P. aeruginosaquorum sensing and virulence genes
Figure 1. Mucins suppress P. aeruginosaquorum sensing genes ..........................................
84
Figure 2. Mucins suppress P. aeruginosavirulence genes ....................................................
85
Appendix D: Mucins suppress P. aeruginosaquorum sensing and virulence genes
Figure 1. Mucins suppress P. aeruginosaattachment to A549 epithelial cells ......................
91
Figure 2. Mucins protect epithelial cells against P. aeruginosacytotoxicity ........................
92
12
Chapter 1
Introduction
Mucus is a biological hydrogel that lines all non-keratinized epithelia in the human body,
including the lungs, intestines, and urogenital tracts. The mucus layer maintains homeostasis
between the host and the environment, acting as a selective barrier that permits passage of
desirable material such as nutrients and oxygen (1), while preventing access of potentially
harmful agents such as toxins or pathogens (2, 3). In recent years, evidence has emerged for a
role of the gel-forming polymers of mucus, the mucins, in regulating interactions of commensal
and pathogenic microbes with the host (2-5). Specifically, mucins have been demonstrated to
suppress virulence traits of opportunistic pathogens, including Pseudomonasaeruginosa(6),
Streptococcus mutans (7), and Candidaalbicans (8), distinguishing them as important candidates
for the regulation of microbial infections on mucosal epithelia. Despite the crucial role of mucus
and mucins in maintaining health (9, 10), their effects on microbial behavior are understudied.
This thesis focuses on the influence of mucins on structured bacterial communities called
biofilms, which are responsible for many human infections. Furthermore, this work examines the
impacts of mucins on both intraspecies and interspecies microbial interactions. Understanding
the interplay between mucus and bacteria contributes to our biological understanding of both
commensal and pathogenic mucosal microbes, and may provide insights for the development of
new strategies to control microbial behavior.
Biofilms
Introduction to Biofilms
Biofilms are structured communities that secrete and encase themselves within a
protective matrix (11-14). They are a common mode of bacterial existence found on both biotic
and abiotic surfaces. Biofilms are often associated with mucosal infections such as dental cavity
formation, cystic fibrosis, and ulcers, where their resistance to host immune defenses and
13
Dispersal
Detachment
Planktonic
DMferentiaton
Maturation
Figure 1. Stages of the biofilm cycle. Free-living planktonic bacteria attach to a surface and become immobilized.
Maturation occurs when the bacteria begin to secrete extracellular polymeric substances that provide structure
and form a protective matrix. Differences in local microenvironments within the biofilm result in physiological
differentiation of bacteria (represented in green). Bacteria can detach as aggregates or disperse by regaining
motility.
antibiotic treatments lead to their persistence (13). Furthermore, biofilms are highly relevant in
the context of the colonization of medical devices. For example, biofilms are found on as many
as 50% of all medical devices (12) and cause 25.6% of all hospital acquired infections (15).
Despite their prevalence, effective strategies which prevent or remove biofilms are lacking.
Extensive studies of biofilms of several model organisms, including P. aeruginosa,
Escherichiacoli, and Staphylococcus aureus, have led to the emergence of a generalized model
for bacterial biofilm development (Figure 1) (14). In brief, biofilm formation begins with the
initial attachment of bacteria to a surface. Bacteria-substrate interactions are dependent on
general physico-chemical properties including charge and hydrophobicity of both the bacteria
and substrate surfaces (16-20), specific bacterial adhesins, and secreted matrix components. For
example, initial surface attachment of Staphylococcus spp. is mediated by the surface protein
Bap (21), extracellular DNA (22), and polysaccharide intercellular adhesin (PIA) (23). For some
bacteria, surface attachment relies on motility, which improves surface accessibility, and motility
appendages, which can act as adhesins (24-27).
Once attached to a surface, the bacteria proliferate and produce a structural and protective
matrix to form a mature biofilm that is resistant to environmental challenges. The biofilm matrix
consists of extracellular polymeric substances (EPS), including polysaccharides, nucleic acids,
lipids, and proteins; the exact composition is specific to each microbial species. Differences in
the composition of the EPS matrix can impact biofilm development and architecture. For
14
example, the thickness and smoothness of P. aeruginosabiofilms differ considerably depending
on the amount of specific EPS polysaccharides Pel, Psl, and alginate that are produced (28-31).
Biofilm and matrix development are also dependent on environmental conditions (32, 33)
including nutrient availability (34, 35), shear stress (36, 37), and quorum sensing (38, 39).
Moreover, the EPS matrix may regulate molecular transport, thereby creating nutrient and
oxygen gradients throughout the biofilm.
Microbes within biofilms are not permanently anchored to the associated community, but
instead can detach or disperse out of the biofilms upon certain perturbations to colonize a new or
more favorable environment (40-43). Detachment occurs when individual cells or groups of
cells are released from the outermost biofilm surfaces, and often results from mechanical forces
or disruption of the biofilm matrix (37). Dispersal is when cells from the biofilm actively migrate
out of the biofilm (40). This process is regulated by physiological pathways such as quorum
sensing (44, 45), prophage activation (46), and response to nutrient availability (47, 48), and
often involves regaining motility (40) or the secretion of rhamnolipids or matrix-hydrolyzing
enzymes (22, 42, 49-5 1). Upon escape from biofilms by active or passive mechanisms, bacteria
can attach to another surface to form a new biofilm (14, 40, 42).
Challenges in treating biofilm infections
Biofilm infections are problematic due to their array of resistance mechanisms that
provide resilience against the immune system and antibiotics. First, the protective barrier
property of the EPS matrix can restrict transport of host antimicrobial peptides (52) and other
antibiotics, including colistin (53), tobramycin (54), and vancomycin (55), into the biofilm.
Moreover, the sheer size of the biofilms can prevent clearance by immune cells, which are more
effective in attacking planktonic microbes. Additionally, physiological and metabolic
differentiation within the biofilm may lead to the emergence of subpopulations of protected
phenotypes. For example, cells that are in a dormant state would be resistant to antibiotics that
target metabolism or growth. Last, the high cell density within the biofilms is conducive to high
frequencies of conjugation, transformation, and transduction, which can result in the horizontal
transfer of antibiotic resistance genes (56).
The poor efficacy of antibiotic treatments for biofilm infections has led researchers to
focus on two main strategies to prevent and combat biofilms. The first is focused on developing
15
surfaces, primarily coatings, that are not readily colonized by bacteria (57). Antibiotics have
been bonded to surfaces to produce bacteriostatic or bactericidal coatings (58). However, the use
of antibiotics can lead to the selection of resistant bacteria and can even stimulate biofilm
formation for some microbes (59). Metals, toxic chemicals, and synthetic materials applied to
coatings are effective in preventing bacterial attachment and viability, but these surfaces can
become coated with biotic material or inactivated in clinical settings (57, 60, 61). Bio-inspired
superhydrophobic surfaces with micron- and nano-scale roughness can also inhibit microbial
attachment, though these effects are transient (62). For established biofilms, researchers have
targeted biofilm detachment and dispersal mechanisms as alternative treatment strategies.
Matrix-hydrolyzing enzymes (63, 64), phage treatments (65), and exposure to mechanical stress
(66, 67) have successfully been used to disrupt biofilms in vitro. Unfortunately, these agents are
often toxic or challenging to apply in a human host, and thus are not ideal for clinical
applications.
The general problems with current anti-biofilms strategies are that they are short-lived,
toxic, or technically difficult to implement. In this thesis, I explore the potential to harness the
intrinsic protective properties of mucus, the material that shields the epithelial lining against
damaging agents and microbes, to prevent biofilm formation and disrupt established biofilms.
Because mucus is naturally present in the body, it is likely to be biocompatible. In the first
chapter of my thesis, I focus on biofilm prevention and explore if mucins can interfere with
surface attachment, the first step in biofilm formation. In the second chapter, I study already
matured biofilms and investigate if mucins can disrupt their integrity and structure.
Mucus and Mucins
Physiology of mucus
The mucus layer covers all wet epithelia in the body, including the ocular, respiratory,
digestive, and reproductive systems. Mucus maintains lubrication and hydration, and forms a
selectively permeable barrier that allows passage of gasses, nutrients and other beneficial
material, while blocking passage of foreign antigens and harmful agents (1-3). In all mucosal
regions, mucus manages the interactions between the epithelia and microorganisms (68, 69).
This regulatory function can in part be attributed to the mucin polymers, which can directly
influence microbial behavior and virulence (discussed later in more detail) (6-8, 70). In addition,
16
mucus harbors immune components like immunoglobulin and antimicrobial peptides that control
bacterial proliferation (3).
Mucus also has specific functions on different anatomical locations and accordingly, can
differ in its composition, properties, and specific functions. For example, in the lungs, the mucus
layer is up to 10 ptm thick (71) and traps particles and microbes that enter the airways during
inhalation. In healthy lungs, this unwanted matter is removed by mucociliary clearance (10).
Dysfunctional respiratory mucus transport leads to chronic infections, which is common in cystic
fibrosis (CF) patients that have dehydrated mucus and impaired mucociliary clearance (72). The
mucus layer in the stomach is over 500 pm thick (73), and protects the epithelia from digestive
gastric acids (74). Irregularities in the gastric mucus lining are associated with development of
peptic ulcers (75). In the intestines, where the mucus layer ranges from 20 gm to 800 Pm in
thickness (76), mucus regulates nutrient absorption (76, 77) and houses the gut commensal
microorganisms. In the female reproductive tract, mucus regulates sperm passage into the cervix
(78) and prevents microbial access to the uterus (68).
Structure and regulation of mucins
The mucus hydrogel is composed of over 90% water, and the remaining content is a
heterogeneous mix of macromolecules, including proteins, lipids, and nucleic acids that are
either secreted or contributed from cellular debris of sloughed epithelial lining. The primary gelforming component of mucus is the mucin glycoprotein, which contributes to both the physical
and functional properties of mucus (2, 3, 10). Mucins are large biopolymers (up to tens of MDa
in size) composed of a protein backbone with protruding oligosaccharides (3, 10). The high
density of glycans causes the peptide backbone to exhibit an extended structural conformation
(Figure 2) (79). Mucins are characterized by the hallmark proline, threonine, and serine (PTS)
rich tandem repeat regions, which are the sites of the extensive 0-glycosylations that contribute
up to 90% of mucins' molecular weight (10, 80). During mucin synthesis, PTS domains are
glycosylated in the Golgi apparatus, where an N-acetylgalactosamine (GalNAc) residue is linked
to a serine or threonine (81). Elongation of the glycan chains results in both branched and linear
oligosaccharides up to 20 residues in length (2). These oligosaccharides are composed of GalNac,
N-acetylglucosamine (GlcNAc), galactose, mannose and fucose residues, and often terminate in
anionic sialic acid or sulfate residues. The composition and arrangement of the oligosaccharide
17
Distribution
Mucin
Secreted mucins
MUC2
MUC5AC
MUC5B
MUC6
MUC7
MUC19
Small intestine, colon, respiratory tract, eye, middle ear epithelium
Respiratory tract, stomach, cervix, eye, middle ear epithelium
Respiratory tract, salivary glands, cervix, gallbladder, seminal fluid, middle ear epithelium
Stomach, duodenum, gallbladder, pancreas, seminal fluid, cervix, middle ear epithelium
Salivary glands, respiratory tract, middle ear epithelium
Sublingual gland, submandibular gland, respiratory tract, eye, middle ear epithelium
Cell-surfa ce mucins
Stomach, breast, gallbladder, cervix, pancreas, respiratory tract, duodenum, colon, kidney, eye, B
MUCi
cells, T cells, dendritic cells, middle ear epithelium
Small intestine, colon, gall bladder, duodenum, middle ear epithelium
MUC3A/B
Respiratory tract, colon, stomach, cervix, eye, middle ear epithelium
MUC4
Colon, small intestine, stomach, pancreas, lung, kidney, prostate, uterus
MUC12
Colon, small intestine, trachea, kidney, appendix, stomach, middle ear epithelium
MUC13
Spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocyte, bone
MUC15
marrow, lymph node, tonsil, breast, fetal liver, lungs, middle ear epithelium
MUC16
Peritoneal mesothelium, reproductive tract, respiratory tract, eye, middle ear epithelium
Small intestine, colon, duodenum, stomach, middle ear epithelium
MUC17
Kidney, placenta, colon, lung, prostate, liver, middle ear epithelium
MUC20
Table 1: Mucin genes and their tissue distributions. Adapted from (2) with permission from Nature Publishing
Group.
chains are dictated by the availability of glycosyltransferases as the mucins move through the
Golgi, resulting in glycan heterogeneity (82, 83).
Mucins are encoded by MUC genes (Table 1), which differ in presence and abundance by
anatomical location. Mucins are categorized into two main classes: cell-surface mucins and
secreted mucins. Cell-surface mucins are tethered to epithelial cell membranes and contribute to
the glycocalyx (84). These mucins are classified by the presence of a transmembrane domain and
a short cytoplasmic domain. Most cell-surface mucins also have epidermal-growth-factor (EGF)
family domains that presumably play a role in signaling with EGF receptors (ERBB) (84). There
is evidence that the cell-surface mucins, MUCI and MUC4, participate in cell signaling events
(84). Additionally, enhanced expression cell-surface mucins have been associated with cancer
(84). Secreted mucins are produced by goblet cells, and most can polymerize to form hydrogels
that provide mucus with its characteristic viscoelastic properties (3, 10). The structure of secreted
18
Mucin Monomer
Mucin Dimer
Mucin Multimer
mysteine Rich
Domain
VWD
Cysteine
Knot
Mucin Gel
e
+- PTS Domain
* 4--O-glycans
Figure 2. Schematic of secreted mucins. Mucin peptides complex with characteristic proline, threonine and serine
(PTS) domains which are the sites of extensive 0-glycosylation. Non-glycosylated domains including von
Willebrand D (VWD) and cysteine rich regions are involved in mucin polymerization into a hydrogel network. New
sketch from (85) based on (2) with permission from Elsevier.
mucins includes cysteine rich domains and D domains (homologous to von-Willebrand factor
dimerization domains) that form disulfide bonds necessary for mucin oligomerization (Figure 2)
(2, 85). Secreted mucins MUC5AC and MUC5B are dominant in the lungs, MUC5AC and
MUC6 are the most prevalent in the stomach, and MUC2 is the primary mucin in the intestines
(Table 1). These mucins are named based on differences in their gene structure, however their
functional differences have not yet been discovered.
Mucin secretion is regulated by both constitutive and regulated pathways. Constitutive
mucin production replenishes and maintains the constantly moving mucus layer. Regulated
mucin production is an innate immune response that is stimulated by neutrophils, inflammatory
cytokines, and other immunogenic material (86). Both routes of mucin regulation are important
for maintaining the health of the mucosal epithelia.
19
Mucin-Microbe Interactions
Types of relationships between mucins and microbes
Mucins contribute to the ability of mucus to protect against unwanted microbial
colonization. Although the physical and biochemical properties of mucins are appreciated as
important for conferring their protective effects, the exact mechanisms of this protection are not
well understood. One hypothesis is that mucins form a physical barrier to microbes. For example,
mucins have been shown to restrict the motility of the gastric bacterium Helicobacterpylori(69),
a well-known mucosal pathogen. Additionally, Johansson et al. demonstrated that the murine
secreted mucin Muc2 was necessary to maintain a region of separation between the commensal
microbes and the intestinal epithelia, and to prevent inflammation that likely stemmed from
microbial contact with the epithelia (Figure 3A) (87). However, mucin gels do not exclusively
hinder bacterial motility, as is seen in the cases of P. aeruginosaand E. coli (6), and therefore are
selective filters rather than impenetrable barriers. Lastly, cell-surface mucin MUC 16 protects
human epithelial cells against S. aureus attachment (88-90), and Muc I reduces Helicobacter
pylori attachment to murine epithelial cells (Figure 3B) (91, 92).
In addition to modulating the transport of bacteria through the mucus, the mucin
biopolymer network provides a selectively permeable barrier that can regulate passage of certain
proteins, small molecules, and other matter which can impact the microbes inhabiting the mucus.
The small pore size (100 nm-400 nm) of the mucin hydrogel mesh network may restrict transport
of certain particles based on size exclusion (93-95). Additionally, hydrophobic domains and the
negatively charged regions of mucins allow mucins to selectively modulate transport through the
gel. Mucins therefore are likely to provide a framework for the presentation of immune factors
like immunoglobulins and antimicrobial peptides in high local concentrations. For example,
airway mucins can bind to cathelicidin LL-37 (96), and colonic mucins bind to a broad array of
antimicrobial peptides including defensins HBD- 1 and -3 and LL-37 without affecting their
efficacy (97). Furthermore, mucins may sequester and alter the availability of nutrients and
extracellular resources utilized by microbes, causing certain regions within the mucus to be more
hospitable than others.
Mucins can also directly modulate the persistence and location of bacteria that reside
within a mucus gel. Certain microbes express cell surface adhesins and mucus-binding proteins
that can interact with mucin-bound glycans (98, 99) and mucin-peptide moieties (100). This
20
Muc2-/-
WT
A
B
S0oClo'
*
40-
I30-
T
Ii
10o.
ii,
10.
Muc1'*
(2x)
MuC1d
n*
Muc I
8
MueP'I
M
ni=4
Mud*
Mouse Genotype
Figure 3. Mucins regulate bacterial attachment to the epithelia. (A) Histological sections of colon
mucosa in WT and Muc2-/- mice show that mucin is necessary to maintain separation between the
epithelium (stained blue) and resident gut bacteria (stained red). Scale bar: 100 pm. Reproduced from
(87) Copyright (2008) National Academy of Sciences, USA. (B) H. pylori attachment to Muc1-/- epithelial
cells is greater than that to WT epithelial cells (Muc +/+). (C) Stomachs of Muc -/- mice are
hypercolonized by H. pylori relative to WT mice. (B, C) Reproduced from (91) with permission from
Elsevier.
binding has been suggested to serve several functions: 1) mucin-bacteria binding could facilitate
mucociliary removal of unwanted bacteria, 2) mucins may serve as competitive ligands for
bacterial adhesins that would otherwise bind to epithelial cells, or 3) bacterial binding to mucin
may promote mucus colonization (2, 3). The purposes of mucin-microbe interactions likely vary
depending on the microorganism, anatomical location, and the nature and strength of mucinmicrobe binding.
Certain species of mucosal bacteria are capable of metabolizing or modifying mucin
molecules and networks. A subset of mucosal microbes can degrade (101-103) and utilize
mucins as a nutrient source (104-107). Some bacteria synergize to metabolize mucins, for
example the oral bacteria Streptococcus oralisand Streptococcus sanguis (108). For pathogenic
21
BiofihMw formon
unprotscted surfaces
Mucins retain cells in
the planktonic stat.
sous subotrals
Mucin polymner
Figure 4. Mucins suppress biofilm formation and maintain bacteria in their planktonic state. Adapted from (6) with
permission from Elsevier.
microbes, disruption of mucin hydrogels permits penetration of the mucus barrier. H. pylori
secretes bicarbonate, which increases local pH and thus reduces mucin polymerization, to
enhance its motility through gastric mucus (69). P. aeruginosaproduces mucolytic enzymes to
facilitate penetration through mucin solutions (109).
Mucins can also impact the expression of microbial virulence genes and traits. For
example, the biopolymers can induce mucin adhesin and virulence gene expression in H. pylori
(70), while they suppress virulent hyphae formation in C. albicans (8). Furthermore, mucins can
interfere with microbial surface attachment and early biofilm formation (Figure 4), for example
in C. albicans (8), S. mutans (7), and P. aeruginosa(6) (Figure 4). Moreover, mucin degradation
can result in the introduction of metabolites that affect microbial pathogenicity. For instance,
mucin utilization by the commensal microbe Bacteroidetes thetaiotamicronleads to the release
of fucose, which in turn suppresses enterohaemorrhagic E coli (EHEC) virulence gene
expression (110). In contrast, cleavage of mucin-associated sialic acids by influenza virus can
exacerbate polymicrobial pneumonia by promoting Streptococcus pneumoniae proliferation
(111). Thus, mucins are an important environmental factor that impact host-associated microbial
behavior and colonization.
Systems for investigating mucin-microbe interactions
Microbial interactions with mucins have been explored using a variety of models. In vivo
animal models in which mucin genes and glycosidases have been altered are useful to study
systemic responses to changes in mucin availability and composition (87, 91, 111-113).
22
Explanted mucosal tissue and mucus-producing cell lines are valuable systems that facilitate
controlled perturbations or the addition of microbes (8, 91). A caveat to these models is that
disruption of mucin and therefore mucus homeostasis may stimulate systemic responses to
compensate for mucin dysregulation. It is difficult to ascertain whether changes in the host and
microbes are a direct result of changes in the mucins, or from downstream effects of altered
mucins. Another important system for studying mucus-microbe interactions is the use of whole
mucus or sputum isolated from human subjects (114-116). However, sample isolation can be
difficult and highly invasive for many mucosal regions. Moreover, due to the complexity of
whole mucus and sputum, it is difficult to pinpoint the specific effects of mucins.
In this work, I use native purified mucins to examine the influence of the biopolymers on
biofilms and microbial interactions. Specifically, I use native mucins purified from fresh pig
gastric mucus which have high homology to human MUC5AC (117), a primary mucin in the
airways and stomach. Native purified mucins, unlike commercially purified mucins, recapitulate
the structural and rheological properties of physiological mucus (118, 119), and have been
previously used to study the behavior of H. pylori (69), P. aeruginosa(6), C. albicans(8), and S.
mutans (7) in mucus environments. This thesis studies mucins in two different configurations
(Figure 5): 1) adsorbed to synthetic surfaces as "2D" thin film coatings, and 2) solubilized in
a "3D" configuration, in which the mucins more closely represent the biochemical and
rheological properties found in native mucus.
Although native purified mucins are an informative and experimentally advantageous
model for mucus, there are limitations of this simplified system. While mucins are a primary gelforming constituent of mucus, other mucus components can likely contribute to its physicochemical and microbial-modulating properties. The large and heterogeneous mucin glycoproteins
are difficult to purify, and the possibility that associated factors may be present in the purified
mucin preparations cannot be eliminated. Furthermore, mucus and mucins in physiological
conditions are constantly replenished, a phenomenon which is not accounted for in some
experiments in this thesis. Despite these caveats, native mucins remain a useful tool in which
experimental conditions can be carefully controlled and perturbed, and are used in this thesis to
study microbial behavior.
23
"3D" Mucin Solution
"2D" Mucin Coating
Figure 5. Schematic of mucin systems studied in this thesis. In Chapter 2, mucins are adsorbed to synthetic
surfaces as "2D" thin film coatings. These mucin coatings are useful for bioengineering applications; however
they do not represent physiological mucus environments. The remainder of this thesis examines mucins in their
solubilized and native "3D" configuration, in which they more closely resemble the biochemical and rheological
properties found in native mucus.
Thesis summary
Mucins have emerged as an important regulator of microbial behavior. Mucins in vivo
can prevent microbial attachment to the epithelia, and mucin gels in vitro suppress microbial
attachment and early biofilm formation on synthetic surfaces. This thesis aims to extend the
current knowledge of how mucins influence microbial communities. Specifically, I investigate
the possibility of exploiting the intrinsic bacteria-modulating properties of mucins to control
biofilms (Chapter 2, Chapter 3 and Appendix A), and the impacts of mucins on bacterial
interactions within planktonic cultures (Appendix B-C) and with the host (Appendix D).
In Chapter 2, 1 explore the application of mucins as surface coatings to reduce bacterial
attachment. The development of anti-adhesive surfaces is desirable to reduce the incidence of
unwanted bacterial colonization, for example on implanted medical devices. Together with
colleagues, I show that thin film coatings of surface adsorbed mucins (Figure 5) suppress
attachment of Streptococcus pneumoniae and S. aureusto polystyrene surfaces. Using
deglycosylated mucins, we illustrate that mucin-bound glycans provide critical structural
properties, and are imperative for the bacteria-repelling capability, of the mucin coatings.
In Chapter 3, 1 study the ability of mucins when presented in their native 3D
configuration to disrupt established P. aeruginosabiofilms. Using reconstituted mucin gels
(Figure 5), I demonstrate that the biopolymers can cause the disassembly and structural
rearrangement of P. aeruginosabiofilms. Appendix A explores the role of the bacteria flagella in
mucin-mediated biofilm disassembly and shows that loss of either the flagellar cap or flagellar
motors renders the biofilms resistant to the dissociative effects of mucins.
24
In Appendix B, I describe the impact of mucins on bacterial interspecies dynamics,
specifically between P. aeruginosaand S. aureus. P. aeruginosakills S. aureus in most
laboratory conditions, however, on native mucosal epithelia these organisms are frequently
found together. I show that mucins support the co-existence of S. aureus with P. aeruginosa,and
that the biopolymers protect S. aureus against the antagonistic effects of P. aeruginosasecreted
pyocyanin.
In Appendix C, I present the finding that mucins can interfere with P. aeruginosaquorum
sensing. Complex mucus environments can affect P. aeruginosagene expression (114, 115, 120),
however the specific effects of mucins were previously unknown. Using transcriptional reporters,
I show preliminary data to illustrate that mucins can downregulate the expression of lasI and rhl
quorum sensing genes, two key regulators of P. aeruginosaquorum sensing, as well as several
downstream virulence genes.
In Appendix D, I address the ability of mucins to shield intact epithelial cells from
infection by P. aeruginosa.Mucins have been described to prevent surface attachment of several
pathogens (89-92). My preliminary experiments show that mucins can also suppress the
attachment of P. aeruginosato epithelial cultured cells and thereby, render the bacteria less
cytotoxic.
Together, this thesis provides evidence that mucins play an important role in regulating
bacterial biofilms, as well as intraspecies and interspecies dynamics. These findings highlight the
importance of mucins and mucus as a consideration when studying mucus-associated microbes.
Furthermore, this work demonstrates the potential for the development of mucin-mediated or
mucin-inspired strategies to regulate microbial behavior.
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34
Chapter 2
Mucin-bound glycans contribute to
bacterial repulsion by mucin coatings
Abstract
Biomaterials that prevent bacterial colonization are needed to reduce the incidence of
microbial colonization of implanted medical devices. This study investigates the use of mucin
biopolymers to prevent attachment of selected respiratory pathogens to polystyrene surfaces. Our
data show that coatings of porcine gastric mucins or bovine submaxillary mucins reduce surface
attachment by Streptococcus pneumoniae and Staphylococcus aureus, but not Pseudomonas
aeruginosa,highlighting the limitations of such coatings as universal antimicrobial surfaces. To
elucidate how mucin coatings repel S. pneumoniae and S. aureus, the molecular components of
mucins are examined. The data presented here suggest that mucin-bound glycans are key
structural contributors of mucin coatings, and are responsible for the repulsive effects toward S.
pneumoniae and S. aureus.
35
Introduction
Microbial colonization is a leading cause of medical device failure. About 50% of
indwelling devices become colonized by microbes (1), causing a significant fraction of hospital
acquired infections (2). Bacterial colonization begins with cell attachment to the device surface.
Attached cells proliferate and mature to form resilient matrix-encased communities called
biofilms. Once established, biofilms are difficult to eradicate due to their resistance to
antimicrobial treatments. Thus, there is a strong focus on developing new surfaces to prevent
bacterial attachment.
This study explores the natural mucus barrier, and specifically its gel-forming mucin
polymers, for strategies to prevent surface attachment. Mucus is the hydrated polymer network
that lines all wet epithelia in the human body, including the respiratory, digestive, and
reproductive tracts. The primary gel-forming components within mucus, the mucins, are highly
glycosylated polymers which exist in secreted and cell-surface forms (3-6). Both mucin types
can protect the underlying mucosal epithelia from microbial infection. For example, gastric
mucin MUC5AC can maintain the bacteria Pseudomonas aeruginosa(7) and the yeast Candida
albicans(8) in a planktonic state and impair subsequent biofilm development. A similar effect
has been observed for human salivary mucin MUC5B toward the bacteria Streptococcus mutans
(9). Moreover, cell-surface mucins can reduce Staphylococcus aureus attachment to corneal
epithelial cells (10) and can limit Helicobacterpyloriattachment to gastric epithelial cells (11).
Given their ability to suppress microbial surface attachment in native conditions, mucins
have also been studied in the context of microbe-repelling surface coatings (12, 13). Although
these thin film coatings do not represent physiological mucin gels in structure or thickness, the
mucin coatings are able to reduce attachment of some microbes to underlying surfaces. Studies
of mucin coatings on polyacrylic acid, poly(methyl methacrylate), silicone, polyurethane, and
polystyrene surfaces show that the function of mucin coatings varies by mucin-type, the surfaces
to which they are adsorbed, and the deposition conditions. For example, bovine submaxillary
mucin coatings reduce Candida albicans attachment to silicon surfaces, but increase its
attachment to poly(acrylic acid-b-methyl methacrylate (PAA-b-PMMA) surfaces. This
variability reflects a gap in our understanding of which functional domains and biochemical
properties of mucin coatings are important in mucin-microbe interactions.
36
This study investigates which molecular components of mucin coatings contribute to
bacterial repulsion, specifically in the context of polystyrene surfaces. The respiratory pathogens
Streptococcuspneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosaoften colonize
medical devices to cause infection (2), and thus were examined in this study. The data show that
coatings made of gastric or submaxillary mucins efficiently prevented surface attachment of S.
pneumoniae and S. aureus, but not of P. aeruginosa. To dissect the role of mucin-bound glycans
in bacterial repulsion, properties of native mucin coatings were compared to those of
deglycosylated mucin coatings. Upon deglycosylation, mucin coatings lost their ability to repel
bacteria and exhibited a thinner and more rigid coating structure. Our findings indicate that
mucin-bound glycans are structural components capable or regulating surface attachment by S.
pneumoniae and S. aureus.
Materials and Methods
Mucins
Bovine submaxillary mucin (Sigma-Aldrich) was dialyzed for 4 days against ultrapure
water using a Spectra/Por Float-A-Lyzer G2 100 kDa MW cutoff dialysis membrane (Spectrum
Labs), then lyophilized for storage. Native porcine gastric mucin was purified as previously
reported (14). Briefly, mucus was scraped from fresh pig stomachs and solubilized in saline
buffer with protease inhibitors and sodium azide. Insoluble material was pelleted by
ultracentrifugation and mucins were purified using size exclusion chromatography on a
Sepharose CL-2B column. Mucin fractions were desalted, concentrated, and lyophilized for
storage. To produce fluorescent mucins, gastric and submaxillary mucins were labeled with
Alexa488 (Invitrogen) following the manufacturer's instructions. Briefly, 10 pl of 10 mg mU1
Alexa488 succinimidyl ester in DMSO was added to 1 ml of 3 mg ml-1 mucins in 0.2 M
bicarbonate buffer (pH 8). After incubation at room temperature for 1 hour, free dye was
separated from the labeled mucin using a Macrosep 100 kDa MW cutoff centrifugal filter (Pall).
Preparationof apo-mucins
Mucin was deglycosylated by treatment with trifluoromethanesulfonic acid (TFMS),
followed by oxidation and beta-elimination of the residual sugars as previously described (15). 5
%
mg lyophilized mucin was cooled on ice and mixed with 375 pl of ice-cold TFMS containing 10
37
(v/v) anisole. The solution was gently stirred on ice for 2 hours then neutralized by the addition
of a solution containing 3 parts pyridine, 1 part methanol and I part water. Precipitates were
dissolved by adding water. The solution was dialyzed for 2 days against ultrapure water using a
20 kDa MW cutoff dialysis membrane (Spectrum Labs). NaCl and acetic acid were then added to
the solution to a final concentration of 0.33 M and 0.1 M, respectively. The solution was adjusted
to pH 4.5 with NaOH. For the oxidation step, ice-cold 200 mM NalO4 was added to the mucin
solution to a final concentration of 100 mM NalO 4 , and incubated at 4 *C for 5 hours in the dark.
The unreacted periodate was destroyed by adding 2 volume of neutralizing solution containing
400 mM Na2 S 2O 3, 100 mM Nal, and 100 mM NaHCO 3. For elimination, the mucin solution was
adjusted to pH 10.5 using 1 M NaOH and incubated at 4 'C for 1 hour. The solution was then
dialyzed overnight at 4 0C against a 5 mM NaHCO 3 buffer (pH 10.5) and further dialyzed for 2
days against ultrapure water. The resulting apo-mucin solution was then concentrated and
dissolved in the appropriate buffer.
Apo-mucins were evaluated for glycan removal using the periodic acid-Schiff assay as
previously described (16). Briefly, in a 96 well plate, 120 ptL of a mixture of 7% acetic acid and
0.06% periodic acid was added to 20 ptL sample. The solution was incubated for 1.5 hours at
370 C. 100 ptL of Schiff reagent was added to the wells, and allowed to react at room temperature
for 10 minutes before measuring absorbance at 550 nm using a SpectraMax M2 Microplate
Reader (Molecular Devices). The ratio of absorbance to mucin mass contained in each sample
was reported.
Mucin coatings
Mucin coatings were generated by incubating 200 pg ml 1 gastric mucins or submaxillary
mucins in 20 mM HEPES (pH 7.4) on the surface of interest. For mucin coatings in 96-well
polystyrene microtiter plates, 200 pg ml- native mucins were incubated in microtiter wells at
room temperature for 1 hour, followed by 3 washes with dH20. For apo-mucin coatings, 200 pg
ml 1 deglycosylated mucins were incubated in wells at room temperature for 1 hour, followed by
3 washes with dH20.
38
Microscopy of mucin coatings
To verify mucin coating homogeneity, mucin coatings were visualized in the polystyrene
microtiter wells. The wells were coated with gastric mucin-Alexa488, submaxillary mucinAlexa488, or their Alexa488-labeled apo-mucin counterparts. A scratch was made in the film
with a pipette tip for reference. The fluorescent coatings were imaged using an Observer ZI
inverted epifluorescence microscope (Zeiss) and a 40x/0.75 NA objective (Zeiss).
Measuringbacterialattachment to mucin and apo-mucin coatings
Bacterial attachment to mucin coatings and modified mucin coatings was evaluated for
Streptococcuspneumoniae, Staphylococcus aureus, and Pseudomonasaeruginosa. S.
pneumoniae TIGR4 serotype 19F was cultured in Todd Hewitt Broth (Becton Dickinson)
supplemented with 0.5% Yeast Extract (Becton Dickinson) in static conditions at 37 0 C with 5%
CO 2. S. aureus UAMS-1 was cultured in Brain Heart Infusion (Becton Dickinson). P.
aeruginosaPAO] was cultured in Luria Broth (Becton Dickinson). S. aureus and P. aeruginosa
were grown shaking at 37 0 C. Bacteria were grown to logarithmic phase, centrifuged and
resuspended in PBS to OD 600 0.4. For the microtiter plate attachment assay, wells were coated
with mucins as described above, or left untreated as a control. Bacteria were prepared as
described for microscopic visualization, and then incubated in wells at 37 0 C for 1.5 hours for S.
aureus and P. aeruginosaor 3 hours for S. pneumoniae. Unattached bacteria were aspirated, and
wells were washed 3 times with dH20. Attached bacteria were fluorescently stained using the
CyQuant Direct Cell Proliferation Assay (Life Technologies) following manufacturer's protocol,
and quantified with a SpectraMax M2 Microplate Reader (Molecular Devices). Fluorescence of
bacteria attached to mucin coated wells were normalized to fluorescence of bacteria attached
uncoated control wells. Experiments were performed in triplicate.
QCM-D
Quartz crystal microbalance with dissipation monitoring (QCM-D, E4 system, Q-Sense)
was used to measure the hydrated mass of mucins adsorbed to a polystyrene-coated quartz
crystal (QSX305, Q-sense). Solutions of 200 pig m1_ native mucins or apo-mucins were adsorbed
to the crystal. The crystal vibration was followed at its fundamental frequency (-5 MHz) and 5
overtones (15, 25, 35, 45, 55 and 65 MHz). Once the excitation was stopped, changes in the
39
resonance frequencies and dissipation of the vibration were followed at the 6 frequencies. When
the adsorbed layers are highly hydrated they usually possess viscoelastic properties requiring the
measurement data to be modeled. Hydrated thickness was calculated using the Q-Tools
3.0.12.518 software that includes the Voigt model (i.e., a spring and dashpot in parallel under no
slip conditions) (17) assuming a density of 1050 kg.m-3 (as validated for a related system (18))
and that the coating is homogeneous in thickness and over the crystal's surface. Measurements
were performed at least in duplicate.
Results
Mucin coatings reduce surface attachment of S. pneumoniae and S. aureus, but not P.
aeruginosa
Surface attachment of three common respiratory pathogens (S. pneumoniae, S. aureus,
and P. aeruginosa)to mucin coatings was evaluated using in-house purified porcine gastric
mucins and commercial bovine submaxillary mucins. Mucins were adsorbed to polystyrene, then
verified using fluorescence microscopy and QCM-D analysis, which confirmed that the mucins
A
C
Wo.
10
B
20
DaD soTim
30
4
50
40
50
(min)
140-
e 20
0
0
10
20
30
Tim (nun)
Figure 1. Mucin coatings are homogeneous. (A, B) Coatings were produced on polystyrene slides of
fluorescently labeled (A) gastric mucins or (B) submaxillary mucins. A scratch was made in the coatings with a
pipette tip for visual comparison with uncoated polystyrene regions. Scale bars: 50 pm. (C, D) Representative
QCM-D data shows saturation of mucins on a polystyrene coated crystal for (C) gastric mucins or (D)
submaxillary mucins.
40
formed relatively homogeneous coatings (Figure 1). To assess bacterial repulsion by mucin
coatings, bacteria bound to mucin-coated or uncoated polystyrene microtiter wells were
fluorescently labelled and measured on a microplate reader. Figure 2B reveals that gastric mucin
coatings reduced attachment of S. pneumoniae by 76.3% and attachment of S. aureus by 81.3%
to underlying polystyrene. Submaxillary mucin coatings were comparably effective, reducing
attachment of S. pneumoniae by 71.5% and of S. aureus by 81.0% to underlying polystyrene. In
contrast, neither surface coating significantly altered P. aeruginosaattachment.
Mucin-bound glycans contribute to repulsion of S. pneumoniae and S. aureus
Previous work demonstrated that mucin-bound glycans within mucin coatings contribute
to the repulsion of mammalian cells (19). To examine if mucin-bound glycans also play a role in
bacterial repulsion, bacterial attachment was tested on coatings made from deglycosylated
mucins, which are henceforth referred to as apo-mucins. Apo-mucins were generated from native
mucins by chemical removal of mucin-associated glycans. Mucin deglycosylation was verified
using the periodic acid-Schiff assay (Figure 3A). Apo-mucins were adsorbed to polystyrene
surfaces to produce coatings, and fluorescence microscopy of Alexa488-labeled apo-mucin
j
1.5-
M Gastrc mucins
E-
Submaxillary mucins
1.0 ----------------------
) OM 0.5-U
E0.0
S. pneumoniae
S. aureus
P. aeruginosa
Figure 2. Mucin coatings reduce bacterial attachment. Bacterial attachment to gastric mucin or submaxillary
coatings generated in polystyrene microtiter wells was quantified using the CyQuant Assay (Life Technologies).
Values represent relative attachment to mucin coatings (bacterial attachment to mucin coatings normalized to
bacteria attached to uncoated polystyrene). Error bars represent standard deviation of 3 biological replicates.
Reduction in attachment of S. pneumoniae and S. aureus to gastric mucin and submaxillary mucin coatings
compared to polystyrene is statistically significant as determined by unpaired t-test with pS0.05.
41
A
C
B
.
0.02
0.01
0.00-
Gastm
acins
Apo
Gastrk
MoinM
Sub-
malary
MXinM
Apo.Submaxdary
Mcins
Figure 3. Apo-mucin coatings are homogeneous. (A) Periodic acid-Schiff (PAS) assay of mucins and apomucins verified deglycosylation. Error bars represent standard deviation of 3 replicates. Reduction in
absorbance/pg protein in apo-mucins relative to their native counterparts is statistically significant as determined
by unpaired t-test with p50.05. (A, B) Coatings were produced on polystyrene slides of fluorescently labeled apogastric mucins (A) or apo-submaxillary mucins (B). A scratch was made in the coatings with a pipette tip for
visual comparison with uncoated polystyrene regions. Scale bars: 50 pm.
coatings confirmed relatively homogeneous surface coverage (Figure 3B, 3C).
The removal of glycans from mucins reduced the ability of the coatings to repel bacteria.
Specifically, apo-gastric mucin coatings exhibited a 4.2 fold increase in S. pneumoniae
attachment and a 10.8 fold increase in S. aureus attachment relative to native gastric mucin
coatings (Figure 4A). Similarly, apo-submaxillary mucin coatings had 3.1 fold more S.
pneumoniae and 8.3 fold more S. aureus attached compared to their glycosylated counterparts
(Figure 4B). These results indicate that mucin-bound glycans are critical for preventing
attachment by S. pneumoniae and S. aureus. In contrast, P. aeruginosaattachment was
comparably efficient on native mucin and apo-mucin coatings, indicating that mucin-bound
A
A-B1.5
1.5 C"
1 Gastric Mucins
IM Apo-Gastric Mucins
L
1.0 -------f3'~----------
Submaxillary Mucins
1101Apo-Submaxilary Mucins
- -------10------0.5
0.5
S. pneumonia. S. aweus
S. pneumoniae S. aureus
P. aeruginosa
P. aerugenosa
Figure 4. Quantification of bacterial attachment to (A) apo-gastric mucin coatings or (B) apo-submaxillary
coatings generated in polystyrene microtiter wells. Values represent relative attachment to mucin coatings
(bacterial attachment to mucin coatings normalized to bacteria attached to uncoated polystyrene). Error bars
represent standard deviation of 3 biological replicates. Increase in attachment of S. pneumoniae and S. aureus
to apo-mucin coatings relative to native mucin coatings is statistically significant as determined by unpaired t-test
with p50.05.
42
glycans, at least in the context of surface coatings, do not measurably affect P. aeruginosa
binding. The glycan compositions of gastric and submaxillary mucins differ considerably (20,
21), suggesting that the repulsive effect of the coatings observed here is dictated not by the
specific glycan components, but instead by general physico-chemical properties conserved
among the different mucin types.
Structural analysis of mucin coatings
The deglycosylation of mucins alters the biochemistry and structure of mucin coatings,
which in turn may affect bacterial attachment (22-24). To better understand the how mucinassociated glycans contribute to bacterial repulsion, QCM-D was used to examine the hydrated
thickness and softness of native mucin and apo-mucin coatings. Figure 5A shows that gastric
mucin coatings were 35.0
9.9 nm thick, while submaxillary mucin coatings were 60.3
3.2 nm
thick. In contrast, apo-gastric mucins and apo-submaxillary mucins both formed thinner coatings
that were less than 4 nm in thickness. QCM-D analysis also provided information about the
softness of the coatings as a measure of energy dissipation of the acoustic waves applied to
analyze the coatings. Figure 5B shows that the energy dissipation was greater for coatings of
native mucins than for their apo-mucin counterparts. Upon deglycosylation, the energy
A s
B 10
C
60-
Gastric
mucins
poSb
Apo-ubgastric maxilary maxlary
muckns mucins mucMs
Gastric
nucins
A.5o
gastric
mucirns
Sub
po-ubmaxilary maxiary
mucins
mucins
C
Apo-mucin coating
Native mucin coating
Figure 5. Mucin glycans contribute structural properties to mucin coatings. (A, B) Coatings produced from native
mucin and apo-mucin coatings were analyzed with QCM-D. Native mucin coatings exhibited (A) thicker coatings
and (B) greater variation in energy dissipation, which is indicative of a softer coating. Error bars represent
standard deviation of at least two replicates. Values for apo-mucins are statistically different from their native
mucin counterparts as determined by unpaired t-test with p50.05, except for variation in dissipation for submaxillary mucins and apo-submaxillary mucins. However, the trend of decreased variation in dissipation for aposubmaxillary mucins was conserved. (C) Model of glycan contributions to the structure of mucin coatings.
43
dissipation measurements decreased 21 fold for gastric mucin coatings and 2.5 fold for bovine
submaxillary mucin coatings, indicating that apo-mucin coatings are stiffer than the native mucin
coatings. These data suggest that mucin-bound glycans influence the thickness and softness of
mucin coatings.
Discussion
Mucins have evolved to help protect the wet epithelia from microbial colonization and
hence, have shown promise as building blocks for anti-microbial surface coatings. Previous work
showed that coatings from submaxillary mucins can prevent S. aureus attachment to underlying
surfaces (12). The current study extends the understanding of mucin coatings by demonstrating
that coatings of submaxillary mucins can also repel S. pneumoniae. In addition the data here
demonstrate that another species of mucins, namely from pig stomachs, is also effective at
repelling both S. aureus and S. pneumoniae, providing a potential additional candidate
biopolymer for the engineering of coatings.
This study also highlights the limitations of mucins as universal bacterial-repelling
surfaces when displayed as 2D coatings. Specifically, neither type of mucin coating significantly
altered P. aeruginosaattachment. When adsorbed to a surface, mucins may lose part of the
biofilm-suppressing functionality, which is exhibited in a three dimensional hydrogel network.
Moreover, mucin-digesting enzymes secreted by P. aeruginosa (25) may damage coating
integrity and its repulsive properties. Last, adhesins on the bacterial surfaces (26, 27) which bind
mucin-bound glycans (28, 29) and mucin-peptide moieties (30) may mediate interactions with
the coatings. Of note is that the mucin coatings generated in this study only repelled Gram
positive S. pneumoniae and S. aureus, but not Gram negative P. aeruginosa.However, previous
work demonstrated that another Gram-negative bacterium, Escherichiacoli, can be repelled by
mucin coatings (13), hence, we conclude that the repulsive effect of mucin coatings may not be
restricted to Gram-positive bacteria.
This study indicates that mucin-bound glycans provide mucin coatings with important
structural properties, specifically thickness and softness. These properties have previously been
shown to increase efficacy of bacterial repulsion by certain classes of polymer coatings, such as
PEG (31-33). One possibility is that the glycans provide coating thickness and softness via their
contributions to the mucin polymers' bottle brush conformation. In solution, deglycosylation
44
causes the mucins' bottle-brush architecture to collapse into a globular protein-like conformation
(Figure 5C) (34). The structural conformation of polymers applied to coatings can determine
antifouling properties, for example glycerol-based brush coatings with bulkier branched side
chains form better antifouling surfaces than those with linear and shorter side chains (35). Thus,
changes in mucin conformation upon deglycosylation may in part explain the apo-mucins
coatings' loss of repulsive ability. The identification of mucin-bound glycans as important
bacteria-repelling, structural components of mucin coatings may inform the development of new
antifouling materials and surfaces that reduce unwanted microbial colonization and prevent
medical device associated infections.
Acknowledgements
This work was completed in collaboration with Dr. Thomas Crouzier, who performed
structural analysis experiments. We thank Dustin Griesemer for his preliminary contributions to
this project. We are grateful to Marc Lipsitch for the donation of S. pneumoniae TIGR4, and
Katherine Lemon for the donation of S. aureus UAMS-l. This work made use of the MRSEC
Shared Experiment Facilities supported by the National Science Foundation under award number
DMR-819762.
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bovine submaxillary mucin coatings: effect of coating deposition conditions. Biofouling
26:387-397.
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O-linked glycoproteins. The deglycosylation of submaxillary and respiratory mucins.
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48
Chapter 3
Mucins trigger disassembly of P. aeruginosa biofilms
Abstract
The opportunistic pathogen Pseudomonas aeruginosaforms problematic biofilms that are
resistant to immune clearance and antimicrobial agents. Mucins, the primary gel-forming
biopolymers of the protective mucus layer, have previously been observed to suppress the
generation of biofilms by a range of different microbes. In this study, I build on this work to
examine if mucins can also disrupt already established biofilms, using P. aeruginosaas a model
organism. Using a hydrodynamic flow cell biofilm system, I show that mucins can cause the
disassembly and structural rearrangement of established P. aeruginosabiofilms. This biofilm
response is dependent on mucin concentration and treatment flow rate. Other viscous polymer
solutions cannot match the biofilm disruption by mucins, suggesting that there is specificity to
mucin-mediated biofilm disruption. The discovery that mucins can disassemble biofilms may
offer insight for the development of novel P. aeruginosabiofilm eradication strategies, both
inside and outside of the body.
49
Introduction
The Gram-negative opportunistic pathogen Pseudomonasaeruginosaforms problematic
biofilms-surface-attached bacterial communities within protective matrices-that cause
morbidity in infections (1, 2) and medical device failures (3, 4). P. aeruginosabiofilms are
challenging to treat because they are highly resistant to antibiotics (5) and harsh chemical
treatments (6). Researchers have targeted biofilm disassembly mechanisms like matrix disruption
(7), phage treatments (8), and exposure to mechanical stress (9-11) as new biofilm eradication
strategies. However, these agents are often toxic or difficult to implement in a human host, and
thus are not ideal for clinical applications. Thus, there is a pressing need for novel biocompatible
biofilm removal methods.
Here, I explore the potential of the protective mucus layer, specifically its gel-forming
mucin biopolymers, to disrupt biofilms. Mucins can promote the clearance of microbes, and have
been demonstrated to impair surface attachment and early biofilm formation in Streptococcus
mutans (12), Candidaalbicans (13), and P. aeruginosa(14). Mucins are thus interesting
candidates for regulating established biofilms. Using a continuous flow cell biofilm model, I
demonstrate that solutions of native purified mucins cause disassembly and structural
rearrangement in P. aeruginosabiofilms. For comparison, other synthetic polymers were also
able to partially disrupt the biofilms, but not to the same extent as the mucins, suggesting that
features specific to the mucins are required to mediate the observed effect on biofilms.
Materials and Methods
Bacterialstrains and culture conditions
The Pseudomonas aeruginosawild type PAO I was used in this study. For constitutive
GFP expression, PAO1 was transformed with the plasmid pBBR1(MCS5)-Plac-gfp by standard
electroporation methods to produce PAO I -GFP. P. aeruginosastrains were grown in Luria
Broth (LB, Difco) with 30 gg/ml gentamicin to maintain the plasmid. For determination of
planktonic cell viability, bacterial cultures were serially diluted in PBS and plated on LB agar for
CFU counts. OD6 0 0 of 0.0025 represents a culture density of~5.0 x 105 CFU/ml.
50
Mucin purification
This study used native porcine gastric mucin, which differ from industrially purified
properties in their rheological properties (15). Native mucins were purified as previously
reported (16). Briefly, mucus was scraped from fresh pig stomachs and solubilized in sodium
chloride buffer with protease inhibitors and sodium azide. Insoluble material was pelleted by
ultracentrifugation and mucins were purified using size exclusion chromatography on a
Sepharose CL-2B column. Mucin fractions were desalted, concentrated, and lyophilized for
storage. Peptide tandem mass spectrometry was used to determine preparation composition.
Microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry on a Thermo
LTQ-Orbitrap mass spectrometer (Harvard Mass Spectrometry and Proteomics Resource
Laboratory) confirmed that these mucin preparations were dominated by MUC5AC. Notably,
MUC2, MUC5B, MUC6 as well as other proteins including actin, histones, and albumin were
also present. To control for effects by contaminants, the mucin preparations were compared to
mucins further purified by CsCl gradient centrifugation (17, 18).
Static adhesion and biofilm assays
PAOl-GFP was inoculated in a 96-well microtiter plate containing 70 pil of LB with or
without 0.5% mucins at an OD 600 of 0.0025. Glass chips cut from a microscope slide were placed
into each well. Cultures were grown for 8, 12, or 16 hours at room temperature. Glass chips were
carefully removed, rinsed 3 times with PBS, and placed on a coverslip. Adherent bacteria were
visualized with epifluorescence microscopy using an Observer ZI inverted epifluorescence
microscope (Zeiss) with a 20x/0.2 NA dry objective. Adherent bacteria were quantified by
measuring the amount of surface coverage per frame, determined using ImageJ. Depicted images
were taken using a 40x/0.75 NA dry objective (Zeiss).
Flow cell biofilm reactor
A PDMS (Polydimethylsiloxane; Sylgard 184; Dow Coming, MI, USA) microfluidic
device was molded from capillaries anchored to a petri dish yielding a negative imprint of 4
straight microchannels (4 x 2 x 35 mm) which was then bonded to a glass slide. A suspension of
PAOI -GFP at OD 60 0 0.0025 was introduced into the microchannels under continuous flow (0.5
gl/min unless indicated otherwise) driven by a syringe pump (PHD Ultra, Harvard Apparatus,
51
MA, USA) for 48 hours at room temperature. Fresh media was introduced for 5 minutes at 25
pl/min before analysis or treatment. Mucins, methylcellulose (15 cP Sigma), PEG 100 kDa
(Sigma), or PEG 600 kDa (Sigma) in LB were introduced into channels at 0.5%, unless
otherwise stated, for the indicated treatment times. Biofilms were imaged before (referred to as
'0 hours') and after treatments at the middle of the channel using a Zeiss 510 confocal laserscanning microscope (CLSM) with 20x/0.5 NA dry objective with 2x or 4x zoom or, 100x/1.4
NA oil immersion objective for high magnification images. Z-stacks were analyzed using
COMSTAT (19) to determine biofilm biomass, average thickness, and surface roughness
coefficient, and to produce thickness heat maps.
Rheology
Rheological tests were performed on an MCR 302 rheometer (Anton-Paar) in a coneplate geometry. The diameter of the cone was 25 mm, the cone angle was V , and the cone
truncation was 51 gm. 90 pl of 0.5% polymer solutions solubilized in LB were applied to the
rheometer. Shear stress was measured at shear rates between Y= 10/s to Y= 100/s. The viscosity
was calculated assuming a Newtonian relationship between the stress and shear rate, -(7)=
y.
Results
Mucins cause P. aeruginosa biofilm disassembly and structural rearrangement
The main goal of this chapter was to test if mucins can disrupt existing biofilms. To
address this question, biofilms were grown in hydrodynamic flow conditions, which are often
found in biomedical and clinical settings. Continuous flow cell biofilm reactors are a useful
system because 1) they better represent biofilms in non-static conditions, and 2) they are
experimentally advantageous because they allow continuous replenishment of mucins, thereby
preventing changes in mucin availability due to degradation or adsorption to biofilm or flow cell
surfaces.
First, PAO1-GFP biofilms were grown under continuous flow at 0.5 pl/min for 48 hours.
Confocal microscopy revealed that these biofilms exhibited a relatively smooth, flat architecture
(Figure IA). The mature biofilms were then exposed to LB containing 0.5% mucins, and after I
hour analyzed by confocal microscopy. Figure IA shows that the initially smooth biofilms began
to fragment after mucin exposure. In contrast, the biofilms remained unchanged in the mucin52
A
Oh
Oh
B
I
30-
0 LB
II~in.L
20
EMucins
]f
10
ih
1h
C
30.
N
-
LB
BEMucins
20.
0
1h
10
Oh
25
20
10..
1h
ELB
0.0l
SMucins
Figure 1. Mucins trigger disassembly and structural reassembly. (A) Confocal scans of 48 hour PA01 -GFP
biofilms before (0h) and after (1h) treatment with 0.5% mucins at 0.5 pl/min. Scale bars = 20 pm. (B,C)
Quantification of biofilm (B) biomass and (C) average thickness of biofilms. (D) Heat map of biofilm thickness
(pm) after 1 hour treatment with 0.5% mucins. (E) Roughness coefficient of biofilms. Error bars represent
standard deviation of biological replicates (n=3). *p50.05 by unpaired t-test.
free LB control treatment (Figure IA). A quantitative analysis of the biofilm structure and
thickness using COMSTAT (19) revealed that exposure to mucins caused a reduction in biomass
by 63.2% and a reduction of thickness by 56.0%, while the biofilms treated with the mucin-free
LB control remained unchanged (Figure l B-C). More detailed characterization of the biofilm
structure after mucin exposure revealed a drastic structural rearrangement. In Figure 1 D, the
biofilm thickness map generated by COMSTAT shows differences in height and topography.
The roughness coefficient, which quantifies the variability in biofilm height within an image
stack, was 9.7 fold higher in mucin-exposed biofilms compared to the control LB-treated
biofilms (Figure 1 E). In addition, I observed that the bacteria within the mucin-exposed biofilms
appeared to assemble in chains. Higher magnification images revealed that the cells were aligned
along their long edges, like cars in a parking lot (Figure 2). This contrasted with the
53
0.5% Mucins
LB
Figure 2. Mucins cause cellular alignment in P. aeruginosa biofilms. Confocal images of 48 hour PA01-GFP
biofilms treated with LB control or 0.5% mucins for 1 hour at 0.5 pl/min. Scale bars = 20 pm.
LB treated control biofilms, in which bacteria did not appear to be directly associated with one
another. Together, these data indicate that mucin treatment causes the biofilm surface to acquire
a more uneven topography and structural rearrangements within the biofilm.
Mucins are not toxic to P. aeruginosa
To determine if mucins disintegrate biofilms by killing the bacteria, I first tested the
toxicity of mucins toward planktonic P. aeruginosacells. The viability of planktonic PAO1-GFP
in 0.5% mucins was evaluated by counting colony forming units (CFU) over the course of 20
hours of exposure. The number of viable PAOI-GFP cells is not reduced in the presence of
mucins, nor are there effects on the growth rate of the planktonic bacteria over this time period
(Figure 3). Hence, the absence of bactericidal or bacteriostatic effects in planktonic cells by
mucins suggests that mucins are generally nontoxic to P. aeruginosa.
54
1011
* LB
101
U Mucins
0109
~10
108
0
1
2
4
6
20
Time (hours)
Figure 3. Mucins are neither bacteriostatic nor bactericidal toward PA01-GFP. Error bars represent standard
deviation of biological replicates (n=3).
It is possible that biofilm bacteria may respond differently than planktonic bacteria to
mucins, thus I next asked if mucin exposure was toxic to the biofilm bacteria. To test this, I
examined if biofilms could continue development after transient mucin exposure. PAO I -GFP
biofilms were treated with 0.5% mucins for 1 hour, after which the mucin-containing medium
was removed and replaced by LB without mucins. Figure 4 shows that after removal of the
mucins, the biofilms resumed growth, increasing their biomass 3-fold and average thickness 2t=20h
A
B
40
'
m
lh mucins +19h LB
M 20h mInUCs
t=h
20-
A~n fn
19h
- mucins
(LB only)
1
20
TIm (hours)
C
+
40-M
lh mucins+19h LB
IM2h mucns
19h
mucins
20910-
20
1
Time (hours)
Figure 4. Biofilms can continue development after mucin treatments. (A) Confocal images of PA01-GFP biofilms
treated with 0.5% mucins for 1 hour, followed by a chase treatment for 19 hours with LB only or 0.5% mucins.
Scale bars = 20 pm. (B) Biofilm biomass and (C) average thickness of biofilms treated for 1 hour with 0.5%
mucins, followed by a chase treatment with LB only or 0.5% mucins (equivalent to 20 hour continuous mucin
treatment). Error bars represent standard deviation of biological replicates (n 3). *p0.05 by unpaired t-test.
55
fold. This result suggests that viable biofilm cells were still present even after exposure to
mucins.
Biofilm disassembly and structural rearrangement are mucin concentration dependent
To further characterize the influence of mucins on P. aeruginosabiofilms, I asked if the
quantity of mucins introduced to the biofilms would affect the extent of disassembly. 48 hour
biofilms were exposed to 0.05%, 0.1%, 0.5%, or 1.0% mucins under continuous flow at 0.5
.i/min for 1 hour, and then analyzed. Confocal images revealed that treatment with 0.05%
mucins caused no visible structural rearrangement in the biofilms (Figure 5A). Biofilms treated
with 0.1% mucins did not appear fragmented, however small clusters of aligned cells were
embedded throughout a flat biofilm architecture that resembled that of the LB control.
COMSTAT analysis of the biofilms showed that the 0.1% and 0.05% mucin treatments did not
significantly disassemble biofilms, as measured by biofilm thickness or biomass (Figure 5C-D),
indicating that there is a threshold mucin concentration that is required for biofilm disruption. 0.5%
and 1% mucin treatments resulted in fragmentation of the biofilm structures (Figure 5A). The 1%
A
Ah treatment
Oh
B
0.05% Mucins
LB
0.1% Mucins
2 ...-----
---- -----------
h
1% Mucins
D
C
130
0.5% Mucins
40
O-
30-
0.3Oh
- ---------
LB 0.05% 0.1% 0.5% 1.0%
LB 0.05% 0.1% 0.5% 1.0%
Mucins
Mucins
LB 0.05% 0.1% 0.5% 1.0%
Mucins
Figure 5. Mucins impact P. aeruginosa biofilms in a concentration-dependent manner. (A) Confocal images of 48
hour PA01-GFP biofilms treated with mucins for 1 hour 0.5 pl/min show mucin concentration-dependent biofilm
fragmentation. Scale bars = 20 pm. (B) Quantification of biofilm (B) biomass, (C) average thickness, and (D)
roughness coefficient after 1 hour mucin treatment. Dotted lines represent mean of 48 hour biofilms prior to mucin
treatments. Error bars represent standard deviation of biological replicates (n 3). p>0.05 by ANOVA, however the
trend of stronger biofilm disruption by higher mucin concentration treatments was reproducible.
56
mucin treatment resulted in stronger disassembly than the 0.5% mucin treatment (Figure 5B-C),
suggesting that mucin-mediated biofilm disruption is dependent on mucin concentration.
Mucin treatment flow rate affects the degree of biofilm disassembly
Because these experiments are performed in a flow cell system, the fluid mechanics of
the bulk liquid experienced by the biofilm surface may influence the biofilm phenotype. Shear
stress, which is dictated by solution viscosity and flow rate, can impact biofilm growth and
disassembly (9-11). 1 next asked if the mucin-mediated biofilm disruption was affected by the
treatment flow rate. 48-hour PAOI -GFP biofilms were treated with 0.5% mucins for I hour at
slow flow (0.5 p]/min) or fast flow (10 pl/min) (Figure 6A). The fast flow mucin treatment
caused enhanced biofilm disassembly, resulting in a biofilm with 31.6% less biomass (Figure 6B)
and a 41.6% decrease in average thickness (Figure 6C) relative to that of the slow flow treatment.
Furthermore, the roughness coefficient of biofilms after the fast flow treatment increased 2-fold
compared to that of the slow flow treatment (Figure 6D). Thus, a faster treatment flow rate
enhances mucin-mediated disassembly and structural changes in P. aeruginosabiofilms.
Ih treatment
A
Slow flow
B
Fast flow
ISUIB
aC
1
0
0.5% Mucins
LB
0.5% Mucins
LB
Oh
B
D
*
E Mucins
Mucins
~930.
oal0.0
Slow
Fast
Fast
Slow
0:
0.
Slow
Fast
Figure 6. Mucin treatment flow rate impacts P.aeruginosa biofilm disassembly and structural rearrangement. (A)
Confocal images of PA01 -GFPbiofilms treated with 0.5% mucins at slow flow (0.5 pl/min) or fast flow (10 pl/min)
for 1 hour. Scale bars = 20 pm. (BC) Quantification of biofilm (B) biomass and (C) average thickness and (D)
structural rearrangement as measured by roughness coefficient after 1 hour of treatment with LB or 0.5% mucins.
Error bars represent standard deviation of biological replicates (n>3). *ps0.05 by unpaired t-test.
57
Mucin specificity of biofilm disassembly
To determine if the biofilm disassembly was specific to mucins, I examined several
commonly studied synthetic polymer solutions for their abilities to disrupt biofilms. Specifically,
I tested methylcellulose, which has been studied as a mucin mimetic due to similarities in its
viscoelastic properties (20, 21), and polyethylene glycol (PEG), which is commonly used in
antifouling coatings (6). Since mucins are heavily glycosylated polymers (22, 23), it is
interesting to compare them to glycan-based polymers like methylcellulose and non-glycan
polymers like PEG to identify if there are general effects of glycans or viscous polymer solutions.
PAOI- GFP biofilms treated with 0.5% methylcellulose solutions or 0.5% PEG solutions (either
600 kDa or 100 kDa) for 1 hour were observed to exhibit some disassembly and structural
rearrangement, however to a lesser extent than the mucin treated biofilms (Figure 7A).
Methylcellulose treatment caused a 29.4% reduction in biomass (Figure 7B) and 33.9%
reduction in average thickness (Figure 7C), and both 600 kDa and 100 kDa PEG treatments
resulted in similar outcomes (Figure 7B-C). These data indicate that biofilm disruption is not
restricted to glycan-based polymers. However, since neither the glycan-based nor non-glycan
synthetic polymers tested here could disassemble the biofilms to the same degree as mucins, it
suggests that there is some specificity to the effects of mucins on the biofilms.
Because the viscosity of the treatment polymers can contribute to biofilm-disrupting
shear stress (9-11), I worked together with a colleague to characterize the rheological properties
of the 0.5% polymer solutions. Data acquired using a rheometer shows that the mucin and 600
kDa PEG solutions had the highest viscosities, followed by methylcellulose, then 100 kDa PEG
(Figure 7D). Although the mucins and the 600kDa PEG solutions had similar viscosities, mucins
caused a greater degree of biofilm disassembly, suggesting that the viscosity of the polymer
solutions is not directly correlated to the extent of biofilm disruption and that there may be
specific biochemical or molecular properties of mucins responsible for the disruption of P.
aeruginosabiofilms.
58
A
_I
LB
B
htreatment
Methylcellulose
Mucins
C
PEG IOOkDa
D
1.5
1.5
1.0
11.0
0.5-.4
PEG 600kDa
1
E
.5
6
4'
Figure 7. P.aeruginosa bioflim disassembly and structural rearrangement by synthetic polymer solutions. (A)
Confocal images of PAG1-GFP biofllms after treatment with 0.5% polymer solutions in LB at 0.5 p1/min for 1 hour.
Scale bars = 20 pm. (B,C) Quantification of bioftlm disassembly measured by biofllm (B) biomass and (C)
thickness. Reported values for biomass and thickness of biofllms after 1 hour treatment normalized to biofllms
pre-treatment. (D) Viscosity of 0.5% polymer solutions measured by a rheometer. Error bars represent standard
deviation of biological replicates (n=3). *Differs from LB control treatment determined by ANOVA, ps0.05.
Discussion
Here, I report the discovery that mucin biopolymers can disrupt P. aeruginosabiofims,
which are highly resistant to antibiotics and other biofim removal strategies. There are several
possibilities by which mucins may be acting upon the biofims. First, the viscoelastic properties
of mucin solutions may contribute to shear stress, a property of solution viscosity and flow rate,
which is known to impact biofilm detachment (9-11). Increasing the flow rate, and thus shear
stress, of mucin solutions resulted in enhanced biofilm disassembly. Notably, a synthetic PEG
solution with similar viscosity to the mucins exhibited a lesser degree of disassembly, suggesting
that there are additional mucin-specific mechanisms of P. aeruginosabiofilm disruption.
In addition to presenting specific rheological properties, mucins may also trigger a physiological
dispersal response in the biofilm bacteria, potentially via direct interactions between mucins and
bacterial signaling systems. For example, P. aeruginosais known to respond to N59
Acetylglucosamine (GIcNAc), a glycan found on mucins, via a two-component regulator (24).
Mucins may also trigger signaling events by altering nutrient availability and environmental
conditions, which are known regulators of P. aeruginosabiofilm dispersal (25, 26). Furthermore,
mucins have charged and hydrophobic moieties that can potentially interact with matrix EPS or
specific bacterial adhesins, and may compete for binding sites that maintain the biofilm structure.
Preliminary experiments investigating how mucins disrupt P. aeruginosabiofilms, reported in
Appendix A, indicate that flagella are involved in the biofilm response to mucins. The specific
role of the flagella, whether via mucin binding, a dispersal signaling response, or bacterial
motility, remains an open question. Mucins likely disrupt P. aeruginosabiofilms by a
combination of these proposed mechanisms.
Mucin exposure gave rise to several interesting new biofilm phenotypes. First,
microcolonies were observed that remained after mucin exposure under high flow. Why do these
portions of the biofilm persist? Physiological differentiation of bacteria in mature biofilms (28)
may lead to the emergence of subpopulations that vary in their expression of mucin interaction
partners. Another notable phenotype within mucin-treated biofilms was a novel bacterial
alignment, in which the rod-shaped cells aligned lengthwise in chains up to tens of cells long,
similar to cars aligned horizontally in a parking lot. The functions and mechanisms of this
phenotype require further investigation.
Together, this work identifies mucins as biofilm-disrupting agents that can disassemble
and prevent subsequent growth of established biofilms. Further characterization of mucinassociated biofilm phenotypes may lead to the development of mucin-based strategies to regulate
these microbial communities. For example, it remains unknown if mucin exposure can impact
biofilms formed by other microorganisms, or can sensitize the biofilms to certain antibiotics.
Better understanding of the extent and mechanisms of mucin-biofilm interactions may provide
foundation for the innovation of novel mucin-inspired materials that can improve the efficacy of
biofilm eradication treatments.
60
Acknowledgements
This work was performed in collaboration with Dr. Nicole Billings, who contributed to
experimental design, data interpretation, and manuscript preparation, and Scott Grindy (HoltenAndersen Lab), who performed and analyzed the viscosity experiments.
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2001. Growth and Detachment of Cell Clusters from Mature Mixed-Species Biofilms. Appl
Environ Microbiol 67:5608-5613.
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Frenkel ES, Ribbeck K. 2015. Salivary Mucins Protect Surfaces from Colonization by
Cariogenic Bacteria. Appi Environ Microbiol 81:332-338.
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Kavanaugh NL, Zhang AQ, Nobile CJ, Johnson AD, Ribbeck K. 2014. Mucins
Suppress Virulence Traits of Candida albicans. mBio 5:e01911-14.
14. Caldara M, Friedlander RS, Kavanaugh NL, Aizenberg J, Foster KR, Ribbeck K.
Mucin Biopolymers Prevent Bacterial Aggregation by Retaining Cells in the FreeSwimming State. Current Biology.
15.
Kocevar-Nared J, Kristl J, Smid-Korbar J. 1997. Comparative rheological investigation
of crude gastric mucin and natural gastric mucus. Biomaterials 18:677-681.
16. Lieleg 0, Lieleg C, Bloom J, Buck CB, Ribbeck K. 2012. Mucin Biopolymers As BroadSpectrum Antiviral Agents. Biomacromolecules.
17. Smith BF, LaMont JT. 1984. Hydrophobic binding properties of bovine gallbladder mucin.
J Biol Chem 259:12170-12177.
18.
Gong DH, Turner B, Bhaskar KR, Lamont JT. 1990. Lipid binding to gastric mucin:
protective effect against oxygen radicals. Am J Physiol 259:G681-686.
19.
Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersboll BK, Molin S.
2000. Quantification of biofilm structures by the novel computer program comstat.
Microbiology 146:2395-2407.
20. Worku ML a, Sidebotham RL b, Baron JH b, Misiewicz JJ c, Logan RPH b,
Keshavarz T d, Karim QN a. 1999. Motility of Helicobacter pylori in a viscous
environment. Journal of Gastroenterology 11:1143-1150.
21. Smith DJ, Gaffney EA, Gadelha H, Kapur N, Kirkman-Brown JC. 2009. Bend
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Rose MC, Voynow JA. 2006. Respiratory Tract Mucin Genes and Mucin Glycoproteins in
Health and Disease. Physiol Rev 86:245-278.
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Thornton DJ, Sheehan JK. 2004. From Mucins to Mucus. Proc Am Thorac Soc 1:54-61.
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188:7344-7353.
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Liu Y, Tay J-H. 2002. The essential role of hydrodynamic shear force in the formation of
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Microb Ecol 40:75-84.
63
64
Appendix A
Mucin-mediated P. aeruginosa biofilm disassembly is
flagella dependent
Introduction
Chapter 3 demonstrated that mucins have the capacity to disrupt established
Pseudomonasaeruginosabiofilms. In this appendix, I explore the role of bacterial flagella in
mucin-mediated biofilm disassembly. Previous studies have demonstrated that flagella play an
important role in P. aeruginosaresponses to mucins (1-3). In addition, the flagella are involved
in bacterial dispersal out of biofilms (11, 12). Here, I study how mucins influence biofilms of
bacteria lacking the flagellar capfliD or flagellar stators motABCD, both of which are impaired
in swimming motility. I found that mucins could not dissociate AfliD biofilms. Moreover,
biofilms of AmotABCD bacteria, which that have intact flagella but are deficient in flagellar
motility, were also resistant to mucin-mediated disassembly. Together these data suggest that P.
aeruginosaflagellar function is important for mucin disruption of biofilms.
Materials and Methods
Bacterialstrainsand culture conditions
Pseudomonas aeruginosastrain PAO I and its derivatives PAO I AfliD and
PAO I AmotABCD from (4) were used in this study. For constitutive GFP expression, P.
aeruginosastrains were transformed with the plasmid pBBRI(MCS5)-Plac-gfp by standard
electroporation methods to produce PAO I -GFP, PAO I AfliD-GFP, and PAO 1 AmotABCD-GFP.
P. aeruginosastrains were grown in Luria Broth (LB, Difco) with 30 p.g/ml gentamicin to
maintain the plasmid.
Mucin purification
This study used native porcine gastric mucins, which differ from industrially purified
properties in their rheological properties (5). Native mucins were purified as previously reported
65
(6). Briefly, mucus was scraped from fresh pig stomachs and solubilized in sodium chloride
buffer with protease inhibitors and sodium azide. Insoluble material was pelleted by
ultracentrifugation and mucins were purified using size exclusion chromatography on a
Sepharose CL-2B column. Mucin fractions were desalted, concentrated, and lyophilized for
storage. Peptide tandem mass spectrometry was used to determine preparation composition.
Microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry on a Thermo
LTQ-Orbitrap mass spectrometer (Harvard Mass Spectrometry and Proteomics Resource
Laboratory) confirmed that these mucin preparations were dominated by MUC5AC. Notably,
MUC2, MUC5B, MUC6 as well as other proteins including actin, histones, and albumin were
also present. To control for effects by contaminants, the mucin preparations were compared to
mucins further purified by CsCl gradient centrifugation (7, 8).
Flow cell biofilm reactor
A PDMS (Polydimethylsiloxane; Sylgard 184; Dow Coming, MI, USA) microfluidic device was
molded from capillaries anchored to a petri dish yielding a negative imprint of 4 straight
microchannels (4 x 2 x 35 mm), which was then bonded to a glass slide. A suspension of PAOIGFP at OD 6 00 0.0025 was introduced into the microchannels under continuous flow (0.5 p1/min)
driven by a syringe pump (PHD Ultra, Harvard Apparatus, MA, USA) for 48 hours at room
temperature. Fresh media was introduced for 5 minutes at 25 gl/min before analysis or treatment.
Biofilms were treated with 0.5% mucins solubilized in LB for Ihour. Biofilms were imaged
before (referred to as '0 hours') and after treatments at the middle of the channel using a Zeiss
510 confocal laser-scanning microscope (CLSM) with 20x/0.5 NA dry objective with 2x or 4x
zoom or, 100 x/1.4 NA oil immersion objective for high magnification images. Z-stacks were
analyzed using COMSTAT (9) to determine biofilm biomass, average thickness, and surface
roughness coefficient.
Results
Mucin-mediated biofilm disruption is dependent on the P. aeruginosa flagellar capfliD
To elucidate the role of FliD in mucin-mediated biofilm disassembly, the effects of mucin
exposure on P. aeruginosafliDmutant biofilms were examined. PAO 1 AfliD-GFP biofilms were
grown for 48 hours, at which point they closely resembled 48 hour PAO1 wildtype (WT)
66
biofilms (Figure IA). Before and after a 1 hour treatment with 0.5% mucins, AfliD biofilms were
analyzed by confocal microscopy. In contrast to WT biofilms, the mucin treatment did not
significantly change the biofilm biomass, average thickness, or roughness coefficient in AfliD
biofilms (Figure IB-D). The resistance of fliD mutant biofilms to mucin-mediated disruption
suggests involvement of FliD in the phenomenon. I hypothesized that FliD played a role in the
biofilm response to mucins by its contribution to a flagellar motility-based dispersal response.
A
Oh
L1 treatment
LB
Mucinsn
B
2.0 0
LB
*1.5
10.5
WT
W
ARID
AmotABCD
WT
s1I
AmotABCD
-
0
.1 11.
0.IT
Z
1M LB
a Mucins
AmotABCD
Wr
AIDAmo BCD
Figure 1. P.aeruginosa swimming fliD is required for mucin-associated biofilm disassembly and structural
rearrangement. (A) Confocal images of 48-hour PA01-GFP or PA01AfiiD-GFP biofilms before (0h) or after 1
hour treatment with LB control or 0.5% mucins at 0.5 pl/min. Scale bars = 20 pm. (B-C) Quantification of (B)
relative biomass, (C) relative thickness, or (D) fold change in roughness coefficient. Reported values are
biomass, thickness, or roughness coefficient of biofilms treated for 1 hour normalized to that of biofilms before
treatment. Error bars represent standard deviation of biological replicates (n23). *p 0.05 by unpaired t-test.
Role of flagellar motility in mucin-mediated biofilm disassembly
To examine whether mucin-mediated biofilm disassembly was dependent on general
flagellar functioning or the presence of FliD specifically, I studied a mutant that lacked the
motABCD flagellar motor genes. The motABCD mutant bacteria produce flagella (10), but like
fliD mutants, are impaired in swimming motility (Figure 2). PAOlAmotABCD-GFP biofilms
were grown in flow cell chambers for 48 hours at room temperature. Confocal microscopy
67
revealed that the AmotABCD biofilms resembled WT biofilms (Figure IA). Similar to the AJiD
biofilms, treatment of PAOI AmotABCD-GFP biofilms with 0.5% mucins for 1 hour did not
trigger biofilm disassembly (Figure 1 A-C) or structural rearrangement (Figure 1 D). This data
indicates that the presence of the flagella is not sufficient for mucin-mediated biofilm
disassembly, and suggests that functional flagella are involved in the biofilm response to mucins.
A
B
E
-30205
wr
AMID
AmotABCD
Figure 2. P.aeruginosa swimming motility. (A) PA01 strains were inoculated into swim agar (M9 medium with
0
0.3% agar) using a toothpick. Plates were imaged after an18 hour incubation at 30 C. (B) Swim zone diameters
were measured for each strain. Error bars represent standard deviation of biological replicates (n=3). Swimming
diameters of AfiD and AmotABCD bacteria were significantly different than WT using the unpaired t-test, p50.05.
Discussion
Biofilms of P. aeruginosabacteria lacking flagellar capfliD or flagella motor stators
motABCD were resistant to disassembly by mucins, suggesting that mucin-mediated biofilm
disruption of P. aeruginosawildtype biofilms is flagella dependent. Based on these results, I
propose that mucins disrupt biofilms by stimulating active dispersal, which in P. aeruginosa
involves swimming motility (11, 12). To disperse out of the biofilm, the sessile bacteria must
regain swimming motility, and thus must have functioning flagella. In this scenario, swimmingimpaired AfliD and AmotABCD bacteria would not be able to disperse in response to mucins,
potentially explaining the resistance of the mutant biofilms to mucin-mediated biofilm
disassembly. This hypothesis is consistent with previous reports of mucin-associated changes in
flagellar-based surface motility in P. aeruginosa(13) and enhancement of swimming motility in
Escherichiacoli (4).
The data presented here point to a role for flagella in mucin disruption of P. aeruginosa
biofilms, however there is a caveat for studies using mutant bacteria. Although the AfliD and
68
AmotABCD strains formed biofilms that visually resembled wildtype biofilms, the possibility
that mutant biofilms differ in matrix composition or bacterial physiology cannot be eliminated.
Further investigation is required to elucidate the mechanisms of mucin-mediated P. aeruginosa
biofilm disassembly.
References
1.
Scharfman A, Arora SK, Delmotte P, Brussel EV, Mazurier J, Ramphal R, Roussel P.
2001. Recognition of Lewis x Derivatives Present on Mucins by Flagellar Components of
Pseudomonas aeruginosa. Infect Immun 69:5243-5248.
2.
Arora SK, Ritchings BW, Almira EC, Lory S, Ramphal R. 1998. The Pseudomonas
aeruginosa Flagellar Cap Protein, FliD, Is Responsible for Mucin Adhesion. Infect Immun
66:1000-1007.
3.
Landry RM, An D, Hupp JT, Singh PK, Parsek MR. 2006. Mucin-Pseudomonas
aeruginosa interactions promote biofilm formation and antibiotic resistance. Molecular
Microbiology 59:142-151.
4.
Caldara M, Friedlander RS, Kavanaugh NL, Aizenberg J, Foster KR, Ribbeck K.
Mucin Biopolymers Prevent Bacterial Aggregation by Retaining Cells in the FreeSwimming State. Current Biology.
5.
Kocevar-Nared J, Kristl J, Smid-Korbar J. 1997. Comparative rheological investigation
of crude gastric mucin and natural gastric mucus. Biomaterials 18:677-681.
6.
Lieleg 0, Lieleg C, Bloom J, Buck CB, Ribbeck K. 2012. Mucin Biopolymers As BroadSpectrum Antiviral Agents. Biomacromolecules.
7.
Smith BF, LaMont JT. 1984. Hydrophobic binding properties of bovine gallbladder mucin.
J Biol Chem 259:12170-12177.
8.
Gong DH, Turner B, Bhaskar KR, Lamont JT. 1990. Lipid binding to gastric mucin:
protective effect against oxygen radicals. Am J Physiol 259:G681-686.
9.
Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersboll BK, Molin S.
2000. Quantification of biofilm structures by the novel computer program comstat.
Microbiology 146:2395-2407.
10.
Toutain CM, Zegans ME, O'Toole GA. 2005. Evidence for Two Flagellar Stators and
Their Role in the Motility of Pseudomonas aeruginosa. J Bacteriol 187:771-777.
I 1.
Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG. 2002. Pseudomonas
aeruginosa Displays Multiple Phenotypes during Development as a Biofilm. J Bacteriol
184:1140-1154.
69
12.
Davies DG, Marques CNH. 2009. A Fatty Acid Messenger Is Responsible for Inducing
Dispersion in Microbial Biofilms. J Bacteriol 191:1393-1403.
13.
Yeung ATY, Parayno A, Hancock REW. 2012. Mucin Promotes Rapid Surface Motility
in Pseudomonas aeruginosa. mBio 3:e00073-12.
70
Appendix B
Mucins protect Staphylococcus aureus against
Pseudomonas aeruginosa pyocyanin
Introduction
Mucosal surfaces are colonized by both commensal and pathogenic microorganisms,
whose interactions can influence the emergence of pathogenesis (1-3). Pseudomonas aeruginosa
and Staphylococcus aureus are common nosocomial pathogens, frequently found together in
wounds (4-6) and in mucosal environments (7-9), such as the thick, stagnant lung mucus of
cystic fibrosis (CF) patients (7, 9). Co-colonization by these two bacteria can result in enhanced
pathogenicity (10-13) and increased antibiotic resistance (14, 15), and cause delayed wound
healing in the host (16). The dynamics of P. aeruginosaand S. aureus interactions inside
mucosal environments are poorly understood, largely because in most laboratory conditions, P.
aeruginosainhibits and kills S. aureus (17-19), making them difficult to study together.
In contrast to common laboratory culture conditions, more complex in vitro and in vivo
wound environments appear to stabilize the co-existence of P. aeruginosaand S. aureus (11, 14).
For example, complex CF sputum alters the timing and degree of P. aeruginosaantistaphylococcal activity (18). These findings indicate that the interactions between P. aeruginosa
and S. aureus depend on the nature and composition of their environment. These observations
point to mucin glycoproteins, the primary gel-forming polymers in the mucus gel, as prime
candidates to influence the interaction dynamics between S. aureus and P. aeruginosa.The
rationale is that mucins can modulate virulent behavioral traits of single species populations,
such as Helicobacterpylori motility (20), Candidaalbicanshyphae formation (21), and P.
aeruginosa(22) and Streptococcus mutans (23) biofilm formation. Hence, it is likely that mucins
can also influence behavior of bacteria in polymicrobial communities.
To test this hypothesis, I studied whether P. aeruginosaand S. aureus population
dynamics are altered by the presence of mucins. My data show that mucins protect S. aureus
against antagonism by P. aeruginosa. I further demonstrate that mucins can suppress the
71
antagonistic effects of pyocyanin, the secreted phenazine which is largely responsible for P.
aeruginosaanti-staphylococcal activity (24). Specifically, mucins reduce the ability of
pyocyanin to inhibit respiration, stimulate reactive oxygen species (ROS) production, and arrest
growth of S. aureus. Preliminary experiments show that mucins can limit pyocyanin diffusion,
hinting that mucin sequestration of pyocyanin may be responsible for the protective effect. Better
understanding of how mucins impact S. aureus and P. aeruginosainteractions can contribute to
the development of better in vitro infection models, and will provide valuable insight to how
polymicrobial communities function in mucosal environments.
Materials and Methods
Bacterialstrainsand culture conditions
S. aureus UAMS-l and P. aeruginosaPA14 were grown in Luria Broth (LB) at 37'C
with shaking. S. aureus and P. aeruginosawere inoculated from overnight cultures at ~107
CFU/mI in monoculture or co-culture (1 S. aureus: 1 P. aeruginosaratio) at a final volume of
100 p1 in polypropylene 1 ml 96-well plates. Cultures were grown shaking at 37'C for 9 hours
unless indicated otherwise. Native purified mucins (purification described below) were added to
media before inoculation at a final concentration of 0.5%. To evaluate bacterial viability,
samples were vortexed with bead bashing for 10 x 2 seconds to break up aggregates, serially
diluted, and plated on LB agar plates for monocultures. For co-cultures, serial dilutions were
plated on LB agar with 10 pg/ml chloramphenicol agar for P. aeruginosaselection and Mannitol
Salt Phenol Red Agar (Sigma) for S. aureus selection.
For pyocyanin challenge experiments, pyocyanin (Cayman Chemicals) was added to
culture media before inoculation with S. aureus. Pyocyanin from a 5 mg/ml DMSO stock was
diluted in culture media to a final concentration of 100 gM. To evaluate bacterial viability,
samples were vortexed with bead bashing for 10 x 2 seconds to break up aggregates, serially
diluted, and plated on LB agar plates.
Mucin purification
This study used native porcine gastric mucins, which differ from industrially purified
properties in their rheological properties (25). Native mucins were purified as previously
reported (26). Briefly, mucus was scraped from fresh pig stomachs and solubilized in sodium
72
chloride buffer with protease inhibitors and sodium azide. Insoluble material was pelleted by
ultracentrifugation and mucins were purified using size exclusion chromatography on a
Sepharose CL-2B column. Mucin fractions were desalted, concentrated, and lyophilized for
storage.
Pyocyanin extraction
P. aeruginosawas inoculated in co-culture with S. aureus in LB with mucins as
described above. Cultures were incubated with shaking in 1 ml polypropylene 96-well plates at
37'C for 9 hours. The cells were then pelleted at 12,000 x g for 5 minutes and the supernatants
retained. One volume of chloroform was added to the supernatants, which was then vortexed for
3 minutes. The upper phase was removed and the chloroform extraction repeated. The
chloroform phases were pooled and 110 pl 0.2 N HCl was added to further extract the pyocyanin.
The pyocyanin extract (the aqueous phase) was transferred to a microplate and quantified by
measuring OD5 20 on a SpectraMax M3 plate reader (Molecular Devices).
Respirationassay
S. aureus cells were inoculated into LB and incubated with shaking at 37'C until in
exponential phase. The cultures were pelleted, washed, and resuspended in PBS. Cells were
diluted to achieve a final concentration of OD 600 0.1 in PBS containing pyocyanin (final
concentration 100 ptM) with or without mucins (final concentration 0.5%). Cells were incubated
in a total 25 pl in a 96 well half area plate. 2.5 pl Alamar Blue respiratory indicator dye (Life
Technologies) was added to samples and incubated at room temperature for 1 hour. Alamar Blue
fluorescence (Ex 540 nm/Em 590 nm) was measured using a SpectraMax M3 plate reader
(Molecular Devices). Cell-free controls of mucins and pyocyanin were incubated with Alamar
Blue as controls.
ROS
S. aureus cells were grown in LB with shaking at 37'C until exponential phase. S. aureus
was inoculated to reach an OD 600 of 1.0 into LB in the presence or absence of pyocyanin (final
concentration 100 RM) and with or without mucins (final concentration 0.5%). Cultures were
incubated for 2 hours with shaking at 370 C. Cells were pelleted and resuspended in PBS
73
containing 25 ptM H2DCF ROS dye (Life Technologies) and incubated for 90 minutes at room
temperature in the dark. Relative ROS production was determined by measuring fluorescence
(Ex 488 nm/Em 515 nm) and normalizing to the OD 600 of the culture. Measurements were
collected using a SpectraMax M3 plate reader (Molecular Devices).
Mucin binding assay
Pyocyanin binding to mucins was investigated using a modified version of the differential
radial capillary action of ligand assay (DRaCALA) as previously described (27). Mucins
solubilized in PBS were mixed with pyocyanin (final concentration 200 pM) and 2 pl were
dropped on a dry nitrocellulose membrane. Pyocyanin was allowed to diffuse until membranes
were dry. Membranes were photographed and analyzed for intensity of the center spot using
ImageJ.
Results
Mucins protect S. aureus from P. aeruginosa antagonism
S. aureus UAMS-1 and P. aeruginosaPA14 were grown as monocultures or co-cultures
in in Luria Broth (LB), in the presence or absence of 0.5% solubilized mucins. In monoculture
conditions, the presence of mucins did not measurably impact the growth of either organism
(Figure 1). When S. aureus and P. aeruginosawere grown together in co-culture without mucins,
S. aureus viability decreased after 6 hours (Figure IA). However, when mucins were present in
the medium, there were nearly 2 orders of magnitude more viable S. aureus cell than in the
mucin-free co-cultures. This result suggests that in the presence of mucins, S. aureus is protected
from the antagonistic effects of P. aeruginosa.
74
A
B
1010
P10
10I
Monoculture LB
+
-o- Monoculture LB+Mucins
10I
-
Co-culture LB
Co-culture LB+Mucins
10
'~1
10j
10ne
5
0
10:
10
5
10
15
Time (hours)
Time (hours)
Figure 1. P. aeruginosa-S. aureus co-culture growth. (A) Growth of S. aureus monocultures and co-cultures with
P. aeruginosa. Mucins significantly increased S. aureus viability in co-culture at 6, 9, and 12 hours using an
unpaired t-test, p50.05. (B) Growth of P. aeruginosa monocultures and co-cultures with S. aureus. Viability was
determined by CFU counts. Error bars represent standard deviation of biological replicates (n=4).
Mucins suppress the antagonistic effects of pyocyanin toward S. aureus
The anti-staphylococcal activity of P. aeruginosahas been largely attributed to its
production of the pigmented phenazine pyocyanin (24). Indeed, P. aeruginosaproduced
pyocyanin during co-cultures with S. aureus in both mucin-containing and mucin-free conditions,
as determined by colorimetric measurement of chloroform extracts of supernatants (Figure 2C).
A
U101
. --t II
101
0104
+ Control LB
-.
Control LB+Mucins
PyocysrunLB
-o- Pyocyanin LB+Mucins
~10I
0
5
10
15
Time (hours)
10
*C
n
*
B
1 6
.0.8.
S0.3-
.OA
0.2I
10.0L
Control
Mucin
Control
Musins
0.0--
LB
Mucne
Figure 2. Mucins suppress the inhibitory effects of pyocyanin. (A) Growth curve of S. aureus UAMS-1 in 100 pM
pyocyanin. Mucins significantly increased S. aureus viability in pyocyanin cultures at 3, 6, and 9 hours using an
unpaired t-test, p50.05. (B) S. aureus respiration, as measured with Alamar Blue. Reported values are the relative
Alamar Blue signal of bacteria treated with 100 pM pyocyanin normalized to control bacteria. (C) S. aureus ROS
production, measured with H2DCF ROS dye. Reported values are fold change in H2DCF signal of bacteria
treated with 100 pM pyocyanin normalized to control bacteria.( D) P. aeruginosa PA14 pyocyanin production.
Pyocyanin was extracted from P. aeruginosa monocultures or co-cultures with S. aureus, and quantified
colorimetrically. Error bars represent standard deviation of biological replicates (n 3). *p5<.5 by unpaired t-test.
75
To determine if mucins protect S. aureus against pyocyanin, I examined the antagonistic effects
of the phenazine in the presence of mucins. First, I evaluated the viability of S. aureus cultures
challenged with 100 pM purified pyocyanin. Without mucins, pyocyanin efficiently suppressed S.
aureus growth (Fig. 2A). However, in the presence of mucins, S. aureus could partially
overcome the pyocyanin-associated growth inhibition, suggesting that mucins can mitigate the
antimicrobial activity of pyocyanin toward S. aureus (Figure 2A).
I next tested if mucins influence the ability of pyocyanin to inhibit aerobic respiration and
stimulate reactive oxygen species (ROS). First, S. aureus respiration was measured in the
presence and absence of mucins using the metabolic indicator Alamar Blue, which fluoresces
upon reduction by the electron transport chain. In the absence of mucins, pyocyanin suppressed
respiration by 65% (Figure 2B). In contrast, in the presence of mucins, pyocyanin reduced
respiration only by 32% (Figure 2B), indicating that mucins can partially prevent pyocyaninmediated inhibition of S. aureus respiration. Similarly, I measured the ROS levels in S. aureus
after pyocyanin exposure using the fluorescent ROS indicator H2DCF. Figure 2C reveals that
pyocyanin exposure stimulated a 5.0 fold increase in S. aureus ROS levels relative to that of the
pyocyanin-free control. However, in the presence of mucins, the same concentration of
pyocyanin only resulted in a 2.3 fold increase in ROS levels. Together these experiments show
that mucins suppress the antagonistic effects of pyocyanin against S. aureus.
Mucins restrict pyocyanin diffusion
How do mucins suppress pyocyanin activity? Mucins may sequester pyocyanin to lower
the effective concentration that can access S. aureus cells. To investigate this hypothesis, I used
the differential radial capillary action of ligand assay (DRaCALA) (27), which exploits the
ability of nitrocellulose membranes to immobilize proteins but not small molecules. 200 gM
pyocyanin in buffer with or without mucins was applied to the membrane. Capillary action
caused unbound pyocyanin to radially migrate from the initial spot of application, whereas in the
presence of mucins, pyocyanin remained associated with the immobile initial spot, indicating an
interaction between mucins and the pyocyanin (Figure 3A). Quantification of pyocyanin pigment
intensity at the center revealed that the degree of pyocyanin retention at the initial spot of
76
A
30Y
00
C20C
s10
0
0.1
0.25
05
Mucin concentration (%)
Figure 3. Mucins restrict pyocyanin diffusion. (A) Schematic of DRaCALA. A mixture of mucins and pyocyanin
are spotted onto a nitrocellulose membrane. Radial capillary action causes unbound pyocyanin to diffuse
outward, while pyocyanin bound to immobilized mucins remain associated with the inner spot. (B)
Quantification of pyocyanin bound to mucins in the inner spot of the membrane, measured by pigment intensity
using ImageJ. Error bars represent standard deviation of biological replicates (n=3). *p 0.05 and **ps0.01 by
ANOVA.
application correlated with the mucin concentration present (Figure 3B). This experiment
suggests that pyocyanin can be bound by mucins. Future work is needed to determine if
pyocyanin-mucin binding indeed leads to the reduced antagonism toward S. aureus.
Discussion
The goal of this study was to test if mucus components, specifically its gel-forming mucin
polymers, influence the interactions between P. aeruginosaand S. aureus. This is an important
question because microbial physiology and polymicrobial communities are often studied in
laboratory conditions that lack essential elements of the native environments where these
microbes are found. The data here show that the presence of mucins can alter P. aeruginosaand
S. aureus dynamics. Specifically, mucins promote co-existence of these organisms, which is
interesting because P. aeruginosaoutcompetes S. aureus in most laboratory culture conditions
(17, 18). This finding may explain why P. aeruginosaand S. aureus are found together on
mucosal epithelia, but not in less complex laboratory broth cultures.
77
While the mechanisms by which mucins affect P. aeruginosaand S. aureus interactions
remain unknown, the data here indicate that neutralization of pyocyanin by mucins may
contribute to the protective effect. Mucins, which are known to interact with a range of particles,
proteins, and small molecules like antibiotics (29, 30), may reduce the antagonistic effects of
pyocyanin by direct binding with the phenazine. Mucin-pyocyanin interactions may lead to
sequestration, effectively lowering the amount of pyocyanin that can access S. aureus cells.
Supporting this hypothesis, the data presented here indicate that mucins can reduce the diffusion
of pyocyanin. Because the pore size of mucin networks are two orders of magnitude larger than
small molecules like pyocyanin (31-33), mucins probably inhibit diffusion via pyocyanin-mucin
binding rather than by size exclusion. Specifically, hydrophobic moieties of pyocyanin may
interact with hydrophobic regions on the mucin peptide backbone. The ability of mucins to
reduce pyocyanin diffusion is somewhat specific, as preliminary experiments show that albumin
cannot achieve this effect.
Another mechanism by which mucins could reduce antagonism of pyocyanin or other P.
aeruginosaanti-staphylococcal systems is by altering the physiology of S. aureus to render it
more resistant. It is possible that a metabolic switch from aerobic respiration to fermentation
could confer resistance to pyocyanin and other respiratory inhibitors, which are only antagonistic
under aerobic conditions (24). This possibility should be further examined, for example by
analysis of oxygen levels and fermentation genes and products in mucin-containing and mucinfree cultures. Other physiological responses of S. aureus to mucins may also provide protection
against P. aeruginosa,for example mucins could stimulate production of factors that degrade or
inactivate pyocyanin or other P. aeruginosatoxins. Further investigation is required to elucidate
the mechanisms of mucin protection of S. aureus against pyocyanin and broader P. aeruginosa
antagonism.
The discovery that mucins impact S. aureus and P. aeruginosa interactions may have
important implications for other mucosal microbes. The ability of mucins to dictate interspecies
dynamics likely extends to other microbe-microbe, as well as microbe-host interactions.
Additionally, since mucins can support the growth of S. aureus in otherwise non-permissive
conditions, mucins may be a helpful tool for isolating other mucosal inhabitants that are difficult
to culture, for example members of the gut microbiota. This work emphasizes the importance of
recreating physiologically relevant conditions to study microbial behavior.
78
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DeLeon S, Clinton A, Fowler H, Everett J, Horswill AR, Rumbaugh KP. 2014.
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Michelsen CF, Christensen A-MJ, Bojer MS, Hoiby N, Ingmer H, Jelsbak L. 2014.
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81
82
Appendix C
Mucins suppress P. aeruginosa quorum sensing genes
Introduction
Virulence of the opportunistic mucosal pathogen Pseudomonasaeruginosais largely
controlled by the quorum sensing cell communication systems. In these systems, bacteria secrete
signaling molecules called autoinducers, which accumulate in the surrounding environment and
are sensed by neighboring cells. Quorum sensing was first described as a bacterial response to
cellular density (7), however recent work revealed that environmental conditions independent of
cell density also regulate P. aeruginosaquorum sensing (8, 9). To better understand P.
aeruginosamucosal infections, it is necessary to study its quorum sensing systems in
physiological environments because 1) autoinducers must diffuse through the extracellular
environment-in this case mucus-to reach neighboring cells, and 2) environmental factors can
impact quorum sensing signaling. Furthermore, recent studies point to mucin glycoproteins, the
primary gel-forming mucus component, as regulators of microbial virulence (13-15). Using
native purified mucins, I demonstrate that mucins suppress P. aeruginosaquorum sensing gene
expression.
Materials and Methods
Bacterialstrains and culture conditions
P. aeruginosaPA14 strains were maintained in M9 media with 1% glucose (w/v) and 1%
casamino acids with 500 pg/ml kanamycin when required. To monitor gene expression, PA14
was transformed with lux reporter plasmids in the pMS402 background (8).
Mucin purification
This study used native porcine gastric mucin, which differ from industrially purified
properties in their rheological properties (17). Native mucins were purified as previously
83
reported (19). Briefly, mucus was scraped from fresh pig stomachs and solubilized in sodium
chloride buffer with protease inhibitors and sodium azide. Insoluble material was pelleted by
ultracentrifugation and mucins were purified using size exclusion chromatography on a
Sepharose CL-2B column. Mucin fractions were desalted, concentrated, and lyophilized for
storage. Lyophilized mucins were reconstituted shaking gently at 4*C overnight in the M9 media.
Transcriptionalreporterassay
P. aeruginosagene expression was monitored in PA14 harboring pMS402 lux-based
reporter plasmids (8). Reporter bacteria were inoculated from exponential growth to OD 600 0.05
in M9 media a black, clear bottom 96 well plate. Cultures were grown shaking at 37'C in a
Tecan Infinite 200 PRO microplate reader, and reporter activity (luminescence, 10OOms
integration time) and bacterial growth (OD 600 ) were measured every 30 minutes for 15 hours.
Results
Mucins suppress P. aeruginosa Las and Rhl quorum sensing genes
To determine the effect mucins on P. aeruginosaquorum sensing, gene expression of
autoinducer synthases (lasI, rh/I) were measured using transcriptional reporters. PAO 1 with
plasmids containing lux promoter fusions were inoculated into M9 media and grown at 37*C
with shaking. Mucins, which did not affect bacterial growth (Figure 1 A), suppressed both lasI
and rh/I quorum sensing genes. The maximum expression of lasI occurred around 5 hours of
growth, and was suppressed by -30% (Figure 1 B) in the presence of mucins. For rhlI, maximum
A
B
10 + M9Media
iX
I
*1
C
+ MMbda
~TTTI~ITIWT~
0.
0.5
o
0.01
0
5
10
Tim. (hours)
15
0.
IM9eMsdia
Iasi
0
10
5
Time (hours)
la
15
0.
rhl
0
10
5
Tim. (hours)
Figure 1. Mucins suppress P. aeruginosa quorum sensing genes. (A) Growth curve of P. aeruginosa with M9 media with
or without 0.5% mucins. (B, C) P. aeruginosa expression of (B) lasi or (C) rhil determined using luminescent
transcriptional reporters. Reported values are luminescence values normalized to the highest value within each
experiment. Error bars represent standard deviation of biological replicates (n23).
84
11
expression was delayed from early stationary phase (9 hours of growth) in the mucin-free control,
to late stationary phase (15 hours of growth) in the presence of mucins (Figure 1 C).
Mucins suppress quorum sensing-regulated virulence genes
Given that P. aeruginosaquorum sensing controls virulence genes, I next used
transcriptional reporters to examine whether mucins could also suppress quorum sensingregulated virulence genes phenazinephzAl, protease aprA, and rhamolipid rhiA. The expression
of all three virulence genes was suppressed in mucins (Figure 2A-C). Of note, the virulence gene
expression in mucins in late stationary phase is similar to transcriptional levels found in mucinfree media. This may be due to a growth-phase dependent mucin response or degradation of
mucins such that the biopolymers can no longer impact quorum sensing or other genes. These
results indicate that mucins suppress P. aeruginosavirulence genes at the level of transcription.
A
-0-
uis--+md 1.0. +Muciris
0
+ M9 Meda
M9 Meda
1
1.0. o-
0.0
C
B
Mg Meda
5
10
Time (hours)
15
0.
0
5
1,0
Tiaw (hours)
is
0.0 0
5
10
Tirm (hours)
r
15
Figure 2. Mucins suppress P. aeruginosa virulence genes. P. aeruginosa expression of (A) aprA, (B) phzA 1, and (C) rhiA
determined using luminescent transcriptional reporters. Reported values are luminescence values normalized to the
highest value within each experiment. Error bars represent standard deviation of biological replicates (n 3).
Discussion
Mucins have long been known to contribute to mucus' barrier properties (10-12). Recent
studies describing mucins as microbial virulence modulators (14) led me to investigate the effect
of mucins on P. aeruginosavirulence, specifically quorum sensing genes. The findings here
indicate that mucins can suppress P. aeruginosaquorum sensing and virulence traits, and
corroborate the broader notion that mucins regulate microbial virulence. One possible
mechanism is that mucins sequester the autoinducer signals, thereby interrupting the positive
feedback quorum sensing systems. Another possibility is that the mucin environment triggers a
physiological change that results in intracellular regulation of quorum sensing signaling and
virulence. Mucin-mediated virulence suppression may be a host defense strategy to keep these
85
opportunists in check. Further work is necessary to understand the mechanisms by which mucins
interfere with P. aeruginosaquorum sensing.
References
1.
Estepa V, Rojo-Bezares B, Torres C, Saenz Y. 2014. Faecal carriage of Pseudomonas
aeruginosa in healthy humans: antimicrobial susceptibility and global genetic lineages.
FEMS Microbiol Ecol 89:15-19.
2.
Kerckhoffs APM, Ben-Amor K, Samsom M, van der Rest ME, de Vogel J, Knol J,
Akkermans LMA. 2011. Molecular analysis of faecal and duodenal samples reveals
significantly higher prevalence and numbers of Pseudomonas aeruginosa in irritable bowel
syndrome. J Med Microbiol 60:236-245.
3.
Govan JR, Deretic V. 1996. Microbial pathogenesis in cystic fibrosis: mucoid
Pseudomonas aeruginosa and Burkholderia cepacia. Microbiological Reviews 60:539 -574.
4.
Apodaca G, Bomsel M, Lindstedt R, Engel J, Frank D, Mostov KE, Wiener-Kronish J.
1995. Characterization of Pseudomonas aeruginosa-induced MDCK cell injury:
glycosylation-defective host cells are resistant to bacterial killing. Infect Immun 63:15411551.
5.
Hatchette TF, Gupta R, Marrie TJ. 2000. Pseudomonas aeruginosa CommunityAcquired Pneumonia in Previously Healthy Adults: Case Report and Review of the
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6.
Bodey GP, Bolivar R, Fainstein V, Jadeja L. 1983. Infections Caused by Pseudomonas
aeruginosa. Clin Infect Dis 5:279-313.
7.
Nealson KH, Hastings JW. 1979. Bacterial bioluminescence: its control and ecological
significance. Microbiol Rev 43:496-518.
8.
Duan K, Surette MG. 2007. Environmental Regulation of Pseudomonas aeruginosa PAO I
Las and Rhl Quorum-Sensing Systems. J Bacteriol 189:4827-4836.
9.
Smith EE, Buckley DG, Wu Z, Saenphimmachak C, Hoffman LR, D'Argenio DA,
Miller SI, Ramsey BW, Speert DP, Moskowitz SM, Burns JL, Kaul R, Olson MV.
2006. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis
patients. Proc Natl Acad Sci USA 103:8487-8492.
10.
Linden SK, Sutton P, Karlsson NG, Korolik V, McGuckin MA. 2008. Mucins in the
mucosal barrier to infection. Mucosal Immunology 1:183-197.
11.
McGuckin MA, Linden SK, Sutton P, Florin TH. 2011. Mucin dynamics and enteric
pathogens. Nat Rev Microbiol 9:265-278.
86
12.
Thornton DJ, Rousseau K, McGuckin MA. 2008. Structure and Function of the
Polymeric Mucins in Airways Mucus. Annual Review of Physiology 70:459-486.
13.
Kawakubo M, Ito Y, Okimura Y, Kobayashi M, Sakura K, Kasama S, Fukuda MN,
Fukuda M, Katsuyama T, Nakayama J. 2004. Natural Antibiotic Function of a Human
Gastric Mucin Against Helicobacter pylori Infection. Science 305:1003-1006.
14.
Kavanaugh NL, Zhang AQ, Nobile CJ, Johnson AD, Ribbeck K. 2014. Mucins
Suppress Virulence Traits of Candida albicans. mBio 5:e01911-14.
15.
Caldara M, Friedlander RS, Kavanaugh NL, Aizenberg J, Foster KR, Ribbeck K.
Mucin Biopolymers Prevent Bacterial Aggregation by Retaining Cells in the FreeSwimming State. Current Biology.
16.
Matsui H, Wagner VE, Hill DB, Schwab UE, Rogers TD, Button B, Taylor RM,
Superfine R, Rubinstein M, Iglewski BH, Boucher RC. 2006. A physical linkage
between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms.
PNAS 103:18131-18136.
17.
Kocevar-Nared J, Kristl J, Smid-Korbar J. 1997. Comparative rheological investigation
of crude gastric mucin and natural gastric mucus. Biomaterials 18:677-681.
18.
Crater JS, Carrier RL. 2010. Barrier properties of gastrointestinal mucus to nanoparticle
transport. Macromol Biosci 10:1473-1483.
19.
Lieleg 0, Lieleg C, Bloom J, Buck CB, Ribbeck K. 2012. Mucin Biopolymers As BroadSpectrum Antiviral Agents. Biomacromolecules.
20.
Hauser AR. 2009. The type III secretion system of Pseudomonas aeruginosa: infection by
injection. Nat Rev Microbiol 7:654-665.
87
88
Appendix D
Mucins reduce P. aeruginosa
epithelial attachment and cytotoxicity
Introduction
Pseudomonas aeruginosacauses infection in mucosal environments, including the
respiratory (1-3) and intestinal tracts (4, 5), where it can cause epithelial injury (6). In vitro
studies of epithelial cytotoxicity by P. aeruginosaprimarily use cell culture models that lack the
protective mucus barrier. However, mucus and specifically its gel-forming polymers, mucins,
play an important role in mediating host-microbe interactions in vivo (7, 8). Previous work
examining the impact of mucins on P. aeruginosabehavior showed that the biopolymers could
reduce bacterial surface attachment to abiotic surfaces (9). In this study, I use A549 lung
epithelial cells as an in vitro model to study the effects of mucins on P. aeruginosaattachment.
The data here demonstrate that mucins reduce P. aeruginosaattachment to epithelial cells.
Furthermore, I investigated how mucins impacted epithelial viability in response to P.
aeruginosaexposure because direct pathogen-host cell contact can lead to epithelial injury (1012). 1 found that mucins can protect epithelial cells against P. aeruginosacytotoxicity. The
findings here support the notion that mucins act as a barrier to protect against microbial invasion
(11-13).
Materials and Methods
Bacterialstrains and culture conditions
P. aeruginosaPA14 strains were maintained in Luria Broth (LB) medium. For
microscopic visualization, PAI4-GFP was made by transforming PA14 with pBBR1(MCS5)Plac-gfp, and grown in 30 pg/ml gentamicin to maintain the plasmid.
89
Mucin purification
This study used native porcine gastric mucins, which differ from industrially purified
properties in their rheological properties (16). Native mucins were purified as previously
reported (17). Briefly, mucus was scraped from fresh pig stomachs and solubilized in sodium
chloride buffer with protease inhibitors and sodium azide. Insoluble material was pelleted by
ultracentrifugation and mucins were purified using size exclusion chromatography on a
Sepharose CL-2B column. Mucin fractions were desalted, concentrated, and lyophilized for
storage. Lyophilized mucins were reconstituted shaking gently at 4'C overnight in the Ham's F12 (Kaighn's) medium (Invitrogen).
Epithelial cell culture
A549 airway epithelial cells were maintained in T25 flasks in Ham's F-12 (Kaighn's)
medium (Invitrogen) with 10% fetal bovine serum (Invitrogen) and 1% antibiotics (25 U/mL
penicillin, 25 pig/mL streptomycin (Invitrogen)). The cells were detached using trypsin-EDTA
(Invitrogen) and passaged before reaching confluence, approximately every 2-3 days.
P. aeruginosaattachment assay
A549 cells were seeded in 96 well plates at a density of 2 x 104 cells/well and incubated
overnight at 37'C with 5% CO 2. I hour prior to infection, wells were washed with PBS, and
incubated in unmodified in Ham's F-12 (Kaighn's) medium (Invitrogen). A549 cells were
exposed to PAI4-GFP in unmodified Ham's F-12 (Kaighn's) medium at multiplicity of infection
(MOI) 10 for 15 minutes at 370 C with 5% CO2 . Non-adherent bacteria were removed by
aspiration and adherent bacteria were visualized with phase contrast and epifluorescence
microscopy using an Axio Observer ZI inverted epifluorescence microscope (Zeiss) with a
40x/0.75 NA dry objective (Zeiss). The number of fluorescent bacteria and epithelial cells in
each frame were manually counted.
Epithelialcytotoxicity assay
A549 cells were seeded in 96-well plates at a density of 2 x 104 cells/well and incubated
overnight at 37'C with 5% CO 2. 1 hour prior to infection, wells were washed with PBS, and
incubated in unmodified in Ham's F-12 (Kaighn's) medium (Invitrogen). PA14-GFP grown to
90
exponential phase in LB was pelleted, and resuspended in serum-free Ham's F-12 (Kaighn's)
medium. PA14 was inoculated in serum-free Ham's F-12 (Kaighn's) medium containing mucin
as indicated. For cytotoxicity experiments, A549 was infected with PA14 at multiplicity of
infection (MOI) 50 for 1 h at 37 0 C with 5% CO 2. Bacteria were removed and replaced with
serum-free Ham's F-12 (Kaighn's) medium with 200 pg/mi gentamicin for 30 min at 370 C with
5% CO2 to kill adherent bacteria. The metabolic indicator dye Alamar Blue was then added to
measure epithelial cell survival as a measure of metabolic activity. After 3 hours of incubation at
370 C with 5% CO 2, viability was determined by measuring Alamar blue fluorescence (Ex. 530
nm/Em. 590 nm).
Results
Mucins reduce P. aeruginosa epithelial attachment
Mucins can prevent P. aeruginosaattachment to synthetic surfaces (9), however the
ability of native mucins to protect epithelial surfaces against P. aeruginosahas not yet been
studied. Using A549 human lung carcinoma cells to model the respiratory epithelium, I
examined epithelial attachment of the GFP-expressing P. aeruginosastrain PA I 4-GFP.
A
Control
0.1% Mucins
I
1.0
-
B
0.0.5-
40.0
Control 1% Mucins
Figure 1. Mucins suppress P. aeruginosa attachment to A549 epithelial cells. (A) Top: Phase contrast
images of A549 epithelial cells. Bottom: Fluorescent images of PA14-GFP attached to A549 epithelial cells.
Scale bar=10 pm. (B) Quantification of P. aeruginosa attachment to A549 epithelial cells. Reported values
are number of attached P. aeruginosa cells normalized to total number of epithelial cells. Error bars represent
standard deviation of biological replicates (n=3). *p0.05 by unpaired t-test.
91
Epithelial cells were incubated with PAl4-GFP for 15 minutes in the presence or absence of 0.5%
mucins, and then non-adherent bacteria were removed. Microscopy of attached bacteria revealed
that P. aeruginosaepithelial attachment was reduced from an average 0.81 bacteria per epithelial
cell in the mucin-free control to 0.35 bacteria per epithelial cell in the presence of mucins (Figure
1).
Mucins protect epithelial cells against P. aeruginosa cytotoxicity
The ability of mucins to reduce P. aeruginosaattachment to epithelial cells suggests that
the polymers may also affect the epithelial viability in response to bacterial exposure. Direct
contact of P. aeruginosawith host cells is known to contribute to epithelial death via the type III
secretion (T3SS) (10). To test if mucins affect P. aeruginosaepithelial cytotoxicity, I used the
fluorescent dye Alamar Blue to measure A549 epithelial cell viability after exposure to PA14 for
1 hour in the presence or absence of the polymers. Figure 2 shows that mucins suppressed P.
aeruginosaepithelial cytotoxicity in a concentration dependent manner: 0.25%, 0.5%, and 1%
mucins reduced cytotoxicity from 80% (mucin-free control) to 43.8%, 37.8%, and 17.4%,
respectively (Figure 2). These experiments suggest that mucins protect epithelial cells from
cytotoxicity by P. aeruginosa.
*
100*
80-
60S4020
0-
1.0
0.5
Control 0.25
Mucn %-
Figure 2. Mucins protect epithelial cells against P. aeruginosa cytotoxicity. A549 epithelial cells were evaluated
for viability using the fluorescent Alamar Blue viability dye after exposure to P. aeruginosa PA14. Reported
values are fluorescence of epithelial cells exposed to P. aeruginosa normalized to untreated control epithelial
cells. Error bars represent standard deviation of biological replicates (n=3). *ps0.05 by ANOVA.
92
Discussion
Mucins have been demonstrated to protect epithelia from attachment by Helicobacter
pylori (18), Staphylococcus aureus (19, 20),, Candida albicans(21), and gut commensals (8).
Here, I show that mucins can suppress P. aeruginosaepithelial attachment, as well as
cytotoxicity. There are several mechanisms by which mucins could confer these protective
effects. Mucins may block bacterial access to the epithelial cells by providing a physical or
biochemical barrier. Furthermore, the biopolymers may prevent epithelial interactions by
occupying bacterial receptors necessary for epithelial binding. Since the P. aeruginosatype 3
secretion system (T3SS), a primary mechanism of epithelial cytotoxicity, requires direct host cell
contact, then suppression of P.aeruginosaepithelial attachment by mucins could contribute to
the reduced levels of cytotoxicity. In addition, mucins might reduce bacteria-induced epithelial
injury by triggering physiological changes in P. aeruginosathat downregulate virulence factors.
This phenomenon has previously been demonstrated for C. albicans, which suppresses
pathogenic traits like hyphal formation in the presence of mucins (21). The results presented here
support the notion that mucins are a key contributor of the ability of mucus to regulate microbial
invasion and toxicity.
References
I.
Estepa V, Rojo-Bezares B, Torres C, Saenz Y. 2014. Faecal carriage of Pseudomonas
aeruginosa in healthy humans: antimicrobial susceptibility and global genetic lineages.
FEMS Microbiol Ecol 89:15-19.
2.
Kerckhoffs APM, Ben-Amor K, Samsom M, van der Rest ME, de Vogel J, Knol J,
Akkermans LMA. 2011. Molecular analysis of faecal and duodenal samples reveals
significantly higher prevalence and numbers of Pseudomonas aeruginosa in irritable bowel
syndrome. J Med Microbiol 60:236-245.
3.
Govan JR, Deretic V. 1996. Microbial pathogenesis in cystic fibrosis: mucoid
Pseudomonas aeruginosa and Burkholderia cepacia. Microbiological Reviews 60:539 -574.
4.
Markou P, Apidianakis Y. 2014. Pathogenesis of intestinal Pseudomonas aeruginosa
infection in patients with cancer. Front Cell Infect Microbiol 3.
5.
Boukadida J, de Montalembert M, Gaillard JL, Gobin J, Grimont F, Girault D, Veron
M, Berche P. 1991. Outbreak of gut colonization by Pseudomonas aeruginosa in
93
immunocompromised children undergoing total digestive decontamination: analysis by
pulsed-field electrophoresis. J Clin Microbiol 29:2068-2071.
6.
Apodaca G, Bomsel M, Lindstedt R, Engel J, Frank D, Mostov KE, Wiener-Kronish J.
1995. Characterization of Pseudomonas aeruginosa-induced MDCK cell injury:
glycosylation-defective host cells are resistant to bacterial killing. Infect Immun 63:15411551.
7.
McGuckin MA, Every AL, Skene CD, Linden SK, Chionh YT, Swierczak A, McAuley
J, Harbour S, Kaparakis M, Ferrero R, Sutton P. 2007. Muc Mucin Limits Both
Helicobacter pylori Colonization of the Murine Gastric Mucosa and Associated Gastritis.
Gastroenterology 133:1210-1218.
8.
Johansson MEV, Phillipson M, Petersson J, Velcich A, Holm L, Hansson GC. 2008.
The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria.
Proc Natl Acad Sci USA 105:15064-15069.
9.
Caldara M, Friedlander RS, Kavanaugh NL, Aizenberg J, Foster KR, Ribbeck K.
Mucin Biopolymers Prevent Bacterial Aggregation by Retaining Cells in the FreeSwimming State. Current Biology.
10.
Hauser AR. 2009. The type III secretion system of Pseudomonas aeruginosa: infection by
injection. Nat Rev Microbiol 7:654-665.
11.
Fleiszig SM, Wiener-Kronish JP, Miyazaki H, Vallas V, Mostov KE, Kanada D, Sawa
T, Yen TS, Frank DW. 1997. Pseudomonas aeruginosa-mediated cytotoxicity and
invasion correlate with distinct genotypes at the loci encoding exoenzyme S. Infect Immun
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Linden SK, Sutton P, Karlsson NG, Korolik V, McGuckin MA. 2008. Mucins in the
mucosal barrier to infection. Mucosal Immunology 1:183-197.
14.
Thornton DJ, Rousseau K, McGuckin MA. 2008. Structure and Function of the
Polymeric Mucins in Airways Mucus. Annual Review of Physiology 70:459-486.
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McGuckin MA, Lind6n SK, Sutton P, Florin TH. 2011. Mucin dynamics and enteric
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McGuckin MA. 2009. MUCI Limits Helicobacter pylori Infection both by Steric
Hindrance and by Acting as a Releasable Decoy. PLoS Pathog 5.
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Chapter 4
Discussion
Mucus and mucins in vivo protect the host epithelia against mucosal microbes by acting
as a physico-chemical barrier and displaying antimicrobial factors (1, 2). Beyond its barrier
function, mucins are emerging as modulators of microbial physiology. This thesis demonstrates
several bacteria-regulating functions of mucins. Specifically, mucins can impact the development
and disassembly of bacterial biofilms and regulate intraspecies and interspecies microbe-microbe
single species and multi-species communities. Whereas most research prior to this thesis focused
on how microbes affect mucins, this work focuses on the opposite question: how do mucins
influence bacterial behavior? Based on my results, I suggest that mucins are potent regulators of
bacterial behavior inside the body, and have strong potential to be used in antimicrobial
strategies in clinical contexts.
In Chapter 2, we showed that purified mucins can be used to generate surface coatings
that suppress the attachment of Streptococcuspneumoniae and Staphylococcus aureus. We
demonstrated through the removal of mucin-associated glycans that these sugars provide
structure to the coatings, and are necessary for bacterial repulsion. These findings implicate both
mucin polymer brush structure and the use of glycans as considerations for the development of
better anti-adhesive coatings.
Mucin-associated glycans also play a role in reducing bacterial attachment in vivo (3),
however, it is unclear whether the mechanisms of repulsion are similar in vivo and for mucin
coatings. While mucins in vivo are anchored to epithelial membranes or polymerized in a
hydrogel network, mucins adsorbed to polystyrene as coatings may differ in their biochemistry
and structure. For coatings, interactions between the mucins and the underlying polystyrene
likely occupy or occlude bacterial binding sites. Additionally, surface adsorption may cause
mucin denaturation, thereby altering mucin conformation and potentially introducing new
binding sites. Thus, our insights to bacterial repulsion by mucin surface coatings cannot be
directly applied to explain microbe-mucin interactions in vivo. Despite this, the success of mucin
97
coatings in our in vitro system suggests that they are valuable tools for hindering bacterial
surface attachment.
In addition to hindering bacterial attachment, the first stage of biofilm formation, my
results in Chapter 3 show that mucins mediate the dissociation of mature P. aeruginosabiofilms.
This finding demonstrates the potential of mucins as anti-biofilm strategies, which is exciting
because new and more efficacious biocompatible strategies are urgently needed. The data
presented here suggests that this effect is specific to mucins, as other synthetic polymers tested in
-
this study could not achieve the same outcome. Although the mechanism behind mucin
mediated biofilm disassembly is not fully understood, I demonstrate in Appendix A the
involvement of the flagella in this phenomenon. Biofilms of swimming-impairedfliD or
motABCD mutants were resistant to disruption by mucins, suggesting that flagellar motility plays
an important role in the response of P. aeruginosabiofilms to mucins. Mucins may stimulate a
chemotactic or biofilm dispersal response, which both engage swimming motility (4-6). To
identify the physiological pathways that are triggered by mucins, transcriptomic or proteomic
analyses of mucin-treated biofilms may be informative. Elucidating the specific mechanisms of
mucin-mediated disassembly could reveal new pathways that can be targeted to control biofilms.
The work presented in Chapter 3 and Appendix A focus on how P. aeruginosabiofilms
respond to mucins, but it is currently unknown whether mucins can disrupt biofilms formed by
other microorganisms. Therefore, more exploration is necessary to understand the extent of the
biofilm-disrupting capability of mucins. Moreover, the specific effects of mucins on biofilms
require further characterization. For example, do mucins sensitize biofilms to antibiotic
treatments? Broader understanding of the impacts of mucins on biofilms may lead to the use of
mucins or mucin-inspired strategies to eradicate biofilms.
After establishing that mucins affect the physiology of monospecies cultures, I next
explored the impacts of mucins on dual species interactions. In Appendix B, I found that mucins
promoted S. aureus survival in the presence of P. aeruginosa.This finding provides an
explanation for why these organisms are often co-isolated from mucosal infections in vivo (7-9),
despite their inability to co-exist in most laboratory conditions (10-12). Furthermore, I
demonstrate that mucins suppress the anti-staphylococcal effects of P. aeruginosapyocyanin.
Binding experiments suggest that mucins may protect S. aureus by sequestering pyocyanin,
however this does not rule out the possibility that a physiological response of S. aureus to
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mucins contributes to a protected state. Although the specific mechanisms by which mucins
protect S. aureus against P. aeruginosaremain unclear, this phenomenon highlights the
importance of mucins and mucus as a consideration when studying mucosal microbes.
Since biofilm formation and interspecies interactions both rely on bacterial
communication, I hypothesized that quorum sensing may be regulated by mucins. Appendix C
reveals that mucins regulate P. aeruginosaintra-species interactions by suppressing Las and Rhl
quorum sensing and related virulence genes. The mechanisms of mucin quorum sensing
regulation have not yet been elucidated, however this finding illustrates a specific microbemicrobe interaction that is modulated by mucins. Since quorum sensing is also used in
interspecies communication (13, 14), it is possible that mucin modulation of quorum sensing
systems can influence the behavior of multispecies communities. Notably, the quorum sensing
regulated phenazine biosynthesis phzAl gene was downregulated by mucins, which contrasts
with the finding in Appendix B that similar levels of pyocyanin are produced in the presence and
absence of mucins. This discrepancy might be explained by compensation for phzA] suppression
by expression of the redundant phenazine biosynthesis gene phzA2 (15). Also, expression levels
of downstream genes in the phenazine biosynthesis pathway, for example phzM or phzS, may be
more important in determining pyocyanin production levels. Another explanation is that
pyocyanin production is upregulated by alternate regulatory pathways. For example the mucin
glycan N-Acetylglucosamine (GIcNAc) is known to stimulate pyocyanin via the two-component
regulator PA0601(16). While the specific pyocyanin biosynthesis genes involved in the PA0601GlcNAc response have not yet been identified, it is possible that activation of this pathway could
compensate for suppression ofphzAl gene expression.
Although the exact mechanisms of mucin-mediated effects on bacterial behavior are
currently unknown, the biopolymers remain an important consideration when examining
mucosal microbes. Mucins may modulate microbial interactions by direct mucin-microbe
interaction or indirectly by affecting the availability of nutrients or signaling molecules. These
interactions are relevant for the study of both commensal and pathogenic microbes associated
with mucus. Commensal microbes are likely well adapted for survival within the mucus layer,
which may explain why many members of the gut microbiome have been difficult to culture in
mucin-free laboratory conditions. The addition of mucins to cultures may better mimic the
physiological environment and improve the ability to isolate otherwise unculturable mucosal
99
microbes. Furthermore, mucins are useful as an in vitro model for complex mucus during
investigations of mucosal microorganisms individually and within polymicrobial communities.
Similarly, mucins are important for investigating interactions between mucosal microbes
and the host epithelia. Although mucins have been shown to prevent epithelial attachment
against pathogens Helicobacterpylori(17, 18) and S. aureus (19, 20), their role in epithelial
protection against P. aeruginosahad not previously been demonstrated. Appendix D confirms
the protective role for mucins against P. aeruginosaepithelial attachment and cytotoxicity.
Mucins also likely impact the behavior of commensal microbes, whose interactions with each
other and the epithelia regulate a multitude of host processes (21). Due to their impacts on
microbial behavior, as well as their influence on the transport of microbial products and
resources, mucins are an important factor to include when studying mucosal microbes.
Overall, this thesis shows that mucins are important regulators of biofilms and other
microbial communities. Mucus is difficult to study due to its complexity and heterogeneity,
however mucins are a useful tool to model physiological mucus conditions. Beyond its barrier
functions, the roles of mucins in regulating microbial physiology are beginning to emerge (2224). As we better understand the interactions of mucins with microbes, there is potential to
translate this knowledge to apply mucins to modulate microbial behavior.
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