Biofilms and bacterial toxin production:
Coordinated metabolic control
Linnea Ista
Biology of Toxins
Spring 2010
What is a biofilm?
Collection of microbes,
small organics and
metabolites growing on
a surface
http://www.bact.wisc.edu/themicrobialworld/Intro.html
Ista, 1999 Appl. Environ, Microbiol. 65:1591
Why we care about biofilms- scientific
curiosity –they are everywhere!
Deep ocean
hydrothermal vent
http://eager2009.files.wordpress.com/2009/06/sc
hrenkbiofilm.png?w=500&h=373
Glacier
•Most abundant form of Human intestines
life on earth
•Total number of
prokaryotic cells on earth
at any given time ~5x1036
•Most bacteria grow in
biofilms.
•We have 10x more
bacterial cells living in
and on us than human
cells.
http://www.morningearth.org/graphicE/BIOSPHERE/Bios-MicrobeImage/M-Fileum.jpg
http://www.unidue.de/imperia/md/images/biofilmcentre/solfatare.jpg
Why we care about biofilms- scientific
curiosity -they are old life!
Fossil stromatolites
(New York State)
http://cas.bellarmine.edu/tietjen/Evolution/
Modern stromotalite (Australia)
http://www.petrifiedseagardens.org/
http://www.photosynthesisresearch.org/
•They are the oldest identified form of life on
earth
•Fossils of stromatolites date to ~ 3.8 Gya
•Stromatolites are layered formations of
minerals and bacteria
•Formed at intersection between ocean
and land
They are the workshop for
biochemical evolution!
 Most of the compounds we know today , with
the exception of some plant toxins have
evolutionary roots in bacteria
 Bacteria in biofilms exchange genes like mad!
 Biofilms might also have been the place where
endosymbiosis occurred.
 Bacteria in ancient biofilms significantly altered
the atmostphere of ancient earth
 They continue to fuel biogeochemical cycles
today
They were probably the
prototype for multicellarity
Sea lettuce development
Pseudomonas biofilm development
Negative Significances of Biofilms Medical
Disease
•Otitis media
•Cystic fibrosis
Device failure
•Catheters
•Heart valves
•Contact lenses
•Stents
Nosocomial infections
•Biofilms serve as reservoirs
•Increase chance of antibiotic resistance
Negative Significances of Biofilms Industrial
Corrosion- lithotrophic
bacteria oxidize the metal
or sulfate reducing
bacteria produce sulfuric
acid! Oil platforms are
vulnerable
Biofilms on ship hulls
increase drag, can
cause corrosion and
recruit macrofoulers
such as barnacles
Heat exchangers
demonstrate up to
75% decreased
efficiency with a
monolayer of
bacteria
Biofilm development


Most of what we know is based on biofilm development in Pseudomonas
aeruginosa strain PAO1
P. aeruginosa is a ubiquitous organism



Soil
Water
Anywhere there are humans


Very adaptive


Can grow aerobically, through anaerobic respiration and fermentatively
Uses many carbon sources


Sometimes indicative of human contamination- for example in Tokyo Harbor
Is a very good bioredediator
Major opportunistic pathogen




Otitis media (ear infection)
Burns – if you survive a motorcycle crash, your next biggest danger is P. aeruginosa
infection of your road rash
Lung infections- most cystic fibrosis patients die of P. aeruginosa pneumonia
Major contaminant of medical devices, especially contact lenses and catheters
How do biofilms form?
Stages:
1. Attachment
2. Adhesion
3. Propagation
4. Maturation
5. Dispersal
Annu. Rev. Microbiol. 2002. 56:187–209
How do biofilms form?
Attachment:
• Planktonic (cells growing in liquid) cells become
associated with surface
• Cells lose their flagella (if they have them)
• In pathogens and commensal organgisms, this is
often mediated by host cell receptors
• In environmental biofilms, this process is thought
to be mediated by the surface tensions of the
attachment substratum, the bacterial surface (or
part of the bacterial surface) and the surrounding
liquid (usually water)
• Depending on the interactions between these
surface tensions, attachment can be
irreversible within minutes or it may take hours.
How do biofilms form?
Adhesion:
•Attached bacteria start making exopolymeric substances (EPS)
•EPS consists mostly of water
•Organic compounds, made by the organism include
•Polysaccharide (in P. aerugonisa-the most famous type is
alginate- which is what clogs the lungs of CF patients)
•Glycoproteins
•Nucleic acid
•Functions:
•Keeps cells on surface under flow (e.g. blood flow, peristalsis
in intestines, air flow in lungs, water flow in streams and soil
•Prevents desiccation of underlying cells
•In opportunistic pathogens, provides protection from white
blood cells and antibiotics
•In environmental organisms, can serve as a storage source of
organic carbon
•In pathogens, is considered a pathogenic mechanism
•Can itself cause immune response
How do biofilms form?
Propagation- cells accumulate on surface
•Under high nutrient conditions, this can actually
be growth
•Recruitment to surface from liquid medium by
soluble factors (quorum sensing-see below)
•Dispersed cells on surface gather by surface
motility mechanisms which are actually super
cool
•Gliding
•Twitching motility (literally they “walk” on pili)
•Rolling motility (held to the surface by EPS,
they roll along the surface
Annu. Rev. Microbiol. 2002. 56:187–209
How do biofilms form?
Maturation
• Biofilm starts building in Z-direction
•Mutant studies and gene expression studies
indicate that cells in different part of the
structure are metabolically and
phenotypically different- i,.e., there is
differentiation occuring
•Processes that make this happen•Cell surface motility over existing cells
(mostly twitching type motility)
•Programmed cell death
•Cell division in certain parts of biofilm
•Cells on can be “dead” , thus
protecting underlying structures
•Leads to a very tissue- like structure with
liquid channels
How do biofilms form?
An important part of the biofilm life
cycle that the canonical view leaves
out: Persistance
•Biofilms can last for years
•In CF patients, 25-30 years
•Some biofilms on rocks at the
deep subsurface are estimated
to be thousands to millions of
years old
•During this time, biofilm structure
can still be dynamic.
•This is the point at which biofilms
can exchange a lot of genetic info!
•This is the least well studied, but
probably most important part of
biofilm development, particularly in
terms of human disease.
Annu. Rev. Microbiol. 2002. 56:187–209
How do biofilms form?
Dispersal:
•Seems to start with a hollowing of the core of
the biofilm
•This part reminds me of blastocyst
formation in human development, but I
may have a vivid imagination
•Cells in the center of the hollow revert to
planktonic growth phenotype
•Regain flagella (if present)
•Lose surface motility structures
• Reactive oxygen and nitrogen species
(especially nitrous oxide) may play a role in
triggering the metabollic conversion
•EPS breaks down in part of the biofilm and
planktonic cells are released.
Annu. Rev. Microbiol. 2002. 56:187–209
So how is this all regulated?
 Two main mechanisms currently recognized:
 Quorum sensing- bacterial hormones
 Involvement in every step of biofilm development
 Seems very evolutionarily conserved
 Both big control and fine tuning
 Internal second messengers
 Controls whether cells are planktonic or biofilm form
 Similar to cAMP signalling in eukaryotes
 Genetic studies indicate that it has evolutionary
relationship with cAMP functioning in eukaryotes
First regulatory pathway
discovered: quorum sensing in
Vibrio fischeri
Vibrio fischeri on a squid
•
ttp://keck.bioimaging.wisc.edu/mcfall-lecture.jpg
Produces bioluminescence but only when
• Attached to surface
• Enough bacteria are there
• Called “quorum sensing” because the
mechanism only operates when a
threshold level of cells are present
• Found to be controlled by acculmulation
of an acyl-homoserine lactone, 3oxohexanoyl-homoserine lactone
Lux protein
http://www.pnas.org/content/102/33/11882/F3.large.jpg
LuxR operon
as a model
for quorum
sensing
•Acyl homoserine lactones have
been found in many Gram
negative organisms.
•Control secreted compounds
(e.g. toxins) and biofilm
formation.
•All AHL systems found so far
have a component that
evolutionarily related to Lux.
http://gcat.davidson.edu/GcatWiki/index.php/Davidson/Missouri_West
ern_iGEM2008
Generalized AHL pathway
How does QS regulate biofilm
formation? An example from
Pseudomonas aeruginosa PAO1
 Has two known quorum sensing systems:
 Las- which is analogous to Lux
 Rhl- controls production of a surfactant called
rhamnolipid.
 Las and Rhl are currently targets of antibiofilm
therapy development
 A third AHL has been discovered but its role is
uncertain
 Las is thought to be the pathway that controls biofilm
formation while Rhl controls soluble secretion
 But these two pathways interact, so it is unclear how
this can be stated definitively.
So how do Las
and Rhl
interact?
http://www.cdc.gov/ncidod/eid/vol4no4/vandel4b.gif
•Activating the Las pathway
produces AHL (3-oxo-C12-HSL)
•AHL triggers more production of
AHL
•AHL also actviates Rhl pathway
which feeds back to the Las
pathway
•Notice that in addition to
inducing biofilm differentiation,
Las also releases compounds that
modify the immune system.
•Rhl induces the production of
pyocyanin (a toxin) and cyanide.
How do quorum sensing
molecules control biofilm
development in P. aeruginosa?

Attachment


Adhesion



Production of a 3 dimensional biofilm requires periodic
release of bacterial DNA to form a scaffold for bacterial
cells- 3-oxo-12-HSL promotes cell death in certain cells
Rhl pathway produces rhamnolipid, which is important in
surface motility.
Persistance– presence of 3-oxo-12-HSL helps maintain the
structure


3-oxo-12-HSL directly activates alginate production
pathway
Maturation


3-oxo-12-HSL may recruit more bacteria to the surface
Promotes loss of flagella
A gradient of AHL is found in the biofilm structure.
Reliease- lack of AHL seems to result in release
TRENDS in MicrobiologyVol.13 No.1 January 2005
Peptide
hormones in
Gram
positive
organisms
Agr= Accessory gene
regulation
Agr D is a prepeptide that
is cyclized and
transported by AgrB
transmembrane protein to
form AIP (auto inducer
protein).
Quorum sensing is common in
prokaryotes
 Present in both eubacteria and archaebacteria
 General wisdom, based mostly on pathogens,
suggests that quorum sensing in Gram negative
organisms procedes through acyl homoserine
lactones and in Gram positive organisms through
peptides
 It seems though that Actinobacteria use AHLs as well
 Recent research shows that quorum sensing can also
effect eukaryotic hosts
 Legume-nitrogen fixing bacteria
 Maybe even human intestinal flora
Intercellular regulation cDGMP
 Cyclic di-guanidine monophosphate
 Promoters binding proteins with GGDEF
turn on cgGMP production
 Promoter sequences are similar to
those turning on cAMP in eukaryotes
 Promotoers binding EAF turn on stuff
that chops up cDGMP.
 Activation of GGDEF promoters results in
biofilm development/maintenance
 Activation of EAF promoters results in
release of biofilm/production of
planktonic cells
NATURE|Vol 441|18 May 2006
A-factor and antibiotic
production in streptomyces
 In Streptopmyces griseus sporulation and antibiotic
production are controlled by “A-factor”
 Some sporulation and antibiotic mutants can be
“cured” by adding A factor into a culture.
 A-factor is a homoserine lactone.
 Other antibiotic producing streptomycetes have
similar factors.
 The “general wisdom” is that only G- organisms use
quorum sensing for metabolic control. I would argue
that it this might not be true for non-pathogens.
Cellular regulation by acyl
homoserine lactones in
Actinomycetes
 Even though Gram positive cells are
not supposed to be regulated by acylhomoserine lactones, actinomytes are.
 Actinomycetes are antibiotic
producers
 Produce about 2/3 of all naturally
occuring antibiotics.
 Produce more antibiotic when growing
in a biofilm than in liquid (i.e. planktonic
culture)
 Extra step in biofilm development- the
formation of exospores.
Streptomyces griseus life
cycle
http://www.bioscience.org/2002/v7/d/horinouc/fig1.jpg

Streptomycetes are soil organisms

This entire cycle takes place on
surfaces (i.e. there is usually not a
planktonic state)

Antibiotic production is concomitant
with formation of aerial hyphae and
sporulation

Acyl homoserine lactones control both
entry into secondary growth and
antibiotic production

AHL production was first disovered in
Steptomyces griseus which makes
streptomycin

Mutants that were unable to make
spores or streptomycin were restored
upon addition of extracts or exudate
from wild type colonies

Compound was called A-factor

Has a Las/Lux sort of control system

For many years biofilm people and
antibiotic people did not talk to each
other so the similarities between biofilm
development and secondary
metabolism in actinomycetes.
Bacterial toxins in pathogens
are, however, downregulated
by compounds involved in
biofilm development!
 I had assumed that since biofilms are considered a “virulence
factor” for pathogens, that biofilm production and bacterial
toxins would be co-regulated

They are– 10 points to Ravenclaw!
 Because they are both involved in virulence, I thought both
biofilm formation and toxin production would be upregulated

They are not- 20 points from Ravenclaw
 Why would this be?
 Pathogens that are making you sick probably want to have lots of
copies of themselves- so they need to dividing rapidly which they
don’t in biofilms
 If bacteria are persisting in an infection (such as P. aeruginosa in cystic
fibrosis patients) they probably don’t want to be detected. Therefore
production of things like toxins are not int their best interests.
 Also-toxins are expensive. Where are the cells in bacteria, which are
not metabolizing rapidly, going to get the energy to make them?
Example 1 Cholera toxin
production in V. cholerae
 Near relative of V. fischerii
in which quorum sensing
was first discovered.
 Lux/Las sort of quorum
sensing system
 Activation of Lux turns on
biofilm formation
 Notice activation of
GGDEF promoters!- QS
interfacing with secondary
messengers
 Deactivation of Lux system
 Shuts down biofilm
formation
 Turns on virulence genes
(HA pathway)
Microbiol Molecular Biol Revi, 2009, 73:. 310–347
.
Gram positive pathogens
 Quorum sensing in Gram
positives is achieved mainly
through peptide hormones
called autoinducer proteins
 Interact through the agr
(accessory gene regulation)
pathway
 Some evidence exists that
genes for Lux-type regulons
are also present in many
Gram positives, but are
dormant
 Most of the Gram positive
organisms studied are
pathogens so there may be
a bias
AIP production in Staphylococcus
aureus. Anal Bioanal Chem (2007) 387:437–444
Staphylococcus aureus
 Once again, activation of
quorum sensing associated
with toxin production (αhemolysin) turns off biofilm
production
 Even better, α-hemolysin itself
down-regulates biofilm
formation.
Microbiol Molecular Biol Revi, 2009, 73:. 310–347
.
So how did regulation evolve?

Both acyl homoserine lactone-based quorum sensing and antibiotic
(halocin) production has been detected in archaebacteria,


Halocins are similar to Gram positive peptide hormones
Not much has been discovered about their production

It has been recently shown that antibiotics in low concentration can function
as signaling pathways between cells.

It has also been shown that some quorum sensing molecules have
antimicrobial activity.

Both quorum sensing and bacterial toxins (particularly antibiotics) tend to be
small molecules, as are some toxins such as α-hemolysin

Computer models show that cooperation by cell-to-cell signaling probably
evolved early as it confers a selective advantage on the population

I suspect that antimicrobial production and quorum sensing might have
coevolved from ancient mechanisms of cell-to-cell communication
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