Question:
• Does Tb interact with periplasm glucans?
If so, through what type of interaction?
A genetic basis for Pseudomonas
aeruginosa biofilm antibiotic
resistance
Thien-Fah Mah, Betsey Pitts, Brett Pellock, Graham C. Walker, Philip S. Stewart & George A. O‘Toole
Presented by Brian Tessler and Dasha D. Pechersky
What is a biofilm?
I just can't go with the flow anymore. I've been
thinking about joining a biofilm.
What is biofilm?
• Extracellular carbohydrate matrix secreted by
sessile (surface attached) bacteria
– slimy, glue-like substance
• Protected growth mode
– Exhibits high levels of antibiotic, host, pH and disinfecting
resistance. 1000x greater antibiotic resistance then
planktonic cells.
Biofilm formation
• 3 stages:
– Attachment
– Biofilm formation
– Persistence and detachment
Biofilm Bacteria
• Biofilm bacterial behavior is more complex than
planktonic behavior
– Once bacteria attach to a surface, a new set of genes “turns
on”. They begin to excrete a slimy material, biofilm.
– There are significant differences in the level of expression of
genes within biofilm bacteria communities.
What is the significance of biofilm?
– Implicated in a significant number of human bacterial
infections
– Biofilms cost the nation billions of dollars yearly in
damage
– Aid in bioremediation of hazardous waste sites, and
protection of soil and groundwater from contamination
Human Infections linked to biofilms
Cystic Fibrosis and Biofilm
• A genetic disorder that leads to
persistent bacterial infections of the
lungs.
• Most CF patients are colonized with
sessile P. aeruginosa
• These antibodies react with their
specific antigens, but the antibodies
are not effective against sessile
bacteria.
• Patients eventually succumb to the
lung damage inflicted by the bacterial
infection, with a life expectancy of
about 30 years.
Tobramycin’s Chemical Structure
• Leading treatment for
cystic fibrosis
• Tb works by binding to
the bacterial 30S
ribosomal subunit,
causing misreading of tRNA
Genetic determinant of resistance
• ~4000 transposon insertion mutants
generated and screened
• Mutant 45E7 displayed decreased resistance
to tobramycin (Tb) in biofilm as compared to
wild type
• In planktonic culture, mutant 45E7 displayed
the same characteristics as wild type
planktonic
Biofilm Antibiotic Sensitivity
• Is the heightened antibiotic resistance solely mediated
by the 45E7 locus?
• Three antibiotics show that 45E7 locus is responsible for
the increased antibiotic resistance of biofilm bacteria
24-h old wild type and mutant biofilm
bacteria staining
Note: Green fluorescence
stains viable cells while red
fluorescence stains dead cells
Biofilm Antibiotic Sensitivity
• Quantatative data conclusively showing mutant 45E7 is
less resistant to Tb when grown in biofilm
48-hr old colonies
Note: Filled bars are wild type
Open bars are mutant
Why is the mutant sensitive to Tb?
What is different between the wild-type and the mutant?
• Map the Gene
• Determine what the gene encodes for
• Determine the function of the gene product
• Show a difference of function between the
mutant and wild-type
Mapping the Gene
The transposon was mapped to the
middle of ORF PA1163
Gene product: ndvB
• The ORF PA1163 has a 58% homology to the
Bradyrhizobium japonicum gene ndvB
• This gene encodes a glucosyltransferase
• Glucosyltransferases function in the transfer of glucose
molecules
• This glucosyltransferase is required for the synthesis of
cyclic-beta-(1,3)-glucans
Proposed Structure of cyclic-B-(1,3)-glucans from
Bradyrhizobium japonicum
Structure: circular polymer of glucose
Location: In the periplasm and secreted
into the extracellular media
Implicated functions of circular polymers
of glucose:
• adaptation to low osmotic pressure
• flagella-mediated motility
• plant-microbe interaction
In a strain of B. japonicum, a planktonic
mutant is more sensitive to
chloramphenicol, an antibiotic
The Principle of Gel Filtration
Separation by size. Biggest molecules come out first!
Periplasmic material was extracted from the bacteria and
purified by gel filtration over a Sephadex G-75 column
Analysis of Sephadex G-75 Column Fractions
Wild-type
ndvB mutant
The difference in the fraction numbers 60-68 shows the mutant
is deficient in what is believed to be the periplasmic glucans.
Monosaccharide Analysis of the 60-68 Fraction
Wild-type
Control
Mutant
• The monosaccharide composition of fractions 6068 were done using Dionex high-preformance
anion-exchange chromatography coupled with
pulsed amperometric detection (HPAEC-PAD)
• The wild-type fraction contains a high amount of
glucose, consistent with the structure of the cyclic
glucans
• The mutant fraction contains minimal glucose,
consistent with the absence of the glucans
How Affinity Chromatography Works
I.
II.
III.
Periplasmic glucans are bound to the column: Tb is filtered through the column
Tb interacts with the periplasmic glucans. All other Tb is washed away
25% acetonitrile disrupts the interaction between the glucans and Tb
Disk Diffusion Assay:
Detecting the Interaction between Periplasmic Glucans
and Tb
Glucans added
Hydrophobic Interaction:
Tb is eluted with 25% acetonitrile
No Glucans added
No hydrophobic interaction:
All Tb is in flow-through and first
wash
This shows that their assay (killed cells) for detecting Tb works
Disk Diffusion Assay: Wild-type v. Mutant
Crude periplasmic extracts
Wild-type
Periplasmic glucans present:
Hydrophobic Interaction with Tb
ndvB
No periplasmic glucans present: No
Hydrophobic Interaction with Tb
This assay shows that something in the periplasm is responsible for
this interaction
Disk Diffusion Assay: Wild-type v. Mutant
Purified Periplasmic Glucans
Wild-type
Periplasmic glucans present:
Hydrophobic Interaction with Tb
ndvB
No periplasmic glucans present:
No Hydrophobic Interaction with Tb
This assay shows the periplasmic glucans are responsible for
interaction with Tb
RT-PCR of wildtype P. aeruginosa
Biofilm Planktonic Genomic
Expression of ndvB occurs
only when in the biofilm
state
ndvB
Controls
rplU
Constitutively Expressed
tolA
Biofilm Control
cupA
Planktonic Control
Two Proposed Mechanisms for Tb
Resistance
• The periplasmic glucans bind to the antibiotics
and sequester them, preventing them from
entering the cytoplasm
• The periplasmic glucans slow the diffusion
of antibiotics into the cytoplasm, giving the
bacteria time to adapt to the antibiotic
Possible Treatment Against These Biofilm
• Co-therapeutic approach with traditional antibiotics
coupled with a drug that can interfere with these
glucans
Problems to Consider: Compensatory Mechanisms
•This paper has shown that mutant biofilms older than 72 hr where
less sensitive to Tb than 24 hr old biofilms. This means that Tb
treatment is more successful on younger biofilms
• Not all strains are as sensitive to Tb as the one used in this
experiment. This group tested three other strains, and found that
mutants from two other strains were not as sensitive to Tb
Flaws of this Paper
•This paper does not analyze the chemical composition of
the biofilm to see if the glucans are also secreted into the
biofilm. It is possible, if these glucans are secreted, the
interaction may occur in the biofilm and not the
periplasm.
• For completeness this paper could have included a
chemical analysis of the planktonic periplasm to show that
the wild-type was sensitive to Tb because it did not have
these periplasmic glucans
• For completeness this paper could have included RTPCR of the mutant showing that ndvB was not expressed
in both the planktonic and biofilm states