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 t RNA

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 Mutant Control • The monosaccharide composition of fractions 60 68 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 No Glucans added Hydrophobic Interaction: Tb is eluted with 25% acetonitrile 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

ndvB

Periplasmic glucans present: Hydrophobic Interaction with Tb 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

ndvB

Periplasmic glucans present: Hydrophobic Interaction with Tb No periplasmic glucans present: No Hydrophobic Interaction with Tb This assay shows the periplasmic glucans are responsible for interaction with Tb

ndvB rplU RT-PCR of wildtype

P. aeruginosa

Biofilm Planktonic Genomic Expression of ndvB occurs only when in the biofilm state Controls 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 RT PCR of the mutant showing that ndvB was not expressed in both the planktonic and biofilm states