The significance of antiactivation in Pseudomonas quorum sensing

Presented by: Shannon Goff

Mentor: Dr. Martin Schuster

Microbiology Department

Oregon State University

Pseudomonas aeruginosa

Human opportunistic pathogen – exploits some break in hosts’

defenses in which to initiate infection

Lives in:

Soil

Water

Plants

Transient colonization of humans

Pseudomonas Antibiotic Resistance

2 methods of resistanceIndividual cell resistance

P. aeruginosa contains multiple methods for resistance in its genome

Biofilm formation

BiofilmsThick gelatinous matrix made of sugars

Protects against antibiotics, disinfectants, desiccation, host immune defenses

The minimal bactericidal concentration can be 100-1000x higher for bacteria in a biofilm than of isolated cells

Form in the lungs of cystic fibrosis patients

Immuno-compromised individuals

Cystic fibrosis infection •less than 1yr – 21%,•greater than 19yrs – 80%•Responsible for death of majority

2nd most common cause of ventilator-associated pneumonia (HAI)

4th most common cause of catheter-associated urinary tract infection (HAI)

Grows sometimes on skin, can infect through injury

Nosocomial infections - originate or occur in a hospital or hospital-like setting[also called hospital-acquired infections (HAI)]

Quorum SensingRegulatory mechanism that controls the ability to produce virulence factors and cause infection

Quorum sensing is transcriptional regulation triggered by a threshold cell density

Controlled by a signal that freely diffuses through the plasma membrane of the cell and accumulates in the cellular environment

Signal diffuses Cells and signal

accumulate Signal binds and

activate LasR, a transcriptional regulator

Virulence gene expression

Quorum Sensing

LasI

LasI

LasR

LasR

LasI

LasR

Signal synthaseSignal receptor

Discovery of anti-activatorQteE – quorum threshold expression element

Function is known: reduces half-life of LasR protein and reduces binding of LasR to signal

In relation to quorum sensing, it is hypothesized that QteE is significant in preventing the signal from binding the LasR inside the same cell (aka short-circuiting)

Quorum Sensing, QteE

LasI

LasI

LasR

LasR

LasI

LasR

Signal synthaseSignal receptor

Signal produced QteE inhibits the

binding of the signal and LasR

Signal accumulates Signal binds with

and activates LasR, the transcriptional regulator

Virulence gene expressionQte

E

QteE

LasR

LasR

Antivirulence drugs

Attack the ability to produce virulence factors

Virulence factors: aid bacteria in causing infection

Proteases, extracellular enzymes, proteins involved in membrane transport for nutrients

Quorum sensing controls biofilm formation as well as production of virulence factors

Using antivirulence drugs doesn’t directly impact the ability of the organism to survive

Less selective pressure

Objective of Research

To determine if QteE prevents short-circuiting in the system of quorum sensing

The increasing population of immunocompromised individuals accompanied by the resistance capabilities of P. aeruginosa make the development of antivirulence drugs a necessary step

These drugs need a well-characterized target, such as quorum sensing

Experimental Plan

To differentiate strains, integrate

mCherry fluorescent protein into

chromosome Insert the GFP plasmid into strains for QS determination

Co-culture strains together

and use microscopy to

identify fluorescence

Integrating mCherry

Confirm integration:

Tecan – measure fluorescent excitation with a machine

Microscopy – check cellular fluorescence with a fluorescent microscope

att site

Microscopy Image

Experimental Preparation

GFP

The green fluorescent protein is controlled by a QS promoter

The cells will fluoresce green when LasR binds the signal and induces transcription of QS genes

plasmid

Experiments: Control & Test

Strain 1 Name (red)

Strain 1 Function

Strain 2 Name

Strain 2 Function

Predicted Results Initial

Predicted Results Final

WT Produces all proteins, QS proceeds as normal

ΔlasI Doesn’t produce LasI signal synthase, QS can only respond to WT-produced signal

Neither strain green

Both turn green at the same time

qteE- Doesn’t produce QteE, can “short-circuit”

lasIqteE-

Double mutant, requires signal from qteE-, depends on diffusion for signal and QS

qteE- is green, lasIqteE- is not

Both green

LasI

LasI

QteE

QteE

Contr

ol

Test

THANK YOU!

HHMI

URISC

CRIPPS scholarship from the College of Science

University Honors College

Dr. Martin Schuster, mentor

Rashmi Gupta, PHD student mentor

Dr. Kevin Ahern, HHMI director

John Levine, assistance with Leica microscope