This presentation discusses the role of quorum sensing in bacterial virulence and its therapeutic potential

1. Molecular Basis of Quorum Sensing in
Virulence of Bacterial Pathogens
Tuesday, May 31, 2016 1
Shezaib Siddiqui
Research Fellow

2.  Introduction
 What is Quorum Sensing?
 Molecular basis of infections
a. Staphylococcus aureus
b. Pseudomonas aeruginosa
 Biosynthesis and degradation of Quorum sensing
 Therapeutic approaches
2

3. 3

4. KEY TERMS
Pathogenicity:
The ability of an organism to cause a disease.
Virulence:
The degree of pathogenicity or disease caused by a pathogen.
OR
Total no. of pathogenic determinants an organism is
collectively termed as virulence.
4

5.  Phenomenon that regulates the gene expression of
bacterial population in unison at particular density in
response to secreted chemical molecules.
OR
 Synchronous gene expression of bacterial population in
an environment.
5

6. HISTORY
o First Identified in late1960s by Nealson et
al.
o Discovered in Vibrio fischeri and Vibrio
harveyii
o V. fischeri is a symbiotic microbiota of
Howaiian bobtail squid light organ.
o V. Harveyi is a free living marine bacterium
o Bioluminescence in Vibrio is control via
Quorum sensing (QS).
o Lux operon was discovered as QS
regulatory coding region.
6
Howaiin bobtail squid
Vibrio fischerii
Vibrio harveyi

7. Quorum Sensing in a Nut Shell
Production and release of signalling molecule (Auto inducer).
Diffusion of Auto inducer (AI) molecule at low cell density leaving the QS
circuit in-active
Activation of QS circuit by AI at higher concentration due to Higher
cell density.
Transcriptional activation of down regulating genes by AI receptor
complex or other activated signalling molecule.
Activation of virulence, biofilm formation, sporulation, competence and
antibiotic production.
7

8. QS IN GRAM NEGATIVE
BACTERIA
 The autoinducers are typically
molecules called acyl-
homoserine lactones
(AHLs).
 AHLs diffuses extracellular
and intracellular via passive
transport.
QS IN GRAM POSITIVE
BACTERIA
 The autoinducers are
oligopeptides, short peptides
typically 8-10 amino acids
long.
 Oligopeptides excretes in
extracellular environment via
active transport.
8

9. Schematic Representation of QS Circuits
Canonical bacterial quorum-sensing (QS) circuits. Autoinducing peptide (AIP) QS in Gram-
positive bacteria by (A) two-component signalling, or (B) an AIP-binding transcription factor.
Small molecule QS in Gram-negative bacteria by (C) a LuxI/LuxR-type system, or (D) two-
component signalling.
Gram
positive
Gram
negative

10. QS Circuit of Vibrio harveyi
Lux Bioluminescence System

11. 11

12. 12

13.  S. aureus is a gram positive opportunistic pathogen.
 Causative agent of many cumbersome respiratory and skin
infections and poisonings.
 Few of high mortality infections and poisonings are Scalded
skin syndrome, toxic shock syndrome, furuncles, carbuncles,
sinusitis, otitis media etc.
 Emerged as Multi drug resistant bug and many hospital setting.
 Prevalent in > 60% of Nosocomial infections
 MRSA has mortality rate of >20%
13

14.  Encode various virulence factor under the control of Quorum
sensing system (Agr operon)
14
Scalded Skin Syndrome Impetigo
Toxic shock syndrome Otitis media

15.  S. aureus has a peptide based quorum sensing system ,encoded
by accessory gene regulator (agr) locus.
 The agr locus controls transcription of two RNA transcripts
RNA II and RNA III from promoter P2 and P3, respectively.
 RNA II encodes Agr A, B, C and D proteins
 RNA III encodes δ-hemolysin and posses regulatory activity.
15
Protein Function
Agr A Transcription factor (Activated
by Agr C)
Agr B Triming and transport of pre-
AIP (Agr D)
Agr C Histidine Kinase recetor of
AIP
Agr D Pre- Auto inducer peptide
Table 1: components of agr system

16. The Agr System
16
QS Circuit of Staphylococcus aureus

17. Role of RNAIII in virulence:
 5’ end upregulates transcription of α- hemolysin.
 3’ end is responsible for the repression of rot (repressor of
toxin) protein and Protein A.
 Rot inhibits the transcription of various toxins.
 Inhibition of rot upregulates the translation of toxins and
increases virulence.
 Repression of Protein A (cell surface protein) decease
adhesion of biofilm.
17

18. Role of Agr in Biofilm control:
 Agr A up regulates the translation of PSMs (Phenol
soluble modulins).
 PSMs are peptide toxin that help in channel formation
in tissues.
 PSMs also acts as surfactants that allow biofilm
dispersal.
 Biofilm dispersal helps S. aureus to colonize and infect
new sites.
18

19. Polymorphism in Agr Locus
 S. aureus can be classified in to 4 distinct groups based
on Agr locus.
19
Agr locus
Agr I Agr II Agr III Agr IV

20. Pseudomonas
aeruginosa
20

21.  P.aeruginosa is a Gram-negative opportunistic pathogen.
 Responsible for severe nosocomial infections cellulitis, UTIs,
endocarditis and epidermidis bullosa.
 Accounts for more than 10% of nosocomial infections.
 Patients with Cystic Fibrosis, injuries (especially burns/boils) are
highly susceptible to P. aeruginosa infections.
 Second leading cause of ventilator associated pneumonia.
 Difficult to treat due to emerging drug resistance.
21

22. 22
Cellulitis Epidermidis bullosa
Endocarditis

23. 23
• Three QS circuits are identified
• LasI/R (3-0C12HL synthase/ Receptor)
• RhlI/R (C4HL synthase/ Receptor)
• Pseudomonas Quinolone Signal (PQS)
Las Inducer/Repressor (I/R) system:
• Auto-induced by LasI
• Controls RhlR operon
• Activates transcription of Las B (Elastase), LasA (Protease), tox A
(Exotoxin A) and aprA (alkaline phosphatase).
• LasI inhibits Rhl R.

24. RhlI/R system:
 Upregulates rhl I
◦
 Positive regulator of Las B (elastase), RhlA and B
(Rhamnosyl transferase), RpoS (Stationary phase σ
factor) and Pyocanin (niche molecule)
24

25. 25

26.  Negative regulation NFκB.
 Induces apoptosis in macrophages.
 Down regulates TNF-α and IL-12.
 Induces IL-8 from human bronchial epithelial cells.
26

27. Biosynthesis and degradation of Acyl Homo serine Lactones
27
Fatty acid
pathway
AHL
synthase

28. 28
Types of Auto inducers in gram negative bacteria
PAME= propionyl amphotericin B methyl ester, DSF= Diffusible signal factor, THMF= tetra hydroxyl methyl furan

29. 29

30.  Novel therapeutic tartest against multi drug resistant bacteria.
 Reported to decrease virulence only, thus empower host
immune system to combat attenuated pathogen.
 Low chances of drug resistance against quorum quenchers as
they do not kill bacteria.
30

31. Strategies for Quroum Sensing Inhibition
1. Elimination of signal
 By stimulating enzymatic degradation of auto inducer
 By inhibition of auto inducer biosynthesis and receptor
molecule biosynthesis
2. Inhibition of signal receptor complex
 By using auto inducer analogues
 By treating with antibodies against auto inducer
31

32. Current Approaches for QS inhibition
 AHL acylases, paraxonases and AHL lactonases are secreted
by many bacteria to degrade AHL , therefore negatively
control QS.
 AHL and AIP analogues have been synthesized to inhibits QS
in S. aureus, P. aeruginosa and other pathogens
 Synthesis of inhibitor molecules against enzymes involved in
AHL biosynthesis, eg: tricolsan inhibits enoyl-ACP reductase.
 Inhibition of auto inducer receptors by using RNAi
technology.
32

33. 33

34. 34
Auto Inducer and Inhibitors of P. aeruginosa

35. 35
Auto inducers and analogue Inhibitors of S. aureus
s

36.  QS circuits serve as new and potential anti virulence
target to control infections with reduces risks of
resistance evolution.
36

37.  Miller, Melissa B., and Bonnie L. Bassler. "Quorum sensing in
bacteria."Annual Reviews in Microbiology 55.1 (2001): 165-199.
 Waters, Christopher M., and Bonnie L. Bassler. "Quorum sensing: cell-to-
cell communication in bacteria." Annu. Rev. Cell Dev. Biol. 21 (2005):
319-346.
 Whiteley, Marvin, Kimberly M. Lee, and E. P. Greenberg. "Identification of
genes controlled by quorum sensing in Pseudomonas
aeruginosa."Proceedings of the National Academy of Sciences 96.24
(1999): 13904-13909.
 Bassler, Bonnie L. "How bacteria talk to each other: regulation of gene
expression by quorum sensing." Current opinion in microbiology 2.6
(1999): 582-587.
 Yarwood, Jeremy M., et al. "Quorum sensing in Staphylococcus aureus
biofilms." Journal of bacteriology 186.6 (2004): 1838-1850.
 Rutherford, Steven T., and Bonnie L. Bassler. "Bacterial quorum sensing:
its role in virulence and possibilities for its control." Cold Spring Harbor
Perspectives in Medicine 2.11 (2012): a012427.
37

38. 38