QUORUM SENSING IN BACTERIAL VIIRULENCE

What is quorum sensing?

• Bacteria can behave either as individual single-celled organisms or as multicellular populations. Bacteria exhibit these behaviors by chemically "talking" to one another through a process called quorum sensing.

• Quorum sensing involves the production, release, and community-wide sensing of molecules called autoinducers ( AI ) that modulate gene expression, and ultimately bacterial behavior, in response to the density of a bacterial population.

A brief overview of the processBacterial genes code for the production of signaling molecules called autoinducers that are released into the bacterium's surrounding environment.

These signaling molecules then bind to signaling receptors either on the bacterial surface or in the cytoplasm.

When these autoinducers reach a critical threshold level, they activate bacterial quorum sensing genes that enable the bacteria to behave as a multicellular population rather than as individual single-celled organisms.

The autoinducer/receptor complex is able to bind to DNA promoters and activate the transcription of quorum sensing-controlled genes in the bacterium.

In this way, individual bacteria within a group are able to benefit from the activity of the entire group.

• MECHANISM IN GRAM NEGATIVE BACTERIA.

• InGram-negative bacteria, the autoinducers are typically molecules called acyl-homoserine lactones or AHL.

• AHLs diffuse readily out of and into bacterial cells where they bind to AHL receptors in the cytoplasm of the bacteria.

• When a critical level of AHL is reached, the cytoplasmic autoinducer/receptor complex functions as a DNA-binding transcriptional activator.

• MECHANISM IN GRAM POSITIVE BACTERIA.

• InGram-positive bacteria, the autoinducers are oligopeptides, short peptides typically 8-10 amino acids long.

• Oligopeptides cannot diffuse in and out of bacteria like AHLs, but rather leave bacteria via specific exporters. They then bind to autoinducer receotors on the surface of the bacterium.

• When a critical level of oligopeptide is reached, the binding of the oligopeptide to its receptor starts a phosphorylation cascade that activates DNA-binding transcriptional regulatory proteins called response regulators.

Canonical bacterial quorum-sensing (QS) circuits.

Canonical bacterial quorum-sensing (QS) circuits. Autoinducing peptide (AIP) QS in Gram-positive bacteria by (A) two-component signaling, 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 signaling.

Gram positive

Gram negative

ADVANTAGES OF MULTICELLULAR BEHAVIOUR

• As the entire population of bacteria simultaneously turn on their virulence genes, the body's immune systems are much less likely to have enough time to counter those virulence factors before harm is done.

• Virulence factors such as exoenzymes and toxins can damage host cells enabling the bacteria in the biofilm to obtain nutrients.

• Quorum sensing enables some of the bacteria to escape the biofilm and return to individual single-celled organism behavior in order to find a new site to colonize.

• Bioluminiscence is also produced by some bacteria by quorum sensing.

Role of quorum sensing in bacterial virulence

Staphylococcus aureus(gram positive)

The accessory gene regulator (agr) quorum sensing system of S. aureus

Important features

• Despite its widespread prevalence in healthy subjects, S. aureus is also a very dangerous opportunistic pathogen which has been increasingly associated with antibiotic resistance.

• S . aureus has multiple virulence factors and can display very rapid transmission, aiding its importance as a human pathogen.

• S. aureus forms biofilms on many surfaces, including indwelling devices such asurethral stents . These indwelling devices, and subsequent biofilms formed on them, pose a serious risk for Staphylococcus infection

Virulence factors of Quorum sensing

• S. aureus has a peptide based quorum sensing system ,encoded by accessory gene regulator (agr) locus.

• The autoinducer (AI) is an oligopeptide termed as Auto inducer peptide (AIP).

• AIP is encoded by agrD.

• Genes involved in agr system are agr A,agrB,agrC,agrD.• agrD, encodes pre-AIP.• AgrB protein (membrane bound) trims and releases AIP from its precursor

form to the extracellular environment.• AgrC (a membrane bound sensor kinase) to which extracellular AIP

binds,leading to autophosphorylation of AgrC- activates AgrA.• AgrA (phosphorylated -> active) , induces transcription at the promoters P2

(higher affinity) and P3.

• Regulation of virulence genes in agr systeem is predominantly involved around promoters P2 & P3 ( oppositely directed).

• P2 promotes transcription of agr operon from the RNAII transcript ,which includes agrA,B,C,D.

• P3 transcription leads to production of RNAIII (regulatory RNA).

• RNAIII is the effector molecule of the agr system.• RNAIII reduces the expression of surface adhesins and increases the

production of capsule toxins and proteases.

**** RNAIII also functions as the mRNA for δ-toxin ( 5’ end upregulates α-haemolysin while 3’ is required for repression of protein A synthesis).4 distinct group of agr polymorphism found agrI,II,III,IV .AIP s of each group cross-inhibits other groups. (exception-group II & IV cross activates each other).

The accessory gene regulator (agr) quorum sensing system of S. aureus

Impact of agr on virulence

• RNAIII has the dual-function of activating production of a-toxin and repressing expression of rot, fibronectin binding proteins A and B, protein A, coagulase, and other surface proteins.

• Repression of rot, which encodes a repressor of toxins, leads to de-repression of additional toxins, proteases, lipases, enterotoxins, superantigens, and urease .

• The net result of this QS regulatory cascade is downregulation of surface virulence factors (such as protein A), and up-regulation of secreted virulence Factors (such as a-toxin).

• Most of the effects of QS on regulation of virulence in S. aureus Are mediated through direct and indirect regulation by RNAIII, however, phosphorylated AgrA also directly activates at least two additional virulence genes encoding phenol-soluble modulines.

• Biofilm formation :• When agr nonfunctional(non active),bacteria S.aureus ha enhanced adhesion abilities

(i.e. first stage of Biofilm formation).• Agr also plays a role in detachment of cells from biofilm ,important for dissemination of

during an infectious process.• Active form triggers virulence .• contribution of agr to disease has been studied in models like septic arthiritis,

osteomyelitis, endopthalmitis, pulmonary infections, toxic shock syndromes.

(GRAM NEGATIVE)Pseudomonas aeruginosa

INTRODUCTION• P.aeruginosa is a Gram-negetive bacterium. • This organism is an opportunistic pathogen & commonly associated with

nosocomial infection & infection of severely burned individuals, and is leading to cause of death in severe respiratory infections, such as chronic lung infections in CF patients.

• Infections with P. aeruginosa are difficult to eradicate due to their high levels of

antibiotic resistance and growth in biofilms. • Pseudomonas aeruginosa is a very versatile organism that can adapt too many

different environments and can cause diseases in plants, animals and humans. • This organism produces a broad range of exoproducts, which are regulated

in a population density-dependent manner via cell-to-cell communication or “quorum sensing".

Quorum sensing in P. aeruginosa: a complex regulatory network resulting in fine signal tuning

In P.aeruginosa at least three interwined quorum sensing (QS) system and one orphan autoinducer receptor affect the ability of this organism to cause disease.

Two intertwined QS systems (the las and the rhl systems),rely on the production of acyl homoserine lactones(AHLs) as the signaling molecules(auto inducers-AIs) have been shown to be involved in virulence, biofilm development, and many other processes in P. aeruginosa.

In the las system lasI gene encodes an enzyme which produce an AIs molecule called N-3-oxododecanoyl-homoserine lactone (3-O-C12-HSL).

In the other hand, the rhlI gene of rhl system encodes an enzyme that produce another AIs molecule called N-butyryl-homoserine lactone (C4-HSL).

Then it regulates its gene expression via different receptor and via feed back regulation.

Where LasR-C12-HSL activates the transcription of rhlR.

Similarly the transcriptional activator of LhlR is activates by binding with C4-HSL (RhlR-C4-HSL).

Both the active LasR & RhlR regulates the production of several extracellular virulence factors

Besides LasR and RhlR, P.aeruginosa encodes an orphan receptor protein, QscR, which can sense 3O-C12-HCL to controls its own regulon.

Besides regulating the expression of virulence factors, some AIs have been shown to directly interact with host cells.

In addition to AHLs, P. aeruginosa releases a 4-quinolone signal molecule into the extra-cellular milieu, the synthesis and bioactivity of which has been reported to be mediated via the las and rhl systems respectively.

It is the third P.aeruginosa AI molecules, this molecule has been

chemically identified as 2-heptyl-3 hydroxy-4(1H)-quinolone and termed the Pseudomonas Quinolone Signal (PQS).

PqsR is a LasR transcriptional factor which is activated by HHQ and PQS, leading to the positive activation of many virulence factors, which includes a large no of genes also controlled by las and rhl.

Besides controlling bacterial virulence, PQS and HHQ have been shown to downregulate the host immune response through NFkB and PQS can also act as an iron chelator, and both the synthesis of PQS and activity of PqsR-PQS are involved in iron homeostasis.

QUORUM SENSING INHIBITION P.aeruginosa elaborates two main sets of QS systems: lasI-LasR and rhlI-

rhlR.

These molecules diffuse out into the environment, and when they reach a putative threshold concentration, they activate the receptors LasR and rhlR.

The mechanism of Quorum Sensing Inhibition (QSI) activity appeared to be a net effect of

(1) the ability of phytochemicals to interfere with the activity of AHL and (2) to modulate the bacterial synthesis of AHL’s. Many natural extracts are

believed to inhibit QS by either interfering with AHL activity by competing with them due to their structural similarity and/or to accelerate the degradation of the LuxR/LasR receptors for the AHL molecules.

Some subinhibitory concentrations of macrolides have been shown to be effective in inhibiting quorum sensing dependent virulence both in vivo and in vitro.

Escherichia coli

(gram negative)

Control of virulence gene expression through quorum sensing and adrenergic signalling in E. coli.

Important features • E.coli is a gram negative ,facultative anaerobic,rod

shaped bacterium.

• Commonly found in the lower intestine warm blooded organisms.

• Most E.coli strains are harmless and resides in gut. Can benefit hosts by producing vit K and preventing establishment of pathogenic bacteria within intestine.

• E . Coli has been used as an indicator organism for faecal contamination.

• Certain strains of E. coli are a major cause of foodborne illness.

• The outbreak started when several people in Germany were infected with enterohemorrhagic E. coli (EHEC) bacteria, leading to hemolytic-uremic syndrome (HUS).

• Escherichia coli E. coli O157:H7 serotype was the cause of major outbreak of HUS in 2011 at Germany.

• Other less common serotypes, such as O104:H4, O121, O26, O103, O111, O145,and O104:H21 can also cause serious infection.

Role of quorum sensing in virulence

• This organism produces a signal molecule termed AI-2.

• the gene responsible for AI-2 (furanosyl borate diester)production was identified and named luxS.

• Genetic studies in enterohaemorrhagic E. coli (EHEC) and enteropathogenic E. coli revealed that LuxS controls the expression of the type-3 secretion system encoded by the locus of enterocyte effacement (LEE) pathogenicity island.

• This virulence determinant causes Attaching & and effacing lesions (AE),caused by pathogens.

• LuxS is a global regulator in EHEC, controlling the expression of over 400 genes

• Many of these genes have functions related to bacterial virulence such as flagellar motility, surface adhesion and Shiga toxin production.

• Later on the molecule responsible for the regulation of LEE and flagellar genes in E. coli was found to be AI-3 and not AI-2.

• The regulatory cascade linking AI-3 sensing and virulence gene expression in E. coli is extremely complex.

• Multiple regulatory genes are controlled by quorum sensing.• Gene systems are namely QseBC, QseEF,QseCD etc.• QseBC - a bacterial two-component signalling system• QseB being the response regulator• QseC the sensor kinase(autophosphorylation).

• The QseBC contols motility expression.• QseC is activated by AI-3. • QseC acts as a receptor for extracellular AI-3.• QseC also functions as the receptor for the host

catecholamine hormones epinephrine and norepinephrine, indicating that small molecule signalling pathways in eukaryotes and bacteria can intertwine.

Control of virulence gene expression through quorum sensing and adrenergic signalling in E. coli.

A second two componentregulatory system

• Histidine kinase QseE and its effector QseF.• involved in the transcriptional control of the

effector EspFu, which is translocated into host cells by EHEC.

• Controls formation of AE lesion.• QseG (outer membrane),aids in typeIII

secretion sytem for translocation of effector molecules in host cells.

Impact of the Qse system on virulence

• Flagellar motility.• Type 3 secretion of potent Shiga toxin (harmful for

intestinal cell lining ,causes HUS)Hemolytic uremic syndrome (HUS) - this condition is characterized by hemolysis (breakup of red blood cells) and kidney failure.

The patient can develop CNS (central nervous system) problems, including seizures, and can also go into a coma.

Additional Roles of Bacterial Auto-inducers (AIs)

• In some bacteria autoinducer AI-2 acts as a metabolic by-product along with its role as a signal molecule.

• in most cases the production of AI-2 is mainly involved in SAM (S-Adenosylmethionine) utilization and SAH (S-Adenosylhomocysteine)detoxification along with its role as a bona fide signal in some bacteria.

Thus it can be said that the synthesis of AI-2 has two connections with bacterial metabolism---• The precursor of AI-2 is a major methyl donor in bacterial cells, acting in multiple metabolic process.• Enzyme LuxS is involved in detoxification of a metabolite intermediate.• In Pseudomonas aeruginosa Pseudomonas Quinolone Signal

(PQS) has iron chelating properties.

Winzer K et al. Microbiology 2002;148:909-922

©2002 by Society for General Microbiology

• Another non-signaling function observed in bacterial signaling molecules is that of Antimicrobials.

• Gram-positive bacteria produce a small peptide namely Lantibiotics which has antibiotic properties. It has also been found that in some cases lantibiotics act as a signaling molecule that regulates its own synthesis, like the activity of AIs as signaling molecule in several bacteri

• Mersacidin and subtilin by Bacillus sp.• Nisin production by Lactobacillus lactis.• Streptin production by Streptococcus pyogenes.

• Bacterial AIs can also help in the competition between species and strains.

• Here these molecules don’t act as signaling molecule rather as signaling inhibitors.

• . It has been observed that different strains of S.aureus produce different peptides. Thus a particular kind of peptide produced from one strain can inhibit the gene expression of other strains.

• Therefore it can be suggested that in S.aureus quorum sensing may function as a way to control the gene expression and also as a mechanism to survive in the competition and allow more efficient host colonization.

Summary • Quorum sensing” (QS) is the phenomenon which allows single bacterial

cells to measure the concentration of bacterial signal molecules. Two principle different QS systems are known, the Autoinducer 1 system (AI-1) for the intraspecies communication using different Acyl

homoserinelactones (AHL) and AI-2 for the interspecies communication.

• AHL were originally identified in marine bacteria, where they play a pivotal role in the regulation of bioluminescence in Vibrio fischeri.

• InGram-negative bacteria, the autoinducers are typically molecules called acyl-homoserine lactones or AHL.

• InGram-positive bacteria, the autoinducers are oligopeptides, short peptides typically 8-10 amino acids long.

• Virulence factors such as exoenzymes and toxins can damage host cells enabling the bacteria in the biofilm to obtain nutrients.

• S. aureus has a peptide based quorum sensing system ,encoded by accessory gene regulator (agr) locus.

• In P.aeruginosa at least three interwined quorum sensing (QS) system (las ,rhl,AHL)and one orphan autoinducer receptor affect the ability of this organism to cause disease.

• In E.coli Lux S have functions related to bacterial virulence such as flagellar motility, surface

adhesion and Shiga toxin production.

Conclusion

From the above foregoing it may be concluded that quorum sensing is a mechanism by which bacteria can not only communicate to each others but also regulate their virulence gene expression in numerous microorganism.

What are the advantages we get by studying quorum sensing mechanism ?• We can understand that how bacteria can communicate to each

others.• We can understand the relationship between bacterial virulence and

quorum sensing.• As quorum sensing controls bacterial virulence, It has been

considered an attractive target for development of new therapeutic strategies.

Attractiveness of using quorum sensing inhibitor over antibiotic • Antibiotics are active on diving/replicating cells but quorum sensing

inhibitors are active on both diving/replicating cells .• Antibiotics have adverse effect on host system but quorum sensing

inhibitors have no adverse effect on host.• eg: QS inhibitor RNAIII-inhibiting peptide (RIP) has been found to

inhibit Staphylococcus aureus virulence without affecting host system.

• Most of the antibiotics failed to inhibit bacteria (ie, bacteria resistent towards antibiotics) but quorum sensing inhibitors are able to inhibit bacterial pathogenesis.

Some of the strategies to combat bacterial virulence based on the inhibition of bacterial quorum sensing (QS) system• To inhibit the production of autoinducer (Ais) molecules• The virulence of Staphylococcus aureus Is destroyed by inhibiting QS

system• Natural QS inhibitors like cyclic sulfur compounds,halogenated

furanones ,penicillin acid,garlic extract, 4-nitro-pyridine-N-oxide were found to inhibit the activation of virulence genes in Pseudomonas aeruginosa

• QS inhibitors from plant species Combretum albiflorum revealed that catechin has a negative impact on the production of QS dependant virulence factor