QS

Question 1

what is QS?

Answer 1

a method of cell-cell communication with secreted molecules that enables single bacterial cells to participate in group behaviours

also a way of monitoring population density

Question 2

3 examples of QS molecules bacteria can produce

Answer 2

AHL’s, proteases and haemolysins

Question 3

proteases break down

Answer 3

host cell proteins such as the immune system

Question 4

haemolysins

Answer 4

lyse red blood cells for iron

Question 5

G- autoinducers are called

Answer 5

AHL’s (acyl homoserine lactones)

made by LuxI/R 2 component system
LuxI makes the AHLs
they diffuse out of the cell
once quorum is reached, AHL diffuses back in and activates LuxR which is a TF
activates QS genes

Question 6

G+ autoinducers are called

Answer 6

AIP’s (auto inducing peptides)
AIP’s do NOT reenter the cell

produced as a propeptide which is activated when it is secreted and cleaved

e.g. AgrC/A 2 component system in S. aureus
AgrB secretes AIP
AgrC is the histidine kinase, binds AIP
AgrA TF phosphorylated, turns on QS genes

Question 7

P. aerugenosa has 3

Answer 7

QS systems

two of them are AHL: LasIR + RlhIR
the third is the PQS system (Pseudomonas quinolone signal)

Question 8

LasIR + RlhIR

Answer 8

LuxI family proteins! conserved produce AHL (slightly different, lactone ring conserved but the carbon tail is different length with different oxygenation), can diffuse freely in aq environment

Question 9

PQS

Answer 9

structurally distinct signal molecule that is extremely hydrophobic in nature - means that it cannot passively diffuse within a bacterial population

it is instead packaged into membrane vesicles and actively trafficked

Question 10

How much of P. aerugenosa’s genome is regulated by QS?

Answer 10

5%

genes activated are potential virulence factors that contribute to infectious process

Question 11

Proteins/molecules produced by the QS regulon in P. ar (

Answer 11

Elastase
Alkaline Protease 
Superoxide dismutase
metalloendopeptidase
HCN 
Rhamnolipids
Pyocyanin

Question 12

what does Elastase do?

Answer 12

degrades elastin and other matrix proteins of the human lung

leading to tissue damage and destruction of lung structure. It is also a potent inflammatory factor.

Question 13

what does Alkaline Protease do?

Answer 13

proteolytically inactivate cytokines and other host defence proteins, thus interfering with the host immune response

Question 14

what does Superoxide dismutase do?

Answer 14

important anti-oxidant protein protecting the cell from oxidative stress

Oxidative stress is a key component of the innate immune response, particularly within phagocytic cells, so the ability of bacteria to resist this stress can promote its survival.

Question 15

what does metalloendopeptidase do?

Answer 15

LasA is a metalloendopeptidase which has strong anti-staphylococcal activity, and can also degrade elastin (bugs can compete against each other)

Question 16

what does HCN do?

Answer 16

is toxic to cells, inhibiting cellular respiration

Within the lungs of CF patients (who are commonly infected with P aeruginosa), the detection of HCN in CF sputum has been directly associated with poorer lung function.

Question 17

what do rhamnolipids do?

Answer 17

haemolytic glycolipids with detergent-like activity. They demonstrate cytolytic activity against monocyte-derived macrophages and neutrophils.

Question 18

what does pyocyanin do?

Answer 18

generates ROS that damage host cells

(this is achieved by disrupting the cellular respiration of the host cells)

It also has diverse immunological effects by disrupting the cytokine and chemokine response to infection

Question 19

what can evade the immune response?

Answer 19

Biofilm architecture

Question 20

QS knockouts in p. aerugenosa have shown

Answer 20

• Less tissue destruction
• Reduced cases of pneumonia
• Reduction in disseminated infection
• Reduced mortality

Question 21

QS has important roles in

Answer 21

biofilm formation

Question 22

which AB can inhibit QS?

Answer 22

Azithromycin

although has no bactericidal or bacteriostatic activity against P. aeruginosa, its use has seen clinical benefits

Question 23

How do inter-species of bacteria communicate?

Answer 23

Similarity between AHL molecules of different species can enable inter-species communication

e.g. P. aeruginosa & B cepacia form mixed biofilms in the CF lung
B. cepacia can respond to the quorum sensing molecules produced by Pseudomonas

Question 24

AHLs Acyl homoserine lactones produced by different bacteria share the same basic structure but differ in:

Answer 24

1. Length of the R-group side-chain

2. Substitution of a carbonyl at the third carbon

Question 25

why is inter-species communication significant?

Answer 25

increase severity of disease

e.g. Non-pathogenic bacteria harboured by the host can modulate the behaviour of pathogenic bacteria
pseudomonas and oropharyngeal bacteria - pPA responds to the autoinducers produced by OF and becomes much more pathogenic

Question 26

QS plays a central role in virulence regulation (including biofilm formation), and as such is an

Answer 26

attractive antimicrobial target

Question 27

Extra reading

Answer 27

Many classes of AIs have been described to date. The most intensely studied AIs are the N-acylhomoserine lactones (AHLs) of Gram-negative bacteria, the peptides of Grampositive bacteria and a class of AIs termed AI-2, whose structures remain unknown in most cases. AHLs are usually detected through binding to and activation of cytoplasmic receptor proteins, which dimerize upon signal detection and can bind to promoter regions of target genes to activate or repress their transcription. Peptides are usually detected through binding to membrane sensor proteins of the two-component system family, although some can also be transported to the cytoplasm before interacting with their receptors. 
On the other hand, AI-2 binds a periplasmic protein and then interacts with either a two component system or a transporter depending on the organism. 
Binding to a membrane-associated sensor kinase causes the activation of a phosphorelay cascade, which results in the activation or repression of a response regulator, culminating in altered gene expression

Question 28

Reading s aureus

Answer 28

S. aureus has multiple virulence factors and can display very rapid transmission, aiding its importance as a human pathogen. Additionally, S. aureus forms biofilms
on many surfaces, including indwelling devices such as
urethral stents. These indwelling devices, and subsequent biofilms formed on them, pose a serious risk for Staphylococcus infection

One of the factors which contribute to S. aureus virulence
is its peptide-based quorum sensing system, encoded by the accessory gene regulator (agr) locus.
The autoinducer in the agr system is an autoinducing peptide (AIP), encoded by agrD.
AIP is trimmed and secreted by AgrB, a membrane-bound protein. The active AIP is 7–9 aa, with a 5-membered thiolactone ring.
AgrC is a membrane-bound sensor kinase to which
extracellular AIP binds, leading to AgrC autophosphorylation and activation of AgrA.
The agr system is intricately involved in the regulation of virulence genes, predominantly from two promoters, P2 and P3, which produce RNAII and RNAIII, respectively

P2 promotes the transcription of the agr operon from the RNAII transcript, which includes agrA, agrB, agrC and agrD. Active AgrA may be a phosphorylated
homodimer that induces transcription at the P2 and P3
promoters, with a higher affinity for P2

Transcription from P3 leads to the production of RNAIII, which is the effector molecule of the agr system. RNAIII is a regulatory RNA, which also functions as the mRNA for the sigma-toxin. The 5’ end is thought to upregulate a-haemolysin, while the 3’ end is required for the repression of protein A synthesis. RNAIII reduces the expression of surface adhesins, and increases the production of capsule, toxins and proteases.

The agr system is thought to regulate over 70 genes, 23 of which are known virulence factors

Of the virulence factors regulated by agr, there are two
classes: the first class contains virulence factors involved in attachment to the host and immune evasion

the second class contains genes involved in the production of exoproteins associated with invasion and toxin production.  It has been thought
that the activation of the agr system essentially switches the bacterium from an adhesive, colonizing commensal to an invasive and aggressive pathogen

It is thought that S. aureus possesses two independent mechanisms of biofilm formation; the first involves an extracellular polysaccharide, polysaccharide intercellular adhesin (PIA), and the second is thought to be PIA-independent, possibly involving adhesive proteins and the sarA and agr global regulators. Because biofilms are thought to play a critical role in S. aureus infection, the role of agr in biofilm formation has been explored. When agr is non-functional, S. aureus has enhanced adhesion abilities.Therefore, when agr is not active, the bacteria remain in the first stage of biofilm formation, adhering to a surface.

RNAIII is the effector of the agr system and is involved in
bacterial virulence, so mechanisms to inhibit RNAIII
have received considerable attention. An RNAIII-inhibiting peptide (RIP) has been found to inhibit S. aureus biofilm formation and toxin production. It is
thought that by inhibiting cell–cell communication, RIP is
able to prevent adherence and virulence of S. aureus.

Question 29

Reading p aerugenosa

Answer 29

P. aeruginosa is a Gram-negative bacterium capable of
surviving in a wide range of environments. This organism is an opportunistic pathogen and it is commonly associated with nosocomial infections and infections of severely burned individuals, and is a leading 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

Quorum sensing regulates the production of several extracellular virulence factors, promotes biofilm maturation and regulates the expression of antibiotic efflux pumps, meaning that it has a key role in the pathogenesis of P. aeruginosa.
The las and rhl systems regulate the timing and production of multiple virulence factors, including elastase, alkaline protease, exotoxin A, rhamnolipids, pyocyanin, lectins and superoxidase dismutase

P. aeruginosa rhlI and lasI mutants cause less
tissue destruction and decrease mortality when compared with wild-type strains in multiple animal models indicating an important role for quorum
sensing in P. aeruginosa pathogenesis.

Apart from regulating the expression of virulence factors,
some of the AIs have been shown to directly interact with host cells. 3las AI’s induces interleukin (IL)-8 secretion from human bronchial epithelial cells. It also inhibits lymphocyte proliferation, downregulates the production of tumour necrosis factor alpha
(TNF-a) and IL-12, and activates T cells. las AI also induces apoptosis in macrophages and neutrophils, suggesting that this molecule not only enhances the expression of virulence genes but also affects
immune responses to infection

Question 30

Quorum sensing in bacterial virulence

Microbiology (2010)

Answer 30

Bacteria communicate through the production of diffusible signal molecules termed autoinducers.

The molecules are produced at basal levels and accumulate during growth. Once a critical
concentration has been reached, autoinducers can activate or repress a number of target genes.

Because the control of gene expression by autoinducers is cell-density-dependent, this
phenomenon has been called quorum sensing.

Quorum sensing controls virulence gene
expression in numerous micro-organisms.

As quorum sensing controls virulence,
it has been considered an attractive target for the development of new therapeutic strategies.

Question 31

Quorum sensing in Staphylococcus aureus virulence

Answer 31

AGR SYSTEM:
One of the factors which contribute to S. aureus virulence is its peptide-based quorum sensing system, encoded by the accessory gene regulator (agr) locus

The agr system is intricately involved in the regulation of virulence genes, predominantly from two promoters, P2 and P3, which produce RNAII and RNAIII, respectively

The autoinducer in the agr system is an oligopeptide that has been termed the autoinducing peptide (AIP), encoded by agrD. AIP is trimmed and secreted by AgrB, a membrane-bound protein

AgrC is a membrane-bound sensor kinase to which
extracellular AIP binds, leading to AgrC autophosphorylation and activation of AgrA

P2 promotes the transcription of the agr operon from the RNAII transcript, which includes agrA, agrB, agrC and agrD

Active AgrA may be a phosphorylated homodimer that induces transcription at the P2 and P3 promoters, with a higher affinity for P2

Transcription from P3 leads to the production of RNAIII, which is the effector molecule of the agr system
RNAIII reduces the expression of surface adhesins, and increases the production of capsule, toxins and proteases

Of the virulence factors regulated by agr, there are two 
classes: the first class contains virulence factors involved in attachment to the host and immune evasion, while the second class contains genes involved in the production of exoproteins associated with invasion and toxin production 

It has been thought that the activation of the agr system essentially switches the bacterium from an adhesive, colonizing commensal to an invasive and aggressive pathogen

BIOFILMS:
One of the ways in which agr is thought to impact
virulence is through its role in biofilm formation

It is thought that S. aureus possesses two independent mechanisms of biofilm formation; the first involves an extracellular polysaccharide, polysaccharide intercellular adhesin (PIA), and the second is thought to be PIA-independent, possibly involving adhesive proteins and the sarA and agr global regulators

When agr is non-functional, S. aureus has enhanced adhesion abilities

It is thought that this role of agr is brought about by the reduction in adhesin production and an increase in the production of both d-haemolysin and proteases

agr is also important for detachment of clusters of cells from the biofilm (dissemination of infection)

Studies have found that agr expression varies during different steps of infection. This suggests that agr may play different roles during the course of infection and this may explain the discrepancy in some of the results regarding its role in bacterial virulence (e.g. in CF patients agr is not active)

DRUG THERAPY:
RNAIII is the effector of the agr system and is involved in bacterial virulence, so mechanisms to inhibit RNAIII
have received considerable attention. An RNAIII-inhibiting peptide (RIP) has been found to inhibit S. aureus biofilm formation and toxin production. By inhibiting cell–cell communication, RIP is able to prevent adherence and virulence of S. aureus.

Question 32

Quorum sensing control of Pseudomonas aeruginosa virulence

Answer 32

P. aeruginosa is a Gram-negative bacterium capable of
surviving in a wide range of environments. This organism is an opportunistic pathogen and it is commonly associated with nosocomial infections and infections of severely burned individuals, and is a leading cause of death in severe respiratory infections, such as chronic lung infections in CF patients

At least three intertwined quorum sensing systems and
one orphan autoinducer receptor affect the ability of P.
aeruginosa to cause disease

LAS/RHL SYSTEMS:
Two of these systems, las and rhl, rely on the production of AHLs as the signalling molecules (AIs)

LAS:
In the las system, N-3-oxododecanoyl-homoserine lactone (3OC12- HSL) is produced by the enzyme encoded by the lasI gene.
When P. aeruginosa reaches a certain threshold density, 3OC12-HSL binds to the transcriptional activator LasR.
LasR, in turn, dimerizes and binds to target promoters to control gene expression

RHL:
in the rhl system, the rhlI gene encodes the enzyme
involved in the production of N-butyryl-homoserine
lactone (C4-HSL). As with 3OC12-HSL, C4-HSL binds to
its cognate transcriptional regulator, RhlR, to control the
activity of target promoters
The rhl system is controlled by the las system at both
transcriptional and post-transcriptional levels

The las and rhl systems regulate the timing and production of multiple virulence factors, including elastase, alkaline protease, exotoxin A, rhamnolipids, pyocyanin, lectins and superoxidase dismutase

QSCR:
Besides LasR and RhlR, P. aeruginosa encodes an
orphan receptor protein, QscR, which can sense 3OC12- HSL to control its own regulon

PQS:
A third P. aeruginosa AI molecule, 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS) has also been identified.
Molecules have been shown to play a role in cell–cell communication in P. aeruginosa. PqsH, which is controlled by the las system
PqsR is a LysR transcriptional factor that is activated
by HHQ and PQS, leading to the positive activation of
many virulence factors, which include a large number of
genes also controlled by las and rhl.
PQS has been shown to affect biofilm formation and to regulate several virulence factors in P. aeruginosa, including elastase, pyocyanin and LecA lectin, and it is considered essential for full virulence in multiple hosts
PQS can also act as an iron chelator, and both the synthesis of PQS and the activity of PqsR–PQS are
involved in iron homeostasis,

DISEASE:
Multiple P. aeruginosa virulence factors are involved in the development of disease, including secreted factors (such as proteases) and cell-associated factors (such as lipopolysaccharide and flagella), as well as the ability to form biofilms

Quorum sensing regulates the production of several extracellular virulence factors, promotes biofilm maturation and regulates the expression of antibiotic efflux pumps

It has been shown that P. aeruginosa rhlI and lasI mutants cause less tissue destruction and decrease mortality when compared with wild-type strains in multiple animal models

Some of the AIs have been shown to directly interact with host cells. 3OC12-HSL induces interleukin (IL)-8 secretion from human bronchial epithelial cells
It also inhibits lymphocyte proliferation, downregulates the production of tumour necrosis factor alpha (TNF-a) and IL-12, and activates T cells to produce gamma-interferon. 3OC12-HSL also induces apoptosis in macrophages and neutrophils, suggesting that this molecule not only enhances the expression of virulence genes but also affects immune responses to infection

Besides controlling bacterial virulence, PQS and HHQ have been shown to downregulate the host immune response through NFkB

DRUG THERAPY:
Natural quorum sensing inhibitors include cyclic sulfur compounds, patulin and penicillin acid

Through the use of a screening system, garlic extracts and 4-nitropyridine-N-oxide were also identified as quorum sensing inhibitors
These molecules specifically inhibited the activation of virulence genes by quorum sensing, reduced biofilm tolerance to tobramycin and also decreased virulence

Molecules derived from the plant species Combretum albiflorum revealed that catechin has a negative impact on the production of quorumsensing-dependent virulence factors

Subinhibitory concentrations of macrolides have
been shown to be effective in inhibiting quorum-sensing dependent virulence
macrolides are not ideal because, like any other antibiotic, excessive usage is associated with bacterial resistance