L5 - Social communication & polymicrobial interactions

Question 1

What is quorum sensing?

Answer 1

• A form of cell-to-cell communication
• Characterized by the secretion & detection of autoinducers
• Detection of autoinducer results in coordinated behaviours within the population

Question 2

QS requires the secretion and detection of what?

Answer 2

Autoinducers

Question 3

What is the concentration of autoinducer is proportional to?

Answer 3

Population density

Question 4

Autoinducer molecules – the AHLs are widespread in what bacterium?

Answer 4

N-acylhomoserine lactone (AHL) QS is widespread in

Gram-ve bacteria

Question 5

Name the autoinducer molecules

Answer 5

• AHL (N-acylhomoserine lactone)
• AI-2 (Autoinducer-2)
• AIP (Autoinducing peptide)
• DSF (Diffusible signal factor)
• PQS (Pseudomonas quinolone signal)

Methods of secretion & detection vary depending on the autoinducer

Question 6

Define the autoinducer molecules

Answer 6

Autoinducer-2 is shared by both Gram-positives & Gram-negatives and is synthesized by the protein LuxS.

AIP-mediated QS activity is a feature of Gram-positives. AIPs differ in their length and amino acid sequence. This peptide is initially produced as a propeptide, which is then processed within the bacterial cell and actively secreted from the cell (rather than passive diffusion). AIPs do not re-enter the cell. Instead, they act on a histidine kinase on the cell surface, that then in turn activates a cytoplasmic response regulator, bringing about a change in gene expression.

DSF-like molecules are commonly found in Gram-negative species. They are also reported to occur in Streptococcus mutans (Gram-positive)

PQS is specific to Pseudomonas aeruginosa, although several species produce related alkyl-quinolone (AQ) molecules.

Question 7

Acyl homoserine lactone (AHL) QS mediated by what?

How does this occur?

Answer 7

Mediated by LuxI & LuxR family proteins
LuxI proteins are AHL synthases; LuxR proteins are transcriptional regulators.

• AHL is produced within the cell and diffuses out. Diffusion gradient favours movement of the AHL from the cytoplasm to the extracellular environment. At high cell density, AHL accumulates in the extracellular environment and the diffusion gradient is reversed. Consequently, the diffusion of AHLs back into cell is favoured. After re-entering the cell, it binds with transcriptional regulator LuxR that then binds to DNA and alters gene expression accordingly.
• When extracellular AHL accumulates, AHL diffuses back into the cell, within the cell AHL binds to transcriptional regulator LuxR, altering its activity and subsequent gene expression

Question 8

PQS QS (P. aeruginosa) mediated by what?

Answer 8

PQS signal molecule is hydrophobic, and is trafficked between cells in outer membrane vesicles.

Due to the very hydrophobic nature of PQS, it is very unlikely to freely diffuse in aqueous environments. Consequently, for trafficking between cells, it appears to be packaged within membrane vesicles. Indeed, by interacting with structural components of the lipid A, PQS promotes its own packaging into vesicles by influencing membrane fluidity and curvature, and promoting vesicle blebbing.

Question 9

AIP QS (Staphylococcus aureus) mediated by what?

Answer 9

• ‘Auto-inducing peptides’ (AIP); derived from the locus agrBDCA
• “Propeptide” is processed and actively secreted by AgrB
• AgrC & AgrA constitute a two-component system that mediates AIP sensing and response.

In the case of Gram-positives like Staphylococcus aureus, the signal is an “auto-inducing peptide” rather than an AHL. This peptide is initially produced as a propeptide, which is then processed within the bacterial cell and actively secreted from the cell (rather than passive diffusion of AHLs). Again, once the concentration of AIP reaches a certain level, it exerts an effect on the bacterial cells. However, AIPs do not re-enter the cell. Instead, they act on a receptor on the cell surface, that then in turn activates a cytoplasmic protein termed a response regulator that alters gene expression. This process is a two-component system, that occurs through histidine kinase activity of the receptor protein and subsequent phosphotransfer to the response regulator.

Question 10

Why do bacteria perform quorum sensing?

Answer 10

Bacteria produce many secreted factors that are important for growth &/or survival, for example:

1. Proteases break down proteins, providing carbon source for growth
2. Haemolysins lyse blood cells, releasing source of iron for bacteria

• Individual bacterial cells cannot produce sufficient quantities to gain much benefit

However, populations of bacteria can produce sufficient quantities if they co-ordinate their behaviour through quorum sensing

Question 11

How many QS systems of P. aeruginosa are there? and what are they?

Answer 11

At least four QS systems exist in P. aeruginosa

1) LasIR system
3-oxo-C12-HSL (AHL)

2) RhlIR system
C4-HSL (AHL)

3) Pseudomonas quinolone signal (PQS)
4) IQS

Within Pseudomonas aeruginosa, there are at least four distinct quorum sensing systems. Two of them are based on the AHL system (LasIR and RlhIR). In these AHL-based systems, LasI and RhlI are the AHL synthases (i.e. the LuxI family proteins), whilst LasR and RhlR are the corresponding transcriptional regulators (the LuxR family proteins).

In addition, there is the PQS system (Pseudomonas quinolone signal). The PQS system uses a structurally distinct signal molecule that is extremely hydrophobic in nature. This hydrophobicity means that for it to effectively diffuse within/through a bacterial population, it is actually packaged into membrane vesicles that bud off from the bacterial cell, and it is these PQS-containing vesicles that enable PQS-mediated cell-cell communication.

The IQS system is the most recently described, so called because it Integrates quorum sensing systems and stress response pathways.

Question 12

Explain the hierarchy of the Pseudomonas AHL systems

Answer 12

LasIR system makes the 3-oxo-C12-HSL. Once the concentration of that HSL exceeds the threshold, it re-enters the cell and binds to LasR. The 3-oxo-C12-HSL-LasR complex binds promoter regions of multiple genes, activating or repressing their transcription. It autoregulates itself (i.e. promoting lasI expression to further promote synthesis of 3-oxo-C12-HSL). It also activates expression of the Rhl system. Both the LasIR and the RhlIR systems regulate sets of virulence genes – some unique to each system, others in common.

The Las system controls the production of multiple virulence factors involved in acute infection and host cell damage, including the LasA & LasB elastases, exotoxin A (toxA), and alkaline protease (AprA). The Rhl system controls expression of several genes, including those responsible for the production of rhamnolipids (rhlAB), and those implicated in secretion systems (XcpR & XcpP).

Question 13

Define the interconnectedness of the four QS signalling networks in P. aeruginosa.

Answer 13

The LasIR system, which makes the 3-oxo-C12-HSL, is generally regarded as being at the top of the hierarchy. The Las system positively-regulates all of the other three systems, as well as regulating itself (i.e. positive feedback loop). The Rhl system also positively-regulates itself.

pqsABCD are involved in the synthesis of PQS. PqsR is a LysR transcriptional regulator that controls expression of the pqsABCD operon, and the expression of pqsR is itself controlled by the LasR system. PqsR is the receptor for PQS and also its co-inducer, as the activity of PqsR in inducing expression of pqsABCD is dramatically increased when PQS is bound. The PQS system enhances expression of the rhl genes, promoting activity of the RhlIR system. However, expression of the pqs operons is inhibited by the activity of the RhlIR system, suggesting that the ratio of concentrations of the 3-oxo-C12-HSL and the C4-HSL play a decisive role in determining the level of PQS activity.

IQS synthesis involves the ambBCDE gene cluster. The IQS system is also tightly controlled by the LasIR system – disruption of either lasI or lasR completely abrogates expression of genes responsible for IQS production.

Beyond those connections identified above, environmental factors can influence activity of QS systems (e.g. IQS is influenced by low phosphate levels), whilst there is evidence to suggest that individual QS systems can compensate for the loss of another (e.g. the Rhl system can compensate for a non-functional Las system by upregulating lasI and promoting production of 3-oxo-C12-HSL).

Question 14

The QS regulon of P. aeruginosa

Answer 14

Elastase 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.

Alkaline Protease can proteolytically inactivate cytokines and other host defence proteins.

Superoxide dismutase is an important anti-oxidant protein, protecting the cell from oxidative stress.

LasA is a metalloendopeptidase which has strong anti-staphylococcal activity, and can also degrade elastin

Hydrogen cyanide (HCN) is toxic to cells, inhibiting cellular respiration. Detection of HCN in CF sputum has been directly associated with poorer lung function.

Rhamnolipids are haemolytic glycolipids with detergent-like activity. They demonstrate cytoltic activity against monocyte-derived macrophages and also polymorphonuclear leukocytes (e.g. neutrophils)
Pyocyanin has very diverse functions.

Question 15

What are the 4 biological activities of pyocyanin from the QS regulon of P. aeruginosa?

Answer 15

REDOX EFFECT
IMMUNOLOGICAL EFFECTS
COMPETITIVENESS
PHYSICAL EFFECTS

Question 16

Define the REDOX EFFECT of pyocyanin

Answer 16

REDOX EFFECT
- Results in production of ROS, inhibiting various cellular processes

• Depletes/inhibits host cell catalase & glutathione
• Depletes ATP/cAMP stores
• Inactivates α1-protease inhibitor, which normally protects tissue from protease-mediated damage

Pyocyanin is redox active, and leads to the production of reactive oxygen species (ROS) and hydrogen peroxide, imposing significant oxidative stress on host cells. A pivotal defence of host cells to oxidative stress includes the role of catalase and glutathione, which oversee the removal of hydrogen peroxide. However, pyocyanin can deplete cellular levels of both catalase and glutathione. In order to generate the ROS, pyocyanin interferes with cellular respiration, resulting in a marked depletion of intracellular cAMP and ATP. This is behind the reduced cilia beating frequency. However, it might also further exacerbate the CF condition by directly altering activity of CFTR (the activity of which relies on ATP-driven conformational changes). Additionally, the redox activity of pyocyanin can inactivate alpha1-protease inhibitor, which is a major defence against protease-mediated cell injury. By inactivating alpha1-protease inhibitor, pyocyanin leaves the lung tissues more susceptible to protease damage.

Question 17

Define the IMMUNOLOGICAL EFFECTS

of pyocyanin

Answer 17

• Promotes IL-8 release, leading to neutrophil influx
• Promotes apoptosis of neutrophils
• Decreases RANTES release (normally resolves inflamm.)
• Inhibits production of IL-2 & IL-2R

It hugely promotes the release of IL-8, which is a major chemoattractant for neutrophils, leading to a large influx of neutrophils. However, it also promotes the apoptosis of those neutrophils and impairs the clearance of those apoptotic cells. Pyocyanin also decreases the release of RANTES, a chemokine that normally functions to resolve inflammation by attracting T-cell monocytes and eosinophils to the inflammation site. Pyocyanin also inhibits the production of IL-2 and the expression of IL-2R. IL-2 enhances the production of other cytokines and is also needed for the proliferation of cytotoxic T-cells. A such, the reduction in IL-2 production could prevent the development of an effective T-cell response and also prevent activation of monocytes and macrophages (through the inhibition of cytokine function).

Question 18

Define the COMPETITIVENESS

of pyocyanin

Answer 18

Pyocyanin has antibiotic activity against other bacteria & fungi

Question 19

Define the PHYSICAL EFFECTS

of pyocyanin

Answer 19

Inhibits ciliary beating

Question 20

What is the importance of QS in P. aeruginosa virulence

Answer 20

lasI gene belongs to the LasIR system.
pqsA gene belongs to the PQS system.
ambB gene belongs to the IQS system.

Note that ∆ambB(ambB) is the complemented form of the ambB mutant (i.e. functional ambB has been reintroduced). Its virulence matches the wildtype.

Question 21

Why are QS-deficient mutants selected during infection?

Answer 21

Mutants are more likely to be ‘signal blind’ (lasR mutants)
It is the response to QS that is energetically expensive, rather than making the AHL itself (5% vs 0.01% cellular ATP pool).

The major energy demands related to QS come from the response to the AHL rather than the production of the AHL itself. Of the total cellular ATP pool, it is estimated that approx. 0.01% is required for synthesis of AHLs, but approx. 5% is required for the subsequent QS response.

QS-deficient mutants are therefore more likely to be blind to the signal molecule (i.e. lasR mutants), rather than being lasI mutants that are unable to make the AHL. However, due to autoregulation of the QS genes, the majority of reported mutants that are deficient in LasR are also unable to make the actual AHL signal molecule, because the production of the signal is itself regulated by QS.

In mixed infections, a 50/50 mix of lasR mutant and wildtype is significantly less virulent than the wildtype alone (and NOT significantly different from the lasR mutant alone). Note that this attenuation is not due to there simply being less wildtype bacteria within the inoculum.

Question 22

Why do QS-deficient mutants need wildtype bacteria?

Answer 22

Mutants are fitter when present as a low proportion of the total population
Mutants make up for the functions they lack by exploiting bacteria that are QS-proficient

Mutants benefit from ‘public goods’ produced by the QS-proficient bacteria, e.g.:

• Proteases
• Haemolysins
• Siderophores
• Biofilm matrix components

Question 23

QS-mediated inter-species communication influences what?

Answer 23

Inter-species interactions between bacteria can profoundly influence their behaviour, including virulence.

Such interactions can occur through numerous mechanisms including:
Sensing of microbial products (e.g. peptidoglycan, siderophores)
Via responding to each others autoinducer molecules

Question 24

How do AHLs mediate inter-species communication?

Answer 24

In some cases, precisely the same autoinducer is produced by multiple species. For example, the 3-oxo-C12-HSL produced by Pseudomonas is produced by some Yersinia species, whilst the C4-HSL produced by Pseudomonas is also produced by some Aeromonas, Serratia and Vibrio species.

Non-identical AHLs can also mediate inter-species communication. A study by Riedel et al (2001) [Microbiol 147, 3249-3262] showed that Burkholderia cepacia can respond to the autoinducers produced by P. aeruginosa, but that P. aeruginosa CANNOT respond to the B. cepacia autoinducers. i.e. the signalling between species is unidirectional.

Question 25

Name 2 bacterial species that respond to one anothers AHLs in vivo

Answer 25

B. cepacia and P. aeruginosa

In a mouse model of chronic lung infection, mice were infected with a mixture of B. cepacia and P. aeruginosa. The P. aeruginosa were either wildtype (left image) or QS-deficient (right image). The P. aeruginosa were labelled with red fluorescent protein. The B. cepacia were genetically engineered to fluoresce green ONLY in response to P. aeruginosa AHLs. The presence of green cells in the left image indicates that B. cepacia IS responding to P. aeruginosa AHLs within the mouse lung. As expected, this is not seen in the right hand image when the P. aeruginosa were QS-deficient (i.e. NOT producing AHLs).

Question 26

Define the Diffusible signal factor (DSF)

Answer 26

DSF signal molecules are cis-2-unsaturated fatty acids, originally identified in the plant pathogen, Xanthomonas

DSF-like molecules were subsequently found in other species including Burkholderia and Pseudomonas
Implicated in traits including virulence and biofilm formation

Question 27

Name 3 Diffusible signal factors (DSF) in bacteria

Answer 27

1) Xanthomonas campestris DSF
2) Burkholderia cenocepacia (BDSF)A
3) Pseudomonas aeruginosa (cis-2 - decanoic acid)

Question 28

Define the DSF perception and inter-species signalling in Xanthomonas and Pseudomonas

Answer 28

In Xanthomonas, DSF is sensed via a two-component system, comprising of the RpfC hybrid histidine kinase and the RpfG response regulator. Note that RpfG is an example of a response regulator that has direct enzymatic activity rather than acting as a transcriptional regulator. The effector domain of RpfG is an HD-GYP domain that has cyclic diGMP phosphodiesterase activity. Its activation results in the reduction of cyclic diGMP levels in the cell, which is an important second messenger.

Similarly, two-component systems that respond to DSF-like signal molecules have been documented in Burkholderia and Pseudomonas. However, intriguingly, whilst P. aeruginosa can respond to both DSF & BDSF via a two-component system (encoded by genes PA1396 and PA1397), the sensor responsible (PA1396) does NOT detect cis-2-decenoic acid (the DSF-like molecule produced by Pseudomonas itself). Consequently, the mechanism by which P. aeruginosa senses its own DSF-like signal molecule remains to be defined.

Question 29

AI-2: A universal language?

Answer 29

AI-2 signal molecule is produced by a wide range of bacterial species
LuxS produces the DPD precursor, which then undergoes a variety of spontaneous rearrangements to produce distinct AI-2 molecules

Over 1/3rd of bacterial genomes reportedly encode a LuxS homologue
Mediator of QS, or simply a metabolic side product?

AI-2 is an interconverting family of extracellular signal molecules. The precursor molecule, DPD, undergoes various rearrangements and additional reactions to form distinct biologically active AI-2 signal molecules. In the figure above, the upper pathway leads to S-THMF-borate, which is made by Vibrio species, whilst the lower pathway results in R-THMF that is made by Salmonella.

DPD, the precursor molecule, is produced by LuxS.

Although LuxS (and therefore AI-2 molecules) are widely distributed across the bacterial kingdom, that doesn’t necessarily mean that AI-2 is performing a QS function in all of those species. It has been suggested that AI-2 is simply a metabolic side product in many species. AI-2-mediated QS activity certainly cannot be inferred simply on the basis of an organism being LuxS-positive.

Question 30

What are the AI-2 virulence-associated traits?

Answer 30

AI-2 has been implicated in virulence traits……
- Biofilm formation

• Motility &/or chemotaxis
• Virulence factor production
• Adherence

In both Gram-positives & Gram-negatives:

• Escherichia coli
• Salmonella species
• Staphylococci (including Staphylococcus aureus)
• Streptococci (including Streptococcus pneumoniae)
• Vibrio cholerae

… including some which don’t make AI-2 themselves!
P. aeruginosa responds to AI-2, but doesn’t encode a LuxS homologue

Question 31

Anti-virulence strategies - Inactivating the AHL molecule

Answer 31

Lactonases and acylases capable of enzymatic inactivation of AHLs are widely distributed in microbes and higher organisms.

Application of an engineered acylase:

The Pseudomonas acylase PvdQ has activity against long-chain AHLs
Koch et al. engineered PvdQ to increase its activity towards shorter chain AHLs, then successfully applied it to inhibit QS and virulence of Burkholderia cenocepacia H111.

Cells of B. cenocepacia H111 were incubated for 24 h with or without PvdQ and tested for C8-HSL production (the AHL of B. cenocepacia) and protease production (protease production is QS-regulated). The C8-HSL levels were quantified using a fluorescent bioreporter strain – the higher the fluorescence (left-hand y-axis) the greater the C8-HSL levels. As shown, addition of PvdQ significantly reduced both C8-HSL levels and protease activity. For comparison, the same studies were performed with a quorum sensing mutant of H111 (“H111 QS-null”).

PvdQ protects Galleria mellonella larvae against B. cenocepacia H111 infection. Larvae were injected with B. cenocepacia H111 that had either been pretreated or not pretreated with PvdQ. After 48 h of incubation, larval survival was assessed. Larvae injected with sterile buffer only showed 100% survival (left hand bar; “control”). Those injected with untreated B. cenocepacia H111 had a lethal outcome, whereas larvae injected with B. cenocepacia pretreated with PvdQ showed nearly 100& survival (comparable to that seen with the QS-null mutant). Pictures of the infected larvae are shown underneath (C).

Question 32

Conclusions

Answer 32

Numerous strategies exist for quorum sensing, albeit with the same general principle
different autoinducer molecules
different methods for autoinducer secretion, trafficking and/or detection

Autoinducers facilitate inter-species communication within mixed bacterial communities, profoundly influencing bacterial behaviour

QS plays a key role in virulence, and as such is recognised as a valid antimicrobial target for future ‘anti-virulence’ strategies