MIC 4124 3.1 - Regulation of Virulence Factors

Are all genes present in a genome expressed?

No, just because a gene is present in the genome, that doesn't mean it's expressed at all times

What are the two main ways bacteria respond to changes in the environment?

- Quorum sensing

- Two-component signalling systems

Bacteria can interact with each other?

Yes, bacteria communicate with each other using chemical signals that allow them to act as communities, able to accomplish tasks that they could not perform as single entities

What organisms did microbiologists study to learn about cell-cell communication between bacteria?

Vibrio fischeri (bioluminescent gram-negative bacteria) which forms a symbiotic relationship with the squid Euprymna scolopes (Hawaiian Bobtail Squid)

V. fischeri response to reaching the critical concentration

They bioluminesce

Symbiotic relationship of V. fischeri and squid

Vibrio fischeri colonize the light organ of the squid

- Living here is beneficial for bacteria because organ is rich in nutrients

When feeding, the squid uses its light organ to provide camouflage by projecting light downwards

- Helps to limit detection by predators

E. scolopes (squid) behaviour

E. scolopes is nocturnal & emerges at dusk to hunt

squid-vibrio symbiosis daily cycle

- At night, the squid light organ is full of V. fischeri - the bioluminescence from this bacterium is used by the squid to camouflage itself from potential predators

- At dawn, the host expels ~95% of the light-organ bacteria into the surrounding

environment.

- The squid then buries beneath the sand and during the day the remaining 5% of bacteria that remain in the light organ grow

What substance, secreted by bacteria, is responsible for facilitating quorum sensing?

Autoinducers

Autoinducer function

Signal to bacteria the density of it surrounding population

- many bacteria produce similar autoinducers, each of which has different functions in the regulation of cellular processes

What type of autoinducers to gram-negative bacteria generally use?

acyl-homoserine lactones (AHLs)

What type of autoinducers to gram-positive bacteria generally use?

auto-inducing peptides (AIPs)

Bacterial cell communication depends on what?

Density of the bacterial population

At low cell densities...?

The autoinducer diffuses out of the cell. The functional outcome is not displayed.

At high cell densities...?

↑ in autoinducer in the external environment

- The diffusion gradient reverses and the autoinducer enters the cell

- Influx of autoinducer promotes signalling events that alter gene expression.

Because the influx of autoinducer is density-dependent, what does it allow single cells to do?

it allows single cells to assess the population density

Quorum

the minimum number of members in an organization needed to execute a role

Quorum sensing

Cell-cell communication that involves the production, detection & response to autoinducers, ultimately allowing bacteria to share information about cell density & to then adjust gene expression in unison

Operon

series of genes controlled by a single promoter usually with related functions

In V. fischeri what must be expressed to produce light?

The luciferase (lux) operon

Components of lux operon

LuxR - upstream of operon important for binding of RNA pol

LuxI - autoinducer synthase, makes AHL

LuxA + LuxB - subunits of luciferase

LuxC, LuxD, + LuxE - encode for polypeptides needed for the substrates of luciferase

V. fischeri quorum sensing (low cell density)

Results in basal level of luxI - low levels of autoinducer, BUT the whole operon is not expressed, so no luciferase being formed. (RNA pol falls off before transcribing other lux genes)

- AHL is quickly secreted out of the cell and does not bind to the lux box promoter region

V. fischeri quorum sensing (high cell density)

High concentration of AHL in the external environment. It will diffuse back into the cell and bind to the LuxR regulator. The LuxR regulator can now recruit RNA pol and it will have high processivity and transcribe the entire gene. Now there is active luciferase and the cell begins to bioluminesce.

Is the expression of virulence factors always done through quorum sensing?

No, but it is a way that bacteria can express virulence factors

How does S. aureus express its virulence factors?

S. aureus is a Gram-positive bacterium that uses a 'two-component' quorum sensing system encoded by the agr locus.

Agr locus components

AgrA - response regulator

AgrB - membrane transporter

AgrC - sensor kinase

AgrD - pre-AIP

S. aureus virulence (Quorum sensing at low concentrations)

At sub-critical autoinducer concentrations, AgrD expression is very low (Agr locus expression is low - basal levels)

S. aureus virulence (Quorum sensing at high concentrations)

AIP binds AgrC (sensor kinase)

AgrC phosphorylates AgrA

Phosphorylated AgrA activates the P2 and P3 promoter

Net result: upregulation of secreted virulence factors (toxins, proteases, lipases, etc.)

Agr locus P2 and P3 promoter functions

P2 - upregulate the expression of the Agr locus

P3 - upregulate the transcription of virulence factors and inhibit rot expression

rot gene function

repressor of toxins gene - inhibits the expression of virulence factors

Two-component signalling system

The TCSS links events occurring outside the cell to the regulation of gene expression

The two components of the Two-component signalling system

1 - Sensor kinase (transmembrane kinase that 'senses' environment)

2 - Response regulator (DNA-binding protein that modulates gene expression)

Sensor kinase response to environmental signal

Recognition of environmental signal results in activation of the kinase 'transmitter' domain.

The SK then auto-phosphorylates itself at a conserved histidine residue.

Sensor kinase response to active transmitter domain

The transmitter domain of the phosphorylated SK interacts with the response regulator, which promotes SK-dependent phosphorylation of a conserved aspartate residue within the response regulator.

Response regulator response to phosphorylation by SK

Phosphorylation of the RR leads to conformational changes in the RR that help mediate specific biological activities, including DNA binding and transcriptional regulation.

The RR therefore can modulate gene expression necessary for adaptation to the original detected stimulus.

Effects that the response regulator can have on gene expression

the RR may activate OR inhibit gene expression

Salmonella Enterica two-component signalling system

The PhoP-PhoQ TCSS is often thought of as the 'master regulator' of Salmonella virulence.

S. enterica pathogenicity islands

S. enterica contains two major pathogenicity islands (Salmonella Pathogenicity Islands). - SPI-1 and SPI-2

What do the SPIs of S. enterica encode for?

Type III Secretion System which allows for secretion of virulence factors

PhoP/PhoQ system (of S. enterica)

Regulates virulence genes directly and indirectly via SsrB/SsrA (SPI-2 genes)

involved in promoting expression of genes that are required for intra-macrophage survival