Lecture 7: Virulence factors - Regulation of gene expression

Question 1

Why is regulation of virulence factors important for bacteria?

Answer 1

Bacteria need virulence factors to initiate and maintain infection. But it is not effective to synthesize virulence factors when they are not necessary, because it takes energy, it is counterproductive and alarms the immune system.

Question 2

How do bacteria regulate virulence?

Answer 2

sensing of the enviromental signals and regulation of gene expression.

Question 3

What is an operon?

Answer 3

a cluster of genes that are expressed as a group from the same promoter.

Question 4

What is a regulon?

Answer 4

a group of genes and/or operons that are controlled by the same transcriptional regulator.

Question 5

Where does the majority of gene regulation take place?

Answer 5

transcriptional control.

Question 6

Why are new genes in bacteria switched on within minutes?

Answer 6

Because transcription and translation are coupled in bacteria.

Question 7

What is the sigma factor?

Answer 7

The beginning part of the RNA polymerase that directs the RNA polymerase to the promotor.

Question 8

What are the 4 signal transfer systems in bacteria?

Answer 8

Cassical regulators (repressor/activator), two-component signal transduction, quorum-sensing and alternative sigma factors.

Question 9

What is the Fur repressor?

Answer 9

(Classical regulator) Many virulence genes are regulated by a Fur repressor. It binds iron, dimerizes and then binds DNA at a FUR box sequence so RNA polymerase can no longer bind and there is no transcription. When there is no iron in the cell there will be transcription.

Question 10

What is the AraC activator?

Answer 10

(Classical regulator) AraC undergoes conformational change upon arabinose-binding, which changes slightly its DNA binding affinity (active), enabling RNA polymerase to bind and start transcription.

Question 11

What are sensor kinases?

Answer 11

(Two-component signal transduction) Sensor kinase binds environmental signal and phosphorylates, and then the response regulator will also be phosphorylated. They can have both activating or repressing effects based on the place that they bind.

Question 12

How is the SPI-2 needle regulated in Salmonella enterica?

Answer 12

The main two component system of SPI-2 is SsrAB. There is also involvement of EnvZ/OmpR & PhoPQ two component systems. The signals are osmolarity, antimicrobial peptides that induce SPI-2 production.

Question 13

How is the SPI-1 needle regulated in Salmonella enterica?

Answer 13

HilA activates the genes encoding T3SS SPI-1 and the effector molecules. HilA is activated by AraC-like activator HilD.

Question 14

What is Quorum Sensing?

Answer 14

Bacteria communicating with each other using diffusible signalling molecules.

Question 15

What processes are regulated by Quorum sensing?

Answer 15

Biofilm production, toxins production, secondary metabolite production, motility and swarming, and conjugation.

Question 16

How does Quorum sensing work?

Answer 16

It is cell to cell communication via a diffusible signal molecule. If there are a lot of bacteria ( high cell density) above the quorate (threshold), they will produce more autoinducer (LuxI and LuxR) and undergo phenotypic change (biofilm, toxin etc).

Question 17

What are Quorum sensing signalling molecules?

Answer 17

Important group of autoinducer molecules are N-acylhomoserine lactones, which are continually synthesized and secreted. At the threshold concentration there will be a signal transduction cascade. There are different quorum sensing signalling modules for gram negative and gram positive bacteria.

Question 18

What are the Quorum sensing cascades in Pseudomonas aeruginosa?

Answer 18

There are 3 cascades, Las, Rhl and RqsR and they also show cross interaction. Together they give rise to different virulence factors.

Question 19

What is the Quorum sensing cheaters mutation in the quorum receptor?

Answer 19

Secreted public goods (toxins, extracellular enzymes) will not be produced by these cheaters and so they will have advantage (spend less energy on production) over the other bacteria who do express the receptor.

Question 20

What are alternative sigma-factors?

Answer 20

They recognise different promoters than the ‘normal’ sigma-factor.

Question 21

What is Sigma E?

Answer 21

An alternative sigma-factor that is induced upon cell envelope stress and stimulates the production of (cell envelope) chaperones and proteases. It is required for intracellular survival of S. enterica.

Question 22

What are the advantages and disadvantages of RNA sequencing?

Answer 22

Advantages are that it is genome wide, very sensitive and Dual-RNA-seq (so you can look at host & pathogen)
Disadvantages of RNA-seq are that there is a high amount of contaminating RNA present (from eukaryotes into the prokaryotes). So a relatively high number of cells and bacteria is required. Only the ‘average’ level of mRNA is determined. This is why it is not good in in vivo experiments, it is usefull to study gene expression of pathogens in cell cultures.

Question 23

What is in vivo expression technology (IVET)?

Answer 23

Select genes that are induced in vivo with the use of an auxothroph marker (cannot produce one of the building blocks that you need for growth) or an antibiotic marker. Can be purA for example, which is a enzyme which is involved in purine biosynthesis.

Question 24

What are the advantages and disadvantages of IVET?

Answer 24

Benefits are positive selection method, not technically demanding, not expensive and a low number of bacteria can be tested.
Disadvantages are that its not genome wide and there is no detection of genes that are regulated post-transcriptionally, already expressed reasonably in vitro or transiently expressed.

Question 25

What are reporter systems?

Answer 25

The promoter of interest is fused to a promoterless reporter gene (LacZ, GFP or luciferase).

Question 26

What is Differential fluorescence induction (DFI)?

Answer 26

A reporter system where you fuse the promotor with a GPF and selective marker by homologous recombination. You measure the GFP with FACS and select for the negative bacteria (take out the ones that are always positive).

Question 27

What is Mig-14?

Answer 27

Mig-14 is a new Salmonella virulence factor found with DFI, this mutant is more resistent to the proinflammatory effects of IFN-y as a result of adaptive immune responses. It is probably horizontally transmitted.