Lecture 6 Stress Responses II Flashcards
Overview of sigma factors
Most genes in E. coli require the standard sigma factors σ70 (or RpoD) for transcription
Alternative sigma factors recognise different promoter sequences
Consequently, expression of gene families can be controlled by regulating the availability of the corresponding sigma factor
Achieved by:
- Changing the rate of synthesis of the sigma factors
- Changing the rate of degradation of the sigma factors
- Through the activity of anti-sigma factors
RpoS – master regulator of general stress response
RpoS (σ38) controls the response to multiple stresses, and its activity can be influenced at every level
Directly or indirectly, RpoS can regulate approx. 10% of the bacterial genome
Multiple stresses can influence the production of RpoS protein, which (as an alternative sigma factor) acts as a master regulator of the general stress response.
A reduced growth rate enhances the rate of RpoS transcription, consistent with the fact that RpoS plays a role in adapting to stationary phase.
The rate of translation of the resulting mRNA is enhanced (often by altering the secondary structure of the mRNA) by multiple stimuli including high cell density, low temperature, high osmolarity and acidic pH.
Ordinarily, the rate of proteolysis of RpoS protein is extremely fast. However, high osmolarity, acidic pH, carbon starvation and high temperature all act to repress the proteolysis of RpoS, thus boosting the levels of active RpoS protein.
Therefore, together, these multiple stresses can function to boost the levels of RpoS and thus promote the activation of RpoS-dependent genes.
Overview of Salmonella infection
Stressors encountered include:
acidic pH
Reactive oxygen & nitrogen species
Nutrient limitation
RpoS phenotypes in Salmonella
In addition to facilitating adaptation to stressors, RpoS can also directly regulate certain virulence factors
Teasing apart the integration of stress responses
Adaptor proteins direct protein substrates to proteases for degradation
RssB is an adaptor protein for RpoS (σS), directing it to the ClpXP protease
IraP, IraM & IraD are anti-adaptor proteins that inhibit RssB, stabilizing RpoS
(p)ppGpp activates IraP, whilst the PhoP-PhoQ TCS activates IraM
A protein called RssB is an adaptor protein for RpoS, the master regulator of general stress.
When phosphorylated, RssB targets RpoS to the ClpXP protease, thus promoting RpoS degradation. The kinase ArcB has been implicated in the phosphorylation of RssB. ArcB monitors the cellular energy state, and so during energy starvation the phosphorylation of RssB by ArcB is reduced, leading to reduced proteolysis of RpoS.
Adaptor proteins can themselves by regulated by anti-adaptor proteins. In the case of RssB, various distinct stress signals promote the synthesis of anti-adaptor proteins that inhibit RssB function. These anti-adaptors include IraP, IraM and IraD. By inhibiting RssB, they promote the stability of RpoS as it is no longer targeted for proteolysis. IraP is activated by the stringent response we discussed previously. IraM is activated by a two-component system PhoPQ that can facilitate adaptation to acidic conditions as well as magnesium limitation.
The RpoE (σE) envelope stress response
Role of RpoE is to maintain integrity & function of cell envelope
RpoE is activated by stresses that trigger envelope stress and generate unfolded envelope proteins
Results in the activation/induction of: Periplasmic folding machinery Proteases Lipid A biosynthesis Lipoproteins Proteins with periplasmic functions
Regulators of RpoE (σE) activity
Cytoplasmic RpoE responds to stress signals in the cell envelope
RseA is the anti-sigma factor of RpoE
RseB stabilizes RseA and enhances its activity
DegS and YaeL form a proteolytic cascade – both are inhibited by their own PDZ domains
Sigma-E is cytoplasmic, but responds to stresses that occur within the periplasmic region of the cell envelope. How is that managed?
This slide shows the factors that regulate sigmaE activation. Ordinarily, in the absence of stress, sigma-E is inhibited by the major negative regulator RseA which acts as an anti-sigma factor. RseB co-operates in this process, through either stabilizing RseA or enhancing RseA activity. DegS and YaeL have proteolytic activity, but they are ordinarily inactive, being inhibited by their own PDZ domains.
Pathway of RpoE (σE ) activation
The envelope stress response is characterized by the accumulation of unfolded outer membrane proteins in the periplasm.
The C-terminal domain of these unfolded proteins interacts with the PDZ domain of DegS, relieving the PDZ-mediated inhibition of DegS proteolytic activity. The active DegS then cleaves the periplasmic domain of RseA. In a similar manner, YaeL cleaves the cytoplasmic domain of RseA, although the precise mechanism by which YaeL becomes activated is unknown. It is possible that the loss of the periplasmic domain of RseA lifts the inhibition that is normally imposed by YaeL’s PDZ domain. This sequential cleavage of RseA releases sigmaE within the cytoplasm
Phenotypes associated with RpoE in Salmonella
The RpoE mutant shows increased sensitivity to hydrogen peroxide compared to the wildtype.
The RpoE mutant is also impaired in its ability to survive within macrophages. The rpoE-comp strain has the rpoE gene restored on a plasmid (i.e. the mutant has been complemented). This confirms that by restoring RpoE function, we can restore the ability of the bacteria to survive within the macrophages.
Phenotypes associated with RpoE in Salmonella
The LD50 (dose required to kill 50% of mice) is significantly higher for the rpoE mutant than for the wildtype, regardless of the route of infection
Dealing with unfolded proteins in the cytoplasm
Elevated temperatures impact protein stability & can lead to unfolding
The ‘heat-shock response’ broadly falls into two categories:
Molecular chaperones
Prevent aggregation of newly synthesized protein and catalyze the correct folding of proteins
- DnaK-DnaJ-GrpE
- GroEL-GroES
Proteases
Remove denatured / irreversibly aggregated proteins
- ClpXP
- Lon, FtsH
There are two central components to the heat-shock response. Molecular chaperones assist the correct folding of proteins, whilst proteases remove denatured/aggregated proteins.
The ClpXP complex consists of the ClpX ATPase and the ClpP protease. The ATP-dependent chaperone activity of ClpX transfers the protein into the proteolytic chamber of the associated self-compartmentalized protease, ClpP. The ATPase provides the energy for protein unfolding and translocation into the ClpP protease complex. Whilst the basic principle is the same, there are other AAA+ proteases that combine the ‘unfoldase’ and protease activities within a single protein (e.g. Lon and FtsH proteases).
GroE chaperone – GroEL & GroES
Provides a sequestered space & functional assistance for protein folding
GroEL comprises 14 identical subunits (2 rings of 7)
Forms a highly hydrophilic chamber
GroES forms a cap on the chamber, creating an enclosed space.
A major component of the heat shock response is the GroE chaperone machine, which comprises of GroEL and GroES. The GroEL complex comprises 14 identical subunits, arranged in two rings of 7 subunits. These rings are stacked back-to-back, forming a chamber-like structure. GroES forms a cap on the chamber, creating an enclosed space. This chamber is highly hydrophilic.
Ordinarily, hydrophobic residues within a correctly folded protein will be buried within the protein. In contrast, a protein that is unfolded (or incorrectly folded) will have exposed hydrophobic residues. The unfolded protein binds GroEL, and is subsequently released into the hydrophilic chamber. This confined hydrophilic space then promotes the proper folding of the polypeptide, so that the hydrophobic residues are buried within the protein. ATP hydrolysis occurs in this ring during folding of the polypeptide.
Both rings of GroEL have chaperone activity, but they work independently of each other (and slightly out-of-phase in a co-ordinated fashion).
RpoH (σ32) regulates the heat-shock response
Basal level of RpoH is present in cells at low temperature
Within 5 min. of shift to high temp, there is a 20-fold increase:
Enhanced rate of translation (2° structure of mRNA)
Increased stability of RpoH (t1/2 1min t1/2 4-5 min)
Although present at a low basal level within the bacterial cell, a shift of the cell to a higher temperature results in the very rapid increase of RpoH (sigma-H) levels within the cell. This is achieved through two mechanisms.
Firstly, there is an enhanced rate of translation of the rpoH mRNA (due to altered secondary structure at the higher temperature).
Additionally, the resulting protein has increased stability, and so has a much longer half-life. Ordinarily the half-life of RpoH is short, due to its association with the DnaK chaperone complex which targets the RpoH protein for degradation by a protease called FtsH. However, under heat-shock conditions, the DnaK chaperone complex preferentially binds to unfolded protein, thus releasing RpoH from its targeted degradation.
Sigma factors - targets for novel antimicrobials?
Pivotal for stress responses & direct regulation of some virulence factors
Bacteria-specific
Not bactericidal, so perhaps lower selective pressure for resistance
Numerous potential strategies (both direct and indirect) for inhibiting sigma factor activity
Blocking holoenzyme assembly
Investigated a plasmid library encoding cyclic peptides (potential inhibitors)
Generate a σE reporter strain of E. coli
Transform this strain with the plasmid library
Do any cyclic peptides inhibit the σE reporter?
Peptide SI24 identified as the first direct inhibitor of a sigma factor
Apparently inhibits assembly of the holoenzyme
However, SI24 was ineffective when added exogenously to bacterial cultures
In an in vitro transcription reaction, SI24 blocks transcription by inhibiting assembly of the holoenzyme (the complex formed by the RNA polymerase enzyme and the sigma factor).
In E. coli, SI24 can be expressed from a plasmid (as it is a peptide, it can be encoded by a gene). This was assessed by using a GFP reporter strain in which the gene encoding GFP was placed under the control of a sigmaE-dependent promoter. In that reporter strain, expression of SI24 reduced GFP expression.
However, purified SI24 fails to inhibit sigmaE activity when added to a culture of E. coli as it is unable to penetrate the cell envelope or unable to accumulate to sufficient concentrations within the cytoplasm
