The σB signalling activation pathway in the enteropathogen Clostridioides difficile

Clostridium difficile is the main cause of antibiotic-associated diarrhoea. Inside the gut, C. difficile must adapt to the stresses it copes with, by inducing protection, detoxification and repair systems that belong to the general stress response involving σ^B. Following stresses, σ^B activation requires a PP2C phosphatase to dephosphorylate the anti-anti-sigma factor RsbV that allows its interaction with the anti-sigma factor RsbW and the release of σ^B. In this work, we studied the signalling pathway responsible for the activation of σ^B in C. difficile. Contrary to other firmicutes, the expression of sigB in C. difficile is constitutive and not autoregulated. We confirmed the partner switching mechanism that involved RsbV, RsbW and σ^B. We also showed that CD2685, renamed RsbZ, and its phosphatase activity are required for RsbV dephosphorylation triggering σ^B activation. While CD0007 and CD0008, whose genes belong to the sigB operon, are not involved in σ^B activity, depletion of the essential iron–sulphur flavoprotein, CD2684, whose gene forms an operon with rsbZ, prevents σ^B activation. Finally, we observed that σ^B is heterogeneously active in a subpopulation of C. difficile cells from the exponential phase, likely leading to a ‘bet-hedging’ strategy allowing a better chance for the cells to survive adverse conditions.     

Non-specific, general and multiple stress resistance of growth-restricted Bacillus subtilis cells by the expression of the σB regulon

Bacillus subtilis cells respond almost immediately to different stress conditions by increasing the production of general stress proteins (GSPs). The genes encoding the majority of the GSPs that are induced by heat, ethanol, salt stress or by starvation for glucose, oxygen or phosphate belong to the σ^B-dependent general stress regulon. Despite a good understanding of the complex regulation of the activity of σ^B and knowledge of a very large number of general stress genes controlled by σ^B, first insights into the physiological role of this non-specific stress response have been obtained only very recently. To explore the physiological role of this regulon, we and others identified σ^B-dependent general stress genes and compared the stress tolerance of wild-type cells with mutants lacking σ^B or general stress proteins. The proteins encoded by σ^B-dependent general stress genes can be divided into at least five functional groups that most probably provide growth-restricted B. subtilis cells with a multiple stress resistance in anticipation of future stress. In particular, sigB mutants are impaired in non-specific resistance to oxidative stress, which requires the σ^B-dependent dps gene encoding a DNA-protecting protein. Protection against oxidative damage of membranes, proteins or DNA could be the most essential component of σ^B-mediated general stress resistance in growth-arrested aerobic Gram-positive bacteria. Other general stress genes have both a σ^B-dependent induction pathway and a second σ^B-independent mechanism of stress induction, thereby partially compensating for a σ^B deficiency in a sigB mutant. In contrast to sigB mutants, null mutations in genes encoding those proteins, such as clpP or clpC, cause extreme sensitivity to salt or heat.     

Polyphosphate kinase 2: a modulator of nucleoside diphosphate kinase activity in mycobacteria

Mycobacteria encode putative class II polyphosphate kinases (PPKs). We report that recombinant PPK2 of Mycobacterium tuberculosis catalyses the synthesis of GTP from GDP using polyphosphate rather than ATP as phosphate donor. Unlike that of PPK1, this is the favoured reaction of PPK2. The sites of autophosphorylation, H115 and H247, as well as G74 were critical for GTP‐synthesizing activity. Compromised survival of a ppk2 knockout (PPK2‐KO) of Mycobacterium smegmatis under heat or acid stress or hypoxia, and the ability of ppk2 of M. tuberculosis to complement this, confirmed that PPK2 plays a role in mycobacterial survival under stress. Intracellular ATP : GTP ratio was higher in PPK2‐KO compared with the wild‐type M. smegmatis, bringing to light a role of PPK2 in regulating the intracellular nucleotide pool. We present evidence that PPK2 does so by interacting with nucleoside diphosphate kinase (Ndk). Pull‐down assays and analysis by surface plasmon resonance demonstrated that the interaction requires G74 of PPK2_MTB and ¹⁰⁹LET¹¹¹ of Ndk_MTB. In summary, we unravel a novel mechanism of regulation of nucleotide pools in mycobacteria. Downregulation of ppk2 impairs survival of M. tuberculosis in macrophages, suggesting that PPK2 plays an important role in the physiology of the bacteria residing within macrophages.     

Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon

clpC ofBacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (P_A and P_B) were mapped upstream of the first gene. P_A resembles promoters recognized by the vegetative RNA polymerase Eσ^A. The other promoter (P_B) was shown to be dependent on σ^B, the general stress σ factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a σ^B-dependent manner. This is the first evidence that σ^B in B, subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by σ³² in Escherichia coli, indicating a new function of σ^B-dependent general stress proteins. P_B deviated from the consensus sequence of σ^B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the σ^B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the σ^A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream σ^A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.     

Bacterial persistence is an active σS stress response to metabolic flux limitation

While persisters are a health threat due to their transient antibiotic tolerance, little is known about their phenotype and what actually causes persistence. Using a new method for persister generation and high‐throughput methods, we comprehensively mapped the molecular phenotype of Escherichia coli during the entry and in the state of persistence in nutrient‐rich conditions. The persister proteome is characterized by σ^S‐mediated stress response and a shift to catabolism, a proteome that starved cells tried to but could not reach due to absence of a carbon and energy source. Metabolism of persisters is geared toward energy production, with depleted metabolite pools. We developed and experimentally verified a model, in which persistence is established through a system‐level feedback: Strong perturbations of metabolic homeostasis cause metabolic fluxes to collapse, prohibiting adjustments toward restoring homeostasis. This vicious cycle is stabilized and modulated by high ppGpp levels, toxin/anti‐toxin systems, and the σ^S‐mediated stress response. Our system‐level model consistently integrates past findings with our new data, thereby providing an important basis for future research on persisters.     

Iron enhances the susceptibility of pathogenic mycobacteria to isoniazid, an antitubercular drug

The catalase-peroxidase KatG of Mycobacterium tuberculosis plays a central role in the mechanism of action of the anti-tubercular drug isoniazid (INH). Like other bacterial catalases, mycobacterial catalase-peroxidases are dual active enzymes with both catalase and peroxidase activities in the same protein molecule. In our previous study, we showed that iron deprivation resulted in the loss of peroxidase activity in several non-pathogenic mycobacterial species. Here we extended the study to pathogenic mycobacteria and showed that the peroxidase activity, associated with iron-sufficient (4 μg Fe/ml) conditions of growth was responsible for INH activation. Upon iron deprivation (0.02 μg Fe/ml), peroxidase activity was abolished and there was no activation of INH, as demonstrated both by INH-mediated NBT reduction (spectrophotometrically and activity staining in gels) and by viability studies as assayed by the microplate Alamar Blue assay (MABA). In the viability assay, iron-sufficient M. tuberculosis, Mycobacterium bovis and Mycobacterium bovis BCG were susceptible to INH and iron-deficient organisms expressing negligible peroxidase survived high concentrations of the drug. It␣is well known that M. tuberculosis is sensitive to low concentrations of INH while the minimum inhibitory concentration of the drug is quite high for other mycobacteria, especially the non-pathogenic species. We showed this difference to be due to the specificity of the peroxidase for the drug. As withholding of iron is one of the host’s mechanisms of controlling an invading pathogen, the implications of these observations on the efficacy of the anti-tubercular drug INH are discussed with reference to the iron status within the human host.     

The Staphylococcus aureus Alternative Sigma Factor ςB Controls the Environmental Stress Response but Not Starvation Survival or Pathogenicity in a Mouse Abscess Model

The role of ς^B, an alternative sigma factor of Staphylococcus aureus, has been characterized in response to environmental stress, starvation-survival and recovery, and pathogenicity. ς^B was mainly expressed during the stationary phase of growth and was repressed by 1 M sodium chloride. A sigB insertionally inactivated mutant was created. In stress resistance studies, ς^B was shown to be involved in recovery from heat shock at 54°C and in acid and hydrogen peroxide resistance but not in resistance to ethanol or osmotic shock. Interestingly, S. aureus acquired increased acid resistance when preincubated at a sublethal pH 4 prior to exposure to a lethal pH 2. This acid-adaptive response resulting in tolerance was mediated via sigB. However, ς^B was not vital for the starvation-survival or recovery mechanisms. ς^B does not have a major role in the expression of the global regulator of virulence determinant biosynthesis, staphylococcal accessory regulator (sarA), the production of a number of representative virulence factors, and pathogenicity in a mouse subcutaneous abscess model. However, SarA upregulates sigB expression in a growth-phase-dependent manner. Thus, ς^B expression is linked to the processes controlling virulence determinant production. The role of ς^B as a major regulator of the stress response, but not of starvation-survival, is discussed.     

ESX‐1 exploits type I IFN‐signalling to promote a regulatory macrophage phenotype refractory to IFNγ‐mediated autophagy and growth restriction of intracellular mycobacteria

The ability of macrophages to eradicate intracellular pathogens is normally greatly enhanced by IFNγ, a cytokine produced mainly after onset of adaptive immunity. However, adaptive immunity is unable to provide sterilizing immunity against mycobacteria, suggesting that mycobacteria have evolved virulence strategies to inhibit the bactericidal effect of IFNγ‐signalling in macrophages. Still, the host–pathogen interactions and cellular mechanisms responsible for this feature have remained elusive. We demonstrate that the ESX‐1 type VII secretion systems of Mycobacterium tuberculosis and Mycobacterium marinum exploit type I IFN‐signalling to promote an IL‐12^low/IL‐10^high regulatory macrophage phenotype characterized by secretion of IL‐10, IL‐27 and IL‐6. This mechanism had no impact on intracellular growth in the absence of IFNγ but suppressed IFNγ‐mediated autophagy and growth restriction, indicating that the regulatory phenotype extends to function. The IFNγ‐refractory phenotype was partly mediated by IL‐27‐signalling, establishing functional relevance for this downstream cytokine. These findings identify a novel macrophage‐modulating function for the ESX‐1 secretion system that may contribute to suppress the efficacy of adaptive immunity and provide mechanistic insight into the antagonistic cross talk between type I IFNs and IFNγ in mycobacterial infection.