Decrypting the H-NS-dependent regulatory cascade of acid stress resistance in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>H-NS regulates the acid stress resistance. The present study aimed to characterize the H-NS-dependent cascade governing the acid stress resistance pathways and to define the interplay between the different regulators.</p> <p>Results</p> <p>We combined mutational, phenotypic and gene expression analyses, to unravel the regulatory hierarchy in acid resistance involving H-NS, RcsB-P/GadE complex, HdfR, CadC, AdiY regulators, and DNA-binding assays to separate direct effects from indirect ones. RcsB-P/GadE regulatory complex, the general direct regulator of glutamate-, arginine- and lysine-dependent acid resistance pathways plays a central role in the regulatory cascade. However, H-NS also directly controls specific regulators of these pathways (e.g. <it>cadC</it>) and genes involved in general stress resistance (<it>hdeAB, hdeD, dps, adiY</it>). Finally, we found that in addition to H-NS and RcsB, a third regulator, HdfR, inversely controls glutamate-dependent acid resistance pathway and motility.</p> <p>Conclusions</p> <p>H-NS lies near the top of the hierarchy orchestrating acid response centred on RcsB-P/GadE regulatory complex, the general direct regulator of glutamate-, arginine- and lysine-dependent acid resistance pathways.</p

Identification of an anti-CRISPR protein that inhibits the CRISPR-Cas type I-B system in Clostridioides difficile

CRISPR-Cas systems provide prokaryotic hosts with adaptive immunity against mobile genetic elements. Many bacteriophages encode anti-CRISPR (Acr) proteins that inhibit host defense. The identification of Acr proteins is challenging due to their small size and high sequence diversity, and only a limited number has been characterized to date. In this study, we report the discovery of a novel Acr protein, AcrIB2, encoded by the φCD38-2 Clostridioides difficile phage that efficiently inhibits interference by the type I-B CRISPR-Cas system of the host and likely acts as a DNA mimic. Most C. difficile strains contain two cas operons, one encoding a full set of interference and adaptation proteins and another encoding interference proteins only. Unexpectedly, we demonstrate that only the partial operon is required for interference and is subject to inhibition by AcrIB2.This work was supported by the Institut Universitaire de France (to O.S.), the Institute for Integrative Biology of the Cell, the University Paris-Saclay, Graduate School Life Sciences and Health, and OI MICROBES funding and Vernadski fellowship (to P.M.). This work was also supported by NIH grant R01 GM10407 (to K.S.), the Russian Science Foundation grant 19-14-00323, and the Ministry of Science and Higher Education of the Russian Science Federation agreement no. 075-10-2021-114

Tissue Compartment Analysis for Biomarker Discovery by Gene Expression Profiling

BACKGROUND:Although high throughput technologies for gene profiling are reliable tools, sample/tissue heterogeneity limits their outcomes when applied to identify molecular markers. Indeed, inter-sample differences in cell composition contribute to scatter the data, preventing detection of small but relevant changes in gene expression level. To date, attempts to circumvent this difficulty were based on isolation of the different cell structures constituting biological samples. As an alternate approach, we developed a tissue compartment analysis (TCA) method to assess the cell composition of tissue samples, and applied it to standardize data and to identify biomarkers. METHODOLOGY/PRINCIPAL FINDINGS:TCA is based on the comparison of mRNA expression levels of specific markers of the different constitutive structures in pure isolated structures, on the one hand, and in the whole sample on the other. TCA method was here developed with human kidney samples, as an example of highly heterogeneous organ. It was validated by comparison of the data with those obtained by histo-morphometry. TCA demonstrated the extreme variety of composition of kidney samples, with abundance of specific structures varying from 5 to 95% of the whole sample. TCA permitted to accurately standardize gene expression level amongst >100 kidney biopsies, and to identify otherwise imperceptible molecular disease markers. CONCLUSIONS/SIGNIFICANCE:Because TCA does not require specific preparation of sample, it can be applied to all existing tissue or cDNA libraries or to published data sets, inasmuch specific operational compartments markers are available. In human, where the small size of tissue samples collected in clinical practice accounts for high structural diversity, TCA is well suited for the identification of molecular markers of diseases, and the follow up of identified markers in single patients for diagnosis/prognosis and evaluation of therapy efficiency. In laboratory animals, TCA will interestingly be applied to central nervous system where tissue heterogeneity is a limiting factor

The Pleiotropic CymR Regulator of Staphylococcus aureus Plays an Important Role in Virulence and Stress Response

We have characterized a novel pleiotropic role for CymR, the master regulator of cysteine metabolism. We show here that CymR plays an important role both in stress response and virulence of Staphylococcus aureus. Genes involved in detoxification processes, including oxidative stress response and metal ion homeostasis, were differentially expressed in a ΔcymR mutant. Deletion of cymR resulted in increased sensitivity to hydrogen peroxide-, disulfide-, tellurite- and copper-induced stresses. Estimation of metabolite pools suggests that this heightened sensitivity could be the result of profound metabolic changes in the ΔcymR mutant, with an increase in the intracellular cysteine pool and hydrogen sulfide formation. Since resistance to oxidative stress within the host organism is important for pathogen survival, we investigated the role of CymR during the infectious process. Our results indicate that the deletion of cymR promotes survival of S. aureus inside macrophages, whereas virulence of the ΔcymR mutant is highly impaired in mice. These data indicate that CymR plays a major role in virulence and adaptation of S. aureus for survival within the host

Type I Toxin-Antitoxin Systems in Clostridia

Type I toxin-antitoxin (TA) modules are abundant in both bacterial plasmids and chromosomes and usually encode a small hydrophobic toxic protein and an antisense RNA acting as an antitoxin. The RNA antitoxin neutralizes toxin mRNA by inhibiting its translation and/or promoting its degradation. This review summarizes our current knowledge of the type I TA modules identified in Clostridia species focusing on the recent findings in the human pathogen Clostridium difficile. More than ten functional type I TA modules have been identified in the genome of this emerging enteropathogen that could potentially contribute to its fitness and success inside the host. Despite the absence of sequence homology, the comparison of these newly identified type I TA modules with previously studied systems in other Gram-positive bacteria, i.e., Bacillus subtilis and Staphylococcus aureus, revealed some important common traits. These include the conservation of characteristic sequence features for small hydrophobic toxic proteins, the localization of several type I TA within prophage or prophage-like regions and strong connections with stress response. Potential functions in the stabilization of genome regions, adaptations to stress conditions and interactions with CRISPR-Cas defence system, as well as promising applications of TA for genome-editing and antimicrobial developments are discussed

RNA-based control mechanisms of Clostridium difficile

Clostridium difficile (CD)-associated diarrhoea is currently the most prevalent nosocomial diarrhoea worldwide. Many characteristics of CD pathogenicity remain poorly understood. Recent data strongly indicate the importance of an RNA network for the control of gene expression in CD. More than 200 regulatory RNAs have been identified by deep sequencing and targeted approaches, including Hfq-dependent trans riboregulators, cis-antisense RNAs, CRISPR RNAs, and c-di-GMP-responsive riboswitches. These regulatory RNAs are involved in the control of major processes in the CD infection cycle, for example motility, biofilm formation, adhesion, sporulation, stress response, and defence against bacteriophages. We will discuss recent advances in elucidation of the original features of RNA-based mechanisms in this important enteropathogen. This knowledge may pave the way for further discoveries in this emergent field

Identification of c-di-GMP-Responsive Riboswitches

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important signaling molecule for community behavior control, cell morphogenesis, and virulence in bacteria. In addition to protein effectors, this second messenger binds RNA molecules that act as riboswitches to control target gene expression. In this chapter, we describe a method for experimental validation of the functionality of c-di-GMP-responsive riboswitches and the analysis of c-di-GMP control of target gene expression by qRT-PCR and Northern blot. This procedure can be used for the studies of in silico-predicted riboswitch candidates, as well as a targeted experimental approach for exploring the data from next-generation sequencing. The examples on the analysis of type I and type II c-di-GMP-responsive riboswitches in Clostridium difficile are provided to illustrate the application of the method

Contrôle de l'expression des gènes dans le processus de motilité chez les bactéries à Gram-négatif

VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF