Shigella MreB promotes polar IcsA positioning for actin tail formation.

Pathogenic Shigella bacteria are a paradigm to address key issues of cell and infection biology. Polar localisation of the Shigella autotransporter protein IcsA is essential for actin tail formation, which is necessary for the bacterium to travel from cell-to-cell; yet how proteins are targeted to the bacterial cell pole is poorly understood. The bacterial actin homologue MreB has been extensively studied in broth culture using model organisms including Escherichia coli, Bacillus subtilis and Caulobacter crescentus, but has never been visualised in rod-shaped pathogenic bacteria during infection of host cells. Here, using single-cell analysis of intracellular Shigella, we discover that MreB accumulates at the cell pole of bacteria forming actin tails, where it colocalises with IcsA. Pharmacological inhibition of host cell actin polymerisation and genetic deletion of IcsA is used to show, respectively, that localisation of MreB to the cell poles precedes actin tail formation and polar localisation of IcsA. Finally, by exploiting the MreB inhibitors A22 and MP265, we demonstrate that MreB polymerisation can support actin tail formation. We conclude that Shigella MreB promotes polar IcsA positioning for actin tail formation, and suggest that understanding the bacterial cytoskeleton during host-pathogen interactions can inspire development of new therapeutic regimes for infection control.This article has an associated First Person interview with the first author of the paper

Septins Recognize and Entrap Dividing Bacterial Cells for Delivery to Lysosomes.

The cytoskeleton occupies a central role in cellular immunity by promoting bacterial sensing and antibacterial functions. Septins are cytoskeletal proteins implicated in various cellular processes, including cell division. Septins also assemble into cage-like structures that entrap cytosolic Shigella, yet how septins recognize bacteria is poorly understood. Here, we discover that septins are recruited to regions of micron-scale membrane curvature upon invasion and division by a variety of bacterial species. Cardiolipin, a curvature-specific phospholipid, promotes septin recruitment to highly curved membranes of Shigella, and bacterial mutants lacking cardiolipin exhibit less septin cage entrapment. Chemically inhibiting cell separation to prolong membrane curvature or reducing Shigella cell growth respectively increases and decreases septin cage formation. Once formed, septin cages inhibit Shigella cell division upon recruitment of autophagic and lysosomal machinery. Thus, recognition of dividing bacterial cells by the septin cytoskeleton is a powerful mechanism to restrict the proliferation of intracellular bacterial pathogens

Etude des gènes de réponse aux stress chez lesbactéries pathogènes à Gram positif

Bacteria have to cope with modifications of their environment, and haveevolved specific survival mechanisms called stress response. In addition to theiradaptive functions, stress proteins are involved in many physiological pathwaysincluding cellular differentiation and virulence development.The main goal of this thesis was to perform a comparative analysis of thestress response in different Gram-positive pathogenic bacteria, and particularly theClp family of proteins.First, we characterized a new mechanism of stress response regulation inStaphylococcus aureus. Indeed, contrary to the situation in the model organismBacillus subtilis, expression of the ubiquitous chaperones encoding operons dnaK andgroESL, is under dual control by the CtsR and HrcA transcriptional regulators.Furthermore, we have shown the existence in streptococci and staphylococci, of anintermediate situation between B. subtilis and S. aureus, in which only the groESLoperon is under dual repression. Finally, we discovered in Streptococcus salivarius thefirst example of a clp gene under dual regulation by CtsR and HrcA.Secondly, we performed a functional analysis of clp genes ofStreptococcus pneumoniae. We have shown that clpC, clpP and clpE are regulated byCtsR and that ClpP is involved in the control of natural competence fortransformation and in resistance to thermal stress. In addition, we demonstrated thatClpE is required for adaptation to stress, and that ClpE and ClpC are both involvedin the virulence of S. pneumoniae.Finally, we characterized a new heat shock gene of Listeria monocytogenes, clpB,which is also a member of the CtsR regulon. Although ClpB is required for virulence,it is not involved in general stress resistance, but is necessary for inducedthermotolerance of this bacterium.Pour survivre aux modifications des conditions du milieu, les bactéries ont dûdévelopper des mécanismes spécifiques, appelés réponses aux stress. En plus de leurrôle adaptatif, les protéines de stress sont impliquées dans diverses fonctionsphysiologiques comme la différenciation cellulaire ou l’expression de facteurs devirulence.L’objectif de cette thèse était de réaliser une analyse comparative de la réponseaux stress chez différentes bactéries pathogènes à Gram-positif, et plusparticulièrement des protéines de la famille Clp.Nous avons ainsi caractérisé un nouveau mécanisme de réponse aux stresschez Staphylococcus aureus. En effet, contrairement à la situation chez le modèleBacillus subtilis, les opérons codant les chaperons ubiquitaires DnaK et GroESL sontdoublement régulés par les répresseurs transcriptionnels CtsR et HrcA. De plus,nous avons montré qu’il existe, chez les streptocoques et les lactocoques, unesituation intermédiaire entre celle de B. subtilis et de S. aureus où seul l’opéron groESLest doublement réprimé. Enfin, nous avons mis en évidence chez Streptococcussalivarius le premier exemple de double régulation par CtsR et HrcA d’un gène clp.D’autre part, nous avons réalisé une analyse fonctionnnelle de gènes clp deStreptococcus pneumoniae. Nous avons montré que les gènes clpC, clpP et clpE sontrégulés par CtsR et que clpP est impliqué dans le contrôle de la compétence naturelle,ainsi que pour la résistance au stress thermique. Par ailleurs, clpE est nécessaire pourl’adaptation au stress, et clpE et clpC sont requis pour la virulence du pneumocoque.Enfin, nous avons mis en évidence chez Listeria monocytogenes, un nouveaugène de choc thermique, clpB, membre du régulon CtsR. Bien que ClpB soitnécessaire au développement de la virulence de ce microorganisme, elle n’est pasrequise pour la résistance générale aux stress, mais joue un rôle dans la résistanceinduite aux stress létaux

Etude des gènes de réponse aux stress chez les bactéries pathogènes à Gram positif

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

clpP of Streptococcus salivarius Is a Novel Member of the Dually Regulated Class of Stress Response Genes in Gram-Positive Bacteria

Nucleotide sequence analysis of the Streptococcus salivarius clpP locus revealed potential binding sites for both the CtsR and HrcA repressors. Dual regulation by HrcA and CtsR was demonstrated by using Bacillus subtilis as a heterologous host, and CtsR was shown to bind directly to the clpP promoter sequence. This is the first example of a clpP gene under the control of HrcA

New Vector for Efficient Allelic Replacement in Naturally Nontransformable, Low-GC-Content, Gram-Positive Bacteria

A shuttle vector designated pMAD was constructed for quickly generating gene inactivation mutants in naturally nontransformable gram-positive bacteria. This vector allows, on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) plates, a quick colorimetric blue-white discrimination of bacteria which have lost the plasmid, greatly facilitating clone identification during mutagenesis. The plasmid was used in Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus to efficiently construct mutants with or without an associated antibiotic resistance gene

Just-in-Time Control of Spo0A Synthesis in Bacillus subtilis by Multiple Regulatory Mechanisms ▿ §

The response regulator Spo0A governs multiple developmental processes in Bacillus subtilis, including most conspicuously sporulation. Spo0A is activated by phosphorylation via a multicomponent phosphorelay. Previous work has shown that the Spo0A protein is not rate limiting for sporulation. Rather, Spo0A is present at high levels in growing cells, rapidly rising to yet higher levels under sporulation-inducing conditions, suggesting that synthesis of the response regulator is subject to a just-in-time control mechanism. Transcription of spo0A is governed by a promoter switching mechanism, involving a vegetative, σA-recognized promoter, Pv, and a sporulation σH-recognized promoter, Ps, that is under phosphorylated Spo0A (Spo0A∼P) control. The spo0A regulatory region also contains four (including one identified in the present work) conserved elements that conform to the consensus binding site for Spo0A∼P binding sites. These are herein designated O1, O2, O3, and O4 in reverse order of their proximity to the coding sequence. Here we report that O1 is responsible for repressing Pv during the transition to stationary phase, that O2 is responsible for repressing Ps during growth, that O3 is responsible for activating Ps at the start of sporulation, and that O4 is dispensable for promoter switching. We also report that Spo0A synthesis is subject to a posttranscriptional control mechanism such that translation of mRNAs originating from Pv is impeded due to RNA secondary structure whereas mRNAs originating from Ps are fully competent for protein synthesis. We propose that the opposing actions of O2 and O3 and the enhanced translatability of mRNAs originating from Ps create a highly sensitive, self-reinforcing switch that is responsible for producing a burst of Spo0A synthesis at the start of sporulation

Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria

International audienceMultiple regulatory mechanisms for coping with stress co-exist in low G+C Gram-positive bacteria. Among these, the HrcA and CtsR repressors control distinct regulons in the model organism, Bacillus subtilis. We recently identified an orthologue of the CtsR regulator of stress response in the major pathogen, Staphylococcus aureus. Sequence analysis of the S. aureus genome revealed the presence of potential CtsR operator sites not only upstream from genes encoding subunits of the Clp ATP-dependent protease, as in B. subtilis, but also, unexpectedly, within the promoter regions of the dnaK and groESL operons known to be specifically controlled by HrcA. The tandem arrangement of the CtsR and HrcA operators suggests a novel mode of dual heat shock regulation by these two repressors. The S. aureus ctsR and hrcA genes were cloned under the control of the PxylA xylose-inducible promoter and used to demonstrate dual regulation of the dnaK and groESL operons by both CtsR and HrcA, using B. subtilis as a heterologous host. Direct binding by both repressors was shown in vitro by gel mobility shift and DNase I footprinting experiments using purified S. aureus CtsR and HrcA proteins. DeltactsR, DeltahrcA and DeltactsRDeltahrcA mutants of S. aureus were constructed, indicating that the two repressors are not redundant but, instead, act together synergistically to maintain low basal levels of expression of the dnaK and groESL operons in the absence of stress. This novel regulatory mode appears to be specific to Staphylococci