Inactivation of pecS restores the virulence of mutants devoid of osmoregulated periplasmic glucans in the phytopathogenic bacterium Dickeya dadantii

International audienceDickeya dadantii is a phytopathogenic enterobacterium that causes soft rot disease in a wide range of plant species. Maceration, an apparent symptom of the disease, is the result of the synthesis and secretion of a set of plant cell wall-degrading enzymes (PCWDEs), but many additional factors are required for full virulence. Among these, osmoregulated periplasmic glucans (OPGs) and the PecS transcriptional regulator are essential virulence factors. Several cellular functions are controlled by both OPGs and PecS. Strains devoid of OPGs display a pleiotropic phenotype including total loss of virulence, loss of motility and severe reduction in the synthesis of PCWDEs. PecS is one of the major regulators of virulence in D. dadantii, acting mainly as a repressor of various cellular functions including virulence, motility and synthesis of PCWDEs. The present study shows that inactivation of the pecS gene restored virulence in a D. dadantii strain devoid of OPGs, indicating that PecS cannot be de-repressed in strains devoid of OPGs

The EnvZ-OmpR Two-Component Signaling System Is Inactivated in a Mutant Devoid of Osmoregulated Periplasmic Glucans in Dickeya dadantii

Osmoregulated periplasmic glucans (OPGs) are general constituents of alpha-, beta-, and gamma-Proteobacteria. This polymer of glucose is required for full virulence of many pathogens including Dickeya dadantii (D. dadantii). The phytopathogenic enterobacterium D. dadantii causes soft-rot disease in a wide range of plants. An OPG-defective mutant is impaired in environment sensing. We previously demonstrated that (i) fluctuation of OPG concentration controlled the activation level of the RcsCDB system, and (ii) RcsCDB along with EnvZ/OmpR controlled the mechanism of OPG succinylation. These previous data lead us to explore whether OPGs are required for other two-component systems. In this study, we demonstrate that inactivation of the EnvZ/OmpR system in an OPG-defective mutant restores full synthesis of pectinase but only partial virulence. Unlike for the RcsCDB system, the EnvZ-OmpR system is not controlled by OPG concentration but requires OPGs for proper activation

Concentration of osmoregulated periplasmic glucans (OPGs) modulates the activation level of the RcsCD RcsB phosphorelay in the phytopathogen bacteria Dickeya dadantii

International audienceOsmoregulated periplasmic glucans (OPGs) are general constituents of many Proteobacteria. Synthesis of these oligosaccharides is repressed by increased osmolarity of the medium. OPGs are important factors required for full virulence in many zoo-or phytopathogens including Dickeya dadantii. The phytopathogen enterobacterium D. dadantii causes soft-rot disease on a wide range of plant species. The total loss of virulence of opg-negative strains of D. dadantii is linked to the constitutive activation of the RcsCD RcsB phosphorelay highlighting relationship between this phosphorelay and OPGs. Here we show that OPGs control the RcsCD RcsB activation in a concentration-dependent manner, are required for proper activation of this phosphorelay by medium osmolarity, and a high concentration of OPGs in planta is maintained to achieve the low level of activation of the RcsCD RcsB phosphorelay required for full virulence in D. dadantii

Acetylation of surface carbohydrates in bacterial pathogens requires coordinated action of a two-domain membrane-bound acyltransferase

Membrane bound acyltransferase-3 (AT3) domain-containing proteins are implicated in a wide range of carbohydrate O-acyl modifications, but their mechanism of action is largely unknown. O-antigen acetylation by AT3 domain-containing acetyltransferases of Salmonella spp. can generate a specific immune response upon infection and can influence bacteriophage interactions. This study integrates in situ and in vitro functional analyses of two of these proteins, OafA and OafB (formerly F2GtrC), which display an "AT3-SGNH fused" domain architecture, where an integral membrane AT3 domain is fused to an extracytoplasmic SGNH domain. An in silico-inspired mutagenesis approach of the AT3 domain identified seven residues which are fundamental for the mechanism of action of OafA, with a particularly conserved motif in TMH1 indicating a potential acyl donor interaction site. Genetic and in vitro evidence demonstrate that the SGNH domain is both necessary and sufficient for lipopolysaccharide acetylation. The structure of the periplasmic SGNH domain of OafB identified features not previously reported for SGNH proteins. In particular, the periplasmic portion of the interdomain linking region is structured. Significantly, this region constrains acceptor substrate specificity, apparently by limiting access to the active site. Coevolution analysis of the two domains suggests possible interdomain interactions. Combining these data, we propose a refined model of the AT3-SGNH proteins, with structurally constrained orientations of the two domains. These findings enhance our understanding of how cells can transfer acyl groups from the cytoplasm to specific extracellular carbohydrates.IMPORTANCE Acyltransferase-3 (AT3) domain-containing membrane proteins are involved in O-acetylation of a diverse range of carbohydrates across all domains of life. In bacteria they are essential in processes including symbiosis, resistance to antimicrobials, and biosynthesis of antibiotics. Their mechanism of action, however, is poorly characterized. We analyzed two acetyltransferases as models for this important family of membrane proteins, which modify carbohydrates on the surface of the pathogen Salmonella enterica, affecting immunogenicity, virulence, and bacteriophage resistance. We show that when these AT3 domains are fused to a periplasmic partner domain, both domains are required for substrate acetylation. The data show conserved elements in the AT3 domain and unique structural features of the periplasmic domain. Our data provide a working model to probe the mechanism and function of the diverse and important members of the widespread AT3 protein family, which are required for biologically significant modifications of cell-surface carbohydrates

Dickeya dadantii : Towards the understanding of the biological role of the osmoregulated periplasmic glucanes

Les glucanes périplasmiques osmorégulés (OPG) sont des oligomères de glucose retrouvés chez la plupart des Proteobacteries. Le squelette glucosidique peut être substitué par différents types de molécules selon les espèces. Ces glucanes sont un des facteurs commun de virulence de nombreuses bactéries zoo- et phyto- pathogènes telles que Salmonella enterica, Pseudomonas aeruginosa ou Dickeya dadantii. D. dadantii est une entérobactérie phytopathogène à large spectre d’hôte. Cette bactérie provoque la maladie de la pourriture molle. Chez cette bactérie, un mutant dépourvu d’OPG montre un phénotype pléïotrope : perte totale de la virulence, diminution de la motilité, baisse de la sécrétion des exoenzymes indispensables à la virulence, induction générale d’une réponse au stress suggérant un sévère défaut de perception de l’environnement. Cette idée est renforcée par le lien unissant des systèmes à deux composants, systèmes clefs de la perception de l’environnement, et les OPG. Une approche exploratoire sur la place des OPG dans la virulence a permis de montrer que dans un mutant dépourvu d’OPG, l’inactivation de pecS, principal répresseur de la virulence, permet de restaurer la virulence sur feuille d’endive. La conservation de plusieurs phénotypes associés au défaut de synthèse d’OPG indique que les deux mutations n’affectent pas une seule et même voie de régulation. Enfin, l’identification du gène codant la succinyl-transférase, enzyme responsable de la substitution des OPG par des résidus succinyles, a permis de compléter le set de gènes impliqués dans la biosynthèse des OPG. Leur rôle reste cependant inconnu.Osmoregulated perisplamic glucans (OPGs) are oligosaccharides accumulated in the envelope of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this glucosidic backbone could be substituted by various kind of molecules depending on the species. Numerous studies indicate that these glucans belong to the common virulence factors for zoo- and phyto- pathogens Proteobacteria as Salmonella enterica or Dickeya dadantii. D. dadantii causes soft rot disease in a wide range of plant species. Strains of this species completely devoid of OPG show a pleiotropic phenotype including increased synthesis of exopolysaccharides, loss of motility, induction of a general stress response and complete loss of virulence. These phenotypes were confirmed by a proteomic analysis and suggest that strains devoid of OPGs are impaired in the perception of their environment. The link between OPGs and two component system, the main system used by bacteria to sense and respond to the environmental variation, strengthen this idea. The study of the place of OPGs in a virulence regulatory network showed that mutation in the major virulence repressor pecS restored virulence in a D. dadantii opg-negative strain. All the phenotypes were not restored indicating that the two mutations didn't affect a same pathway. Finally, the identification of opgC, was investigated, encoding a membrane-bound protein required for succinylation of OPG. However, the role of succinyl residues remains unknown

Dickeya dadantii (Vers la compréhension du rôle biologique des glucanes périplasmiques osmorégulés)

Les glucanes périplasmiques osmorégulés (OPG) sont des oligomères de glucose retrouvés chez la plupart des Proteobacteries. Le squelette glucosidique peut être substitué par différents types de molécules selon les espèces. Ces glucanes sont un des facteurs commun de virulence de nombreuses bactéries zoo- et phyto- pathogènes telles que Salmonella enterica, Pseudomonas aeruginosa ou Dickeya dadantii. D. dadantii est une entérobactérie phytopathogène à large spectre d hôte. Cette bactérie provoque la maladie de la pourriture molle. Chez cette bactérie, un mutant dépourvu d OPG montre un phénotype pléïotrope : perte totale de la virulence, diminution de la motilité, baisse de la sécrétion des exoenzymes indispensables à la virulence, induction générale d une réponse au stress suggérant un sévère défaut de perception de l environnement. Cette idée est renforcée par le lien unissant des systèmes à deux composants, systèmes clefs de la perception de l environnement, et les OPG. Une approche exploratoire sur la place des OPG dans la virulence a permis de montrer que dans un mutant dépourvu d OPG, l inactivation de pecS, principal répresseur de la virulence, permet de restaurer la virulence sur feuille d endive. La conservation de plusieurs phénotypes associés au défaut de synthèse d OPG indique que les deux mutations n affectent pas une seule et même voie de régulation. Enfin, l identification du gène codant la succinyl-transférase, enzyme responsable de la substitution des OPG par des résidus succinyles, a permis de compléter le set de gènes impliqués dans la biosynthèse des OPG. Leur rôle reste cependant inconnu.Osmoregulated perisplamic glucans (OPGs) are oligosaccharides accumulated in the envelope of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this glucosidic backbone could be substituted by various kind of molecules depending on the species. Numerous studies indicate that these glucans belong to the common virulence factors for zoo- and phyto- pathogens Proteobacteria as Salmonella enterica or Dickeya dadantii. D. dadantii causes soft rot disease in a wide range of plant species. Strains of this species completely devoid of OPG show a pleiotropic phenotype including increased synthesis of exopolysaccharides, loss of motility, induction of a general stress response and complete loss of virulence. These phenotypes were confirmed by a proteomic analysis and suggest that strains devoid of OPGs are impaired in the perception of their environment. The link between OPGs and two component system, the main system used by bacteria to sense and respond to the environmental variation, strengthen this idea. The study of the place of OPGs in a virulence regulatory network showed that mutation in the major virulence repressor pecS restored virulence in a D. dadantii opg-negative strain. All the phenotypes were not restored indicating that the two mutations didn't affect a same pathway. Finally, the identification of opgC, was investigated, encoding a membrane-bound protein required for succinylation of OPG. However, the role of succinyl residues remains unknown.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

What do we know about osmoadaptation of Yersinia pestis?

International audienc

Two Different Virulence-Related Regulatory Pathways in <i>Borrelia burgdorferi</i> Are Directly Affected by Osmotic Fluxes in the Blood Meal of Feeding <i>Ixodes</i> Ticks

<div><p>Lyme disease, caused by <i>Borrelia burgdorferi</i>, is a vector-borne illness that requires the bacteria to adapt to distinctly different environments in its tick vector and various mammalian hosts. Effective colonization (acquisition phase) of a tick requires the bacteria to adapt to tick midgut physiology. Successful transmission (transmission phase) to a mammal requires the bacteria to sense and respond to the midgut environmental cues and up-regulate key virulence factors before transmission to a new host. Data presented here suggest that one environmental signal that appears to affect both phases of the infective cycle is osmolarity. While constant in the blood, interstitial fluid and tissue of a mammalian host (300 mOsm), osmolarity fluctuates in the midgut of feeding <i>Ixodes scapularis</i>. Measured osmolarity of the blood meal isolated from the midgut of a feeding tick fluctuates from an initial osmolarity of 600 mOsm to blood-like osmolarity of 300 mOsm. After feeding, the midgut osmolarity rebounded to 600 mOsm. Remarkably, these changes affect the two independent regulatory networks that promote acquisition (Hk1-Rrp1) and transmission (Rrp2-RpoN-RpoS) of <i>B</i>. <i>burgdorferi</i>. Increased osmolarity affected morphology and motility of wild-type strains, and lysed Hk1 and Rrp1 mutant strains. At low osmolarity, <i>Borrelia</i> cells express increased levels of RpoN-RpoS-dependent virulence factors (OspC, DbpA) required for the mammalian infection. Our results strongly suggest that osmolarity is an important part of the recognized signals that allow the bacteria to adjust gene expression during the acquisition and transmission phases of the infective cycle of <i>B</i>. <i>burgdorferi</i>.</p></div

A Widefield Light Microscopy-Based Approach Provides Further Insights into the Colonization of the Flea Proventriculus by Yersinia pestis

International audienceYersinia pestis (the agent of flea-borne plague) must obstruct the flea's proventriculus to maintain transmission to a mammalian host. To this end, Y. pestis must consolidate a mass that entrapped Y. pestis within the proventriculus very early after its ingestion. We developed a semiautomated fluorescent image analysis method and used it to monitor and compare colonization of the flea proventriculus by a fully competent flea-blocking Y. pestis strain, a partially competent strain, and a noncompetent strain. Our data suggested that flea blockage results primarily from the replication of Y. pestis trapped in the anterior half of the proventriculus. However, consolidation of the bacteria-entrapping mass and colonization of the entire proventricular lumen increased the likelihood of flea blockage. The data also showed that consolidation of the bacterial mass is not a prerequisite for colonization of the proventriculus but allowed Y. pestis to maintain itself in a large flea population for an extended period of time. Taken as the whole, the data suggest that a strategy targeting bacterial mass consolidation could significantly reduce the likelihood of Y. pestis being transmitted by fleas (due to gut blockage), but also the possibility of using fleas as a long-term reservoir. IMPORTANCE Yersinia pestis (the causative agent of plague) is one of the deadliest bacterial pathogens. It circulates primarily among rodent populations and their fleas. Better knowledge of the mechanisms leading to the flea-borne transmission of Y. pestis is likely to generate strategies for controlling or even eradicating this bacillus. It is known that Y. pestis obstructs the flea's foregut so that the insect starves, frantically bites its mammalian host, and regurgitates Y. pestis at the bite site. Here, we developed a semiautomated fluorescent image analysis method and used it to document and compare foregut colonization and disease progression in fleas infected with a fully competent flea-blocking Y. pestis strain, a partially competent strain, and a noncompetent strain. Overall, our data provided new insights into Y. pestis' obstruction of the proventriculus for transmission but also the ecology of plague

Expression of genes encoding putative ion transporters.

<p>(A) Expression analyses of different ion transporter genes of <i>B</i>. <i>burgdorferi</i> grown in BSK-II at 250, 450 and 650 mOsm. (B) Same as A except measured in unfed, partially fed and replete <i>I</i>. <i>scapularis</i> infected with <i>B</i>. <i>burgdorferi</i>. Gene expression was normalized to <i>enoS</i>.</p