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International audience; Yersinioses caused by Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are significant concerns in human and veterinary health. The link between virulence and the potent LcrV antigen has prompted the latter's selection as a major component of anti-Yersinia vaccines. Here, we report that (i) the group of Yersinia species encompassing Y. pestis and Y. pseudotuberculosis produces at least five different clades of LcrV and (ii) vaccination of mice with an LcrV-secreting Lactococcus lactis only protected against Yersinia strains producing the same LcrV clade as that of used for vaccination. By vaccinating with engineered LcrVs and challenging mice with strains producing either type of LcrV or a LcrV mutated for regions of interest, we highlight key polymorphic residues responsible for the absence of cross-protection. Our results show that an anti-LcrV-based vaccine should contain multiple LcrV clades if protection against the widest possible array of Yersinia strains is sought.

International audience; 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.

International audience; Polyamines are an essential class of metabolites found throughout all kingdoms in life. Borrelia burgdorferi harbors no enzymes to synthesize or degrade polyamines yet does contain a polyamine uptake system, potABCD. In this report, we describe the initial characterization of this putative transport system. After several unsuccessful attempts to inactivate potABCD, we placed the operon under the control of an inducible LacI promoter expression system. Analyses of this construct confirmed that potABCD was required for in vitro survival. Additionally, we demonstrated that the potABCD operon were upregulated in vitro by low osmolarity. Previously, we had shown that low osmolarity triggers the activation of the Rrp2/RpoN/RpoS regulatory cascade, which regulates genes essential for the transmission of spirochetes from ticks to mammalian hosts. Interestingly, induction of the pot operon was only affected in an rpoS mutant but not in a rpoN mutant, suggesting that the genes were RpoS dependent and RpoN independent. Furthermore, potABCD was upregulated during tick feeding concomitant with the initiation of spirochete replication. Finally, uptake experiments determined the specificity of B. burgdorferi's PotABCD for spermidine.

International audience; Lyme disease, caused by Borrelia burgdorferi, 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 Ixodes scapularis. 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 B. burgdorferi. Increased osmolarity affected morphology and motility of wild-type strains, and lysed Hk1 and Rrp1 mutant strains. At low osmolarity, Borrelia 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 B. burgdorferi.

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International audience; Osmoregulated periplasmic glucans (OPGs) are a family of periplasmic oligosaccharides found in the envelope of most Proteobacteria. They are required for virulence of zoo- and phyto-pathogens. The glucose backbone of OPGs is substituted by various kinds of molecules depending on the species, O-succinyl residues being the most widely distributed. In our model, Dickeya dadantii, a phytopathogenic bacteria causing soft rot disease in a wide range of plant species, the backbone of OPGs is substituted by O-succinyl residues in media of high osmolarity and by O-acetyl residues whatever the osmolarity. The opgC gene encoding a transmembrane protein required for the succinylation of the OPGs in D. dadantii was found after an in silico search of a gene encoding a protein with the main characteristics recovered in the two previously characterized OpgC of E. coli and R. sphaeroides, i.e. 10 transmembrane segments and one acyl-transferase domain. Characterization of the opgC gene revealed that high osmolarity expression of the succinyl transferase is controlled by both the EnvZ-OmpR and RcsCDB phosphorelay systems. The loss of O-succinyl residue did not affect the virulence of D. dadantii, suggesting that only the glucose backbone of OPGs is required for virulence.

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. Here we show that OPGs control the RcsCDB activation in a concentration-dependant manner, are required for proper activation of this phosphorelay by medium osmolarity, and that a high concentration of OPGs in planta is maintained to achieve the low level of activation of the RcsCDB phosphorelay required for full virulence in D. dadantii. The EnvZ-OmpR system are activated in strain devoid OPG suggesting a role of OPGs in the adaptation of osmolarity variation. The level of activation of the envelope stress system CpxAR was not affected by the loss of OPGs. These results were completed by membrane proteomic analysis and by microscopic pictures experiments but they didn't show any structural perturbation of the envelope in the strain devoid of OPGs. 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.; Les glucanes périplasmiques osmorégulés (OPG) sont des oligomères de glucose retrouvés chez la plupart des Protéobacteries. 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.

Borrelia species are amino acid auxotrophs that utilize di- and tri- peptides obtained through their oligopeptide transport system to supply amino acids for replicative growth during their enzootic cycles. However, Borrelia species from both the Lyme disease (LD) and relapsing fever (RF) groups harbor an amino acid transport and catabolism system, the Arginine Deiminase System (ADI), that could potentially augment intracellular L-arginine required for growth. RF spirochetes contain a “complete”, four gene ADI (arcA, B, D, and C) while LD spirochetes harbor arcA, B, and sometimes D but lack arcC (encoding carbamate kinase). In this study, we evaluated the role of the ADI system in bacterial survival and virulence and discovered important differences in RF and LD ADIs. Both in vitro and in a murine model of infection, B. hermsii cells significantly reduced extracellular L-arginine levels and that reduction was dependent on arginine deiminase expression. Conversely, B. burgdorferi did not reduce the concentration of L-arginine during in vitro growth experiments nor during infection of the mammalian host, suggesting a fundamental difference in the ability to directly utilize L-arginine compared to B. hermsii. Further experiments using a panel of mutants generated in both B. burgdorferi and B. hermsii, identified important differences in growth characteristics and ADI transcription and protein expression. We also found that the ADI system plays a key role in blood and spleen colonization in RF spirochetes. In this study we have identified divergent metabolic strategies in two closely related human pathogens, that ultimately impacts the host-pathogen interface during infection.

International audience; The post-translational modification of proteins has been shown to be extremely important in prokaryotes. Using a highly sensitive mass spectrometry-based proteomics approach, we have characterized the acetylome of B. burgdorferi. As previously reported for other bacteria, a relatively low number (5%) of the potential genome-encoded proteins of B. burgdorferi were acetylated. Of these, the vast majority were involved in central metabolism and cellular information processing (transcription, translation, etc.). Interestingly, these critical cell functions were targeted during both ML (mid-log) and S (stationary) phases of growth. However, acetylation of target proteins in ML phase was limited to single lysine residues while these same proteins were acetylated at multiple sites during S phase. To determine the acetyl donor in B. burgdorferi, we used mutants that targeted the sole acetate metabolic/anabolic pathway in B. burgdorferi (lipid I synthesis). B. burgdorferi strains B31-A3, B31-A3 ackA (acetyl-P − and acetyl-CoA −) and B31-A3 pta (acetyl-P + and acetyl-CoA −) were grown to S phase and the acetylation profiles were analyzed. While only two proteins were acetylated in the ackA mutant, 140 proteins were acetylated in the pta mutant suggesting that acetyl-P was the primary acetyl donor in B. burgdorferi. Using specific enzymatic assays, we were able to demonstrate that hyperacetylation of proteins in S phase appeared to play a role in decreasing the enzymatic activity of at least two glycolytic proteins. Currently, we hypothesize that acetylation is used to modulate enzyme activities during different stages of growth. This strategy would allow the bacteria to post-translationally stimulate the activity of key glycolytic enzymes by deacetylation rather than expending excessive energy synthesizing new proteins. This would be an appealing, low-energy strategy for a bacterium with limited metabolic capabilities. Future work focuses on identifying potential protein deacetylase(s) to complete our understanding of this important biological process.

The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis . When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii , opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli . A Δ opgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥37°C, except when the plague bacillus expressed opgGH . Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary.