Genome-wide analysis of Brucella melitensis genes required throughout intranasal infection in mice

Brucellae are facultative intracellular Gram-negative coccobacilli that chronically infect various mammals and cause brucellosis. Human brucellosis is among the most common bacterial zoonoses and the vast majority of cases are attributed to B .melitensis .Using transposon sequencing (Tn-seq) analysis, we showed that among 3369 predicted genes of the B .melitensis genome, 861 are required for optimal growth in rich medium and 186 additional genes appeared necessary for survival of B .melitensis in RAW 264.7 macrophages in vitro .As the mucosal immune system represents the first defense against Brucella infection, we investigated the early phase of pulmonary infection in mice. In situ analysis at the single cell level indicates a succession of killing and growth phases, followed by heterogenous proliferation of B .melitensis in alveolar macrophages during the first 48 hours of infection. Tn-seq analysis identified 94 additional genes that are required for survival in the lung at 48 hours post infection. Among them, 42 genes are common to RAW 264.7 macrophages and the lung conditions, including the T4SS and purine synthesis genes. But 52 genes are not identified in RAW 264.7 macrophages, including genes implicated in lipopolysaccharide (LPS) biosynthesis, methionine transport, tryptophan synthesis as well as fatty acid and carbohydrate metabolism. Interestingly, genes implicated in LPS synthesis and β oxidation of fatty acids are no longer required in Interleukin (IL)-17RA -/- mice and asthmatic mice, respectively. This demonstrates that the immune status determines which genes are required for optimal survival and growth of B .melitensis in vivo .info:eu-repo/semantics/publishe

Role of two component regulatory system in plague cycle

Le bacille de la peste, Yersinia pestis, a une vie parasitaire au cours de laquelle il oscille le plus souvent d’un hôte mammifère à l’autre par l’intermédiaire des puces, et plus rarement par voie aéroportée. Comme tel, Yersinia pestis doit rapidement ressentir et répondre aux variations brutales de son environnement afin se maintenir dans la nature. C’est pourquoi, nous avons étudié le rôle des systèmes de régulation à deux composants dans la peste compte tenu que ces systèmes sont connus pour avoir un rôle clef dans l’adaptation des bactéries aux changements environnementaux. En plus du système PhoP-PhoQ dont l’importance chez le mammifère et la puce a été précédemment révélée, nous avons découvert que quatre systèmes sont requis pour le cycle de la peste. Plus particulièrement, l'un d'entre eux permet une colonisation optimale du tube digestif de la puce alors que les 3 autres systèmes sont impliqués dans la production de biofilm, un processus indispensable à une transmission optimale du bacille par les puces. Nous avons aussi découvert que le système OmpR-EnvZ est l’unique système, en plus du système PhoP-PhoQ, requis pour la production de la peste bubonique, septicémique et pulmonaire. Nos travaux, menés in vitro, ex-vivo et in vivo suggèrent que le rôle du système OmpR-EnvZ serait de protéger la bactérie contre les effecteurs toxiques sécrétés par les polynucléaires neutrophiles dans les tissus et, ceci tout au long du processus infectieux.Plague bacillus, Yersinia pestis has a parasitic lifestyle in which it is mainly transmitted between mammilian hosts through the bite of infected fleas, and in rare cases through infected droplets. Thus, Yersinia pestis must rapidly sense and respond to wide and brutal changes of its environment in order to survive. We aimed at decipher the role of two component regulatory systems in plague, as they are known to be key players in bacterial adaptation to the environment. In addition to the already described PhoP-PhoQ system, we found out that four systems are required for plague cycle. We showed that one of these systems is important for an optimal colonization of the flea's digestive tract, while the three others are required for biofilm production, an essential step in the bacillus transmission by the fleas. We also found out that OmpR-EnvZ, in addition to PhoP-PhoQ, is the only one to be important to produce bubonic, septicemic and pulmonary plague. Our in vitro, ex-vivo and in vivo works suggest that the OmpR-EnvZ system would be to protect bacterial against toxic effectors that are produced by polymorphonuclear leukocytes all along the infectious process

New Insights into How <i>Yersinia pestis</i> Adapts to Its Mammalian Host during Bubonic Plague

<div><p>Bubonic plague (a fatal, flea-transmitted disease) remains an international public health concern. Although our understanding of the pathogenesis of bubonic plague has improved significantly over the last few decades, researchers have still not been able to define the complete set of <i>Y. pestis</i> genes needed for disease or to characterize the mechanisms that enable infection. Here, we generated a library of <i>Y. pestis</i> mutants, each lacking one or more of the genes previously identified as being up-regulated <i>in vivo</i>. We then screened the library for attenuated virulence in rodent models of bubonic plague. Importantly, we tested mutants both individually and using a novel, “per-pool” screening method that we have developed. Our data showed that in addition to genes involved in physiological adaption and resistance to the stress generated by the host, several previously uncharacterized genes are required for virulence. One of these genes (<i>ympt1.66c</i>, which encodes a putative helicase) has been acquired by horizontal gene transfer. Deletion of <i>ympt1.66c</i> reduced <i>Y. pestis</i>' ability to spread to the lymph nodes draining the dermal inoculation site – probably because loss of this gene decreased the bacteria's ability to survive inside macrophages. Our results suggest that (i) intracellular survival during the early stage of infection is important for plague and (ii) horizontal gene transfer was crucial in the acquisition of this ability.</p></div

Nutrient depletion may trigger the Yersinia pestis OmpR‐EnvZ regulatory system to promote flea‐borne plague transmission

International audienc

Stress, metabolic and uncharacterized genes are needed for resistance and/or growth in serum.

<p>The ability of selected mutants to grow in fresh serum was evaluated after 6 and 21Δ<i>rseC</i>, Δ<i>ypo0337</i>, Δ<i>gpmA</i>, Δ<i>ibpA</i>, Δ<i>ypo3369</i>, Δ<i>ypo0988</i>, Δ<i>amn</i>, Δ<i>ypo0617-0618</i>, Δ<i>ypo2586-2587</i> and Δ<i>ypo0426</i> mutants differed significantly from that of the wild-type strain (p<0.05 in a two-way analysis of variance).</p

PdeA is required for the rod shape morphology of Brucella abortus

Cyclic-di-GMP plays crucial role in the cell cycle regulation of the α-Proteobacterium Caulobacter crescentus. Here we investigated its role in the α-Proteobacterium Brucella abortus, a zoonotic intracellular pathogen. Surprisingly, deletion of all predicted cyclic-di-GMP synthesizing or degrading enzymes did not drastically impair the growth of B. abortus, nor its ability to grow inside cell lines. As other Rhizobiales, B. abortus displays unipolar growth from the new cell pole generated by cell division. We found that the phosphodiesterase PdeA, the ortholog of the essential polar growth factor RgsP of the Rhizobiale Sinorhizobium meliloti, is required for rod shape integrity but is not essential for B. abortus growth. Indeed, the radius of the pole is increased by 31 ± 1.7% in a ΔpdeA mutant, generating a coccoid morphology. A mutation in the cyclic-di-GMP phosphodiesterase catalytic site of PdeA does not generate the coccoid morphology and the ΔpdeA mutant kept the ability to recruit markers of new and old poles. However, the presence of PdeA is required in an intra-nasal mouse model of infection. In conclusion, we propose that PdeA contributes to bacterial morphology and virulence in B. abortus, but it is not crucial for polarity and asymmetric growth.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis

International audienceThe 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 ⌬opgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macro-phages, 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

Incidence of plague in immunocompetent rats (A), immunocompetent mice (B), and neutropenic mice (C) injected intradermally with ∼20 WT <i>Y. pestis</i> (white circles), Δ<i>ypmt1.66c</i> (black circles) or complemented mutant (grey circles).

<p>The mutant was significantly (p<0.05) less virulent than the WT strain in immunocompetent rats (A), immunocompetent mice (B and C) and neutropenic mice (C). The survival curves of mice infected with the complemented mutant harboring the <i>ypmt1.66c</i> gene on a high copy number plasmid (pCR2) and the wild-type (B) were significantly different (p<0.05). The virulence of <i>Y. pestis</i> lacking <i>ympt1.66c</i> was no greater in neutropenic mice than it was in immunocompetent mice (dashed lines) (p>0.05). Data were obtained from groups of 8 or 9 animals. P values were determined using the Gehan-Breslow-Wilcoxon test.</p

YPMT1.66c is required for intracellular survival in macrophages (A) and for optimal growth in serum (B).

<p>Shown are the data obtained with the wild-type (white squares and bars), the Δ<i>ypmt1.66c</i> (grey squares and bars) and the complemented mutant (black squares and bars) strains. Data are quoted as the mean (SD) from three independent experiments using macrophages (A) and 5 independent experiments using serum from 5 different healthy donors (B). The mutant's survival (A) and growth curves (B) differed significantly from those of the WT and the complemented strain (p<0.05 in a two-way analysis of variance). The complemented mutant's and the WT's survival (A) and growth curves (B) did not differ significantly (p>0.05 in a two-way analysis of variance).</p

List of mutants selected using the per-pool screening method.

†<p>, data were obtained from groups of 10 rats inoculated intradermally with a pool of 5 mutants (with 20 CFU of each mutant).</p