The signaling peptide PapR is required for the activity of the quorum-sensor PlcRa in Bacillus thuringiensis

International audienceThe transcriptional regulator PlcR, its cognate cell-cell signaling heptapeptide PapR7 , and the oligopeptide permease OppABCDF, required for PapR7 import, form a quorum-sensing system that controls the expression of virulence factors in Bacillus cereus and Bacillus thuringiensis species. In B. cereus strain ATCC 14579, the transcriptional regulator PlcRa activates the expression of abrB2 gene, which encodes an AbrB-like transcriptional regulator involved in cysteine biosynthesis. PlcRa is a structural homolog of PlcR: in particular, its C-terminal TPR peptide-binding domain could be similarly arranged as in PlcR. The signaling peptide of PlcRa is not known. As PlcRa is a PlcR-like protein, the cognate PapR7 peptide (ADLPFEF) is a relevant candidate to act as a signaling peptide for PlcRa activation. Also, the putative PapRa7 peptide (CSIPYEY), encoded by the papRa gene adjacent to the plcRa gene, is a relevant candidate as addition of synthetic PapRa7 induces a dose-dependent increase of abrB2 expression. To address the issue of peptide selectivity of PlcRa, the role of PapR and PapRa peptides in PlcRa activity was investigated in B. thuringiensis 407 strain, by genetic and functional complementation analyses. A transcriptional fusion between the promoter of abrB2 and lacZ was used to monitor the PlcRa activity in various genetic backgrounds. We demonstrated that PapR was necessary and sufficient for PlcRa activity. We showed that synthetic PapRs from pherogroups II, III and IV and synthetic PapRa7 were able to trigger abrB2 expression, suggesting that PlcRa is less selective than PlcR. Lastly, the mode of binding of PlcRa was addressed using an in silico approach. Overall, we report a new role for PapR as a signaling peptide for PlcRa activity and show a functional link between PlcR and PlcRa regulons in B. thuringiensis

Clostridioides difficile chez des équidés autopsiés, Constitution et caractérisation de la collection de souches CloDifEqui

National audienceClostridioides difficile est un entéro‐pathogène majeur, anaérobie et sporulant. Il provoque, par sestoxines, des diarrhées post‐antibiotiques difficiles à traiter et des récidives fréquentes dans lesquellesles biofilms pourraient jouer un rôle. Le portage asymptomatique de souches toxinogènes quin’expriment pas les gènes de toxines participe à leur persistence et leur virulence pourra s’exprimerchez un autre hôte. C. difficile pose un problème de santé humaine, à l’hôpital et dans la communauté,mais aussi vétérinaire. Chez les animaux qui peuvent disséminer des spores et contaminer l’homme,l’importance de C. difficile reste mal évaluée : sa recherche n’est pas systématique, et celle de sestoxines encore plus rare..Le projet CloDifEqui de l’Anses et l’INRAe, qui est financé notamment par l’Institut Français du Chevalet de l’Equitation, consiste à évaluer la fréquence de C. difficile chez les équidés autopsiés de 2019 à2021 à l’Anses quelle que soit la cause de la mort.A chaque autopsie, les données de l’animal (individuelles, cliniques, épidémiologiques) sontenregistrées et du contenu intestinal est prélèvé pour rechercher C. difficile. Les souches isoléesd’espèce confirmée constituent la collection CloDifEqui. Leurs principaux caractères génotypiques(gènes des toxines, ribotype …) et phénotypiques (sporulation, mobilité, résistance aux antibiotiques,formation de biofilm …) sont en cours d’étude et seront comparés à ceux de souches d’origine humaineou animale. En parallèle, un diagnostic d’infection est établi en collaboration avec le CNR C. difficile parla recherche des toxines dans les contenus intestinaux présentant C. difficile et provenant d’équidésavec des signes d’infection intestinale. Cela permettra d’établir la fréquence de C. difficile chez leséquidés, à la fois en cas d’infection et de portage asymptomatique. Les résultats de caractérisation dessouches seront croisés avec les données individuelles et cliniques des animaux autopsiés, notammentleurs traitements antibiotiques antérieurs éventuels.Après 80 autopsies, la collection CloDifEqui comprend 20 souches isolées d’animaux différents etcaractériéses. L’analyse croisée de l’ensemble des données et résultats est en cours. Le projet devraitcontribuer à mieux comprendre l’épidémiologie de C. difficile chez les équidés et à évaluer lacirculation des souches, leur potentiel de transmission zoonotique et de persistance

The CodY-dependent <i>clhAB2</i> operon is involved in cell shape, chaining and autolysis in <i>Bacillus cereus</i> ATCC 14579

<div><p>The Gram-positive pathogen <i>Bacillus cereus</i> is able to grow in chains of rod-shaped cells, but the regulation of chaining remains largely unknown. Here, we observe that glucose-grown cells of <i>B</i>. <i>cereus</i> ATCC 14579 form longer chains than those grown in the absence of glucose during the late exponential and transition growth phases, and identify that the <i>clhAB</i>_<i>2</i> operon is required for this chain lengthening phenotype. The <i>clhAB</i>_<i>2</i> operon is specific to the <i>B</i>. <i>cereus</i> group (i.e., <i>B</i>. <i>thuringiensis</i>, <i>B</i>. <i>anthracis</i> and <i>B</i>. <i>cereus</i>) and encodes two membrane proteins of unknown function, which are homologous to the <i>Staphylococcus aureus</i> CidA and CidB proteins involved in cell death control within glucose-grown cells. A deletion mutant (Δ<i>clhAB</i>_<i>2</i>) was constructed and our quantitative image analyses show that Δ<i>clhAB</i>_<i>2</i> cells formed abnormal short chains regardless of the presence of glucose. We also found that glucose-grown cells of Δ<i>clhAB</i>_<i>2</i> were significantly wider than wild-type cells (1.47 μm ±CI_95% 0.04 vs 1.19 μm ±CI_95% 0.03, respectively), suggesting an alteration of the bacterial cell wall. Remarkably, Δ<i>clhAB</i>_<i>2</i> cells showed accelerated autolysis under autolysis-inducing conditions, compared to wild-type cells. Overall, our data suggest that the <i>B</i>. <i>cereus clhAB</i>_<i>2</i> operon modulates peptidoglycan hydrolase activity, which is required for proper cell shape and chain length during cell growth, and down-regulates autolysin activity. Lastly, we studied the transcription of <i>clhAB</i>_<i>2</i> using a <i>lacZ</i> transcriptional reporter in wild-type, <i>ccpA</i> and <i>codY</i> deletion-mutant strains. We found that the global transcriptional regulatory protein CodY is required for the basal level of <i>clhAB</i>_<i>2</i> expression under all conditions tested, including the transition growth phase while CcpA, the major global carbon regulator, is needed for the high-level expression of <i>clhAB</i>_<i>2</i> in glucose-grown cells.</p></div

Development of a Remote‐Controlled Implantable Rat Sacral Nerve Stimulation System

International audienc

Clostridioides difficile in necropsied equidae, Isolation and caracterisation of the CloDifEqui collection of strains

International audienceBackground: Clostridioides difficile is a sporulating anaerobe and a major entero-pathogen of both humans and animals. It produces toxins and is responsible for post-antibiotic diarrhoea and colitis that are difficult to treat and can be recurrent. In animals, the importance of C. difficile is not well evaluated. In cases of diarrhoea, its presence is not necessarily tested for. Moreover, diagnosis of C. difficile infection is difficult, because toxinogenic strains can be asymptomatically carried, if they do not produce toxins. They nevertheless remain able, after spread and transmission, to cause new, and notably community-acquired, infections. Objectives: The project aimed to i) build up and characterise a collection of C. difficile strains isolated from necropsied equidae, and ii) evaluate the frequency of C. difficile carriers in this whole equine population, together with its prevalence as a cause of infectious disease. Study design: Post-mortem survey Methods: The presence of C. difficile was systematically monitored in equideae that had been necropsied at Anses since 2019, whatever the cause of their death. Animal data (individual, clinical, epidemiological, cause of death) were registered and intestinal contents sampled to search for C. difficile. C. difficile strains were isolated and stored, forming the CloDifEqui collection. They were characterised by molecular genotyping and phenotyping. The intestinal contents of equideae displaying a toxinogenic strain were analysed for the presence of the toxins, in order to establish a diagnosis of infection. Results: Twenty strains were isolated in 80 necropsies. Conclusions: C difficile is prevalent in equine intestinal contents. Further work on molecular epidemiology and to compare these findings with the animal data is needed

Inter-constriction cell arrangements and cell width measures in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and Δ<i>clhAB2ΩclhAB</i>_<i>2</i> populations in the presence or absence of glucose.

<p>(A) Close view of <i>Bc</i>, Δ<i>clhAB2</i>, and Δ<i>clhAB2</i>,<i>ΩclhAB2</i> chains at <i>t</i>₀ using fluorescence microscopy. Cells were grown in LB and LB with 0.35% glucose (LBG). Division septa and cytoplasmic membranes were imaged using the FM4-64 lipophilic dye. Chains exhibited constrictions that occurred at septa spaced 4 cells apart in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and Δ<i>clhAB</i>_<i>2</i><i>ΩclhAB</i>_<i>2</i> in LB medium and in Δ<i>clhAB</i>_<i>2</i> in LBG medium. They also exhibited constrictions that occurred at septa spaced 8 cells apart in <i>Bc</i> and Δ<i>clhAB</i>_<i>2</i>,Ω<i>clhAB</i>_<i>2</i> in LBG medium. (B) Distributions of “short” (≤4) and “long” (>4) inter-constriction cell types in the <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and complemented mutant populations (N>250 cell arrangements). Two inter-constriction arrangement types in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and Δ<i>clhAB</i>_<i>2</i>Ω<i>clhAB</i>_<i>2</i> populations were defined (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials and methods</a>). The first type, containing cell arrangements with two to four cells ("short") and the second type, including cell arrangements with six to eight cells ("long"). The significant effects of glucose (dashed line) and <i>clhAB</i>_<i>2</i> mutation (solid line) are based on a Binomial analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials and methods</a>) with <i>P</i> <0.01** and <0.05*. (C) Cell width measures in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i> and Δ<i>clhAB</i>_<i>2</i>Ω<i>clhAB</i>_<i>2</i> populations (N>250 bacilli) in LB and LBG media. The significant effect of <i>clhAB</i>_<i>2</i> mutation in LBG medium is based on a Student’s t test and ANOVA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials and methods</a>) with <i>P</i> <0.001***. A <i>P</i> value close to the cutoff 0.05 was considered as non significant (NS). Mean ± CI _95% is depicted.</p

CodY-dependent regulation of the <i>clhAB</i>_<i>2</i> operon in the presence or absence of glucose.

<p>Effect of <i>codY</i> mutation on the expression of <i>clhAB</i>_<i>2</i>. Cells of <i>Bc</i> and isogenic mutant strains (Δ<i>codY</i>, <i>codY</i>-complemented mutant), which all harbored the transcriptional P_{<i>clhAB2</i>}’-<i>lacZ</i> fusion construct, were grown in LB (closed symbols) or in LBG (open symbols) media. See legend of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g002" target="_blank">Fig 2</a> for additional informations.</p

Chain lengths in <i>Bc</i>, Δ<i>clhAB</i>_<i>2</i>, and Δ<i>clhAB</i>_<i>2</i><i>ΩclhAB</i>_<i>2</i> populations in the presence or absence of glucose.

<p>(A) Phase-contrast images of cell-chains at <i>t</i>₀ of wild-type (<i>Bc</i>), Δ<i>clhAB</i>_<i>2</i> (Δ), complemented (ΔΩ) and pHT315 (Δp) mutant strains grown in LB medium and LB medium supplemented with glucose (LBG). Images of chains revealed strong constrictions (deeper invaginations) corresponding to cells undergoing separation. Scale bar (5μm) is shown for each image. (B) Fluorescent micrographs of <i>Bc</i>, Δ, and ΔΩ cell-chains at <i>t</i>₀ in LB and LBG. Division septa and cytoplasmic membranes were imaged using the FM4-64 lipophilic dye. Scale bar (5μm) is shown for each image. (C) Box plots of chain length (number of cells per chain) at <i>t</i>₀ in <i>Bc</i> (blue), Δ<i>clhAB</i>_<i>2</i> (red), Δ<i>clhAB</i>_<i>2</i>Ω<i>clhAB</i>_<i>2</i> (green) populations. Between 90 and 130 chains from at least three independent cultures were analysed. No fewer than 1,000 cells were quantitated for each strain represented in the graph using fluorescence micrographs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials and methods</a>). Median (strong line in the box), interquartile range (IQR; box), whiskers (1.5 x IQR) and outliers (dot) are presented. Significance is based on two tests, Mann-Whitney and Two-Sample Fisher-Pitman Permutation, with a <i>P</i> of <0.01**. Non significative (NS), <i>P</i>> 0.05.</p

The <i>clhAB</i>_<i>2</i> expression requires the presence of a 15-bp putative CodY-binding motif.

<p>(A) The sequence of the <i>clhAB</i>_<i>2</i> regulatory region. GM38 primer (sequence in blue) and GM39 primer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#sec002" target="_blank">Materials and methods</a>) used for the <i>clhAB</i>_<i>2</i>-<i>lacZ</i> fusion construction are located. The CodY binding motif (in green) is identified 178 bp upstream from the likely initiation codon (in bold). Sequences used for SOE primers design are in bold. (B) Schematic drawing of the PCR-driven overlap extension method used for mutagenesis analysis. Primers sequences are shown. CodY binding motif, green box. (C) Effect of CodY binding motif deletion on <i>clhAB</i>_<i>2</i> expression. <i>Bc</i> cells harboring either P_{<i>clhAB2</i>}’-<i>lacZ</i> fusion or P_CodYBm’-<i>lacZ</i> fusion, were grown in LB (closed symbols) or LBG (open symbols) media. See legend of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g002" target="_blank">Fig 2</a> for additional information. Representative experiment of n = 2 experiments are shown.</p