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Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. During gemination, spores must degrade their cortex layer, which is a thick, protective layer of modified peptidoglycan. Cortex degradation depends on the presence of the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C. difficile, MAL production depends on the CwlD amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind Zn2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to Zn2+, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of Zn2+ co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought., Author summary Spore germination is essential for many spore-forming pathogens to initiate infection. In order for spores to germinate, they must degrade a thick, protective layer of cell wall known as the cortex. The enzymes that digest this layer selectively recognize the spore-specific cell wall modification, muramic-∂-lactam (MAL). MAL is made in part through the activity of the CwlD amidase, which is found in all spore-forming bacteria. While Bacillus subtilis CwlD appears to have amidase activity on its own, Clostridioides difficile CwlD activity depends on its binding partner, the GerS lipoprotein. To understand why C. difficile CwlD requires GerS, we determined the X-ray crystal structure of the CwlD:GerS complex and discovered that GerS binds to a site distant from CwlD’s active site. We also found that GerS stabilizes CwlD binding to its co-factor, Zn2+, indicating that GerS allosterically activates CwlD amidase. Notably, regulation at the level of Zn2+ binding has not previously been described for bacterial amidases, and GerS is the first protein to be shown to allosterically activate an amidase. Since binding partners of bacterial amidases were only first discovered 15 years ago, our results suggest that diverse mechanisms remain to be discovered for these critical enzymes.

Volume 11, no. 5, e01559-20, 2020. [https://doi.org/10.1128/mBio.01559-20][1]. On PDF page 8, [Fig. 5][2] should be replaced by the figure below. In the supplemental material, Fig. S7 has been replaced online. The figure legends are not modified. These changes do not change the conclusions of the

Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile. This enteropathogen has developed efficient strategies to detoxify O2. In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile. These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract., Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2. O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon.

A relação entre parasitoses intestinais, alergia e asma ainda não está completamente conhecida. Diversos estudos tem identificado infecções por A. lumbricoides como fator de risco para asma e alergia. O presente trabalho visou investigar o perfil de reatividade IgE a tropomiosina de diferentes fontes, e por diferentes métodos, e avaliar a relevância clínica de nova estratégia para o diagnóstico de alergia, através do ImmunoCAP-ISAC, uma vez que tropomiosinas são pan-alérgenos em invertebrados, incluindo baratas, ácaros, camarão, outros crustáceos e moluscos, Ascaris lumbricoides e outros parasitas. Tropomiosinas induzem resposta IgE que está associada a reatividade cruzada imunológica. Além disso, foi avaliado se a estratégia de diagnóstico por microarray pode melhorar o diagnóstico de alergia a baratas, pois a sensibilização a tropomiosina de barata ocorre em 50% a 62% dos casos, indicando que a identificação de outros alérgenos é necessária para melhorar o diagnóstico de alergia a barata entre pacientes com asma e/ou rinite. Métodos: Avaliamos 40 pacientes alérgicos a barata com asma e/ou rinite e 10 indivíduos controles não alérgicos. Foram realizados testes cutâneos com extratos comerciais de aeroalérgenos e tropomiosinas recombinantes de A. lumbricoides e barata P. americana (rPer a 7), além de medidas de IgE específica para tropomiosinas de A. lumbricoides e P. americana (rPer a 7) por ELISA. O diagnóstico de alergia por componentes foi feito por técnica de microarray, através do ImmunoCAP-ISAC; avaliamos IgE para tropomiosinas presentes nesse painel de componentes nBla g 7, tropomiosina natural da barata B. germanica e rAni s 3, tropomiosina recombinante do parasita A. simplex, que apresentam 96-97% de identidade de sequencia com tropomiosinas de barata Per a 7 e de A. lumbricoides Asc l 3, respectivamente. Avaliamos também IgE para componentes de barata rBla g 1, rBla g 2, e rBla g 5. Resultados: Avaliação da resposta IgE para tropomiosinas de Ascaris e barata mostrou excelente concordância por métodos in vivo e in vitro. Essa possível reatividade cruzada IgE em pacientes sem evidência de infecção parasitária, nos permite formular a hipótese de que em nosso meio, o contato com parasitas cedo na infância, poderia induzir sensibilização IgE a tropomiosina, e a exposição, ainda durante a infância, a tropomiosinas de outras fontes, como ácaros e baratas, por via inalatória, poderia levar ao desenvolvimento de doenças alérgicas como asma e rinite e/ou potencializar o efeito da resposta a estes aeroalérgenos. A maioria (55%) dos pacientes apresentou positividade para o alérgeno rBla g 7 (tropomiosina) de barata. Apenas 3 pacientes foram positivos para os outros alérgenos recombinantes de barata, indicando que a estratégia de diagnóstico por componentes mostrou valor adicional limitado, quando comparado aos métodos de teste cutâneo e IgE específica para barata, para investigar alergia a barata. The relationship of intestinal parasitosis, allergy and asthma is not completely known. Several studies have identified Ascaris lumbricoides infections as a risk factor for asthma and allergy. The present work aimed to investigate the IgE reactivity profile to tropomyosin from different sources, and by different methods, and to evaluate the clinical relevance of a new strategy for the diagnosis of allergy through ImmunoCAP-ISAC, since tropomyosins are pan-allergens in invertebrates, including cockroaches, mites, shrimp, other crustaceans and mollusks, A. lumbricoides and other parasites. Tropomyosins induce IgE response and are associated with immunological cross reactivity. In addition, it was evaluated whether the microarray diagnostic strategy can improve the diagnosis of cockroach allergy, as cockroach tropomyosin sensitization occurs in 50% to 62% of cases, indicating that identification of other allergens is necessary to improve diagnosis of cockroach allergy among patients with asthma and/or rhinitis. Methods: We evaluated 40 cockroach allergic patients with asthma and/or rhinitis and 10 non-allergic control subjects. Skin tests were carried out with commercial extracts of airborne allergens, and measurements of IgE to A. lumbricoides and cockroach Periplaneta americana (rPer to 7) tropomyosins were carried out by ELISA. IgE antibodies to tropomyosins from Blattella germanica (Bla g 7, 96% identity to Per a 7) and Anisakis simplex (Ani s 3, 97% identity to A. lumbricoides tropomyosin) were assessed by Immuno Solidphase Allergen Chip (ImmunoCAP-ISAC). We also evaluated IgE to the cockroach components rBla g 1, rBla g 2, and rBla g 5. Results: Evaluation of IgE response to Ascaris and cockroach tropomyosins showed excellent agreement by in vivo and in vitro methods. This possible IgE cross-reactivity in patients with no evidence of parasitic infection allows us to hypothesize that in our area, contact with parasites early in childhood could induce tropomyosin IgE sensitization and exposure to tropomyosins from other sources, such as mites and cockroaches, by inhalation, even during childhood, could lead to the development of allergic diseases such as asthma and rhinitis and/or to potentiate the effect of response to these aeroallergens. Most (55%) patients were positive presented IgE response to the cockroach rBla g 7 (tropomyosin) allergen. Only 3 patients were positive to the other recombinant cockroach allergens, indicating that the component diagnostic strategy showed limited additional value when compared to cockroach-specific IgE and skin test methods to investigate cockroach allergy.

International audience; Clostridium difficile is the main cause of antibiotic-associated diarrhoea. Inside the gut, C. difficile must adapt to the stresses it copes with, by inducing protection, detoxification and repair systems that belong to the general stress response involving σB . Following stresses, σB activation requires a PP2C phosphatase to dephosphorylate the anti-anti-sigma factor RsbV that allows its interaction with the anti-sigma factor RsbW and the release of σB . In this work, we studied the signalling pathway responsible for the activation of σB in C. difficile. Contrary to other firmicutes, the expression of sigB in C. difficile is constitutive and not autoregulated. We confirmed the partner switching mechanism that involved RsbV, RsbW and σB . We also showed that CD2685, renamed RsbZ, and its phosphatase activity are required for RsbV dephosphorylation triggering σB activation. While CD0007 and CD0008, whose genes belong to the sigB operon, are not involved in σB activity, depletion of the essential iron-sulphur flavoprotein, CD2684, whose gene forms an operon with rsbZ, prevents σB activation. Finally, we observed that σB is heterogeneously active in a subpopulation of C. difficile cells from the exponential phase, likely leading to a 'bet-hedging' strategy allowing a better chance for the cells to survive adverse conditions.

Clostridium difficile is the main cause of antibiotic-associated diarrhoea. Inside the gut, C. difficile must adapt to the stresses it copes with, by inducing protection, detoxification and repair systems that belong to the general stress response involving σ

International audience; The strict anaerobe Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, the coat and exosporium, enable the spores to resist physical and chemical stress. However, little is known about the mechanisms of their assembly. In this study, we characterized a new spore protein, CotL, which is required for the assembly of the spore coat. The cotL gene was expressed in the mother cell compartment under the dual control of the RNA polymerase sigma factors, σE and σK . CotL was localized in the spore coat, and the spores of the cotL mutant had a major morphologic defect at the level of the coat/exosporium layers. Therefore, the mutant spores contained a reduced amount of several coat/exosporium proteins and a defect in their localization in sporulating cells. Finally, cotL mutant spores were more sensitive to lysozyme and were impaired in germination, a phenotype likely to be associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.

Background Drugs administered during the anaesthetic procedure and postoperative period belong to different pharmacological groups and are to ensure the best possible conditions for surgery and maximum safety of patients. Their adverse side effects are often dependent on immune responses. The diversity and number of the agents administered hinder precise determination of the drug eliciting the adverse drug reaction.