Visualization of a substrate-induced productive conformation of the catalytic triad of the Neisseria meningitidis peptidoglycan O-acetylesterase reveals mechanistic conservation in SGNH esterase family members.

Peptidoglycan O-acetylesterase (Ape1), which is required for host survival in Neisseria sp., belongs to the diverse SGNH hydrolase superfamily, which includes important viral and bacterial virulence factors. Here, multi-domain crystal structures of Ape1 with an SGNH catalytic domain and a newly identified putative peptidoglycan-detection module are reported. Enzyme catalysis was performed in Ape1 crystals and key catalytic intermediates along the SGNH esterase hydrolysis reaction pathway were visualized, revealing a substrate-induced productive conformation of the catalytic triad, a mechanistic detail that has not previously been observed. This substrate-induced productive conformation of the catalytic triad shifts the established dogma on these enzymes, generating valuable insight into the structure-based design of drugs targeting the SGNH esterase superfamily.

New Approach Methods to Assess the Enteropathogenic Potential of Strains of the Bacillus cereus Group, including Bacillus thuringiensis.

Bacillus cereus (Bc) is a wide group of Gram-positive and spore-forming bacteria, known to be the etiological agents of various human infections, primarily food poisoning. The Bc group includes enteropathogenic strains able to germinate in the digestive tract and to produce enterotoxins such as Nhe, Hbl, and CytK. One species of the group, Bacillus thuringiensis (Bt), has the unique feature of producing insecticidal crystals during sporulation, making it an important alternative to chemical pesticides to protect crops from insect pest larvae. Nevertheless, several studies have suggested a link between the ingestion of pesticide strains and human cases of food poisoning, calling their safety into question. Consequently, reliable tools for virulence assessment are worth developing to aid decision making in pesticide regulation. Here, we propose complementary approaches based on two biological models, the human intestinal Caco-2 cell line and the insect Drosophila melanogaster, to assess and rank the enteric virulence potency of Bt strains in comparison with other Bc group members. Using a dataset of 48 Bacillus spp. strains, we showed that some Bc group strains, including Bt, were able to induce cytotoxicity in Caco-2 cells with concomitant release of IL-8 cytokine, a landmark of pro-inflammatory response. In the D. melanogaster model, we were able to sort a panel of 39 strains into four different classes of virulence, ranging from no virulence to strong virulence. Importantly, for the most virulent strains, mortality was associated with a loss of intestinal barrier integrity. Interestingly, although strains can share a common toxinotype, they display different degrees of virulence, suggesting the existence of specific mechanisms of virulence expression in vivo in the intestine.

Peptidoglycan maturation enzymes affect flagellar functionality in bacteria.

The flagellar machinery is a highly complex organelle composed of a free rotating flagellum and a fixed stator that converts energy into movement. The assembly of the flagella and the stator requires interactions with the peptidoglycan layer through which the organelle has to pass for externalization. Lytic transglycosylases are peptidoglycan degrading enzymes that cleave the sugar backbone of peptidoglycan layer. We show that an endogenous lytic transglycosylase is required for full motility of Helicobacter pylori and colonization of the gastric mucosa. Deficiency of motility resulted from a paralysed phenotype implying an altered ability to generate flagellar rotation. Similarly, another Gram-negative pathogen Salmonella typhimurium and the Gram-positive pathogen Listeria monocytogenes required the activity of lytic transglycosylases, Slt or MltC, and a glucosaminidase (Auto), respectively, for full motility. Furthermore, we show that in absence of the appropriate lytic transglycosylase, the flagellar motor protein MotB from H. pylori does not localize properly to the bacterial pole. We present a new model involving the maturation of the surrounding peptidoglycan for the proper anchoring and functionality of the flagellar motor.

Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila.

Bacillus thuringiensis subsp. kurstaki (Btk) is a strong pathogen toward lepidopteran larvae thanks to specific Cry toxins causing leaky gut phenotypes. Hence, Btk and its toxins are used worldwide as microbial insecticide and in genetically modified crops, respectively, to fight crop pests. However, Btk belongs to the B. cereus group, some strains of which are well known human opportunistic pathogens. Therefore, ingestion of Btk along with food may threaten organisms not susceptible to Btk infection. Here we show that Cry1A toxins induce enterocyte death and intestinal stem cell (ISC) proliferation in the midgut of Drosophila melanogaster, an organism non-susceptible to Btk. Surprisingly, a high proportion of the ISC daughter cells differentiate into enteroendocrine cells instead of their initial enterocyte destiny. We show that Cry1A toxins weaken the E-Cadherin-dependent adherens junction between the ISC and its immediate daughter progenitor, leading the latter to adopt an enteroendocrine fate. Hence, although not lethal to non-susceptible organisms, Cry toxins can interfere with conserved cell adhesion mechanisms, thereby disrupting intestinal homeostasis and endocrine functions.

Identification of Genetic Markers for the Detection of Bacillus thuringiensis Strains of Interest for Food Safety.

Bacillus thuringiensis (Bt), belonging to the Bacillus cereus (Bc) group, is commonly used as a biopesticide worldwide due to its ability to produce insecticidal crystals during sporulation. The use of Bt, especially subspecies aizawai and kurstaki, to control pests such as Lepidoptera, generally involves spraying mixtures containing spores and crystals on crops intended for human consumption. Recent studies have suggested that the consumption of commercial Bt strains may be responsible for foodborne outbreaks (FBOs). However, its genetic proximity to Bc strains has hindered the development of routine tests to discriminate Bt from other Bc, especially Bacillus cereus sensu stricto (Bc ss), well known for its involvement in FBOs. Here, to develop tools for the detection and the discrimination of Bt in food, we carried out a genome-wide association study (GWAS) on 286 complete genomes of Bc group strains to identify and validate in silico new molecular markers specific to different Bt subtypes. The analyses led to the determination and the in silico validation of 128 molecular markers specific to Bt, its subspecies aizawai, kurstaki and four previously described proximity clusters associated with these subspecies. We developed a command line tool based on a 14-marker workflow, to carry out a computational search for Bt-related markers from a putative Bc genome, thereby facilitating the detection of Bt of interest for food safety, especially in the context of FBOs.

A M23B family metallopeptidase of Helicobacter pylori required for cell shape, pole formation and virulence.

The molecular basis of the regulation of specific shapes and their role for the bacterial fitness remain largely unknown. We focused in this study on the Gram-negative and spiral-shaped Helicobacter pylori. To colonize its unique niche, H. pylori needs to reach quickly the human gastric mucosa, by swimming to and through the mucus layer. For that reason, the specific shape of H. pylori is predicted to be necessary for optimal motility in vivo, and consequently for its colonization ability. Here, we describe the involvement of a PG-modifying enzyme, HdpA (HP0506), in the mouse colonization ability of this bacterium, by regulating its shape. Indeed, the inactivation of the hp0506 gene led to a stocky and branched phenotype, affecting H. pylori colonization capacity despite a normal motility phenotype in vitro. In contrast, the overexpression of the hp0506 gene induced the transformation of H. pylori from rod to dividing cocci shaped bacteria. Furthermore, we demonstrated by PG analysis and enzymology, that HdpA carried both d,d-carboxypeptidase and d,d-endopeptidase activities. Thus, HdpA is the first enzyme belonging to the M23-peptidase family able to perform the d,d-carboxypeptidation and regulate cell shape.

Comparative phenotypic, genotypic and genomic analyses of Bacillus thuringiensis associated with foodborne outbreaks in France.

Bacillus thuringiensis (Bt) belongs to the Bacillus cereus (Bc) group, well known as an etiological agent of foodborne outbreaks (FBOs). Bt distinguishes itself from other Bc by its ability to synthesize insecticidal crystals. However, the search for these crystals is not routinely performed in food safety or clinical investigation, and the actual involvement of Bt in the occurrence of FBOs is not known. In the present study, we reveal that Bt was detected in the context of 49 FBOs declared in France between 2007 and 2017. In 19 of these FBOs, Bt was the only microorganism detected, making it the most likely causal agent. Searching for its putative origin of contamination, we noticed that more than 50% of Bt isolates were collected from dishes containing raw vegetables, in particular tomatoes (48%). Moreover, the genomic characterization of isolates showed that most FBO-associated Bt isolates exhibited a quantified genomic proximity to Bt strains, used as biopesticides, especially those from subspecies aizawai and kurstaki. Taken together, these results strengthen the hypothesis of an agricultural origin for the Bt contamination and call for further investigations on Bt pesticides.

Crosstalk between Helicobacter pylori and gastric epithelial cells is impaired by docosahexaenoic acid.

H. pylori colonizes half of the world's population leading to gastritis, ulcers and gastric cancer. H. pylori strains resistant to antibiotics are increasing which raises the need for alternative therapeutic approaches. Docosahexaenoic acid (DHA) has been shown to decrease H. pylori growth and its associated-inflammation through mechanisms poorly characterized. We aimed to explore DHA action on H. pylori-mediated inflammation and adhesion to gastric epithelial cells (AGS) and also to identify bacterial structures affected by DHA. H. pylori growth and metabolism was assessed in liquid cultures. Bacterial adhesion to AGS cells was visualized by transmission electron microscopy and quantified by an Enzyme Linked Immunosorbent Assay. Inflammatory proteins were assessed by immunoblotting in infected AGS cells, previously treated with DHA. Bacterial total and outer membrane protein composition was analyzed by 2-dimensional gel electrophoresis. Concentrations of 100 µM of DHA decreased H. pylori growth, whereas concentrations higher than 250 µM irreversibly inhibited bacteria survival. DHA reduced ATP production and adhesion to AGS cells. AGS cells infected with DHA pre-treated H. pylori showed a 3-fold reduction in Interleukin-8 (IL-8) production and a decrease of COX2 and iNOS. 2D electrophoresis analysis revealed that DHA changed the expression of H. pylori outer membrane proteins associated with stress response and metabolism and modified bacterial lipopolysaccharide phenotype. As conclusions our results show that DHA anti-H. pylori effects are associated with changes of bacteria morphology and metabolism, and with alteration of outer membrane proteins composition, that ultimately reduce the adhesion of bacteria and the burden of H. pylori-related inflammation.

The effect of bulgecin A on peptidoglycan metabolism and physiology of Helicobacter pylori.

Helicobacter pylori, a human-specific bacterial pathogen responsible for severe gastric diseases, constitutes a major public health issue. In the last decade, rates of H. pylori resistance to antibiotics were increasing drastically, requiring alternative therapeutic strategies to deal with eradication failures. Therefore, we evaluated the potential of bulgecin A, a glycosidic inhibitor of the lytic transglycosylase (LTG) Slt70 of Escherichia coli, as a new therapeutic approach against the H. pylori infection. In this study, we show that bulgecin A is able to specifically inactivate the H. pylori LTG Slt, but not its ortholog MltD. Moreover, bulgecin A synergized with amoxicillin, an inhibitor of penicillin binding proteins, inducing strong morphological alterations, cellular damages, and cell death. Similarly, the simultaneous inactivation of the peptidoglycan (PG) peptidase HdpA and Slt led to inhibition of H. pylori growth, highlighting the strong potential of targeting the PG biosynthetic pathway at different biochemical steps to enhance our therapeutic approaches against bacteria. Hence, we propose that bulgecin A constitutes an attractive compound for the development of new therapeutic strategies against H. pylori combined with other inhibitors of PG biosynthetic enzymes.