Search

International audience; The disruption of gut microbiota homeostasis has been associated with numerous diseases and with a disproportionate inflammatory response, including overproduction of nitric oxide (NO) in the intestinal lumen. However, the influence of NO on the human gut microbiota has not been well characterized yet. We used in vitro fermentation systems inoculated with human fecal samples to monitor the effect of repetitive NO pulses on the gut microbiota. NO exposure increased the redox potential and modified the fermentation profile and gas production. The overall metabolome was modified, reflecting less strict anaerobic conditions and shifts in amino acid and nitrogen metabolism. NO exposure led to a microbial shift in diversity with a decrease in Clostridium leptum group and Faecalibacterium prausnitzii biomass and an increased abundance of the Dialister genus. Escherichia coli, Enterococcus faecalis, and Proteus mirabilis operational taxonomic unit abundance increased, and strains from those species isolated after NO stress showed resistance to high NO concentrations. As a whole, NO quickly changed microbial fermentations, functions, and composition in a pulse-and dose-dependent manner. NO could shift, over time, the trophic chain to conditions that are unfavorable for strict anaerobic microbial processes, implying that a prolonged or uncontrolled inflammation has detrimental and irreversible consequences on the human microbiome.IMPORTANCE Gut microbiota dysbiosis has been associated with inflammatory diseases. The human inflammatory response leads to an overproduction of nitric oxide (NO) in the gut. However, so far, the influence of NO on the human gut microbiota has not been characterized. In this study, we used in vitro fermentation systems with human fecal samples to understand the effect of NO on the microbiota: NO modified the microbial composition and its functionality. High NO concentration depleted the microbiota of beneficial butyrate-producing species and favored potentially deleterious species (E. coli, E. faecalis, and P. mirabilis), which we showed can sustain high NO concentrations. Our work shows that NO may participate in the vicious circle of inflammation, leading to detrimental and irreversible consequences on human health.

Nitric oxide (NO) is present in various organisms from humans, to plants, fungus and bacteria. NO is a fundamental signaling molecule implicated in major cellular functions. The role of NO ranges from an essential molecule to a potent mediator of cellular damages. The ability of NO to react with a broad range of biomolecules allows on one hand its regulation and a gradient concentration and on the other hand to exert physiological as well as pathological functions. In humans, NO is implicated in cardiovascular homeostasis, neurotransmission and immunity. However, NO can also contribute to cardiovascular diseases (CVDs) or septic shock. For certain denitrifying bacteria, NO is part of their metabolism as a required intermediate of the nitrogen cycle. However, for other bacteria, NO is toxic and harmful. To survive, those bacteria have developed processes to resist this toxic effect and persist inside their host. NO also contributes to maintain the host/microbiota homeostasis. But little is known about the impact of NO produced during prolonged inflammation on microbiota integrity, and some pathogenic bacteria take advantage of the NO response to colonize the gut over the microbiota. Taken together, depending on the environmental context (prolonged production, gradient concentration, presence of partners for interaction, presence of oxygen, etc.), NO will exert its beneficial or detrimental function. In this review, we highlight the dual role of NO for humans, pathogenic bacteria and microbiota, and the mechanisms used by each organism to produce, use or resist NO.

International audience; Bacterial response to nitric oxide (NO) is of major importance for bacterial survival. NO stress is a main actor of the eukaryotic immune response and several pathogenic bacteria have developed means for detoxification and repair of the damages caused by NO. However, bacterial mechanisms of NO resistance by Gram-positive bacteria are poorly described. In the opportunistic foodborne pathogen Bacillus cereus, genome sequence analyses did not identify homologs to known NO reductases and transcriptional regulators, such as NsrR, which orchestrate the response to NO of other pathogenic or non-pathogenic bacteria. Using a transcriptomic approach, we investigated the adaptation of B. cereus to NO stress. A cluster of 6 genes was identified to be strongly up-regulated in the early phase of the response. This cluster contains an iron-sulfur cluster repair enzyme, a nitrite reductase and three enzymes involved in siroheme biosynthesis. The expression pattern and close genetic localization suggest a functional link between these genes, which may play a pivotal role in the resistance of B. cereus to NO stress during infection.

The authors thank the Centre Technologique des Microstructures of the Lyon 1 University for providing access to its TEM facilities, and the MIMA2 platform Jouy en Josas for access to electron microscopy equipment (MIMA2, INRAE, 2018. Microscopy and Imaging Facility for Microbes, Animals and Foods, https://doi.org/10.15454/1. 5572348210007727E12). JV thanks Maria-Vesna Nikolic (University of Belgrade, Serbia) for English editing.; International audience; Paper-based DNA biosensors are powerful tools in point-of-care diagnostics since they are affordable, portable, user-friendly, rapid and robust. However, their sensitivity is not always as high as required to enable DNA quantification. To improve the response of standard dot blots, we have applied a new enhancement strategy that increases the sensitivity of assays based on the use of biotinylated silica-nanoparticles (biotin-Si-NPs). After immobilization of a genomic Campylobacter DNA onto a paper membrane, and addition of a biotinylated-DNA detection probe, hybridization was evidenced using streptavidin-conjugated to horseradish peroxidase (HRP) in the presence of luminol and H2O2. Replacement of the single biotin by the biotin-Si-NPs boosted on average a 30 fold chemiluminescent read-out of the biosensor. Characterization of biotin-Si-NPs onto a paper with immobilized DNA was done using a scanning electron microscope. A limit of detection of 3 pg/μL of DNA, similar to the available qPCR kits, is achieved, but it is cheaper, easier and avoids inhibition of DNA polymerase by molecules from the food matrices. We demonstrated that the new dot blot coupled to biotin-Si-NPs successfully detected Campylobacter from naturally contaminated chicken meat, without needing a PCR step. Hence, such an enhanced dot blot paves the path to the development of a portable and multiplex paper based platform for point-of-care screening of chicken carcasses for Campylobacter.

https://doi.org/10.1016/j.fm.2021.103759 B. cereusis a human pathogen associated withfood poisoningleading to gastrointestinal disorders, as well as local and severe systemic infections. The pathogenic spectrum ofB. cereusranges from strains used as probiotics in humans to lethal highly toxic strains. In this study, we gathered a collection of 100 strains representative of the pathological diversity ofB. cereusin humans, and characterized these strains for their cytotoxic potential towards human cells. We analyzed the correlation between cytotoxicity to epithelial and macrophage cells and the combination of 10 genes suspected to play a role duringB. cereusvirulence. We highlight genetic differences among isolates and studied correlations between genetic signature, cytotoxicity and strain pathological status. We hope that our findings will improve our understanding of thepathogenicityofB. cereus,thereby making it possible to improve both clinical diagnosis and food safety.

Paper-based DNA biosensors are powerful tools in point-of-care diagnostics since they are affordable, portable, user-friendly, rapid and robust. However, their sensitivity is not always as high as required to enable DNA quantification. To improve the response of standard dot blots, we have applied a new enhancement strategy that increases the sensitivity of assays based on the use of biotinylated silica-nanoparticles (biotin-Si-NPs). After immobilization of a genomic Campylobacter DNA onto a paper membrane, and addition of a biotinylated-DNA detection probe, hybridization was evidenced using streptavidin-conjugated to horseradish peroxidase (HRP) in the presence of luminol and H2O2. Replacement of the single biotin by the biotin-Si-NPs boosted on average a 30 fold chemiluminescent read-out of the biosensor. Characterization of biotin-Si-NPs onto a paper with immobilized DNA was done using a scanning electron microscope. A limit of detection of 3 pg/μL of DNA, similar to the available qPCR kits, is achieved, but it is cheaper, easier and avoids inhibition of DNA polymerase by molecules from the food matrices. We demonstrated that the new dot blot coupled to biotin-Si-NPs successfully detected Campylobacter from naturally contaminated chicken meat, without needing a PCR step. Hence, such an enhanced dot blot paves the path to the development of a portable and multiplex paper based platform for point-of-care screening of chicken carcasses for Campylobacter.Graphical abstract

Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians.

The &lsquo, One Health&rsquo, approach recommended by WHO recognizes the inseparable link between human, animal and environmental health [...]

Production of reactive nitrogen species is an important component of the host immune defence against bacteria. Here, we show that the bacterial protein Mfd (Mutation frequency decline), a highly conserved and ubiquitous bacterial protein involved in DNA repair, confers bacterial resistance to the eukaryotic nitrogen response produced by macrophage cells and during mice infection. In addition, we show that RecBC is also necessary to survive this stress. The inactivation of recBC and mfd genes is epistatic showing that Mfd follows the RecBC repair pathway to protect the bacteria against the genotoxic effect of nitrite. Surprisingly given the role of Mfd in transcription-coupled repair, UvrA is not necessary to survive the nitrite response. Taken together, our data reveal that during the eukaryotic nitrogen response, Mfd is required to maintain bacterial genome integrity in a NER-independent but RecBC-dependent pathway.

How pathogenic bacteria infect and kill their host is currently widely investigated. In comparison, the fate of pathogens after the death of their host receives less attention. We studied Bacillus thuringiensis (Bt) infection of an insect host, and show that NprR, a quorum sensor, is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes, including many encoding degradative enzymes or proteins involved in the synthesis of a nonribosomal peptide named kurstakin. These degradative enzymes are essential in vitro to degrade several substrates and are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We show that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. We report that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to spore spreading.

The aim of this study was to identify and characterise Bacillus cereus from a unique national collection of 564 strains associated with 140 strong-evidence food-borne outbreaks (FBOs) occurring in France during 2007 to 2014. Starchy food and vegetables were the most frequent food vehicles identified; 747 of 911 human cases occurred in institutional catering contexts. Incubation period was significantly shorter for emetic strains compared with diarrhoeal strains A sub-panel of 149 strains strictly associated to 74 FBOs and selected on Coliphage M13-PCR pattern, was studied for detection of the genes encoding cereulide, diarrhoeic toxins (Nhe, Hbl, CytK1 and CytK2) and haemolysin (HlyII), as well as panC phylogenetic classification. This clustered the strains into 12 genetic signatures (GSs) highlighting the virulence potential of each strain. GS1 (nhe genes only) and GS2 (nhe, hbl and cytK2), were the most prevalent GS and may have a large impact on human health as they were present in 28% and 31% of FBOs, respectively. Our study provides a convenient molecular scheme for characterisation of B. cereus strains responsible for FBOs in order to improve the monitoring and investigation of B. cereus-induced FBOs, assess emerging clusters and diversity of strains.

Production of reactive nitrogen species (NO) is a key step in the immune response following infections. NO induces lesions to bacterial DNA, thus limiting bacterial growth within hosts. Using two pathogenic bacteria, Bacillus cereus and Shigella flexneri, we show that the DNA-repair protein Mfd (Mutation-Frequency-Decline) is required for bacterial resistance to the host-NO-response. In both species, a mutant deficient for mfd does not survive to NO, produced in vitro or by phagocytic cells. In vivo, the ∆mfd mutant is avirulent and unable to survive the NO-stress. Moreover, NO induces DNA-double-strand-breaks and point mutations in the Δmfd mutant. In overall, these observations demonstrate that NO damages bacterial DNA and that Mfd is required to maintain bacterial genomic integrity. This unexpected discovery reveals that Mfd, a typical housekeeping gene, turns out to be a true virulence factor allowing survival and growth of the pathogen in its host, due to its capacity to protect the bacterium against NO, a key molecule of the innate immune defense. As Mfd is widely conserved in the bacterial kingdom, these data highlight a mechanism that may be used by a large spectrum of bacteria to overcome the host immune response and especially the mutagenic properties of NO.

Shigella, the causative agent of bacillary dysentery, invades epithelial cells. Upon bacterial–cell contact, the type III bacterial effector IpaA binds to the cytoskeletal protein vinculin to promote actin reorganization required for efficient bacterial uptake. We show that the last 74 C-terminal residues of IpaA (A559) bind to human vinculin (HV) and promotes its association with actin filaments. Polymerisation experiments demonstrated that A559 was sufficient to induce HV-dependent partial capping of the barbed ends of actin filaments. These results suggest that IpaA regulates actin polymerisation/depolymerisation at sites of Shigella invasion by modulating the barbed end capping activity of vinculin.

Some bacteria of the Bacillus cereus group are enteropathogens. The first cells encountered by bacteria following oral contamination of the host are epithelial cells. We studied the capacity of these bacteria to adhere to epithelial cells and the consequences of this interaction. We found that cell adhesion is strain dependent and that a strain mutated in flhA, which encodes a component of flagellum-apparatus formation, is impaired in adhesion, suggesting that flagella are important virulence factors. The bacteria are cytotoxic to epithelial cells and induce substantial cytoplasmic and membrane alterations. However, direct contact between cells and bacteria is not required for cytotoxicity. The determinants of this cytotoxicity are secreted and their expression depends on the pleiotropic regulator PlcR. Adhesion and cytotoxicity of B. cereus to epithelial cells might explain the diarrhea caused by these pathogens. Our findings provide further insight into the pathogenicity of B. cereus group members.

The Bacillus cereus group contains seven valid species. The strains are affiliated to species either on the basis of genetic clustering or due to remarkable phenotypes: anthrax illness in animals and human is attributed to B. anthracis, δ-endotoxin synthesis and virulence to insects – B. thuringiensis, rhizoidal growth – B. mycoides, psychrotolerance – B. weihenstephanensis and thermophily – B. cytotoxicus. Recent advances in the group phylogeny identified four separated clades although other classifications also persist. Apart from anthrax the bacteria of this group can cause serious clinical cases, like pulmonary, eye or wound infections. They are also notorious food pathogens causing diarrhoea or vomiting. Emetic isolates usually represent a narrow lineage and synthesize thermostable plasmid-encoded toxin. The grounds of clinical infections or diarrhoea are less obvious although great progress has been made recently to understand the molecular machinery of the food-related pathogenesis. The toxins NHE, HBL and CytK have been rather well characterized. The global regulator PlcR has been intensively studied and unambiguously related to regulation of toxins and multiple virulence factors. Such issues are discussed as the group taxonomy, strain identification, genomic studies, application of integrative biology methods, toxin action mechanisms and their regulation.

Gastric infections by Helicobacter pylori are characteristically associated with an intense inflammation and infiltration of mainly polymorphonuclear lymphocytes (PMNs) and monocytes. The inflammatory response by infiltrated immune cells appears to be a primary cause of the damage to surface epithelial layers and may eventually result in gastritis, peptic ulcer, gastric cancer and/or MALT-associated gastric lymphoma. Our analysis of the interaction between H. pylori and PMNs and monocytes revealed that H. pylori inhibits its own uptake by these professional phagocytes. To some degree, this effect resembles antiphagocytosis by Yersinia enterocolitica. Increasing numbers of bacteria associated per cell are more efficient at blocking their own engulfment. In H. pylori, bacterial protein synthesis is necessary to block phagocytic uptake, as shown by the time and concentration dependence of the bacteriostatic protein synthesis inhibitor chloramphenicol. Furthermore, H. pylori appears broadly to inhibit the phagocytic function of monocytes and PMNs, as infection with H. pylori abrogates the phagocytes' ability to engulf latex beads or adherent Neisseria gonorrhoeae cells. This antiphagocytic phenotype depends on distinct virulence (vir) genes, such as virB7 and virB11, encoding core components of a putative type IV secretion apparatus. Our data indicate that H. pylori exhibits an antiphagocytic activity that may play an essential role in the immune escape of this persistent pathogen.

absent

Background: Bacillus cereus is an environmental pathogen whose spores resist the usual cleaning procedure applied by the food industry and hospitals. We reported a cluster of severe intestinal infections due to B cereus in 2 very low birth weight neonates from a neonatal intensive care unit. Methods: Environmental sampling and bacteriological analysis of pooled breast milk (PBM) were performed. Practices for preparation and administration of milk were observed and additional laboratory experiments performed. Strains were typed using M13-polymerase chain reaction and their virulence tested using cellular and insect models. Results: B cereus strains were exclusively isolated from intestinal tracts and PBM. No environmental culture yielded any viable B cereus. Although genotypically diverse, both clinical and food strains exhibited high virulence potency. These findings pointed out the pathogenic potency of B cereus in neonates; the putative role of PBM as a reservoir for pathogenic B cereus and the triggered effect of a defective care procedure, which allowed the growth of bacteria in pasteurized PBM. Well described from dried formula, the presence of pathogenic B cereus strains in PBM was not previously reported. Conclusions: Careful monitoring of conditions during collection, conservation, and administration of PBM should be implemented in high-risk populations such as premature neonates.

Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. We have previously shown that B. cereus Haemolysin II (HlyII) induces macrophage cell death by apoptosis. In this work, we investigated the regulation of the hlyII gene. We show that HlyIIR, the negative regulator of hlyII expression in B. cereus, is especially active during the early bacterial growth phase. We demonstrate that glucose 6P directly binds to HlyIIR and enhances its activity at a post-transcriptional level. Glucose 6P activates HlyIIR, increasing its capacity to bind to its DNA-box located upstream of the hlyII gene, inhibiting its expression. Thus, hlyII expression is modulated by the availability of glucose. As HlyII induces haemocyte and macrophage death, two cell types that play a role in the sequestration of nutrients upon infection, HlyII may induce host cell death to allow the bacteria to gain access to carbon sources that are essential components for bacterial growth.

The Bacillus cereus pathogenic spectrum ranges from strains used as probiotics to human-lethal strains. However, prediction of the pathogenic potential of a strain remains difficult. Here, we show that food poisoning and clinical strains can be differentiated from harmless strains on the basis of host colonization phenotypes.

Bacillus thuringiensis (Bt) is a spore-forming entomopathogen broadly used in agriculture crop. The haemolysin HlyII is an important Bt virulence factor responsible for insect death. In this work, we focused on the regulation of the hlyII gene throughout the bacterial growth in vitro and in vivo during insect infection. We show that hlyII regulation depends on the global regulator Fur. This regulation occurs independently of HlyIIR, the other known regulator of hlyII gene expression. Moreover, we show that hlyII is highly expressed when iron is depleted in vivo. As HlyII induces haemocyte and macrophage death, which are involved in the sequestration of iron upon infection, HlyII may induce host cell death to allow bacteria to gain access to iron. (C) 2013 Elsevier Inc. All rights reserved.

During bacterial infection, professional phagocytes are attracted to the site of infection, where they constitute a first line of host cell defense. Their function is to engulf and destroy the pathogens. Thus, bacteria must withstand the bactericidal activity of professional phagocytes, including macrophages to counteract the host immune system. Bacillus cereus infections are characterized by bacteremia despite the accumulation of inflammatory cells at the site of infection. This implies that the bacteria have developed means of resisting the host immune system. Bacillus cereus spores survive, germinate, and multiply in contact with macrophages, eventually producing toxins that kill these cells. However, the exact mechanism by which B. cereus evades immune attack remains unclear. This review addresses the interaction between B. cereus and macrophages, highlighting, in particular, the ways in which the bacteria escape the microbicidal activities of professional phagocytes.

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system(1). Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides(2,3). In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals(1). Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections. Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis(4-7). Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model(8-11). Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality. G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 °C to 30 °C) and infection studies can be conducted between 15 °C to above 37 °C(12,13), allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD50(14), measurement of bacterial survival(15,16) and examination of the infection process(17). Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections(18,19).

The Bacillus cereus sensu lato complex has recently been divided into several phylogenetic groups with clear differences in growth temperature range. However, only a few studies have investigated the actual pathogenic potential of the psychrotolerant strains of the B. cereus group at low temperature, and little information is available concerning gene expression at low temperature. We found that vegetative cells of the psychrotolerant B. weihenstephanensis strain KBAB4 were pathogenic against the model insect Galleria mellonella at 15°C but not at 30°C. A similar temperature-dependent difference also was observed for the supernatant, which was cytotoxic to Vero epithelial cell lines and to murine macrophage J774 cells at 15°C but not at 30°C. We therefore determined the effect of low temperature on the production of various proteins putatively involved in virulence using two-dimensional protein gel electrophoresis, and we showed that the production of the Hbl enterotoxin and of two proteases, NprB and NprP2, was greater at a growth temperature of 15°C than at 30°C. The quantification of the mRNA levels for these virulence genes by real-time quantitative PCR at both temperatures showed that there was also more mRNA present at 15°C than at 30°C. We also found that at 15°C, hbl mRNA levels were maximal in the mid- to late exponential growth phase. In conclusion, we found that the higher virulence of the B. cereus KBAB4 strain at low temperature was accompanied by higher levels of the production of various known PlcR-controlled virulence factors and by a higher transcriptional activity of the corresponding genes.

How pathogenic bacteria infect and kill their host is currently widely investigated. In comparison, the fate of pathogens after the death of their host receives less attention. We studied Bacillus thuringiensis (Bt) infection of an insect host, and show that NprR, a quorum sensor, is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes, including many encoding degradative enzymes or proteins involved in the synthesis of a nonribosomal peptide named kurstakin. These degradative enzymes are essential in vitro to degrade several substrates and are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We show that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. We report that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to spore spreading., Author Summary Bacillus thuringiensis (Bt) is a well known entomopathogenic bacterium successfully used as a biopesticide for fifty years. The insecticidal properties of Bt are mainly due to specific toxins forming a crystal inclusion associated with the spore. After ingestion by susceptible insect larvae, toxins could induce favorable conditions for spore germination. The bacteria multiply in the insect and coordinate their behavior using signaling molecules involved in quorum sensing. The activation of the quorum sensor PlcR leads to the production of virulence factors allowing the bacteria to kill the insect host. Here we show that, in the cadaver, Bt shifts from a virulent to a necrotrophic lifestyle during which a second quorum sensor (NprR) becomes functional. NprR activates genes encoding degradative enzymes (proteases, lipases and chitinases) and a lipopeptide (kurstakin) involved in swarming and biofilm formation. The kurstakin is also essential for the survival of Bt after insect death. This suggests that NprR allows the bacteria to survive and eventually to sporulate in the host cadaver, thus improving their ability to disseminate in the environment. Altogether these results show that the pathogenic and necrotrophic lifestyles of Bt are tightly controlled by two quorum-sensing systems acting sequentially during the infection process.

Insects are the largest group of animals on earth. Like mammals, virus, fungi, bacteria and parasites infect them. Several tissue barriers and defense mechanisms are common for vertebrates and invertebrates. Therefore some insects, notably the fly Drosophila and the caterpillar Galleria mellonella, have been used as models to study host-pathogen interactions for several insect and mammal pathogens. They are excellent tools to identify pathogen determinants and host tissue cell responses. We focus here on the comparison of effectors used by two different groups of bacterial insect pathogens to accomplish the infection process in their lepidopteran larval host: Bacillus thuringiensis and the nematode-associated bacteria, Photorhabdus and Xenorhabdus. The comparison reveals similarities in function and expression profiles for some genes, which suggest that such factors are conserved during evolution in order to attack the tissue encountered during the infection process.

To achieve widespread dissemination in the host, Bacillus anthracis cells regulate their attachment to host endothelium during infection. Previous studies identified BslA (Bacillus anthracis S-layer Protein A), a virulence factor of B. anthracis, as necessary and sufficient for adhesion of vegetative cells to human endothelial cells. While some factors have been identified, bacteria-specific contributions to BslA mediated adhesion remain unclear. Using the attenuated vaccine Sterne 7702 strain of B. anthracis, we tested the hypothesis that InhA (immune inhibitor A), a B. anthracis protease, regulates BslA levels affecting the bacteria's ability to bind to endothelium. To test this, a combination of inhA mutant and complementation analysis in adhesion and invasion assays, Western blot and InhA inhibitor assays were employed. Results show InhA downregulates BslA activity reducing B. anthracis adhesion and invasion in human brain endothelial cells. BslA protein levels in ΔinhA bacteria were significantly higher than wild-type and complemented strains showing InhA levels and BslA expression are inversely related. BslA was sensitive to purified InhA degradation in a concentration- and time-dependent manner. Taken together these data support the role of InhA regulation of BslA-mediated vegetative cell adhesion and invasion.

International audience; Hemorrhagic meningitis is a fatal complication of anthrax, but its pathogenesis remains poorly understood. The present study examined the role of B. anthracis-secreted metalloprotease InhA on monolayer integrity and permeability of human brain microvasculature endothelial cells (HBMECs) which constitute the blood-brain barrier (BBB). Treatment of HBMECs with purified InhA resulted in a time-dependent decrease in trans-endothelial electrical resistance (TEER) accompanied by zonula occluden-1 (ZO-1) degradation. An InhA-expressing B. subtilis exhibited increased permeability of HBMECs, which did not occur with the isogenic inhA deletion mutant (Delta inhA) of B. anthracis, compared with the corresponding wild-type strain. Mice intravenously administered with purified InhA or nanoparticles-conjugated to InhA demonstrated a time-dependent Evans Blue dye extravasation, leptomeningeal thickening, leukocyte infiltration, and brain parenchymal distribution of InhA indicating BBB leakage and cerebral hemorrhage. Mice challenged with vegetative bacteria of the Delta inhA strain of B. anthracis exhibited a significant decrease in leptomeningeal thickening compared to the wildtype strain. Cumulatively, these findings indicate that InhA contributes to BBB disruption associated with anthrax meningitis through proteolytic attack on the endothelial tight junctional protein zonula occluden (ZO)-1.

Trypan blue is a dye that has been widely used for selective staining of dead tissues or cells. Here, we show that the pore-forming toxin HlyII of Bacillus cereus allows trypan blue staining of macrophage cells, despite the cells remaining viable and metabolically active. These findings suggest that the dye enters viable cells through the pores. To our knowledge, this is the first demonstration that trypan blue may enter viable cells. Consequently, the use of trypan blue staining as a marker of vital status should be interpreted with caution. The blue coloration does not necessarily indicate cell lysis, but may rather indicate pore formation in the cell membranes and more generally increased membrane permeability.

Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recr ...

Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. The precise mechanisms and the bacterial factors allowing B. cereus to circumvent host immune responses remain to be elucidated. We have previously shown that B. cereus induces macrophage cell death by an unknown mechanism. Here we identified the toxic component from the B. cereus supernatant. We report that Haemolysin II (HlyII) provokes macrophage cell death by apoptosis through its pore-forming activity. The HlyII-induced apoptotic pathway is caspase 3 and 8 dependent, thus most likely mediated by the death receptor pathway. Using insects and mice as in vivo models, we show that deletion of hlyII strongly reduces virulence. In addition, we show that after infection of Bombyx mori larvae, the immune cells are apoptotic, demonstrating that HlyII induces apoptosis of phagocytic cells in vivo. Altogether, our results clearly unravel HlyII as a novel virulence protein that induces apoptosis in phagocytic cells in vitro and in vivo.

Bacillus cereus EntFM displays an NlpC/P60 domain, characteristic of cell wall peptidases. The protein is involved in bacterial shape, motility, adhesion to epithelial cells, biofilm formation, vacuolization of macrophages, and virulence. These data provide new information on this, so far, poorly studied toxin and suggest that this protein is a cell wall peptidase, which we propose to rename CwpFM.