RESUMO
Bacillus cereus is a spore forming bacteria recognized among the leading agents responsible for foodborne outbreaks in Europe. B. cereus is also gaining notoriety as an opportunistic human pathogen inducing local and systemic infections. The real incidence of such infection is likely underestimated and information on genetic and phenotypic characteristics of the incriminated strains is generally scarce. We have recently analyzed a large strain collection of varying pathogenic potential. Screening for biomarkers to differentiate among clinical and non-clinical strains, a gene encoding an alcohol dehydrogenase-like protein was identified among the leading candidates. This family of proteins has been demonstrated to be involved in the virulence of several bacterial species. The relevant gene was knocked out to elucidate its function with regards to resistance to host innate immune response, both in vitro and in vivo. Our results demonstrate that the adhB gene plays a significant role in resistance to nitric oxide and oxidative stress in vitro, as well as its pathogenic ability with regards to in vivo toxicity. These properties may explain the pathogenic potential of strains carrying this newly identified virulence factor.
Assuntos
Álcool Desidrogenase/metabolismo , Bacillus cereus/patogenicidade , Proteínas de Bactérias/metabolismo , Biomarcadores/metabolismo , Imunidade Inata/fisiologia , Virulência/genética , Álcool Desidrogenase/genética , Animais , Bacillus cereus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Peróxido de Hidrogênio/farmacologia , Insetos/crescimento & desenvolvimento , Insetos/microbiologia , Larva/imunologia , Larva/microbiologia , Mutação , Óxido Nítrico/farmacologia , Estresse Oxidativo/efeitos dos fármacosRESUMO
OBJECTIVES: Bacillus cereus is responsible for food poisoning and rare but severe clinical infections. The pathogenicity of strains varies from harmless to lethal strains. However, there are currently no markers, either alone or in combination, to differentiate pathogenic from non-pathogenic strains. The objective of the study was to identify new genetic biomarkers to differentiate non-pathogenic from clinically relevant B. cereus strains. METHODS: A first set of 15 B. cereus strains were compared by RNAseq. A logistic regression model with lasso penalty was applied to define combination of genes whose expression was associated with strain pathogenicity. The identified markers were checked for their presence/absence in a collection of 95 B. cereus strains with varying pathogenic potential (food-borne outbreaks, clinical and non-pathogenic). Receiver operating characteristic area under the curve (AUC) analysis was used to determine the combination of biomarkers, which best differentiate between the "disease" versus "non-disease" groups. RESULTS: Seven genes were identified during the RNAseq analysis with a prediction to differentiate between pathogenic and non-pathogenic strains. The validation of the presence/absence of these genes in a larger collection of strains coupled with AUC prediction showed that a combination of four biomarkers was sufficient to accurately discern clinical strains from harmless strains, with an AUC of 0.955, sensitivity of 0.9 and specificity of 0.86. CONCLUSIONS: These new findings help in the understanding of B. cereus pathogenic potential and complexity and may provide tools for a better assessment of the risks associated with B. cereus contamination to improve patient health and food safety.
Assuntos
Bacillus cereus , Microbiologia de Alimentos , Marcadores Genéticos , Bacillus cereus/genética , Bacillus cereus/isolamento & purificação , Filogenia , RNA-Seq , VirulênciaRESUMO
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.
Assuntos
Bacillus cereus/metabolismo , Proteínas de Bactérias/metabolismo , Heme/análogos & derivados , Ferro/metabolismo , Óxido Nítrico/toxicidade , Nitrito Redutases/metabolismo , Estresse Oxidativo , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/genética , Bacillus cereus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Heme/biossíntese , Transcrição GênicaRESUMO
B. cereus is a human pathogen associated with food poisoning leading to gastrointestinal disorders, as well as local and severe systemic infections. The pathogenic spectrum of B. cereus ranges 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 of B. cereus in 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 during B. cereus virulence. 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 the pathogenicity of B. cereus, thereby making it possible to improve both clinical diagnosis and food safety.
Assuntos
Bacillus cereus/patogenicidade , Doenças Transmitidas por Alimentos/microbiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Animais , Bacillus cereus/classificação , Bacillus cereus/genética , Bacillus cereus/isolamento & purificação , Linhagem Celular , Células Epiteliais/microbiologia , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Humanos , Macrófagos/microbiologia , Filogenia , VirulênciaRESUMO
OBJECTIVES: Bacillus cereus is responsible for food poisoning and rare but severe clinical infections. The pathogenicity of B. cereus strains varies from harmless to lethal strains. The objective of this study was to characterize three B. cereus isolates isolated from the same patient and identify their virulence potentials. METHODS: Three isolates of B. cereus were isolated from various blood samples from a patient who developed sepsis following a central venous catheter infection. The three isolates were compared by WGS, genotyping and SNP analysis. Furthermore, the isolates were compared by phenotypical analysis including bacterial growth, morphology, germination efficacy, toxin production, antibiotic susceptibility and virulence in an insect model of infection. RESULTS: According to WGS and genotyping, the 3 isolates were shown to be identical strains. However, the last recovered strain had lost the mega pAH187_270 plasmid. This last strain showed different phenotypes compared to the first isolated strain, such as germination delay, different antibiotic susceptibility and a decreased virulence capacity towards insects. A 50- kbp region of pAH187_270 plasmid was involved in the virulence potential and could thus be defined as a new pathogenicity island of B. cereus. CONCLUSIONS: These new findings help in the understanding of B. cereus pathogenic potential and complexity and provide further hints into the role of large plasmids in the virulence of B. cereus strains. This may provide tools for a better assessment of the risks associated with B. cereus hospital contamination to improve hygiene procedure and patient health.
Assuntos
Bacillus cereus , Doenças Transmitidas por Alimentos , Bacillus cereus/genética , Doenças Transmitidas por Alimentos/microbiologia , Ilhas Genômicas , Humanos , Plasmídeos/genética , Virulência/genéticaRESUMO
The commensal bacterium Enterococcus faecalis is a common cause of nosocomial infections worldwide. The increasing prevalence of multi-antibiotic resistant E. faecalis strains reinforces this public health concern. Despite numerous studies highlighting several pathology-related genetic traits, the molecular mechanisms of E. faecalis virulence remain poorly understood. In this work, we studied 23 bacterial proteins that could be considered as virulence factors or involved in the Enterococcus interaction with the host. We systematically tested their interactions with human proteins using the Human ORFeome library, a set of 12,212 human ORFs, in yeast. Among the thousands of tested interactions, one involving the E. faecalis virulence factor ElrA and the human protein FHL2 was evidenced by yeast two-hybrid and biochemically confirmed. Further molecular characterizations allowed defining an FHL2-interacting domain (FID) of ElrA. Deletion of the FID led to an attenuated in vivo phenotype of the mutated strain clearly indicating that this interaction is likely to contribute to the multifactorial virulence of this opportunistic pathogen. Altogether, our results show that FHL2 is the first host cellular protein directly targeted by an E. faecalis virulence factor and that this interaction is involved in Enterococcus pathogenicity.
Assuntos
Proteínas de Bactérias/metabolismo , Enterococcus faecalis/fisiologia , Infecções por Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Interações Hospedeiro-Patógeno , Proteínas com Homeodomínio LIM/metabolismo , Proteínas Musculares/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Linhagem Celular , Enterococcus faecalis/patogenicidade , Feminino , Humanos , Proteínas com Homeodomínio LIM/química , Camundongos , Proteínas Musculares/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/química , Fatores de Virulência/química , Fatores de Virulência/genéticaRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Exodesoxirribonuclease V/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Nitrogênio/farmacologia , Fatores de Transcrição/metabolismo , Animais , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/genética , Bacillus cereus/fisiologia , Proteínas de Bactérias/genética , Células HeLa , Humanos , Camundongos , Viabilidade Microbiana , Mutação , Óxido Nítrico/metabolismo , Células RAW 264.7 , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Lactobacillus delbrueckii subsp. lactis CNRZ327 is a dairy bacterium with anti-inflammatory properties both in vitro and in vivo. Here, we report the genome sequence of this bacterium, which appears to contain no less than 215 insertion sequence (IS) elements, an exceptionally high number regarding the small genome size of the strain.
RESUMO
"Candidatus Arthromitus" sp. strain SFB-mouse-NL (SFB, segmented filamentous bacteria) is a commensal bacterium necessary for inducing the postnatal maturation of homeostatic innate and adaptive immune responses in the mouse gut. Here, we report the genome sequence of this bacterium, which sets it apart from earlier sequenced mouse SFB isolates.
RESUMO
BACKGROUND: Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus are lactic acid producing bacteria that are largely used in dairy industries, notably in cheese-making and yogurt production. An earlier in-depth study of the first completely sequenced ssp. bulgaricus genome revealed the characteristics of a genome in an active phase of rapid evolution, in what appears to be an adaptation to the milk environment. Here we examine for the first time if the same conclusions apply to the ssp. lactis, and discuss intra- and inter-subspecies genomic diversity in the context of evolutionary adaptation. RESULTS: Both L. delbrueckii ssp. show the signs of reductive evolution through the elimination of superfluous genes, thereby limiting their carbohydrate metabolic capacities and amino acid biosynthesis potential. In the ssp. lactis this reductive evolution has gone less far than in the ssp. bulgaricus. Consequently, the ssp. lactis retained more extended carbohydrate metabolizing capabilities than the ssp. bulgaricus but, due to high intra-subspecies diversity, very few carbohydrate substrates, if any, allow a reliable distinction of the two ssp. We further show that one of the most important traits, lactose fermentation, of one of the economically most important dairy bacteria, L. delbruecki ssp. bulgaricus, relies on horizontally acquired rather than deep ancestral genes. In this sense this bacterium may thus be regarded as a natural GMO avant la lettre. CONCLUSIONS: The dairy lactic acid producing bacteria L. delbrueckii ssp. lactis and ssp. bulgaricus appear to represent different points on the same evolutionary track of adaptation to the milk environment through the loss of superfluous functions and the acquisition of functions that allow an optimized utilization of milk resources, where the ssp. bulgaricus has progressed further away from the common ancestor.
Assuntos
Evolução Molecular , Genes Bacterianos , Lactobacillus delbrueckii/genética , Aminoácidos/biossíntese , Proteínas de Bactérias/genética , Metabolismo dos Carboidratos , Fermentação , Transferência Genética Horizontal , Genoma Bacteriano , Tipagem de Sequências Multilocus , Proteoma/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli-B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/biossíntese , Clonagem Molecular/métodos , Proteínas de Membrana/biossíntese , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Flagelina/biossíntese , Flagelina/imunologia , Flagelina/metabolismo , Expressão Gênica , Células HT29 , Humanos , Imunidade Celular , Proteínas de Membrana/genética , Metagenômica , Plasmídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/genéticaRESUMO
Our knowledge of species and functional composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about variation across the world. By combining 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets, here we identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific. We also confirmed the enterotypes in two published, larger cohorts, indicating that intestinal microbiota variation is generally stratified, not continuous. This indicates further the existence of a limited number of well-balanced host-microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis to understand microbial communities. Although individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers.
Assuntos
Bactérias/classificação , Intestinos/microbiologia , Metagenoma , Bactérias/genética , Técnicas de Tipagem Bacteriana , Biodiversidade , Biomarcadores/análise , Europa (Continente) , Fezes/microbiologia , Feminino , Humanos , Masculino , Metagenômica , FilogeniaRESUMO
Natural competence is active in very diverse species of the bacterial kingdom and probably participates in horizontal gene transfer. Recently, the genome sequence of various species, including Lactococcus lactis, revealed the presence of homologues of competence genes in bacteria, which were not previously identified as naturally transformable. We investigated the conservation among lactococcal strains of key components of the natural competence process in streptococci: (i) comX which encodes a sigma factor, allowing the expression of the late competence genes involved in DNA uptake, (ii) its recognition site, the cin-box and (iii) dprA which encodes a protein shown to determine the fate of incoming DNA. The comX and dprA genes and the cin-box appeared conserved among strains, although some L. lactis ssp. lactis strains presented an inactivated dprA gene. We established that ComX controls the expression of the late competence genes in L. lactis. In conclusion, our work strongly suggests that ComX has the same role in streptococci and L. lactis, i.e. the regulation of late competence genes. It also allowed the identification of a set of L. lactis strains and the construction of a comX overexpression system, which should facilitate the investigation of the natural competence activity in lactococci.
Assuntos
Proteínas de Bactérias , Sequência Conservada , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/classificação , Lactococcus lactis/genética , Fator sigma , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Variação Genética , Lactococcus lactis/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Análise de Sequência de DNA , Fator sigma/química , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The two csp-like genes from the lactic acid bacterium Lactobacillus delbrueckii ssp. bulgaricus were characterized and designated cspA and cspB. The gene cspA has been identified using a polymerase chain reaction (PCR)-based approach with degenerated primers and further characterized using an inverse PCR strategy. cspA encodes a protein of 65 amino acid residues which displays between 81 and 77% identity with proteins CspL and CspP of Lactobacillus plantarum. cspB has been identified as a cspA ortholog using the partial sequence of the L. bulgaricus ATCC11842. cspB encodes a protein of 69 amino acids which has 42% identity with CspA. Northern blot analyses showed that cspA is transcribed as a single gene and that its transcription increased after a temperature downshift from 42 to 25 degrees C. In contrast, cspB is part of an operon transcribed at constant level irrespective of the temperature. These results indicate that cspA encodes the only Csp-like protein of L. bulgaricus induced by a downshift of temperature.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Temperatura Baixa , Genes Bacterianos , Proteínas de Choque Térmico/genética , Lactobacillus/genética , Lactobacillus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Bacteriano/análise , Homologia de Sequência de Aminoácidos , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
Transcription of a new catabolic operon in Bacillus subtilis, involved in the late stages of galacturonic acid utilization, has been studied. The operon consists of four genes: kdgR, encoding the putative regulator protein; kdgK, encoding 2-keto-3-deoxygluconate kinase; kdgA, encoding 2-keto-3-deoxygluconate-6-phosphate aldolase; and kdgT, encoding a transporter. These four genes are organized in one transcriptional unit and map at 198 degrees of the B. subtilis chromosome. Primer extension experiments and Northern blot analysis show that an active sigmaA-dependent promoter precedes kdgR and transcription is terminated at the putative p-independent terminator downstream of kdgT. The operon is negatively regulated by the kdgR and ccpA gene products, which belong to the LacI family of transcription regulators. The expression of the genes in this operon can be induced by galacturonate and strongly repressed when glucose is present in the growth medium. Knockout mutations in genes kdgR and ccpA remove, respectively, the effects of galacturonate and glucose on the transcription of this operon.