Characterization of a small PlcR-regulated gene co-expressed with cereolysin O

<p>Abstract</p> <p>Background</p> <p>In the human pathogen <it>Bacillus cereus</it>, the expression of most extracellular virulence factors is controlled by the transcriptional activator PlcR. Among these virulence factors, cereolysin O (Clo) is an haemolysin belonging to the cholesterol-dependant cytolysins, a protein family extensively studied in Gram-positive bacteria.</p> <p>Results</p> <p>In the genomes of bacteria belonging to the <it>B. cereus </it>group, including <it>Bacillus anthracis </it>and <it>Bacillus thuringiensis</it>, a small gene encoding a 26-amino acid peptide was present in multicopy. One copy was always found upstream from the gene encoding Clo. In <it>B. cereus </it>ATCC 14579, the small gene and the <it>clo </it>gene are co-transcribed. Transcriptional fusions showed that the three paralogues identified in this strain were expressed in a PlcR-dependent manner. We propose to name these peptides Spp for small PlcR-regulated peptides. We show that a synthetic peptide corresponding to the deduced product of the <it>spp </it>genes displayed antibacterial activity.</p> <p>Conclusion</p> <p>The co-expression of <it>spp</it>, a small PlcR-regulated multicopy gene with <it>clo </it>suggests a yet unidentified relationship between Spp and the cholesterol-dependent cytolysin in bacteria belonging to the <it>B.cereus </it>group.</p

Whole Genome DNA Methylation (Methylome) Analysis of an Entomopathogenic Bacterium

Background: DNA methylation is an epigenetic mechanism involved in the pathogenicity of several major bacterial pathogens. It can decrease the affinity of some transcriptional regulators to their binding site, leading to sub-populations expressing or not various genes, depending on the DNA methylation state. Dam DNA methyltransferase is widespread in Gammaproteobacteria and methylates the adenine of GATC sites. Objectives: The role of Dam was investigated in Photorhabdus luminescens during its symbiosis with a soil nematode and during its pathogenic stage in insects.Methods: SMRT sequencing (PacBio) and Bisulfite-seq were performed to identify the DNA methylation of the whole genome (methylome). In addition, RNAseq and phenotypic analysis were performed in a P. luminescens strain overexpressing Dam.Results: Dam overexpression caused a decrease in motility whereas it increased biofilm formation. While symbiosis ability of the Dam overexpressing strain was not significantly different from that of a control strain, the nemato-bacterial complex displayed an impaired pathogenicity in insect, as also observed after direct insect injection of the bacteria alone. Transcriptomic analysis revealed that the observed phenotypes were related to differences at the transcriptional level. More than 99% of the GATC sites of the genome were found methylated and DNA methylation levels did not change over growth kinetics. However, the Dam-overexpressing strain displayed more methylated GATC sites than the control and most of these sites were located in promoter regions. These sites may be involved in the observed differences in phenotypes and gene expression and provide clues to understand the involvement of Dam DNA methylation in P. luminescens life-cycle

Toxin production in a rare and genetically remote cluster of strains of the Bacillus cereus group

<p>Abstract</p> <p>Background</p> <p>Three enterotoxins are implicated in diarrhoeal food poisoning due to <it>Bacillus cereus</it>: Haemolysin BL (Hbl), Non-haemolytic enterotoxin (Nhe), and Cytotoxin K (CytK). Toxin gene profiling and assays for detection of toxin-producing stains have been used in attempts to evaluate the enterotoxic potential of <it>B. cereus </it>group strains. <it>B. cereus </it>strain NVH 391/98, isolated from a case of fatal enteritis, was genetically remote from other <it>B. cereus </it>group strains. This strain lacked the genes encoding Hbl and Nhe, but contains CytK-1. The high virulence of this strain is thought to be due to the greater cytotoxic activity of CytK-1 compared to CytK-2, and to a high level of <it>cytK </it>expression. To date, only three strains containing <it>cytK-1 </it>have been identified; <it>B. cereus </it>strains NVH 391/98, NVH 883/00, and INRA AF2.</p> <p>Results</p> <p>A novel gene variant encoding Nhe was identified in these three strains, which had an average of 80% identity in protein sequence with previously identified Nhe toxins. While culture supernatants containing CytK and Nhe from NVH 391/98 and INRA AF2 were highly cytotoxic, NVH 883/00 expressed little or no CytK and Nhe and was non-cytotoxic. Comparative sequence and expression studies indicated that neither the PlcR/PapR quorum sensing system, nor theYvrGH and YvfTU two-component systems, were responsible for the observed difference in toxin production. Additionally, phylogenetic analysis of 13 genes showed that NVH 391/98, NVH 883/00, and INRA AF2 comprise a novel cluster of strains genetically distant from other <it>B. cereus </it>group strains.</p> <p>Conclusion</p> <p>Due to its divergent sequence, the novel <it>nhe </it>operon had previously not been detected in NVH 391/98 using PCR and several monoclonal antibodies. Thus, toxigenic profiling based on the original <it>nhe </it>sequence will fail to detect the toxin in this group of strains. The observation that strain NVH 883/00 carries <it>cytK-1 </it>but is non-cytotoxic indicates that the detection of this gene variant is not a sufficient criterion for identification of highly cytotoxic strains. The presence of the novel <it>nhe </it>operon and the <it>cytK-1 </it>gene variant in this cluster of strains reflect their phylogenetically remote relationship towards other <it>B. cereus </it>group strains.</p

Role of the Photorhabdus Dam methyltransferase during interactions with its invertebrate hosts

Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens during the whole bacterial life cycle including during symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that during the life cycle of P. luminescens, Dam is not involved the bacterial symbiosis with the nematode H. bacteriophora, but it contributes to the pathogenicity of the nemato-bacterial complex

Extending the cereus group genomics to putative food-borne pathogens of different toxicity

The cereus group represents sporulating soil bacteriacontaining pathogenic strains which may cause diarrheic or emetic foodpoisoning outbreaks. Multiple locus sequence typing revealed a presencein natural samples of these bacteria of about thirty clonal complexes.Application of genomic methods to this group was however biased due tothe major interest for representatives closely related to B. anthracis.Albeit the most important food-borne pathogens were not yet defined,existing dataindicate that they are scattered all over the phylogenetictree. The preliminary analysis of the sequences of three genomesdiscussed in this paper narrows down the gaps in our knowledge of thecereus group. The strain NVH391-98 is a rare but particularly severefood-borne pathogen. Sequencing revealed that the strain must be arepresentative of a novel bacterial species, for which the name Bacilluscytotoxis is proposed. This strain has a reduced genome size compared toother cereus group strains. Genome analysis revealed absence of sigma Bfactor and the presence of genes encoding diarrheic Nhe toxin, notdetected earlier. The strain B. cereus F837/76 represents a clonalcomplex close to that of B. anthracis. Including F837/76, three such B.cereus strains had been sequenced. Alignment of genomes suggests that B.anthracis is their common ancestor. Since such strains often emerge fromclinical cases, they merit a special attention. The third strain, KBAB4,is a typical psychrotrophe characteristic to unbiased soil communities.Phylogenic studies show that in nature it is the most active group interms of gene exchange. Genomic sequence revealed high presence ofextra-chromosomal genetic material (about 530 kb) that may account forthis phenomenon. Genes coding Nhe-like toxin were found on a big plasmidin this strain. This may indicate a potential mechanism of toxicityspread from the psychrotrophic strain community. The results of thisgenomic work and ecological compartments of different strains incite toconsider a necessity of creating prophylactic vaccines against bacteriaclosely related to NVH391-98 and F837/76. Presumably developing of suchvaccines can be based on the properties of non-pathogenic strains such asKBAB4 or ATCC14579 reported here or earlier. By comparing the proteincoding genes of strains being sequenced in this project to others weestimate the shared proteome in the cereus group to be 3,000?b200 genesand the total proteome to be 20-25,000 genes

DNA Methylation in the entomopathogenic bacterium Photorhabdus luminescens

Première réunion du groupe REID - «Hétérogénéité clonale chez les procaryotes et interactions micro-organismes -hôte »DNA Methylation in the entomopathogenic bacterium [i]Photorhabdus luminescens[/i]. Hétérogénéité clonale chez les procaryotes et interactions micro-organismes -hôt

Guide pratique du risque toxique lié au bricolage

AIX-MARSEILLE2-BU Pharmacie (130552105) / SudocSudocFranceF

Caractérisation de cytolysines chez les bactéries entomopathogènes des genres Xenorhabdus et Photorhabdus, et étude de leur rôle dans la relation bactérie-insecte

Les entérobactéries des genres Xenorhabdus et Photorhabdus associées symbiotiquement à des nématodes des sols sont pathogènes par injection chez de nombreux insectes. Ces bactéries produisent in vitro des hémolysines, protéines souvent impliquées dans la virulence chez d'autres bactéries pathogènes, entraînant la cytolyse d'hématies et parfois d'autres cellules. L'objectif de ce travail a été de caractériser de tels facteurs, puis d'évaluer leur rôle dans la virulence chez l'insecte. Des études in vitro ont mis en évidence la présence de deux activités cytolytiques (C1 et C2) pour les hémocytes d'insectes (cellules immunocompétentes capables de phagocytose et d'encapsulement) dans les surnageants de culture des souches de Xenorhabdus. Chez X. nematophila F1, ces activités ciblent les deux types d'hémocytes majoritaires du lépidoptère Spodoptera littoralis, ainsi que des hématies de mammifères. Afin de caractériser le(s) gène(s) codant pour C1, le criblage d'une banque du prophage de X. nematophila a permis de cloner un locus de 5,7 kb qui entraîne une activité hémolytique chez E. coli. Ce locus, qui en fait induit l'activité hémolytique cryptique SheA d'E. coli, code pour une holine fonctionnelle, protéine phagique formant des pores dans la membrane externe des bactéries. L'hypothèse de son implication dans la sécrétion de l'activité C1 est discutée. Chez P. luminiscens, une activité cytolytique associée aux bactéries et s'exerçant sur hématies de cheval est produite in vitro. Les gènes phlA et phlB codant pour cette activité ont été identifiées dans la séquence du génome de P. luminiscens TT01. Ces gènes présentent des similarités de séquences avec les cytolysines de la famille des SAST (Secretion and Activation Serratia Type of Hemolysin). L'activité cytolytique est induite en conditions carencées en fer, et des fusions de promoteurs avec des gènes rapporteurs ont révélé une augmentation transcriptionnelle. par ailleurs, la transcription des gènes in vivo dans l'hémolymphe d'insecte a été vérifiée. Le mutant phlA- construit par échange allélique présente le même pouvoir pathogène que la souche sauvage. Cependant, il est moins compétitif que la souche sauvage au cours des septicémies chez l'insecte, indiquant que ce facteur de virulence ne joue pas un rôle majeur dans le pouvoir pathogène de la bactérie.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Comparison of cytotoxin cytK promoters from Bacillus cereus strain ATCC 14579 and from a B. cereus food-poisoning strain

The cytotoxin CytK produced by Bacillus cereus is believed to be involved in food-borne diseases. The transcriptional activity of the cytK promoter region in a food-poisoning strain was studied using a reporter gene and compared with that in the reference B. cereus strain ATCC 14579. In the food-poisoning strain, cytK is more strongly transcribed, possibly explaining the pathogenicity. The global regulator PlcR in B. cereus controls several putative virulence factors. It was found that PlcR regulates cytK in this clinical strain despite a mismatch in the PlcR recognition site, as currently defined. This suggests that the PlcR box consensus should be reconsidered and that the PlcR regulon might be larger than suspected. It is also shown that the high level of cytK transcription is not caused by a modification in the PlcR recognition site