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

CodY Regulates the Activity of the Virulence Quorum Sensor PlcR by Controlling the Import of the Signaling Peptide PapR in Bacillus thuringiensis

In Gram-positive bacteria, cell–cell communication mainly relies on cytoplasmic sensors of the RNPP family. Activity of these regulators depends on their binding to secreted signaling peptides that are imported into the cell. These quorum sensing regulators control important biological functions in bacteria of the Bacillus cereus group, such as virulence and necrotrophism. The RNPP quorum sensor PlcR, in complex with its cognate signaling peptide PapR, is the main regulator of virulence in B. cereus and Bacillus thuringiensis(Bt). Recent reports have shown that the global stationary phase regulator CodY, involved in adaptation to nutritional limitation, is required for the expression of virulence genes belonging to the PlcR regulon. However, the mechanism underlying this regulation was not described. Using genetics and proteomics approaches, we showed that CodY regulates the expression of the virulence genes through the import of PapR. We report that CodY positively controls the production of the proteins that compose the oligopeptide permease OppABCDF, and of several other Opp-like proteins. It was previously shown that the pore components of this oligopeptide permease, OppBCDF, were required for the import of PapR. However, the role of OppA, the substrate-binding protein (SBP), was not investigated. Here, we demonstrated that OppA is not the only SBP involved in the recognition of PapR, and that several other OppA-like proteins can allow the import of this peptide. Altogether, these data complete our model of quorum sensing during the lifecycle of Bt and indicate that RNPPs integrate environmental conditions, as well as cell density, to coordinate the behavior of the bacteria throughout growt

Iron acquisition in Bacillus cereus: the roles of IlsA and bacillibactin in exogenous ferritin iron mobilization

9siIn host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis.openopenSegond D; Abi Khalil E; Buisson C; Daou N; Kallassy M; Lereclus D; Arosio P; Bou-Abdallah F; Nielsen Le Roux C.Segond, D; Abi Khalil, E; Buisson, C; Daou, N; Kallassy, M; Lereclus, D; Arosio, Paolo; Bou Abdallah, F; Nielsen Le Roux, C

PEPTIDES EXTRACTED FROM ARTEMISIA HERBA ALBA HAVE ANTIMICROBIAL ACTIVITY AGAINST FOODBORNE PATHOGENIC GRAM-POSITIVE BACTERIA

Background: Artemisia herba alba, classified into the family of Asteraceae, is an aromatic herb that is traditionally used as a purgative and anti-pyretic folk medicine by rural people of south Tunisia. This study reports the first identification of antimicrobial peptides from this medicinal plant that inhibited the growth of several food-borne pathogenic bacteria. Materials and methods: The extraction and purification of peptidic agents from Artemisia herba alba, have been performed using precipitation by ammonium sulfate of a phosphate buffer crude extract obtained from the plant leaves, followed by reverse-phase HPLC on a C18 column. The mass of the peptides was estimated by SDS-PAGE electrophoresis, followed by a gel overlay assay and ultra-filtration through a 5 kDa cut-off membrane. Fractions from every purification steps were sampled and assayed for activity towards different food-borne bacterial strains pathogenic and non pathogenic to humans. Results: The phosphate buffer crude extract, as well as its ammonium sulfate precipitate, designated AS-P, inhibited the growth of Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus sensu stricto and the new approved species Bacillus cytotoxicus. AS-P MICs (minimum inhibitory concentrations) ranged from 0.241 to 3.8 mg/ml proteins for L. monocytogenes and B. cereus sensu stricto (strains ATCC10987 and IP5832), respectively. The bioactive AS-P molecules were stable up to 10 minutes heating at 120°C and they resisted organic solvent effects. Antimicrobial activity of A. herba alba AS-P decreased to 40 and 60% after proteolytic treatment with trypsin and proteinase K, respectively, suggesting peptides being responsible for the A. herba alba AS-P activity. The mass of antibacterial A. herba alba peptides was estimated below 5 kDa. Two AS-P fractions, eluted at 40 and 37% acetonitrile, showed antibacterial activity when assayed against L. monocytogenes. Conclusion: A. herba alba could make a new source of novel natural anti-infective agents that could be used in food bio-preservation as natural additives or in human infectious disease treatments against multi-drug resistant pathogens

The PlcR Virulence Regulon of Bacillus cereus

PlcR is a Bacillus cereus transcriptional regulator, which activates gene expression by binding to a nucleotidic sequence called the ‘PlcR box’. To build a list of all genes included in the PlcR regulon, a consensus sequence was identified by directed mutagenesis. The reference strain ATCC14579 sequenced genome was searched for occurrences of this consensus sequence to produce a virtual regulon. PlcR control of these genes was confirmed by comparing gene expression in the reference strain and its isogenic Δ-plcR strain using DNA microarrays, lacZ fusions and proteomics methods. The resulting list included 45 genes controlled by 28 PlcR boxes. Forty of the PlcR controlled proteins were exported, of which 22 were secreted in the extracellular medium and 18 were bound or attached to cell wall structures (membrane or peptidoglycan layer). The functions of these proteins were related to food supply (phospholipases, proteases, toxins), cell protection (bacteriocins, toxins, transporters, cell wall biogenesis) and environment-sensing (two-component sensors, chemotaxis proteins, GGDEF family regulators). Four genes coded for cytoplasmic regulators. The PlcR regulon appears to integrate a large range of environmental signals, including food deprivation and self cell-density, and regulate the transcription of genes designed to overcome obstacles that hinder B. cereus growth within the host: food supply, host barriers, host immune defenses, and competition with other bacterial species. PlcR appears to be a key component in the efficient adaptation of B. cereus to its host environment

Necrotrophism Is a Quorum-Sensing-Regulated Lifestyle in Bacillus thuringiensis

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

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

IlsA, A Unique Surface Protein of Bacillus cereus Required for Iron Acquisition from Heme, Hemoglobin and Ferritin

The human opportunistic pathogen Bacillus cereus belongs to the B. cereus group that includes bacteria with a broad host spectrum. The ability of these bacteria to colonize diverse hosts is reliant on the presence of adaptation factors. Previously, an IVET strategy led to the identification of a novel B. cereus protein (IlsA, Iron-regulated leucine rich surface protein), which is specifically expressed in the insect host or under iron restrictive conditions in vitro. Here, we show that IlsA is localized on the surface of B. cereus and hence has the potential to interact with host proteins. We report that B. cereus uses hemoglobin, heme and ferritin, but not transferrin and lactoferrin. In addition, affinity tests revealed that IlsA interacts with both hemoglobin and ferritin. Furthermore, IlsA directly binds heme probably through the NEAT domain. Inactivation of ilsA drastically decreases the ability of B. cereus to grow in the presence of hemoglobin, heme and ferritin, indicating that IlsA is essential for iron acquisition from these iron sources. In addition, the ilsA mutant displays a reduction in growth and virulence in an insect model. Hence, our results indicate that IlsA is a key factor within a new iron acquisition system, playing an important role in the general virulence strategy adapted by B. cereus to colonize susceptible hosts

Récents développements de la recherche sur la génétique de Bacillus thuringiensis Berliner

International audienc