Role of mprF1 and mprF2 in the Pathogenicity of Enterococcus faecalis

Aujourd hui, Enterococcus faecalis est considéré comme l un des plus importants agents pathogènes causant des maladies nosocomiales. En raison de sa résistance innée et acquise aux antibiotiques, l identification de nouvelles cibles pour le traitement de cette bactérie est une grande priorité. Le facteur Multiple Peptide Résistance (MprF), qui a été décrit en premier chez Staphylococcus aureus, modifie le phosphatidylglycérol avec de la lysine et réduit ainsi la charge négative de l enveloppe cellulaire. Ceci a comme conséquence d augmenter la résistance aux peptides antimicrobiens cationiques (PAC). Deux gènes paralogues putatifs (mprF1 et mprF2) ont été identifiés chez E. faecalis par recherche BLAST en utilisant le gène décrit chez S. aureus. Une caractérisation de ces deux gènes d E. faecalis ainsi que des mécanismes conduisant à une résistance aux PAC, pourrait aider à développer des nouvelles stratégies thérapeutiques contre ce pathogène. Deux mutants de délétion et un double mutant ont été construits par recombinaison homologue chez E. faecalis. L analyse des phospholipides des membranes cytoplasmiques des deux mutants mprF1 et mprF2 par chromatographie sur couche mince a montré que seule l inactivation de mprF2 inhibe la synthèse de trois amino-phosphatidlyglycérol distincts (comme la Lysine-PG, l Alanine-PG et l Arginine-PG). De plus, le mutant mprF2 est également plus sensible aux PAC que la souche sauvage. La capacité de formation d un biofilm est généralement considérée comme un facteur important de virulence, ce qui est également le cas pour les entérocoques. Le mutant mprF2 montre une capacité accrue dans ce phénomène. Ceci semble être du à une augmentation de la concentration d ADN extracellulaire dans le biofilm formé par ce mutant. Curieusement, cette augmentation est indépendante d une autolyse. Le mutant mprF2 est également plus résistant à l opsonophagocytose. Cependant, le gène mprF2 ne joue aucun rôle dans les bactériémies de souris et les endocardites de rats.En revanche, aucun phénotype n a été trouvé pour un mutant mprF1 jusqu à présent. Cette mutation ne modifie ni la synthèse de l aminoacyl-PG en condition de laboratoire ni la résistance aux PAC et à l opsonophagocytose. Par conséquent, il semble que mprF2 soit le seul gène mprF fonctionnel chez E. faecalis. Néanmoins, contrairement à d autres bactéries, mprF2 ne semble pas être un facteur de virulence majeur pour cette espèce.Enterococcus faecalis is regarded nowadays as one of the most important nosocomial pathogens. Due to its innate and acquired resistance to antibiotics, identification of new targets for antimicrobial treatment of E. faecalis is a high priority. The multiple peptides resistance factor (MprF), which was first described in Staphylococcus aureus, modifies phosphatidylglycerol with lysine and reduces the negative charge of the membrane, thus increasing resistance to cationic antimicrobial peptides (CAMPs). Two putative mprF paralogs (mprF1 and mprF2) were identified in E. faecalis by Blast search using the well-described S. aureus gene as a lead. A better understanding of these two genes and mechanisms leads to enterococcal resistance to CAMPs might help designing therapeutic strategies against this bacteria. Two single deletion mutants and double mutant in E. faecalis were created by homologues recombination. Analysis of cell membrane phospholipids from both mutants by thin-layer chromatography showed that inactivation of mprF2 abolished the synthesis of three distinct amino-phosphatidylglycerol (mostly likely Lysin-PG, Alanine-PG and Argine-PG). The CAMPs testing assay demonstrated that the deletion mutant of mprF2 was more susceptible to CAMPs than the wild type. Biofilm formation is usually regarded as a virulence factor which provides an important way for enterococci to cause infections. Inactivation of mprF2 led to increase the biofilm formation which we showed that it was due to the accumulation of eDNA in the biofilm, but the release of eDNA is independent from autolysis. The mprF2 mutant was resistance to killing by opsonophagocytosis more than wild type. However, the mprF2 gene plays no role in bacteremia in mice and rat endocarditis. Our results showed that non polar effect mprF1 mutant does not affect in the synthesis of aminoacyl-PG in the laboratory condition. It also has no effect on susceptible to CAMPs, opsonic killing and autolysis. Therefore, it seems that mprF2 is the only functional mprF gene in E. faecalis in the laboratory condition. Unlike mprF found in other bacteria, mprF does not seem to be a major virulence factor in enterococci.CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

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

Molecular basis for group-specific activation of the virulence regulator PlcR by PapR heptapeptides

The transcriptional regulator PlcR and its cognate cell–cell signalling peptide PapR form a quorum-sensing system that controls the expression of extra-cellular virulence factors in various species of the Bacillus cereus group. PlcR and PapR alleles are clustered into four groups defining four pherotypes. However, the molecular basis for group specificity remains elusive, largely because the biologically relevant PapR form is not known. Here, we show that the in vivo active form of PapR is the C-terminal heptapeptide of the precursor, and not the pentapeptide, as previously suggested. Combining genetic complementation, anisotropy assays and structural analysis we provide a detailed functional and structural explanation for the group specificity of the PlcR–PapR quorum-sensing system. We further show that the C-terminal helix of the PlcR regulatory domain, specifically the 278 residue, in conjunction with the N-terminal residues of the PapR heptapeptide determines this system specificity. Variability in the specificity-encoding regions of plcR and papR genes suggests that selection and evolution of quorum-sensing systems play a major role in adaptation and ecology of Bacilli

Disinfection of Ocular Cells and Tissues by Atmospheric-Pressure Cold Plasma

Background: Low temperature plasmas have been proposed in medicine as agents for tissue disinfection and have received increasing attention due to the frequency of bacterial resistance to antibiotics. This study explored whether atmospheric-pressure cold plasma (APCP) generated by a new portable device that ionizes a flow of helium gas can inactivate ocular pathogens without causing significant tissue damage. Methodology and Principal Findings: We tested the APCP effects on cultured Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Herpes simplex virus-1, ocular cells (conjunctival fibroblasts and keratocytes) and ex-vivo corneas. Exposure to APCP for 0.5 to 5 minutes significantly reduced microbial viability (colony-forming units) but not human cell viability (MTT assay, FACS and Tunel analysis) or the number of HSV-1 plaque-forming units. Increased levels of intracellular reactive oxygen species (ROS) in exposed microorganisms and cells were found using a FACS-activated 2',7'-dichlorofluorescein diacetate probe. Immunoassays demonstrated no induction of thymine dimers in cell cultures and corneal tissues. A transient increased expression of 8-OHdG, genes and proteins related to oxidative stress (OGG1, GPX, NFE2L2) was determined in ocular cells and corneas by HPLC, qRT-PCR and Western blot analysis. Conclusions: A short application of APCP appears to be an efficient and rapid ocular disinfectant for bacteria and fungi without significant damage on ocular cells and tissues, although the treatment of conjunctival fibroblasts and keratocytes caused a time-restricted generation of intracellular ROS and oxidative stress-related responses

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

Bloodstream-To-Eye Infections Are Facilitated by Outer Blood-Retinal Barrier Dysfunction

This work was funded by National Institutes of Health (NIH; http://www.nih.gov) Grants R01EY024140 and R21EY022466 (to M.C.C.) and R01EY019494 (to M.H.E.). Our research is also funded in part by NIH Core Grant P30EY021725 (to Robert E. Anderson, OUHSC) and an unrestricted grant from Research to Prevent Blindness Inc. (http://www.rpbusa.org) to the Dean A. McGee Eye Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.We thank Bolanle Adebayo (Cameron University, Lawton OK), Craig Land (Oklahoma State University, Stillwater OK), Nathan Jia (Oklahoma Christian University, Edmond OK), Kobbe Wiafe (Oklahoma School of Science and Mathematics, Oklahoma City OK), and Amanda Roehrkasse and Madhu Parkunan (OUHSC) for intellectual discussions and technical assistance. The authors also acknowledge thank Nanette Wheatley, Dr. Feng Li, and Mark Dittmar (OUHSC Live Animal Imaging Core, P30EY021725) for their invaluable technical assistance.This work was presented in part at the 2014 Association for Research in Vision and Ophthalmology Annual Conference in Orlando FL.The blood-retinal barrier (BRB) functions to maintain the immune privilege of the eye, which is necessary for normal vision. The outer BRB is formed by tightly-associated retinal pigment epithelial (RPE) cells which limit transport within the retinal environment, maintaining retinal function and viability. Retinal microvascular complications and RPE dysfunction resulting from diabetes and diabetic retinopathy cause permeability changes in the BRB that compromise barrier function. Diabetes is the major predisposing condition underlying endogenous bacterial endophthalmitis (EBE), a blinding intraocular infection resulting from bacterial invasion of the eye from the bloodstream. However, significant numbers of EBE cases occur in non-diabetics. In this work, we hypothesized that dysfunction of the outer BRB may be associated with EBE development. To disrupt the RPE component of the outer BRB in vivo, sodium iodate (NaIO3) was administered to C57BL/6J mice. NaIO3-treated and untreated mice were intravenously injected with 108 colony forming units (cfu) of Staphylococcus aureus or Klebsiella pneumoniae. At 4 and 6 days postinfection, EBE was observed in NaIO3-treated mice after infection with K. pneumoniae and S. aureus, although the incidence was higher following S. aureus infection. Invasion of the eye was observed in control mice following S. aureus infection, but not in control mice following K. pneumoniae infection. Immunohistochemistry and FITC-dextran conjugate transmigration assays of human RPE barriers after infection with an exoprotein-deficient agr/sar mutant of S. aureus suggested that S. aureus exoproteins may be required for the loss of the tight junction protein, ZO-1, and for permeability of this in vitro barrier. Our results support the clinical findings that for both pathogens, complications which result in BRB permeability increase the likelihood of bacterial transmigration from the bloodstream into the eye. For S. aureus, however, BRB permeability is not required for the development of EBE, but toxin production may facilitate EBE pathogenesis.Yeshttp://www.plosone.org/static/editorial#pee

Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase

Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence