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International audience; Recent data support the hypothesis that Gram-positive bacteria (monoderms) arose from Gram-negative ones (diderms) through loss of the outer membrane (OM), but how this happened remains unknown. As tethering of the OM is essential for cell envelope stability in diderm bacteria, its destabilization may have been involved in this transition. In the present study, we present an in-depth analysis of the four known main OM-tethering systems across the Tree of Bacteria (ToB). We show that the presence of such systems follows the ToB with a bimodal distribution matching the deepest phylogenetic divergence between Terrabacteria and Gracilicutes. Whereas the lipoprotein peptidoglycan-associated lipoprotein (Pal) is restricted to the Gracilicutes, along with a more sporadic occurrence of OmpA, and Braun's lipoprotein is present only in a subclade of Gammaproteobacteria, diderm Terrabacteria display, as the main system, the OmpM protein. We propose an evolutionary scenario whereby OmpM represents a simple, ancestral OM-tethering system that was later replaced by one based on Pal after the emergence of the Lol machinery to deliver lipoproteins to the OM, with OmpA as a possible transition state. We speculate that the existence of only one main OM-tethering system in the Terrabacteria would have allowed the multiple OM losses specifically inferred in this clade through OmpM perturbation, and we provide experimental support for this hypothesis by inactivating all four ompM gene copies in the genetically tractable diderm Firmicute Veillonella parvula. High-resolution imaging and tomogram reconstructions reveal a non-lethal phenotype in which vast portions of the OM detach from the cells, forming huge vesicles with an inflated periplasm shared by multiple dividing cells. Together, our results highlight an ancient shift of OM-tethering systems in bacterial evolution and suggest a mechanism for OM loss and the multiple emergences of the monoderm phenotype from diderm ancestors.

International audience; Bacterial surface proteins constitute an amazing repertoire of molecules with important functions such as adherence, invasion, signalling and interaction with the host immune system or environment. In Gram-positive bacteria, many surface proteins of the "LPxTG" family are anchored to the peptidoglycan (PG) by an enzyme named sortase. While this anchoring mechanism has been clearly deciphered, less is known about the spatial organization of cell wall-anchored proteins in the bacterial envelope. Here, we review the question of the precise spatial and temporal positioning of LPxTG proteins in subcellular domains in spherical and ellipsoid bacteria (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae and Enterococcus faecalis) and in the rod-shaped bacterium Listeria monocytogenes. Deposition at specific sites of the cell wall is a dynamic process tightly connected to cell division, secretion, cell morphogenesis and levels of gene expression. Studying spatial occupancy of these cell wall-anchored proteins not only provides information on PG dynamics in responses to environmental changes, but also suggests that pathogenic bacteria control the distribution of virulence factors at specific sites of the surface, including pole, septa or lateral sites, during the infectious process.

International audience; Lantibiotics are antimicrobial peptides from the bacteriocin family, secreted by Gram-positive bacteria. These peptides differ from other bacteriocins by the presence of (methyl)lanthionine residues, which result from enzymatic modification of precursor peptides encoded by structural genes. Several groups of lantibiotics have been distinguished, the largest of which is the lacticin 481 group. This group consists of at least 16 members, including lacticin 481, streptococcin A-FF22, mutacin II, nukacin ISK-1, and salivaricins. We present the first review devoted to this lantibiotic group, knowledge of which has increased significantly within the last few years. After updating the group composition and defining the common properties of these lantibiotics, we highlight the most recent developments. The latter concern: transcriptional regulation of the lantibiotic genes; understanding the biosynthetic machinery, in particular the ability to perform in vitro prepeptide maturation; characterization of a novel type of immunity protein; and broad application possibilities. This group differs in many aspects from the best known lantibiotic group (nisin group), but shares properties with less-studied groups such as the mersacidin, cytolysin and lactocin S groups.

International audience; Methanolic extracts prepared from the leaves, twigs and the roots of Vismia laurentii De Wild as well as nine compounds isolated from these crude extracts, were tested for their antimicrobial activity against Gram-positive bacteria (six species), Gram-negative bacteria (12 species) and two Candida species using disc diffusion and well micro-dilution methods. The disc diffusion assay indicated that the crude extract was active against all the pathogens tested, whereas isolated compounds showed selective activities. The degree of sensitivity of the test organisms to purified compounds varied from 25 to 90%. Fridelin (8) was found to be the most active compound, while Bivismiaquinone (3) was the least active. The lowest minimum inhibition concentration (MIC) values as obtained by the micro-dilution assays were 19.53 and 1.22 microg/ml for the crude extracts and purified compounds, respectively. The lowest value for the purified compounds (1.22 microg/ml) was obtained with O(1)-demethyl-3',4'-deoxypsorospermin-3',4'-diol (6) on Candida gabrata and Bacillus subtilis; 1,8-dihydroxy-6-methoxy-3-methylanthraquinone (5) on Bacillus subtilis and 6-deoxyisojacareubin (7) on Bacillus stearothermophilus. These results provide promising baseline information for the potential use of these crude extracts as well as some of the isolated compounds in the treatment of bacterial and fungal infections.

International audience; In the last decade, pili, which are encoded within pathogenicity islands, have been found in many Gram-positive bacteria, including the major streptococcal and enterococcal pathogens. These long proteinaceous polymers extending from the bacterial surface are constituted of covalently linked pilin subunits, which play major roles in adhesion and host colonization. They are also involved in biofilm formation, a characteristic life-style of the bacteria constituting the oral flora. Pili are highly immunogenic structures that are under the selective pressure of host immune responses. Indeed, pilus expression was found to be heterogeneous in several bacteria with the co-existence of two subpopulations expressing various levels of pili. The molecular mechanisms underlying this complex regulation are poorly characterized except for Streptococcus pneumoniae. In this review, we will discuss the roles of Gram-positive bacteria pili in adhesion to host extracellular matrix proteins, tissue tropism, biofilm formation, modulation of innate immune responses and their contribution to virulence, and in a second part the regulation of their expression. This overview should help to understand the rise of pili as an intensive field of investigation and pinpoints the areas that need further study.

Contains fulltext : 96277.pdf (Publisher’s version ) (Open Access) BACKGROUND: Two NF-kappaB signaling pathways, Toll and immune deficiency (imd), are required for survival to bacterial infections in Drosophila. In response to septic injury, these pathways mediate rapid transcriptional activation of distinct sets of effector molecules, including antimicrobial peptides, which are important components of a humoral defense response. However, it is less clear to what extent macrophage-like hemocytes contribute to host defense. METHODOLOGY/PRINCIPAL FINDINGS: In order to dissect the relative importance of humoral and cellular defenses after septic injury with three different gram-positive bacteria (Micrococcus luteus, Enterococcus faecalis, Staphylococcus aureus), we used latex bead pre-injection to ablate macrophage function in flies wildtype or mutant for various Toll and imd pathway components. We found that in all three infection models a compromised phagocytic system impaired fly survival--independently of concomitant Toll or imd pathway activation. Our data failed to confirm a role of the PGRP-SA and GNBP1 Pattern Recognition Receptors for phagocytosis of S. aureus. The Drosophila scavenger receptor Eater mediates the phagocytosis by hemocytes or S2 cells of E. faecalis and S. aureus, but not of M. luteus. In the case of M. luteus and E. faecalis, but not S. aureus, decreased survival due to defective phagocytosis could be compensated for by genetically enhancing the humoral immune response. CONCLUSIONS/SIGNIFICANCE: Our results underscore the fundamental importance of both cellular and humoral mechanisms in Drosophila immunity and shed light on the balance between these two arms of host defense depending on the invading pathogen.

A novel strictly anaerobic, moderately halophilic and mesophilic bacterium, designated strain SOL3f37T, was isolated from a hydrocarbon-polluted soil surrounding a deep petroleum environment located in south Tunisia. Cells of strain SOL3f37T stained Gram-positive and were motile, straight and spore-forming. Strain SOL3f37T had a typical Gram-positive-type cell-wall structure, unlike the thick, multilayered cell wall of its closest relative Clostridiisalibacter paucivorans. The major fatty acids were iso-C15 : 0 (41 %), iso-C14 : 0 3-OH and/or iso-C15 : 0 dimethyl acetal (21.6 %), iso-C13 : 0 (4.4 %), anteiso-C15 : 0 (3.9 %) and iso-C15 : 1 (2.8 %). Strain SOL3f37T grew between 20 and 48 °C (optimum 40 °C) and at pH 6.2–8.1 (optimum pH 6.9). Strain SOL3f37T required at least 0.5 g NaCl l−1 and grew in the presence of NaCl concentrations up to 150 g l−1 (optimum 40 g l−1). Yeast extract (2 g l−1) was required for degradation of pyruvate, fumarate, fructose, glucose and mannitol. Also, strain SOL3f37T grew heterotrophically on yeast extract, peptone and bio-Trypticase, but was unable to grow on Casamino acids. Sulfate, thiosulfate, sulfite, elemental sulfur, fumarate, nitrate and nitrite were not reduced. The DNA G+C content was 30.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SOL3f37T was a member of the family Clostridiaceae in the order Clostridiales; strain SOL3f37T was related to members of various genera of the family Clostridiaceae. It exhibited highest 16S rRNA gene sequence similarity (93.4 %) with Clostridiisalibacter paucivorans 37HS60T, 91.8 % with Thermohalobacter berrensis CTT3T and 91.7 % with Caloranaerobacter azorensis MV1087T. On the basis of genotypic, phenotypic and phylogenetic data, it is suggested that strain SOL3f37T represents a novel species in a new genus. The name Sporosalibacterium faouarense gen. nov., sp. nov. is proposed, with SOL3f37T (=DSM 21485T =JCM 15487T) as the type strain of Sporosalibacterium faouarense.

International audience; The serine-rich repeat family of fimbriae play important roles in the pathogenesis of streptococci and staphylococci. Despite recent attention, their finer structural details and precise adhesion mechanisms have yet to be determined. Fap1 (Fimbriae-associated protein 1) is the major structural subunit of serine-rich repeat fimbriae from Streptococcus parasanguinis and plays an essential role in fimbrial biogenesis, adhesion, and the early stages of dental plaque formation. Combining multidisciplinary, high resolution structural studies with biological assays, we provide new structural insight into adhesion by Fap1. We propose a model in which the serine-rich repeats of Fap1 subunits form an extended structure that projects the N-terminal globular domains away from the bacterial surface for adhesion to the salivary pellicle. We also uncover a novel pH-dependent conformational change that modulates adhesion and likely plays a role in survival in acidic environments.

International audience; Over the last 10 years, DNA sequences of more than 600 bacterial species have been deposited in databases and are now available to search any gene, motif, or regulatory sequence of interest. Although genome data are instrumental in phylogenetic analysis and in silico design of metabolic and regulatory networks, only a very small fraction of the information has been experimentally validated. A striking example is lipoproteins predicted from genome sequences. Despite the predominance of this class of surface proteins in bacteria (up to 0.5 to 8% of the proteome), very few of these proteins have been identified as lipoproteins by biochemical methods (19). In this issue of the Journal of Bacteriology, Baumgärtner et al. (2) report a systematic analysis of lipoproteins of Listeria monocytogenes, a facultative gram-positive intracellular bacterial pathogen that causes severe infections (listeriosis) in both human and animals. These authors used an L. monocytogenes mutant defective in lipoprotein diacylglyceryl transferase (Lgt), an enzyme involved in lipoprotein processing. Three aspects of their study should be highlighted: (i) new findings concerning the roles of Lgt and lipoprotein-specific signal peptidase II (Lsp) during lipoprotein processing (22); (ii) the identification of 26 of the 68 lipoproteins predicted in the initial annotation of the L. monocytogenes strain EGD-e genome (7); and (iii) experimental evidence that a few of these lipoproteins are regulated by PrfA, the master virulence regulator of L. monocytogenes (8). Below, we discuss the significance of these findings separately.

The complement system is a major humoral portion of the innate immune system, playing a significant role in host defence against microorganisms. The biological importance of this system is underlined by the fact that at least three different pathways for its activation exist, the classical, the MBL and the alternative pathway. To elucidate the involvement of the classical and/or the MBL pathway during bacterial septicemia, 32 patients with gram-positive and 30 patients with gram-negative bacterial infections were investigated. In patients with gram-positive bacteria, a significant consumption of C1q (p=0.005) but not of mannose-binding lectin (MBL) (p=0.2) was found during the acute phase of infection. In contrast, in patients with gram-negative bacterial infections, a significant reduction of MBL (p=0.002) and only a moderate, less significant reduction of C1q (p=0.03) were observed. As a model for the binding of MBL to gram-negative bacteria, Salmonella strains with defined mutations in their lipopolysaccharide (LPS) structure were used. The comparison of the binding of MBL to these Salmonella strains with that of the corresponding isolated LPS forms bound to microtiter plates revealed a similar binding pattern, supporting the interpretation that LPS on the surface of gram-negative bacteria is the major acceptor molecule for MBL on these bacteria, which according to our results obviously also takes place during gram-negative bacterial septicaemia. Furthermore, we were able to demonstrate that MBL bound to LPS was able to initiate activation of the complement cascade as measured by the occurrence of the cleavage product C4c.

International audience; In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters. Recombinant Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. The activity of Cg-Def was retained in vitro at a salt concentration similar to that of seawater. The Cg-Def structure shares the so-called cystine-stabilized alpha-beta motif (CS-alphabeta) with arthropod defensins but is characterized by the presence of an additional disulfide bond, as previously observed in the mussel defensin (MGD-1). Nevertheless, despite a similar global fold, the Cg-Def and MGD-1 structures mainly differ by the size of their loops and by the presence of two aspartic residues in Cg-Def. Distribution of Cg-def mRNA in various oyster tissues revealed that Cg-def is mainly expressed in mantle edge where it was detected by mass spectrometry analyses. Furthermore, we observed that the Cg-def messenger concentration was unchanged after bacterial challenge. Our results suggest that Cg-def gene is continuously expressed in the mantle and would play a key role in oyster by providing a first line of defense against pathogen colonization.

International audience; Bacterial surface proteins constitute a diverse group of molecules with important functions, such as adherence, invasion, signaling and interaction with the host immune system or the environment. In Gram-positive bacteria, many surface proteins are anchored to the cell wall envelope by an enzyme named sortase, which recognizes a conserved carboxylic sorting motif. The sequence of the prototype staphylococcal SrtA has been widely used to identify homologs in bacterial genomes, revealing a profusion of sortases in almost all Gram-positive bacteria, often with more than one sortase-like protein per genome [M.J. Pallen, A.C. Lam, M. Antonio, K. Dunbar, Trends Microbiol. 9 (2001) 97-102]. In light of increasing reports on the identification and/or characterization of paralogous sortase genes, a classification of sortases now appears necessary. This report provides an analysis of sixty-one sortases from complete Gram-positive genomes, and suggests the existence of four structural groups of sortases. We propose the classification of sortases into 4 classes designated A, B, C and D. This classification should help to discriminate between sortases in the future.

The activities of short synthetic, nonhemolytic peptides derived from the C-terminal region of myotoxin 11, a catalytically inactive phospholipase A(2) homologue present in the venom of the snake Bothrops asper, have been shown to reproduce the bactericidal activity of the parent protein. They combine cationic and hydrophobic-aromatic amino acids, thus functionally resembling the antimicrobial peptides of innate defenses. This study evaluated the antimicrobial and antiendotoxic properties of a 13-mer derivative peptide of the C-terminal sequence from positions 115 to 129 of myotoxin II, named pEM-2. This peptide (KKWRWWLKALAKK) showed bactericidal activity against both gram-positive and gram-negative bacteria. In comparison to previously described peptide variants derived from myotoxin II, the toxicity of pEM-2 toward eukaryotic cells in culture was significantly reduced, being similar to that of lactoferricin B but lower than that of polymyxin B. The all-D enantiomer of pEM-2 [pEM-2 (D)] retained the same bactericidal potency Of its L-enantiomeric counterpart, but it showed an enhanced ability to counteract the lethal activity of an intraperitoneal lipopolysaccharide challenge in mice, which correlated with a significant reduction of the serum tumor necrosis factor alpha levels triggered by this endotoxin. Lethality induced by intraperitoneall infection of mice with Eschelichia coli or Salmonella enterica serovar Typhimurium was reduced by the administration of pEM-2 (D). These results demonstrate that phospholipase A(2)-derived peptides may have the potential to counteract microbial infections and encourage further evaluations of their actions in vivo. Las actividades de péptidos sintéticos cortos, no hemolíticos, derivados de la región C-terminal de la miotoxina II, un homólogo de la fosfolipasa A2 catalíticamente inactivo presente en el veneno de la serpiente Bothrops asper, han han demostrado que reproducen la actividad bactericida de la proteína madre. Combinan aminoácidos catiónicos e hidrofóbicos-aromáticos, asemejándose así funcionalmente a los péptidos antimicrobianos de las defensas innatas. En este estudio evaluó las propiedades antimicrobianas y antiendotóxicas de un péptido derivado de 13 meros de la secuencia C-terminal C-terminal de las posiciones 115 a 129 de la miotoxina II, denominado pEM-2. Este péptido (KKWRWLKALAKK) mostró actividad bactericida contra bacterias tanto grampositivas como gramnegativas. En comparación con las variantes de péptidos péptido derivado de la miotoxina II, la toxicidad del pEM-2 para las células eucariotas en cultivo se redujo significativamente fue significativamente reducida, siendo similar a la de la lactoferricina B pero inferior a la de la polimixina B. El enantiómero todo-D de pEM-2 [pEM-2 (D)] conservó la misma potencia bactericida que su homólogo enantiomérico L pero mostró una mayor capacidad para contrarrestar la actividad letal de una provocación con lipopolisacárido intraperitoneal de lipopolisacárido intraperitoneal en ratones, lo que se correlaciona con una reducción significativa de los niveles séricos del factor de necrosis tumoral alfa desencadenados por esta endotoxina. La letalidad inducida por la infección intraperitoneal de ratones con Escherichia coli o Salmonella enterica serovar Typhimurium se redujo con la administración de pEM-2 (D). Estos resultados demuestran que los péptidos derivados de la fosfolipasa A2 pueden tener el potencial de contrarrestar las infecciones microbianas y y animan a seguir evaluando sus acciones in vivo. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

International audience; clpP and clpC of Bacillus subtillis encode subunits of the Clp ATP-dependent protease and are required for stress survival, including growth at high temperature. They play essential roles in stationary phase adaptive responses such as the competence and sporulation developmental pathways, and belong to the so-called class III group of heat shock genes, whose mode of regulation is unknown and whose expression is induced by heat shock or general stress conditions. The product of ctsR, the first gene of the clpC operon, has now been shown to act as a repressor of both clpP and clpC, as well as clpE, which encodes a novel member of the Hsp100 Clp ATPase family. The CtsR protein was purified and shown to bind specifically to the promoter regions of all three clp genes. Random mutagenesis, DNasel footprinting and DNA sequence deletions and comparisons were used to define a consensus CtsR recognition sequence as a directly repeated heptad upstream from the three clp genes. This target sequence was also found upstream from clp and other heat shock genes of several Gram-positive bacteria, including Listeria monocytogenes, Streptococcus salivarius, S. pneumoniae, S. pyogenes, S. thermophilus, Enterococcus faecalis, Staphylococcus aureus, Leuconostoc oenos, Lactobacillus sake, Lactococcus lactis and Clostridium acetobutylicum. CtsR homologues were also identified in several of these bacteria, indicating that heat shock regulation by CtsR is highly conserved in Gram-positive bacteria.

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