Search

Some of the bacterial cells in isogenic populations behave differently from others. We describe here how a new type of phenotypic heterogeneity relating to resistance to cationic antimicrobial peptides (CAMPs) is determinant for the pathogenic infection process of the entomopathogenic bacterium Photorhabdus luminescens. We demonstrate that the resistant subpopulation, which accounts for only 0.5% of the wild-type population, causes septicemia in insects. Bacterial heterogeneity is driven by the PhoPQ two-component regulatory system and expression of pbgPE, an operon encoding proteins involved in lipopolysaccharide (LPS) modifications. We also report the characterization of a core regulon controlled by the DNA-binding PhoP protein, which governs virulence in P. luminescens. Comparative RNAseq analysis revealed an upregulation of marker genes for resistance, virulence and bacterial antagonism in the pre-existing resistant subpopulation, suggesting a greater ability to infect insect prey and to survive in cadavers. Finally, we suggest that the infection process of P. luminescens is based on a bet-hedging strategy to cope with the diverse environmental conditions experienced during the lifecycle.

International audience; Comparison of the proteomes of wild-type Photorhabdus luminescens and its hcaR derivative, grown in insect hemolymph, showed that hcaR disruption decreased the production of toxins (tcdA1, mcf and pirAB) and proteins involved in oxidative stress response (SodA, AhpC, Gor). The disruption of hcaR did not affect growth rate in insects, but did delay the virulence of P. luminescens in Bombyx mori and Spodoptera littoralis larvae. This delayed virulence was associated with a lower toxemia rather than delay in bacteremia. The disruption of hcaR also increased bacterial sensitivity to hydrogen peroxide. A sodA mutant and an hcaR mutant had similar phenotypes in terms of sensitivity to hydrogen peroxide, virulence, toxin gene expression, and growth rate in insects. Thus, the two processes affected by hcaR disruption - toxemia and oxidative stress response - appear to be related. Besides, expression of toxin genes tcdA1, mcf, and pirAB was decreased by paraquat challenge. We provide here the first demonstration of the importance of toxemia for P. luminescens virulence. Our results also highlight the power of proteomic analysis for detecting unexpected links between different, concomitant processes in bacteria

Extraintestinal pathogenic (ExPEC) Escherichia coli strains of serotype O18:K1:H7 are mainly responsible for neonatal meningitis and sepsis in humans and belong to a limited number of closely related clones. The same serotype is also frequently isolated from the extraintestinal lesions of colibacillosis in poultry, but it is not well known to what extent human and avian strains of this particular serotype are related. Twenty-two ExPEC isolates of human origin and 33 isolates of avian origin were compared on the basis of their virulence determinants, lethality for chicks, pulsed-field gel electrophoresis (PFGE) patterns, and classification in the main phylogenetic groups. Both avian and human isolates were lethal for chicks and harbored similar virulence genotypes. A major virulence pattern, identified in 75% of the isolates, was characterized by the presence of F1 variant fimbriae; S fimbriae; IbeA; the aerobactin system; and genomic fragments A9, A12, D1, D7, D10, and D11 and by the absence of P fimbriae, F1C fimbriae, Afa adhesin, and CNF1. All but one of the avian and human isolates also belonged to major phylogenetic group B2. However, various subclonal populations could be distinguished by PFGE in relation to animal species and geographical origin. These results demonstrate that very closely related clones can be recovered from extraintestinal infections in humans and chickens and suggest that avian pathogenic E. coli isolates of serotype O18:K1:H7 are potential human pathogens.

Entomopathogenic bacteria of the genus Photorhabdu s harbor a type III secretion system. This system was probably acquired prior to the separation of the species within this genus. Furthermore, the core components of the secretion machinery are highly conserved but the predicted effectors differ between Photorhabdus luminescens and P. asymbiotica , two highly related species with different hosts.

The genus Xenorhabdus consists of the specific bacterial symbionts of the entomopathogenic nematodes of the family Steinernematidae (40) and was separated from the genus Photorhabdus (11), which contains the symbionts of the entomopathogenic nematodes of the family Heterorhabditidae. Both genera are entomopathogenic gram-negative bacteria belonging to the Enterobacteriaceae. The nematodes carry their bacterial symbionts monoxenically in a special vesicle of the infective stage (L3 juveniles) in Steinernematidae (8) and throughout the whole intestine of Heterorhabditidae (20). These bacteria are transported by their nematode hosts into the hemocoel of the insect prey, which is killed, probably via a combination of toxin action and septicemia. The bacterial symbionts also contribute to the symbiotic relationship by establishing and maintaining suitable conditions for nematode reproduction (31). Recently, isolation of some Photorhabdus strains from infected humans in Australia and the United States was reported (21, 30), and the strains from the United States were classified as Photorhabdus asymbiotica (23). The form of the bacterium that is normally isolated from symbiotic infective-stage nematodes is referred to as phase I. Like many pathogenic bacteria, Xenorhabdus and Photorhabdus strains spontaneously produce colonial variants which have been called phase II variants (10). The two variants of the bacteria have generally been shown to be equally pathogenic for the larvae of the greater wax moth, Galleria mellonella (3). However, Volgyi et al. (42) described for the first time a phase II variant that showed reduced virulence in the tobacco hornworm, Manduca sexta. Xenorhabdus nematophila and Photorhabdus luminescens are highly pathogenic to insects, and 50% insect mortality has been reported with direct infection with fewer than 20 bacteria per larva (5). The bacterial factors involved in killing of the insect or in overcoming the insect immune reactions are still under investigation. Following invasion of the insect host by the nematodes, both bacteria produce potential virulence factors, including lipase, protease, lecithinase, and lipopolysaccharides (LPSs), in the hemocoel (for a review, see reference 24). It was shown that purified LPS, Photorhabdus protease fractions, or Xenorhabdus lecithinase isomers showed no toxic effect following injection into insect hemocoel (12, 16, 39). Recently, a novel toxin complex with both oral and injectable activities against a wide range of insects was identified in a supernatant of P. luminescens (13). Purified toxin complex a (Tca) has specific effects on the midgut epithelium of the insect (9). In order to study Xenorhabdus and Photorhabdus virulence in insects, a genetic approach was also used. Avirulent mutants of X. nematophila have been isolated by transposon mutagenesis (Tn5). These mutants were pleiotropic, but all five mutants that tested as avirulent in G. mellonella were nonmotile and partially impaired in blood hemolysis (43). It was also shown that a homoserine lactone autoinducer restored virulence to one avirulent X. nematophila strain and stimulated the level of bacterial lipase activity (17). Recently we reported that flhDC, the flagellar master operon of X. nematophila, controls flagellin expression. Furthermore we revealed that lipolytic and extracellular hemolysin activity is flhD dependent. We also showed that the flhD null mutant displayed an attenuated virulence phenotype in the common cutworm, Spodoptera littoralis, compared to the wild-type strain (25). The recently published partial genome sequence of P. luminescens (22) revealed a diverse array of genes that putatively encodes potential virulence factors. These factors include exoenzymes (proteases, lipases, and chitinases), a type III secretion system (Yop homolog), and several classes of toxins (insecticidal toxin complex, Rtx-like toxins, and hemolysin and cytotoxin homologs) (22). Until now, studies examining hemolytic activity of both genera have not been reported. Cytolysins are proteins which cause lysis of red blood cells (RBC) as well as nucleated cell types by hydrolysis (lipases, phospholipases, or proteases) or by forming pores in the plasma membrane. Surfactants may also cause cytolysis by solubilization of the target cell membrane. Bacterial cytolysins are usually recognized as hemolysin on blood agar where a transparent zone appears around colonies. The production by a few strains of Xenorhabdus and Photorhabdus of hemolysin has been detected on agar supplemented with sheep blood (6, 21, 25). Apart from their phoretic location inside infective juvenile nematodes, these bacteria are only observed in insect hemolymph, where they enter their growth cycle. Here the bacteria are in contact with hemocytes which achieve defense reactions in insects. Some of these cells are immunocompetent cells able to engulf (phagocytosis) or to isolate and kill (nodule formation) bacteria. We hypothesize that hemolytic activities could target the immunocompetent cells in insect hemolymph. In this study, we report that different cytolytic activities were found in supernatants of Xenorhabdus whereas none was detected in supernatants of various strains of Photorhabdus. We have studied the kinetics of the production of cytolytic activities over the course of in vitro bacterial growth. We also provide evidence on the characteristics and on the specificity of each of these cytolytic activities against mammalian RBC and insect hemocyte types.

International audience; Terrestrial isopods can be killed by some entomopathogenic bacteria among Xenorhabdus and Photorhabdus species even with no or very limited multiplication. This suggests that toxemia and not septicemia is the major cause of entomopathogenic bacteria pathogenicity against these crustaceans. In this paper, we revealed that the injection of stationary phase culture supernatant of P. luminescens TT01, in which toxins can be accumulated, led alone to a rapid decrease in the number of host immune cells and killed most of the Armadillidium vulgare individuals within 48 h. The pathogenicity was strongly attenuated when supernatant was heated and totally suppressed after 100-kDa filtration suggesting that the toxin responsible for killing A. vulgare would be a protein above this size. Additionally, we tested the culture supernatant of two TT01 mutants that have been previously shown as being altered in their pathogenicity against lepidopteran insects one of them being known as exhibiting lower expression of some toxins. However, the supernatants of the mutants was as pathogenic for A. vulgare as the wild type strains suggesting that the toxins involved in killing A. vulgare may be different than previously described ones.

UMR 1334 AGAP : Equipe AFEF ‘Architecture et Fonctionnement des Espèces fruitières’ ; Team AFFS ‘Architecture and Functioning of Fruit Species’ Contact: Elisabeth.Chevreau@angers.inra.fr, chevreau@angers.inra.fr Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; The bacterial pathogen Erwinia amylovora causes the devastating disease known as fire blight in some rosaceous plants including apple and pear. One of the pathogenicity factors affecting fire blight development is the production of a siderophore, desferrioxamine, which overcomes the limiting conditions in plant tissues and also protects bacteria against active oxygen species. In this paper we examine the effect of an iron chelator protein encoded by the pea ferritin gene on the fire blight susceptibility of pear (Pyrus communis). Transgenic pear clones expressing this gene controlled either by the constitutive promoter CaMV 355 or by the inducible promoter sgd24 promoter were produced. The transgenic clones produced were analysed by Q-RT-PCR to determine the level of expression of the pea transgene. A pathogen-inducible pattern of expression of the pea transgene was observed in sgd24-promoter transformants. Adaptation to iron deficiency in vitro was tested in some transgenic clones and different iron metabolism parameters were measured. No strong effect on iron and chlorophyll content, root reductase activity and fire blight susceptibility was detected in the transgenic lines tested. No transformants showed a significant reduction in susceptibility to fire blight in greenhouse conditions when inoculated with E. amylovora

International audience; Cycle inhibiting factors (Cif) constitute a broad family of cyclomodulins present in bacterial pathogens of invertebrates and mammals. Cif proteins are thought to be type III effectors capable of arresting the cell cycle at G2/M phase transition in human cell lines. We report here the first direct functional analysis of CifPl, from the entomopathogenic bacterium Photorhabdus luminescens, in its insect host. The cifPl gene was expressed in P. luminescens cultures in vitro. The resulting protein was released into the culture medium, unlike the well characterized type III effector LopT. During locust infection, cifPl was expressed in both the hemolymph and the hematopoietic organ, but was not essential for P. luminescens virulence. CifPl inhibited proliferation of the insect cell line Sf9, by blocking the cell cycle at the G2/M phase transition. It also triggered host cell death by apoptosis. The integrity of the CifPl catalytic triad is essential for the cell cycle arrest and pro-apoptotic activities of this protein. These results highlight, for the first time, the dual role of Cif in the control of host cell proliferation and apoptotic death in a non-mammalian cell line.

International audience; Xenorhabdus nematophila/Steinernema carpocapsae and Photorhabdus luminescens/Heterorhabditis bacteriophora are nemato-bacterial complexes highly pathogenic for insects. Using a syringe as artificial vector, we have analyzed the effects of the two bacteria, X. nematophila and P. luminescens on the genetic tool insect, Drosophila melanogaster. Both bacteria were found to kill adult flies in a dose dependent manner with X. nematophila being the fastest. On the other hand, when an injection of non-pathogenic bacteria, Escherichia coli, is performed 1 day before challenge with the entomopathogenic bacteria, then the survival of Drosophila flies was prolonged by at least 20 h. After injection of entomopathogenic bacteria, Drosophila mutant Dif1, affected on the Toll pathway, showed a similar phenotype than wild-type flies whereas Drosophila mutant DreddD55, affected on the imd pathway, was not protected by a prior injection of E. coli. This suggested that members of the imd pathway might be targets of these entomopathogenic bacteria albeit synthesis of antimicrobial peptides through this signaling pathway was induced by X. nematophila as well as P. luminescens. Finally, P. luminescens phoP mutant, an avirulent mutant in the Lepidopteran insect, Spodoptera littoralis, was found poorly virulent for D. melanogaster. phoP mutant partially protected D. melanogaster flies if injected 1 day before the injection of P. luminescens wild-type TT01 to the same extent than the E. coli-induced protection. However, phoP recovered a level of pathogenicity comparable to P. luminescens wild-type TT01 when injected to Drosophila flies affected on the imd pathway.

Correspondance auteur: R. Zumbihl E-mail: zumbihl@univ-montp2.fr; International audience; Photorhabdus asymbiotica is unique among the entomopathogenic bacteria of this genus in also being able to infect humans, leading to its isolation from some clinical samples. Recent comparative genomics data and the results of studies of interactions between bacteria and cells provide insight into the adaptation of this bacterium to its new niche, the human body

We used the information from a set of concatenated sequences from four genes (recA,gyrB,dnaNandgltX) to investigate the phylogeny of the generaPhotorhabdusandXenorhabdus(entomopathogenic bacteria associated with nematodes of the generaHeterorhabditisandSteinernema, respectively). The robustness of the phylogenetic tree obtained by this multigene approach was significantly better than that of the tree obtained by a single gene approach. The comparison of the topologies of single gene phylogenetic trees highlighted discrepancies which have implications for the classification of strains and new isolates; in particular, we propose the transfer ofPhotorhabdus luminescenssubsp.thracensistoPhotorhabdus temperatasubsp.thracensiscomb. nov. (type strain CIP 108426T=DSM 15199T). We found that, within the genusXenorhabdus, strains or isolates that shared less than 97 % nucleotide identity (NI), calculated on the concatenated sequences of the four gene fragments (recA,gyrB,dnaNandgltX) encompassing 3395 nucleotides, did not belong to the same species. Thus, at the 97 % NI cutoff, we confirm the current 20 species of the genusXenorhabdusand propose the description of a novel species,Xenorhabdus vietnamensissp. nov. (type strain VN01T= CIP 109945T=DSM 22392T). Within each of the three current species of the genusPhotorhabdus,P. asymbiotica,P. luminescensandP. temperata, strains or isolates which shared less than 97 % NI did not belong to the same subspecies. Comparisons of the four gene fragments plus therplBgene fragment analysed separately led us to propose four novel subspecies:Photorhabdus luminescenssubsp.caribbeanensissubsp. nov. (type strain HG29T=CIP 109949T=DSM 22391T),P. luminescenssubsp.hainanensissubsp. nov. (type strain C8404T= CIP 109946T=DSM 22397T),P. temperatasubsp.khaniisubsp. nov. (type strain C1T=NC19T=CIP 109947T=DSM 3369T), andP. temperatasubsp.tasmaniensissubsp. nov. (type strain T327T= CIP 109948T=DSM 22387T).

Article Open Access Correspondance auteur: J.M. Escoubas E-mail: Jean-Michel.Escoubas@univ-montp2.fr; International audience; Background: X-tox proteins are a family of immune-related proteins only found in Lepidoptera and characterized by imperfectly conserved tandem repeats of several defensin-like motifs. Previous phylogenetic analysis of X-tox genes supported the hypothesis that X-tox have evolved from defensins in a lineage-specific gene evolution restricted to Lepidoptera. In this paper, we performed a protein study in which we asked whether X-tox proteins have conserved the antimicrobial functions of their ancestral defensins and have evolved as defensin reservoirs. Methodology/Principal Findings: We followed the outcome of Spod-11-tox, an X-tox protein characterized in Spodoptera frugiperda, in bacteria-challenged larvae using both immunochemistry and antimicrobial assays. Three hours post infection, the Spod-11-tox protein was expressed in 80% of the two main classes of circulating hemocytes (granulocytes and plasmatocytes). Located in secretory granules of hemocytes, Spod-11-tox was never observed in contact with microorganisms entrapped within phagolyzosomes showing that Spod-11-tox is not involved in intracellular pathogen killing. In fact, the Spod-11-tox protein was found to be secreted into the hemolymph of experimentally challenged larvae. In order to determine antimicrobial properties of the Spod-11-tox protein, it was consequently fractionated according to a protocol frequently used for antimicrobial peptide purification. Over the course of purification, the anti-Spod-11-tox immunoreactivity was found to be dissociated from the antimicrobial activity. This indicates that Spod-11-tox is not processed into bioactive defensins in response to a microbial challenge. Conclusions/Significance: Altogether, our results show that X-tox proteins have not evolved as defensin reservoirs and have lost the antimicrobial properties of the ancestral insect defensins. The lepidopteran X-tox protein family will provide a valuable and tractable model to improve our knowledge on the molecular evolution of defensins, a class of innate immune effectors largely distributed over the three eukaryotic kingdoms

Correspondance auteur: E. Oswald E-mail: e.oswald@envt.fr Numéro article: e4855; International audience; The cycle inhibiting factor (Cif) produced by enteropathogenic and enterohemorrhagic Escherichia coli was the first cyclomodulin to be identified that is injected into host cells via the type III secretion machinery. Cif provokes cytopathic effects characterized by G(1) and G(2) cell cycle arrests, accumulation of the cyclin-dependent kinase inhibitors (CKIs) p21(waf1/cip1) and p27(kip1) and formation of actin stress fibres. The X-ray crystal structure of Cif revealed it to be a divergent member of a superfamily of enzymes including cysteine proteases and acetyltransferases that share a conserved catalytic triad. Here we report the discovery and characterization of four Cif homologs encoded by different pathogenic or symbiotic bacteria isolated from vertebrates or invertebrates. Cif homologs from the enterobacteria Yersinia pseudotuberculosis, Photorhabdus luminescens, Photorhabdus asymbiotica and the beta-proteobacterium Burkholderia pseudomallei all induce cytopathic effects identical to those observed with Cif from pathogenic E. coli. Although these Cif homologs are remarkably divergent in primary sequence, the catalytic triad is strictly conserved and was shown to be crucial for cell cycle arrest, cytoskeleton reorganization and CKIs accumulation. These results reveal that Cif proteins form a growing family of cyclomodulins in bacteria that interact with very distinct hosts including insects, nematodes and humans

International audience; Lysozyme is an important component of the insect non-specific immune response against bacteria that is characterized by its ability to break down bacterial cell-walls. By searching an EST database from the fall armyworm, Spodoptera frugiperda (Negre et al., 2006), we identified five sequences encoding proteins of the lysozyme family. The deduced protein sequences corresponded to three classical c-type lysozymes Sf-Lys1, Sf-Lys2 and Sf-Lys3, and two lysozyme-like proteins, Sf-LLP1 and Sf-LLP2. Sf-Lys1 was purified from the hemolymph of Escherichia coli-challenged S. frugiperda larvae. The mature protein had a molecular mass of 13.975 Da with an isoelectric point of 8.77 and showed 98.3% and 96.7% identity with lysozymes from Spodoptera litura and Spodoptera exigua, respectively. As the other insect lysozymes, Sf-Lys1 was active against Gram positive bacteria such as Micrococcus luteus but also induced a slight permeabilization of the inner membrane of E. coli. Genes encoding these five Sf-Lys or Sf-LLPs were differentially up-regulated in three immune-competent tissues (hemocytes, fat body and gut) after challenges with non-pathogenic bacteria, E. coli and M. luteus, or entomopathogenic bacterium, Photorhabdus luminescens. Sf-Lys1 and Sf-Lys2 were mainly induced in fat body in the presence of E. coli or P. luminescens. Sf-Lys3, which had an acidic isoelectric point, was found to be the most up-regulated of all five Sf-Lys or Sf-LLPs in hemocytes and gut after challenge with P. luminescens. More molecular data are now available to investigate differences in physiological functions of these different members of the lysozyme superfamily.

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus , which is both an opportunistic human pathogen and an insect pathogen. The Δ dlt Bc mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda . Δ dlt Bc was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis . We detected the dlt operon in three gram-negative genera: Erwinia ( Erwinia carotovora ), Bordetella ( Bordetella pertussis , Bordetella parapertussis , and Bordetella bronchiseptica ), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in Δ dlt Bc ). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.

Correspondance auteur: S. Jaoua E-mail: samir.jaoua@cbs.rnrt.tn; International audience; Photorhabdus temperata and Bacillus thuringiensis are entomopathogenic bacteria exhibiting toxicities against different insect larvae. Vegetative Insecticidal Protein Vip3LB is a Bacillus thuringiensis insecticidal protein secreted during the vegetative growth stage exhibiting lepidopteran specificity. In this study, we focused for the first time on the heterologous expression of vip3LB gene in Photorhabdus temperata strain K122. Firstly, Western blot analyses of whole cultures of recombinant Photorhabdus temperata showed that Vip3LB was produced and appeared lightly proteolysed. Cellular fractionation and proteinase K proteolysis showed that in vitro-cultured recombinant Photorhabdus temperata K122 accumulated Vip3LB in the cell and appeared not to secrete this protein. Oral toxicity of whole cultures of recombinant Photorhabdus temperata K122 strains was assayed on second-instar larvae of Ephestia kuehniella, a laboratory model insect, and the cutworm Spodoptera littoralis, one of the major pests of many important crop plants. Unlike the wild strain K122, which has no effect on the larval growth, the recombinant bacteria expressing vip3LB gene reduced or stopped the larval growth. These results demonstrate that the heterologous expression of Bacillus thuringiensis vegetative insecticidal protein-encoding gene vip3LB in Photorhabdus temperata could be considered as an excellent tool for improving Photorhabdus insecticidal activities

Photorhabdus species are gram-negative entomopathogenic bacteria of the family Enterobacteriaceae . Among the different members of the genus, one species, Photorhabdus asymbiotica , is a pathogen of both insects and humans. The pathogenicity mechanisms of this bacterium are unknown. Here we show that P. asymbiotica is a facultative intracellular pathogen that is able to replicate inside human macrophage-like cells. Furthermore, P. asymbiotica was shown for the first time in an intracellular location after insect infection. We also demonstrated that among Australian and American clinical isolates, only the Australian strains were able to invade nonphagocytic human cells. In cell culture infection experiments, Australian clinical isolates as well as cell-free bacterial culture supernatant induced strong apoptosis of a macrophage cell line at 6 h postinfection. American isolates also induced cellular death, but much later than that induced by Australian ones. Mammalian cultured cells analyzed for key features of apoptosis displayed apoptotic nuclear morphology, activation of the initiator caspases 8 and 9 and the executioner caspases 3 and 7, and poly(ADP-ribose) polymerase proteolysis, suggesting activation of both the intrinsic and extrinsic apoptotic pathways.

International audience; Isolation and identification of native nematode-bacterial associations in the field are necessary for successful control of endemic pests in a particular location. No study has yet been undertaken to recover and identify EPN in metropolitan France. In the present paper, we provide results of a survey of EPN and their symbiotic bacteria conducted in Herault and Gard regions in Southern France. Molecular characterization of isolated nematodes depicted three different Steinernema species and one Heterorhabditis species, H. bacteriophora. Steinernema species recovered were identified as: S. feltiae and S. affine and an undescribed species. Xenorhabdus symbionts were identified as X. bovienii for both S. feltiae and S. affine. Phylogenetic analysis placed the new undescribed Steinernema sp. as closely related to S. arenarium but divergent enough to postulate that it belongs to a new species within the "glaseri-group"., The Xenorhabdus symbiont from this Steinernema sp. was identified as X. kozodoii. All Heterorhabditis isolates recovered were diagnosed as H. bacteriophoro and their bacterial symbionts were identified as Photorhabdus luminescens. Molecular characterization of these nematodes enabled the distinction of two different H. bacteriophora strains. Bacterial symbiontic, strains of these two H. bacteriophora strains were identified as P. lurninescens ssp. kayaii and P. luminescens ssp. laumondii

International audience; Xenorhabdus nematophila, a member of the Enterobacteriaceae, kills many species of insects by strongly depressing the immune system and colonizing the entire body. A peptide cytotoxin has been purified from X. nematophila broth growth, and the cytolytic effect on insect immunocytes and hemolytic effect on mammalian red blood cells of this toxin have been described (Ribeiro, C., Vignes, M., and Brehelin, M. (2003) J. Mal. Chem. 278, 3030-3039). We show here that this toxin, Xenorhabdus alpha-xenorhabdolysin (Xax), triggers apoptosis in both insect and mammalian cells. We also report the cloning and sequencing of two genes, xaxAB, encoding this toxin in X. nematophila. The expression of both genes in recombinant Escherichia coli led to the production of active cytotoxin/hemolysin. However, hemolytic activity was observed only if the two peptides were added in the appropriate order. Furthermore, we report here that inactivation of xaxAB genes in X. nematophila abolished the major cytotoxic activity present in broth growth, called C I. We also show that these genes are present in various entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus, in Pseudomonas entomophila, in the human pathogens Yersinia enterocolitica and Proteus mirabilis, and in the plant pathogen Pseudomonas syringae. This toxin cannot be classified in any known family of cytotoxins on the basis of amino acid sequences, locus organization, and activity features. It is, therefore, probably the prototype of a new family of binary toxins.

International audience; The free-living soil nematode Caenorhabditis elegans interacts with diverse microorganisms in its natural habitat. These microorganisms may serve as a food source or represent a harmful threat. As such, they constitute one of the most important ecological factors of the worm's natural environment. In this study, we examined the interaction between two natural isolates of C. elegans and three natural isolates of the entomopathogenic bacterium Photorhabdus luminescens. Two of the tested P. luminescens strains were clearly pathogenic. They consistently reduced the worm's developmental, survival and reproductive rate, most likely through the production of nematicidal compounds. The third natural isolate tested did not decrease worm fitness. These differences could be associated with a deletion of one gene (tcdA4) (∼7kb) of the toxin complex D pathogenicity island, which was found in only the non-pathogenic strain. Our study additionally highlights the importance of behaviour in defence. Caenorhabditis elegans showed two distinct avoidance behaviours towards the pathogenic but not the non-pathogenic P. luminescens strains. In particular, nematodes physically escaped and also reduced the ingestion of pathogenic bacteria. Our results strongly suggest that C. elegans possesses the capacity to distinguish between and subsequently respond to harmful and non-harmful strains of the same bacterial species. The underlying mechanisms of such interactions are currently unknown. Their dissection will represent a major challenge for future research and should enhance our knowledge of the ecology of this important model organism in biological research

Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae , several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis , L. garvieae , Lactococcus l ., S. iniae , S. dysgalactiae , S. parauberis , S. agalactiae , Carnobacterium spp., the Enterococcus “ faecium ” group, a heterogeneous Enterococcus -like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus , V. salmoninarum , and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately.

Bacterial virulence is an integrative process that may involve quorum sensing. In this work, we compared by global expression profiling the wild-type entomopathogenic Photorhabdus luminescens subsp. laumondii TT01 to a luxS- deficient mutant unable to synthesize the type 2 quorum-sensing inducer AI-2. AI-2 was shown to regulate more than 300 targets involved in most compartments and metabolic pathways of the cell. AI-2 is located high in the hierarchy, as it controls the expression of several transcriptional regulators. The regulatory effect of AI-2 appeared to be dose dependent. The luxS- deficient strain exhibited decreased biofilm formation and increased type IV/V pilus-dependent twitching motility. AI-2 activated its own synthesis and transport. It also modulated bioluminescence by regulating the synthesis of spermidine. AI-2 was further shown to increase oxidative stress resistance, which is necessary to overcome part of the innate immune response of the host insect involving reactive oxygen species. Finally, we showed that the luxS- deficient strain had attenuated virulence against the lepidopteran Spodoptera littoralis . We concluded that AI-2 is involved mainly in early steps of insect invasion in P. luminescens .

The bacterium Photorhabdus establishes a highly specific association with Heterorhabditis , its nematode host. Photorhabdus strains associated with Heterorhabditis bacteriophora or Heterorhabditis megidis were compared using a Photorhabdus DNA microarray. We describe 31 regions belonging to the Photorhabdus flexible gene pool. Distribution analysis of regions among the Photorhabdus genus identified loci possibly involved in nematode specificity.

The composition and activities of the faecal microbiota in twelve healthy subjects analysed in a single open study were monitored before (1-week baseline step), during (10d supplementation step) and after (10d follow-up step) the ingestion of a fermented milk containingLactobacillus caseiDN-114001. Fluorescentin situhybridisation with group-specific DNA probes, real-time PCR usingL. paracaseigroup-specific primers and temporal temperature gradient gel electrophoresis (TTGE) using group-specific primers were carried out, together with bacterial enzyme activity and metabolite analyses to monitor the structure and activities of the faecal microbiota.L. caseiDNA was detected in the faeces of all of the subjects by TTGE after 10d supplementation. Its quantification by real-time PCR showed a 1000-fold increase during the test step compared with initial levels. No major modification in either the dominant members of the faecal microbiota or their activities was observed during the trial. In conclusion, the short-term consumption of a milk product containingL. caseiDN-114001 was accompanied by a high, transient increase in the quantity of this strain in the faeces of all of the subjects without markedly affecting biochemical or bacteriological factors.

We investigated the diversity of a collection of 76Xenorhabdusstrains, isolated from at least 27 species ofSteinernemanematodes and collected in 32 countries, using three complementary approaches: 16S rRNA gene sequencing, molecular typing and phenotypic characterization. The 16S rRNA gene sequences of theXenorhabdusstrains were highly conserved (similarity coefficient >95 %), suggesting that the common ancestor of the genus probably emerged between 250 and 500 million years ago. Based on comparisons of the 16S rRNA gene sequences, we identified 13 groups and seven unique sequences. This classification was confirmed by analysis of molecular typing profiles of the strains, leading to the classification of new isolates into theXenorhabdusspecies described previously and the description of ten novelXenorhabdusspecies:Xenorhabdus cabanillasiisp. nov. (type strain USTX62T=CIP 109066T=DSM 17905T),Xenorhabdus doucetiaesp. nov. (type strain FRM16T=CIP 109074T=DSM 17909T),Xenorhabdus griffiniaesp. nov. (type strain ID10T=CIP 109073T=DSM 17911T),Xenorhabdus hominickiisp. nov. (type strain KE01T=CIP 109072T=DSM 17903T),Xenorhabdus koppenhoeferisp. nov. (type strain USNJ01T=CIP 109199T=DSM 18168T),Xenorhabdus kozodoiisp. nov. (type strain SaVT=CIP 109068T=DSM 17907T),Xenorhabdus mauleoniisp. nov. (type strain VC01T=CIP 109075T=DSM 17908T),Xenorhabdus miraniensissp. nov. (type strain Q1T=CIP 109069T=DSM 17902T),Xenorhabdus romaniisp. nov. (type strain PR06-AT=CIP 109070T=DSM 17910T) andXenorhabdus stockiaesp. nov. (type strain TH01T=CIP 109067T=DSM 17904T). TheXenorhabdusstrains studied here had very similar phenotypic patterns, but phenotypic features nonetheless differentiated the following species:X. bovienii,X. cabanillasii,X. hominickii,X. kozodoii,X. nematophila,X. poinariiandX. szentirmaii. Based on phenotypic analysis, we identified two major groups of strains. Phenotypic group GAcomprised strains able to grow at temperatures of 35–42 °C, whereas phenotypic group GBcomprised strains that grew at temperatures below 35 °C, suggesting that someXenorhabdusspecies may be adapted to tropical or temperate regions and/or influenced by the growth and development temperature of their nematode host.

Dickeya dadantii ( Erwinia chrysanthemi ) is a phytopathogenic bacterium causing soft rot diseases on many crops. The sequencing of its genome identified four genes encoding homologues of the Cyt family of insecticidal toxins from Bacillus thuringiensis , which are not present in the close relative Pectobacterium carotovorum subsp. atrosepticum . The pathogenicity of D. dadantii was tested on the pea aphid Acyrthosiphon pisum , and the bacterium was shown to be highly virulent for this insect, either by septic injury or by oral infection. The lethal inoculum dose was calculated to be as low as 10 ingested bacterial cells. A D. dadantii mutant with the four cytotoxin genes deleted showed a reduced per os virulence for A. pisum , highlighting the potential role of at least one of these genes in pathogenicity. Since only one bacterial pathogen of aphids has been previously described ( Erwinia aphidicola ), other species from the same bacterial group were tested. The pathogenic trait for aphids was shown to be widespread, albeit variable, within the phytopathogens, with no link to phylogenetic positioning in the Enterobacteriaceae . Previously characterized gut symbionts from thrips ( Erwinia / Pantoea group) were also highly pathogenic to the aphid, whereas the potent entomopathogen Photorhabdus luminescens was not. D. dadantii is not a generalist insect pathogen, since it has low pathogenicity for three other insect species ( Drosophila melanogaster , Sitophilus oryzae , and Spodoptera littoralis ). D. dadantii was one of the most virulent aphid pathogens in our screening, and it was active on most aphid instars, except for the first one, probably due to anatomical filtering. The observed difference in virulence toward apterous and winged aphids may have an ecological impact, and this deserves specific attention in future research.

Correspondance: jean-francois.bernardet@jouy.inra.fr; International audience; Members of most Chryseottacterium species occur in aquatic environments or food products, while strains of some other species are pathogenic to humans and animals. A collection of 52 Chryseobacterium sp. strains isolated from diseased fish, one frog isolate and 22 reference strains were included in a polyphasic taxonomy study. Fourteen clusters of strains were delineated following the comparison of whole-cell protein profiles. Most of these clusters were confirmed when the phenotypic and RAPD profiles and the 16S rRNA gene sequences were compared. Fatty acid composition helped differentiate the Chryseobacterium strains from members of related genera. None of the fish isolates could be allocated to the two species previously reported from fish but two isolates belonged to C. joostei, while the frog isolate was identified as Elizabethkingia meningoseptica, a human pathogen previously included in the genus Chryseobacterium. Three clusters grouping from 3 to 13 isolates will probably constitute the core of new Chryseobacterium species but all other isolates occupied separate or uncertain positions in the genus. This study further demonstrated the overall high similarity displayed by most Chryseottacterium strains whatever the technique used and the resulting difficulty in delineating new species in the genus. Members of this bacterial group should be considered potential emergent pathogens in various fish and frog species, farming conditions and geographical areas

Correspondance auteur: M. Brehélin brehelin@univ-montp2.fr; Haemocytes are the main immunocompetent cells in insect cellular immune reactions. Here, we show that in Spodoptera littoralis, granular haemocytes are the primary phagocyte haemocytes, both in vivo and in vitro. The "trigger" and "zipper" modes of engulfment known in mammal macrophages are active, in vivo, in S. littoralis granular haemocytes, together with macropinocytosis. Lipopolysaccharide as well as lipoteichoic acid inhibit the binding of both Gram-positive (Corynebacterium xerosis) and Gramnegative (Escherichia coli) bacteria on granular haemocytes. In addition, different ligands can inhibit the binding of E coli. Most of these inhibitors are known as ligands of scavenger receptors in mammal macrophages and we hypothesise that one of the receptors present on S. littoralis granular haemocytes could be a scavenger-like receptor

The entomopathogenic nematodeSteinernema carpocapsaeis mutualistically associated with the bacteriumXenorhabdus nematophila. Infective Juveniles (IJs) transportX. nematophilacells that provide them with good conditions to reproduce within the insect. In the laboratory, long term stationary-phase culture conditions sometimes leadX. nematophila's variant 1 cells, which were previously isolated from the worms, to spontaneously and irreversibly change into a new phenotypic variant (variant 2). In this paper, we tested the ability of each phenotypic variant to (i) be transmitted by IJs, (ii) to optimize the worm's fitness within the insect, and (iii) to counteract the effect of closely related antagonistic bacteria previously shown as being able to totally preventS. carpocapsae's reproduction within the insect. We found that IJs did associate with cells of both phenotypes but that the variant 2 cells were preferentially retained by the nematodes when both variants were present in the insect. Both phenotypic variants led to the same fitness ofS. carpocapsaein insects not infected by antagonistic bacteria. In insects infected by antagonistic bacteria, both variants were able to provide protection toS. carpocapsae. Nevertheless, this protection depended on the phenotypic variant and the antagonistic bacteria that were co-injected into the insect. Further analysis conducedin vitroshowed that this variability could be partly linked to the sensitivity of each antagonistic bacterium to xenorhabdicin, produced byX. nematophila.

The level of specialization of the entomopathogenic nematode Steinernema scapterisci with its native Xenorhabdus symbiont was investigated by testing (1) the influence of non-native bacterial strains on nematode fitness within an insect-host (Galleria mellonella) and (2) specificity of the association between the nematode infective juveniles and non-native bacteria. All non-native Xenorhabdus spp. or Photorhabdus spp. strains tested were mutualistically associated with other entomopathogenic nematodes in nature. We showed that most of the Xenorhabdus spp. strains tested led to an insignificant difference of the nematode's fitness compared to the one obtained with the native bacterium. Conversely, Photorhabdus spp. strains almost entirely abolished nematode reproduction. The phylogenetic analysis of bacterial strains tested, showed that there was a negative correlation between S. scapterisci's reproduction rate with a bacterial strain and the genetic distance of this bacterial strain from the native one. We also showed that the native bacterium was the only one which was transmitted by S. scapterisci's infective juveniles. All these results, suggested a specialization between S. scapterisci and its native Xenorhabdus. As the same phenomenon was already demonstrated in the association between S. carpocapsae and X. nematophila, specialization between partners would not be an exception in entomopathogenic nematode-bacteria interactions. Nevertheless, S. scapterisci showed a dramatically higher compatibility with non-native Xenorhabdus spp. strains than did S. carpocapsae, suggesting differences in the co-evolutionary processes between nematodes and bacteria in these two model systems.

International audience; Photorhabdus is an entomopathogenic bacterium belonging to the Enterobacteriaceae. The genome of the TT01 strain of Photorhabdus luminescens was recently sequenced and a large number of toxin-encoding genes were found. Genomic analysis predicted the presence on the chromosome of genes encoding a type three secretion system (TTSS), the main role of which is the delivery of effector proteins directly into eukaryotic host cells. We report here the functional characterization of the TTSS. The locus identified encodes the secretion/translocation apparatus, gene expression regulators and an effector protein - LopT - homologous to the Yersinia cysteine protease cytotoxin YopT. Heterologous expression in Yersinia demonstrated that LopT was translocated into mammal cells in an active form, as shown by the appearance of a form of the RhoA GTPase with modified electrophoretic mobility. In vitro study showed that recombinant LopT was able to release RhoA and Rac from human and insect cell membrane. In vivo assays of infection of the cutworm Spodoptera littoralis and the locust Locusta migratoria with a TT01 strain carrying a translational fusion of the lopT gene with the gfp reporter gene revealed that the lopT gene was switched on only at sites of cellular defence reactions, such as nodulation, in insects. TTSS-mutant did not induce nodule formation and underwent phagocytosis by insect macrophage cells, suggesting that the LopT effector plays an essential role in preventing phagocytosis and indicating an unexpected link between TTSS expression and the nodule reaction in insects

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus . When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria , also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.

50 ref.; International audience; Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.

Lysogeny has previously been described in the entomopathogenic bacteria of the genus Xenorhabdus. Screening of a X. nematophila prophage DNA library on blood agar resulted in the identification of a 5.7-kb locus that caused a haemolytic phenotype when cloned in Escherichia coli, but not in the E. coli sheA null mutant, lacking the SheA cryptic haemolysin. This locus exhibited similarity to lysis genes from lambdoid phages. In particular, it encoded a functional holin able to complement a λ Sam7 mutant. It is the second time that a locus encoding a functional holin is shown to reveal the SheA haemolytic phenotype in E. coli. The possible role of the holin in extracellular release of SheA is discussed.

Article en Open Access; International audience; The trade-off hypothesis proposes that the evolution of pathogens' virulence is shaped by a link between virulence and contagiousness. This link is often assumed to come from the fact that pathogens are contagious only if they can reach high parasitic load in the infected host. In this paper we present an experimental test of the hypothesis that selection on fast replication can affect virulence. In a serial passage experiment, we selected 80 lines of the bacterial insect-pathogen Xenorhabdus nematophila to multiply fast in an artificial culture medium. This selection resulted in shortened lag phase in our selected bacteria. We then injected these bacteria into insects and observed an increase in virulence. This could be taken as a sign that virulence in Xenorhabdus is linked to fast multiplication. But we found, among the selected lineages, either no link or a positive correlation between lag duration and virulence: the most virulent bacteria were the last to start multiplying. We then surveyed phenotypes that are under the control of the flhDC super regulon, which has been shown to be involved in Xenorhabdus virulence. We found that, in one treatment, the flhDC regulon has evolved rapidly, but that the changes we observed were not connected to virulence. All together, these results indicate that virulence is, in Xenorhabdus as in many other pathogens, a multifactorial trait. Being able to grow fast is one way to be virulent. But other ways exist which renders the evolution of virulence hard to predict

International audience; A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif) family of effector proteins, identified in a growing number of pathogens that harbour functional type III secretion systems and have a wide host range, arrest the eukaryotic cell cycle. Here, the crystal structures of Cifs from the insect pathogen/nematode symbiont Photorhabdus luminescens (a γ-proteobacterium) and human pathogen Burkholderia pseudomallei (a β-proteobacterium) are presented. Both of these proteins adopt an overall fold similar to the papain sub-family of cysteine proteases, as originally identified in the structure of a truncated form of Cif from Enteropathogenic E. coli (EPEC), despite sharing only limited sequence identity. The structure of an N-terminal region, referred to here as the ‘tail-domain’ (absent in the EPEC Cif structure), suggests a surface likely to be involved in host-cell substrate recognition. The conformation of the Cys-His-Gln catalytic triad is retained, and the essential cysteine is exposed to solvent and addressable by small molecule reagents. These structures and biochemical work contribute to the rapidly expanding literature on Cifs, and direct further studies to better understand the molecular details of the activity of these proteins.