Xenorhabdus nematophila bacteria shift from mutualistic to virulent Lrp-dependent phenotypes within the receptacles of Steinernema carpocapsae insect-infective stage nematodes.

Xenorhabdus nematophila bacteria are mutualists of Steinernema carpocapsae nematodes and pathogens of insects. Xenorhabdus nematophila exhibits phenotypic variation between insect virulence (V) and the mutualistic (M) support of nematode reproduction and colonization initiation in the infective juvenile (IJ) stage nematode that carries X. nematophila between insect hosts. The V and M phenotypes occur reciprocally depending on levels of the transcription factor Lrp: high-Lrp expressors are M+V- while low-Lrp expressors are V+M-. We report here that variable (wild type) or fixed high-Lrp expressors also are optimized, relative to low- or no-Lrp expressors, for colonization of additional nematode stages: juvenile, adult and pre-transmission infective juvenile (IJ). In contrast, we found that after the bacterial population had undergone outgrowth in mature IJs, the advantage for colonization shifted to low-Lrp expressors: fixed low-Lrp expressors (M-V+) and wild type (M+V+) exhibited higher average bacterial CFU per IJ than did high-Lrp (M+V-) or no-Lrp (M-V-) strains. Further, the bacterial population becomes increasingly low-Lrp expressing, based on expression of an Lrp-dependent fluorescent reporter, as IJs age. These data support a model that virulent X. nematophila have a selective advantage and accumulate in aging IJs in advance of exposure to insect hosts in which this phenotype is necessary.

Virulent secondary metabolites of entomopathogenic bacteria genera, Xenorhabdus and Photorhabdus, inhibit phospholipase A2 to suppress host insect immunity.

BACKGROUND: Xenorhabdus and Photorhabdus are entomopathogenic bacteria that cause septicemia and toxemia in insects. They produce secondary metabolites to induce host immunosuppression. Their metabolite compositions vary among bacterial species. Little is known about the relationship between metabolite compositions and the bacterial pathogenicity. The objective of this study was to compare pathogenicity and production of secondary metabolites of 14 bacterial isolates (species or strains) of Xenorhabdus and Photorhabdus. RESULTS: All bacterial isolates exhibited insecticidal activities after hemocoelic injection to Spodoptera exigua (a lepidopteran insect) larvae, with median lethal doses ranging from 168.8 to 641.3 CFU per larva. Bacterial infection also led to immunosuppression by inhibiting eicosanoid biosynthesis. Bacterial culture broth was fractionated into four different organic extracts. All four organic extracts of each bacterial species exhibited insecticidal activities and resulted in immunosuppression. These organic extracts were subjected to GC-MS analysis which predicted 182 compounds, showing differential compositions for 14 bacteria isolates. There were positive correlations between total number of secondary metabolites produced by each bacterial culture broth and its bacterial pathogenicity based on immunosuppression and insecticidal activity. From these correlation results, 70 virulent compounds were selected from secondary metabolites of high virulent bacterial isolates by deducting those of low virulent bacterial isolates. These selected virulent compounds exhibited significant immunosuppressive activities by inhibiting eicosanoid biosynthesis. They also exhibited relatively high insecticidal activities. CONCLUSION: Virulence variation between Xenorhabdus and Photorhabdus is determined by their different compositions of secondary metabolites, of which PLA2 inhibitors play a crucial role.

ngrA-dependent natural products are required for interspecies competition and virulence in the insect pathogenic bacterium Xenorhabdus szentirmaii.

Xenorhabdus species are symbionts of entomopathogenic nematodes and pathogens of susceptible insects. Nematodes enter insect hosts and perforate the midgut to invade the haemocoel where Xenorhabdus bacteria are released transitioning to their pathogenic stage. During nematode invasion microbes from the insect gut translocate into the haemocoel. Different species of nematodes carrying specific strains of Xenorhabdus can also invade the same insect. Xenorhabdus species thereby compete for nutrients and space with both related strains and non-related gut microbes. While Xenorhabdus species produce diverse antimicrobial compounds in complex media, their functions in insect hosts are not well understood. We show that Xenorhabdus szentirmaii produced ngrA-dependent antibiotics that were active against both gut-derived microbes and Xenorhabdus nematophila whereas antibiotics of X. nematophila were not active against X. szentirmaii. X. nematophila growth was inhibited in co-cultures with wild-type X. szentirmaii in medium that mimics insect haemolymph. An antibiotic-deficient strain of X. szentirmaii was created by inactivating the ngrA gene that encodes the enzyme that attaches the 4' phosphopantetheinyl moiety to non-ribosomal peptide synthetases involved in antibiotic biosynthesis. X. nematophila growth was not inhibited in co-cultures with the ngrA strain. The growth of X. nematophila was suppressed in Manduca sexta co-injected with wild-type X. szentirmaii and X. nematophila. In contrast, growth of X. nematophila was not suppressed in M. sexta co-injected with the ngrA strain. Two unique compounds were detected by MALDI-TOF MS analysis in haemolymph infected with the wild-type but not with the ngrA strain. Finally, killing of M. sexta was delayed in insects infected with the ngrA strain. These findings indicate that in the insect host X. szentirmaii produces ngrA-dependent products involved in both interspecies competition and virulence.

Variation in pathogenicity of different strains of Xenorhabdus nematophila; Differential immunosuppressive activities and secondary metabolite production.

Xenorhabdus nematophila, an entomopathogenic bacterium, is mutualistic with the nematode Steinernema carpocapsae. The bacterium produces secondary metabolites to inhibit target insect phospholipase A2 (PLA2) and induce immunosuppression, which is required for the pathogenicity of this bacterium-nematode complex. However, it was unclear if immunosuppressive intensity of the bacteria was correlated with their insecticidal potency. We compared six different X. nematophila strains inhibiting the immune responses of the beet armyworm (Spodoptera exigua) to explain their virulence variations. In addition to four known strains obtained from the Korean Agricultural Culture Collection, we identified two new strains (SK1 and SK2) of X. nematophila from two different isolates of S. carpocapsae. Although all six strains were virulent, they showed significant variation in median lethal bacterial dosage (LD50). The LD50 of most strains was 15-30 CFU/larva, however, the LD50 of the SK1 strain was more than two-fold higher against S. exigua larvae. Immunosuppressive activities of the six strains were measured by comparing hemocyte-spreading behavior and nodule formation; the SK1 strain was significantly less potent than other bacterial strains. These suppressed hemocyte behaviors were recovered by adding arachidonic acid (a catalytic product of PLA2) into all six strains. Bacterial culture broth was fractionated with different organic solvents and the ability to inhibit immune response and PLA2 activity were assessed. All organic extracts had immunosuppressive activities and PLA2-inhibitory activities. GC-MS analysis showed that these organic extracts possessed a total of 87 different compounds. There were variations in chemical components among the six bacterial strains. Organic extracts of SK1 strain, which exhibited the lowest virulence, contained the least number of secondary metabolites.

Sand crickets (Gryllus firmus) have low susceptibility to entomopathogenic nematodes and their pathogenic bacteria.

The entomopathogenic nematode, Steinernema scapterisci, a specialist parasite of crickets, has been successfully used to combat the southern mole cricket, Neoscapteriscus borellii, which is an invasive pest of turf grass. As an entomopathogenic nematode, S. scapterisci causes rapid death of the insects it infects and uses bacteria to facilitate its parasitism. However, our understanding of the relative contributions of the nematode, S. scapterisci, and its bacterial symbiont, Xenorhabdus innexi, to parasitism remains limited. Here we utilized the sand cricket, Gryllus firmus, as a model host to evaluate the contributions of the EPNs S. scapterisci and S. carpocapsae, as well as their symbiotic bacteria, X. innexi and X. nematophila, respectively, to the virulence of the nematode-bacterial complex. We found that G. firmus has reduced susceptibility to infection from both S. scapterisci and the closely related generalist parasite S. carpocapsae, but that S. scapterisci is much more virulent than S. carpocapsae. Further, we found that N. borellii has reduced susceptibility to X. nematophila, and that G. firmus has reduced susceptibility to X. nematophila, X. innexi, and Serratia marcescens, much more so than other insects that have been studied. We found that the reduced susceptibility of G. firmus to bacterial infection is dependent on development, with adults being less susceptible to infection than nymphs. Our data provide evidence that unlike other EPNs, the virulence of S. scapterisci to crickets is dependent on the nematode rather than the bacterial symbiont that it carries and we speculate that S. scapterisci may be evolving independence from X. innexi.

Screening a fosmid library of Xenorhabdus stockiae HN_xs01 reveals SrfABC toxin that exhibits both cytotoxicity and injectable insecticidal activity.

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with the nematodes of the Steinernematid family, are known to produce several toxic proteins that interfere with the cellular immune responses of insects. In order to identify novel cytotoxins from Xenorhabdus spp., a fosmid library of X. stockiae HN_xs01 strain was constructed and the cytotoxicity of fosmid clones was tested against insect midgut CF-203 cells. An FS2 clone bearing the srfABC operon, originally identified in Salmonella enterica, exhibited excellent cytotoxicity against CF-203 cells. The srfABC operon alone exhibited cytotoxic effects and all three components of SrfABC toxin were essential for full cytotoxicity. Immunofluorescence studies showed that SrfABC toxin could depolymerize microtubules and disrupt mitochrondria. Flow cytometer analysis demonstrated that SrfABC toxin significantly induced G2/M phase arrest and apoptosis in CF-203 cells. Furthermore, SrfABC toxin exhibits highly injectable insecticidal activity against Helicoverpa armigera larvae. As is often found in host-associated microorganisms, SrfABC toxin is thought to play an important role in host colonization.

Differential immunosuppression by inhibiting PLA2 affects virulence of Xenorhabdus hominickii and Photorhabdus temperata temperata.

Immunity negatively influences bacterial pathogenicity. Eicosanoids mediate both cellular and humoral immune responses in insects. This study tested a hypothesis that differential bacterial virulence of Xenorhabdus/Photorhabdus is dependent on their inhibitory activity against phospholipase A2 (PLA2) activity. P. temperata subsp. temperata ('Ptt') was more than 40 times more potent than X. hominickii ('Xh'). Although both bacteria suppressed cellular immune responses, Ptt infection suppressed hemocyte nodule formation much more than Xh infection. Their differential immunosuppression appeared to be induced by their secondary metabolites because organic extracts of Ptt-cultured broth exhibited higher inhibitory activities against cellular immune responses than Xn-cultured broth extracts. Humoral immune responses were analyzed by measuring expression levels of 11 antimicrobial peptide (AMP) genes. Among inducible AMPs in hemocytes and fat body, higher number and more kinds of AMPs exhibited lower expression levels in Ptt infection than those in Xh infection. Suppressed immune responses induced by Ptt or Xh infection were significantly rescued by the addition of a catalytic product of PLA2, suggesting that PLA2 was a common inhibitory target. In fact, Ptt infection inhibited PLA2 activity more strongly than Xh infection. RNA interference of a PLA2 gene decreased its expression and significantly increased bacterial virulence. Moreover, addition of PLA2 inhibitor to Xh infection enhanced its virulence, similar to virulence level of Ptt infection. These results suggest that variation in Xenorhabdus/Photorhabdus bacterial virulence can be explained by their differential inhibitory activities against host insect PLA2.

The insect pathogenic bacterium Xenorhabdus innexi has attenuated virulence in multiple insect model hosts yet encodes a potent mosquitocidal toxin.

BACKGROUND: Xenorhabdus innexi is a bacterial symbiont of Steinernema scapterisci nematodes, which is a cricket-specialist parasite and together the nematode and bacteria infect and kill crickets. Curiously, X. innexi expresses a potent extracellular mosquitocidal toxin activity in culture supernatants. We sequenced a draft genome of X. innexi and compared it to the genomes of related pathogens to elucidate the nature of specialization. RESULTS: Using green fluorescent protein-expressing X. innexi we confirm previous reports using culture-dependent techniques that X. innexi colonizes its nematode host at low levels (~3-8 cells per nematode), relative to other Xenorhabdus-Steinernema associations. We found that compared to the well-characterized entomopathogenic nematode symbiont X. nematophila, X. innexi fails to suppress the insect phenoloxidase immune pathway and is attenuated for virulence and reproduction in the Lepidoptera Galleria mellonella and Manduca sexta, as well as the dipteran Drosophila melanogaster. To assess if, compared to other Xenorhabdus spp., X. innexi has a reduced capacity to synthesize virulence determinants, we obtained and analyzed a draft genome sequence. We found no evidence for several hallmarks of Xenorhabdus spp. toxicity, including Tc and Mcf toxins. Similar to other Xenorhabdus genomes, we found numerous loci predicted to encode non-ribosomal peptide/polyketide synthetases. Anti-SMASH predictions of these loci revealed one, related to the fcl locus that encodes fabclavines and zmn locus that encodes zeamines, as a likely candidate to encode the X. innexi mosquitocidal toxin biosynthetic machinery, which we designated Xlt. In support of this hypothesis, two mutants each with an insertion in an Xlt biosynthesis gene cluster lacked the mosquitocidal compound based on HPLC/MS analysis and neither produced toxin to the levels of the wild type parent. CONCLUSIONS: The X. innexi genome will be a valuable resource in identifying loci encoding new metabolites of interest, but also in future comparative studies of nematode-bacterial symbiosis and niche partitioning among bacterial pathogens.

Identification and occurrence of the hydroxamate siderophores aerobactin, putrebactin, avaroferrin and ochrobactin C as virulence factors from entomopathogenic bacteria.

Effective iron acquisition and fine-tuned intracellular iron storage systems are the main prerequisites for a successful host invasion by a pathogen. Bacteria have developed several different strategies to sequester this essential element from their environment, one relies on the secretion of low molecular weight compounds with high affinity for ferric iron, the so-called siderophores. Here, we report hydroxamate siderophore structures produced by entomopathogenic bacteria of the species Xenorhabdus and Photorhabdus, which are known for their potential to produce bioactive natural products, required for their role as nematode symbiont and insect pathogen. Four siderophores could be identified, namely aerobactin, putrebactin, avaroferrin and ochrobactin C, which was found previously only in marine bacteria. While the putrebactin and avaroferrin producing biosynthesis gene cluster (BGC) is more widespread and most likely was present in a common ancestor of these bacteria, the aerobactin and ochrobactin producing BGC was probably taken up by a few strains individually. For aerobactin a role in virulence towards Galleria mellonella larvae is shown.

Variable virulence phenotype of Xenorhabdus bovienii (γ-Proteobacteria: Enterobacteriaceae) in the absence of their vector hosts.

Xenorhabdus bovienii bacteria have a dual lifestyle: they are mutualistic symbionts to many species of Steinernema nematodes and are pathogens to a wide array of insects. Previous studies have shown that virulence of X.bovienii-Steinernema spp. pairs decreases when the nematodes associate with non-cognate bacterial strains. However, the virulence of the X. bovienii strains alone has not been fully investigated. In this study, we characterized the virulence of nine X. bovienii strains in Galleria mellonella and Spodoptera littoralis and performed a comparative genomic analysis to correlate observed phenotypes with strain genotypes. Two X. bovienii strains were found to be highly virulent against the tested insect hosts, while three strains displayed attenuated insect virulence. Comparative genomic analyses revealed the presence of several clusters present only in virulent strains, including a predicted type VI secretion system (T6SS). We performed intra-species-competition assays, and showed that the virulent T6SS+ strains generally outcompeted the less virulent T6SS- strains. Thus, we speculate that the T6SS in X. bovienii may be another addition to the arsenal of antibacterial mechanisms expressed by these bacteria in an insect, where it could potentially play three key roles: (1) competition against the insect host microbiota; (2) protection of the insect cadaver from necrotrophic microbial competitors; and (3) outcompeting other Xenorhabdus species and/or strains when co-infections occur.

The Global Transcription Factor Lrp Is both Essential for and Inhibitory to Xenorhabdus nematophila Insecticidal Activity.

In the entomopathogenic bacterium Xenorhabdus nematophila, cell-to-cell variation in the abundance of the Lrp transcription factor leads to virulence modulation; low Lrp levels are associated with a virulent phenotype and suppression of antimicrobial peptides (AMPs) in Manduca sexta insects, while cells that lack lrp or express high Lrp levels are virulence attenuated and elicit AMP expression. To better understand the basis of these phenotypes, we examined X. nematophila strains expressing fixed Lrp levels. Unlike the lrp-null mutant, the high-lrp strain is fully virulent in Drosophila melanogaster, suggesting that these two strains have distinct underlying causes of virulence attenuation in M. sexta Indeed, the lrp-null mutant was defective in cytotoxicity against M. sexta hemocytes relative to that in the high-lrp and low-lrp strains. Further, supernatant derived from the lrp-null mutant but not from the high-lrp strain was defective in inhibiting weight gain when fed to 1st instar M. sexta These data suggest that contributors to the lrp-null mutant virulence attenuation phenotype are the lack of Lrp-dependent cytotoxic and extracellular oral growth inhibitory activities, which may be particularly important for virulence in D. melanogaster In contrast, the high-Lrp strain was sensitive to the antimicrobial peptide cecropin, had a transient survival defect in M. sexta, and had reduced extracellular levels of insecticidal activity, measured by injection of supernatant into 4th instar M. sexta Thus, high-lrp strain virulence attenuation may be explained by its hypersensitivity to M. sexta host immunity and its inability to secrete one or more insecticidal factors.IMPORTANCE Adaptation of a bacterial pathogen to host environments can be achieved through the coordinated regulation of virulence factors that can optimize success under prevailing conditions. In the insect pathogen Xenorhabdus nematophila, the global transcription factor Lrp is necessary for virulence when injected into Manduca sexta or Drosophila melanogaster insect hosts. However, high levels of Lrp, either naturally occurring or artificially induced, cause attenuation of X. nematophila virulence in M. sexta but not D. melanogaster Here, we present evidence suggesting that the underlying cause of high-Lrp-dependent virulence attenuation in M. sexta is hypersensitivity to host immune responses and decreased insecticidal activity and that high-Lrp virulence phenotypes are insect host specific. This knowledge suggests that X. nematophila faces varied challenges depending on the type of insect host it infects and that its success in these environments depends on Lrp-dependent control of a multifactorial virulence repertoire.

High Levels of the Xenorhabdus nematophila Transcription Factor Lrp Promote Mutualism with the Steinernema carpocapsae Nematode Host.

Xenorhabdus nematophila bacteria are mutualistic symbionts of Steinernema carpocapsae nematodes and pathogens of insects. The X. nematophila global regulator Lrp controls the expression of many genes involved in both mutualism and pathogenic activities, suggesting a role in the transition between the two host organisms. We previously reported that natural populations of X. nematophila exhibit various levels of Lrp expression and that cells expressing relatively low levels of Lrp are optimized for virulence in the insect Manduca sexta The adaptive advantage of the high-Lrp-expressing state was not established. Here we used strains engineered to express constitutively high or low levels of Lrp to test the model in which high-Lrp-expressing cells are adapted for mutualistic activities with the nematode host. We demonstrate that high-Lrp cells form more robust biofilms in laboratory media than do low-Lrp cells, which may reflect adherence to host tissues. Also, our data showed that nematodes cultivated with high-Lrp strains are more frequently colonized than are those associated with low-Lrp strains. Taken together, these data support the idea that high-Lrp cells have an advantage in tissue adherence and colonization initiation. Furthermore, our data show that high-Lrp-expressing strains better support nematode reproduction than do their low-Lrp counterparts under both in vitro and in vivo conditions. Our data indicate that heterogeneity of Lrp expression in X. nematophila populations provides diverse cell populations adapted to both pathogenic (low-Lrp) and mutualistic (high-Lrp) states.IMPORTANCE Host-associated bacteria experience fluctuating conditions during both residence within an individual host and transmission between hosts. For bacteria that engage in evolutionarily stable, long-term relationships with particular hosts, these fluctuations provide selective pressure for the emergence of adaptive regulatory mechanisms. Here we present evidence that the bacterium Xenorhabdus nematophila uses various levels of the transcription factor Lrp to optimize its association with its two animal hosts, nematodes and insects, with which it behaves as a mutualist and a pathogen, respectively. Building on our previous finding that relatively low cellular levels of Lrp are optimal for pathogenesis, we demonstrate that, conversely, high levels of Lrp promote mutualistic activities with the Steinernema carpocapsae nematode host. These data suggest that X. nematophila has evolved to utilize phenotypic variation between high- and low-Lrp-expression states to optimize its alternating behaviors as a mutualist and a pathogen.

Identification and bacterial characteristics of Xenorhabdus hominickii ANU101 from an entomopathogenic nematode, Steinernema monticolum.

An entomopathogenic nematode, Steinernema monticolum, was collected in Korea. Its identity was confirmed by morphological and molecular characters. Its symbiotic bacterium, Xenorhabdus hominickii ANU101, was isolated and assessed in terms of bacterial characteristics. Sixty-eight different carbon sources were utilized by X. hominickii ANU101 out of 95 different sources from a Biolog assay. Compared to other Xenorhabdus species, X. hominickii ANU101 was relatively susceptible to high temperatures and did not grow above 34°C. Furthermore, its growth rate was much slower than other Xenorhabdus species. X. hominickii exhibited insecticidal activities against coleopteran, dipteran, and lepidopteran insect pests. The bacterial virulence was not correlated with its host nematode virulence with respect to relative insecticidal activity against target insects. X. hominickii ANU101 exhibited antibiotics tolerance. The bacterium possesses four different plasmids (Xh-P1 (104,132bp), Xh-P2 (95,975bp), Xh-P3 (88,536bp), and Xh-P4 (11,403bp)) and encodes 332 open reading frames. Subsequent predicted genes include toxin/antitoxins comprising a multidrug export ATP-binding/permease. This study reports bacterial characters of X. hominickii and its entomopathogenicity.

An entomopathogenic bacterium, Xenorhabdus hominickii ANU101, produces oxindole and suppresses host insect immune response by inhibiting eicosanoid biosynthesis.

An entomopathogenic bacterium, Xenorhabdus hominickii ANU101, was isolated from an entomopathogenic nematode, Steinernema monticolum. X. hominickii exhibited significant insecticidal activities at ≥6.6×102 colony-forming units per larva against a lepidopteran insect, Spodoptera exigua with hemocoelic injection. The insecticidal activity of X. hominickii was reduced by an addition of arachidonic acid (AA, a catalytic product of PLA2), but enhanced by an addition by dexamethasone (DEX, a specific inhibitor of PLA2). S. exigua could defend the bacterial infection by forming hemocyte nodules. However, live X. hominickii significantly reduced the hemocytic nodulation compared to similar treatment with heat-killed X. hominickii. An addition of AA to live X. hominickii significantly rescued the immunosuppression. X. hominickii also inhibited phenoloxidase activity in hemolymph of S. exigua larvae. Furthermore, the bacteria suppressed gene expressions of antimicrobial peptides, such as attacin-1, attacin-2, defensin, gallerimycin and transferrin-1 of S. exigua. An organic extract of X. hominickii-cultured broth with ethyl acetate possessed oxindole and significantly suppressed hemocyte nodulation. Again, an addition of AA diminished the inhibitory activity of the organic extract against hemocyte nodulation. Oxindole alone inhibited hemocyte nodulation and PLA2 enzyme activity. These results suggest that the entomopathogenicity of X. hominickii comes from its inhibitory activity against eicosanoid biosynthesis of target insects.

The Global Transcription Factor Lrp Controls Virulence Modulation in Xenorhabdus nematophila.

UNLABELLED: The bacterium Xenorhabdus nematophila engages in phenotypic variation with respect to pathogenicity against insect larvae, yielding both virulent and attenuated subpopulations of cells from an isogenic culture. The global regulatory protein Lrp is necessary for X. nematophila virulence and immunosuppression in insects, as well as colonization of the mutualistic host nematode Steinernema carpocapsae, and mediates expression of numerous genes implicated in each of these phenotypes. Given the central role of Lrp in X. nematophila host associations, as well as its involvement in regulating phenotypic variation pathways in other bacteria, we assessed its function in virulence modulation. We discovered that expression of lrp varies within an isogenic population, in a manner that correlates with modulation of virulence. Unexpectedly, although Lrp is necessary for optimal virulence and immunosuppression, cells expressing high levels of lrp were attenuated in these processes relative to those with low to intermediate lrp expression. Furthermore, fixed expression of lrp at high and low levels resulted in attenuated and normal virulence and immunosuppression, respectively, and eliminated population variability of these phenotypes. These data suggest that fluctuating lrp expression levels are sufficient to drive phenotypic variation in X. nematophila. IMPORTANCE: Many bacteria use cell-to-cell phenotypic variation, characterized by distinct phenotypic subpopulations within an isogenic population, to cope with environmental change. Pathogenic bacteria utilize this strategy to vary antigen or virulence factor expression. Our work establishes that the global transcription factor Lrp regulates phenotypic variation in the insect pathogen Xenorhabdus nematophila, leading to attenuation of virulence and immunosuppression in insect hosts. Unexpectedly, we found an inverse correlation between Lrp expression levels and virulence: high levels of expression of Lrp-dependent putative virulence genes are detrimental for virulence but may have an adaptive advantage in other aspects of the life cycle. Investigation of X. nematophila phenotypic variation facilitates dissection of this phenomenon in the context of a naturally occurring symbiosis.

Genome sequence and comparative analysis of a putative entomopathogenic Serratia isolated from Caenorhabditis briggsae.

BACKGROUND: Entomopathogenic associations between nematodes in the genera Steinernema and Heterorhabdus with their cognate bacteria from the bacterial genera Xenorhabdus and Photorhabdus, respectively, are extensively studied for their potential as biological control agents against invasive insect species. These two highly coevolved associations were results of convergent evolution. Given the natural abundance of bacteria, nematodes and insects, it is surprising that only these two associations with no intermediate forms are widely studied in the entomopathogenic context. Discovering analogous systems involving novel bacterial and nematode species would shed light on the evolutionary processes involved in the transition from free living organisms to obligatory partners in entomopathogenicity. RESULTS: We report the complete genome sequence of a new member of the enterobacterial genus Serratia that forms a putative entomopathogenic complex with Caenorhabditis briggsae. Analysis of the 5.04 MB chromosomal genome predicts 4599 protein coding genes, seven sets of ribosomal RNA genes, 84 tRNA genes and a 64.8 KB plasmid encoding 74 genes. Comparative genomic analysis with three of the previously sequenced Serratia species, S. marcescens DB11 and S. proteamaculans 568, and Serratia sp. AS12, revealed that these four representatives of the genus share a core set of ~3100 genes and extensive structural conservation. The newly identified species shares a more recent common ancestor with S. marcescens with 99% sequence identity in rDNA sequence and orthology across 85.6% of predicted genes. Of the 39 genes/operons implicated in the virulence, symbiosis, recolonization, immune evasion and bioconversion, 21 (53.8%) were present in Serratia while 33 (84.6%) and 35 (89%) were present in Xenorhabdus and Photorhabdus EPN bacteria respectively. CONCLUSION: The majority of unique sequences in Serratia sp. SCBI (South African Caenorhabditis briggsae Isolate) are found in ~29 genomic islands of 5 to 65 genes and are enriched in putative functions that are biologically relevant to an entomopathogenic lifestyle, including non-ribosomal peptide synthetases, bacteriocins, fimbrial biogenesis, ushering proteins, toxins, secondary metabolite secretion and multiple drug resistance/efflux systems. By revealing the early stages of adaptation to this lifestyle, the Serratia sp. SCBI genome underscores the fact that in EPN formation the composite end result - killing, bioconversion, cadaver protection and recolonization- can be achieved by dissimilar mechanisms. This genome sequence will enable further study of the evolution of entomopathogenic nematode-bacteria complexes.

Xenorhabdus bovienii CS03, the bacterial symbiont of the entomopathogenic nematode Steinernema weiseri, is a non-virulent strain against lepidopteran insects.

Xenorhabdus bacteria (γ-proteobacteria: Enterobacteriaceae) have dual lifestyles. They have a mutualistic relationship with Steinernema nematodes (Nematoda: Steinernematidae) and are pathogenic to a wide range of insects. Each Steinernema nematode associates with a specific Xenorhabdus species. However, a Xenorhabdus species can have multiple nematode hosts. For example, Xenorhabdus bovienii (Xb) colonizes at least nine Steinernema species from two different phylogenetic clades. The Steinernema-Xb partnership has been found in association with different insect hosts. Biological and molecular data on the Steinernema jollieti-Xb strain SS-2004 pair have recently been described. In particular, the Xb SS-2004 bacteria are virulent alone after direct injection into insect, making this strain a model for studying Xb virulence. In this study, we searched for Xb strains attenuated in virulence. For this purpose, we underwent infection assays with five Steinernema spp.-Xb pairs with two insects, Galleria mellonella (Lepidoptera: Pyralidae) and Spodoptera littoralis (Lepidoptera: Noctuidae). The S. weiseri-Xb CS03 pair showed attenuated virulence and lower fitness in S. littoralis in comparison to the other nematode-bacteria pairs. Furthermore, when injected alone into the hemolymph of G. mellonella or S. littoralis, the Xb CS03 bacterial strain was the only non-virulent strain. By comparison with the virulent Xb SS-2004 strain, Xb CS03 showed an increased sensitivity to the insect antimicrobial peptides, suggesting an attenuated response to the insect humoral immunity. To our current knowledge, Xb CS03 is the first non-virulent Xb strain identified. We propose this strain as a new model for studying the Xenorhabdus virulence.

Pdl1 is a putative lipase that enhances Photorhabdus toxin complex secretion.

The Toxin Complex (TC) is a large multi-subunit toxin first characterized in the insect pathogens Photorhabdus and Xenorhabdus, but now seen in a range of pathogens, including those of humans. These complexes comprise three protein subunits, A, B and C which in the Xenorhabdus toxin are found in a 4:1:1 stoichiometry. Some TCs have been demonstrated to exhibit oral toxicity to insects and have the potential to be developed as a pest control technology. The lack of recognisable signal sequences in the three large component proteins hinders an understanding of their mode of secretion. Nevertheless, we have shown the Photorhabdus luminescens (Pl) Tcd complex has been shown to associate with the bacteria's surface, although some strains can also release it into the surrounding milieu. The large number of tc gene homologues in Pl make study of the export process difficult and as such we have developed and validated a heterologous Escherichia coli expression model to study the release of these important toxins. In addition to this model, we have used comparative genomics between a strain that releases high levels of Tcd into the supernatant and one that retains the toxin on its surface, to identify a protein responsible for enhancing secretion and release of these toxins. This protein is a putative lipase (Pdl1) which is regulated by a small tightly linked antagonist protein (Orf53). The identification of homologues of these in other bacteria, linked to other virulence factor operons, such as type VI secretion systems, suggests that these genes represent a general and widespread mechanism for enhancing toxin release in gram negative pathogens.

Microbial population dynamics in the hemolymph of Manduca sexta infected with Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae.

Xenorhabdus nematophila engages in a mutualistic association with the nematode Steinernema carpocapsae. The nematode invades and traverses the gut of susceptible insects. X. nematophila is released in the insect blood (hemolymph), where it suppresses host immune responses and functions as a pathogen. X. nematophila produces diverse antimicrobials in laboratory cultures. The natural competitors that X. nematophila encounters in the hemolymph and the role of antimicrobials in interspecies competition in the host are poorly understood. We show that gut microbes translocate into the hemolymph when the nematode penetrates the insect intestine. During natural infection, Staphylococcus saprophyticus was initially present and subsequently disappeared from the hemolymph, while Enterococcus faecalis proliferated. S. saprophyticus was sensitive to X. nematophila antibiotics and was eliminated from the hemolymph when coinjected with X. nematophila. In contrast, E. faecalis was relatively resistant to X. nematophila antibiotics. When injected by itself, E. faecalis persisted (~10(3) CFU/ml), but when coinjected with X. nematophila, it proliferated to ~10(9) CFU/ml. Injection of E. faecalis into the insect caused the upregulation of an insect antimicrobial peptide, while the transcript levels were suppressed when E. faecalis was coinjected with X. nematophila. Its relative antibiotic resistance together with suppression of the host immune system by X. nematophila may account for the growth of E. faecalis. At higher injected levels (10(6) CFU/insect), E. faecalis could kill insects, suggesting that it may contribute to virulence in an X. nematophila infection. These findings provide new insights into the competitive events that occur early in infection after S. carpocapsae invades the host hemocoel.

Enzymatic characterization of a serralysin-like metalloprotease from the entomopathogen bacterium, Xenorhabdus.

We investigated the enzymatic properties of a serralysin-type metalloenzyme, provisionally named as protease B, which is secreted by Xenorhabdus bacterium, and probably is the ortholog of PrA peptidase of Photorhabdus bacterium. Testing the activity on twenty-two oligopeptide substrates we found that protease B requires at least three amino acids N-terminal to the scissile bond for detectable hydrolysis. On such substrate protease B was clearly specific for positively charged residues (Arg and Lys) at the P1 substrate position and was rather permissive in the others. Interestingly however, it preferred Ser at P1 in the oligopeptide substrate which contained amino acids also C-terminal to the scissile bond, and was cleaved with the highest k(cat)/K(M) value. The pH profile of activity, similarly to other serralysins, has a wide peak with high values between pH 6.5 and 8.0. The activity was slightly increased by Cu(2+) and Co(2+) ions, it was not sensitive for serine protease inhibitors, but it was inhibited by 1,10-phenanthroline, features shared by many Zn-metalloproteases. At the same time, EDTA inhibited the activity only partially even either after long incubation or in excess amount, and Zn(2+) was inhibitory (both are unusual among serralysins). The 1,10-phenanthroline inhibited activity could be restored with the addition of Mn(2+), Cu(2+) and Co(2+) up to 90-200% of its original value, while Zn(2+) was inefficient. We propose that both the Zn inhibition of protease B activity and its resistance to EDTA inhibition might be caused by an Asp in position 191 where most of the serralysins contain Asn.