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Abstract Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C) and human (37°C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of EPS properties. Despite its abundance and conservation in the genus, we find no evidence for a role of Pam in either virulence or symbiosis.

Abstract Background The Gram-negative bacterium Photorhabdus asymbiotica (Pa) has been recovered from human infections in both North America and Australia. Recently, Pa has been shown to have a nematode vector that can also infect insects, like its sister species the insect pathogen P. luminescens (Pl). To understand the relationship between pathogenicity to insects and humans in Photorhabdus we have sequenced the complete genome of Pa strain ATCC43949 from North America. This strain (formerly referred to as Xenorhabdus luminescens strain 2) was isolated in 1977 from the blood of an 80 year old female patient with endocarditis, in Maryland, USA. Here we compare the complete genome of Pa ATCC43949 with that of the previously sequenced insect pathogen P. luminescens strain TT01 which was isolated from its entomopathogenic nematode vector collected from soil in Trinidad and Tobago. Results We found that the human pathogen Pa had a smaller genome (5,064,808 bp) than that of the insect pathogen Pl (5,688,987 bp) but that each pathogen carries approximately one megabase of DNA that is unique to each strain. The reduced size of the Pa genome is associated with a smaller diversity in insecticidal genes such as those encoding the Toxin complexes (Tc's), Makes caterpillars floppy (Mcf) toxins and the Photorhabdus Virulence Cassettes (PVCs). The Pa genome, however, also shows the addition of a plasmid related to pMT1 from Yersinia pestis and several novel pathogenicity islands including a novel Type Three Secretion System (TTSS) encoding island. Together these data suggest that Pa may show virulence against man via the acquisition of the pMT1-like plasmid and specific effectors, such as SopB, that promote its persistence inside human macrophages. Interestingly the loss of insecticidal genes in Pa is not reflected by a loss of pathogenicity towards insects. Conclusion Our results suggest that North American isolates of Pa have acquired virulence against man via the acquisition of a plasmid and specific virulence factors with similarity to those shown to play roles in pathogenicity against humans in other bacteria.

Photorhabdus is a highly effective insect pathogen and symbiont of insecticidal nematodes. To exert its potent insecticidal effects, it elaborates a myriad of toxins and small molecule effectors. Among these, the Photorhabdus Virulence Cassettes (PVCs) represent an elegant self-contained delivery mechanism for diverse protein toxins. Importantly, these self-contained nanosyringes overcome host cell membrane barriers, and act independently, at a distance from the bacteria itself. In this study, we demonstrate that Pnf, a PVC needle complex associated toxin, is a Rho-GTPase, which acts via deamidation and transglutamination to disrupt the cytoskeleton. TEM and Western blots have shown a physical association between Pnf and its cognate PVC delivery mechanism. We demonstrate that for Pnf to exert its effect, translocation across the cell membrane is absolutely essential., Competing Interests: IV, AH, JH, KS, GY, NW No competing interests declared, (© 2019, Vlisidou et al.)

H 2 O 2 is an early danger cue required for innate immune cell recruitment to wounds. To date, little is known about whether H 2 O 2 is required for the migration of human adaptive immune cells to sites of inflammation. However, oxidative stress is known to impair T cell activity, induce actin stiffness, and inhibit cell polarization. In this study, we show that low oxidative concentrations of H 2 O 2 also impede chemokinesis and chemotaxis of previously activated human T cells to CXCL11, but not CXCL10 or CXCL12. We show that this deficiency in migration is due to a reduction in inflammatory chemokine receptor CXCR3 surface expression and cellular activation of lipid phosphatase SHIP-1. We demonstrate that H 2 O 2 acts through an Src kinase to activate a negative regulator of PI3K signaling, SHIP-1 via phosphorylation, providing a molecular mechanism for H 2 O 2 -induced chemotaxis deficiency. We hypothesize that although H 2 O 2 serves as an early recruitment trigger for innate immune cells, it appears to operate as an inhibitor of T lymphocyte immune adaptive responses that are not required until later in the repair process., (Copyright © 2017 The Authors.)

Photorhabdus are highly effective insect pathogenic bacteria that exist in a mutualistic relationship with Heterorhabditid nematodes. Unlike other members of the genus, Photorhabdus asymbiotica can also infect humans. Most Photorhabdus cannot replicate above 34°C, limiting their host-range to poikilothermic invertebrates. In contrast, P. asymbiotica must necessarily be able to replicate at 37°C or above. Many well-studied mammalian pathogens use the elevated temperature of their host as a signal to regulate the necessary changes in gene expression required for infection. Here we use RNA-seq, proteomics and phenotype microarrays to examine temperature dependent differences in transcription, translation and phenotype of P. asymbiotica at 28°C versus 37°C, relevant to the insect or human hosts respectively. Our findings reveal relatively few temperature dependant differences in gene expression. There is however a striking difference in metabolism at 37°C, with a significant reduction in the range of carbon and nitrogen sources that otherwise support respiration at 28°C. We propose that the key adaptation that enables P. asymbiotica to infect humans is to aggressively acquire amino acids, peptides and other nutrients from the human host, employing a so called "nutritional virulence" strategy. This would simultaneously cripple the host immune response while providing nutrients sufficient for reproduction. This might explain the severity of ulcerated lesions observed in clinical cases of Photorhabdosis. Furthermore, while P. asymbiotica can invade mammalian cells they must also resist immediate killing by humoral immunity components in serum. We observed an increase in the production of the insect Phenol-oxidase inhibitor Rhabduscin normally deployed to inhibit the melanisation immune cascade. Crucially we demonstrated this molecule also facilitates protection against killing by the alternative human complement pathway.

Drosophila melanogaster has been extensively used to study the humoral arm of innate immunity because of the developmental and functional parallels with mammalian innate immunity. However, the fly cellular response to infection is far less understood. Investigative work on Drosophila haemocytes, the immunosurveillance cells of the insect, has revealed that they fulfil roles similar to mammalian monocytes and macrophages. They respond to wound signals and orchestrate the coagulation response. In addition, they phagocytose and encapsulate invading pathogens, and clear up apoptotic bodies controlling inflammation. This review briefly describes the Drosophila haematopoietic system and discusses what is currently known about the contribution of haemocytes to the immune response upon infection and wounding, during all stages of development., (© 2015 FEBS.)

Beyond their role in cell metabolism, development, and reproduction, hormones are also important modulators of the immune system. In the context of inflammatory disorders, systemic administration of pharmacological doses of synthetic glucocorticoids (GCs) is widely used as an anti-inflammatory treatment [1, 2]. However, not all actions of GCs are immunosuppressive, and many studies have suggested that physiological concentrations of GCs can have immunoenhancing effects [3-7]. For a more comprehensive understanding of how steroid hormones regulate immunity and inflammation, a simple in vivo system is required. The Drosophila embryo has recently emerged as a powerful model system to study the recruitment of immune cells to sterile wounds [8] and host-pathogen dynamics [9]. Here we investigate the immune response of the fly embryo to bacterial infections and find that the steroid hormone 20-hydroxyecdysone (20-HE) can regulate the quality of the immune response and influence the resolution of infection in Drosophila embryos., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The outcome of any bacterial infection, whether it is clearance of the infecting pathogen, establishment of a persistent infection, or even death of the host, is as dependent on the host as on the pathogen (Finlay and Falkow 1989). To infect a susceptible host bacterial pathogens express virulence factors, which alter host cell physiology and allow the pathogen to establish a nutrient-rich niche for growth and avoid clearance by the host immune response. However survival within the host often results in tissue damage, which to some cases accounts for the disease-specific pathology. For many bacterial pathogens the principal determinants of virulence and elicitors of host tissue damage are soluble exotoxins, which allow bacteria to penetrate into deeper tissue or pass through a host epithelial or endothelial barrier. Therefore, exploring the complex interplay between host tissue and bacterial toxins can help us to understand infectious disease and define the contributions of the host immune system to bacterial virulence. In this chapter, we describe a new model, the Drosophila embryo, for addressing a fundamental issue in bacterial pathogenesis, the elucidation of the in vivo targets of bacterial toxins and the monitoring of the first moments of the infection process in real-time. To develop this model, we used the insect and emerging human pathogen Photorhabdus asymbiotica and more specifically we characterised the initial cross-talk between the secreted cytotoxin Mcf1 and the embryonic hemocytes. Mcf1 is a potent cytotoxin which has been detected in all Photorhabdus strains isolated so far, which can rapidly kill insects upon injection. Despite several in vitro tissue culture studies, the biology of Mcf1 in vivo is not well understood. Furthermore, despite the identification of many Photorhabdus toxins using recombinant expression in E. coli (Waterfield et al. 2008), very few studies address the molecular mechanism of action of these toxins in relation to specific immune responses in vivo in the insect model.

Many bacteria persist within phagocytes, deploying complex sets of tightly regulated virulence factors to manipulate and survive within host cells. So far, no single factor has been identified that is sufficient to allow intracellular persistence of an otherwise non-pathogenic bacterium. Here we report that the two-component KdpD/KdpE sensor kinase/response regulator of the insect and human pathogen Photorhabdus asymbiotica (Pa) is sufficient to allow a harmless laboratory strain of E. coli to resist phagocytic killing and persist within insect hemocytes, ultimately killing the insect. Screening of a cosmid library of Pa in E. coli by injection into the moth Manduca sexta, previously identified three overlapping clones which caused the insect to cease feeding and subsequently die. Transposon mutagenesis revealed a cosmid encoded kdp high affinity potassium pump regulon was responsible for this phenotype. Gentamycin protection assays and confocal microscopy revealed the cosmid clones were persisting inside insect hemocytes far longer than control bacteria. Cloning and expression of PakdpD/kdpE alone into E. coli recapitulated the phenotype. Bioassay results and transcriptional analysis of various E. coli kdp mutants harboring the Pa kdp genes confirmed that Pa KdpD/KdpE was able to induce the E. coli kdp pump structural genes in response to exposure to insect hemocytes but not blood plasma alone. The finding that Pa KdpD/KdpE can facilitate resistance of E. coli to phagocytic killing suggests a central role for potassium in this process, supporting previous work implicating potassium sensing in virulence of other bacteria and also in the normal process of protease killing of engulfed bacteria by neutrophils., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica) and non-pathogenic (Escherichia coli) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid 'freezing' phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors.

Prokaryotic microorganisms are widespread in all environments on Earth, establishing diverse interactions with many eukaryotic taxa, including insects. These associations may be symbiotic, pathogenic and vectoring. Independently of the type of interaction, each association starts with the adhesion of the microorganism to the host, entry and "invasion" of the host, then progresses to establishment and dissemination within the host, by avoiding host immune responses, and concludes with transmission back to the environment or to a new host. Advances in genomics and genetics have allowed the dissection of these processes and provided important information on the elements driving the shaping of the members of each association. Furthermore, many mechanisms involved in the establishment of the associations have been scrutinised, along with the development of new methods for the management of insect populations.

Enterohaemorrhagic Escherichia coli (EHEC) infections in humans are frequently associated with direct or indirect contact with ruminant faeces and may result in haemorrhagic colitis and severe renal and neurological sequelae. Broadly cross-protective vaccines for control of EHEC do not yet exist and the molecular mechanisms that influence bacterial persistence in the intestines of ruminants are incompletely understood. We sought to determine the role in colonisation and protective efficacy of EspA, which forms a filamentous extension of the locus of enterocyte effacement-encoded type III secretion system that injects EHEC proteins into enterocytes. A non-polar deletion of espA severely impaired the ability of E. coli O157:H7 to colonise the intestines of calves. Vaccination of calves with highly purified recombinant EspA induced high-titre antigen-specific IgG1 (also reactive to native EspA) and salivary IgA responses, however these responses did not protect calves against intestinal colonisation by E. coli O157:H7 upon experimental infection.

Enterohaemorrhagic Escherichia coli (EHEC) causes bloody diarrhoea in humans and deploys a type III secretion system (T3SS) encoded by the locus of enterocyte effacement to elicit the formation of attaching and effacing (AE) lesions on intestinal epithelia. Here, we report the identification of a new secreted substrate of this system, z1829, which is encoded by cryptic prophage CP-933N. Elevated secretion of a beta-lactamase-z1829 fusion protein was detected upon mutation of sepD in EHEC O157:H7 and the fusion protein was translocated into infected epithelial cells in a T3SS-dependent manner; accordingly, we named the protein EspK. In common with the related Salmonella enterica type III secreted effector GogB, we observed that EspK localized to the cytoplasm when transiently expressed in COS-7 cells using EspK-specific antiserum. Inactivation of espK did not impair adherence or actin nucleation during infection of HeLa cells but affected persistence of EHEC O157:H7 in the intestines of orally inoculated calves. Inactivation of an orthologue of espK in the murine AE pathogen Citrobacter rodentium did not impair intestinal colonization in mice.

Enteropathogenic Escherichia coli contain a large chromosomal gene (lifA) that encodes lymphostatin, a predicted 365 kDa protein that inhibits the mitogen-activated proliferation of peripheral blood lymphocytes and lamina propria mononuclear cells and the synthesis of proinflammatory cytokines. Non-O157 serotypes of enterohaemorrhagic E. coli (EHEC) contain a highly homologous gene, designated efa1 (EHEC factor for adherence), which influences adherence to epithelial cells in vitro and intestinal colonization in calves. Serotype O157:H7 EHEC strains contain a truncated version of this gene (efa1') and a pO157-encoded homologue of lifA/efa1 (toxB). Here we report for the first time that efa1 inhibits mitogen-activated proliferation of bovine peripheral blood lymphocytes by EHEC O103:H2, but that E. coli K-12 strains expressing the N-terminal and central portions of the protein lack activity. While a Shiga toxin-negative E. coli O157:H7 strain was shown to possess lymphostatin-like activity, deletion of efa1' or toxB, singly or in combination, failed to significantly relieve the inhibitory effect.

Intimin facilitates intestinal colonization by enterohemorrhagic Escherichia coli O157:H7; however, the importance of intimin binding to its translocated receptor (Tir) as opposed to cellular coreceptors is unknown. The intimin-Tir interaction is needed for optimal actin assembly under adherent bacteria in vitro, a process which requires the Tir-cytoskeleton coupling protein (TccP/EspF(U)) in E. coli O157:H7. Here we report that E. coli O157:H7 tir mutants are at least as attenuated as isogenic eae mutants in calves and lambs, implying that the role of intimin in the colonization of reservoir hosts can be explained largely by its binding to Tir. Mutation of tccP uncoupled actin assembly from the intimin-Tir-mediated adherence of E. coli O157:H7 in vitro but did not impair intestinal colonization in calves and lambs, implying that pedestal formation may not be necessary for persistence. However, an E. coli O157:H7 tccP mutant induced typical attaching and effacing lesions in a bovine ligated ileal loop model of infection, suggesting that TccP-independent mechanisms of actin assembly may operate in vivo.

The role of the neuroendocrine environment in the pathogenesis of enteric bacterial infections is increasingly being recognized. Here we report that norepinephrine augments Escherichia coli O157:H7-induced intestinal inflammatory and secretory responses as well as bacterial adherence to intestinal mucosa in a bovine ligated ileal loop model of infection. Norepinephrine modulation of enteritis and adherence was dependent on the ability of E. coli O157:H7 to form attaching and effacing lesions.

Protective monoclonal antibodies (MAbs) to the major outer membrane protein (MOMP) of species of the family Chlamydiaceae, which is the primary vaccine candidate antigen, recognize nonlinear epitopes conferred by the oligomeric conformation of the molecule. Protective MAbs failed to recognize oligomeric MOMP of the variant strain LLG, which bears amino acid substitutions in variable segments (VSs) 1, 2, and 4, and competed with monomer-specific MAbs mapping to these VSs in reference strain 577. The results suggest that multiple sites located in the three VSs contribute to the epitope of protective MAbs.