RESUMO
Borrelia burgdorferi causes Lyme disease, the most common tick-transmitted illness in North America. When Ixodes scapularis feed on an infected vertebrate host, spirochetes enter the tick gut along with the bloodmeal and colonize the vector. Here, we show that a secreted tick protein, I. scapularisprotein disulfide isomerase A3 (IsPDIA3), enhances B. burgdorferi colonization of the tick gut. I. scapularis ticks in which ispdiA3 has been knocked down using RNA interference have decreased spirochete colonization of the tick gut after engorging on B. burgdorferi-infected mice. Moreover, administration of IsPDIA3 antiserum to B. burgdorferi-infected mice reduced the ability of spirochetes to colonize the tick when feeding on these animals. We show that IsPDIA3 modulates inflammatory responses at the tick bite site, potentially facilitating spirochete survival at the vector-host interface as it exits the vertebrate host to enter the tick gut. These data provide functional insights into the complex interactions between B. burgdorferi and its arthropod vector and suggest additional targets to interfere with the spirochete life cycle.
Assuntos
Borrelia burgdorferi/fisiologia , Ixodes/metabolismo , Doença de Lyme/transmissão , Isomerases de Dissulfetos de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Vetores Aracnídeos/microbiologia , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Imunidade Humoral , Inflamação/enzimologia , Inflamação/genética , Inflamação/metabolismo , Ixodes/enzimologia , Ixodes/genética , Proteínas de Membrana/metabolismo , Camundongos , Filogenia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Interferência de RNA , Proteínas Recombinantes , Alinhamento de Sequência , Spirochaetales/fisiologiaRESUMO
Ticks and tick-borne diseases are on the rise world-wide and vaccines to prevent transmission of tick-borne diseases is an urgent public health need. Tick transmission of pathogens to the mammalian host occurs during tick feeding. Therefore, it is reasoned that vaccine targeting of tick proteins essential for feeding would thwart tick feeding and consequently prevent pathogen transmission. The phenomenon of acquired tick-immunity, wherein, repeated tick infestations of non-natural hosts results in the development of host immune responses detrimental to tick feeding has served as a robust paradigm in the pursuit of tick salivary antigens that may be vaccine targeted. While several salivary antigens have been identified, immunity elicited against these antigens have only provided modest tick rejection. This has raised the possibility that acquired tick-immunity is directed against tick components other than tick salivary antigens. Using Ixodes scapularis, the blacklegged tick, that vectors several human pathogens, we demonstrate that immunity directed against tick salivary glycoproteins is indeed sufficient to recapitulate the phenomenon of tick-resistance. These observations emphasize the utility of tick salivary glycoproteins as viable vaccine targets to thwart tick feeding and direct our search for anti-tick vaccine candidates.
Assuntos
Proteínas de Artrópodes/química , Glicoproteínas/química , Ixodes/química , Saliva/química , Animais , Feminino , Cobaias , Ixodes/crescimento & desenvolvimento , Ninfa/química , Ninfa/crescimento & desenvolvimento , CoelhosRESUMO
Lyme disease is a common tick-borne infection caused by the spirochete Borrelia burgdorferi sensu stricto (s.s.). B. burgdorferi s.s. may utilize chemotaxis, the directional migration towards or away from a chemical stimulus, for transmission, acquisition, and infection. However, the specific signals recognized by the spirochete for these events have not been defined. In this study, we identify an Ixodes scapularis salivary gland protein, Salp12, that is a chemoattractant for the spirochete. We demonstrate that Salp12 is expressed in the I. scapularis salivary glands and midgut and expression is not impacted by B. burgdorferi s.s. infection. Knockdown of Salp12 in the salivary glands or passive immunization against Salp12 reduces acquisition of the spirochete by ticks but acquisition is not completely prevented. Knockdown does not impact transmission of B. burgdorferi s.s. This work suggests a new role for chemotaxis in acquisition of the spirochete and suggests that recognition of Salp12 contributes to this phenomenon.
Assuntos
Vetores Aracnídeos/microbiologia , Proteínas de Artrópodes/fisiologia , Borrelia burgdorferi/fisiologia , Quimiotaxia , Ixodes/microbiologia , Animais , Doença de Lyme/transmissão , Glândulas Salivares/fisiologiaRESUMO
Bacterial symbionts can affect several biotic interactions of their hosts, including their competition with other species. Nematodes in the genus Steinernema utilize Xenorhabdus bacterial symbionts for insect host killing and nutritional bioconversion. Here, we establish that the Xenorhabdus bovienii bacterial symbiont (Xb-Sa-78) of Steinernema affine nematodes can impact competition between S. affine and S. feltiae by a novel mechanism, directly attacking its nematode competitor. Through co-injection and natural infection assays we demonstrate the causal role of Xb-Sa-78 in the superiority of S. affine over S. feltiae nematodes during competition. Survival assays revealed that Xb-Sa-78 bacteria kill reproductive life stages of S. feltiae. Microscopy and timed infection assays indicate that Xb-Sa-78 bacteria colonize S. feltiae nematode intestines, which alters morphology of the intestine. These data suggest that Xb-Sa-78 may be an intestinal pathogen of the non-native S. feltiae nematode, although it is a nonharmful colonizer of the native nematode host, S. affine. Screening additional X. bovienii isolates revealed that intestinal infection and killing of S. feltiae is conserved among isolates from nematodes closely related to S. affine, although the underlying killing mechanisms may vary. Together, these data demonstrate that bacterial symbionts can modulate competition between their hosts, and reinforce specificity in mutualistic interactions.
Assuntos
Proteínas de Artrópodes/imunologia , Interações Hospedeiro-Patógeno/imunologia , Doenças Transmitidas por Carrapatos/prevenção & controle , Carrapatos/imunologia , Vacinas/imunologia , Animais , Antígenos/imunologia , Proteínas de Artrópodes/uso terapêutico , Descoberta de Drogas , Humanos , Insetos Vetores/imunologia , Proteômica , Infestações por Carrapato/prevenção & controle , Infestações por Carrapato/terapia , Doenças Transmitidas por Carrapatos/terapia , Transcriptoma/imunologiaRESUMO
New strategies are needed to combat antibiotic resistance, especially against pathogens such as methicillin-resistant Staphylococcus aureus A tick antifreeze glycoprotein, IAFGP, possesses potent antibiofilm properties against a variety of clinical pathogens, including S. aureus Synergy between IAFGP, or a peptide (P1) representative of a repeat region of the protein, with different antibiotics was assessed in vitro Antibiotics that synergized with either IAFPG or P1 were further evaluated in vivo using vertebrate and invertebrate infection models. IAFGP readily enhanced the efficacy of antibiotics against S. aureus Synergy with daptomycin, an antibiotic used to treat methicillin-resistant S. aureus, was observed in vitro and in vivo using iafgp-transgenic mice and flies. Furthermore, synergy with ciprofloxacin or gentamicin, antibiotics not generally used to treat S. aureus, was also perceived. The combined effect of the antibiotic and IAFGP was associated with improved permeation of the antibiotic into the cell. Our results highlight that synergy of IAFGP with antibiotics traditionally used to treat this pathogen, and enhancement of the potency of antibiotics not commonly used against this microbe, can provide novel alternative therapeutic strategies to combat bacterial infections.
Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Carrapatos/microbiologia , Animais , Proteínas Anticongelantes/metabolismo , Biofilmes/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
R-type bacteriocins are contractile phage tail-like structures that are bactericidal towards related bacterial species. The C-terminal region of the phage tail fiber protein determines target-binding specificity. The mutualistic bacteria Xenorhabdus nematophila and X. bovienii produce R-type bacteriocins (xenorhabdicins) that are selectively active against different Xenorhabdus species. We analyzed the P2-type remnant prophage clusters in draft sequences of nine strains of X. bovienii The C-terminal tail fiber region in each of the respective strains was unique and consisted of mosaics of modular units. The region between the main tail fiber gene (xbpH1) and the sheath gene (xbpS1) contained a variable number of modules encoding tail fiber fragments. DNA inversion and module exchange between strains was involved in generating tail fiber diversity. Xenorhabdicin-enriched fractions from three different X. bovienii strains isolated from the same nematode species displayed distinct activities against each other. In one set of strains, the strain that produced highly active xenorhabdicin was able to eliminate a sensitive strain. In contrast, xenorhabdicin activity was not a determining factor in the competitive fitness of a second set of strains. These findings suggest that related strains of X. bovienii use xenorhabdicin and additional antagonistic molecules to compete against each other.
Assuntos
Bacteriocinas/metabolismo , Proteínas da Cauda Viral/genética , Xenorhabdus/genética , Xenorhabdus/metabolismo , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antibiose , Bacteriocinas/química , Bacteriocinas/genética , Bacteriocinas/farmacologia , Bacteriófagos , DNA Bacteriano/genética , Genoma Bacteriano , Proteínas da Cauda Viral/química , Xenorhabdus/citologia , Xenorhabdus/efeitos dos fármacosRESUMO
Phage-mediated horizontal gene transfer (HGT) is common in free-living bacteria, and many transferred genes can play a significant role in their new bacterial hosts. However, there are few reports concerning phage-mediated HGT in endosymbionts (obligate intracellular bacteria within animal or plant hosts), such as Wolbachia. The Wolbachia-infecting temperate phage WO can actively shift among Wolbachia genomes and has the potential to mediate HGT between Wolbachia strains. In the present study, we extend previous findings by validating that the phage WO can mediate transfer of non-phage genes. To do so, we utilized bioinformatic, phylogenetic, and molecular analyses based on all sequenced Wolbachia and phage WO genomes. Our results show that the phage WO can mediate HGT between Wolbachia strains, regardless of whether the transferred genes originate from Wolbachia or other unrelated bacteria.
RESUMO
Sortase mediated transpeptidation reactions play a significant role in covalent attachment of surface proteins to the cell wall of Gram-positive bacteria. Earlier studies have shown that sortase A (StrA) is required for the virulence of Staphylococci. The human pathogen Staphylococcus simulans CJ16 carries a putative sortase A (SsiStrA) encoding gene, but neither transpeptidation activity nor biochemical characteristics of SsiStrA have been investigated. Here, we identified and characterized StrA from coagulase-negative Staphylococci. SsiStrA was cloned and overexpressed in Escherichia coli BL21 in a soluble form. Size-exclusion chromatography, cross-linking and dynamic light scattering demonstrated that SsiStrA existed as monomer-dimer equilibrium in vitro. We further demonstrated that SsiStrA has sortase activity, and it recognized and cleaved the sorting motif LXPTG. H117, C180 and R193 residues were critical for enzyme activity, and calcium ions enhanced activity.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Cisteína Endopeptidases/metabolismo , Staphylococcus/enzimologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Aminoaciltransferases/química , Aminoaciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Cálcio/metabolismo , Domínio Catalítico , Parede Celular/genética , Cromatografia em Gel , Dicroísmo Circular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Escherichia coli/genética , Immunoblotting , Cinética , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Staphylococcus/genética , Especificidade por SubstratoRESUMO
BACKGROUND: Xenorhabdus bacteria engage in a beneficial symbiosis with Steinernema nematodes, in part by providing activities that help kill and degrade insect hosts for nutrition. Xenorhabdus strains (members of a single species) can display wide variation in host-interaction phenotypes and genetic potential indicating that strains may differ in their encoded symbiosis factors, including secreted metabolites. METHODS: To discern strain-level variation among symbiosis factors, and facilitate the identification of novel compounds, we performed a comparative analysis of the genomes of 10 Xenorhabdus bovienii bacterial strains. RESULTS: The analyzed X. bovienii draft genomes are broadly similar in structure (e.g. size, GC content, number of coding sequences). Genome content analysis revealed that general classes of putative host-microbe interaction functions, such as secretion systems and toxin classes, were identified in all bacterial strains. In contrast, we observed diversity of individual genes within families (e.g. non-ribosomal peptide synthetase clusters and insecticidal toxin components), indicating the specific molecules secreted by each strain can vary. Additionally, phenotypic analysis indicates that regulation of activities (e.g. enzymes and motility) differs among strains. CONCLUSIONS: The analyses presented here demonstrate that while general mechanisms by which X. bovienii bacterial strains interact with their invertebrate hosts are similar, the specific molecules mediating these interactions differ. Our data support that adaptation of individual bacterial strains to distinct hosts or niches has occurred. For example, diverse metabolic profiles among bacterial symbionts may have been selected by dissimilarities in nutritional requirements of their different nematode hosts. Similarly, factors involved in parasitism (e.g. immune suppression and microbial competition factors), likely differ based on evolution in response to naturally encountered organisms, such as insect hosts, competitors, predators or pathogens. This study provides insight into effectors of a symbiotic lifestyle, and also highlights that when mining Xenorhabdus species for novel natural products, including antibiotics and insecticidal toxins, analysis of multiple bacterial strains likely will increase the potential for the discovery of novel molecules.
Assuntos
Variação Genética , Genoma Bacteriano/genética , Simbiose/fisiologia , Xenorhabdus/genética , Simbiose/genéticaRESUMO
UNLABELLED: Microbial symbionts provide benefits that contribute to the ecology and fitness of host plants and animals. Therefore, the evolutionary success of plants and animals fundamentally depends on long-term maintenance of beneficial associations. Most work investigating coevolution and symbiotic maintenance has focused on species-level associations, and studies are lacking that assess the impact of bacterial strain diversity on symbiotic associations within a coevolutionary framework. Here, we demonstrate that fitness in mutualism varies depending on bacterial strain identity, and this is consistent with variation shaping phylogenetic patterns and maintenance through fitness benefits. Through genome sequencing of nine bacterial symbiont strains and cophylogenetic analysis, we demonstrate diversity among Xenorhabdus bovienii bacteria. Further, we identified cocladogenesis between Steinernema feltiae nematode hosts and their corresponding X. bovienii symbiont strains, indicating potential specificity within the association. To test the specificity, we performed laboratory crosses of nematode hosts with native and nonnative symbiont strains, which revealed that combinations with the native bacterial symbiont and closely related strains performed significantly better than those with more divergent symbionts. Through genomic analyses we also defined potential factors contributing to specificity between nematode hosts and bacterial symbionts. These results suggest that strain-level diversity (e.g., subspecies-level differences) in microbial symbionts can drive variation in the success of host-microbe associations, and this suggests that these differences in symbiotic success could contribute to maintenance of the symbiosis over an evolutionary time scale. IMPORTANCE: Beneficial symbioses between microbes and plant or animal hosts are ubiquitous, and in these associations, microbial symbionts provide key benefits to their hosts. As such, host success is fundamentally dependent on long-term maintenance of beneficial associations. Prolonged association between partners in evolutionary time is expected to result in interactions in which only specific partners can fully support symbiosis. The contribution of bacterial strain diversity on specificity and coevolution in a beneficial symbiosis remains unclear. In this study, we demonstrate that strain-level differences in fitness benefits occur in beneficial host-microbe interactions, and this variation likely shapes phylogenetic patterns and symbiotic maintenance. This highlights that symbiont contributions to host biology can vary significantly based on very-fine-scale differences among members of a microbial species. Further, this work emphasizes the need for greater phylogenetic resolution when considering the causes and consequences of host-microbe interactions.
Assuntos
Variação Genética , Nematoides/microbiologia , Simbiose , Xenorhabdus/genética , Xenorhabdus/fisiologia , Adaptação Biológica , Animais , Evolução Biológica , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Xenorhabdus/classificaçãoRESUMO
The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization-defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I-E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression.
Assuntos
Proteínas de Bactérias/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , RNA Bacteriano/metabolismo , Rabditídios/microbiologia , Simbiose , Xenorhabdus/fisiologia , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Elementos de DNA Transponíveis , Intestinos/microbiologia , Dados de Sequência Molecular , Mutagênese Insercional , RNA Bacteriano/genética , Rabditídios/fisiologia , Xenorhabdus/genéticaRESUMO
The specificity of a horizontally transmitted microbial symbiosis is often defined by molecular communication between host and microbe during initial engagement, which can occur in discrete stages. In the symbiosis between Steinernema nematodes and Xenorhabdus bacteria, previous investigations focused on bacterial colonization of the intestinal lumen (receptacle) of the nematode infective juvenile (IJ), as this was the only known persistent, intimate and species-specific contact between the two. Here we show that bacteria colonize the anterior intestinal cells of other nematode developmental stages in a species-specific manner. Also, we describe three processes that only occur in juveniles that are destined to become IJs. First, a few bacterial cells colonize the nematode pharyngeal-intestinal valve (PIV) anterior to the intestinal epithelium. Second, the nematode intestine constricts while bacteria initially remain in the PIV. Third, anterior intestinal constriction relaxes and colonizing bacteria occupy the receptacle. At each stage, colonization requires X. nematophila symbiosis region 1 (SR1) genes and is species-specific: X. szentirmaii, which naturally lacks SR1, does not colonize unless SR1 is ectopically expressed. These findings reveal new aspects of Xenorhabdus bacteria interactions with and transmission by theirSteinernema nematode hosts, and demonstrate that bacterial SR1 genes aid in colonizing nematode epithelial surfaces.
Assuntos
Rabditídios/crescimento & desenvolvimento , Rabditídios/microbiologia , Simbiose , Xenorhabdus/isolamento & purificação , Xenorhabdus/fisiologia , Animais , Sistema Digestório/microbiologia , Células Epiteliais/microbiologia , Xenorhabdus/classificaçãoRESUMO
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic (1-3). One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis (4). Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects (5). For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage (6-8). Recently, several other nematode species have been shown to utilize bacteria to kill insects (9-13), and investigations have begun examining the interactions between the nematodes and bacteria in these systems (9). We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila (14). Similar methods have been used to investigate other nematode-bacterium associations (9) (,) (15-18)and the approach therefore is generally applicable. The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization (14) (,) (16) (,) (19). Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues (14) (,) (16) (,) (19-21). This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication (22)or grinding (23), which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes (21) (,) (24). Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization (17) (,) (18), and is less laborious than other methods, including sonication (22) (,) (25-27)and individual nematode dissection (28) (,) (29).
Assuntos
Microscopia de Fluorescência/métodos , Rabditídios/microbiologia , Xenorhabdus/química , Animais , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Xenorhabdus/genética , Xenorhabdus/metabolismoRESUMO
Nematodes are ubiquitous organisms that have a significant global impact on ecosystems, economies, agriculture, and human health. The applied importance of nematodes and the experimental tractability of many species have promoted their use as models in various research areas, including developmental biology, evolutionary biology, ecology, and animal-bacterium interactions. Nematodes are particularly well suited for the investigation of host associations with bacteria because all nematodes have interacted with bacteria during their evolutionary history and engage in a variety of association types. Interactions between nematodes and bacteria can be positive (mutualistic) or negative (pathogenic/parasitic) and may be transient or stably maintained (symbiotic). Furthermore, since many mechanistic aspects of nematode-bacterium interactions are conserved, their study can provide broader insights into other types of associations, including those relevant to human diseases. Recently, genome-scale studies have been applied to diverse nematode-bacterial interactions and have helped reveal mechanisms of communication and exchange between the associated partners. In addition to providing specific information about the system under investigation, these studies also have helped inform our understanding of genome evolution, mutualism, and innate immunity. In this review we discuss the importance and diversity of nematodes, "omics"' studies in nematode-bacterial systems, and the wider implications of the findings.
Assuntos
Bactérias/crescimento & desenvolvimento , Fenômenos Fisiológicos Bacterianos , Nematoides/microbiologia , Nematoides/fisiologia , Simbiose , Animais , Bactérias/química , Bactérias/genética , Genômica/métodos , Metabolômica/métodos , Proteômica/métodos , TranscriptomaRESUMO
Xenorhabdus bovienii (SS-2004) bacteria reside in the intestine of the infective-juvenile (IJ) stage of the entomopathogenic nematode, Steinernema jollieti. The recent sequencing of the X. bovienii genome facilitates its use as a model to understand host - symbiont interactions. To provide a biological foundation for such studies, we characterized X. bovienii in vitro and host interaction phenotypes. Within the nematode host X. bovienii was contained within a membrane bound envelope that also enclosed the nematode-derived intravesicular structure. Steinernema jollieti nematodes cultivated on mixed lawns of X. bovienii expressing green or DsRed fluorescent proteins were predominantly colonized by one or the other strain, suggesting the colonizing population is founded by a few cells. Xenorhabdus bovienii exhibits phenotypic variation between orange-pigmented primary form and cream-pigmented secondary form. Each form can colonize IJ nematodes when cultured in vitro on agar. However, IJs did not develop or emerge from Galleria mellonella insects infected with secondary form. Unlike primary-form infected insects that were soft and flexible, secondary-form infected insects retained a rigid exoskeleton structure. Xenorhabdus bovienii primary and secondary form isolates are virulent towards Manduca sexta and several other insects. However, primary form stocks present attenuated virulence, suggesting that X. bovienii, like Xenorhabdus nematophila may undergo virulence modulation.