How the insect pathogen bacteria Bacillus thuringiensis and Xenorhabdus/Photorhabdus occupy their hosts

Insects are the largest group of animals on earth. Like mammals, virus, fungi, bacteria and parasites infect them. Several tissue barriers and defense mechanisms are common for vertebrates and invertebrates. Therefore some insects, notably the fly Drosophila and the caterpillar Galleria mellonella, have been used as models to study host-pathogen interactions for several insect and mammal pathogens. They are excellent tools to identify pathogen determinants and host tissue cell responses. We focus here on the comparison of effectors used by two different groups of bacterial insect pathogens to accomplish the infection process in their lepidopteran larval host: Bacillus thuringiensis and the nematode-associated bacteria, Photorhabdus and Xenorhabdus. The comparison reveals similarities in function and expression profiles for some genes, which suggest that such factors are conserved during evolution in order to attack the tissue encountered during the infection process.     

Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.     

Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination

Photorhabdus and Xenorhabdus bacteria colonize the intestines of the infective soil-dwelling stage of entomophagous nematodes, Heterorhabditis and Steinernema, respectively. These nematodes infect susceptible insect larvae and release the bacteria into the insect blood. The bacteria kill the insect larvae and convert the cadaver into a food source suitable for nematode growth and development. After several rounds of reproduction the nematodes are recolonized by the bacteria before emerging from the insect cadaver into the soil to search for a new host. Photorhabdus and Xenorhabdus bacteria therefore engage in both pathogenic and mutualistic interactions with different invertebrate hosts as obligate components of their life cycle. In this review we aim to describe current knowledge of the molecular mechanisms utilized by Photorhabdus and Xenorhabdus to control their host-dependent interactions. Recent work has established that there is a trade-off between pathogenicity and mutualism in both these species of bacteria suggesting that the transition between these interactions must be under regulatory control. Despite the superficial similarity between the life cycles of these bacteria, it is now apparent that the molecular components of the regulatory networks controlling pathogenicity and mutualism in Photorhabdus and Xenorhabdus are very different.     

Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen

Pathogenicity and symbiosis are central to bacteria-host interactions. Although several human pathogens have been subjected to functional genomic analysis, we still understand little about bacteria-invertebrate interactions despite their ecological prevalence. Advances in our knowledge of this area are often hindered by the difficulty of isolating and working with invertebrate pathogenic bacteria and their hosts. Here we review studies on pathogenicity and symbiosis in an insect pathogenic bacterium Photorhabdus and its entomopathogenic nematode vector and model insect hosts. Whilst switching between these hosts, Photorhabdus changes from a state of symbiosis with its nematode vector to one of pathogenicity towards its new insect host and both the bacteria and the nematode then cooperatively exploit the dying insect. We examine candidate genes involved in symbiosis and pathogenicity, their secretion and expression patterns in culture and in the host, and begin to dissect the extent of their genetic coregulation. We describe the presence of several large genomic islands, putatively involved in pathogenicity or symbiosis, within the otherwise Yersinia-like backbone of the Photorhabdus genome. Finally, we examine the emerging comparative genomics of the Photorhabdus group and begin to describe the interrelationship between anti-invertebrate virulence factors and those used against vertebrates.     

Effect of the insect pathogenic bacterium Photorhabdus on insect phagocytes

Photorhabdus are insect pathogenic bacteria that replicate within the insect haemocoel following release from their entomopathogenic nematode symbionts. To investigate how they escape the cellular immune response we examined the effects of two strains of Photorhabdus, W14 and K122, on Manduca sexta phagocytes (haemocytes), in vitro and in vivo. Following injection of Esherichia coli into Manduca larvae, these non-pathogenic bacteria are rapidly cleared from the haemolymph and the number of free haemocytes transiently increases. In contrast, following injection of either strain of pathogenic Photorhabdus, the bacteria grow rapidly while the number of haemocytes decreases dramatically. In vitro incubation of haemocytes with either Photorhabdus supernatant reduced haemocyte viability, and the W14 supernatant caused distinct changes in the actin cytoskeleton morphology of different haemocyte cell types. In phagocytosis assays both Photorhabdus strains can inhibit their own phagocytosis whether the bacterial cells are alive or dead. Further, the supernatant of W14 also contains a factor capable of inhibiting the phagocytosis of labelled E. coli. Together these results suggest that Photorhabdus evades the cellular immune response by killing haemocytes and suppressing phagocytosis by mechanisms that differ between strains.     

A nematode symbiont sheds light on invertebrate immunity

Photorhabdus bacteria live in a 'symbiosis of pathogens' with nematodes that invade and kill insects. Recent work has begun to use the power of the model insect Drosophila to dissect the molecular basis of the invertebrate immune response to the combined insult of the worms and their symbiotic bacterial pathogens. By using RNA interference, it is now also possible to dissect this complex tripartite interaction in a range of both model and non-model hosts.     

The regulation of pathogenicity and mutualism in Photorhabdus

Photorhabdus is a genus of insect-pathogenic bacteria that also maintains a mutualistic interaction with Heterorhabditid nematodes. Bacteria in this genus are members of the family Enterobacteriaceae and are, therefore, closely related to many important mammalian pathogens. This bacteria-nematode complex has been exploited as a biocontrol agent that is active against several insect pests. However, this model system is also uniquely placed to address important fundamental questions about pathogenicity and mutualism. Indeed, recent genetic studies have suggested that there is a significant overlap in the genetic requirements of Photorhabdus for these contrasting interactions. In addition, the identification of key regulators of pathogenicity and symbiosis only serves to highlight the similarities between Photorhabdus, a genus of bacteria that infects invertebrate hosts, and closely related mammalian enteric pathogens.     

Host-derived extracellular nucleic acids enhance innate immune responses, induce coagulation, and prolong survival upon infection in insects

Extracellular nucleic acids play important roles in human immunity and hemostasis by inducing IFN production, entrapping pathogens in neutrophil extracellular traps, and providing procoagulant cofactor templates for induced contact activation during mammalian blood clotting. In this study, we investigated the functions of extracellular RNA and DNA in innate immunity and hemolymph coagulation in insects using the greater wax moth Galleria mellonella a reliable model host for many insect and human pathogens. We determined that coinjection of purified Galleria-derived nucleic acids with heat-killed bacteria synergistically increases systemic expression of antimicrobial peptides and leads to the depletion of immune-competent hemocytes indicating cellular immune stimulation. These activities were abolished when nucleic acids had been degraded by nucleic acid hydrolyzing enzymes prior to injection. Furthermore, we found that nucleic acids induce insect hemolymph coagulation in a similar way as LPS. Proteomic analyses revealed specific RNA-binding proteins in the hemolymph, including apolipoproteins, as potential mediators of the immune response and hemolymph clotting. Microscopic ex vivo analyses of Galleria hemolymph clotting reactions revealed that oenocytoids (5-10% of total hemocytes) represent a source of endogenously derived extracellular nucleic acids. Finally, using the entomopathogenic bacterium Photorhabdus luminescens as an infective agent and Galleria caterpillars as hosts, we demonstrated that injection of purified nucleic acids along with P. luminescens significantly prolongs survival of infected larvae. Our results lend some credit to our hypothesis that host-derived nucleic acids have independently been co-opted in innate immunity of both mammals and insects, but exert comparable roles in entrapping pathogens and enhancing innate immune responses.     

The lumicins: novel bacteriocins from Photorhabdus luminescens with similarity to the uropathogenic-specific protein (USP) from uropathogenic Escherichia coli

Bacteriocins are proteins produced by bacteria to destroy other bacteria occupying their ecological niche. Photorhabdus luminescens is an insect pathogenic bacterium carried by an entomopathogenic nematode and occupies several different niches in its life cycle. The nematode enters the insect and releases a single strain of P. luminescens. The bacteria then kill the host and the bacteria and nematodes replicate within the cadaver. Strikingly, at the end of the infection the cadaver is still occupied by a single strain of bacterium, suggesting that P. luminescens can destroy other bacteria entering, or present within, the insect. Here we describe four loci encoding 'lumicins' in P. luminescens subsp. akhurstii strain W14. The lumicins are novel bacteriocins capable of killing other strains of Photorhabdus and Escherichia coli. These loci predict killer proteins and multiple dual type immunity proteins with domains similar to pyocins and colicins. The killer proteins are chimeric in nature with multiple domains, one of which is similar to the uropathogenic-specific protein (USP) described from uropathogenic E. coli. The implications of these novel bacteriocins for the lifestyle of Photorhabdus and the potential role of USP as a bacteriocin in E. coli are discussed.     

共生菌与昆虫的免疫

共生菌可通过产生抗菌物质、调控宿主免疫相关基因和微生物种间竞争作用等方式保护昆虫宿主免受病原体的侵染。为维持共生关系,昆虫进化出精细的调控机制避免对共生菌的过激免疫应答,共生菌通过免疫识别信号多态性或化学拟态来降低或躲避宿主免疫系统对自身的伤害。本文在分析共生菌对宿主免疫的功能及其机制的基础上,探讨宿主对免疫应答的精准调控以及共生体系的协同进化,以期为共生菌对宿主免疫影响的深入研究提供参考。     

pfaB products determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis

Insect blood (hemolymph) contains prophenoloxidase, a proenzyme that is activated to protective phenoloxidase when the insect is damaged or challenged with microorganisms. The Gram-negative bacterium Photorhabdus luminescens kills the lepidopteron insect Manduca sexta by using a variety of toxins. We screened P. luminescens and Photorhabdus asymbiotica cosmid libraries in an Escherichia coli host against previously activated M. sexta hemolymph phenoloxidase and identified three overlapping cosmid clones from P. luminescens and five from P. asymbiotica that suppressed the activity of the enzyme both in vitro and in vivo . Genome alignments of cosmid end sequences from both species confirmed that they contained orthologous loci. We examined one of the cosmids from P. luminescens in detail: it induced the formation of significantly fewer melanotic nodules, proliferated faster within the insect host and was significantly more virulent towards fifth-stage larvae than E. coli control bacteria. Insertional mutagenesis of this cosmid yielded 11 transposon mutants that were no longer inhibitory. All of these were insertions into a single 5.5-kb locus, which contained three ORFs and was homologous to the maltodextrin phosphorylase locus of E. coli . The implications of this novel inhibitory factor of insect phenoloxidase for Photorhabdus virulence are discussed.     

Direct infection of Spodoptera litura by Photorhabdus luminescens encapsulated in alginate beads

Actively growing cultures of Photorhabdus luminescens were encapsulated in sodium alginate beads and examined for their ability to infect insect hosts. These beads, containing approximately 2.5 x 10(7)Photorhabdus cells per bead, when mixed with sterilized soil and exposed to Spodoptera litura larvae resulted in 100% mortality in 48 h, while the use of alginate encapsulated Heterorhabditis nematode resulted in 40% mortality after 72 h. The bacteria were reisolated from the dead insect thus proving Koch's postulates and demonstrating the ability of P. luminescens to kill the insect host on their own, independent of the symbiont nematode. The LC(50) dose of Photorhabdus cells was estimated at 1010 cells per larva for killing S. litura 6th instar larvae in 48 h.     

Masters of conquest and pillage: Xenorhabdus nematophila global regulators control transitions from virulence to nutrient acquisition

Invertebrate animal models are experimentally tractable and have immunity and disease symptoms that mirror those of vertebrates. Therefore they are of particular utility in understanding fundamental aspects of pathogenesis. Indeed, artificial models using human pathogens and invertebrate hosts have revealed conserved and novel molecular mechanisms of bacterial infection and host immune responses. Additional insights may be gained from investigating interactions between invertebrates and pathogens they encounter in their natural environments. For example, enteric bacteria in the genera Photorhabdus and Xenorhabdus are pathogens of insects that also mutualistically associate with nematodes in the genera Heterorhabditis and Steinernema respectively. These bacteria serve as models to understand naturally occurring symbiotic associations that result in disease in or benefit for animals. Xenorhabdus nematophila is the best-studied species of its genus with regard to the molecular mechanisms of its symbiotic associations. In this review, we summarize recent advances in understanding X. nematophila –host interactions. We emphasize regulatory cascades involved in coordinating transitions between various stages of the X. nematophila life cycle: infection, reproduction and transmission.     

An In Vitro Inhibition Test That Predicts Toxicity of Bacterial Pathogen Combinations in the Colorado Potato Beetle

Many insect bacterial pathogens are not toxic enough for field control. Combinations of bacteria may increase toxicity. Bacteria toxic to Colorado potato beetle , Photorhabdus luminescens, Chromobacterium violaceum and Serratia marcescens , were tested in pair-wise combinations in an in vitro double streak test to determine bacterial compatibility. Only C. violaceum and S. marcescens grew to confluency. Their combined toxicity in vivo was additive. Other bacterial combinations had clear zones between bacterial streaks indicating inhibition. In the insect, the combined toxicity was less than the most toxic bacteria of the pair. For these strains, this in vitro test predicted compatibility in the insect.     

不同昆虫寄主对昆虫病原线虫共生菌的敏感性比较

用菜青虫、棉铃虫、甜菜夜蛾、玉米螟、粘虫、黄粉虫等 6种昆虫对 1 0株昆虫病原线虫共生菌进行了敏感性测定。结果表明 :供试菌株对 6种昆虫都有胃毒活性 ,不同菌株对同一种昆虫的毒力差别较大 ,同一菌株对不同昆虫差别也很大。 1 0株菌在 1 2 0h对菜青虫的校正死亡率和体重抑制率均最高 ,显然是最敏感的寄主。在 1 0株共生菌中 ,XenorhabdusnematophilaHB3 1 0 5 9菌株的胃毒活性最高。     

The exbD gene of Photorhabdus temperata is required for full virulence in insects and symbiosis with the nematode Heterorhabditis

Photorhabdus are bacteria found colonizing the gut of a specialized stage of the nematode Heterorhabditis, called the infective juvenile (IJ). The IJ is a free-living stage of the nematode that seeks out and infects insect larvae. Once inside the insect the IJ release Photorhabdus into the haemolymph where the bacteria rapidly proliferate, killing the insect within 48-72 h. The nematodes grow and reproduce in the insect cadaver by feeding on the Photorhabdus biomass. In this study we use Photorhabdus temperata K122 to show that genes involved in iron acquisition play a key role during the course of the tripartite bacteria-nematode-insect interaction. We show that a strain carrying a mutation in a gene with homology to exbD, encoding a component of the TonB complex, is unable to grow well in conditions where iron is not freely available. In addition, this mutant, BMM417, requires a longer time to kill the insect larvae than the wild-type bacteria and this defect in pathogenicity is complemented by the co-injection of iron. Moreover, the increase in LT(50) observed with BMM417 is correlated with a significantly slower in vivo growth rate suggesting that iron is limiting in the insect. We also show that BMM417 is unable to support the growth and development of the Heterorhabditis nematode. Addition of exogenous iron to the growth media restores nematode growth and development on BMM417, suggesting that aspects of iron metaboism in Photorhabdus are important during the symbiosis with the nematode.     

Identification and sequence of an unstable DNA element in the entomopathogenic bacteria Photorhabdus temperata strain K122

AIMS: A search was conducted for a difference in genome composition between phenotypic variants of the insect pathogenic bacteria, Photorhabdus temperata. METHODS: An unstable 300 bp fragment of DNA was identified by amplified fragment length polymorphism (AFLP) analysis, which was not, however, associated with phenotypic variation. RESULTS: During prolonged culturing of the bacteria, one copy of the repeated fragment was deleted and a restriction site linked to one of the copies was lost or gained. The sequence did not show substantial identity to any in the database, but a 16-bp region was identical to part of the marR gene of Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The work has implications for the understanding of genetic instability in this and other pathogenic species of bacteria. In addition, the complete unstable element may be useful as a genetic tool in Photorhabdus spp.     

A genomic sample sequence of the entomopathogenic bacterium Photorhabdus luminescens W14: potential implications for virulence

Photorhabdus luminescens is a pathogenic bacterium that lives in the guts of insect-pathogenic nematodes. After invasion of an insect host by a nematode, bacteria are released from the nematode gut and help kill the insect, in which both the bacteria and the nematodes subsequently replicate. However, the bacterial virulence factors associated with this "symbiosis of pathogens" remain largely obscure. In order to identify genes encoding potential virulence factors, we performed approximately 2,000 random sequencing reads from a P. luminescens W14 genomic library. We then compared the sequences obtained to sequences in existing gene databases and to the Escherichia coli K-12 genome sequence. Here we describe the different classes of potential virulence factors found. These factors include genes that putatively encode Tc insecticidal toxin complexes, Rtx-like toxins, proteases and lipases, colicin and pyocins, and various antibiotics. They also include a diverse array of secretion (e.g., type III), iron uptake, and lipopolysaccharide production systems. We speculate on the potential functions of each of these gene classes in insect infection and also examine the extent to which the invertebrate pathogen P. luminescens shares potential antivertebrate virulence factors. The implications for understanding both the biology of this insect pathogen and links between the evolution of vertebrate virulence factors and the evolution of invertebrate virulence factors are discussed.     

Bacterial strategies to overcome insect defences

Recent genetic and molecular analyses have revealed how several strategies enable bacteria to persist and overcome insect immune defences. Genetic and genomic tools that can be used with Drosophila melanogaster have enabled the characterization of the pathways that are used by insects to detect bacterial invaders and combat infection. Conservation of bacterial virulence factors and insect immune repertoires indicates that there are common strategies of host invasion and pathogen eradication. Long-term interactions of bacteria with insects might ensure efficient dissemination of pathogens to other hosts, including humans.