Photorhabdus luminescens toxin-induced permeability change in Manduca sexta and Tenebrio molitor midgut brush border membrane and in unilamellar phospholipid vesicle

Photorhabdus luminescens, a Gram-negative bacterium, secretes a protein toxin (PL toxin) that is toxic to insects. In this study, the effects of the PL toxin on large receptor-free unilamellar phospholipid vesicles (LUVs) of Manduca sexta and on brush border membrane vesicles (BBMVs) of M. sexta and Tenebrio molitor were examined. Cry1Ac served as a positive control in our experiments due to its known channel-forming activity on M. sexta. Voltage clamping assays with dissected midguts of M. sexta and T. molitor clearly showed that both Cry1Ac and PL toxin caused channel formation in the midguts, although channel formation was not detected for T. molitor midguts under Cry1Ac and it was less sensitive to PL toxin than to Cry1Ac for M. sexta midguts. Calcein release experiments showed that both toxins made LUVs (unilamellar lipid vesicles) permeable, and at some concentrations of the toxins such permeabilizing effects were pH-dependent. The lowest concentrations of PL toxin were more than 600-fold and 24-fold lower to induce BBMV permeability of T. molitor and M. sexta than those to induce calcein release from LUVs of M. sexta. These further support that PL toxin is responsible for channel formation in the larvae midguts. The lower concentration to induce permeability in BBMV than in LUV is, probably, attributable to that BBMV has PL toxin receptors that facilitate the toxin to induce permeabilization. Furthermore, our results indicate that the effects of PL toxin on BBMV permeability of M. sexta were not significantly influenced by Gal Nac, but those of Cry1Ac were. This implies that PL toxin and Cry1Ac might use different molecular binding sites in BBMV to cause channel formation.     

A Novel Insecticidal Toxin from Photorhabdus luminescens, Toxin Complex a (Tca), and Its Histopathological Effects on the Midgut of Manduca sexta

Photorhabdus luminescens is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from Bacillus thuringiensis (δ-endotoxins and Vip’s), P. luminescens toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran Manduca sexta. In view of the difference in the normal mode of delivery of P. luminescens toxin and the apparent communality in the histopathological effects of other gut-active toxins from B. thuringiensis, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of M. sexta. Here we report that the histopathology of the M. sexta midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by M. sexta, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.     

Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection

During insect infection Photorhabdus luminescens emits light and expresses virulence factors, including insecticidal toxin complexes (Tcs) and an RTX-like metalloprotease (Prt). Using quantitative PCR and protein assays, we describe the expression patterns of these factors both in culture and during insect infection and compare them to the associated bacterial growth curves. In culture, light and active Prt protease are produced in stationary phase. Tca also appears in stationary phase, whereas Tcd is expressed earlier. These patterns seen in a culture flask are strikingly similar to those observed during insect infection. Thus, in an infected insect, bacteria grow exponentially until the time of insect death at approximately 48 h, when both light and the virulence factors Prt protease and Tca are produced. In contrast, Tcd appears much earlier in insect infection. However, at present, the biological significance of this difference in timing of the production of the two toxins in unclear. This is the first documentation of the expression of Tcs and Prt in an insect and highlights the malleability of Photorhabdus as a model system for bacterial infection.     

A metalloprotease secreted by the insect pathogen Photorhabdus luminescens induces melanization

Photorhabdus luminescens is a gram-negative insect pathogen that enters the hemocoel of infected hosts and produces a number of secreted proteins that promote colonization and subsequent death of the insect. In initial studies to determine the exact role of individual secreted proteins in insect pathogenesis, concentrated culture supernatants from various P. luminescens strains were injected into the tobacco hornworm Manduca sexta. Culture supernatants from P. luminescens TT01, the genome-sequenced strain, stimulated a rapid melanization reaction in M. sexta. Comparison of the profiles of secreted proteins from the various Photorhabdus strains revealed a single protein of approximately 37 kDa that was significantly overrepresented in the TT01 culture supernatant. This protein was purified by DEAE ion-exchange and Superdex 75 gel filtration chromatography and identified by matrix-assisted laser desorption ionization-time of flight analysis as the product of the TT01 gene plu1382 (NCBI accession number NC_005126); we refer to it here as PrtS. PrtS is a member of the M4 metalloprotease family. Injection of PrtS into larvae of M. sexta and Galleria mellonella and into adult Drosophila melanogaster and D. melanogaster melanization mutants (Bc) confirmed that the purified protein induced the melanization reaction. The prtS gene was transcribed by P. luminescens injected into M. sexta before death of the insect, suggesting that the protein was produced during infection. The exact function of this protease during infection is not clear. The bacteria might survive inside the insect despite the melanization process, or it might be that the bacterium is specifically activating melanization in an attempt to circumvent this innate immune response.     

Biophysical characterization of an insect lysozyme from Manduca sexta

Insect lysozyme from Manduca sexta (MS-lys) was overexpressed in E. coli and refolded to obtain active protein. Recombinant MS-lys presented a globular structure, with an alpha-helical content of 57% as assessed by circular dichroism spectroscopy. Light scattering studies showed that in solution MS-lys has a quasi-monodisperse size distribution, with a rod-like structure similar to nucleation clusters reported in egg lysozyme pre-crystallization stages. These results show that MS-lys is an excellent candidate for crystallization, folding and denaturation studies.     

Expression of a glycosylphosphatidylinositol-linked Manduca sexta aminopeptidase N in insect cells.

Aminopeptidase N (APN; EC 3.4.11.2) is an exopeptidase that is attached to cell membranes by a hydrophobic amino-terminal stalk in vertebrates or a glycosylphosphatidylinositol (GPI) anchor in insects. In this study, we report the cloning, expression, and characterization of an aminopeptidase N from Manduca sexta midgut. The full-length aminopeptidase N cDNA (APN1a) encodes a 995-amino-acid protein. The predicted amino acid sequence differs by 8 amino acids from M. sexta APN1. These different amino acids do not modify any putative glycosylation or glycosylphosphatidylinositol anchor sites. The full-length cDNA was cloned into an expression plasmid, pHSP-HR5, and transiently expressed in an insect cell line derived from Spodoptera frugiperda (Sf21 cells). Immunoblot analysis with anti-APN antiserum showed that APN1a expressed in Sf21 cells is the same size (120 kDa) as APN found in midgut brush border membranes. After treatment with phosphatidylinositol-specific phospholipase C (PIPLC), anti-cross-reacting determinant antibody specific for PIPLC cleavage products recognized the expressed 120-kDa APN1a, but not endogenous Sf21 proteins, indicating that APN1a has an intact glycosylphosphatidylinositol anchor. These results are evidence that Sf21 cells synthesize few, if any, endogenous GPI-linked proteins. Immunofluorescence staining showed that the expressed APN1a was located on the surface of Sf21 cells.     

Structure of a paralytic peptide from an insect, Manduca sexta.

Paralytic peptide 1 (PP1) from a moth, Manduca sexta, is a 23-residue peptide (Glu-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Tyr-Leu-Arg-Thr-Ala-Asp-Gly-Arg -Cys-Lys-Pro-Thr-Phe) that was first found to have paralytic activity when injected into M. sexta larvae. Recent studies demonstrated that PP1 also stimulated the spreading and aggregation of a blood cell type called plasmatocytes and inhibited bleeding from wounds. We determined the solution structure of PP1 by two-dimensional 1H NMR spectroscopy to begin to understand structural-functional relationships of this peptide. PP1 has an ordered structure, which is composed of a short antiparallel beta-sheet at residues Tyr11-Thr14 and Arg18-Pro21, three beta turns at residues Phe3-Gly6, Ala8-Tyr11 and Thr14-Gly17, and a half turn at the carboxyl-terminus (residues Lys20-Phe23). The well-defined secondary and tertiary structure was stabilized by hydrogen bonding and side-chain hydrophobic interactions. In comparison with two related insect peptides, whose structures have been solved recently, the amino-terminal region of PP1 is substantially more ordered. The short antiparallel beta-sheet of PP1 has a folding pattern similar to the carboxyl-terminal subdomain of epidermal growth factor (EGF). Therefore, PP1 may interact with EGF receptor-like molecules to trigger its different biological activities.     

The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme

Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of alpha-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 degrees C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.     

Mass Production of the Beneficial Nematode Heterorhabditis bacteriophora and Its Bacterial Symbiont Photorhabdus luminescens

Entomoparasitic nematodes (EPNs) are being commercialized as a biocontrol measure for crop insect pests, as they provide advantages over common chemical insecticides. Mass production of these nematodes in liquid media has become a major challenge for commercialization. Producers are not willing to share the trade secrets of mass production and by doing so, have made culturing EPNs extremely difficult to advance existing technologies. Theoretically, mass production in liquid media is an ideal culturing method as it increases cost efficiency and nematode quantity. This paper will review current culturing methodologies and suggest basic culturing parameters for mass production. This review is focused on Heterorhabditis bacteriophora; however, this information can be useful for other nematode species.     

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species^1-5, as well as the wealth of information available regarding the insect''s immune system^6-8, and the pending genome sequence⁹ make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter¹⁰. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking¹¹ and oral toxicity assays¹². Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides⁷; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR¹³. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes⁶. To analyze these responses, injected insects can be dissected and visualized by microscopy^{13, 14}.     

Crystal Structures of Cif from Bacterial Pathogens Photorhabdus luminescens and Burkholderia pseudomallei

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

Site-specific antiphagocytic function of the Photorhabdus luminescens type III secretion system during insect colonization

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

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.     

Central nervous system features of a nicotine-resistant insect, the tobacco hornworm Manduca sexta

The purpose of this study is to look for structural correlates of the demonstrated nicotine-insensitivity of larval Manduca sexta CNS, an insensitivity which is only slightly perturbed by desheathing (a technique used to disrupt perineurial diffusion barriers). The general organization of the hornworm ganglion is found to conform to the conventional insect pattern, but the following points are noted and discussed in terms of their potential relationship to nicotine-insensitivity: the damage caused to perineurial cells by desheathing is extremely localized, with cells immediately adjacent to the torn region showing good ultrastructural integrity; ionic lanthanum does not gain access to the subperineurial extracellular space following desheathing; lanthanum penetrates the ganglion in the cytoplasm of tracheal cells damaged peripherally during desheathing, but is excluded from the extracellular space surrounding such tracheal cells; smooth endoplasmic reticulum is much in evidence in perineurial cells and tracheal cells, sites where it might be implicated in nicotine detoxification; individual basal perineurial cells appear to cover extensive regions of the ganglion, thereby limiting intercellular diffusion.     

Survival of insect pathogenic and human clinical isolates of Photorhabdus luminescens in previously sterile soil.

Most characterized strains of the bacterium Photorhabdus luminescens are symbionts of entomopathogenic nematodes, whereas other strains have been isolated from human clinical specimens. The ability of P. luminescens strains to survive and grow in soil has received limited attention, with some studies indicating these bacteria have little or no ability to persist in soil. Survival and (or) growth of P. luminescens strains in previously sterilized soil, and examination of different soil amendments on their numbers in soil, have not been previously reported. Entomopathogenic P. luminescens (ATCC 29999) and a human clinical isolate (ATCC 43949) were introduced into a soil that had been sterilized by autoclaving, with or without different soil amendments, and bacterial numbers were estimated over time by viable plate count. In the previously sterilized soil receiving no exogenous amendments, numbers fell drastically over a week's time, followed by an increase in numbers by day 30. Treatments involving the addition of calcium carbonate and gelatin or casamino acids to soil usually resulted in higher bacterial numbers. For some sampling dates and soil treatments, there were statistically significant differences between the numbers of the two bacterial strains recovered from soil. The two strains of P. luminescens used in this study were able to survive and grow after being inoculated into previously sterilized soil.     

Two new subspecies of Photorhabdus luminescens, isolated from Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae): Photorhabdus luminescens subsp. kayaii subsp. nov. and Photorhabdus luminescens subsp. thracensis subsp. nov

Bacterial isolates from nematodes from Turkish soil samples were initially characterized by molecular methods and seven members of the genus Photorhabdus identified to the species level, using riboprint analyses and metabolic properties. Strain 07-5 (DSM 15195) was highly related to the type strain of Photorhabdus luminescens subsp. laumondii DSM 15139T, and was regarded a strain of this subspecies. Strains 1121T (DSM 15194T), 68-3 (DSM 15198) and 47-10 (DSM 15197) formed one, strain 39-8T (DSM 15199T), 39-7 (DSM 15196) and 01-12 (DSM 15193) formed a second cluster that branched intermediate the three subspecies of Photorhabdus luminescens. Based upon moderate 16S rRNA gene sequence similarities and differences in metabolic properties among themselves and with type strains of the three subspecies we consider the two clusters to represent two new subspecies of Photorhabdus luminescens for which the names Photorhabdus luminescens subsp. kayaii, type strain 1121T (DSM 15194T, NCIMB 13951T), and Photorhabdus luminescens subsp. thracensis subsp. nov., type strain 39-8T (DSM 15199T, NCIMB 13952T) are proposed.     

Expression and activity of a probable toxin from Photorhabdus luminescens

As an insect pathogen, Photorhabdus luminescens possesses an arsenal of toxins. Here we cloned and expressed a probable toxin from P. luminescens subsp. akhurstii YNd185, designated as Photorhabdus insecticidal toxin (Pit). The pit gene shares 94% nucleotide and 98% predicted amino acid sequence identity with plu1537, a predicted ORF from P. luminescens subsp. laumondii TT01 and 30% predicted amino acid sequence similarity to a fragment of a 13.6 kDa insecticidal crystal protein gene of Bacillus thuringiensis (Bt). The pit was expressed as a GST-Pit fusion protein in E. coli, most of which was insoluble and sequestered into inclusion bodies. The inclusion bodies were harvested and dissolved. The resultant protein was purified and the Pit was cleaved from the fusion protein by thrombin and purified from GST then used for bioassay. Pit killed Galleria mellonella (LD₅₀, 30 ng/larva) and Spodoptera litura (LD₅₀, 191 ng/larva) via hemocoel injection. Relative to a control that lacked toxin, Pit did not significantly increase mortality of S. litura and Helicoverpa armigera when introduced orally, but the treatment did inhibit growth of the insects. The present study demonstrated that Pit possessed insecticidal activity.