Comparison of Proteolytic Activities Produced by Entomopathogenic
Photorhabdus
Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes
Abstract:Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (ϳ74, ϳ55, ϳ54, and ϳ37 kDa) could be separated. The N-terminal sequences of three of t… Show more
“…Finally, extracellular activities, such as hemolysis and lipase and protease activities, were found to be weaker in the phenotypic variants (5). The activities of two metalloproteases, PrtA and PrtS, were attenuated in the supernatant of the P. temperata K122 phenotypic variant (9,49). Our data suggest that the transcription of prtA (plu0655) is not modified but that the prtS gene (plu1382) and its downstream gene (plu1381), which probably belongs to the same transcription unit, are less strongly transcribed in VAR* than in TT01␣.…”
Photorhabdus luminescens lives in a mutualistic association with entomopathogenic nematodes and is pathogenic for insects. Variants of Photorhabdus frequently arise irreversibly and are studied because they have altered phenotypic traits that are potentially important for the host interaction. VAR* is a colonial and phenotypic variant displaying delayed pathogenicity when directly injected into the insect, Spodoptera littoralis. In this study, we evaluated the role of transcriptomic modulation in determining the phenotypic variation and delayed pathogenicity of VAR* with respect to the corresponding wild-type form, TT01␣. A P. luminescens microarray identified 148 genes as differentially transcribed between VAR* and TT01␣. The net regulator status of VAR* was found to be significantly modified. We also observed in VAR* a decrease in the transcription of genes supporting certain phenotypic traits, such as pigmentation, crystalline inclusion, antibiosis, and protease and lipase activities. Three genes encoding insecticidal toxins (pit and pirB) or putative insecticidal toxins (xnp2) were less transcribed in VAR* than in the TT01␣. The overexpression of these genes was not sufficient to restore the virulence of VAR* to the levels of ⌻⌻01␣, which suggests that the lower virulence of VAR* does not result from impaired toxemia in insects. Three loci involved in oxidative stress responses (sodA, katE, and the hca operon) were found to be downregulated in VAR*. This is consistent with the greater sensitivity of VAR* to H 2 O 2 and may account for the impaired bacteremia in the hemolymph of S. littoralis larvae observed with VAR*. In conclusion, we demonstrate here that some phenotypic traits of VAR* are regulated transcriptionally and highlight the multifactorial nature of pathogenicity in insects.Photorhabdus luminescens is a member of the Enterobacteriaceae that lives in a mutualistic association with entomopathogenic nematodes and is pathogenic for a wide range of insects. Studies on Photorhabdus pathogenicity have shown this bacterium to be highly virulent, due to the production of an array of insecticidal toxins and the induction of host immune depression (6,18, 25,28,41,67).
“…Finally, extracellular activities, such as hemolysis and lipase and protease activities, were found to be weaker in the phenotypic variants (5). The activities of two metalloproteases, PrtA and PrtS, were attenuated in the supernatant of the P. temperata K122 phenotypic variant (9,49). Our data suggest that the transcription of prtA (plu0655) is not modified but that the prtS gene (plu1382) and its downstream gene (plu1381), which probably belongs to the same transcription unit, are less strongly transcribed in VAR* than in TT01␣.…”
Photorhabdus luminescens lives in a mutualistic association with entomopathogenic nematodes and is pathogenic for insects. Variants of Photorhabdus frequently arise irreversibly and are studied because they have altered phenotypic traits that are potentially important for the host interaction. VAR* is a colonial and phenotypic variant displaying delayed pathogenicity when directly injected into the insect, Spodoptera littoralis. In this study, we evaluated the role of transcriptomic modulation in determining the phenotypic variation and delayed pathogenicity of VAR* with respect to the corresponding wild-type form, TT01␣. A P. luminescens microarray identified 148 genes as differentially transcribed between VAR* and TT01␣. The net regulator status of VAR* was found to be significantly modified. We also observed in VAR* a decrease in the transcription of genes supporting certain phenotypic traits, such as pigmentation, crystalline inclusion, antibiosis, and protease and lipase activities. Three genes encoding insecticidal toxins (pit and pirB) or putative insecticidal toxins (xnp2) were less transcribed in VAR* than in the TT01␣. The overexpression of these genes was not sufficient to restore the virulence of VAR* to the levels of ⌻⌻01␣, which suggests that the lower virulence of VAR* does not result from impaired toxemia in insects. Three loci involved in oxidative stress responses (sodA, katE, and the hca operon) were found to be downregulated in VAR*. This is consistent with the greater sensitivity of VAR* to H 2 O 2 and may account for the impaired bacteremia in the hemolymph of S. littoralis larvae observed with VAR*. In conclusion, we demonstrate here that some phenotypic traits of VAR* are regulated transcriptionally and highlight the multifactorial nature of pathogenicity in insects.Photorhabdus luminescens is a member of the Enterobacteriaceae that lives in a mutualistic association with entomopathogenic nematodes and is pathogenic for a wide range of insects. Studies on Photorhabdus pathogenicity have shown this bacterium to be highly virulent, due to the production of an array of insecticidal toxins and the induction of host immune depression (6,18, 25,28,41,67).
“…When the protease inhibitor was removed, the protein underwent an apparent autocatalytic event, clipping off the N-terminal 48 amino acids. We determined the N-terminal sequence of the active rPrtS to be SSDDS, which corresponds to the N-terminal sequence determined for this protein by Marokhazi et al (13). The apparent molecular weight of the cleaved rPrtS correlated with that of the native PrtS found in culture supernatants; we are confident that this result reflects the actual N-terminal sequence of the active protein.…”
Section: Discussionmentioning
confidence: 55%
“…We use the PrtS designation here. The levels of PrtS produced by Photorhabdus strains vary (13), and for undetermined reasons this protein is made in abundance by TT01.…”
Section: Discussionmentioning
confidence: 99%
“…Many potential bacterial products are implicated in this killing, including a toxin termed "makes caterpillars floppy," hemolysins, RTX (repeats in toxin) family members, and large toxin complexes (Tc) (1-3, 5, 7). Photorhabdus also secretes numerous proteases and lipases and is able to inhibit competitor bacteria by synthesizing various antibiotics and bacteriocins (7)(8)(9)13).…”
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.
“…Protease activity was detected by the gelatin hydrolysis assay on plates as described by Marokhazi et al (26). Gelatin nutrient agar plates were spot-inoculated with 20 µL of partially purified enzyme preparations.…”
Section: Detection Of Protease Activity Of the Partially Purified Enzmentioning
Phospholipases C (PLCs) from Pseudomonas aeruginosa D183 and Bacillus cereus D101, 2 clinical isolates from 2 pus specimens, were partially purified by ammonium sulfate precipitation followed by dialysis and used to study the possible role of PLC in the virulence of the isolates. Partially purified PLC from both isolates induced lysis of Vero cells in the presence and absence of the producing bacterial cells. Noncytolytic dilutions of the partially purified PLC from Pseudomonas aeruginosa increased adherence of the producing cells to Vero cells but did not affect internalization. However, Bacillus cereus cells neither adhered to nor were internalized within the Vero cells in the presence or absence of noncytolytic dilutions of partially purified PLC from the isolate. Both PLC preparations were hemolytic to human red blood cells but did not induce human platelet aggregation. Thus, we can conclude that PLCs from Pseudomonas aeruginosa and Bacillus cereus are important virulence factors. PLC production by the 2 isolates was found to be chromosomal-mediated rather than plasmid-mediated.
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