1 Department of Entomology, University of Wisconsin-Madison, Madison, USA
2 Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK
3 Institut de Génétique et Microbiologie, CNRS UMR 8621, Bâtiment 360, Université Paris XI, 91405 Orsay Cedex, France
Correspondence
Mark Blight
mark.blight{at}mac-mail.igmors.u-psud.fr
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ABSTRACT |
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The GenBank accession numbers for the W14 and K122 prtA clone sequences reported in this paper are AY230749 and AY230750 respectively.
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INTRODUCTION |
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Repeats-in-toxin (RTX) toxins are a large family of calcium-dependent, pore-forming cytotoxins produced by different genera of the Enterobacteriaceae and Pasteurellaceae (Welch, 1991). RTX toxins are metzincin metalloendopeptidases secreted to the external medium via a Type I pathway. These proteases have been identified in Erwinia chrysanthemi (Dahler et al., 1990
; Letoffe et al., 1990
), Pseudomonas aeruginosa (Duong et al., 1992
; Guzzo et al., 1990
, 1991a
, b
), Pseudomonas fluorescens (Ahn et al., 1999
; Kawai et al., 1999
) and Serratia marcescens (Letoffe et al., 1991
; Nakahama et al., 1986
). In each case the protease is secreted via an ABC transporter, often linked to the protease structural gene in the same operon. However, the genetic organization of the protease (prt) operon varies, as does the number of proteases secreted (Ahn et al., 1999
; Akatsuka et al., 1995
, 1997
; Delepelaire & Wandersman, 1990
; Duong et al., 1992
; Ghigo & Wandersman, 1992a
, b
; Guzzo et al., 1991b
; Kawai et al., 1999
).
The aim of the current study was to clone and characterize the zinc alkaline metalloprotease secreted by two species of Photorhabdus, Photorhabdus luminescens and Photorhabdus temperata (Fischer-Le Saux et al., 1999), represented by the strains, P. luminescens subsp. akhurstii strain W14 (Bowen et al., 1998
) and Photorhabdus temperata strain K122 (Waterfield et al., 2001
), hereafter referred to simply as strains W14 and K122 for clarity. Photorhabdus is an entomopathogenic member of the Enterobactericeae found in a symbiotic relationship with entomopathogenic nematodes of the family Heterorhabditiae (Forst & Clarke, 2001
; Forst et al., 1997
). The bacteria occupy the intestinal tract of the infective stage of the nematode, the infective juvenile. The nematodes live in the soil where they actively seek out potential insect larval hosts. On finding a host, the nematode enters the larva and regurgitates the bacteria into the insect haemocoel (open circulatory system). The bacteria then grow rapidly producing a wide range of toxins and hydrolytic exoenzymes that kill the insect and aid in the conversion of the cadaver into food (bioconversion) for both the bacteria and developing nematodes (ffrench-Constant et al., 2003
).
Previous attempts to characterize proteolytic activities secreted by either Photorhabdus or Xenorhabdus have led to considerable confusion in the literature. Thus earlier workers have either purified a proteolytic fraction from culture broth and inferred it to have a role in toxicity, via analogy with proteases produced by other insect pathogens (Ong & Chang, 1997; Schmidt et al., 1988
; Yamanaka et al., 1992
), or they have examined the toxicity of cell-free culture supernatants to insects via injection and then suggested that toxicity is correlated with the protease activity also observed in the culture broth (Jarosz et al., 1991
). In our earlier work on protease purification from strain W14 we have demonstrated that proteolytic activity in the supernatant is independent of the high-molecular-mass insecticidal toxin complexes (Bowen et al., 2000
). We have also shown that protease activity can be resolved into three fractions, one of
55 kDa and two of
40 kDa (Bowen et al., 2000
). To further clarify the likely role of secreted protease, here we describe the biochemistry of the purified PrtA protein (corresponding to the
55 kDa fraction) and the cloning of the associated prtA gene locus from two different Photorhabdus strains.
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METHODS |
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Protein analysis and protease purification.
Proteins were analysed by SDS-PAGE and protease activity was assessed by electrophoresis enzymograms, as described previously (Bowen et al., 2000). Culture supernatant fractions were prepared by centrifuging bacterial cultures at 14 000 g and 4 °C for 5 min and precipitating supernatant proteins with 10 % trichloroacetic acid (TCA) for 30 min on ice. Precipitated proteins were pelleted at 14 000 g and 4 °C for 30 min and washed once with 500 µl cold (-20 °C) 80 % acetone, followed by resuspension in SDS-PAGE sample buffer and analysis by SDS-PAGE and enzymography. Protease activity was measured as the increase of BODIPY-FL fluorescence released from cleaved BODIPY-FL-labelled casein using the EnzChek Protease Assay kit (Molecular Probes). Measurements were made in a Biolumin microplate fluorescence spectrophotometer (Molecular Dynamics) with excitation and emission wavelengths of 505 and 513 nm, respectively. Purified protease in 20 mM Tris/HCl, 5 mM CaCl2, pH 8·0, was assayed in a volume of 200 µl with BODIPY-FL casein at a final concentration of 5 µg ml-1. Analysis of protease autocatalytic degradation was performed in 20 mM Tris/HCl with the addition of either 10 mM CaCl2 or 10 mM EDTA (pH 8·0) at either 30, 37, 42 or 60 °C at a protein concentration of 1 mg purified PrtA ml-1. Aliquots were removed at time intervals and analysed by SDS-PAGE. Estimation of degradation half-life was made from the densitometric analysis of the peak surface area of intact mature 55 kDa protease.
Protease purification was performed as described previously (Valens et al., 2002). The cytotoxicity of purified PrtA was tested against a tissue culture of mammalian (COS7) cells by adding serial dilutions of protease to the growth medium and incubating overnight. Cells were then fixed and stained with FITC-labelled phalloidin to examine the integrity of the cells and their actin cytoskeleton.
Insect infection and in vivo analysis of protease activity.
Fifth instar larvae of the Greater wax moth (Galleria mellonella) (Mr G. Dumond, Earl La Teigne Dorée, France) were injected ventrally with 100 Photorhabdus luminescens cells (counted by plating c.f.u.) in sterile PBS. Larvae were maintained at 28 °C during subsequent infection. Larval extracts were prepared at intervals by homogenizing single larvae in 1 ml PBS in a hand-held 10 ml teflon homogenizer. Tissue debris was removed by centrifugation at 2000 g and 4 °C for 5 min. Supernatants were precipitated with 10 % TCA on ice for 30 min and then centrifuged at 14 000 g and 4 °C for 15 min. Precipitated pellets were washed once with 500 µl cold (-20 °C) 80 % acetone and resuspended in SDS-PAGE sample buffer prior to analysis by SDS-PAGE enzymography.
Library construction and screening.
Approximately 150 µg strain W14 phase variant I genomic DNA, partially digested with Sau3A, was size-fractionated on a NaCl density gradient (a linear gradient from 1·25 to 5 M NaCl in Tris/EDTA). The gradient was generated using a Bio-Rad EconoSystem, programming the pump to generate the gradient and pumping it into a 4 ml centrifuge tube. Gradients were centrifuged for 3·5 h at 44 000 r.p.m. in an SW 60 swinging bucket rotor at 18 °C and were fractionated by puncturing the bottom of the tube and collecting 100 µl fractions (3540 fractions). DNA was precipitated by adding ethanol to a final concentration of 66 %. The precipitated DNA was dissolved in 10 µl sterile water and 1 µl from every fraction was analysed by agarose gel electrophoresis to check size and concentration. Fragments larger than 12 kb were ligated into pBluescript KS+ (Stratagene) and transformed into library component E. coli XL-1 Blue MRF' cells (Stratagene). The cells were allowed to recover for 1 h in LB at 30 °C and were then plated on gelatin agar with 100 µg ampicillin ml-1. Similarly, a library of strain K122 variant I genomic DNA was constructed in BamHI-digested, dephosphorylated pUC18 (Amersham-Pharmacia Biotech) comprising fragments of between 10 and 15 kb isolated following sucrose density-gradient centrifugation of Sau3AI partially digested chromosomal DNA as above. Recombinant clones were isolated following chemical transformation of E. coli DH5
library competent cells (Life Technologies) and selection on LB agar containing 100 µg ampicillin ml-1. The three positive clones secreting protease activity from the W14 genomic library were end-sequenced and their relative degree of overlap was determined by restriction mapping. Clone pPRT1-W14 was chosen since it was the smallest clone still retaining secreted proteolytic activity. Deletions, based upon suitable restriction sites, were then made within pPRT1-W14 both for further nucleotide sequencing and also for re-assay for protease activity on gelatin agar plates. The sequence of both strands of the complete W14 protease operon, and its associated Type I transporter, was finished by primer walking.
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RESULTS |
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The predicted PrtA proteins from the two different strains are 87·6 % identical and 92·1 % similar. The predicted amino acid sequence of PrtA confirms that it is an RTX-like zinc metalloprotease similar to proteases from Erwinia chrysanthemi and Serratia marcescens. Alignments (Table 1) show the conservation of the RTX repeats with the Prosite motif D-x-[li]-x(4)-g-x-D-x-[LI]-x-G-G-x(3)-D (Prosite Motif PDOC00293) implicated in Ca2+ binding and a putative zinc-binding motif (position 184193, TFTHEIGHTL) with the consensus sequence [GSTALIVN]-x(2)-H-E-[LIVMFYW]-(DEHRKP)-H-x-[LIVMFYWGSPQ] (Prosite Motif PDOC00129). The protease inhibitor is most similar to the inh gene of Erwinia chrysanthemi and is predicted to possess an N-terminal secretion signal (cleavage between residues 2627) consistent with export to the periplasm via the sec-dependent pathway, as confirmed previously (Valens et al., 2002
). The members of the putative Type I trans-envelope transporter are most similar to the apr genes of the Pseudomonas aeruginosa RTX-like zinc metalloprotease transporter and include an ATP-binding cassette (ABC) protein, PrtB, a membrane-fusion protein (MFP), PrtC and an outer-membrane protein (OMP), PrtD. A series of six internal deletions within the W14 prt operon on plasmid pPRT1-W14 were obtained using internal restriction endonuclease cleavage sites (Fig. 1c
) and all six clones were defective for the secretion of active protease. This indicates that all of the genes in the prt operon are required for the synthesis and secretion of PrtA to the culture medium
Analysis of PrtA secretion from Photorhabdus luminescens and recombinant E. coli
Photorhabdus strains exhibit phase variation whereby individual strains show phenotypic variation between phase I and phase II which differ in colony colour and morphology. To examine possible differences in expression between these variants, phase I and II colonies, from both strains W14 and K122, together with E. coli DH5 harbouring either plasmid pPRT-K122 or pPRT1-W14 were grown to stationary phase in LB medium at 30 °C with agitation. Culture supernatants were prepared and analysed by SDS-PAGE by both Coomassie blue staining (Fig. 2
a) and gelatin enzymogram (Fig. 2b
). The Coomassie-blue-stained profile shows large numbers of proteins in the medium, secreted by both phase I and II variants of both strains W14 and K122 (Fig. 2a
, lanes 1, 3, 5 and 6). Both W14 and K122 phase I cells secrete qualitatively and quantitatively different profiles (lanes 1 and 3) and this difference is also reflected between the phase II variant cells (lanes 5 and 6). Moreover, in both strains, phase I cells secrete considerably more proteins than the phase II cells. The entire prt operon of W14 and K122 is present on recombinant clones pPRT1-W14 and pPRT-K122, including 845 and 1798 bp of upstream DNA sequence, respectively. However, secretion of a protein with an apparent molecular mass of 55 kDa from E. coli DH5
harbouring pPRT1-W14 is clearly evident (lane 4), whereas no abundant 55 kDa band is present in the supernatant from E. coli DH5
harbouring pPRT-K122. Protease activity is associated with a minor 55 kDa band in supernatants of pPRT-K122 (lane 2), indicating that, unlike the W14 clone pPRT-W14, the K122 operon harboured in pPRT-K122 secretes substantially less protease to the supernatant when expressed in E. coli DH5
.
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DISCUSSION |
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We purified native PrtA from strain K122, as this strain produces less extracellular polysaccharide than W14, thus facilitating purification of PrtA directly from the supernatant. We recovered recombinant PrtA-expressing clones from strain W14 as clones from this strain produced more protease and thus could be detected in library screens, whereas clones from K122 libraries could not. The genomic organization of the prtA locus from strain W14, with the structural gene for the metalloprotease and its associated type I transporter being found together, differs from that seen in other bacteria. Only one protease-encoding ORF, prtA1, is associated with the operon, unlike that of Erwinia chrysanthemi B374 which has four associated protease-encoding genes (Delepelaire & Wandersman, 1990; Ghigo & Wandersman, 1992a
, b
). The prtA gene is termed prtA1 as the available genomic sequence predicts a homologue of prtA1 (prtA2) elsewhere in the W14 genome. The prt operon architecture in strain W14 resembles most closely that of the Pseudomonas aeruginosa apr operon. Indeed, BLAST analysis of the SWISS-PROT database indicates that the Photorhabdus luminescens protease, ABC transporter, MFP and OMP are most closely related at the amino acid sequence level to the Apr functional homologues. The exception is that of the gene encoding the inhibitor, inh, which is most closely related to that from Erwinia chrysanthemi.
Purification of native PrtA protease from strain K122 was achieved directly from stationary-phase culture supernatants. The protease is remarkably stable and resistant to high temperature, detergent and the presence of reducing agents. Biochemical analysis using protease inhibitors indicated that the enzyme is a zinc-dependent alkaline metalloprotease and autocatalytic cleavage peptide profiles in the presence and absence of calcium suggested that PrtA requires bound calcium to stabilize its structure, as for other RTX proteins. Both primary- and secondary-phase variants secrete PrtA to the medium as detected by enzymography. However, direct measurements of PrtA activity in culture supernatants of both K122 and W14 indicate that the actual secreted native activity from phase II cells is approximately 25-fold lower than that of phase I cells (data not shown). Such a difference is not as manifest in SDS-PAGE enzymograms. This may be due to an activation of inherently inhibited phase II secreted protease following treatment with SDS, reducing agent and heating. Previous studies have suggested that primary-phase variants of Photorhabdus temperata produce more active lipase enzyme than the secondary variants, whilst levels of lipase gene transcription remain the same (Wang & Dowds, 1993). This led to the hypothesis that the difference in the specific activity of the lipase between the two phenotypic variants may be associated with post-translational regulation (Wang & Dowds, 1993
). Whilst we did not examine levels of PrtA gene transcription in the two variants, we note that differences in the association of PrtA with its candidate inhibitor represent a potential mechanism for generating the apparent differences in expression we observed in this study. In this respect, a recent report describes the secretion of a broad-spectrum protease inhibitor from Photorhabdus luminescens isolated from Heterorhabditis bacteriophora HP88 nematodes (Wee et al., 2000
). The authors describe the purification of a 12 kDa protein, secreted into the medium by phase II variant cells, able to inhibit endogenous secreted Photorhabdus luminescens protease activity. The small size of HP88 Inh is similar to that of the predicted mature size of the K122 and W14 Inh protein of 11 kDa. The K122 and W14 Inh is predicted to possess a classical sec pathway-dependent N-terminal secretion signal, and is targeted to the periplasm in both phase I and II cells (Valens et al., 2002
). Although the N-terminal amino acid sequence of HP88 Inh (STGIVTFKNDXGEDIV) (Wee et al., 2000
) is not similar to the predicted mature N terminus of K122 and W14 Inh (SSLVLPHASELKGVWQL), we cannot exclude the possibility that they are of similar origin and function. If phase II variant cells release Inh to the medium from the periplasm then we would predict that it would inhibit secreted PrtA and thus lead to the observation of reduced levels of specific PrtA activity in phase II culture supernatants as compared to those of phase I cells. Moreover, it is also likely that such an interaction would be disrupted upon SDS-PAGE, thus permitting the detection of elevated levels of PrtA activity in enzymograms as observed in this and earlier reports (Wee et al., 2000
).
Analysis of protease activity in G. mellonella insect larvae infected with phase I or phase II K122 cells reveals that a protease species of the correct size for PrtA is detectable late in insect infection and shortly prior to larval death. Moreover, active enzyme is produced by the phase II variant cells much later than the phase I variant. Both phase I and II variants are equally virulent to G. mellonella larvae with death occurring at approximately 24 h post-infection in both cases. Combined with the observation that injected protease is not directly toxic to G. mellonella, this suggests that PrtA is not a major virulence factor, but that it may have an alternative role in host bioconversion. Purified PrtA did however show detrimental effects on mammalian cells at low concentrations, although we noted that more non-specific proteases such as trypsin may promote a similar effect. We also noted that earlier immunocytochemistry studies with an anti-PrtA antibody have show PrtA immunoreactivity associated with the basal lamina of tissues within the insect, suggesting that the protease may attack these membranes that surround individual organs within the insect (Silva et al., 2002).
In summary, here we have described the biochemical characterization of a Photorhabdus extracellular protease, PrtA, and its associated Type I secretion apparatus. PrtA is a zinc metalloprotease of the RTX family and is encoded by the prtA gene which lies within a locus also encoding its own inhibitor and associated ABC transporter. Study of the expression of PrtA within infected insects supports its suggested role in host bioconversion as the active enzyme appears in the infected cadaver after the insect has already been killed (Daborn et al., 2001).
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ACKNOWLEDGEMENTS |
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Received 6 December 2002;
revised 7 March 2003;
accepted 14 March 2003.