A new broad-spectrum protease inhibitor from the entomopathogenic bacterium Photorhabdus luminescens

Abstract

A new protease inhibitor was purified to apparent homogeneity from a culture medium of Photorhabdus luminescens by ammonium sulfate precipitation and preparative isoelectric focusing followed by affinity chromatography. Ph. luminescens, a bacterium symbiotically associated with the insect-parasitic nematode Heterorhabditis bacteriophora, exists in two morphologically distinguishable phases (primary and secondary). It appears that only the secondary-phase bacterium produces this protease inhibitor. The protease inhibitor has an M r of approximately 12 000 as determined by SDS-PAGE. Its activity is stable over a pH range of 35-11 and at temperatures below 50 SC. The N-terminal 16 amino acids of the protease inhibitor were determined as STGIVTFKND(X)GEDIV and have a very high sequence homology with the N-terminal region of an endogenous inhibitor (IA-1) from the fruiting bodies of an edible mushroom, Pleurotus ostreatus. The purified protease inhibitor inactivated the homologous protease with an almost 1 :1 stoichiometry. It also inhibited proteases from a related insect-nematode-symbiotic bacterium, Xenorhabdus nematophila. Interestingly, when present at a molar ratio of 5 to 1, this new protease inhibitor completely inactivated the activity of both trypsin and elastase. The activity of proteinase A and cathepsin G was partially inhibited by this bacterial protease inhibitor, but it had no effect on chymotrypsin, subtilisin, thermolysin and cathepsins B and D. The newly isolated protease inhibitor from the secondary-phase bacteria and its specific inhibition of its own protease provides an explanation as to why previous investigators failed to detect the presence of protease activity in the secondary-phase bacteria. The functional implications of the protease inhibitor are also discussed in relation to the physiology of nematode-symbiotic bacteria.

Comparison of Proteolytic Activities Produced by Entomopathogenic Photorhabdus Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes

“…At the same time, of the four secondary-phase Photorhabdus strains (Hm, HP88, W14, and K122) that have been tested so far for protease production, only strain Hm was not found to secrete protease activity (1). Later, however, this strain and the other three secondaryphase variants were reported to produce at least some proteolytic activity (6,23,32).…”

“…laumondii HP88, and Photorhabdus temperata subsp. temperata K122) and X. nematophila proved to be alkaline metalloproteases (5)(6)(7)32). The gene for the protease in P. luminescens W14 was found to encode an enzyme that belongs to the RTX (repeatsin-toxin) family and was designated PrtA after a similar protease in Erwinia chrysanthemi (6,13).…”

Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus

“…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.…”

“…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).…”

“…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 broadspectrum 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.…”