Proteolytic Enzyme Production by Strains of the Insect Pathogen Xenorhabdus and Characterization of an Early-Log-Phase-Secreted Protease as a Potential Virulence Factor

As a comparison to a similar study on Photorhabdus strains, 15 Xenorhabdus bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of Xenorhabdus strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of Xenorhabdus-infected Galleria mellonella larvae from 15 h postinfection. The purified protease B hydrolyzed in vitro seven proteins in the hemolymph of Manduca sexta that were also cleaved by PrtA peptidase from Photorhabdus. The N-terminal sequence of protease B showed similarity to a 55-kDa serralysin type metalloprotease in Xenorhabdus nematophila, which had been identified as an orthologue of Photorhabdus PrtA peptidase.Xenorhabdus and Photorhabdus bacteria are highly virulent, fatal pathogens for insects. Phylogenetically, they are sister genera in the family Enterobacteriaceae (3, 4). There are some differences between Xenorhabdus and Photorhabdus in their biology (e.g., light production), and they also differ in their interaction with their symbiotic nematode partners, which are in the Steinernematidae and Heterorhabditidae genera, respectively (8, 9). At the same time, they also have several properties in common. For example, due to their similar strategy of infection, their entrance into the hemocoel is absolutely dependent on the invasion of insects by their symbiotic nematode partners. An interesting feature of both genera is that they have two phenotypic (form) variants, primary and secondary (9). The primary form is natural, while the secondary form can be observed (generated) mostly in the laboratory. They differ in, for example, antibiotic production, outer membrane proteins, and cell surface structures (fimbriae and flagellae [23], symbiotic capabilities with nematode partners, and exoenzyme production [9]). The secondary form variants were found, with nonbiochemical detection methods, to produce less or no proteolytic activity compared to the primary phenotypic variants (see references 9 and 23 and references therein). The high pathogenicity makes Xenorhabdus and Photorhabdus good model organisms of infection, which can be exploited—by studying the function of their virulence factors—for the investigation of the immune system of insects and the mechanisms the pathogens use to cope with the immune defense of hosts. The comparative analysis of these bacterial partners provides an opportunity to study the question of how similar the infection mechanisms can be at the molecular level of two evolutionarily different insect pathogen bacterium-nematode complexes that, at the same time, have similar infection strategies.Of the virulence factors, we have been interested in secreted proteases that may be used by the pathogens during the first stage of infection in the penetration of the tissues of host or in the suppression of its immune response. The secretion and biochemistry of these enzymes are better studied in Photorhabdus, where four secreted proteases could be detected in a screen of 20 strains by a combination of five methods (15). The earliest secreted Photorhabdus protease is PrtA peptidase, a metzincin in the M10B family of serralysins. The others are PhpC (Photorhabdus protease C), which belongs to the M4 metallopeptidase family of thermolysin-like proteases, OpdA, a collagen peptidase in the family of thimet oligopeptidases and PhpD, a furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY)-cleaving enzyme, the identity of which is still unknown. In contrast, although a number of Xenorhabdus strains were tested for proteolytic activity with simple bacteriological plate assays (2, 25), only one (Xenorhabdus nematophila) was investigated by a biochemical detection method of protease activities, zymography. Two activities have been found by this method, and one of these activities has been partially characterized (5).As an approach to establish the similarity between Xenorhabdus and Photorhabdus in the mechanism of infection regarding the type and role of proteolytic enzymes, we investigated 15 Xenorhabdus strains for the secretion of proteases employing the same five detection methods that we had previously used for Photorhabdus strains. Two of the strains (Xenorhabdus nematophila AN6 and Xenorhabdus cabanillassii RIO-HU) were represented with their phenotypic variant pairs.