Proteolytic enzyme production by strains of the insect pathogen xenorhabdus and characterization of an early-log-phase-secreted protease as a potential virulence factor

Abstract

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.

FIG. 1.

Zymographic detection of Xenorhabdus proteases in culture. (A and B) Enzyme activities produced by X. kozodoii Morocco and Intermedium strains were monitored with zymography coupled to SDS-PAGE (A) and zymography coupled to native PAGE (B). (A) The activities were tested with both casein (a) and gelatin (b) as the substrate. The positions of enzyme activity bands A, B, and C are shown to the right of the gels. (B) Protease production of X. kozodoii Morocco strain is shown using casein as the substrate. The positions of enzyme activity bands 1 and 2 are shown to the left of the gel. (C) Effects of inhibitors of catalytic serine (PMSF) and metal ion (EDTA and 1,10-phenanthroline) are shown on the activity of ∼5.0 pmol of protease B (arrow), purified from X. kozodoii Morocco strain. The gel slices are as follows: a, enzyme not treated (control); b, PMSF-treated enzyme; c, EDTA-treated enzyme; d, 1,10-phenanthroline-treated enzyme. The concentration of inhibitors during both sample treatment and gel slice incubation was 5.0 mM.

FIG. 2.

Culture growth and detection of proteolytic activities with chromogenic substrates. Growth of the culture growth was monitored by measuring the optical density at 600 nm (OD₆₀₀) (filled symbols • and ▪ for X. kozodoii Morocco and Intermedium strains, respectively). The data shown are representative of four measurements for both cultures. Enzyme activities were measured with Fua-LGPA and Fua-ALVY substrates (open symbols ⋄ and ▵, respectively). The data are representative of two measurements on both substrates in the culture of X. kozodoii Morocco strain. The activities were measured using 50 μl of culture supernatant at a final concentration of substrate of 50 μM.

FIG. 3.

Detection of protease B in insects and insect mortality on Xenorhabdus infection. (A) SDS-PAGE-coupled zymographic detection with casein substrate of protease B production during G. mellonella infection (the detected activity is indicated by the arrow). The numbers above the lanes indicate the time the sample was taken in hours postinfection. Purified protease B was also run in the gels as a standard (lanes B1 and B2, ∼10.0 and ∼20.0 pmol of enzyme loaded, respectively). Lane C1, control hemolymph from PBS-injected insects; lane C2, control hemolymph from naïve insects. (B) Survival rate of G. mellonella larvae injected with ∼100 cells of X. kozodoii Morocco strain (□) or with PBS (▵). Data from four experiments are averaged. For the conditions of infection and sample preparation, see Materials and Methods.

FIG. 4.

Cleavage with protease B of proteins in the hemolymph of M. sexta. The in vitro digestion of hemolymph fractions with purified protease B is shown. The hemolymph fraction (fractions A to F) (see Materials and Methods) and whether the hemolymph fraction was incubated in the presence (+) or absence (−) of protease B are indicated above the gels. The incubation times (in minutes) are shown immediately above the gels. The bands from protease B are indicated by asterisks on the gels.

FIG. 5.

Comparison of the N-terminal sequence of protease B to several N-terminal sequences in the serralysin (M10B) subfamily of metalloproteases. Amino acids that are identical to the amino acid in protease B are shown by capital letters. UniProt identifiers are given in parentheses.