Burkholderia cenocepacia ZmpB is a broad-specificity zinc metalloprotease involved in virulence

Abstract

In previous studies we characterized the Burkholderia cenocepacia ZmpA zinc metalloprotease. In this study, we determined that B. cenocepacia has an additional metalloprotease, which we designated ZmpB. The zmpB gene is present in the same species as zmpA and was detected in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria, and B. pyrrocinia but was absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The zmpB gene was expressed, and ZmpB was purified from Escherichia coli by using the pPROEXHTa His(6) Tag expression system. ZmpB has a predicted preproenzyme structure typical of thermolysin-like proteases and is distantly related to Bacillus cereus bacillolysin. ZmpB was expressed as a 63-kDa preproenzyme precursor that was autocatalytically cleaved into mature ZmpB (35 kDa) and a 27-kDa prepropeptide. EDTA, 1,10-phenanthroline, and Zn(2+) cations inhibited ZmpB enzyme activity, indicating that it is a metalloprotease. ZmpB had proteolytic activity against alpha-1 proteinase inhibitor, alpha(2)-macrogobulin, type IV collagen, fibronectin, lactoferrin, transferrin, and immunoglobulins. B. cenocepacia zmpB and zmpA zmpB mutants had no proteolytic activity against casein and were less virulent in a rat agar bead chronic infection model, indicating that zmpB is involved in B. cenocepacia virulence. Expression of zmpB was regulated by both the CepIR and CciIR quorum-sensing systems.

FIG. 1.

Expression of ZmpA and ZmpB in K56-2 zmpB and K56-2 zmpA zmpB mutants. Supernatant proteins were precipitated with 10% trichloroacetic acid, separated by Tricine-14% SDS-PAGE, and blotted to PVDF membranes. A. Coomassie blue-stained gel. B. Western blot reacted with anti-ZmpB. C. Western blot reacted with anti-ZmpA. Lanes 1, K56-2; lanes 2, K56-2 zmpB; lanes 3, K56-2 zmpB(pBS6); lanes 4, K56-2 zmpA zmpB; lanes 5, K56-2 zmpA; lanes 6, recombinant ZmpB; lanes 7, recombinant ZmpA. Solid arrow, ZmpA; open arrow, ZmpB.

FIG. 2.

Expression and purification of ZmpB. Lane M, molecular weight markers (in thousands); lane 1, cell lysate after induction with IPTG; lane 2, uninduced crude cell extract; lane 3, purified inclusion bodies; lane 4, purified recombinant ZmpB after refolding and elution from an Ni-NTA column. Solid arrow, His-prepro-ZmpB fusion protein; open arrow, indicates the His-prepropeptide. The mature ZmpB is indicated.

FIG. 3.

Substrate specificity of ZmpB. Substrates were digested with ZmpB, ZmpAH465G (which has no proteolytic activity), or in the absence of enzyme (no protease) for 24 to 48 h. Proteins were separated by SDS-PAGE and stained with Coomassie blue. Collagen, fibronectin, and α₂-macroglobulin were electrophoresed by SDS-7.5% PAGE. All other substrates were electrophoresed by SDS-12.5% PAGE.

FIG. 4.

Regulation of zmpB by the cepIR and cciIR quorum-sensing genes. Plasmid pBS9 containing a zmpB::luxCDABE fusion was introduced into K56-2 quorum-sensing mutants. Cultures were grown for 20 h, and luminescence in counts per second (CPS) and turbidity at an absorbance of 600 nm were measured using a Wallac Victor² 1420 multilabel counter. Values shown are the mean relative CPS (±standard deviation) for triplicate cultures and are representative of three independent experiments. *, significantly different from result for K56-2 (P < 0.05 by analysis of variance).

FIG. 5.

Distribution of ZmpB in the B. cepacia complex. A. Southern hybridization analysis of the zmpB gene. A 0.8-kb ³²P-labeled MluI fragment encoding from Arg-35 to Asn-307 of the 323-amino-acid mature ZmpB from K56-2 was used to probe NotI-digested genomic DNA. Lane 1, K56-2; lane 2, B. cepacia LMG17997; lane 3, B. multivorans LMG13010; lane 4, B. cenocepacia J2315; lane 5, B. stabilis LMG14294; lane 6, B. stabilis C7322; lane 7, B. vietnamiensis PC259; lane 8, B. dolosa LMG18943; lane 9, B. ambifaria Cep996; lane 10, B. anthina LMG20980; lane 11, B. pyrrocinia LMG21822. B. Detection of ZmpB by Western blotting of culture supernatants. Trichloroacetic acid-precipitated culture supernatants were separated by Tricine 14%-PAGE and blotted onto PVDF membrane. Blots were reacted with anti-ZmpB. Lane 1, K56-2; lane 2, B. cepacia LMG17997; lane 3, B. multivorans LMG13010; lane 4, B. cenocepacia C6433; lane 5, B. stabilis LMG18888; lane 6, B. vietnamiensis Pc259; lane 7, B. dolosa LMG18943; lane 8, B. ambifaria CEP996; lane 9, B. anthina LMG20980; lane 10, B. pyrrocinia LMG 21822; lane 11, recombinant ZmpB.