Distribution and Expression of the ZmpA Metalloprotease in the Burkholderia cepacia Complex

Abstract

The distribution of the metalloprotease gene zmpA was determined among strains of the Burkholderia cepacia complex (Bcc). The zmpA gene was present in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria and B. pyrrocinia but absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The presence of zmpA generally correlated with extracellular proteolytic activity with the exception of five strains, which had zmpA but had no detectable proteolytic activity when skim milk agar was used as a substrate (zmpA protease deficient). Western immunoblot experiments with anti-ZmpA antibodies suggest that the zmpA protease-deficient strains do not secrete or accumulate detectable ZmpA. Transcriptional zmpA::lacZ fusions were introduced in selected strains of the Bcc. zmpA::lacZ was expressed in all strains, but expression was generally lower in the zmpA proteasedeficient strains than in the zmpA protease-proficient strains. Quantitative reverse transcriptase real-time PCR demonstrated that zmpA protease-deficient strains did express zmpA mRNA, although at various levels. ZmpA has previously been shown to be positively regulated by the CepIR quorum-sensing system. Addition of exogenous AHLs did not restore extracellular protease production to any of the zmpA protease-deficient strains; however, introduction of cepR in trans complemented protease activity in two of five strains. Extracellular proteolytic activity was restored by the presence of zmpA in trans in two of the five strains. These studies suggest that although some strains of the Bcc contain the zmpA gene, multiple factors may influence its expression.

Identification of Specific and Universal Virulence Factors in Burkholderia cenocepacia Strains by Using Multiple Infection Hosts

“…The production of extracellular proteolytic activity is stringently QS regulated in BCC strains (23,34). Two zinc metalloproteases, encoded by zmpA and zmpB, are positively regulated by CepR and CciR (34,48,63).…”

The Burkholderia cenocepacia LysR-Type Transcriptional Regulator ShvR Influences Expression of Quorum-Sensing, Protease, Type II Secretion, and afc Genes

“…Analysis of sequenced genomes at http://www.burkholderia.com (60) indicates that B. cenocepacia J2315 is the only Burkholderia strain which lacks gspL, which is part of the gspG operon (21). B. cenocepacia J2315 produces little or no protease activity on skim milk agar despite detection of high zmpA expression levels using qRT-PCR (17). QS regulation of the T2SS is observed in Pseudomonas aeruginosa and Burkholderia glumae.…”

“…ZmpA and ZmpB extracellular proteases each contribute to virulence in a rat chronic respiratory infection model (26). Several studies highlight the importance of the T2SS, encoded by the gsp genes, for secretion of proteases and other extracellular enzymes (14,17,27,52). Protease, phospholipase C, and hemolysin activities were reduced due to a gspN mutation in Burkholderia vietnamiensis CEP40 (14).…”

The Type 2 Secretion Pseudopilin, gspJ , Is Required for Multihost Pathogenicity of Burkholderia cenocepacia AU1054

“…However, for the ET12 strain K56-2, the T2SS-dependent protease ZmpA is required for persistence in the rat lung, and both ZmpA and ZmpB are required for K56-2-induced inflammation in the same rat model (40). Furthermore, strain K56-2 with a functional T2SS was more virulent to nematodes and produces more protease activity than strain J2315 with a nonfunctional T2SS (10,25). These observations suggest that the ancestral environmental B. cenocepacia had a functional T2SS during infection of the cystic fibrosis lung and that cheater strains with nonfunctional T2SS frequently occur during chronic lung infections.…”