Identification and Expression of a Burkholderia pseudomallei Collagenase in Escherichia coli

Abstract

Extracellular protein profiles were compared for broth-grown cultures of Burkholderia pseudomallei and its avirulent close relative Burkholderia thailandensis. A number of protein bands were present in the B. pseudomallei profile but absent or less abundant in the B. thailandensis profile. Four such prominent secreted proteins were identified by using N-terminal sequencing coupled to searches of the B. pseudomallei genome sequence database. The genes for two proteins with similarity to carbohydrate-binding proteins, and a further protein homologous to known bacterial collagenases, were present in both B. pseudomallei and B. thailandensis. The putative collagenase gene was cloned and expressed as a fusion protein in Escherichia coli. Cell lysates from Escherichia coli containing the recombinant protein exhibited detectable gelatinase and collagenase activities.

Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM

“…Three of these proteins, BURPS668_2041, BURPS668_A0759, and BURPS668_1946, were identified in this study as members of the T2SS secretome ( Table 2). The last protein, BURPS668_ A1216, was a protein with homology to known bacterial collagenases (47). We found two putative collagenases, including BURPS668_A1216, that were present in roughly equal amounts in the rich-medium supernatants of MSHR668 and 668 ⌬gspD, suggesting that they are secreted in a T2SS-independent manner (Table 3).…”

“…In 2004, Rainbow et al (47) performed N-terminal sequencing on four proteins that were present in the supernatant of pathogenic B. pseudomallei but absent in the supernatant of nonpathogenic B. thailandensis. Three of these proteins, BURPS668_2041, BURPS668_A0759, and BURPS668_1946, were identified in this study as members of the T2SS secretome ( Table 2).…”

Nonadaptive radiation of the gut microbiome in an adaptive radiation of Cyprinodon pupfishes with minor shifts for scale-eating

“…Burkholderiaceae is a family of proteobacteria which contains many human and animal pathogens (diCenzo et al 2019), plant and insect symbionts (Gyaneshwar et al 2011;Takeshita and Kikuchi 2017), and can be found in soil, water, and polluted environments (Coenye and Vandamme 2003;Estrada-de los Santos et al 2016). They also include some collagenase-producing bacteria, such as Burkholderia pseudomallei (Uni-ProtKB -A3P3M6; Rainbow et al 2004), which is the causative agent of melioidosis in humans (Holden et al 2004).…”

Nonadaptive radiation of the gut microbiome in an adaptive radiation ofCyprinodonpupfishes with minor shifts for scale-eating

“…Burkholderiaceae is a family of proteobacteria which contains many human and animal pathogens (diCenzo et al 2019), plant and insect symbionts (Gyaneshwar et al 2011; Takeshita and Kikuchi 2017), and can be found in soil, water, and polluted environments (Coenye and Vandamme 2003; Estrada-de los Santos et al 2016). They also include some collagenase-producing bacteria, such as Burkholderia pseudomallei (UniProtKB – A3P3M6; Rainbow et al 2004), which is the causative agent of melioidosis in humans (Holden et al 2004).…”