Burkholderia collagen-like protein 8, Bucl8, is a unique outer membrane component of a putative tetrapartite efflux pump in Burkholderia pseudomallei and Burkholderia mallei

PLoS One. 2020 Nov 23;15(11):e0242593. doi: 10.1371/journal.pone.0242593. eCollection 2020.

Abstract

Bacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identified in silico Burkholderia collagen-like protein 8 (Bucl8) in the hazardous pathogens Burkholderia pseudomallei and Burkholderia mallei. We hypothesize that Bucl8, which contains two predicted tandem outer membrane efflux pump domains, is a component of a putative efflux pump. Unique to Bucl8, as compared to other outer membrane proteins, is the presence of an extended extracellular region containing a collagen-like (CL) domain and a non-collagenous C-terminus (Ct). Molecular modeling and circular dichroism spectroscopy with a recombinant protein, corresponding to this extracellular CL-Ct portion of Bucl8, demonstrated that it adopts a collagen triple helix, whereas functional assays screening for Bucl8 ligands identified binding to fibrinogen. Bioinformatic analysis of the bucl8 gene locus revealed it resembles a classical efflux-pump operon. The bucl8 gene is co-localized with downstream fusCDE genes encoding fusaric acid (FA) resistance, and with an upstream gene, designated as fusR, encoding a LysR-type transcriptional regulator. Using reverse transcriptase (RT)-qPCR, we defined the boundaries and transcriptional organization of the fusR-bucl8-fusCDE operon. We found exogenous FA induced bucl8 transcription over 80-fold in B. pseudomallei, while deletion of the entire bucl8 locus decreased the minimum inhibitory concentration of FA 4-fold in its isogenic mutant. We furthermore showed that the putative Bucl8-associated pump expressed in the heterologous Escherichia coli host confers FA resistance. On the contrary, the Bucl8-associated pump did not confer resistance to a panel of clinically-relevant antimicrobials in Burkholderia and E. coli. We finally demonstrated that deletion of the bucl8-locus drastically affects the growth of the mutant in L-broth. We determined that Bucl8 is a component of a novel tetrapartite efflux pump, which confers FA resistance, fibrinogen binding, and optimal growth.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Outer Membrane Proteins / metabolism
  • Bacterial Outer Membrane Proteins / physiology
  • Burkholderia / genetics
  • Burkholderia / metabolism
  • Burkholderia mallei / genetics
  • Burkholderia mallei / metabolism*
  • Burkholderia pseudomallei / genetics
  • Burkholderia pseudomallei / metabolism*
  • Collagen / metabolism
  • Drug Resistance, Multiple, Bacterial / genetics
  • Escherichia coli / genetics
  • Escherichia coli Proteins / genetics
  • Genes, Bacterial / drug effects
  • Membrane Transport Proteins / metabolism*
  • Operon / drug effects
  • Transcription Factors / metabolism

Substances

  • Bacterial Outer Membrane Proteins
  • Escherichia coli Proteins
  • Membrane Transport Proteins
  • Transcription Factors
  • Collagen