Burkholderia collagen-like protein 8, Bucl8, is a unique outer membrane component of a putative tetrapartite efflux pump in Burkholderia pseudomallei and Burkholderia mallei (217 views)
Plosone (ISSN: 1932-6203linking, 1932-6203electronic), 2020 Nov 23; 15(11): e0242593-N/D.
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Paper type: Journal Article, Research Support, N. I. H. , Extramural, Non-U. S. Gov'T,
Impact factor: 2.74, 5-year impact factor: 4.383
Url: Not available.
Keywords: Bacterial Outer Membrane Proteins Metabolism Physiology, Burkholderia Genetics Metabolism, Burkholderia Mallei Genetics Metabolism, Burkholderia Pseudomallei Genetics Metabolism, Collagen Metabolism, Drug Resistance, Multiple, Bacterial Genetics, Escherichia Coli Genetics, Escherichia Coli Proteins Genetics, Bacterial Drug Effects, Membrane Transport Proteins Metabolism, Operon Drug Effects, Transcription Factors Metabolism,
Affiliations:
*** IBB - CNR ***
Department of Microbiology, Immunology and Cell Biology, School of Medicine, West Virginia University, Morgantown, WV, United States of America.
Cancer Center, West Virginia University, Morgantown, WV, United States of America.
Bioinformatics Core, West Virginia University, Morgantown, WV, United States of America.
Department of Pathology, West Virginia University, Morgantown, WV, United States of America.
Bacteriology Division, The United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Frederick, MD, United States of America.
Institute of Biostructures and Bioimaging, National Research Council, Naples, Italy.
References:
Not available.
Plosone (ISSN: 1932-6203linking, 1932-6203electronic), 2020 Nov 23; 15(11): e0242593-N/D.
Export to BibTeX Export to EndNote
Paper type: Journal Article, Research Support, N. I. H. , Extramural, Non-U. S. Gov'T,
Impact factor: 2.74, 5-year impact factor: 4.383
Url: Not available.
Keywords: Bacterial Outer Membrane Proteins Metabolism Physiology, Burkholderia Genetics Metabolism, Burkholderia Mallei Genetics Metabolism, Burkholderia Pseudomallei Genetics Metabolism, Collagen Metabolism, Drug Resistance, Multiple, Bacterial Genetics, Escherichia Coli Genetics, Escherichia Coli Proteins Genetics, Bacterial Drug Effects, Membrane Transport Proteins Metabolism, Operon Drug Effects, Transcription Factors Metabolism,
Affiliations:
*** IBB - CNR ***
Department of Microbiology, Immunology and Cell Biology, School of Medicine, West Virginia University, Morgantown, WV, United States of America.
Cancer Center, West Virginia University, Morgantown, WV, United States of America.
Bioinformatics Core, West Virginia University, Morgantown, WV, United States of America.
Department of Pathology, West Virginia University, Morgantown, WV, United States of America.
Bacteriology Division, The United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Frederick, MD, United States of America.
Institute of Biostructures and Bioimaging, National Research Council, Naples, Italy.
References:
Not available.
Burkholderia collagen-like protein 8, Bucl8, is a unique outer membrane component of a putative tetrapartite efflux pump in Burkholderia pseudomallei and Burkholderia mallei
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.
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.
Burkholderia collagen-like protein 8, Bucl8, is a unique outer membrane component of a putative tetrapartite efflux pump in Burkholderia pseudomallei and Burkholderia mallei
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