Inactivation of bpsl1039-1040 ATP-binding cassette transporter reduces intracellular survival in macrophages, biofilm formation and virulence in the murine model of Burkholderia pseudomallei infection.

Peechanika Pinweha,Sam Willcocks,Paiboon Vattanaviboon,Veerachat Muangsombut,Niramol Jitprasutwit,Usa Boonyuen,Sunee Korbsrisate,Parameth Thiennimitr,Jon Cuccui,Pornpan Pumirat,Gregory J Bancroft,Felipe Cia,Varintip Srinon,Brendan W Wren

doi:10.1371/journal.pone.0196202

Abstract

Burkholderia pseudomallei, a gram-negative intracellular bacillus, is the causative agent of a tropical infectious disease called melioidosis. Bacterial ATP-binding cassette (ABC) transporters import and export a variety of molecules across bacterial cell membranes. At present, their significance in B. pseudomallei pathogenesis is poorly understood. We report here characterization of the BPSL1039-1040 ABC transporter. B. pseudomallei cultured in M9 medium supplemented with nitrate, demonstrated that BPSL1039-1040 is involved in nitrate transport for B. pseudomallei growth under anaerobic, but not aerobic conditions, suggesting that BPSL1039-1040 is functional under reduced oxygen tension. In addition, a nitrate reduction assay supported the function of BPSL1039-1040 as nitrate importer. A bpsl1039-1040 deficient mutant showed reduced biofilm formation as compared with the wild-type strain (P = 0.027) when cultured in LB medium supplemented with nitrate under anaerobic growth conditions. This reduction was not noticeable under aerobic conditions. This suggests that a gradient in oxygen levels could regulate the function of BPSL1039-1040 in B. pseudomallei nitrate metabolism. Furthermore, the B. pseudomallei bpsl1039-1040 mutant had a pronounced effect on plaque formation (P < 0.001), and was defective in intracellular survival in both non-phagocytic (HeLa) and phagocytic (J774A.1 macrophage) cells, suggesting reduced virulence in the mutant strain. The bpsl1039-1040 mutant was found to be attenuated in a BALB/c mouse intranasal infection model. Complementation of the bpsl1039-1040 deficient mutant with the plasmid-borne bpsl1039 gene could restore the phenotypes observed. We propose that the ability to acquire nitrate for survival under anaerobic conditions may, at least in part, be important for intracellular survival and has a contributory role in the pathogenesis of B. pseudomallei.

Highlights

Burkholderia pseudomallei is a non-spore-forming, gram-negative bacillus that causes a severe, human, tropical infectious disease called melioidosis [1]
This result is similar to other bacteria [22], in which ATP-binding cassette (ABC) transporter genes are often clustered in an operon
The upstream gene of bpsl1039 and downstream gene of bpsl1040 are annotated to be transcribed in an opposite direction, suggesting that only bpsl1039 and bpsl1040 are expressed in the same operon. This Reverse Transcriptase (RT)-PCR result suggests that the B. pseudomallei 6H4 mutant has a mutation in bpsl1039 and bpsl1040

Summary

Introduction

Burkholderia pseudomallei is a non-spore-forming, gram-negative bacillus that causes a severe, human, tropical infectious disease called melioidosis [1]. This saprophytic and facultative intracellular bacterium is found in the soil and water within endemic areas, including Southeast Asia and Northern Australia. Limmathurotsakul et al [2] estimated that there are 165,000 human melioidosis cases worldwide annually, from which 89,000 people die, demonstrating the underappreciated importance of this severe infectious disease. Treatment with ineffective antimicrobials may result in case fatality rates exceeding 70% [2]. There is a need to develop a deeper understanding of B. pseudomallei pathogenesis and to identify potential vaccines to prevent this life-threatening bacterial disease

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