Burkholderia mallei and Burkholderia pseudomallei Cluster 1 Type VI Secretion System Gene Expression Is Negatively Regulated by Iron and Zinc

Mary N Burtnick,Paul J Brett,Ronald Mark Wooten

doi:10.1371/journal.pone.0076767

Abstract

Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM) were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

Highlights

Burkholderia mallei, the etiologic agent of glanders, is a Gramnegative bacillus that primarily infects horses, mules and donkeys, and occasionally humans
We describe the use of minimal media formulations that facilitate Hcp1 production and demonstrate that virG is required for T6SS-1 gene expression during growth in this media
For growth of the Burkholderia strains in minimal media, the following formulations were used: Difco M9 Minimal Salts supplemented with 0.4% glucose (M9G); M9G supplemented with 0.5% (w/v) Bacto Casamino Acids (M9CG); or M9G supplemented with 0.5% Bacto Tryptone (M9TG)

Summary

Introduction

Burkholderia mallei, the etiologic agent of glanders, is a Gramnegative bacillus that primarily infects horses, mules and donkeys, and occasionally humans. Chronically infected horses are believed to be the only reservoir of this host-adapted pathogen [1,2,3,4]. Disease in equines presents as chronic or acute illnesses that are commonly characterized by the presence of caseous pulmonary nodules, nasal ulcerations and cutaneous abscess formation [4,8,11]. Due to the high risk of aerosol infection and the historical use of this organism as a biological weapon, B. mallei is currently listed as a Tier 1 Select Agent by the Centers for Disease Control and Prevention (CDC) [4,12,14,15]

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