Genetic and Transcriptional Analysis of the Siderophore Malleobactin Biosynthesis and Transport Genes in the Human Pathogen Burkholderia pseudomallei K96243

Abstract

Burkholderia pseudomallei is a gram-negative facultative intracellular pathogen that causes melioidosis, an invasive disease of humans and animals. To address the response of this bacterium to iron-limiting conditions, we first performed a global transcriptional analysis of RNA extracted from bacteria grown under iron-limiting and iron-rich conditions by microarrays. We focused our study on those open reading frames (ORFs) induced under iron limitation, which encoded predicted proteins that could be involved in the biosynthesis and uptake of the siderophore malleobactin. We purified this siderophore and determined that it consisted of at least three compounds with different molecular weights. We demonstrated that ORFs BPSL1776 and BPSL1774, designated mbaA and mbaF, respectively, are involved in the biosynthesis of malleobactin, while BPSL1775, named fmtA, is involved in its transport. These genes are in an operon with two other ORFs (mbaJ and mbaI) whose transcription is under the control of MbaS, a protein that belongs to the extracytoplasmic function sigma factors. Interestingly, the transcription of the mbaA, fmtA, and mbaS genes is not controlled by the availability of the siderophore malleobactin.Burkholderia pseudomallei is a gram-negative facultative intracellular pathogen that is the causative agent of melioidosis. Although this invasive disease is endemic to southeast Asia and northern Australia, its worldwide availability, high rate of mortality, and aerosol infectivity resulted in its classification as a select agent (12, 65). B. pseudomallei is also a saprophytic organism that has developed high resistance to many hostile environmental conditions such as acidic environments, dehydration, prolonged nutrient starvation, and antiseptic and detergent solutions (12).Despite increasing knowledge on the epidemiology of this disease, very little is known on the molecular mechanisms of the infection; only a few virulence factors, i.e., the capsular polysaccharide, lipopolysaccharide, type IV pilus, and a type III secretion apparatus, have been described with animal models (55,64). This pathogen can invade phagocytic and nonphagocytic cells using the type III secretion apparatus to promote its escape into the cytoplasm, where the bacterium induces polar actin polymerization (56, 57). Intracellular B. pseudomallei cells can induce host cell membrane protrusions that may facilitate cell-to-cell spread of the bacteria and induce the fusion of adjacent cells forming giant multinucleated cells (24,25,54). Sequencing of the two B. pseudomallei chromosomes (22) has generated a significant amount of data expected to yield information on new virulence factors and putative targets for vaccine development.A potential virulence factor in B. pseudomallei that so far has not been studied is the ability to utilize iron, a feature that may play an important role in the pathogenesis of the disease caused by this bacterium. Iron is required for the growth of nearly all microorganisms in the environment, as well as in biologica...

In vivo expression technology identifies a type VI secretion system locus in Burkholderia pseudomallei that is induced upon invasion of macrophages

“…The presence of a Fur box overlapping the putative promoter for bhuR suggests that the haem uptake system of B. pseudomallei is induced in response to iron starvation. Consistent with this, microarray analysis has shown that transcription of BPSS0243 (bhuS) and BPSS0244 (bhuR) is induced under iron-limiting conditions (Tuanyok et al, 2005;Alice et al, 2006). It has been observed previously that iron acquisition genes are required by other intracellular pathogens, such as Legionella pneumophila, for growth within macrophages (Pope et al, 1996).…”

Iron acquisition mechanisms of the Burkholderia cepacia complex

“…However, in contrast to the pyochelin receptor, synthesis of which is inducible by its cognate iron-loaded siderophore (Heinrichs and Poole 1996;Michel et al 2005), orbA gene expression is not stimulated by the presence of ferric-ornibactin, and the ability to utilise ornibactin is not a prerequisite for production of ornibactin (Sokol et al 2000). Similar observations have been made regarding the malleobactin receptor of B. pseudomallei (Alice et al 2006). Transport of ornibactin across the cytoplasmic membrane is likely to be specified by the orbB, orbC and orbD genes (BCAL1694, BCAL1691 and BCAL1692, respectively).…”

“…The TonBdependent pyochelin outer membrane receptor, FptA, and/or its gene, have been identified in P. aeruginosa, P. fluorescens, B. cenocepacia and B. pseudomallei (Heinrichs et al 1991;Ankenbauer 1992;Ankenbauer and Quan 1994;Visser et al 2004;Paulsen et al 2005;Alice et al 2006). Additionally, Southern hybridisation experiments have identified the presence of the fptA gene in the genomes of P. aureofaciens, P. putida and B. cepacia 25416, although P. aureofaciens and P. putida are not known to produce pyochelin (Castignetti 1997).…”

“…The nature of the species that did not produce ornibactin in the Darling et al study are not known (P. Sokol, personal communication). More recently, strong evidence has been provided that the siderophore malleobactin, produced by B. pseudomallei, is, in fact, ornibactin (Alice et al 2006). Indeed, ornibactin appears to be the most prevalent siderophore among the Burkholderia, and is produced by a much greater proportion of clinical isolates than is pyochelin (Darling et al 1998).…”

“…Among a panel of 14 B. vietnamiensis strains, none were shown to produce pyochelin (Meyer et al 1995). Recently, it has been shown that the B. pseudomallei genome encodes the machinery necessary for the biosynthesis and transport of pyochelin (Holden et al 2004;Tuanyok et al 2005;Alice et al 2006). The pathway for the biosynthesis of pyochelin in P. aeruginosa has been the subject of extensive investigation (Quadri 2000).…”