Genome-scale analysis of the genes that contribute to Burkholderia pseudomallei biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster.

Grace I Borlee,Mary N Burtnick,Herbert P Schweizer,Mihnea R Mangalea,Paul J Brett,Bradley R Borlee,John T Belisle,M Nurul Islam,Ivo Steinmetz,Kevin H Martin,Nawarat Somprasong,David P Aucoin,Dean C Crick,Brooke A Plumley,Nicholas P Day

doi:10.1371/journal.pntd.0005689

Abstract

Burkholderia pseudomallei, the causative agent of melioidosis, is an important public health threat due to limited therapeutic options for treatment. Efforts to improve therapeutics for B. pseudomallei infections are dependent on the need to understand the role of B. pseudomallei biofilm formation and its contribution to antibiotic tolerance and persistence as these are bacterial traits that prevent effective therapy. In order to reveal the genes that regulate and/or contribute to B. pseudomallei 1026b biofilm formation, we screened a sequence defined two-allele transposon library and identified 118 transposon insertion mutants that were deficient in biofilm formation. These mutants include transposon insertions in genes predicted to encode flagella, fimbriae, transcriptional regulators, polysaccharides, and hypothetical proteins. Polysaccharides are key constituents of biofilms and B. pseudomallei has the capacity to produce a diversity of polysaccharides, thus there is a critical need to link these biosynthetic genes with the polysaccharides they produce to better understand their biological role during infection. An allelic exchange deletion mutant of the entire B. pseudomallei biofilm-associated exopolysaccharide biosynthetic cluster was decreased in biofilm formation and produced a smooth colony morphology suggestive of the loss of exopolysaccharide production. Conversely, deletion of the previously defined capsule I polysaccharide biosynthesis gene cluster increased biofilm formation. Bioinformatics analyses combined with immunoblot analysis and glycosyl composition studies of the partially purified exopolysaccharide indicate that the biofilm-associated exopolysaccharide is neither cepacian nor the previously described acidic exopolysaccharide. The biofilm-associated exopolysaccharide described here is also specific to the B. pseudomallei complex of bacteria. Since this novel exopolysaccharide biosynthesis cluster is retained in B. mallei, it is predicted to have a role in colonization and infection of the host. These findings will facilitate further advances in understanding the pathogenesis of B. pseudomallei and improve diagnostics and therapeutic treatment strategies.

Highlights

B. pseudomallei, an environmental saprophyte, is the etiological agent of melioidosis and has been traditionally described as being endemic to Northern Australia and Southeast Asia [1]
A better understanding of the role of biofilm formation during pathogenesis will aid in melioidosis diagnosis and the development of new therapeutics and vaccines
Relapsing melioidosis is correlated with biofilm formation and the role of biofilm growth during chronic human infections has been widely accepted

Summary

Introduction

B. pseudomallei, an environmental saprophyte, is the etiological agent of melioidosis and has been traditionally described as being endemic to Northern Australia and Southeast Asia [1]. B. pseudomallei is an important global pathogen, as indicated by a recently published study that predicts approximately 165,000 human cases of melioidosis with greater than 50% mortality annually in 79 countries where the pathogen is probably endemic [6]. Bacteria growing as a biofilm are embedded in a matrix comprised of self-produced extracellular polymeric substances (EPS) that include polysaccharides, proteins, lipids, and nucleic acids. This matrix is thought to serve as a scaffold to hold biofilm cells together and protect from some antimicrobials (see [10] for recent review). Additional capsular polysaccharides and exopolysaccharides remain to be identified and Burkholderia pseudomallei biofilm-associated genes characterized. In the absence of information linking the identity, structural composition, and expression of these EPS components, it will not be possible to determine their role in the establishment and progression of disease

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Conclusion