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

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.

类鼻疽伯克霍尔德菌（Burkholderia pseudomallei）是类鼻疽病的致病菌，由于其临床治疗手段有限，已成为重要的公共卫生威胁。针对类鼻疽伯克霍尔德菌感染的治疗方案优化研究，依赖于对该菌生物膜形成的作用、以及其对抗生素耐受与持续感染的贡献的深入理解——因为这些细菌特性是阻碍有效治疗的关键因素。为了揭示调控或参与类鼻疽伯克霍尔德菌1026b生物膜形成的基因，本研究筛选了序列定义的双等位基因转座子文库，鉴定出118株生物膜形成缺陷的转座子插入突变体。这些突变体的转座子插入位点涉及预测编码鞭毛、菌毛、转录调控因子、多糖以及假设蛋白（hypothetical protein）的基因。多糖是生物膜的核心组成成分，类鼻疽伯克霍尔德菌具备合成多种多糖的能力，因此亟需将这些多糖生物合成基因与其所合成的多糖建立关联，以更好地解析其在感染过程中的生物学功能。针对类鼻疽伯克霍尔德菌全基因组中与生物膜相关的胞外多糖（exopolysaccharide）生物合成基因簇构建的等位交换缺失突变体，其生物膜形成能力显著下降，且菌落形态呈光滑型，提示该突变体丧失了胞外多糖合成能力。与之相反，敲除此前已明确的荚膜I多糖生物合成基因簇后，菌株的生物膜形成能力反而增强。生物信息学分析结合免疫印迹分析与部分纯化胞外多糖的糖基组成研究结果表明，本研究关联的生物膜相关胞外多糖既非西帕聚糖（cephacian），亦非此前报道的酸性胞外多糖。本次报道的生物膜相关胞外多糖仅特异性存在于类鼻疽伯克霍尔德菌复合群中。由于该新型胞外多糖生物合成基因簇在马鼻疽伯克霍尔德菌（B. mallei）中也存在保守序列，因此推测其在宿主定植与感染过程中发挥重要作用。本研究结果将有助于进一步推动类鼻疽伯克霍尔德菌致病机制的解析，并为优化诊断方法与治疗策略提供理论支撑。