Data_Sheet_1_Rv1717 Is a Cell Wall - Associated β-Galactosidase of Mycobacterium tuberculosis That Is Involved in Biofilm Dispersion.pdf

Understanding the function of conserved hypothetical protein (CHP)s expressed by a pathogen in the infected host can lead to better understanding of its pathogenesis. The present work describes the functional characterization of a CHP, Rv1717 of Mycobacterium tuberculosis (Mtb). Rv1717 has been previously reported to be upregulated in TB patient lungs. Rv1717 belongs to the cupin superfamily of functionally diverse proteins, several of them being carbohydrate handling proteins. Bioinformatic analysis of the amino acid sequence revealed similarity to glycosyl hydrolases. Enzymatic studies with recombinant Rv1717 purified from Escherichia coli showed that the protein is a β-D-galactosidase specific for pyranose form rather than the furanose form. We expressed the protein in Mycobacterium smegmatis (Msm), which lacks its ortholog. In MsmRv1717, the protein was found to localize to the cell wall (CW) with a preference to the poles. MsmRv1717 showed significant changes in colony morphology and cell surface properties. Most striking observation was its unusual Congo red colony morphotype, reduced ability to form biofilms, pellicles and autoagglutinate. Exogenous Rv1717 not only prevented biofilm formation in Msm, but also degraded preformed biofilms, suggesting that its substrate likely exists in the exopolysaccharides of the biofilm matrix. Presence of galactose in the extracellular polymeric substance (EPS) has not been reported before and hence we used the galactose-specific Wisteria floribunda lectin (WFL) to test the same. The lectin extensively bound to Msm and Mtb EPS, but not the bacterium per se. Purified Rv1717 also hydrolyzed exopolysaccharides extracted from Msm biofilm. Eventually, to decipher its role in Mtb, we downregulated its expression and demonstrate that the strain is unable to disperse from in vitro biofilms, unlike the wild type. Biofilms exposed to carbon starvation showed a sudden upregulation of Rv1717 transcripts supporting the potential role of Rv1717 in Mtb dispersing from a deteriorating biofilm.

解析病原体在感染宿主中表达的保守假想蛋白（conserved hypothetical protein, CHP）的功能，有助于更深入阐释其致病机制。本研究针对结核分枝杆菌（Mycobacterium tuberculosis, Mtb）的CHP Rv1717开展功能表征。既往研究已证实，Rv1717在结核病患者肺部的表达水平显著上调。Rv1717隶属于功能多样的杯状蛋白超家族，该超家族中诸多蛋白为糖类加工相关蛋白。对其氨基酸序列的生物信息学分析显示，该蛋白与糖基水解酶具有序列相似性。以从大肠杆菌中纯化获得的重组Rv1717开展酶学实验，结果表明其为特异性识别吡喃糖型而非呋喃糖型的β-D-半乳糖苷酶。我们将该蛋白在缺乏其同源蛋白的耻垢分枝杆菌（Mycobacterium smegmatis, Msm）中进行异源表达。在表达Rv1717的耻垢分枝杆菌（MsmRv1717）中，该蛋白定位于细胞壁（cell wall, CW），且偏好定位于菌体两极。MsmRv1717的菌落形态与细胞表面特性出现显著改变，最突出的表型为异常的刚果红菌落形态，同时其形成生物被膜、菌膜的能力以及自凝集能力均显著下降。外源添加的Rv1717不仅可抑制耻垢分枝杆菌的生物被膜形成，还能降解已形成的生物被膜，提示其底物可能存在于生物被膜基质的胞外多糖中。此前尚无研究报道胞外聚合物（extracellular polymeric substance, EPS）中存在半乳糖，因此我们采用半乳糖特异性的紫藤凝集素（Wisteria floribunda lectin, WFL）开展验证实验。该凝集素可广泛结合耻垢分枝杆菌与结核分枝杆菌的EPS，但并不直接结合菌体本身。纯化获得的Rv1717同样可水解从耻垢分枝杆菌生物被膜中提取的胞外多糖。为进一步解析Rv1717在结核分枝杆菌中的功能，我们下调了其表达水平，结果显示该突变菌株无法像野生型菌株一样从体外生物被膜中解离。在碳饥饿条件下培养的生物被膜中，Rv1717的转录本水平显著上调，这进一步支持了Rv1717在结核分枝杆菌从衰退生物被膜中解离过程中发挥潜在作用的结论。