Data_Sheet_7_Origin of the Mobile Di-Hydro-Pteroate Synthase Gene Determining Sulfonamide Resistance in Clinical Isolates.PDF

Sulfonamides are synthetic chemotherapeutic agents that work as competitive inhibitors of the di-hydro-pteroate synthase (DHPS) enzyme, encoded by the folP gene. Resistance to sulfonamides is widespread in the clinical setting and predominantly mediated by plasmid- and integron-borne sul1-3 genes encoding mutant DHPS enzymes that do not bind sulfonamides. In spite of their clinical importance, the genetic origin of sul1-3 genes remains unknown. Here we analyze sul genes and their genetic neighborhoods to uncover sul signature elements that enable the elucidation of their genetic origin. We identify a protein sequence Sul motif associated with sul-encoded proteins, as well as consistent association of a phosphoglucosamine mutase gene (glmM) with the sul2 gene. We identify chromosomal folP genes bearing these genetic markers in two bacterial families: the Rhodobiaceae and the Leptospiraceae. Bayesian phylogenetic inference of FolP/Sul and GlmM protein sequences clearly establishes that sul1-2 and sul3 genes originated as a mobilization of folP genes present in, respectively, the Rhodobiaceae and the Leptospiraceae, and indicate that the Rhodobiaceae folP gene was transferred from the Leptospiraceae. Analysis of %GC content in folP/sul gene sequences supports the phylogenetic inference results and indicates that the emergence of the Sul motif in chromosomally encoded FolP proteins is ancient and considerably predates the clinical introduction of sulfonamides. In vitro assays reveal that both the Rhodobiaceae and the Leptospiraceae, but not other related chromosomally encoded FolP proteins confer resistance in a sulfonamide-sensitive Escherichia coli background, indicating that the Sul motif is associated with sulfonamide resistance. Given the absence of any known natural sulfonamides targeting DHPS, these results provide a novel perspective on the emergence of resistance to synthetic chemotherapeutic agents, whereby preexisting resistant variants in the vast bacterial pangenome may be rapidly selected for and disseminated upon the clinical introduction of novel chemotherapeuticals.

磺胺类药物（sulfonamides）是一类合成化疗剂，可作为二氢蝶酸合酶（di-hydro-pteroate synthase, DHPS）的竞争性抑制剂，该酶由folP基因编码。磺胺类药物耐药性在临床环境中广泛流行，主要由质粒和整合子携带的sul1-3基因介导，这些基因编码无法与磺胺类药物结合的突变型DHPS酶。尽管这类基因具有重要的临床价值，但其遗传起源至今仍不明确。本研究通过分析sul基因及其遗传邻区，挖掘可用于阐明其遗传起源的sul特征元件。本研究鉴定出与sul编码蛋白相关的蛋白质序列Sul基序（Sul motif），同时发现磷酸葡萄糖胺变位酶基因（glmM）与sul2基因存在稳定的共关联关系。我们在红杆菌科（Rhodobiaceae）和钩端螺旋体科（Leptospiraceae）两个细菌科中，鉴定出携带上述遗传标记的染色体编码folP基因。对FolP/Sul及GlmM蛋白质序列进行贝叶斯系统发育推断，结果明确证实sul1-2和sul3基因分别起源于红杆菌科和钩端螺旋体科中folP基因的水平移动事件，同时表明红杆菌科的folP基因源自钩端螺旋体科。对folP/sul基因序列的GC含量分析结果支持上述系统发育推断，同时表明染色体编码的FolP蛋白中Sul基序的出现起源久远，远早于临床中磺胺类药物的使用。体外实验显示，在对磺胺类药物敏感的大肠杆菌（Escherichia coli）宿主背景中，红杆菌科和钩端螺旋体科的FolP蛋白均可赋予耐药性，而其他相关染色体编码的FolP蛋白则无此效果，这表明Sul基序与磺胺类药物耐药性相关。鉴于目前尚未发现任何靶向DHPS的天然磺胺类药物，本研究结果为合成化疗剂的耐药性产生机制提供了全新视角：在临床引入新型化疗药物后，庞大细菌泛基因组中预先存在的耐药变异株可被快速筛选并传播扩散。