Conventional antibiotic-based therapies for the treatment of Staphylococcus pseudintermedius infections have become less reliable in recent years due to the increased prevalence of methicillin resistance and multidrug resistance. Therefore, development of alternate therapeutic strategies is a high priority. Treatments based on sortase inhibition hold potential to address this shortcoming. Sortase A (SrtA) is a transpeptidase, commonly produced by Gram positive bacteria, that interacts with proteins baring a C-terminal Leu-Pro-X-Thr-Gly (LPXTG) motif, anchoring them on the peptidoglycan cell wall. SrtA substrates include numerous virulence factors that may overcome the host immune response including protein A, fibrinogen binding proteins and fibronectin binding proteins. In the present study, the srtA gene from S. pseudintermedius was identified and its conservation and level of transcription determined in isolates representing major clonal complexes. The gene was synthesized and then expressed in Escherichia coli and the recombinant enzyme’s activity measured using a synthetic substrate. A three dimensional homology model of SrtA was generated and used in virtual screening libraries of chemicals for potential inhibitors. Four compounds that showed 50% or greater inhibition of SrtA activity were identified. A thermal shift assay confirmed binding of one inhibitor to the active site of SrtA. SrtA inhibitor was active in preventing protein A anchoring in bacterial cultures. The results confirm the function of the srtA encoded protein and indicate that SrtA inhibition has potential for treatment of S. pseudintermedius infections.

近年来，由于甲氧西林耐药性和多药耐药性的增加，用于治疗假中间葡萄球菌感染的传统抗生素疗法变得不太可靠。因此，开发替代治疗策略是当务之急。基于分选酶抑制的治疗有可能解决这个缺点。Sortase A (SrtA) 是一种转肽酶，通常由革兰氏阳性细菌产生，它与带有 C 端 Leu-Pro-X-Thr-Gly (LPXTG) 基序的蛋白质相互作用，将它们锚定在肽聚糖细胞壁上。SrtA 底物包括许多可以克服宿主免疫反应的毒力因子，包括蛋白 A、纤维蛋白原结合蛋白和纤连蛋白结合蛋白。在本研究中，鉴定了来自S.pseudintermedius 的srtA基因，并在代表主要克隆复合体的分离物中确定了其保守性和转录水平。该基因被合成，然后在大肠杆菌中表达，并使用合成底物测量重组酶的活性。生成了 SrtA 的三维同源模型，并将其用于潜在抑制剂的化学物质虚拟筛选库。鉴定了对 SrtA 活性具有 50% 或更大抑制作用的四种化合物。热位移测定证实了一种抑制剂与 SrtA 的活性位点的结合。SrtA 抑制剂在阻止蛋白质 A 锚定在细菌培养物中具有活性。结果证实了srtA的功能编码蛋白质并表明 SrtA 抑制具有治疗假中间链球菌感染的潜力。