Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
mBio. 2024 Apr 10;15(4):e0338323. doi: 10.1128/mbio.03383-23. Epub 2024 Feb 28.
We previously demonstrated that mutation of in limits biofilm formation, cytotoxicity for osteoblasts and osteoclasts, and virulence in osteomyelitis, and that all of these phenotypes can be attributed to the increased production of extracellular proteases. Here we extend these studies to assess the individual importance of these proteases alone and in combination with each other using the methicillin-resistant USA300 strain LAC, the methicillin-susceptible USA200 strain UAMS-1, and isogenic mutants that were also unable to produce aureolysin (Aur), staphopain A (ScpA), staphylococcal serine protease A (subsp.), staphopain B (SspB), and the staphylococcal serine protease-like proteins A-F (SplA-F). Biofilm formation was restored in LAC and UAMS-1 mutants by subsequent mutation of and , while mutation of had the greatest impact on cytotoxicity to mammalian cells, particularly with conditioned medium (CM) from the more cytotoxic strain LAC. However, SDS-PAGE and western blot analysis of CM confirmed that mutation of was also required to mimic the phenotype of mutants unable to produce any extracellular proteases. Nevertheless, in a murine model of post-traumatic osteomyelitis, mutation of and had the greatest impact on restoring the virulence of LAC and UAMS-1 mutants, with concurrent mutation of and the operon having relatively little effect. These results demonstrate that the increased production of Aur and ScpA in combination with each other is a primary determinant of the reduced virulence of mutants in diverse clinical isolates including both methicillin-resistant and methicillin-susceptible strains.IMPORTANCEPrevious work established that SarA plays a primary role in limiting the production of extracellular proteases to prevent them from limiting the abundance of virulence factors. Eliminating the production of all 10 extracellular proteases in the methicillin-resistant strain LAC has also been shown to enhance virulence in a murine sepsis model, and this has been attributed to the specific proteases Aur and ScpA. The importance of this work lies in our demonstration that the increased production of these same proteases largely accounts for the decreased virulence of mutants in a murine model of post-traumatic osteomyelitis not only in LAC but also in the methicillin-susceptible human osteomyelitis isolate UAMS-1. This confirms that -mediated repression of Aur and ScpA production plays a critical role in the posttranslational regulation of virulence factors in diverse clinical isolates and diverse forms of infection.
我们之前的研究表明，突变 中的 SarA 会限制生物膜的形成、破骨细胞和成骨细胞的细胞毒性以及骨髓炎的毒力，所有这些表型都可以归因于细胞外蛋白酶产量的增加。在这里，我们使用耐甲氧西林的 USA300 菌株 LAC、甲氧西林敏感的 USA200 菌株 UAMS-1 以及无法产生胞外溶葡萄球菌素 (Aur)、葡萄球菌蛋白酶 A (ScpA)、葡萄球菌丝氨酸蛋白酶 A (亚种)、葡萄球菌蛋白酶 B (SspB) 和葡萄球菌丝氨酸蛋白酶样蛋白 A-F (SplA-F) 的同基因 突变体，扩展了这些研究，以评估这些蛋白酶单独和相互组合的重要性。生物膜形成在 LAC 和 UAMS-1 突变体中通过随后突变 和 得以恢复，而突变 对哺乳动物细胞的细胞毒性影响最大，尤其是在用更具细胞毒性的 LAC 菌株的条件培养基 (CM) 处理时。然而，CM 的 SDS-PAGE 和 Western blot 分析证实，突变 也是模拟无法产生任何细胞外蛋白酶的 突变体表型所必需的。尽管如此，在创伤后骨髓炎的小鼠模型中，突变 和 对恢复 LAC 和 UAMS-1 突变体的毒力影响最大，而同时突变 和 操纵子的影响相对较小。这些结果表明，Aur 和 ScpA 的产量增加相互结合是降低包括耐甲氧西林和甲氧西林敏感菌株在内的各种临床分离株中 突变体毒力的主要决定因素。
先前的工作表明，SarA 在限制细胞外蛋白酶的产生以防止它们限制 毒力因子的丰度方面发挥主要作用。在耐甲氧西林的 LAC 菌株中消除所有 10 种细胞外蛋白酶的产生也已被证明可增强小鼠败血症模型中的毒力，这归因于特定的蛋白酶 Aur 和 ScpA。这项工作的重要性在于，我们证明了在创伤后骨髓炎的小鼠模型中，这些相同蛋白酶产量的增加在很大程度上解释了 突变体在 LAC 以及耐甲氧西林的人类骨髓炎分离株 UAMS-1 中的毒力降低。这证实了 SarA 介导的 Aur 和 ScpA 产生的抑制在不同临床分离株和不同形式的 感染中 毒力因子的翻译后调控中起着关键作用。
