Supplementary Material for: Interference with Lipoprotein Maturation Sensitizes Methicillin-Resistant <b><i>Staphylococcus aureus</i></b> to Human Group IIA-Secreted Phospholipase A₂ and Daptomycin

Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA-secreted phospholipase A₂ (hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, including <i>S. aureus</i>. To determine hGIIA-resistance mechanisms of MRSA, we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role of <i>lspA</i>, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in both in vitro assays and an infection model in hGIIA-transgenic mice. Increased susceptibility of the <i>lspA</i> mutant was associated with enhanced activity of hGIIA on the cell membrane. Moreover, <i>lspA</i> deletion increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. MRSA wild type could be sensitized to hGIIA and daptomycin killing through exposure to LspA-specific inhibitors globomycin and myxovirescin A1. Analysis of &gt;26,000 <i>S. aureus</i> genomes showed that LspA is highly sequence-conserved, suggesting universal application of LspA inhibition. The role of LspA in hGIIA resistance was not restricted to MRSA since <i>Streptococcus mutans</i> and <i>Enterococcus faecalis</i> were also more hGIIA-susceptible after <i>lspA</i> deletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological interference with LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically applied antibiotics.

耐甲氧西林金黄色葡萄球菌（Methicillin-resistant Staphylococcus aureus，MRSA）已被世界卫生组织列为高优先级病原体，凸显了开发新型治疗药物以应对其感染的迫切需求。人类IIA型分泌型磷脂酶A₂（human group IIA-secreted phospholipase A₂，hGIIA）是对抗包括金黄色葡萄球菌在内的革兰氏阳性菌的最有效杀菌蛋白之一。为阐明MRSA对hGIIA的耐药机制，我们利用亚致死浓度的重组hGIIA对内布拉斯加转座子突变体库（Nebraska Transposon Mutant Library）进行了筛选。通过体外实验与hGIIA转基因小鼠感染模型，我们鉴定并证实了编码脂蛋白信号肽酶LspA的lspA基因作为新型hGIIA耐药基因的功能。lspA突变株的敏感性升高与hGIIA在其细胞膜上的活性增强密切相关。此外，lspA基因缺失还会提升MRSA对达托霉素的敏感性——达托霉素是治疗MRSA感染的最后一线抗生素。经LspA特异性抑制剂globomycin与黏病毒霉素A1处理后，野生型MRSA可对hGIIA和达托霉素的杀伤作用变得敏感。对超过26000株金黄色葡萄球菌基因组的分析显示，LspA的序列高度保守，这表明LspA抑制策略具备普适性。LspA在hGIIA耐药中的作用并非仅局限于MRSA：变形链球菌（Streptococcus mutans）与粪肠球菌（Enterococcus faecalis）分别经lspA基因缺失或LspA抑制处理后，同样对hGIIA的敏感性显著升高。综上，本研究结果表明，通过药理学手段干预LspA，可削弱包括MRSA在内的革兰氏阳性病原菌的防御能力，从而增强先天宿主防御分子与临床应用抗生素的清除效果。

搜集汇总

背景与挑战

背景概述

该数据集是研究耐甲氧西林金黄色葡萄球菌（MRSA）抵抗人类IIA组分泌型磷脂酶A2（hGIIA）机制的补充材料，发现lspA基因编码的脂蛋白信号肽酶LspA是关键抵抗因子，其缺失或抑制可增强MRSA对hGIIA和抗生素达托霉素的敏感性，且这一机制在其他革兰氏阳性菌中同样适用，为开发新疗法提供了潜在靶点。