Supplementary Material for: The Janus Face of a-Toxin: A Potent Mediator of Cytoprotection in Staphylococci-Infected Macrophages

After phagocytosis by macrophages, <i>Staphylococcus aureus</i> evades killing in an a-toxin-dependent manner, and then prevents apoptosis of infected cells by upregulating expression of antiapoptotic genes like <i>MCL-1 (myeloid cell leukemia-1)</i>. Here, using purified a-toxin and a set of <i>hla</i>-deficient strains, we show that a-toxin is critical for the induction of <i>MCL-1 </i>expression and the cytoprotection of infected macrophages. Extracellular or intracellular treatment of macrophages with a-toxin alone did not induce cytoprotection conferred by increased Mcl-1, suggesting that the process is dependent on the production of a-toxin by intracellular bacteria. The increased expression of <i>MCL-1</i> in infected cells was associated with enhanced NFκB activation, and subsequent IL-6 secretion. This effect was only partially inhibited by blocking TLR2, which suggests the participation of intracellular receptors in the specific recognition of <i>S. aureus </i>strains secreting a-toxin. Thus, <i>S. aureus</i> recognition by intracellular receptors and/or activation of downstream pathways leading to Mcl-1 expression is facilitated by a-toxin released by intracellular bacteria which permeabilize phagosomes, ensuring pathogen access to the cytoplasmatic compartment. Given that the intracellular survival of <i>S. aureus</i> depends on a-toxin, we propose a novel role for this agent in the protection of the intracellular niche, and further dissemination of staphylococci by infected macrophages.

巨噬细胞吞噬金黄色葡萄球菌（Staphylococcus aureus）后，该菌可通过依赖α毒素（a-toxin）的方式逃逸杀伤，并通过上调MCL-1（髓系细胞白血病因子1，myeloid cell leukemia-1）等抗凋亡基因的表达，抑制感染细胞的凋亡。本研究利用纯化的α毒素及一系列hla缺陷菌株，证实α毒素对于诱导MCL-1表达及保护感染巨噬细胞至关重要。仅通过胞外或胞内单独施加α毒素处理巨噬细胞，均无法诱导由Mcl-1表达上调所介导的细胞保护效应，提示该过程依赖胞内细菌合成的α毒素。感染细胞中MCL-1的表达上调，与核因子κB（NFκB）活化增强及后续白细胞介素6（IL-6）的分泌密切相关。该效应仅可被Toll样受体2（TLR2）部分阻断抑制，表明胞内受体参与了对分泌α毒素的金黄色葡萄球菌菌株的特异性识别。因此，胞内细菌释放的α毒素可使吞噬体膜穿孔，使病原体得以进入胞浆腔室，从而促进胞内受体对金黄色葡萄球菌的识别，以及激活下游通路以诱导Mcl-1的表达。鉴于金黄色葡萄球菌的胞内存活依赖α毒素，本研究提出该毒素在保护胞内生存微环境，以及介导受感染巨噬细胞进一步传播葡萄球菌方面，具有此前未被报道的新功能。