Microarray analysis of genes involved in Streptolysin S immunity

Group A Streptococcus (GAS) is one of the world’s most successful pathogens, causing a multitude of common infections such as pharyngitis, cellulitis, and impetigo. It is also responsible for more severe diseases such as rheumatic fever, necrotizing fasciitis, and toxic shock syndrome. Group A Streptococcus produces a powerful peptide toxin known as streptolysin S (SLS). Although recent advances have begun to uncover the structure of SLS, many questions remain as to its route of synthesis, export and function. A fundamental yet unanswered question about SLS is the mechanism employed by GAS to resist the effects of its own toxin. To address this question, we developed a unique microarray-based approach aimed at identifying bacterial genes involved in SLS immunity. We measured changes in gene expression in a non-SLS producing GAS strain (∆SLS) after exposure to active SLS, as well as to an inactive SLS isoform. Initial exposure of a non-SLS producing GAS to the native SLS toxin resulted in significant upregulation of several gene candidates. Proteins encoded by these gene candidates were produced and tested for their ability to neutralize SLS toxin activity in vitro. The protein encoded by the gene Spy_0787 decreased the cytolytic activity of wt SLS by 50%. Bacterial immunity is still a relatively unknown and unexplained phenomena. Insights into how toxin-producing microorganisms achieve resistance against the effects of their own toxin could uncover important therapeutic targets to neutralize virulence factors such as SLS, as well as other related peptide toxins. The microarray technique described here can be leveraged to other microbial systems to understand how microorganisms in general react to antibacterial and cytotoxic compounds for which the mechanism of action is unknown. Supernatants containing wt and S39A forms of SLS secreted by GAS strains were collected from 50 ml of overnight cultures of each strain at 37 degrees C in Todd Hewett broth supplemented with 10 mg/ml of bovine serum albumin fraction V (Sigma chemicals) in order to stabilize the toxin. The supernatant was then isolated and subjected to filter sterilization. For SLS exposure conditions, bacterial pellets containing GAS ∆SagA mutants were incubated with 1. supernatants containing wtSLS; 2. supernatants containing SLS S39A isoform; or 3. sterile TH media with 10mg/ml of BSA. The bacteria pellets were quickly resuspended after which the bacterial pellets were recollected for RNA isolation. A total of three exposure timepoints were used: 0h (immediately after exposure to SLS-containing supernatants), 3h post-exposure, and 6h post-exposure. The pellets were frozen at -80C until used for RNA extraction. Total RNA extraction and purification from the bacterial pellets were performed using the RNeasy isolation kit per manufacturer's instructions (Qiagen). RNA samples were processed following standard Roche NimbleGen gene expression analysis protocols. Double-stranded cDNA was synthesized from 10 μg of total RNA using random hexamer primers with the SuperScript cDNA synthesis kit (Invitrogen). One μg of cDNA was labeled with Cy3-random nonamers (Roche NimbleGen) for microarray hybridization.

A群链球菌（Group A Streptococcus, GAS）是全球最成功的致病菌之一，可引发咽炎、蜂窝织炎、脓疱疮等多种常见感染，还可导致风湿热、坏死性筋膜炎、中毒性休克综合征等重症疾病。该菌可分泌一种强效肽类毒素——链球菌溶血素S（streptolysin S, SLS）。尽管近年来的研究进展已逐步解析了SLS的结构，但其合成、分泌途径与功能仍有诸多未解之谜。关于SLS的一个核心且尚未解答的问题是：A群链球菌如何抵御自身分泌的毒素的作用。

为解答这一问题，本研究开发了一种基于微阵列（microarray）的独特实验方法，旨在筛选参与SLS免疫的细菌基因。我们检测了非产SLS的A群链球菌菌株（∆SLS）分别暴露于活性SLS与失活SLS亚型后，其基因表达的变化情况。最初将非产SLS的A群链球菌暴露于天然SLS毒素后，多个候选基因出现了显著的上调表达。我们对这些候选基因编码的蛋白进行了重组表达，并在体外实验中检测其中和SLS毒素活性的能力。其中，基因Spy_0787编码的蛋白可将野生型（wild type, wt）SLS的溶细胞活性降低50%。

细菌免疫仍是一个相对未知且难以解释的现象。解析产毒微生物如何抵御自身毒素的作用机制，可为中和SLS等毒力因子及其他相关肽类毒素提供重要的治疗靶点。本研究所述的微阵列技术可推广应用于其他微生物系统，以解析微生物普遍对作用机制不明的抗菌剂与细胞毒性化合物的响应机制。

我们从各菌株50 mL的过夜培养物中收集含野生型与S39A突变型SLS的上清液：培养条件为37℃、Todd-Hewitt肉汤（添加10 mg/mL V型牛血清白蛋白（Sigma Chemicals）以稳定毒素）。随后分离上清液并进行过滤除菌处理。

在SLS暴露实验中，我们将携带GAS ∆SagA突变体的细菌沉淀分别与以下试剂孵育：1. 含野生型SLS的上清液；2. 含S39A突变型SLS亚型的上清液；3. 添加10 mg/mL BSA的无菌TH培养基。孵育结束后快速重悬细菌沉淀，随后重新收集沉淀以进行RNA提取。本实验共设置三个暴露时间点：0 h（暴露于含SLS上清液后即刻）、暴露后3 h、暴露后6 h。收集的细菌沉淀于-80℃冷冻保存，直至用于RNA提取。

使用RNeasy总RNA提取试剂盒（Qiagen），按照厂商说明书完成细菌沉淀的总RNA提取与纯化。RNA样本按照Roche NimbleGen标准基因表达分析流程进行处理。使用SuperScript cDNA合成试剂盒（Invitrogen），以随机六聚体引物从10 μg总RNA中合成双链cDNA。取1 μg cDNA，用Cy3标记的随机九聚体引物（Roche NimbleGen）进行标记，用于微阵列杂交。