Department of Microbiology and Immunology, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201, USA.
Infect Immun. 2011 Jan;79(1):33-43. doi: 10.1128/IAI.00771-10. Epub 2010 Oct 25.
Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm and are required for virulence. While many effectors have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effector proteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and its effectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat) or regulatory (ΔssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excluding the translocon components SseBCD, all SPI-2 effectors were biased for identification in the ΔssaL mutant, substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled our secretome data with a machine learning algorithm (SIEVE, SVM-based identification and evaluation of virulence effectors) and selected 12 candidate proteins for further characterization. Using CyaA' reporter fusions, we identified six novel type III effectors and two additional proteins that were secreted into J774 macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolar deletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Six mutants were significantly attenuated for spleen colonization. Our results also suggest that non-type III secretion mechanisms are required for full Salmonella virulence.
鼠伤寒沙门氏菌血清型是全世界急性肠胃炎的主要原因。这种病原体有两个 III 型分泌系统（TTSS），编码在沙门氏菌致病岛 1 和 2（SPI-1 和 SPI-2）中，将毒力因子（效应子）输送到宿主细胞质中，是毒力所必需的。虽然已经鉴定出许多效应子，并对其进行了至少部分特征描述，但完整的效应子库尚未被编目。在这项蛋白质组学研究中，我们鉴定了分泌到设计用于诱导 SPI-2 TTSS 及其效应子表达的明确最小培养基中的效应蛋白。我们比较了野生型菌株与缺失 SPI-2 TTSS 必需（ssaK::cat）或调节（ΔssaL）成分的菌株的分泌组。我们鉴定出了 20 种已知的 SPI-2 效应子。除了转运体成分 SseBCD 外，所有 SPI-2 效应子在 ΔssaL 突变体中更偏向于鉴定，这证实了 SsaL 在 TTS 中的调节作用。为了鉴定新的效应蛋白，我们将分泌组数据与机器学习算法（SIEVE，基于 SVM 的毒力效应因子的识别和评估）相结合，选择了 12 种候选蛋白进行进一步表征。使用 CyaA'报告融合物，我们鉴定出了六个新的 III 型效应子和另外两个在没有 TTSS 的情况下分泌到 J774 巨噬细胞中的蛋白。为了评估它们在毒力中的作用，我们构建了非极性缺失突变体，并在腹膜内感染 129/SvJ 小鼠中进行了竞争指数分析。六个突变体在脾脏定植方面明显减弱。我们的结果还表明，非 III 型分泌机制是沙门氏菌完全毒力所必需的。
