C. difficile induces antibiotic-associated diarrhea due to the action of two secreted toxins, TcdA and TcdB. A considerable range of virulence among C. difficile strains has been widely reported. During a hospital outbreak, 46 isolates were collected that belonged to different genotypes. Of those, the majority corresponded to two virulent strains, the globally distributed Sequence Type 1 (ST1)_North American Pulsotype 1 (NAP1) and the endemic ST54_NAP_CR1 genotypes, respectively. Whereas the virulence of the latter has been attributed to increased secretion of toxins and production of a highly cytotoxic TcdB, these characteristics do not explain the increased lethality of the former. We undertook a proteomic comparative approach of the isolates participating in the outbreak to look for proteins present in the exoproteome of the ST1_NAP1and ST54_NAP_CR1 strains. We used a low virulent ST2_NAP4 strain isolated also in the outbreak as control. Dendrograms constructed using the exoproteomes of the strains were very similar to those created using genomic information, suggesting an association between secreted proteins and relative virulence of the strains. By 2D electrophoresis and mass spectrometry it was found that approximately half of the proteins are shared among strains of different genotypes. From the identified proteins, the surface-located SlpA draw our attention due to its detection in ST54_NAP_CR1 exoproteomes. Biochemical analysis indicated that the processing of SlpA is different in the ST54_NAP_CR1 strain and confirmed that this strain secretes more SlpA than its counterparts. Furthermore, SlpA from the ST54_NAP_CR1 strain exerted an increased proinflammatory activity. Altogether, these results indicate that the exoproteome composition correlates with the C. difficile genotype and suggest that particular proteins secreted by some strains could synergize with the effects of TcdA and TcdB increasing their virulence.

由于两种分泌的毒素TcdA和TcdB的作用，艰难梭菌诱导了与抗生素相关的腹泻。艰难梭菌菌株之间的毒力范围已广为报道。在医院爆发期间，收集了46个属于不同基因型的分离株。其中，大多数对应于两个强毒株，即全球分布的序列类型1（ST1）_北美脉冲型1（NAP1）和地方性ST54_NAP _CR1基因型，分别。尽管后者的毒力归因于毒素分泌的增加和高细胞毒性TcdB的产生，但这些特征不能解释前者的致死性增加。我们对参加此次暴发的分离株采取了蛋白质组学比较方法，以寻找存在于ST1_NAP1和ST54_NAP _CR1的蛋白质组中的蛋白质株。我们使用在爆发中也分离出的低毒力ST2_NAP4菌株作为对照。使用菌株的外蛋白质组构建的树状图与使用基因组信息创建的树状图非常相似，表明分泌的蛋白质与菌株的相对毒力之间存在关联。通过2D电泳和质谱分析，发现大约一半的蛋白质在不同基因型的菌株之间共享。从鉴定出的蛋白质中，表面定位的SlpA由于在ST54_NAP _CR1外蛋白质组中被检测到而引起了我们的注意。生化分析表明，ST54_NAP _CR1菌株对SlpA的加工不同，并证实该菌株比其同种菌株分泌更多的SlpA。此外，ST54_NAP的SlpA_CR1株发挥增强的促炎活性。总而言之，这些结果表明外蛋白质组组成与艰难梭菌基因型相关，并暗示某些菌株分泌的特定蛋白质可能与TcdA和TcdB的毒力作用协同作用。