Culture of Campylobacter jejuni with Sodium Deoxycholate induces Virulence Gene Expression

C. jejuni, a spiral-shaped gram-negative bacterium, is a leading bacterial cause of human foodborne illness. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Further, maximal host cell invasion requires the secretion of proteins termed Campylobacter invasion antigens (Cia). As bile acids are known to alter the pathogenic behavior of other gastrointestinal pathogens, we hypothesized that the virulence potential of Campylobacter may be triggered by the bile acid deoxycholate (DOC). In support of this hypothesis, culturing C. jejuni with a physiologically relevant concentration of DOC significantly altered the kinetics of cell invasion as evidenced by gentamicin-protection assays. In contrast to C. jejuni harvested from Mueller-Hinton (MH) agar plates, C. jejuni harvested from MH agar plates supplemented with DOC demonstrated Cia secretion as judged by metabolic labeling experiments. DOC was also found to induce the expression of the ciaB gene as judged by B-galactosidase reporter assays and real-time RT-PCR. Microarray analysis revealed that DOC induced the expression of virulence genes (i.e., ciaB, cmeABC, dccR, and tlyA). In summary, we demonstrate that it is possible to enhance the pathogenic behavior of C. jejuni by modifying the culture conditions. These results provide a foundation to identify genes expressed by C. jejuni in response to in vivo-like culture conditions. Keywords: Stress response Overall design: For the expression profiling arrays, an indirect comparison of gene expression was performed, where the expression profile of the C. jejuni F38011 cultured in the presence and absence of DOC was measured separately on different slides as described previously (26). Briefly, Cy5 labeled reference DNA from the C. jejuni F38011 strain was mixed with Cy3 labeled test cDNA (C. jejuni F38011 cultured in the presence or absence of DOC) and hybridized to the Campylobacter cDNA array (26) on separate slides. DNA microarrays were scanned using an Axon GenePix 4000B microarray laser scanner (Axon Instruments, Union City, CA) and the data for spot and background intensities were processed using the GenePix 4.0 software. To compensate for any effect of the amount of template and uneven Cy-dye incorporation, data normalization was performed as previously described (26). For the comparison of genes differentially expressed in the presence and absence of DOC,six hybridization measurements were generated per biological experiment (three technical replicate arrays and two replicate features per array).

空肠弯曲菌（Campylobacter jejuni），一种螺旋形革兰氏阴性菌，是人类食源性疾病的细菌性主要病因。急性疾病与空肠弯曲菌对肠上皮的侵袭有关。此外，最大程度的主细胞侵袭需要分泌被称为弯曲菌侵袭抗原（Cia）的蛋白质。鉴于胆汁酸已知能够改变其他胃肠道病原体的致病行为，我们假设弯曲菌的致病潜力可能被胆汁酸脱氧胆酸（DOC）所触发。为此假设提供支持的是，使用与生理相关浓度的DOC培养空肠弯曲菌，显著改变了细胞侵袭动力学，这一现象通过庆大霉素保护实验得到证实。与从Mueller-Hinton（MH）琼脂平板上收集的空肠弯曲菌相比，从补充DOC的MH琼脂平板上收集的空肠弯曲菌通过代谢标记实验显示出Cia分泌。通过β-半乳糖苷酶报告实验和实时RT-PCR分析，DOC也被发现能诱导ciaB基因的表达。微阵列分析揭示了DOC诱导了致病基因（如ciaB、cmeABC、dccR和tlyA）的表达。总之，我们证明了通过改变培养条件可以增强空肠弯曲菌的致病行为。这些结果为识别在体内类似培养条件下表达的空肠弯曲菌基因奠定了基础。关键词：应激反应 整体设计：对于表达谱阵列，进行了基因表达的非直接比较，其中分别在不同载玻片上测量了空肠弯曲菌F38011在有DOC和没有DOC存在下的表达谱，如前所述（26）。简而言之，Cy5标记的空肠弯曲菌F38011菌株参考DNA与Cy3标记的测试cDNA（空肠弯曲菌F38011在有DOC或没有DOC存在下培养）混合，并分别与Campylobacter cDNA阵列（26）在单独的载玻片上杂交。使用Axon GenePix 4000B微阵列激光扫描仪（Axon Instruments，Union City，CA）扫描DNA微阵列，并使用GenePix 4.0软件处理斑点和背景强度的数据。为了补偿模板数量和Cy染料非均匀结合的影响，数据进行了标准化，如前所述（26）。对于比较DOC存在与否差异表达的基因，每个生物学实验生成了六个杂交测量值（三个技术重复阵列和每个阵列两个重复特征）。