Signaling of Neisseria meningitidis MC58 mutants to primary human umbilical vein endothelial cells (HUVEC)

We examined the adherence-mediated signaling of meningococci to human cells by comparing gene expression profiles of primary human umbilical vein endothelial cells (HUVEC) infected by piliated and adherent wild-type (WT), frpC/frpA-deficient mutant, or the non-adherent (ΔpilD) N. meningitidis MC58 bacteria defective in production of the type IV pilus, respectively. Surprisingly, no significant difference was found between the transcriptomes of HUVECs infected by bacteria producing, or not the RTX FrpC and FrpA proteins, thus failing to provide any hints on their biological activity. In contrast, pili-mediated adhesion of meningococci resulted in alterations of expression levels of human genes known to regulate apoptosis, cell proliferation, inflammatory response or adhesion. In particular, genes for signaling pathway proteins involved in early embryonic development, such as transforming growth factor-β (TGF-β)/Smad, Wnt/β-catenin, and Notch/Jagged were found to be upregulated upon adhesion of N. meningitidis together with genes for a number of transcription factors. This reveals that adhering piliated meningocci manipulate signaling pathways controlling human cell proliferation, survival and defense mechanisms, while establishing a commensal relationship with the host. Keywords: time course, infection response, genetic modification Time courses of gene expression profiles of primary human umbilical vein endothelial cells (HUVEC) during infection with piliated and adherent wild-type (WT), frpC/frpA-deficient mutant, or the non-adherent (ΔpilD) Neisseria meningitidis MC58 bacteria defective in production of the type IV pilus, were analyzed respectively. Total RNA isolated from uninfected HUVEC (reference) and HUVEC after 1, 4 or 6 hours of infection with N. meningitidis mutants was labeled according to standard Affymetrix protocol and hybridized to HG-U133A GeneChips. Data were analyzed by Robust Multi-array Analysis algorithm. For every condition at least two biological replicates were analyzed, totally representing 23 Affymetrix GeneChips.

本研究通过比较不同脑膜炎奈瑟菌（Neisseria meningitidis）感染的原代人脐静脉内皮细胞（primary human umbilical vein endothelial cells, HUVEC）的基因表达谱，探究了脑膜炎奈瑟菌黏附介导的人细胞信号转导过程。所使用的菌株分别为：携带菌毛、具有黏附能力的野生型（wild-type, WT）菌株、frpC/frpA缺陷突变株，以及IV型菌毛（type IV pilus）合成缺陷的非黏附型（ΔpilD）脑膜炎奈瑟菌MC58。

令人意外的是，表达RTX结构域FrpC与FrpA蛋白的细菌与不表达该类蛋白的细菌所感染的HUVEC转录组之间未发现显著差异，因此无法为这两种蛋白的生物学活性提供任何线索。与之相反，脑膜炎奈瑟菌通过菌毛介导的黏附过程，可引起已知参与调控细胞凋亡、细胞增殖、炎症应答及细胞黏附的人类基因表达水平发生改变。具体而言，与早期胚胎发育相关的信号通路蛋白编码基因，如转化生长因子-β（transforming growth factor-β, TGF-β）/Smad、Wnt/β-连环蛋白（Wnt/β-catenin）以及Notch/Jagged通路相关基因，与多种转录因子编码基因一道，在脑膜炎奈瑟菌黏附后呈现上调表达。上述结果表明，携带菌毛的黏附型脑膜炎奈瑟菌可通过调控控制人细胞增殖、存活及防御机制的信号通路，与宿主建立共生关系。

关键词：时间进程、感染应答、基因修饰

本研究分析了上述不同菌株感染HUVEC过程中的基因表达谱时间进程：从未感染的HUVEC（作为对照样本）以及感染脑膜炎奈瑟菌突变株1、4、6小时后的HUVEC中提取总RNA，按照标准Affymetrix实验流程进行标记，并与HG-U133A基因芯片进行杂交。数据采用稳健多阵列分析（Robust Multi-array Analysis）算法进行处理。每个实验条件至少设置2次生物学重复，共计使用23张Affymetrix基因芯片。