Eichelberg K, Ginocchio C C, Galán J E
Department of Microbiology, School of Medicine, SUNY Stony Brook 11794-5222.
J Bacteriol. 1994 Aug;176(15):4501-10. doi: 10.1128/jb.176.15.4501-4510.1994.
Entry into intestinal epithelial cells is an essential step in the pathogenesis of Salmonella infections. Our laboratory has previously identified a genetic locus, inv, that is necessary for efficient entry of Salmonella typhimurium into cultured epithelial cells. We have carried out a molecular and functional analysis of invB and invC, two members of this locus. The nucleotide sequence of these genes indicated that invB and invC encode polypeptides with molecular masses of 15 and 47 kDa, respectively. Polypeptides with the predicted sizes were observed when these genes were expressed under the control of a T7 promoter. Strains carrying nonpolar mutations in these genes were constructed, and their phenotypes were examined in a variety of assays. A mutation in invC rendered S. typhimurium defective in their ability to enter cultured epithelial cells, while mutations in invB did not. Comparison of the predicted sequences of InvB and InvC with translated sequences in GenBank revealed that these polypeptides are similar to the Shigella spp. proteins Spa15 and Spa47, which are involved in the surface presentation of the invasion protein antigens (Ipa) of these organisms. In addition, InvC showed significant similarity to a protein family which shares sequence homology with the catalytic beta subunit of the F0F1 ATPase from a number of microorganisms. Consistent with this finding, purified preparations of InvC showed significant ATPase activity. Site-directed mutagenesis of a residue essential for the catalytical function of this family of proteins resulted in a protein devoid of ATPase activity and unable to complement an invC mutant of S. typhimurium. These results suggest that InvC may energize the protein export apparatus encoded in the inv locus which is required for the surface presentation of determinants needed for the entry of Salmonella species into mammalian cells. The role of InvB in this process remains uncertain.
进入肠道上皮细胞是沙门氏菌感染发病机制中的一个关键步骤。我们实验室先前已鉴定出一个基因位点inv，它对于鼠伤寒沙门氏菌有效进入培养的上皮细胞是必需的。我们对该位点的两个成员invB和invC进行了分子和功能分析。这些基因的核苷酸序列表明，invB和invC分别编码分子量为15 kDa和47 kDa的多肽。当这些基因在T7启动子的控制下表达时，观察到了预测大小的多肽。构建了在这些基因中携带非极性突变的菌株，并在各种试验中检查了它们的表型。invC中的突变使鼠伤寒沙门氏菌进入培养上皮细胞的能力出现缺陷，而invB中的突变则没有。将InvB和InvC的预测序列与GenBank中的翻译序列进行比较，发现这些多肽与志贺氏菌属的Spa15和Spa47蛋白相似，它们参与这些生物体侵袭蛋白抗原（Ipa）的表面呈现。此外，InvC与一个蛋白家族具有显著相似性，该家族与多种微生物的F0F1 ATP酶的催化β亚基具有序列同源性。与此发现一致，纯化的InvC制剂显示出显著的ATP酶活性。对该蛋白家族催化功能所必需的一个残基进行定点诱变，产生了一种没有ATP酶活性且无法互补鼠伤寒沙门氏菌invC突变体的蛋白。这些结果表明，InvC可能为inv位点编码的蛋白输出装置提供能量，该装置是沙门氏菌属进入哺乳动物细胞所需决定簇表面呈现所必需的。InvB在此过程中的作用仍不确定。
