Yang Zi, Duncan-Lowey Jeffrey K, Roy Craig R
Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA.
Infect Immun. 2025 Apr 8;93(4):e0044824. doi: 10.1128/iai.00448-24. Epub 2025 Mar 12.
is a gram-negative, obligate intracellular pathogen that causes human Q fever. Within host cells, proliferates in a spacious, acidic, lysosome-derived -containing vacuole (CCV) by a process that requires the Dot/Icm type IVB secretion system to deliver effectors that manipulate host cell functions. A previous transposon mutagenesis screen identified the gene as being important for intracellular replication of . Here, the function of Cbu0937 was investigated. The ::Tn mutant had no detectable defect replicating in the axenic acidified citrate cysteine medium 2. Additionally, intracellular replication of the ::Tn mutant was not restored by co-infection of host cells with an isogenic wild-type strain of . Thus, the ::Tn mutant has a cell-intrinsic intracellular replication defect. Intracellular replication of the ::Tn mutant was restored by complementing the gene with a plasmid encoding either untagged or an epitope-tagged version of Cbu0937. Analysis of the predicted structure of the Cbu0937 protein using AlphaFold revealed high similarity between Cbu0937 and several bacterial porins. Fractionation studies and surface labeling of producing a functional epitope-tagged protein indicated the localization of Cbu0937 to the bacterial outer membrane. From these data, we conclude that encodes a porin that plays an essential role in supporting intracellular replication, which likely involves the acquisition of an important metabolite in the CCV lumen.
是一种革兰氏阴性专性细胞内病原体，可引起人类Q热。在宿主细胞内，它通过一种需要Dot/Icm IVB型分泌系统来传递操纵宿主细胞功能的效应子的过程，在一个宽敞的、酸性的、源自溶酶体的含泡（CCV）中增殖。先前的转座子诱变筛选确定该基因对在细胞内复制很重要。在此，研究了Cbu0937的功能。::Tn突变体在无细胞酸化柠檬酸盐半胱氨酸培养基2中复制时没有可检测到的缺陷。此外，宿主细胞与的同基因野生型菌株共感染并不能恢复::Tn突变体的细胞内复制。因此，::Tn突变体具有细胞内在的细胞内复制缺陷。通过用编码未标记或表位标记版本的Cbu0937的质粒补充该基因，可恢复::Tn突变体的细胞内复制。使用AlphaFold对Cbu0937蛋白的预测结构进行分析，发现Cbu0937与几种细菌孔蛋白高度相似。对产生功能性表位标记蛋白的进行分级分离研究和表面标记，表明Cbu0937定位于细菌外膜。从这些数据中，我们得出结论，编码一种孔蛋白，该孔蛋白在支持细胞内复制中起重要作用，这可能涉及在CCV腔内获取一种重要的代谢物。
