Plasmids and strains used in this study.

The multi-host pathogen Rhodococcus equi is a parasite of macrophages preventing maturation of the phagolysosome, thus creating a hospitable environment supporting intracellular growth. Virulent R. equi isolated from foals, pigs and cattle harbor a host-specific virulence plasmid, pVAPA, pVAPB and pVAPN respectively, which encode a family of 17 Vap proteins belonging to seven monophyletic clades. We examined all 17 Vap proteins for their ability to complement intracellular growth of a R. equi ΔvapA strain, and show that only vapK1, vapK2 and vapN support growth in murine macrophages of this strain. We show that only the clade-1 proteins VapA, VapK1, VapK2 and VapN are located on the R. equi cell surface. The pVAPB plasmid encodes three clade-1 proteins: VapK1, VapK2 and VapB. The latter was not able to support intracellular growth and was not located on the cell surface. We previously showed that the unordered N-terminal VapA sequence is involved in cell surface localisation of VapA. We here show that although the unordered N-terminus of the 17 Vap proteins is highly variable in length and sequence, it is conserved within clades, which is consistent with our observation that the N-terminus of clade-1 Vap proteins plays a role in cell surface localisation.

多宿主病原菌马红球菌（Rhodococcus equi）是一种巨噬细胞寄生菌，可阻断吞噬溶酶体的成熟过程，进而构建适宜胞内增殖的微环境。从马驹、猪和牛中分离的强毒马红球菌分别携带有宿主特异性毒性质粒pVAPA、pVAPB和pVAPN，这些质粒编码由17种Vap蛋白组成的蛋白家族，该家族隶属于7个单系进化枝。我们针对全部17种Vap蛋白，检测其对马红球菌ΔvapA缺失菌株胞内生长的互补恢复能力，结果显示仅vapK1、vapK2和vapN可恢复该菌株在小鼠巨噬细胞内的生长。研究同时证实，仅进化枝1的VapA、VapK1、VapK2和VapN四种蛋白定位于马红球菌的细胞表面。pVAPB质粒编码3种进化枝1蛋白：VapK1、VapK2与VapB，其中VapB既无法恢复菌株的胞内生长，也未定位于细胞表面。我们此前的研究表明，VapA的无序N末端序列参与其细胞表面定位过程。本研究发现，尽管17种Vap蛋白的无序N末端在长度与序列上均存在高度多样性，但在各自所属的进化枝内保守，这与我们观察到的进化枝1 Vap蛋白N末端参与细胞表面定位的结果一致。