Newton Victoria, McKenna Shawn L, De Buck Jeroen
Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, Canada T2N 4N1.
Vet Microbiol. 2009 Mar 30;135(3-4):394-400. doi: 10.1016/j.vetmic.2008.09.066. Epub 2008 Sep 24.
Johne's disease or paratuberculosis in cattle is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Although the stages of infection have been well described, very few virulence factors of MAP have been studied in detail. We aimed to study the localization and immunogenicity of members of the polymorphic PPE protein family which is unique to Mycobacteria and has been linked to virulence in Mycobacterium tuberculosis (Mtb). The presence of PPE proteins in the cell wall was investigated by enzymatic digest of surface exposed proteins of live MAP bacteria and analysis by LC-MS/MS. Polyclonal antisera were generated against a recombinant fragment of one PPE protein and a synthetic peptide of the other to confirm their surface exposure. Sera from naturally infected cows were investigated for the presence of specific antibodies against the recombinant PPE protein. Two PPE proteins, Map3420c and Map1506, were detected by mass spectrometry and confirmed to be surface exposed on live MAP cells by immunohistochemistry. The sera from naturally infected animals contained specific antibodies against recombinantly expressed Map3420c as demonstrated by western blotting. These findings show the in vitro expression of two PPE proteins. Additionally the surface exposure and immunogenicity of PPE proteins of MAP was demonstrated.
牛的副结核或约内氏病由鸟分枝杆菌副结核亚种（MAP）引起。尽管感染阶段已有详细描述，但对MAP的毒力因子却鲜有深入研究。我们旨在研究多态性PPE蛋白家族成员的定位和免疫原性，该家族是分枝杆菌特有的，且与结核分枝杆菌（Mtb）的毒力有关。通过对活MAP细菌表面暴露蛋白进行酶消化并用LC-MS/MS分析，研究细胞壁中PPE蛋白的存在情况。针对一种PPE蛋白的重组片段和另一种的合成肽制备了多克隆抗血清，以确认它们在表面的暴露情况。对自然感染奶牛的血清进行检测，以确定是否存在针对重组PPE蛋白的特异性抗体。通过质谱检测到两种PPE蛋白Map3420c和Map1506，并通过免疫组织化学证实它们在活MAP细胞表面暴露。蛋白质印迹法表明，自然感染动物的血清中含有针对重组表达的Map3420c的特异性抗体。这些发现显示了两种PPE蛋白的体外表达。此外，还证实了MAP的PPE蛋白在表面的暴露及其免疫原性。
