Woo Seng-Ryong, Heintz Joseph A, Albrecht Ralph, Barletta Raúl G, Czuprynski Charles J
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive West, Madison, WI 53706, USA.
Microb Pathog. 2007 Aug-Sep;43(2-3):106-13. doi: 10.1016/j.micpath.2007.04.004. Epub 2007 May 10.
We previously reported that the number of acid-fast bacilli within Mycobacterium paratuberculosis-infected bovine monocytes increased steadily during an 8-day incubation period in vitro, despite a decrease in the number of viable bacilli as estimated by a radiometric method. In this study, we used differential live/dead staining of bacilli from infected monocytes to show that the percentage of viable bacilli decreased during an 8-day incubation period. We observed poor phagosome-lysosome fusion in monocytes that had ingested viable M. paratuberculosis (30% phagosome-lysosome fusion), while monocytes that ingested heat killed M. paratuberculosis exhibited 94% phagosome-lysosome fusion at 24h after infection. Treatment with the selective Ca(2+)/CaM and PI3 kinase inhibitors (i.e. KN62 and Wortmannin) in combination increased the survival of M. paratuberculosis in bovine monocytes without significantly altering phagosome-lysosome fusion. Scanning electron microscopy suggested that M. paratuberculosis-infected monocytes were less differentiated (smaller and less spreading) than uninfected monocytes at 4 and 8 days of infection. Overall, these data suggest that both multiplication and killing of intracellular M. paratuberculosis occur concomitantly in bovine monocytes. Monocytes in turn may be adversely affected by the bacilli, their products, or factors released from infected monocytes.
我们之前报道过，尽管通过放射性方法估计，副结核分枝杆菌感染的牛单核细胞内的抗酸杆菌数量在体外8天的培养期内稳步增加，但活菌数量却有所减少。在本研究中，我们对感染单核细胞中的杆菌进行了活/死鉴别染色，结果显示活菌百分比在8天培养期内下降。我们观察到，摄取活的副结核分枝杆菌的单核细胞中吞噬体-溶酶体融合较差（吞噬体-溶酶体融合率为30%），而摄取热灭活副结核分枝杆菌的单核细胞在感染后24小时的吞噬体-溶酶体融合率为94%。联合使用选择性Ca(2+)/CaM和PI3激酶抑制剂（即KN62和渥曼青霉素）进行处理，可提高副结核分枝杆菌在牛单核细胞中的存活率，且不会显著改变吞噬体-溶酶体融合。扫描电子显微镜显示，在感染4天和8天时，感染副结核分枝杆菌的单核细胞比未感染的单核细胞分化程度更低（更小且铺展性更差）。总体而言，这些数据表明，牛单核细胞内副结核分枝杆菌的增殖和杀伤是同时发生的。反过来，单核细胞可能会受到杆菌、其产物或感染单核细胞释放的因子的不利影响。
