Soni Shilpa, Ernst Robert K, Muszyński Artur, Mohapatra Nrusingh P, Perry Malcolm B, Vinogradov Evgeny, Carlson Russell W, Gunn John S
Center for Microbial Interface Biology, Department of Molecular Virology, Immunology and Medical Genetics, and Department of Internal Medicine, Division of Infectious Diseases, The Ohio State University Columbus, OH, USA.
Front Microbiol. 2010 Nov 19;1:129. doi: 10.3389/fmicb.2010.00129. eCollection 2010.
Francisella tularensis is a CDC Category A biological agent and a potential bioterrorist threat. There is no licensed vaccine against tularemia in the United States. A long-standing issue with potential Francisella vaccines is strain phase variation to a gray form that lacks protective capability in animal models. Comparisons of the parental strain (LVS) and a gray variant (LVSG) have identified lipopolysaccharide (LPS) alterations as a primary change. The LPS of the F. tularensis variant strain gains reactivity to F. novicida anti-LPS antibodies, suggesting structural alterations to the O-antigen. However, biochemical and structural analysis of the F. tularensis LVSG and LVS LPS demonstrated that LVSG has less O-antigen but no major O-antigen structural alterations. Additionally, LVSG possesses structural differences in both the core and lipid A regions, the latter being decreased galactosamine modification. Recent work has identified two genes important in adding galactosamine (flmF2 and flmK) to the lipid A. Quantitative real-time PCR showed reduced transcripts of both of these genes in the gray variant when compared to LVS. Loss of flmF2 or flmK caused less frequent phase conversion but did not alter intramacrophage survival or colony morphology. The LVSG strain demonstrated an intramacrophage survival defect in human and rat but not mouse macrophages. Consistent with this result, the LVSG variant demonstrated little change in LD(50) in the mouse model of infection. Furthermore, the LVSG strain lacks the protective capacity of F. tularensis LVS against virulent Type A challenge. These data suggest that the LPS of the F. tularensis LVSG phase variant is dramatically altered. Understanding the mechanism of blue to gray phase variation may lead to a way to inhibit this variation, thus making future F. tularensis vaccines more stable and efficacious.
土拉弗朗西斯菌是美国疾病控制与预防中心（CDC）认定的A类生物制剂，也是一种潜在的生物恐怖威胁。美国尚无获批的兔热病疫苗。潜在的弗朗西斯菌疫苗长期存在的一个问题是菌株会变异为灰色形态，这种形态在动物模型中缺乏保护能力。亲本菌株（LVS）与灰色变体（LVSG）的比较已确定脂多糖（LPS）改变是主要变化。土拉弗朗西斯菌变体菌株的LPS对新凶手弗朗西斯菌抗LPS抗体产生反应，表明O抗原发生了结构改变。然而，对土拉弗朗西斯菌LVSG和LVS LPS的生化及结构分析表明，LVSG的O抗原较少，但没有主要的O抗原结构改变。此外，LVSG在核心区和脂质A区都存在结构差异，后者的氨基半乳糖修饰减少。最近的研究确定了两个在脂质A中添加氨基半乳糖（flmF2和flmK）方面很重要的基因。定量实时PCR显示，与LVS相比，灰色变体中这两个基因的转录本均减少。flmF2或flmK的缺失导致相变频率降低，但并未改变细胞内生存能力或菌落形态。LVSG菌株在人和大鼠巨噬细胞中表现出细胞内生存缺陷，但在小鼠巨噬细胞中没有。与这一结果一致，LVSG变体在感染小鼠模型中的半数致死剂量（LD50）变化不大。此外，LVSG菌株缺乏土拉弗朗西斯菌LVS对强毒A型攻击的保护能力。这些数据表明，土拉弗朗西斯菌LVSG相变变体的LPS发生了显著改变。了解从蓝色到灰色相变的机制可能会找到抑制这种变异的方法，从而使未来的土拉弗朗西斯菌疫苗更稳定、更有效。
