Department of Natural Sciences and Mathematics, West Liberty University, West Liberty, West Virginia, USA.
Department of Natural Sciences and Mathematics, West Liberty University, West Liberty, West Virginia, USA
J Bacteriol. 2020 Jun 25;202(14). doi: 10.1128/JB.00048-20.
is an intracellular pathogen and the causative agent of tularemia. The type six secretion system (T6SS) is required for a number of host-pathogen interactions, including phagolysosomal escape and invasion of erythrocytes. One known effector of the T6SS, OpiA, has recently been shown to be a phosphatidylinositol-3 kinase. To investigate the role of OpiA in erythrocyte invasion, we constructed an -null mutant in the live vaccine strain, LVS. OpiA was not required for erythrocyte invasion; however, deletion of affected growth of LVS in broth cultures in a medium-dependent manner. We also found that influenced cell size, gentamicin sensitivity, bacterial viability, and the lipid content of A fluorescently tagged OpiA (OpiA-emerald-green fluorescent protein [EmGFP]) accumulated at the cell poles of , which is consistent with the location of the T6SS. However, OpiA-EmGFP also exhibited a highly dynamic localization, and this fusion protein was detected in erythrocytes and THP-1 cells , further supporting that OpiA is secreted. Similar to previous reports with , our data demonstrated that had a minimal effect on intracellular replication of in host immune cells However, THP-1 cells infected with the mutant produced modestly (but significantly) higher levels of the proinflammatory cytokine tumor necrosis factor alpha compared to these host cells infected with wild-type bacteria. We conclude that, in addition to its role in host-pathogen interactions, our results reveal that the function of is central to the biology of bacteria. is a pathogenic intracellular pathogen that is of importance for public health and strategic defense. This study characterizes the gene of LVS, an attenuated strain that has been used as a live vaccine but that also shares significant genetic similarity to related strains that cause human disease. The data presented here provide the first evidence of a T6SS effector protein that affects the physiology of , namely, the growth, cell size, viability, and aminoglycoside resistance of LVS. This study also adds insight into our understanding of OpiA as a determinant of virulence. Finally, the fluorescence fusion constructs presented here will be useful tools for dissecting the role of OpiA in infection.
是一种细胞内病原体，也是土拉热的病原体。 型六型分泌系统（T6SS）是许多宿主-病原体相互作用所必需的，包括吞噬体逃逸和红细胞入侵。T6SS 的一种已知效应物 OpiA 最近被证明是一种磷酸肌醇 3 激酶。为了研究 OpiA 在红细胞入侵中的作用，我们在活疫苗株 LVS 中构建了一个 -null 突变体。OpiA 不参与红细胞入侵；然而， 的缺失以培养基依赖的方式影响 LVS 在肉汤培养物中的生长。我们还发现， 影响细胞大小、庆大霉素敏感性、细菌活力和荧光标记的 OpiA（OpiA-emerald-green 荧光蛋白 [EmGFP]）的脂质含量 在 LVS 的细胞极积累，这与 T6SS 的位置一致。然而，OpiA-EmGFP 也表现出高度动态的定位，并且这种融合蛋白在红细胞和 THP-1 细胞中被检测到 ，这进一步支持 OpiA 是分泌的。与以前的 报告类似，我们的数据表明， 对宿主免疫细胞内 的复制几乎没有影响 然而，与野生型细菌感染的这些宿主细胞相比，感染 突变体的 THP-1 细胞产生的促炎细胞因子肿瘤坏死因子 α 的水平略高（但显著）。我们得出结论，除了在宿主-病原体相互作用中的作用外，我们的结果还揭示了 的功能对 细菌的生物学至关重要。 是一种重要的致病性细胞内病原体，对公共卫生和战略防御具有重要意义。本研究描述了 LVS 中的 基因，LVS 是一种减毒活疫苗，但与引起人类疾病的相关 菌株具有显著的遗传相似性。本文提供的资料首次证明了 T6SS 效应蛋白会影响 的生理学，即 LVS 的生长、细胞大小、活力和氨基糖苷类抗性。本研究还增加了我们对 OpiA 作为毒力决定因素的理解。最后，本文提供的荧光融合构建体将是剖析 OpiA 在感染中的作用的有用工具。
