The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1, Yoshida, Yamaguchi, 753-8515, Japan; Department of Pathology and Parasitology, Patuakhali Science and Technology University, Babugonj, Barisal, 8210, Bangladesh.
Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, 162-8640, Japan.
Microb Pathog. 2018 Oct;123:74-81. doi: 10.1016/j.micpath.2018.06.046. Epub 2018 Jun 30.
Tularemia is a zoonosis caused by CDC-declared Tier 1 threat agent Francisella tularensis. F. tularensis subsp. novicida (F. novicida) is virulent in mice but non-pathogenic in immunocompetent humans and serves as a potential surrogate organism. In a recent study, we established a silkworm (Bombyx mori) model of infection for F. novicida. Francisella secretes its virulence factors through various mechanisms that modify the intracellular environment to ensure its replication and survival. To identify new pathogenic factors, we focused on the type I secretory system (T1SS) of Francisella. In silico analysis revealed a RtxA (Repeats-in-toxin) like protein in the Francisella genome. The characteristics of RtxA like protein were investigated using mutant analysis. Firstly, the role of rtxA in silkworms was investigated by infecting them with F. novicida strains into the hemocoel. The rtxA mutant failed to kill the silkworms, whereas F. novicida wild-type (WT) strain killed silkworms within 3-7 days post infection. The arrested growth of the mutant strain in silkworms was observed using a whole-body CFU count assay. We also investigated the growth characteristics of the rtxA mutant in hemocytes, one of the primary multiplication sites of Francisella within silkworms. Interrupted growth of the rtxA mutant with significantly reduced cytotoxicity was observed in hemocytes via confocal microscopy. Next, we analyzed the effect of rtxA in human monocyte cell line THP-1. The mutant strain showed significantly decreased growth and reduced cytotoxicity compared with its parental strain in THP-1 cells. This study newly identified RtxA like protein of F. novicida as an important lethal pathogenic factor in silkworm and mammalian cells.
兔热病是由疾病预防控制中心宣布的一级威胁因子弗朗西斯菌引起的一种人畜共患病。F. tularensis 亚种 novicida（F. novicida）在小鼠中具有毒力，但在免疫功能正常的人类中无致病性，可作为潜在的替代生物体。在最近的一项研究中，我们建立了 F. novicida 感染家蚕（Bombyx mori）的模型。弗朗西斯菌通过多种机制分泌其毒力因子，改变细胞内环境，以确保其复制和存活。为了鉴定新的致病因子，我们专注于弗朗西斯菌的 I 型分泌系统（T1SS）。通过计算机分析，在弗朗西斯菌基因组中发现了一种类似于 Repeats-in-toxin（RtxA）的蛋白。通过突变分析研究了类似于 RtxA 蛋白的特征。首先，通过将 F. novicida 菌株感染到家蚕血腔中来研究 rtxA 在蚕中的作用。rtxA 突变体未能杀死家蚕，而 F. novicida 野生型（WT）菌株在感染后 3-7 天内杀死了家蚕。通过对整个生物体 CFU 计数测定，观察到突变株在家蚕中的生长停滞。我们还研究了 rtxA 突变体在血淋巴细胞中的生长特性，血淋巴细胞是弗朗西斯菌在家蚕体内的主要增殖部位之一。通过共聚焦显微镜观察到 rtxA 突变体在血淋巴细胞中的生长中断，且细胞毒性显著降低。接下来，我们分析了 rtxA 在人单核细胞系 THP-1 中的作用。与亲本菌株相比，突变株在 THP-1 细胞中的生长明显减少，细胞毒性降低。这项研究新发现 F. novicida 的类似于 RtxA 蛋白是家蚕和哺乳动物细胞中的一个重要致死性致病因子。
