VARP and Rab9 Are Dispensable for the Rab32/BLOC-3 Dependent Salmonella Killing.
VARP and Rab9 Are Dispensable for the Rab32/BLOC-3 Dependent Salmonella Killing.
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DOI：
10.3389/fcimb.2020.581024
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发表时间：
2020
影响因子：
5.7
通讯作者：
Spanò S
中科院分区：
文献类型：
--
作者：
Balci A;Solano-Collado V;Baldassarre M;Spanò S
伤寒沙门氏菌（S.伤寒（Typhi）是伤寒的病原体，伤寒是一种每年导致约20万人死亡的疾病。以前，我们发现了一种依赖于Rab 32和BLOC-3（BRAM）的抗菌途径，这对杀死S至关重要。鼠巨噬细胞中的伤寒。BLOC-3复合物由两个亚基HPS 1和HPS 4组成，并对Rab 32表现出鸟嘌呤-核苷酸交换因子（GEF）活性。在黑素细胞中，Rab 9已被证明与HPS 4和RUTBC 1（Rab 32 GTP酶激活（GAP）蛋白）相互作用，并调节Rab 32介导的黑素体生物发生。有趣的是，Rab 9缺陷型黑素细胞表现出色素减退，这是与Rab 32或BLOC-3缺陷型黑素细胞相似的表型。此外，VPS 9-锚定重复蛋白（VARP）已显示通过与Rab 32相互作用调节黑素细胞酶运输到黑素体中。尽管Rab 32、Rab 9和VARP是黑素细胞中黑素生成的一部分，但Rab 9和VARP是否是BRAM介导的巨噬细胞杀伤所必需的目前尚不清楚。在这里，我们发现HPS 4被募集到含沙门氏菌的空泡（SCV）中，并且BLOC-3的过表达显著增加Rab 32阳性细菌空泡。我们发现SCV获得Rab 9，然而过量表达Rab 9并不改变HPS 4在细菌液泡上的定位。重要的是，我们使用shRNA敲低巨噬细胞中的Rab 9和VARP，并表明这些蛋白质是Rab 32募集到SCV的关键。此外，我们评估了S.鼠伤寒沙门氏菌在Rab 9或VARP缺陷的巨噬细胞，并证明这些蛋白质不是必不可少的BRAM途径依赖性杀伤。
Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever, a disease that kills an estimated 200,000 people annually. Previously, we discovered an antimicrobial pathway dependent on Rab32 and BLOC-3 (BRAM) that is critical to kill S. Typhi in murine macrophages. The BLOC-3 complex is comprised of the two sub-units HPS1 and HPS4 and exhibits guanine-nucleotide exchange factor (GEF) activity to Rab32. In melanocytes, Rab9 has been shown to interact with HPS4 and RUTBC1, a Rab32 GTPase activating (GAP) protein, and regulate the Rab32-mediated melanosome biogenesis. Intriguingly, Rab9-deficient melanocytes exhibit hypopigmentation, a similar phenotype to Rab32 or BLOC-3 deficient melanocytes. Additionally, VPS9-ankyrin-repeat-protein (VARP) has been shown to regulate melanocytic enzyme trafficking into the melanosomes through interaction with Rab32. Although Rab32, Rab9 and VARP are a part of melanogenesis in melanocytes, whether Rab9 and VARP are required for the BRAM mediated killing in macrophages is currently unknown. Here we showed that HPS4 is recruited to the Salmonella-containing vacuoles (SCV) and over-expression of BLOC-3 significantly increased Rab32-positive bacteria vacuoles. We found that SCV acquire Rab9, however over-expressing Rab9 did not change HPS4 localization on bacteria vacuoles. Importantly, we used shRNA to knock-down Rab9 and VARP in macrophages and showed that these proteins are dispensable for Rab32 recruitment to the SCV. Furthermore, we assessed the survival of S. Typhimurium in macrophages deficient for Rab9 or VARP and demonstrated that these proteins are not essential for BRAM pathway-dependent killing.