VARP and Rab9 Are Dispensable for the Rab32/BLOC-3 Dependent Salmonella Killing

Abstract

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.

Hermansky-Pudlak syndrome type 1 causes impaired anti-microbial immunity and inflammation due to dysregulated immunometabolism

“…Since BLOC-3 acts as a guanine nucleotide exchange factor for Rab32 12 , we would expect that HPS1 deficiency would result in an accumulation of Rab32-GDP, which could stimulate mTORC1 signaling. Intriguingly, BLOC-3 overexpression increases Rab32 localization to Salmonella-containing vacuoles, lending credence to the hypothesis that BLOC-3 controls Rab32 localization for bacterial clearance 48 . While HPS1 and HPS4 can be recovered in phagosomes, Rab32 localizes to both phagosomes and lipid droplets 44,49 .…”

Mitochondrial A‐kinase anchoring proteins in cardiac ventricular myocytes

“…Attempts to obtain immunofluorescence signals using Rab32 and WAVE‐1 antibodies were not successful (Figures S2 and S3 ). The Rab32 antibody has been used with immunofluorescence techniques in cell lines (Balci et al, 2020 ; Hu et al, 2019 ), so levels of Rab32 might not have been enough for detection in isolated ARVMs. The WAVE‐1 antibody is specific for epitopes near the N‐terminal domain, which in part, regulates subcellular localization of the protein and could potentially be masked to the antibody.…”

Determination of the Salmonella intracellular lifestyle by the diversified interaction of Type III secretion system effectors and host GTPases

“…Rab32 is activated in response to Rab9-dependent recruitment of BLOC-3 and regulates the downstream signaling (Ohishi et al, 2019). However, Rab9 is dispensable for Rab32 recruitment to the SCV and is not essential for Rab32/BLOC-3 pathway-dependent bacterial killing (Balci et al, 2020).…”