Salmonella Typhimurium is an intracellular pathogen that causes gastroenteritis in humans. Aided by a battery of effector proteins, S. Typhimurium resides intracellularly in a specialized vesicle, called the Salmonella-containing vacuole (SCV) that utilizes the host endocytic vesicular transport pathway (VTP). Here, we probed the possible role of SUMOylation, a post-translation modification pathway, in SCV biology. Proteome analysis by complex massspectrometry (MS/MS) revealed a dramatically altered SUMOproteome (SUMOylome) in S. Typhimurium-infected cells. RAB7, a component of VTP, was key among several crucial proteins identified in our study. Detailed MS/MS assays, in vitro SUMOylation assays and structural docking analysis revealed SUMOylation of RAB7 (RAB7A) specifically at lysine 175. A SUMOylation-deficient RAB7 mutant (RAB7 K175R) displayed longer half-life, was beneficial to SCV dynamics and functionally deficient. Collectively, the data revealed that RAB7 SUMOylation blockade by S. Typhimurium ensures availability of long-lived but functionally compromised RAB7, which was beneficial to the pathogen. Overall, this SUMOylation-dependent switch of RAB7 controlled by S. Typhimurium is an unexpected mode of VTP pathway regulation, and unveils a mechanism of broad interest well beyond Salmonella-host crosstalk. This article has an associated First Person interview with the first author of the paper.

Highlights d The deSUMOylase SENP7 contributes to IBD pathophysiology d SENP7 function and interactome modulate epithelial-immune crosstalk d SIAH2 negatively regulates SENP7 by ubiquitination in healthy, but not inflamed, cells d Epithelial SENP7 upregulation triggers proinflammatory mechanisms via gd T cells

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Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier coupled with microbial dysbiosis has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease Chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7 dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

Background Intestinal crypts form a pristine gut biogeographical niche, homing the intestinal stem cells and being the closest neighbors to underlying lamina propria. Initially believed to be sterile, the crypt lumen was recently shown to inhabit a conserved microbial population. However, the identity of the crypt-associated microbiota (CAM) remains elusive. The present study is among the first to illustrate the CAM composition in health and its deviation during ulcerative colitis (UC), and the effect of faecal microbiota transplantation along with an anti-inflammatory diet (FMT-AID) on the structure of this community. Methods Recto-sigmoidal biopsies from controls, and from patients with mild-moderate UC before and after FMT-AID (n=26), were subjected to methacarn-fixation, followed by paraffin-embedding, sectioning and laser-capture microdissection-assisted crypt isolation. DNA isolated from micro-dissected samples was subjected to 16S rRNA gene sequencing. The bacterial presence in colonic crypts was confirmed using fluorescence in-situ hybridization(FISH). Microbiome data analysis was carried out by using QIIME2 and R packages. Results FISH performed using pan-bacterial probes revealed the presence of sparse microbial clusters in colonic crypts, distinct from the overlying layer of Mucosa-associated microbiota (MAM), in both controls as well as in UC(Fig.1a and 1b). While MAM is dominated by members of phyla Firmicutes (45%) and Bacteroidetes (26%),CAM is comprised predominantly of aerobic members of Actinobacteria(54%) and Proteobacteria(38%), followed by minor proportions of Firmicutes(4%), Acidobacteria and Cyanobacteria (2% each). The significant members of CAM included aerobic genera -Cutibacterium(35%),Sphingobium(13%), Paracoccus(11%), Micrococcus(3%), Lawsonella(3%), Rothia(2%), Prauserella(2%), Kocuria, Corynebacterium, Acinetobacter and Brevundimonas (1% each)(Fig.1c and 1d). Analysis of CAM diversity in controls, and in patients with UC before and after FMT-AID, showed no significant alterations in the α- and β diversity matrices(Fig.1e and 1f).CAM demonstrated UC-associated dysbiosis of specific taxa which was restored after FMT-AID(Fig.2a and 2b). These FMT-restored CAM taxa correlated negatively with disease-associated parameters - Fecal calprotectin (FCP), Simple Clinical Colitis Activity Index (SCCAI) and Ulcerative Colitis Endoscopic Index of Severity (UCEIS)(Fig.2c and 2d). The positive effects of FMT-AID further refurbished the CAM-MAM interaction networks, which were obliterated in UC.(Fig.2e) Conclusion A gut bacterial community, enriched in aerobic bacteria, resides in the colonic crypts, and undergoes taxa-level alterations during UC and in response to FMT.