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    Salmonella Engages Host MicroRNAs To Modulate SUMOylation: a New Arsenal for Intracellular Survival. Verma Smriti,Mohapatra Gayatree,Ahmad Salman Mustfa,Rana Sarika,Jain Swati,Khalsa Jasneet Kaur,Srikanth C V Molecular and cellular biology Posttranslational modifications (PTMs) can alter many fundamental properties of a protein. One or combinations of them have been known to regulate the dynamics of many cellular pathways and consequently regulate all vital processes. Understandably, pathogens have evolved sophisticated strategies to subvert these mechanisms to achieve instantaneous control over host functions. Here, we present the first report of modulation by intestinal pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) of host SUMOylation, a PTM pathway central to all fundamental cellular processes. Both in cell culture and in a mouse model, we observed that S. Typhimurium infection led to a dynamic SUMO-conjugated proteome alteration. The intracellular survival of S. Typhimurium was dependent on SUMO status as revealed by reduced infection and Salmonella-induced filaments (SIFs) in SUMO-upregulated cells. S. Typhimurium-dependent SUMO modulation was seen as a result of depletion of crucial SUMO pathway enzymes Ubc-9 and PIAS1, at both the protein and the transcript levels. Mechanistically, depletion of Ubc-9 relied on upregulation of small noncoding RNAs miR30c and miR30e during S. Typhimurium infection. This was necessary and sufficient for both down-modulation of Ubc-9 and a successful infection. Thus, we demonstrate a novel strategy of pathogen-mediated perturbation of host SUMOylation, an integral mechanism underlying S. Typhimurium infection and intracellular survival. 10.1128/MCB.00397-15
    A SUMOylation-dependent switch of RAB7 governs intracellular life and pathogenesis of Typhimurium. Mohapatra Gayatree,Gaur Preksha,Mujagond Prabhakar,Singh Mukesh,Rana Sarika,Pratap Shivendra,Kaur Navneet,Verma Smriti,Krishnan Vengadesan,Singh Nirpendra,Srikanth C V Journal of cell science Typhimurium is an intracellular pathogen that causes gastroenteritis in humans. Aided by a battery of effector proteins, Typhimurium resides intracellularly in a specialized vesicle, called the -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 mass-spectrometry (MS/MS) revealed a dramatically altered SUMO-proteome (SUMOylome) in Typhimurium-infected cells. RAB7, a component of VTP, was key among several crucial proteins identified in our study. Detailed MS/MS assays, SUMOylation assays and structural docking analysis revealed SUMOylation of RAB7 (RAB7A) specifically at lysine 175. A SUMOylation-deficient RAB7 mutant (RAB7) displayed longer half-life, was beneficial to SCV dynamics and functionally deficient. Collectively, the data revealed that RAB7 SUMOylation blockade by 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 Typhimurium is an unexpected mode of VTP pathway regulation, and unveils a mechanism of broad interest well beyond -host crosstalk. This article has an associated First Person interview with the first author of the paper. 10.1242/jcs.222612
    Prevention of gastrointestinal lead poisoning using recombinant Lactococcus lactis expressing human metallothionein-I fusion protein. Xiao Xue,Zhang Changbin,Liu Dajun,Bai Weibin,Zhang Qihao,Xiang Qi,Huang Yadong,Su Zhijian Scientific reports Low-level lead poisoning is an insidious disease that affects millions of children worldwide, leading to biochemical and neurological dysfunctions. Blocking lead uptake via the gastrointestinal tract is an important prevention strategy. With this in mind, we constructed the recombinant Lactococcus lactis strain pGSMT/MG1363, which constitutively expressed the fusion protein glutathione S-transferase (GST)-small molecule ubiquitin-like modifier protein (SUMO)-metallothionein-I (GST-SUMO-MT). The thermodynamic data indicated that the average number of lead bound to a GST-SUMO-MT molecule was 3.655 and this binding reaction was a spontaneous, exothermic and entropy-increasing process. The total lead-binding capacity of pGSMT/MG1363 was 4.11 ± 0.15 mg/g dry mass. Oral administration of pGSMT/MG1363 (1 × 10(10) Colony-Forming Units) to pubertal male rats that were also treated with 5 mg/kg of lead acetate daily significantly inhibited the increase of blood lead levels, the impairment of hepatic function and the decrease of testosterone concentration in the serum, which were all impaired in rats treated by lead acetate alone. Moreover, the administration of pGSMT/MG1363 for 6 weeks did not affect the serum concentration of calcium, magnesium, potassium or sodium ions. This study provides a convenient and economical biomaterial for preventing lead poisoning via the digestive tract. 10.1038/srep23716
    Sumoylation by Ubc9 regulates the stem cell compartment and structure and function of the intestinal epithelium in mice. Demarque Maud D,Nacerddine Karim,Neyret-Kahn Hélène,Andrieux Alexandra,Danenberg Esther,Jouvion Grégory,Bomme Perrine,Hamard Ghislaine,Romagnolo Béatrice,Terris Benoît,Cumano Ana,Barker Nick,Clevers Hans,Dejean Anne Gastroenterology BACKGROUND & AIMS:Small ubiquitin-like modifiers (SUMOs) are attached to other proteins to regulate their function (sumoylation). We investigated the role of Ubc9, which covalently attaches SUMOs to proteins, in the gastrointestinal tract of adult mice. METHODS:We investigated the effects of decreased sumoylation in adult mammals by generating mice with an inducible knockout (by injection of 4-hydroxytamoxifen) of the E2 enzyme Ubc9 (Ubc9fl/-/ROSA26-CreERT2 mice). We analyzed the phenotypes using a range of histologic techniques. RESULTS:Loss of Ubc9 from adult mice primarily affected the small intestine. Ubc9fl/-/ROSA26-CreERT2 mice died within 6 days of 4-hydroxytamoxifen injection, losing 20% or less of their body weight and developing severe diarrhea on the second day after injection. Surprisingly, other epithelial tissues appeared to be unaffected at that stage. Decreased sumoylation led to the depletion of the intestinal proliferative compartment and to the rapid disappearance of stem cells. Sumoylation was required to separate the proliferative and differentiated compartments from the crypt and control differentiation and function of the secretory lineage. Sumoylation was required for nucleus positioning and polarized organization of actin in the enterocytes. Loss of sumoylation caused detachment of the enterocytes from the basal lamina, as observed in tissue fragility diseases. We identified the intermediate filament keratin 8 as a SUMO substrate in epithelial cells. CONCLUSIONS:Sumoylation maintains intestinal stem cells and the architecture, mechanical stability, and function of the intestinal epithelium of mice. 10.1053/j.gastro.2010.10.002
    Crohn's Disease-Associated Adherent-Invasive Escherichia coli Manipulate Host Autophagy by Impairing SUMOylation. Dalmasso Guillaume,Nguyen Hang T T,Faïs Tiphanie,Massier Sébastien,Barnich Nicolas,Delmas Julien,Bonnet Richard Cells The intestinal mucosa of Crohn's disease (CD) patients is abnormally colonized with adherent-invasive (AIEC) that are able to adhere to and to invade intestinal epithelial cells (IECs), to survive in macrophages, and to induce a pro-inflammatory response. AIEC persist in the intestine, and induce inflammation in CEABAC10 transgenic mice expressing human CAECAM6, the receptor for AIEC. SUMOylation is a eukaryotic-reversible post-translational modification, in which SUMO, an ubiquitin-like polypeptide, is covalently linked to target proteins. Here, we investigated the role of SUMOylation in host responses to AIEC infection. We found that infection with the AIEC LF82 reference strain markedly decreased the levels of SUMO-conjugated proteins in human intestinal epithelial T84 cells. This was also observed in IECs from LF82-infected CEABAC10 transgenic mice. LF82-induced deSUMOylation in IECs was due in part to increased level of microRNA (miR)-18, which targets mRNA encoding a protein involved in SUMOylation. Over-expression of SUMOs in T84 cells induced autophagy, leading to a significant decrease in the number of intracellular LF82. Consistently, a decreased expression of UBC9, a protein necessary for SUMOylation, was accompanied with a decrease of LF82-induced autophagy, increasing bacterial intracellular proliferation and inflammation. Finally, the inhibition of miR-18 significantly decreased the number of intracellular LF82. In conclusion, our results suggest that AIEC inhibits the autophagy response to replicate intracellularly by manipulating host SUMOylation. 10.3390/cells8010035
    Sumoylation controls host anti-bacterial response to the gut invasive pathogen Shigella flexneri. Fritah Sabrina,Lhocine Nouara,Golebiowski Filip,Mounier Joëlle,Andrieux Alexandra,Jouvion Grégory,Hay Ronald T,Sansonetti Philippe,Dejean Anne EMBO reports Shigella flexneri, the etiological agent of bacillary dysentery, invades the human colonic epithelium and causes its massive inflammatory destruction. Little is known about the post-translational modifications implicated in regulating the host defense pathway against Shigella. Here, we show that SUMO-2 impairs Shigella invasion of epithelial cells in vitro. Using mice haploinsufficient for the SUMO E2 enzyme, we found that sumoylation regulates intestinal permeability and is required to restrict epithelial invasion and control mucosal inflammation. Quantitative proteomics reveals that Shigella infection alters the sumoylation status of a restricted set of transcriptional regulators involved in intestinal functions and inflammation. Consistent with this, sumoylation restricts the pro-inflammatory transcriptional response of Shigella-infected guts. Altogether, our results show that the SUMO pathway is an essential component of host innate protection, as it reduces the efficiency of two key steps of shigellosis: invasion and inflammatory destruction of the intestinal epithelium. 10.15252/embr.201338386
    Ubc9 overexpression and SUMO1 deficiency blunt inflammation after intestinal ischemia/reperfusion. Karhausen Jörn,Bernstock Joshua D,Johnson Kory R,Sheng Huaxin,Ma Qing,Shen Yuntian,Yang Wei,Hallenbeck John M,Paschen Wulf Laboratory investigation; a journal of technical methods and pathology The intestinal epithelium constitutes a crucial defense to the potentially life-threatening effects of gut microbiota. However, due to a complex underlying vasculature, hypoperfusion and resultant tissue ischemia pose a particular risk to function and integrity of the epithelium. The small ubiquitin-like modifier (SUMO) conjugation pathway critically regulates adaptive responses to metabolic stress and is of particular significance in the gut, as inducible knockout of the SUMO-conjugating enzyme Ubc9 results in rapid intestinal epithelial disintegration. Here we analyzed the pattern of individual SUMO isoforms in intestinal epithelium and investigated their roles in intestinal ischemia/reperfusion (I/R) damage. Immunostaining revealed that epithelial SUMO2/3 expression was almost exclusively limited to crypt epithelial nuclei in unchallenged mice. However, intestinal I/R or overexpression of Ubc9 caused a remarkable enhancement of epithelial SUMO2/3 staining along the crypt-villus axis. Unexpectedly, a similar pattern was found in SUMO1 knockout mice. Ubc9 transgenic mice, but also SUMO1 knockout mice were protected from I/R injury as evidenced by better preserved barrier function and blunted inflammatory responses. PCR array analysis of microdissected villus-tip epithelia revealed a specific epithelial contribution to reduced inflammatory responses in Ubc9 transgenic mice, as key chemotactic signaling molecules such as IL17A were significantly downregulated. Together, our data indicate a critical role particularly of the SUMO2/3 isoforms in modulating responses to I/R and provide the first evidence that SUMO1 deletion activates a compensatory process that protects from ischemic damage. 10.1038/s41374-018-0035-6
    SUMOylation pathway alteration coupled with downregulation of SUMO E2 enzyme at mucosal epithelium modulates inflammation in inflammatory bowel disease. Mustfa Salman Ahmad,Singh Mukesh,Suhail Aamir,Mohapatra Gayatree,Verma Smriti,Chakravorty Debangana,Rana Sarika,Rampal Ritika,Dhar Atika,Saha Sudipto,Ahuja Vineet,Srikanth C V Open biology Post-translational modification pathways such as SUMOylation are integral to all cellular processes and tissue homeostasis. We investigated the possible involvement of SUMOylation in the epithelial signalling in Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD). Initially in a murine model of IBD, induced by dextran-sulfate-sodium (DSS mice), we observed inflammation accompanied by a lowering of global SUMOylation of colonic epithelium. The observed SUMOylation alteration was due to a decrease in the sole SUMO E2 enzyme (Ubc9). Mass-spectrometric analysis revealed the existence of a distinct SUMOylome (SUMO-conjugated proteome) in DSS mice with alteration of key cellular regulators, including master kinase Akt1. Knocking-down of Ubc9 in epithelial cells resulted in dramatic activation of inflammatory gene expression, a phenomenon that acted via reduction in Akt1 and its SUMOylated form. Importantly, a strong decrease in Ubc9 and Akt1 was also seen in endoscopic biopsy samples ( = 66) of human CD and UC patients. Furthermore, patients with maximum disease indices were always accompanied by severely lowered Ubc9 or SUMOylated-Akt1. Mucosal tissues with severely compromised Ubc9 function displayed higher levels of pro-inflammatory cytokines and compromised wound-healing markers. Thus, our results reveal an important and previously undescribed role for the SUMOylation pathway involving Ubc9 and Akt1 in modulation of epithelial inflammatory signalling in IBD. 10.1098/rsob.170024