Post Translational 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 (ST) of host SUMOylation, a PTM pathway central to all fundamental cellular processes. Both in cell culture and in mouse model, we observed ST infection led to a dynamic SUMO-conjugateted proteome alteration. The intracellular survival of ST was dependent on SUMO status as revealed by reduced infection and Salmonella induced filaments (SIFs) in SUMO-upregulated cells. ST dependent SUMO modulation was seen as a result of depletion of crucial SUMO-pathway enzymes Ubc-9 and PIAS1, both at the protein and transcript levels. Mechanistically, depletion of Ubc-9 relied on upregulation of small non-coding RNA miR30c and miR30e during ST 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 ST infection and intracellular survival.

The thymus generates cells of the T cell lineage that seed the lymphatic and blood systems. Transcription factor regulatory networks control the lineage programming and maturation of thymic precursor cells. Whether extrathymic antigenic events, such as the microbial colonization of the mucosal tract also shape the thymic T cell repertoire is unclear. We show here that intestinal microbes influence the thymic homeostasis of PLZF-expressing cells in early life. Impaired thymic development of PLZF⁺ innate lymphocytes in germ-free (GF) neonatal mice is restored by colonization with a human commensal, Bacteroides fragilis, but not with a polysaccharide A (PSA) deficient isogenic strain. Plasmacytoid dendritic cells influenced by microbes migrate from the colon to the thymus in early life to regulate PLZF⁺ cell homeostasis. Importantly, perturbations in thymic PLZF⁺ cells brought about by alterations in early gut microbiota persist into adulthood and are associated with increased susceptibility to experimental colitis. Our studies identify a pathway of communication between intestinal microbes and thymic lymphocytes in the neonatal period that can modulate host susceptibility to immune-mediated diseases later in life. Significance The burgeoning intestinal microbiota pose a unique challenge to the developing immune system of the newborn. Extensive crosstalk between mucosal immune cells and microbes is necessary to establish healthy microbial communities and promote the development of productive mucosal immunity. However, it is yet unknown whether intestinal microbes also influence the development of lymphocytes themselves in the thymus. Here, we report that thymic distribution of transcription factor PLZF-expressing innate lymphocytes is regulated by intestinal microbes in early life. Migratory plasmacytoid dendritic cells are one conveyor of microbial information to developing thymic cells and perturbation of this entero-thymic communication in early life impacts disease susceptibility in adulthood. Our data highlight a novel regulation of thymic lymphocyte development by early-life microbiota.

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 ( N = 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.

Cytochrome P450s (CYP450s) are hemoproteins catalysing diverse biochemical reactions important for metabolism of xenobiotics and synthesis of physiologically important compounds such as sterols. Therefore, they are functionally important for survival of invading pathogens. One such opportunistic pathogen Leishmania donovani causes visceral leishmaniasis worldwide, which is an important public health problem due to significant disease burden. The parasite genome database, Gene DB, annotates 3 CYP450s in Leishmania, however, the functional role of cytochrome P450 enzymes in Leishmania spp. remains elusive. A CYP450-like gene cloned from Leishmania donovani was identified as a novel CYP450, the CYP5122A1. Upon co-localization with organelle specific markers, CYP5122A1 distribution was shown to be localized in the promastigote ER, mitochondria and the glycosomes. Replacement of one allele of CYP5122A1 with either neomycin or hygromycin gene by homologous recombination in Leishmania promastigotes induced substantial reduction of CYP5122A1 expression. These parasites showed impaired growth, lower mitochondrial Ca²⁺ and membrane potential resulting in low ATP generation. Also, these parasites were less infective in vitro and in vivo than their wild-type counterparts as assessed by incubation of Leishmania promastigotes with macrophages in vitro as well as through administration of parasites into hamsters. The HKOs were more susceptible to drugs like miltefosine and antimony, but showed reduced sensitivity to amphotericin B. Removal of two alleles of CYP5122A1 did not allow the parasites to survive. The mutant parasites showed 3.5 times lower ergosterol level as compared to the wild-type parasites when estimated by Gas chromatography/mass spectrometry. Complementation of CYP5122A1 through episomal expression of protein by using pXG-GFP+2 vector partially rescued CYP5122A1 expression and restored ergosterol levels by 1.8 times. Phenotype reversal included restored growth pattern and lesser drug susceptibility. In summary, this study establishes CYP5122A1 as an important molecule linked to processes like cell growth, infection and ergosterol biosynthesis in Leishmania donovani.

Anemia is a frequent complication of many inflammatory disorders, including inflammatory bowel disease. Although the pathogenesis of this problem is m