Atri Ta

Physiological TLR5 expression in the intestine is regulated by differential DNA binding of Sp1/Sp3 through simultaneous Sp1 dephosphorylation and Sp3 phosphorylation by two different PKC isoforms

Toll-like receptor 5 (TLR5) expression in the intestinal epithelial cells (IECs) is critical to maintain health, as underscored by multiple intestinal and extra-intestinal diseases in mice genetically engineered for IEC-specific TLR5 knockout. A gradient of expression exists in the colonic epithelial cells from the cecum to the distal colon. Intriguingly, an identical gradient for the dietary metabolite, butyrate also exists in the luminal contents. However, both being critical for intestinal homeostasis and immune response, no studies examined the role of butyrate in the regulation of TLR5 expression. We showed that butyrate transcriptionally upregulates TLR5 in the IECs and augments flagellin-induced immune responses. Both basal and butyrate-induced transcription is regulated by differential binding of Sp-family transcription factors to the GC-box sequences over the TLR5 promoter. Butyrate activates two different protein kinase C isoforms to dephosphorylate/acetylate Sp1 by serine/threonine phosphatases and phosphorylate Sp3 by ERK-MAPK, respectively. This resulted in Sp1 displacement from the promoter and binding of Sp3 to it, leading to p300 recruitment and histone acetylation, activating transcription. This is the first study addressing the mechanisms of physiological TLR5 expression in the intestine. Additionally, a novel insight is gained into Sp1/Sp3-mediated gene regulation that may apply to other genes.

Caveolin-1 is transcribed from a hypermethylated promoter to mediate colonocyte differentiation and apoptosis.

Caveolin-1(CAV1) is a tyrosine-phosphorylated scaffold protein of caveolae with multiple interacting partners. It functions both as an oncogene and a tumour suppressor depending upon the cellular contexts. In the early stage of colorectal cancers (CRC), CAV1 suppresses tumour progression, while over-expression of CAV1 reduced the tumourigenicity of colon carcinoma cells. In contrast, elevated level of CAV1 was reported in stage III CRC. To address this ambiguity, we studied the functional role and the regulation of CAV1 expression during colonocyte differentiation and apoptosis. Here, we reported for the first time that CAV1 expression was increased during colonocyte differentiation and mediated butyrate-induced differentiation and apoptosis of HT29 cells. CAV1 expression was silenced by promoter hypermethylation in HT-29 cells and reactivated by prolonged histone hyperacetylation of the promoter upon treatment of the cells with butyrate. However, the methylation status was unaltered by butyrate. We for the first time showed that HDAC inhibitor-mediated transactivation of CAV1 was regulated by methylation density of the promoter. Our study further explains the underlying mechanisms of the anti-cancer property of butyrate in CRC.

Ribavirin suppresses bacterial virulence by targeting LysR-type transcriptional regulators.

Targeting bacterial virulence mechanisms without compromising bacterial growth is a promising strategy to prevent drug resistance. LysR-type transcriptional regulators (LTTRs) possess structural conservation across bacterial species and regulate virulence in numerous pathogens, making them attractive targets for antimicrobial agents. We targeted AphB, a Vibrio cholerae LTTR, which regulates the expression of genes encoding cholera toxin and toxin-co-regulated pilus for inhibitor designing. Since AphB ligand is unknown, we followed a molecular fragment-based approach for ligand designing using FDA-approved drugs and subsequent screen to identify molecules that exhibited high-affinity binding to AphB ligand-binding pocket. Among the identified compounds, ribavirin, an anti-viral drug, antagonized AphB functions. Ribavirin perturbed Vibrio cholerae pathogenesis in animal models. The inhibitory effects of the drug was limited to the bacteria expressing wild type AphB, but not its constitutively active mutant (AphBN100E), which represents the ligand-bound state, suggesting that ribavirin binds to the active site of AphB to exert its inhibitory role and there exists no AphB-independent mechanism of its action. Similarly, ribavirin suppressed the functions of Salmonella Typhi LTTR Hrg, indicating its broad spectrum efficacy. Moreover, ribavirin did not affect the bacterial viability in culture. This study cites an example of drug repurposing for anti-infective therapy.

An AIL family protein promotes type three secretion system-1-independent invasion and pathogenesis of Salmonella enterica serovar Typhi

Adhesion and invasion of Intestinal Epithelial Cells (IECs) are critical for the pathogenesis of Salmonella Typhi, the aetiological agent of human typhoid fever. While type three secretion system‐1 (T3SS‐1) is a major invasion apparatus of Salmonella, independent invasion mechanisms were described for non‐typhoidal Salmonellae. Here, we show that T2942, an AIL‐like protein of S. Typhi Ty2 strain, is required for adhesion and invasion of cultured IECs. That invasion was T3SS‐1 independent was proved by ectopic expression of T2942 in the non‐invasive E. coli BL21 and double‐mutant Ty2 (Ty2Δt2942ΔinvG) strains. Laminin and fibronectin were identified as the host‐binding partners of T2942 with higher affinity for laminin. Standalone function of T2942 was confirmed by cell adhesion of the recombinant protein, while the protein or anti‐T2942 antiserum blocked adhesion/invasion of S. Typhi, indicating specificity. A 20‐amino acid extracellular loop was required for invasion, while several loop regions of T2942 contributed to adhesion. Further, T2942 cooperates with laminin‐binding T2544 for adhesion and T3SS‐1 for invasion. Finally, T2942 was required and synergistically worked with T3SS‐1 for pathogenesis of S. Typhi in mice. Considering wide distribution of T2942 among clinical strains, the protein or the 20‐mer peptide may be suitable for vaccine development.

Suppression of Spleen Tyrosine Kinase (Syk) by Histone Deacetylation Promotes, Whereas BAY61-3606, a Synthetic Syk Inhibitor Abrogates Colonocyte Apoptosis by ERK Activation

Spleen tyrosine kinase (Syk), a non‐receptor tyrosine kinase, regulates tumor progression, either negatively or positively, depending on the tissue lineage. Information about the role of Syk in colorectal cancers (CRC) is limited, and conflicting reports have been published. We studied Syk expression and its role in differentiation and apoptosis of the colonocytes. Here, we reported for the first time that expression of two transcript variants of Syk is suppressed in colonocytes during butyrate‐induced differentiation, which mediates apoptosis of HT‐29 cells. Despite being a known HDAC inhibitor, butyrate deacetylates histone3/4 around the transcription start site (TSS) of Syk. Histone deacetylation precludes the binding of RNA Polymerase II to the promoter and inhibits transcription. Since butyrate is a colonic metabolite derived from undigested fibers, our study offers a plausible explanation of the underlying mechanisms of the protective role of butyrate as well as the dietary fibers against CRC through the regulation of Syk. We also report that combined use of butyrate and highly specific Syk inhibitor BAY61‐3606 does not enhance differentiation and apoptosis of colonocytes. Instead, BAY completely abolishes butyrate‐induced differentiation and apoptosis in a Syk‐ and ERK1/2‐dependent manner. While butyrate dephosphorylates ERK1/2 in HT‐29 cells, BAY re‐phosphorylates it, leading to its activation. This study describes a novel mechanism of butyrate action in CRC and explores the role of Syk in butyrate‐induced differentiation and apoptosis. In addition, our study highlights those commercial small molecule inhibitors, although attractive drug candidates should be used with concern because of their frequent off‐target effects. J. Cell. Biochem. 118: 191–203, 2017.

An Inducible and Secreted Eukaryote-Like Serine/Threonine Kinase of Salmonella enterica Serovar Typhi Promotes Intracellular Survival and Pathogenesis

ABSTRACT Eukaryote-like serine/threonine kinases (eSTKs) constitute an important family of bacterial virulence factors. Genome analysis had predicted putative eSTKs in Salmonella enterica serovar Typhi, although their functional characterization and the elucidation of their role in pathogenesis are still awaited. We show here that the primary sequence and secondary structure of the t4519 locus of Salmonella Typhi Ty2 have all the signatures of eukaryotic superfamily kinases. t4519 encodes a ∼39-kDa protein (T4519), which shows serine/threonine kinase activities in vitro. Recombinant T4519 (rT4519) is autophosphorylated and phosphorylates the universal substrate myelin basic protein. Infection of macrophages results in decreased viability of the mutant (Ty2Δt4519) strain, which is reversed by gene complementation. Moreover, reactive oxygen species produced by the macrophages signal to the bacteria to induce T4519, which is translocated to the host cell cytoplasm. That T4519 may target a host substrate(s) is further supported by the activation of host cellular signaling pathways and the induction of cytokines/chemokines. Finally, the role of T4519 in the pathogenesis of Salmonella Typhi is underscored by the significantly decreased mortality of mice infected with the Ty2Δt4519 strain and the fact that the competitive index of this strain for causing systemic infection is 0.25% that of the wild-type strain. This study characterizes the first eSTK of Salmonella Typhi and demonstrates its role in promoting phagosomal survival of the bacteria within macrophages, which is a key determinant of pathogenesis. This, to the best of our knowledge, is the first study to describe the essential role of eSTKs in the in vivo pathogenesis of Salmonella spp.

Retinoic acid pre-treatment down regulates V. cholerae outer membrane vesicles induced acute inflammation and enhances mucosal immunity

Bacterial outer membrane vesicles have been extensively investigated and considered as a next generation vaccine. Recently, we have demonstrated that the cholera pentavalent outer membrane vesicles (CPMVs) immunogen induced adaptive immunity and had a strong protective efficacy against the circulating V. cholerae strains in a mouse model. In this present study, we are mainly focusing on reducing outer membrane vesicle (OMV) -mediated toxicity without altering its antigenic property. Therefore, we have selected All-trans Retinoic Acid (ATRA), active metabolites of vitamin A, which have both anti-inflammatory and mucosal adjuvant properties. Pre-treatment of ATRA significantly reduced CPMVs induced TLR2 mediated pro-inflammatory responses in vitro and in vivo. Furthermore, we also found ATRA pre-treatment significantly induced mucosal immune response and protective efficacy after two doses of oral immunization with CPMVs (75µg). This study can help to reduce OMV based vaccine toxicity and induce better protective immunity where children and men suffered from malnutrition mainly in developing countries.

Antimicrobial Peptides in Cervicovaginal Lavage of Women Married to HIV Sero-Reactive Men are not Associated with Resistance to HIV butModulate Mucosal Pro-Inflammatory Response

Objectives: It is critical to understand the HIV transmission dynamics in women, who are married to HIV seropositive men and either go into HIV concordant relationship (WCR) or remain HIV discordant (WDR) over a period of time. Antimicrobial peptides (AMPs) are known to possess anti-HIV activities and may offer protection against acquisition of HIV. We investigated if AMP expression is associated with HIV sero-negative status of the WDR population, and how the co-regulated innate immune responses at the genital mucosa differ between WDR and WCR individuals in eastern-India. Methods: Levels of human beta-defensins, human neutrophil peptides 1-3 and IL-8 were measured by ELISA in the cervicovaginal lavarge (CVL) collected from WCR and WDR individuals. Moreover, we studied the activation of TLR signalling pathways and TNF-alpha induction in THP-1 cells by the microbial components of CVLs and their modulation by pre-treatment of the cells with recombinant AMPs. Results: Antimicrobial peptide expression in the CVLs showed no significant association with resistance to HIV infection. We observed a shift toward significant co-regulated expression of hBD2 and hBD3 (r=0.52, p<0.0001) and hBD3 and IL-8 (r=-0.26, p=0.03) in the WCR group compared with the WDR (r=0.30, p=0.13 for hBD2 and hBD3; r=-0.15, p=0.44 for hBD3 and IL-8) population. We also found that hBD3 inhibited the pro-inflammatory immune responses induced by CVL, perhaps through the inhibition of TLR4 and TLR1/2 signalling. This was in agreement with the negative correlation between hBD3 and IL-8 expression in the WCR population. Conclusions: Previous studies identified different microbial composition of CVLs between women living with and without HIV. This might result in a difference in the co-regulated expression of AMPs and inflammatory mediators between the WCR and WDR populations. hBD3 may be critical to control inflammation at the cervico-vaginal mucosa, indicating its novel therapeutic potential in HIV infection.

Double-stranded RNA induces cathelicidin expression in the intestinal epithelial cells through phosphatidylinositol 3-kinase-protein kinase Cζ-Sp1 pathway and ameliorates shigellosis in mice

Cathelicidin antimicrobial peptide is a key component of the host innate immune system. It is constitutively expressed by the intestinal epithelial cells, but induced at further higher levels by different host-derived and microbial stimuli, including the ligands for Toll-like receptors (TLRs). While the underlying mechanisms of cathelicidin expression remain incompletely understood, altered expression may be associated with gastro-intestinal infections and inflammatory diseases. We demonstrate here that viral double-stranded RNA and its synthetic analog poly(I:C) are potent and tissue-specific inducers of cathelicidin mRNA and protein expression in the mouse as well as human intestinal epithelial cells. Reporter assays showed that poly(I:C) transcriptionally regulates murine cathelicidin-related antimicrobial peptide (mCRAMP) by recruiting Sp1 transcription factor to the GC-box cis-regulatory element at -71bp of the mCRAMP putative promoter. Sp1 recruitment to the endogenous mCRAMP promoter was confirmed by chromatin immunoprecipitation (ChIP) assays. Immunoblotting, qPCR, ChIP and siRNA-mediated gene knockdown studies revealed that the activation of phosphatidylinositol 3-kinase/protein kinase Cζ pathways in poly(I:C)-stimulated cells underlies Sp1 phosphorylation and recruitment to the mCRAMP promoter, leading to enhanced transcription. We further showed that intra-rectal poly(I:C) administration in mice reduces intestinal bacterial load and mucosal inflammation following Shigella flexneri 2a infection by inducing mCRAMP expression in the colonic epithelial cells. This study reports novel regulatory mechanisms of cathelicidin expression that may be targeted to treat gastro-intestinal infections.

Polo-like kinase 1 expression is suppressed by CCAAT/enhancer-binding protein α to mediate colon carcinoma cell differentiation and apoptosis

BACKGROUND Human polo-like kinase 1 (PLK1), a highly conserved serine/threonine kinase is a key player in several essential cell-cycle events. PLK1 is considered an oncogene and its overexpression often correlates with poor prognosis of cancers, including colorectal cancer (CRC). However, regulation of PLK1 expression in colorectal cells was never studied earlier and it is currently unknown if PLK1 regulates differentiation and apoptosis of CRC. METHODS PLK1 expression was analyzed by real-time PCR and western blotting. Transcriptional regulation was studied by reporter assay, gene knock-down, EMSA and ChIP. RESULTS PLK1 expression was down-regulated during butyrate-induced differentiation of HT-29 and other CRC cells. Also, PLK1 down-regulation mediated the role of butyrate in CRC differentiation and apoptosis. We report here a novel transcriptional regulation of PLK1 by butyrate. Transcription factors CCAAT/enhancer-binding protein α (C/EBPα) and Oct-1 share an overlapping binding site over the PLK1 promoter. Elevated levels of C/EBPα by butyrate treatment of CRC cells competed out the activator protein Oct-1 from binding to the PLK1 promoter and sequestered it. Binding of C/EBPα was associated with increased deacetylation near the transcription start site (TSS) of the PLK1 promoter, which abrogated transcription through reduced recruitment of RNA polymerase II. We also found a synergistic role between the synthetic PLK1-inhibitor SBE13 and butyrate on the apoptosis of CRC cells. CONCLUSION This study offered a novel p53-independent regulation of PLK1 during CRC differentiation and apoptosis. GENERAL SIGNIFICANCE Down-regulation of PLK1 is one of the mechanisms underlying the anti-cancer role of dietary fibre-derived butyrate in CRC.