Bidirectional regulation between AP-1 and SUMOylation pathway genes modulates inflammatory signaling during Salmonella infection.

Post-translational modifications (PTMs), such as SUMOylation, are known to modulate fundamental processes of a cell. Infectious agents such as Salmonella Typhimurium (STm), which causes gastroenteritis, utilize the PTM mechanism SUMOylation to hijack the host cell. STm suppresses host SUMO pathway genes UBC9 (also known as UBE2I) and PIAS1 to perturb SUMOylation for an efficient infection. In the present study, the regulation of SUMO pathway genes during STm infection was investigated. A direct binding of c-Fos (encoded by FOS), a component of activator protein-1 (AP-1), to promoters of both UBC9 and PIAS1 was observed. Experimental perturbation of c-Fos led to changes in the expression of both UBC9 and PIAS1. STm infection of fibroblasts with SUMOylation-deficient c-Fos (c-FOS-KOSUMO-def-FOS) resulted in uncontrolled activation of target genes, leading to massive immune activation. Infection of c-FOS-KOSUMO-def-FOS cells favored STm replication, indicating misdirected immune mechanisms. Finally, chromatin immunoprecipitation assays confirmed a context-dependent differential binding and release of AP-1 to and from target genes due to its phosphorylation and SUMOylation, respectively. Overall, our data point towards the existence of a bidirectional cross-talk between c-Fos and the SUMO pathway and highlight their importance in AP-1 function in STm infection and beyond. This article has an associated First Person interview with the first author of the paper.

A multi-omics analysis reveals that the lysine deacetylase ABHD14B influences glucose metabolism in mammals.

The sirtuins and histone deacetylases are the best characterized members of the lysine deacetylase (KDAC) enzyme family. Recently, we annotated the "orphan" enzyme ABHD14B (α/ß-hydrolase domain containing protein # 14B) as a novel KDAC and showed this enzyme's ability to transfer an acetyl-group from protein lysine residue(s) to coenzyme-A to yield acetyl-coenzyme-A, thereby, expanding the repertoire of this enzyme family. However, the role of ABHD14B in metabolic processes is not fully elucidated. Here, we investigated the role of this enzyme using mammalian cell knockdowns in a combined transcriptomics and metabolomics analysis. We found from these complementary experiments in vivo that the loss of ABHD14B results in significantly altered glucose metabolism, specifically the decreased flux of glucose through glycolysis and the citric acid cycle. Further, we show that depleting hepatic ABHD14B in mice also results in defective systemic glucose metabolism, particularly during fasting. Taken together, our findings illuminate the important metabolic functions that the KDAC ABHD14B plays in mammalian physiology and poses new questions regarding the role of this hitherto cryptic metabolism-regulating enzyme.

SUMOylation of Jun fine-tunes the Drosophila gut immune response.

Post-translational modification by the small ubiquitin-like modifier, SUMO can modulate the activity of its conjugated proteins in a plethora of cellular contexts. The effect of SUMO conjugation of proteins during an immune response is poorly understood in Drosophila. We have previously identified that the transcription factor Jra, the Drosophila Jun ortholog and a member of the AP-1 complex is one such SUMO target. Here, we find that Jra is a regulator of the Pseudomonas entomophila induced gut immune gene regulatory network, modulating the expression of a few thousand genes, as measured by quantitative RNA sequencing. Decrease in Jra in gut enterocytes is protective, suggesting that reduction of Jra signaling favors the host over the pathogen. In Jra, lysines 29 and 190 are SUMO conjugation targets, with the JraK29R+K190R double mutant being SUMO conjugation resistant (SCR). Interestingly, a JraSCR fly line, generated by CRISPR/Cas9 based genome editing, is more sensitive to infection, with adults showing a weakened host response and increased proliferation of Pseudomonas. Transcriptome analysis of the guts of JraSCR and JraWT flies suggests that lack of SUMOylation of Jra significantly changes core elements of the immune gene regulatory network, which include antimicrobial agents, secreted ligands, feedback regulators, and transcription factors. Mechanistically, SUMOylation attenuates Jra activity, with the TFs, forkhead, anterior open, activating transcription factor 3 and the master immune regulator Relish being important transcriptional targets. Our study implicates Jra as a major immune regulator, with dynamic SUMO conjugation/deconjugation of Jra modulating the kinetics of the gut immune response.

SUMO conjugation regulates immune signalling.

Post-translational modifications (PTMs) are critical drivers and attenuators for proteins that regulate immune signalling cascades in host defence. In this review, we explore functional roles for one such PTM, the small ubiquitin-like modifier (SUMO). Very few of the SUMO conjugation targets identified by proteomic studies have been validated in terms of their roles in host defence. Here, we compare and contrast potential SUMO substrate proteins in immune signalling for flies and mammals, with an emphasis on NFκB pathways. We discuss, using the few mechanistic studies that exist for validated targets, the effect of SUMO conjugation on signalling and also explore current molecular models that explain regulation by SUMO. We also discuss in detail roles of evolutionary conservation of mechanisms, SUMO interaction motifs, crosstalk of SUMO with other PTMs, emerging concepts such as group SUMOylation and finally, the potentially transforming roles for genome-editing technologies in studying the effect of PTMs.

SUMO-Enriched Proteome for Drosophila Innate Immune Response.

Small ubiquitin-like modifier (SUMO) modification modulates the expression of defense genes in Drosophila, activated by the Toll/nuclear factor-κB and immune-deficient/nuclear factor-κB signaling networks. We have, however, limited understanding of the SUMO-modulated regulation of the immune response and lack information on SUMO targets in the immune system. In this study, we measured the changes to the SUMO proteome in S2 cells in response to a lipopolysaccharide challenge and identified 1619 unique proteins in SUMO-enriched lysates. A confident set of 710 proteins represents the immune-induced SUMO proteome and analysis suggests that specific protein domains, cellular pathways, and protein complexes respond to immune stress. A small subset of the confident set was validated by in-bacto SUMOylation and shown to be bona-fide SUMO targets. These include components of immune signaling pathways such as Caspar, Jra, Kay, cdc42, p38b, 14-3-3ε, as well as cellular proteins with diverse functions, many being components of protein complexes, such as prosß4, Rps10b, SmD3, Tango7, and Aats-arg. Caspar, a human FAF1 ortholog that negatively regulates immune-deficient signaling, is SUMOylated at K551 and responds to treatment with lipopolysaccharide in cultured cells. Our study is one of the first to describe SUMO proteome for the Drosophila immune response. Our data and analysis provide a global framework for the understanding of SUMO modification in the host response to pathogens.

An immunomodulatory role for the Mycobacterium tuberculosis region of difference 1 locus proteins PE35 (Rv3872) and PPE68 (Rv3873).

The pathogenesis of Mycobacterium tuberculosis involves the coordinate action of multiple bacillary components that modulate host immune responses to ensure its survival. One such group of factors is the multigenic PE_PPE protein family, several members of which have been implicated in host immune evasion. Here we investigate the function of the PE-PPE gene pair PE35 (Rv3872)-PPE68 (Rv3873), located in the region of difference 1, encoding a specialized mycobacterial secretion system that is deleted in all vaccine strains of Mycobacterium bovis BCG. We report that this gene pair is co-operonic in M. tuberculosis, and demonstrate that its gene products interact with each other. Stimulation of THP-1 macrophages with recombinant PE35 and PPE68, singly or in combination, led to a dose-dependent increase in levels of the anti-inflammatory cytokine interleukin (IL)-10 and the chemokine monocyte chemoattractant protein-1, and caused a reciprocal decrease in levels of the proinflammatory cytokine IL-12. PE35/PPE68-stimulated production of IL-10 and monocyte chemoattractant protein-1 was observed to be dependent on toll-like receptor 2, as receptor blockade caused a significant reduction in their levels. Pharmacological inhibition indicated that this induction involved activation of the mitogen-activated protein kinase signalling axis. In a transwell migration assay, culture supernatants from PE35/PPE68-treated THP-1 cells were observed to stimulate the migration of monocytes. Our findings suggest that the PE35-PPE68 gene pair plays an important immunomodulatory role in regulating the pathophysiology of M. tuberculosis. STRUCTURED DIGITAL ABSTRACT: TLR2 physically interacts with PPE68 by anti bait coimmunoprecipitation (View interaction) PE35 binds to PPE68 by pull down (View interaction) PE35 physically interacts with PPE68 by anti tag coimmunoprecipitation (View interaction) TLR2 physically interacts with PE35 by anti bait coimmunoprecipitation (View interaction) PPE68 and PE35 physically interact by dihydrofolate reductase reconstruction (View interaction).

Evaluation of LAMP assay using phenotypic tests and conventional PCR for detection of blaNDM-1 and blaKPC genes among carbapenem-resistant clinical Gram-negative isolates.

Carbapenem-resistant pathogens cause infections associated with significant morbidity and mortality. This study evaluates the use of the loop-mediated isothermal amplification (LAMP) assay for rapid and cost-effective detection of bla(NDM-1) and bla(KPC) genes among carbapenem-resistant Gram-negative bacteria in comparison with conventional PCR and existing phenotypic methods. A total of 60 carbapenem-resistant clinical isolates [Escherichia coli (15), Klebsiella pneumoniae (22), Acinetobacter baumannii (23)] were screened for the presence of carbapenemases (bla(KPC) and bla(NDM-1)) using phenotypic methods such as the modified Hodge test (MHT) and combined disc test (CDT) and molecular methods such as conventional PCR and LAMP assay. In all, 47/60 isolates (78.3%) were MHT positive while 48 isolates were positive by CDT [46.6% positive with EDTA, 30% with 3' aminophenylboronic acid (APB) plus EDTA and 1.6% with APB alone]. Isolates showing CDT positivity with EDTA or APB contained bla(NDM-1) and bla(KPC) genes, respectively. bla(NDM-1) was present as a lone gene in 28 isolates (46.7%) and present together with the bla(KPC) gene in 19 isolates (31.7%). Only one E. coli isolate had a lone bla(KPC) gene. The LAMP assay detected either or both bla(NDM-1) and bla(KPC) genes in four isolates that were missed by conventional PCR. Neither gene could be detected in 12 (20%) isolates. The LAMP assay has greater sensitivity, specificity and rapidity compared to the phenotypic methods and PCR for the detection of bla(NDM-1) and bla(KPC). With a turnaround time of only 2-3 h, the LAMP assay can be considered a point-of-care assay.