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 abstractTLR2 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)

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.

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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 39 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.

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