SummarySecretins are a family of large bacterial outer membrane channels that serve as exit ports for folded proteins, filamentous phage and surface structures. Despite the large size of their substrates, secretins do not compromise the barrier function of the outer membrane, implying a gating mechanism. The region in the primary structure that forms the putative gate has not previously been determined for any secretin. To identify residues involved in gating the pIV secretin of filamentous bacteriophage f1, we used random mutagenesis of the gene followed by positive selection for mutants with compromised barrier function ('leaky' mutants). We identified mutations in 34 residues, 30 of which were clustered into two regions located in the centre of the conserved C-terminal secretin family domain: GATE1 (that spanned 39 residues) and GATE2 (that spanned 14 residues). An internal deletion constructed in the GATE2 region resulted in a severely leaky phenotype. Three of the four remaining mutations are located in the region that encodes the N-terminal, periplasmic portion of pIV and could be involved in triggering gate opening. Two missense mutations in the 24-residue region that separates GATE1 and GATE2 were also constructed. These mutant proteins were unstable, defective in multimerization and non-functional.

Mucosal-associated invariant T (MAIT) cells are abundant innate-like T lymphocytes in mucosal tissues and recognize a variety of riboflavin-related metabolites produced by the microbial flora. Relevant issues are whether MAIT cells are heterogeneous in the colon, and whether the local environment influences microbial metabolism thereby shaping MAIT cell phenotypes and responses. We found discrete MAIT cell populations in human colon, characterized by the diverse expression of transcription factors, cytokines and surface markers, indicative of activated and precisely controlled lymphocyte populations. Similar phenotypes were rare among circulating MAIT cells and appeared when circulating MAIT cells were stimulated with the synthetic antigens 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil, and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil. Furthermore, bacteria grown in colon-resembling conditions with low oxygen tension and harvested at stationary growth phase, potently activated human MAIT cells. The increased activation correlated with accumulation of the above antigenic metabolites as indicated by mass spectrometry. Thus, the colon environment contributes to mucosal immunity by directly affecting bacterial metabolism, and indirectly controlling the stimulation and differentiation of MAIT cells.

Non-polymorphic MHC class I-related molecule MR1 presents antigenic bacterial metabolites to mucosal-associated invariant T (MAIT) cells and self-antigens to MR1-restricted T (MR1T) cells. Both MR1-restricted T cell populations are readily identified in healthy individuals, with MAIT cells accounting for 1-10% of circulating T cells, while MR1T cells have frequencies comparable to peptide-specific T cells (<0.1%). Self-reactive MR1T cells display a heterogeneous phenotype, and are capable of releasing both T H1 and T H2 cytokines, supporting not only activation of inflammation but also contributing to its regulation. Importantly, MR1T cells recognize and kill a diverse range of MR1-expressing tumor cells. On the other hand, evidence suggests MAIT cells augment cancer growth and metastases. This review addresses the potential role of MR1-restricted T cells in controlling tumor cells, facilitating their elimination and regulating cancer immunity. We also discuss therapeutic opportunities surrounding MR1-restricted T cells in cancer.

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The phytohormone gibberellin and the DELLA proteins act together to control key aspects of plant development. Gibberellin induces degradation of DELLA proteins by recruitment of an F-box protein using a molecular switch: a gibberellin-bound nuclear receptor interacts with the N-terminal domain of DELLA proteins, and this event primes the DELLA C-terminal domain for interaction with the F-box protein. However, the mechanism of signalling between the N- and C-terminal domains of DELLA proteins is unresolved. In the present study, we used in vivo and in vitro approaches to characterize di- and tri-partite interactions of the DELLA protein RGL1 (REPRESSOR OF GA1-3-LIKE 1) of Arabidopsis thaliana with the gibberellin receptor GID1A (GIBBERELLIC ACID-INSENSITIVE DWARF-1A) and the F-box protein SLY1 (SLEEPY1). Deuterium-exchange MS unequivocally showed that the entire N-terminal domain of RGL1 is disordered prior to interaction with the GID1A; furthermore, association/dissociation kinetics, determined by surface plasmon resonance, predicts a two-state conformational change of the RGL1 N-terminal domain upon interaction with GID1A. Additionally, competition assays with monoclonal antibodies revealed that contacts mediated by the short helix Asp-Glu-Leu-Leu of the hallmark DELLA motif are not essential for the GID1A–RGL1 N-terminal domain interaction. Finally, yeast two- and three-hybrid experiments determined that unabated communication between N- and C-terminal domains of RGL1 is required for recruitment of the F-box protein SLY1.

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Background: Antimicrobial combinations have been proven as a promising approach in the confrontation with multi-drug resistant bacterial pathogens. In the present study, we identify and characterize a synergistic interaction of broad-spectrum nitroreductase-activated prodrugs 5-nitrofurans, with a secondary bile salt, Sodium Deoxycholate (DOC) in growth inhibition and killing of enterobacteria. Results: Using checkerboard assay, we show that combination of nitrofuran furazolidone (FZ) and DOC generates a profound synergistic effect on growth inhibition in several enterobacterial species including Escherichia coli, Salmonella enterica, Citrobacter gillenii and Klebsiella pneumoniae. The Fractional Inhibitory Concentration Index (FICI) for DOC-FZ synergy ranges from 0.125 to 0.35 that remains unchanged in an ampicillin-resistant E. coli strain containing a β-lactamase-producing plasmid. Findings from the time-kill assay further highlight the synergy with respect to bacterial killing in E. coli and Salmonella. We further characterize the mechanism of synergy in E. coli K12, showing that disruption of the tolC or acrA genes that encode components of multidrug efflux pumps causes, respectively, a complete or partial loss, of the DOC-FZ synergy. This finding indicates the key role of TolC-associated efflux pumps in the DOC-FZ synergy. Overexpression of Nitric Oxide-detoxifying enzyme Hmp results in a threefold increase in FICI for DOC-FZ interaction, suggesting a role of nitric oxide in the synergy. We further demonstrate that DOC-FZ synergy is largely independent of NfsA and NfsB, the two major activation enzymes of the nitrofuran prodrugs. Conclusions: This study is to our knowledge the first report of nitrofuran-deoxycholate synergy against Gramnegative bacteria, offering potential applications in antimicrobial therapeutics. The mechanism of DOC-FZ synergy involves FZ-mediated inhibition of TolC-associated efflux pumps that normally remove DOC from bacterial cells. One possible route contributing to that effect is via FZ-mediated nitric oxide production.

MR1T lymphocytes are a recently identified population of T cells that recognize unknown self-antigens presented by the non-polymorphic MHC-I-related molecule, MR1. MR1T cells can kill tumor cells and modulate the functions of other immune cells with promising therapeutic applications. By integrating genetic, pharmacological and biochemical approaches we identified carbonyl stress and alterations of nucleobase metabolism in tumor target cells that promote recognition by MR1 T cells. We dissected these pathways and found that nucleobase adduct-containing metabolites are self-antigens stimulating MR1T cells. Several nucleobase adducts are presented by MR1 molecules and stimulate individual MR1T cells. Our data suggest that MR1T cells are surveyor of cellular metabolic alterations occurring in conditions of metabolic stress, such as cancer, and lay the groundwork for the development of novel HLA-unrestricted T cell-based therapies.