Amuc_1100 pretreatment alleviates acute pancreatitis in a mouse model through regulating gut microbiota and inhibiting inflammatory infiltration.

Amuc_1100 is a membrane protein from Akkermansia muciniphila, which has been found to play a role in host immunological homeostasis in the gastrointestinal tract by activating TLR2 and TLR4. In this study we investigated the effects and underlying mechanisms of Amuc_1100 on acute pancreatitis (AP) induced in mice by intraperitoneal injection of caerulein and lipopolysaccharide (LPS). The mice were treated with the protein Amuc_1100 (3 µg, i.g.) for 20 days before caerulein injection. Cecal contents of the mice were collected for 16S rRNA sequencing. We found that pretreatment with Amuc_1100 significantly alleviated AP-associated pancreatic injury, reduced serum amylase and lipase. Amuc_1100 pretreatment significantly inhibited the expression of proinflammatory cytokines (TNF-α, IL-1ß, IFN-γ and IL-6) in spleen and pancreas through inhibiting NF-κB signaling pathway. Moreover, Amuc_1100 pretreatment significantly decreased the inflammatory infiltration, accompanied by the reduction of Ly6C+ macrophages and neutrophils in the spleen of AP mice. Gut microbiome analysis showed that the abundance of Bacteroidetes, Proteobacteria, Desulfobacterota and Campilobacterota was decreased, while the proportion of Firmicutes and Actinobacteriota was increased in AP mice pretreated with Amuc_1100. We further demonstrated that Amuc_1100 pretreatment restored the enrichment of tryptophan metabolism, which was mediated by intestinal flora. These results provide new evidence that Amuc_1100 lessens the severity of AP through its anti-inflammatory properties with a reduction of macrophages and neutrophil infiltration, as well as its regulation of the composition of intestinal flora and tryptophan metabolism.

Identification of a Potential Antimycobacterial Drug Sensitizer Targeting a Flavin-Independent Methylenetetrahydrofolate Reductase.

The increasing antibiotic resistance of Mycobacterium tuberculosis and pathogenic nontuberculosis mycobacteria highlights the urgent need for new prevention and treatment strategies. Recently, the cocrystal structure of a Mycobacterium smegmatis flavin-independent 5,10-methylenetetrahydrofolate reductase (MsmMTHFR) that binds with a reduced nicotinamide adenine dinucleotide (NADH) has been well-determined, providing a structural basis for the screening of antimycobacterial leads targeting MsmMTHFR, a new enzyme involved in tetrahydrofolic acid (THF) biosynthesis. In this study, we identified compound AB131 as a promising candidate that fits well into the NADH binding pocket of MsmMTHFR through virtual screening. We discovered that AB131 and its derivatives (13 and 14) can sensitize the antimycobacterial activity of the antitubercular drug para-aminosalicyclic acid (PAS) by 2-5-fold against various species of mycobacteria. Although the compounds themselves do not exhibit any antimycobacterial activity, the high binding affinity of AB131 with MsmMTHFR or Rv2172c was evaluated by microscale thermophoresis analysis. Additionally, we predicted and validated the key residues (V115, V117, P118, and R163) of MsmMTHFR that are involved in the interaction with AB131 by using molecular docking and mutagenesis analysis. These findings offer a potential exploitable target for developing potent and specific antimycobacterial drug sensitizers.

In vitro antifungal and antibiofilm activities of auranofin against itraconazole-resistant Aspergillus fumigatus.

BACKGROUND: Infections caused by azole-resistant Aspergillus are a rising public health threat with high mortality rates, high treatment costs and limited available antifungals, indicating an urgent need for new antifungals or strategies. Our aim was to investigate antifungal and antibiofilm activities of auranofin, an FDA-approved anti-antirheumatic drug. METHODS: Fungal susceptibility testing for auranofin was carried out by the broth-based microdilution methods. Cell viability treated by auranofin was tested by resazurin dye testing. The synergistic effect of auranofin and antifungal drugs was evaluated using checkboard assay. The inhibitory of biofilms were measured by crystal violet staining. Gene expression level analysis and enzyme activity was investigated with qRT-PCR analysis and DTNB assay. The key amino acid residues in the binding of auranofin with A. fumigatus thioredoxin reductase (AfTrxR) were indicated by structural analyses, site-directed mutagenesis, and microscale thermophoresis (MST) assays. RESULTS: Auranofin has fungicidal activity and in vitro antifungal spectrum including Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus, Aspergillus niger, even itraconazole (ITC)-resistant A. fumigatus. Additionally, it has antibiofilm activities against ITC-resistant A. fumigatus by reducing the expression level of SomA and MedA. Moreover, we discovered a synergistic effect of auranofin and ITC or amphotericin B against ITC-resistant A. fumigatus. Auranofin downregulated the gene transcription of AfTrxR, and strongly inhibited the enzyme activity of AfTrxR through interacting with residues C145 and C148. CONCLUSIONS: Auranofin has fungicidal and antibiofilm activities in Aspergillus spp. and is also a potentiator of ITC or amphotericin B in vitro.

Investigations into the antibacterial effects and potential mechanism of gambogic acid and neogambogic acid.

The growing threat of antibiotic-resistant bacterial infections to public health necessitates the development of novel antibacterial agents. Inhibiting bacterial cell wall synthesis has remained a key focus for antibiotic development. Our search for inhibitors of undecaprenyl diphosphate synthase (UPPS), an essential enzyme required for bacterial cell wall formation, revealed that two primary components of gamboge, gambogic acid (GA) and neogambogic acid (NGA), significantly inhibited the activity of Enterococcus faecalis UPPS (EfaUPPS) with the half maximal inhibitory concentrations (IC50) of 3.08 µM and 3.07 µM, respectively. In the in vitro antibacterial assay, both GA and NGA also exhibited inhibitory activities against E. faecalis with the minimal inhibitory concentrations (MICs) of 2 µg/mL. Using microscale thermophoresis, molecular docking, and enzymatic assays, we further confirmed that GA and NGA occupy the substrate binding pocket of EfaUPPS with micro-molar binding affinity, preventing the natural substrates farnesyl diphosphate (FPP) from entering. Mutagenesis analysis revealed that L91 and L146 are two key residues in the binding between GA/NGA and UPPS. Furthermore, we also demonstrated that GA and NGA can improve E. faecalis-induced undesirable inflammation in a mouse infection model. Taken together, our findings provide a basis for structural optimization of GA/NGA to develop improved antibiotic leads and enhance treatment success rates in clinical practice.

Structural basis of three different transcription activation strategies adopted by a single regulator SoxS.

Transcription activation is established through extensive protein-protein and protein-DNA interactions that allow an activator to engage and remodel RNA polymerase. SoxS, a global transcription activator, diversely regulates subsets of stress response genes with different promoters, but the detailed SoxS-dependent transcription initiation mechanisms remain obscure. Here, we report cryo-EM structures of three SoxS-dependent transcription activation complexes (SoxS-TACI, SoxS-TACII and SoxS-TACIII) comprising of Escherichia coli RNA polymerase (RNAP), SoxS protein and three representative classes of SoxS-regulated promoters. The structures reveal that SoxS monomer orchestrates transcription initiation through specific interactions with the promoter DNA and different conserved domains of RNAP. In particular, SoxS is positioned in the opposite orientation in SoxS-TACIII to that in SoxS-TACI and SoxS-TACII, unveiling a novel mode of transcription activation. Strikingly, two universally conserved C-terminal domains of alpha subunit (αCTD) of RNAP associate with each other, bridging SoxS and region 4 of σ70. We show that SoxS interacts with RNAP directly and independently from DNA, remodeling the enzyme to activate transcription from cognate SoxS promoters while repressing transcription from UP-element containing promoters. Our data provide a comprehensive summary of SoxS-dependent promoter architectures and offer new insights into the αCTD contribution to transcription control in bacteria.

Structural basis of transcription activation by Rob, a pleiotropic AraC/XylS family regulator.

Rob, which serves as a paradigm of the large AraC/XylS family transcription activators, regulates diverse subsets of genes involved in multidrug resistance and stress response. However, the underlying mechanism of how it engages bacterial RNA polymerase and promoter DNA to finely respond to environmental stimuli is still elusive. Here, we present two cryo-EM structures of Rob-dependent transcription activation complex (Rob-TAC) comprising of Escherichia coli RNA polymerase (RNAP), Rob-regulated promoter and Rob in alternative conformations. The structures show that a single Rob engages RNAP by interacting with RNAP αCTD and σ70R4, revealing their generally important regulatory roles. Notably, by occluding σ70R4 from binding to -35 element, Rob specifically binds to the conserved Rob binding box through its consensus HTH motifs, and retains DNA bending by aid of the accessory acidic loop. More strikingly, our ligand docking and biochemical analysis demonstrate that the large Rob C-terminal domain (Rob CTD) shares great structural similarity with the global Gyrl-like domains in effector binding and allosteric regulation, and coordinately promotes formation of competent Rob-TAC. Altogether, our structural and biochemical data highlight the detailed molecular mechanism of Rob-dependent transcription activation, and provide favorable evidences for understanding the physiological roles of the other AraC/XylS-family transcription factors.

Structural basis of transcription activation by the global regulator Spx.

Spx is a global transcriptional regulator in Gram-positive bacteria and has been inferred to efficiently activate transcription upon oxidative stress by engaging RNA polymerase (RNAP) and promoter DNA. However, the precise mechanism by which it interacts with RNAP and promoter DNA to initiate transcription remains obscure. Here, we report the cryo-EM structure of an intact Spx-dependent transcription activation complex (Spx-TAC) from Bacillus subtilis at 4.2 Å resolution. The structure traps Spx in an active conformation and defines key interactions accounting for Spx-dependent transcription activation. Strikingly, an oxidized Spx monomer engages RNAP by simultaneously interacting with the C-terminal domain of RNAP alpha subunit (αCTD) and σA. The interface between Spx and αCTD is distinct from those previously reported activators, indicating αCTD as a multiple target for the interaction between RNAP and various transcription activators. Notably, Spx specifically wraps the conserved -44 element of promoter DNA, thereby stabilizing Spx-TAC. Besides, Spx interacts extensively with σA through three different interfaces and promotes Spx-dependent transcription activation. Together, our structural and biochemical results provide a novel mechanistic framework for the regulation of bacterial transcription activation and shed new light on the physiological roles of the global Spx-family transcription factors.