Host-derived peptide signals regulate Pseudomonas aeruginosa virulence stress via the ParRS and CprRS two-component systems.

Pseudomonas aeruginosa, a versatile bacterium, has dual significance because of its beneficial roles in environmental soil processes and its detrimental effects as a nosocomial pathogen that causes clinical infections. Understanding adaptability to environmental stress is essential. This investigation delves into the complex interplay of two-component system (TCS), specifically ParRS and CprRS, as P. aeruginosa interprets host signals and navigates stress challenges. In this study, through phenotypic and proteomic analyses, the nuanced contributions of ParRS and CprRS to the pathogenesis and resilience mechanisms were elucidated. Furthermore, the indispensable roles of the ParS and CprS extracellular sensor domains in orchestrating signal perception remain unknown. Structural revelations imply a remarkable convergence of TCS sensors in interacting with host peptides, suggesting evolutionary strategies for bacterial adaptation. This pioneering work not only established links between cationic antimicrobial peptide (CAMP) resistance-associated TCSs and virulence modulation in nosocomial bacteria, but also transcended conventional boundaries. These implications extend beyond clinical resistance, permeating into the realm of soil revitalization and environmental guardianship. As it unveils P. aeruginosa intricacies, this study assumes a mantle of guiding strategies to mitigate clinical hazards, harness environmental advantages, and propel sustainable solutions forward.

Nucleotide binding as an allosteric regulatory mechanism for Akkermansia muciniphila ß-N-acetylhexosaminidase Am2136.

ß-N-acetylhexosaminidases (EC3.2.1.52), which belong to the glycosyl hydrolase family GH20, are important enzymes for oligosaccharides modification. Numerous microbial ß-N-acetylhexosaminidases have been investigated for applications in biology, biomedicine and biotechnology. Akkermansia muciniphila is an anaerobic intestinal commensal bacterium which possesses specific ß-N-acetylhexosaminidases for gut mucosal layer colonization and mucin degradation. In this study, we assessed the in vitro mucin glycan cleavage activity of the A. muciniphila ß-N-acetylhexosaminidase Am2136 and demonstrated its ability that hydrolyzing the ß-linkages joining N-acetylglucosamine to a wide variety of aglycone residues, which indicated that Am2136 may be a generalist ß-N-acetylhexosaminidase. Structural and enzyme activity assay experiments allowed us to probe the essential function of the inter-domain interactions in ß23-ß33. Importantly, we revealed that the hydrolysis activity of Am2136 was enhanced by nucleotides. We further speculated that this activation mechanism might be associated with the conformational motions between domain III and IV. To our knowledge, this is the first report of nucleotide effector regulated ß-N-acetylhexosaminidase, to reveal its novel biological functions. These findings contribute to understanding the distinct properties within the GH20 family and lay a certain foundation to develop controllable glycan hydrolyzing catalysts.Abbreviations: OD600 - optical cell densities at 600 nm; LB - Luria-Bertani; IPTG - isopropyl ß-D-1-thiogalactopyranoside; PMSF - phenylmethanesulfonyl fluoride; rmsd - root mean square deviation; GlcNAc - N-acetyl-ß-D-glucosamine; GalNAc - N-acetyl-ß-D-galactosamine; Gal - galactose.

Structural analysis of the sulfatase AmAS from Akkermansia muciniphila.

Akkermansia muciniphila, an anaerobic Gram-negative bacterium, is a major intestinal commensal bacterium that can modulate the host immune response. It colonizes the mucosal layer and produces nutrients for the gut mucosa and other commensal bacteria. It is believed that mucin desulfation is the rate-limiting step in the mucin-degradation process, and bacterial sulfatases that carry out mucin desulfation have been well studied. However, little is known about the structural characteristics of A. muciniphila sulfatases. Here, the crystal structure of the premature form of the A. muciniphila sulfatase AmAS was determined. Structural analysis combined with docking experiments defined the critical active-site residues that are responsible for catalysis. The loop regions I-V were proposed to be essential for substrate binding. Structure-based sequence alignment and structural superposition allow further elucidation of how different subclasses of formylglycine-dependent sulfatases (FGly sulfatases) adopt the same catalytic mechanism but exhibit diverse substrate specificities. These results advance the understanding of the substrate-recognition mechanisms of A. muciniphila FGly-type sulfatases. Structural variations around the active sites account for the different substrate-binding properties. These results will enhance the understanding of the roles of bacterial sulfatases in the metabolism of glycans and host-microbe interactions in the human gut environment.

Structural and biochemical analysis of 1-Cys peroxiredoxin ScPrx1 from Saccharomyces cerevisiae mitochondria.

BACKGROUND: ScPrx1 is a yeast mitochondrial 1-Cys peroxiredoxins (Prx), a type of Prx enzyme which require thiol-containing reducing agents to resolve its peroxidatic cysteine. ScPrx1 plays important role in protection against oxidative stress. Mitochondrial thioredoxin ScTrx3 and glutathione have been reported to be the physiological electron donor for ScPrx1. However, the mechanism underlying their actions, especially the substrate recognition of ScPrx1 requires additional elucidation. METHODS: The structure of ScPrx1 was obtained through crystallization experiments. The oligomeric state of ScPrx1 was monitored by Blue-Native PAGE. Mutations were generated by the QuikChange PCR-based method. The ScPrx1 activity assay was carried out by measuring the change of 340 nm absorption of the NADPH oxidation. RESULTS: ScPrx1 exist as a homodimer in solution. The structure adopts a typical Prx-fold core which is preceded by an N-terminal ß-hairpin and has a C-terminal extension. Mutations (Glu94Ala, Arg198Ala and Trp126) close to the active site could enhance the catalytic efficiency of ScPrx1 while His83Ala and mutations on α4-ß6 region exhibited reduced activity. The biochemical data also show that the deletion or mutations on ScPrx1 C-terminal have 2-4.56 fold increased activity. CONCLUSION: We inferred that conformational changes of ScPrx1 C-terminal segment were important for its reaction, and the α4-ß6 loop regions around the ScPrx1 active sites were important for the catalytic function of ScPrx1. Collectively, these structural features provides a basis for understanding the diverse reductant species usage in different 1-Cys Prxs.