Structural and functional characterization of itaconyl-CoA hydratase and citramalyl-CoA lyase involved in itaconate metabolism of Pseudomonas aeruginosa.

Itaconate is a key anti-inflammatory/antibacterial metabolite in pathogen-macrophage interactions that induces adaptive changes in Pseudomonas aeruginosa-exposed airways. However, the impact and mechanisms underlying itaconate metabolism remain unclear. Our study reveals that itaconate significantly upregulates the expression of pyoverdine in P. aeruginosa and enhances its tolerance to tobramycin. Notably, the enzymes responsible for efficient itaconate metabolism, PaIch and PaCcl, play crucial roles in both utilizing itaconate and clearing its toxic metabolic intermediates. By using protein crystallography and molecular dynamics simulations analyses, we have elucidated the unique catalytic center and substrate-binding pocket of PaIch, which contribute to its highly efficient catalysis. Meanwhile, analysis of PaCcl has revealed how interactions between domains regulate the conformational changes of the active sites and binding pockets, influencing the catalytic process. Overall, our research uncovers the significance and mechanisms of PaIch and PaCcl in the efficient metabolism of itaconate by P. aeruginosa.

Direct and Continuous Monitoring of Multicomponent Antibiotic Gentamicin in Blood at Single-Molecule Resolution.

Point-of-care monitoring of small molecules in biofluids is crucial for clinical diagnosis and treatment. However, the inherent low degree of recognition of small molecules and the complex composition of biofluids present significant obstacles for current detection technologies. Although nanopore sensing excels in the analysis of small molecules, the direct detection of small molecules in complex biofluids remains a challenge. In this study, we present a method for sensing the small molecule drug gentamicin in whole blood based on the mechanosensitive channel of small conductance in Pseudomonas aeruginosa (PaMscS) nanopore. PaMscS can directly detect gentamicin and distinguish its main components with only a monomethyl difference. The 'molecular sieve' structure of PaMscS enables the direct measurement of gentamicin in human whole blood within 10 min. Furthermore, a continuous monitoring device constructed based on PaMscS achieved continuous monitoring of gentamicin in live rats for approximately 2.5 h without blood consumption, while the drug components can be analyzed in situ. This approach enables rapid and convenient drug monitoring with single-molecule level resolution, which can significantly lower the threshold for drug concentration monitoring and promote more efficient drug use. Moreover, this work also lays the foundation for the future development of continuous monitoring technology with single-molecule level resolution in the living body.

Molecular mechanism of the one-component regulator RccR on bacterial metabolism and virulence.

The regulation of carbon metabolism and virulence is critical for the rapid adaptation of pathogenic bacteria to host conditions. In Pseudomonas aeruginosa, RccR is a transcriptional regulator of genes involved in primary carbon metabolism and is associated with bacterial resistance and virulence, although the exact mechanism is unclear. Our study demonstrates that PaRccR is a direct repressor of the transcriptional regulator genes mvaU and algU. Biochemical and structural analyses reveal that PaRccR can switch its DNA recognition mode through conformational changes triggered by KDPG binding or release. Mutagenesis and functional analysis underscore the significance of allosteric communication between the SIS domain and the DBD domain. Our findings suggest that, despite its overall structural similarity to other bacterial RpiR-type regulators, RccR displays a more complex regulatory element binding mode induced by ligands and a unique regulatory mechanism.

Pseudomonas aeruginosa regulator PvrA binds simultaneously to multiple pseudo-palindromic sites for efficient transcription activation.

Tetracycline repressor (TetR) family regulators (TFRs) are the largest group of DNA-binding transcription factors and are widely distributed in bacteria and archaea. TFRs play vital roles in controlling the expression of various genes and regulating diverse physiological processes. Recently, a TFR protein Pseudomonas virulence regulator A (PvrA), was identified from Pseudomonas aeruginosa as the transcriptional activator of genes involved in fatty acid utilization and bacterial virulence. Here, we show that PvrA can simultaneously bind to multiple pseudo-palindromic sites and upregulate the expression levels of target genes. Cryo-electron microscopy (cryo-EM) analysis indicates the simultaneous DNA recognition mechanism of PvrA and suggests that the bound DNA fragments consist of a distorted B-DNA double helix. The crystal structure and functional analysis of PvrA reveal a hinge region that secures the correct domain motion for recognition of the promiscuous promoter. Additionally, our results showed that mutations disrupting the regulatory hinge region have differential effects on biofilm formation and pyocyanin biosynthesis, resulting in attenuated bacterial virulence. Collectively, these findings will improve the understanding of the relationship between the structure and function of the TetR family and provide new insights into the mechanism of regulation of P. aeruginosa virulence.

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.

High-fidelity biosensing of dNTPs and nucleic acids by controllable subnanometer channel PaMscS.

Current tools for dNTP analysis mainly rely on expensive fluorescent labeling, mass spectrometry or electrochemistry. Single-molecule assay by protein nanopores with an internal diameter of ca. 1-3.6 nm provides a useful tool for dNTP sensing. However, the most commonly used protein nanopores require additional modifications to enable dNTP detection. In this study, the PaMscS channel (mechanosensitive channel of small conductance from Pseudomonas aeruginosa) embedded in the bilayer lipid membrane (BLM) of E. coli polar lipid extract was applied as a nanopore for single molecular sensing. Two mutants of PaMscS nanopores on the side portal region (PaMscS W130A and PaMscS K180R) were selected for direct dNTP or pyrophosphoric acid (PPi) detection without aptamer or protein modification. Notably, the PaMscS mutant pore can be adjusted by regulation of osmolarity differences, which is crucial for the optimal detection of specific molecules. In addition, we established a PaMscS-based diagnosis method for the rapid sensing of disease-associated nucleic acids by monitoring the consumption of dNTPs, with 86% specificity and 100% sensitivity among 22 clinical samples. This protein nanopore, without aptamer or modification, paves a new way for dNTPs, PPi direct sensing and nucleic acid detection with low cost but high versatility.

Type II Antitoxin HigA Is a Key Virulence Regulator in Pseudomonas aeruginosa.

Bacterial type II toxin-antitoxin (TA) systems are abundant genetic elements and are involved in a diverse array of physiological processes. These systems encode an antitoxin protein that directly binds and effectively neutralizes the protein toxin. Recent studies have highlighted the key roles of type II TA modules in bacterial virulence and pathogenesis, but the underlying mechanisms remain unclear. Here, we investigated the antitoxin HigA in Pseudomonas aeruginosa infection. Proteomic analysis of the higA deletion strain revealed an enhanced expression of pathogenic proteins. We further verified that HigA negatively controlled T3SS and T6SS expression by directly interacting with the promoter regions of the regulators amrZ and exsA, respectively. In other words, the reversal of HigA-mediated transcriptional inhibition on stress stimulation could induce virulence genes. These findings confirm the crucial roles of the type II antitoxin in bacterial infection, which highlights the potential of the HigBA TA system as an antibacterial treatment target.

Structural characterization of PaFkbA: A periplasmic chaperone from Pseudomonas aeruginosa.

Bacterial Mip-like FK506-binding proteins (FKBPs) mostly exhibit peptidyl-prolyl-cis/trans-isomerase (PPIase) and chaperone activities. These activities are associated with various intracellular functions with diverse molecular mechanisms. Herein, we report the PA3262 gene-encoded crystal structure of the Pseudomonas aeruginosa PAO1's Mip-like protein PaFkbA. Biochemical characterization of PaFkbA demonstrated PaFkbA's chaperone activity for periplasmic protein MucD, a negative regulator of alginate biosynthesis. Furthermore, structural analysis of PaFkbA was used to describe the key features of PaFkbA chaperone activity. The outcomes of this analysis showed that the hinge region in the connecting helix of PaFbkA leads to the crucial conformational state transition for PaFkbA activity. Besides, the N-terminal domains participated in dimerization, and revealed its potential connection with FKBP domain and substrate binding. Mutagenesis and chaperone activity assay supported the theory that inter-domain motions are essential for PaFkbA function. These results provide biochemical and structural insights into the mechanism for FKBP's chaperone activity and establish a plausible correlation between PaFkbA and P. aeruginosa MucD.

Molecular Basis of the Versatile Regulatory Mechanism of HtrA-Type Protease AlgW from Pseudomonas aeruginosa.

AlgW, a membrane-bound periplasmic serine protease belonging to the HtrA protein family, is a key regulator of the regulated intramembrane proteolysis (RIP) pathway and is responsible for transmitting the envelope stress signals in Pseudomonas aeruginosa The AlgW PDZ domain senses and binds the C-terminal of mis-localized outer membrane proteins (OMPs) or periplasmic protein MucE, leading to catalytic activation of the protease domain. While AlgW is functionally well studied, its exact activation mechanism remains to be elucidated. Here, we show that AlgW is a novel HtrA protease that can be biochemically activated by both peptide and lipid signals. Compared with the corresponding homologue DegS in Escherichia coli, AlgW exhibits a distinct substrate specificity and regulation mechanism. Structural, biochemical, and mutagenic analyses revealed that, by specifically binding to the C-terminal decapeptide of MucE, AlgW could adopt more relaxed conformation and obtain higher activity than with tripeptide activation. We also investigated the regulatory mechanism of the LA loop in AlgW and proved that the unique structural feature of this region was responsible for the distinct enzymatic property of AlgW. These results demonstrate the unique and diverse activation mechanism of AlgW, which P. aeruginosa may utilize to enhance its adaptability to environmental stress.IMPORTANCE HtrA-family proteases are commonly employed to sense the protein folding stress and activate the regulated intramembrane proteolysis (RIP) cascade in Gram-negative bacteria. Here, we reveal the unique dual-signal activation and dynamic regulation properties of AlgW, an HtrA-type protease triggering the AlgU stress-response pathway, which controls alginate production and mucoid conversion in Pseudomonas aeruginosa The structural and functional data offer insights into the molecular basis underlying the transition of different activation states of AlgW in response to different effectors. Probing these unique features provides an opportunity to correlate the diverse regulation mechanism of AlgW with the high adaptability of P. aeruginosa to environmental changes during infection.

Effect of Urate-Lowering Therapy on the Progression of Kidney Function in Patients With Asymptomatic Hyperuricemia: A Systematic Review and Meta-Analysis.

Background: Hyperuricemia is involved in the risk of chronic kidney disease (CKD). However, whether urate-lowering therapy (ULT) can influence the progression of kidney function in patients with asymptomatic hyperuricemia is still controversial. We conducted a systematic review and meta-analysis to evaluate the effect of ULT on the progression of kidney function in asymptomatic hyperuricemia patients. Methods: The MEDLINE, EMBASE and Cochrane databases were searched without language, national or ethnic restrictions for randomized controlled trials published prior to November 30, 2020, that compared ULT with controlled therapy in patients with asymptomatic hyperuricemia. Results: Eleven studies were included for qualitative synthesis. ULT did not ameliorate eGFR slopes (WMD 0.36 ml/min/1.73 m2 per year, 95% CI: -0.31, 1.04), or lead to reductions in kidney events (RR 1.26; 95% CI: 0.80, 2.00) or all-cause mortality (RR 1.00; 95% CI: 0.65, 1.55), although ULT resulted in a decrease in serum uric acid levels (WMD -2.73 mg/dl; 95% CI: -3.18, -2.28) and lowered the incidence of gout episodes (0.9 vs 2.7%, RR 0.38; 95% CI: 0.17, 0.86). Conclusion: In patients with asymptomatic hyperuricemia, ULT did not decay the progression of kidney function. Long-term and larger sample studies are needed to verify the results. Systematic Review Registration: [www.crd.york.ac.uk/PROSPERO/#recordDetails], identifier [CRD42020204482].

Molecular basis of the lipid-induced MucA-MucB dissociation in Pseudomonas aeruginosa.

MucA and MucB are critical negative modulators of sigma factor AlgU and regulate the mucoid conversion of Pseudomonas aeruginosa. Previous studies have revealed that lipid signals antagonize MucA-MucB binding. Here we report the crystal structure of MucB in complex with the periplasmic domain of MucA and polyethylene glycol (PEG), which unveiled an intermediate state preceding the MucA-MucB dissociation. Based on the biochemical experiments, the aliphatic side chain with a polar group was found to be of primary importance for inducing MucA cleavage. These results provide evidence that the hydrophobic cavity of MucB is a primary site for sensing lipid molecules and illustrates the detailed control of conformational switching within MucA-MucB in response to lipophilic effectors.

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.