vitamin-c-assisted-fenton-oxidation-improves-oxidative-killing

Vitamin C-Assisted Fenton Oxidation Improves Oxidative Killing of <em>Helicobacter pylori In Vitro</em>

Helicobacter pylori infection remains a major global health burden, and the increasing prevalence of antibiotic resistance underscores the need for ...

West China Hospital, Sichuan University

West China Hospital of Sichuan University

 is a virulence-associated gene in , making it an attractive target for anti drug development. The encoded protein, aminopeptidases P (Pa-PepP), is a type of X-prolyl peptidase that possesses diverse biological functions. The crystal structure verified its canonical pita-bread fold and functional tetrameric assembly, and the functional studies measured the influences of different metal ions on the activity. A trimetal manganese cluster was observed at the active site, elucidating the mechanism of inhibition by metal ions. Additionally, a loop extending from the active site appeared to be important for specific large-substrate binding. Based on the structural comparison and bacterial invasion assays, we showed that this non-conserved surface loop was critical for  virulence. Taken together, these findings can extend our understanding of the catalytic mechanism and virulence-related functions of Pa-PepP and provide a solid foundation for the design of specific inhibitors against pathogenic-bacterial infections.

Structure-Function Relationship of Aminopeptidase P from .

Biofilm formation is a critical determinant in the pathopoiesis of  It could significantly increase bacterial resistance to drugs and host defense. Thus, inhibition of biofilm matrix production could be regarded as a promising attempt to prevent colonization of  and the subsequent infection. PpgL, a periplasmic gluconolactonase, has been reported to be involved in  quorum-sensing (QS) system regulation. However, the detailed function and catalysis mechanism remain elusive. Here, the crystal structure of PpgL is described in the current study, along with biochemical analysis, revealing that PpgL is a typical β-propeller enzyme with unique metal-independent lactone hydrolysis activity. Consequently, comparative analysis of seven-bladed propeller lactone-catalyzing enzymes and mutagenesis studies identify the critical sites which contribute to the diverse catalytic and substrate recognition functions. In addition, the reduced biofilm formation and attenuated invasion phenotype resulting from deletion of  confirm the importance of PpgL in  pathogenesis. These results suggest that PpgL is a potential target for developing new agents against the diseases caused by .

Structural and Functional Insights into PpgL, a Metal-Independent β-Propeller Gluconolactonase That Contributes to  Virulence.

The lipopolysaccharide O-antigen structure expressed by the European Helicobacter pylori model strain G27 encompasses a trisaccharide, an intervening glucan-heptan and distal Lewis antigens that promote immune escape. However, several gaps still remain in the corresponding biosynthetic pathway. Here, systematic mutagenesis of glycosyltransferase genes in G27 combined with lipopolysaccharide structural analysis, uncovered HP0102 as the trisaccharide fucosyltransferase, HP1283 as the heptan transferase, and HP1578 as the GlcNAc transferase that initiates the synthesis of Lewis antigens onto the heptan motif. Comparative genomic analysis of G27 lipopolysaccharide biosynthetic genes in strains of different ethnic origin revealed that East-Asian strains lack the HP1283/HP1578 genes but contain an additional copy of HP1105 and JHP0562. Further correlation of different lipopolysaccharide structures with corresponding gene contents led us to propose that the second copy of HP1105 and the JHP0562 may function as the GlcNAc and Gal transferase, respectively, to initiate synthesis of the Lewis antigen onto the Glc-Trio-Core in East-Asian strains lacking the HP1283/HP1578 genes. In view of the high gastric cancer rate in East Asia, the absence of the HP1283/HP1578 genes in East-Asian H. pylori strains warrants future studies addressing the role of the lipopolysaccharide heptan in pathogenesis.

East-Asian Helicobacter pylori strains synthesize heptan-deficient lipopolysaccharide.

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.

Molecular basis of the lipid-induced MucA-MucB dissociation in 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  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 , 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 L 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  may utilize to enhance its adaptability to environmental stress. 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  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  to environmental changes during infection.

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

 infection is an infectious disease. Given the alarmingly high antibiotic resistance in , gastroenterologists should change the empiric  treatment paradigm to an antimicrobial susceptibility testing-guided precision treatment. Antimicrobial stewardship programs for  should be implemented locally, regionally, and nationally to monitor the antibiotic resistance pattern.

 infection is an infectious disease and the empiric therapy paradigm should be changed.

Increasing  resistance to antibiotics has ledthat molecular testing is appropriate as a sub to adoption of seven different bismuth quadruple therapies (BQT) in China without differentiation of first-line or second-line regimens. The objective of this study was to evaluate the efficacy of susceptibility-guided BQT for patients who had experienced previous treatment failures. A total of 133 patients was included and  was successfully cultured from 101 patients (75.9%) for subsequent antimicrobial susceptibility testing (AST). Based on the AST results, 88 patients completed one of five AST-guided 14-day BQT regimens: esomeprazole and bismuth colloidal pectin, along with either, amoxicillin and clarithromycin (EBAC), amoxicillin and levofloxacin (EBAL), amoxicillin and furazolidone (EBAF), amoxicillin and tetracycline (EBAT), or tetracycline and furazolidone (EBTF).  eradication rates were 100% for EBAC (5/5), EBAL (13/13), EBAF (14/14), and EBTF (43/43), but 76.9% for EBAT (10/13). The three patients that failed the EBAT regimen were all cured after subsequent treatment with the EBTF regimen. Our study demonstrates the excellent efficacy of the AST-guided BQT for referred  patients, and that the current EBAT regimen, used in clinics, needs to be optimized. In addition, 57 of the isolates were subjected to whole-genome sequencing. Analysis of the sequences revealed that point mutations in 23S rRNA correlated well with the phenotypic clarithromycin resistance with a concordance of 91.2%, while the concordance between phenotypic levofloxacin resistance and  point mutations was 82.3%. This suggests that molecular testing is appropriate as a substitute for AST as a more rapid and cost-effective method for determining clarithromycin and levofloxacin resistance in Chinese patients.

Susceptibility-guided bismuth quadruple therapies for resistant  infections.

The prevalence of Helicobacter pylori antibiotic susceptibility in the Tibet Autonomous Region, China is not determined. This study aimed to evaluate the antibiotic resistance patterns of H. pylori isolates there.

Antibiotic resistance patterns of Helicobacter pylori strains isolated from the Tibet Autonomous Region, China.

 has a unique lipopolysaccharide structure that is essential in maintaining its cell envelope integrity and imbues the bacterium with natural resistance to cationic antimicrobial peptides (CAMPs). Our group has recently elucidated the complete set of LPS glycosyltransferase genes in  reference strain G27. Here, with a series of eight systematically constructed LPS glycosyltransferase gene mutants (G27Δ, G27Δ, G27Δ, G27Δ, G27Δ, G27Δ, G27Δ and G27Δ), we investigated the roles of  LPS glycosyltransferases in maintaining cell morphology, cell wall permeability, and antimicrobial susceptibilities. We demonstrated that deletion of these LPS glycosyltransferase genes did not interfere with bacterial cell wall permeability, but resulted in significant morphological changes (coccoid, coiled "c"-shape, and irregular shapes) after 48 h growth as compared to the rod-like cell shape of the wild-type strain. Moreover, as compared with the wild-type, none of the LPS mutants had altered susceptibility against clarithromycin, levofloxacin, amoxicillin, tetracycline, and metronidazole. However, the deletion of the conserved LPS glycosyltransferases, especially the O-antigen-initiating enzyme WecA, displayed a dramatic increase in susceptibility to the CAMP polymyxin B and rifampicin. Taken together, our findings suggest that the LPS glycosyltransferases play critical roles in the maintenance of the typical spiral morphology of , as well as resistance to CAMPs and rifampicin. The LPS glycosyltransferases could be promising targets for developing novel anti- drugs.

Roles of Lipopolysaccharide Glycosyltransferases in Maintenance of  Morphology, Cell Wall Permeability, and Antimicrobial Susceptibilities.

Helicobacter pylori lipopolysaccharide (LPS) structures vary among strains of different geographic origin. The aim of this study was to characterize the LPS O-antigen profiles of H. pylori strains isolated from Southwest China, and to further analyze the association of Lewis antigen expression with clinical outcomes and antibiotic resistance.

Lipopolysaccharide O-antigen profiles of Helicobacter pylori strains from Southwest China.

The common adverse effects and the complicated administration of tetracycline and metronidazole greatly affect the clinical application of the classical bismuth quadruple therapy (BQT) for  eradication. This pilot study aimed to evaluate the efficacy and safety of minocycline/amoxicillin-based BQT for  eradication. Firstly, consecutive  isolates collected at West China Hospital of Sichuan University between 2018 and 2021 were included for susceptibility testing of tetracycline and minocycline using E-test strips. Secondly, both treatment-naïve and experienced patients were included to receive a 14-day minocycline/amoxicillin-based BQT: esomeprazole 40 mg or vonoprazan 20 mg, bismuth colloidal pectin 300 mg, amoxicillin 1000 mg, and minocycline 100 mg, all given twice daily. Among a total of 101  isolates, tetracycline resistance was 3.0%, whereas minocycline resistance was nil. A total of 114 patients (treatment-naïve/experienced, 72/42) received the minocycline/amoxicillin-based BQT. The overall intention-to-treat (ITT) and per protocol (PP) eradication rates were 94.7% (108/114) and 97.3% (108/111), respectively. The ITT and PP eradication rates were 91.7% (66/72) and 95.7% (66/69) among the treatment-naïve patients, and both were 100.0% among the treatment-experienced patients. No serious adverse event was recorded. This pilot study suggests that minocycline/amoxicillin-based BQT is an excellent therapy for  eradication.

Minocycline/Amoxicillin-Based Bismuth Quadruple Therapy for  Eradication: A Pilot Study.

 has been recognized not only as a causative agent of a spectrum of gastroduodenal diseases including chronic gastritis, peptic ulcer, mucosa-associated lymphoid tissue lymphoma, and gastric cancer, but also as the culprit in several extra-gastric diseases. However, the association of  infection with extra-gastric diseases remains elusive, prompting a reevaluation of the role of -derived outer membrane vesicles (OMVs). Like other gram-negative bacteria,  constitutively sheds biologically active OMVs for long-distance delivery of bacterial virulence factors in a concentrated and protected form, averting the need of direct bacterial contact with distant host cells to induce extra-gastric diseases associated with this gastric pathogen. Additionally, -derived OMVs contribute to bacterial survival and chronic gastric pathogenesis. Moreover, the immunogenic activity, non-replicable nature, and anti-bacterial adhesion effect of  OMVs make them a desirable vaccine candidate against infection. The immunogenic potency and safety concerns of the OMV contents are challenges in the development of  OMV-based vaccines. In this review, we discuss recent advances regarding  OMVs, focusing on new insights into their biogenesis mechanisms and biological functions.

 Outer Membrane Vesicles: Biogenesis, Composition, and Biological Functions.

 To investigate the correlation between different histological subtypes (adenosquamous carcinoma, adenocarcinoma, and squamous cell carcinoma) and the prognosis of cervical cancer.  In this retrospective cohort analysis, patients with cervical cancer who underwent radical surgery followed by either concurrent chemoradiotherapy (CCRT) or radiotherapy (RT) at West China Hospital of Sichuan University between 2009 and 2018 were enrolled. The study included patients with confirmed pathological diagnoses of cervical adenosquamous carcinoma (ASC), adenocarcinoma (AC), and squamous cell carcinoma (SCC). To ensure a balanced representation, 1:3 propensity score matching (PSM) between cervical adenosquamous carcinoma (ASC) or adenocarcinoma (AC) and squamous cell carcinoma (SCC) was performed. The prognosis of different pathological subtypes, including 5-year overall survival (OS), 5-year disease-free survival (DFS), and treatment failure patterns in terms of recurrence and metastasis, were evaluated between groups.  This study enrolled a total of 714 patients between 2009 and 2018, of whom 614 (86%) were diagnosed with SCC. In a 1:3 ratio propensity score matching, 34 cases of ASC were matched with 102 cases of SCC, while 66 cases of AC were paired with another 198 cases of SCC. Baseline demographic and disease characteristics were well-balanced among the treatment groups. During a median follow-up period of 41 months (range: 14 to 122 months), a total of 40 patients experienced disease recurrence. The primary recurrence pattern was distant metastasis, observed in 36 out of 40 cases. Among these cases, recurrence occurred in 28 patients (9.3%) diagnosed with SCC, 10 patients (15.2%) with AC, and 2 patients (5.9%) with ASC. In the AC group, local failure and distant failure were observed in 2% and 12% of cases, respectively. In comparison, the corresponding rates in the paired SCC group were 0.6% and 8.7%. The 5-year OS and DFS rates in the AC group were 82.1% and 79.2%, respectively, compared to the paired SCC group, which had rates of 95.2% and 92.8% respectively (<0.05). Conversely, in the ASC group, the 5-year OS and DFS rates were 96.3% and 92.6%, while the paired SCC group displayed OS and DFS rates of 93.4% and 81.2% respectively, with no statistically significant difference observed.  By comparing the prognostic outcomes of different histological subtypes, we concluded that AC histology was linked to a poor prognosis and an increased risk of distant recurrence. ASC histology had a similar outcome to SCC histology rather than AC. Given the poor prognosis for patients diagnosed with AC after adjusting for prognostic factors, it becomes imperative to explore alternative treatment options beyond the current conventional therapy for this condition.

Article	Year
Structure-Function Relationship of Aminopeptidase P from . Frontiers in microbiology\| PubMed ID: 29259588	2017
Structural and Functional Insights into PpgL, a Metal-Independent β-Propeller Gluconolactonase That Contributes to Virulence. Infection and immunity\| PubMed ID: 30642898	2019
East-Asian Helicobacter pylori strains synthesize heptan-deficient lipopolysaccharide. PLoS genetics\| PubMed ID: 31747390	2019
Molecular basis of the lipid-induced MucA-MucB dissociation in Pseudomonas aeruginosa. Communications biology\| PubMed ID: 32747658	2020
Molecular Basis of the Versatile Regulatory Mechanism of HtrA-Type Protease AlgW from Pseudomonas aeruginosa. mBio\| PubMed ID: 33622718	2021
infection is an infectious disease and the empiric therapy paradigm should be changed. Precision clinical medicine\| PubMed ID: 35692450	2019
Susceptibility-guided bismuth quadruple therapies for resistant infections. Precision clinical medicine\| PubMed ID: 35692608	2020
Antibiotic resistance patterns of Helicobacter pylori strains isolated from the Tibet Autonomous Region, China. BMC microbiology\| PubMed ID: 35964011	2022
Roles of Lipopolysaccharide Glycosyltransferases in Maintenance of Morphology, Cell Wall Permeability, and Antimicrobial Susceptibilities. International journal of molecular sciences\| PubMed ID: 37511140	2023
Lipopolysaccharide O-antigen profiles of Helicobacter pylori strains from Southwest China. BMC microbiology\| PubMed ID: 37993791	2023
Minocycline/Amoxicillin-Based Bismuth Quadruple Therapy for Eradication: A Pilot Study. Microorganisms\| PubMed ID: 38543480	2024
Outer Membrane Vesicles: Biogenesis, Composition, and Biological Functions. International journal of biological sciences\| PubMed ID: 39113715	2024
Comparison of Survival Between Different Histological Subtypes in Cervical Cancer Patients: A Retrospective and Propensity Score-matched Analysis. Journal of Cancer\| PubMed ID: 39513111	2024

Yalin Shen

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