An Extended Spectrum of Target Proteins and Modification Sites in the General O-Linked Protein Glycosylation System in Neisseria gonorrhoeae

2012 ◽  
Vol 11 (12) ◽  
pp. 5781-5793 ◽  
Author(s):  
Jan Haug Anonsen ◽  
Åshild Vik ◽  
Wolfgang Egge-Jacobsen ◽  
Michael Koomey
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Protein Glycosylation ◽  
Target Proteins ◽  
Extended Spectrum

scholarly journals Lactobacillus plantarum WCFS1 O-linked protein glycosylation: An extended spectrum of target proteins and modification sites detected by mass spectrometry

Glycobiology ◽  
2013 ◽  
Vol 23 (12) ◽  
pp. 1439-1451 ◽  
Author(s):  
Lasse Fredriksen ◽  
Anders Moen ◽  
Alexei A Adzhubei ◽  
Geir Mathiesen ◽  
Vincent GH Eijsink ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lactobacillus Plantarum ◽  
Protein Glycosylation ◽  
Target Proteins ◽  
Extended Spectrum

scholarly journals A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases

2017 ◽  
Author(s):  
Jennifer A. Schoborg ◽  
Jasmine Hershewe ◽  
Jessica C. Stark ◽  
Weston Kightlinger ◽  
James E. Kath ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Cellular Membrane ◽  
Production Method ◽  
Protein Glycosylation ◽  
Campylobacter Coli ◽  
Amino Acid Residues ◽  
Target Proteins ◽  
A Cell ◽  
Campylobacter Lari

AbstractProtein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 440 µg/mL for the single-subunit OST enzyme, ‘Protein glycosylation B’ (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

scholarly journals Molecular Analyses of TEM Genes and Their Corresponding Penicillinase-Producing Neisseria gonorrhoeae Isolates in Bangkok, Thailand

2011 ◽  
Vol 56 (2) ◽  
pp. 916-920 ◽  
Author(s):  
Shu-ichi Nakayama ◽  
Chanwit Tribuddharat ◽  
Sasiprapa Prombhul ◽  
Ken Shimuta ◽  
Somporn Srifuengfung ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Public Health Problem ◽  
Major Public Health Problem ◽  
Sexual Network ◽  
Genetic Event ◽  
Content Type ◽  
Extended Spectrum ◽  
Resistance To Penicillin ◽  
High Level ◽  
International Awareness

ABSTRACTNeisseria gonorrhoeaeis a major public health problem globally, especially because the bacterium has developed resistance to most antimicrobials introduced for first-line treatment of gonorrhea. In the present study, 96N. gonorrhoeaeisolates with high-level resistance to penicillin from 121 clinical isolates in Thailand were examined to investigate changes related to their plasmid-mediated penicillin resistance and their molecular epidemiological relationships. A β-lactamase (TEM) gene variant,blaTEM-135, that may be a precursor in the transitional stage of a traditionalblaTEM-1gene into an extended-spectrum β-lactamase (ESBL), possibly causing high resistance to all extended-spectrum cephalosporins inN. gonorrhoeae, was identified. Clonal analysis using multilocus sequence typing (MLST) andN. gonorrhoeaemultiantigen sequence typing (NG-MAST) revealed the existence of a sexual network among patients from Japan and Thailand. Molecular analysis of theblaTEM-135gene showed that the emergence of this allele might not be a rare genetic event and that the allele has evolved in different plasmid backgrounds, which results possibly indicate that it is selected due to antimicrobial pressure. The presence of theblaTEM-135allele in the penicillinase-producingN. gonorrhoeaepopulation may call for monitoring for the possible emergence of ESBL-producingN. gonorrhoeaein the future. This study identified ablaTEMvariant (blaTEM-135) that is a possible intermediate precursor for an ESBL, which warrants international awareness.

scholarly journals Penicillinase-Producing Plasmid Types in Neisseria gonorrhoeae Clinical Isolates from Australia

2014 ◽  
Vol 58 (12) ◽  
pp. 7576-7578 ◽  
Author(s):  
David Whiley ◽  
Ella Trembizki ◽  
Cameron Buckley ◽  
Kevin Freeman ◽  
Andrew Lawrence ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Clinical Isolates ◽  
Content Type ◽  
Stepping Stone ◽  
Extended Spectrum

ABSTRACTPenicillinase-producingNeisseria gonorrhoeae(PPNG) carrying theblaTEM-135gene is of particular concern, as it is considered a stepping stone toward resistance to extended-spectrum cephalosporins. Here, we sought to characterize plasmid types and the occurrence of theblaTEM-135gene forN. gonorrhoeaeclinical isolates from Australia. We found thatblaTEM-135was prevalent in Australian PPNG and was detected on all three major plasmid types.

scholarly journals Susceptibility trends of zoliflodacin against multidrug-resistant Neisseria gonorrhoeae clinical isolates in Nanjing, China (2014-2018)

2020 ◽  
Author(s):  
Wenjing Le ◽  
Xiaohong Su ◽  
Xiangdi Lou ◽  
Xuechun Li ◽  
Xiangdong Gong ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Dna Sequencing ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Drug Resistant ◽  
Extended Spectrum ◽  
High Level ◽  
Level Resistance

ABSTRACTPreviously, we reported potent activity of a novel spiropyrimidinetrione, zoliflodacin, against N. gonorrhoeae isolates from symptomatic men in Nanjing, China, collected in 2013. Here, we investigated trends of susceptibilities of zoliflodacin in 986 gonococcal isolates collected from men between 2014 and 2018. N. gonorrhoeae isolates were tested for susceptibility to zoliflodacin and seven other antibiotics. Mutations in gyrA, gyrB, parC and parE genes were determined by PCR and DNA sequencing. The MIC of zoliflodacin for N. gonorrhoeae ranged from ≤0.002 to 0.25 mg/L; the overall MIC50s and MIC90s were 0.06 mg/L and 0.125mg/L in 2018, increasing two-fold from 2014. However, the percent of isolates with lower zoliflodacin MICs declined in each year sequentially while the percent with higher MICs increased yearly (P≤0.00001). All isolates were susceptible to spectinomycin but resistant to ciprofloxacin (MIC ≥1 μg/ml); 21.2% (209/986) were resistant to azithromycin (≥1 μg/ml), 43.4% (428/986) were penicillinase-producing (PPNG), 26.9% (265/986) tetracycline-resistant (TRNG) and 19.4% (191/986) were multi-drug resistant (MDR) isolates. Among 143 isolates with higher zoliflodacin MICs (0.125-0.25 mg/L), all had quinolone resistance associated double or triple mutations in gyrA; 139/143 (97.2%) also had mutations in parC. There were no D429N/A and/or K450T mutations in GyrB identified in the 143 isolates with higher zoliflodacin MICs; a S467N mutation in GyrB was identified in one isolate. We report that zoliflodacin has excellent in vitro activity against clinical gonococcal isolates, including those with high-level resistance to ciprofloxacin, azithromycin and extended spectrum cephalosporins.

scholarly journals Equations To Predict Antimicrobial MICs in Neisseria gonorrhoeae Using Molecular Antimicrobial Resistance Determinants

2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Walter Demczuk ◽  
Irene Martin ◽  
Pam Sawatzky ◽  
Vanessa Allen ◽  
Brigitte Lefebvre ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Training Data ◽  
Sequencing Analysis ◽  
Regression Equations ◽  
Validation Data ◽  
Data Set ◽  
Content Type ◽  
Resistance Determinants ◽  
Extended Spectrum

ABSTRACT The emergence of Neisseria gonorrhoeae strains that are resistant to azithromycin and extended-spectrum cephalosporins represents a public health threat, that of untreatable gonorrhea infections. Multivariate regression modeling was used to determine the contributions of molecular antimicrobial resistance determinants to the overall antimicrobial MICs for ceftriaxone, cefixime, azithromycin, tetracycline, ciprofloxacin, and penicillin. A training data set consisting of 1,280 N. gonorrhoeae strains was used to generate regression equations which were then applied to validation data sets of Canadian (n = 1,095) and international (n = 431) strains. The predicted MICs for extended-spectrum cephalosporins (ceftriaxone and cefixime) were fully explained by 5 amino acid substitutions in PenA, A311V, A501P/T/V, N513Y, A517G, and G543S; the presence of a disrupted mtrR promoter; and the PorB G120 and PonA L421P mutations. The correlation of predicted MICs within one doubling dilution to phenotypically determined MICs of the Canadian validation data set was 95.0% for ceftriaxone, 95.6% for cefixime, 91.4% for azithromycin, 98.2% for tetracycline, 90.4% for ciprofloxacin, and 92.3% for penicillin, with an overall sensitivity of 99.9% and specificity of 97.1%. The correlations of predicted MIC values to the phenotypically determined MICs were similar to those from phenotype MIC-only comparison studies. The ability to acquire detailed antimicrobial resistance information directly from molecular data will facilitate the transition to whole-genome sequencing analysis from phenotypic testing and can fill the surveillance gap in an era of increased reliance on nucleic acid assay testing (NAAT) diagnostics to better monitor the dynamics of N. gonorrhoeae.

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