Abstract
The sexually transmitted disease gonorrhea (causative agent: Neisseria gonorrhoeae) remains an urgent public health threat globally because of its reproductive health repercussions, high incidence, widespread antimicrobial resistance (AMR), and absence of a vaccine. To mine gonorrhea antigens and enhance our understanding of gonococcal AMR at the proteome level, we performed the first large-scale proteomic profiling of a diverse panel (n = 15) of gonococcal strains, including the 2016 World Health Organization (WHO) reference strains. These strains show all existing AMR profiles - established through phenotypic characterization and reference genome publication - and are intended for quality assurance in laboratory investigations. Herein, these isolates were subjected to subcellular fractionation and labeling with tandem mass tags coupled to mass spectrometry and multi-combinatorial bioinformatics. Our analyses detected 904 and 723 common proteins in cell envelope and cytoplasmic subproteomes, respectively. We identified nine novel gonorrhea vaccine candidates. Expression and conservation of new and previously selected antigens were investigated. In addition, established gonococcal AMR determinants were evaluated for the first time using quantitative proteomics. Six new proteins, WHO_F_00238, WHO_F_00635c, WHO_F_00745, WHO_F_01139, WHO_F_01144c, and WHO_F_01126, were differentially expressed in all strains, suggesting that they represent global proteomic AMR markers, indicate a predisposition toward developing or compensating gonococcal AMR, and/or act as new antimicrobial targets. Finally, phenotypic clustering based on the isolates' defined antibiograms and common differentially expressed proteins yielded seven matching clusters between established and proteome-derived AMR signatures. Together, our investigations provide a reference proteomics data bank for gonococcal vaccine and AMR research endeavors, which enables microbiological, clinical, or epidemiological projects and enhances the utility of the WHO reference strains.
Highlights
Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea
To address the need for discovery of additional gonorrhea vaccine and drug candidates and to enhance our understanding of antimicrobial resistance (AMR) at the proteome level, we examined the 2016 World Health Organization (WHO) N. gonorrhoeae reference strains [50] and the FA6140 strain [51]
As the reference strain for protein identification and quantitation because it has the largest genome (2,292,467 bp) and proteome (2,450 open reading frames (ORFs)) among the 2016 WHO reference strains [50] and FA6140 [68], and it is susceptible to most antimicrobials currently or historically used for gonorrhea treatment
Summary
Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea. All WHO panel strains have been subjected to extensive phenotypic, genomic, and genetic analyses to establish diagnostic markers, molecular epidemiological characteristics, reference genomes, and AMR profiles (phenotypic and genetic) for all antimicrobials currently and previously used for gonorrhea treatment, in addition to novel antimicrobials considered for future interventions This panel includes WHO X, the first extensively drug-resistant gonococcal strain identified with highlevel resistance to ceftriaxone, as well as additional strains with different levels of resistance to ceftriaxone, azithromycin and any additional therapeutic antimicrobials. Our study is the first to investigate the global proteomic profiles of 15 N. gonorrhoeae reference strains using subcellular fractionation to separate cytoplasmic (C) and cell envelope (CE) associated proteomes, which were measured with tandem mass tags coupled to liquid chromatography and tandem mass spectrometry [TMT-LC-MS/MS; [56]], a highly reproducible and sensitive technique. Differential protein expression in four proteomics data sets (CE and C fractions in two biological replicates) was designated by fold changes ≥1.5 or ≤0.667 in reference to strain. The raw mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE [66] partner repository with the data set identifier PXD008412
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