Abstract
The sexually transmitted disease gonorrhea (causative agent: Neisseria gonorrhoeae) remains an urgent public health threat globally due to the repercussions on reproductive health, 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, previously described in regard to phenotypic and reference genome characteristics, 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 901 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_00635, WHO_F_00745, WHO_F_01139, WHO_F_01144, and WHO_F_01226, 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 databank for gonococcal vaccine and AMR research endeavors, which enables microbiological, clinical, or epidemiological projects and enhances the utility of the WHO reference strains.
- The abbreviations used are
- ACN
- Acetonitrile
- AGC
- Automatic gain control
- AMR
- Antimicrobial resistance
- C
- Cytoplasmic
- CDC
- Centers for Disease Control and Prevention
- CE
- Cell envelope
- COG
- Cluster of orthologous genes
- cRAP
- Common repository of adventitious proteins
- FDR
- False discovery rate
- GCB
- Gonococcal base agar
- GCBL
- Gonococcal base liquid medium
- KEGG
- Kyoto encyclopedia of genes and genomes
- LPS
- Lipopolysaccharide
- OMV
- Outer membrane vesicle
- ORF
- Open reading frame
- WHO
- World Health Organization
Footnotes
↵** Joint senior authors
Author contributions: A.E.S. designed research with input from M.U.; R.A.Z. performed the experiments; F.E.E. performed computational data analysis; F.E.E., A.E.S., T.W., and M. U. interpreted the data; F.E.E., A.E.S., and M.U. wrote the paper. All authors commented on and approved the final version of the paper.