Quantitative Proteomics of the 2016 WHO Neisseria gonorrhoeae Reference Strains Surveys Vaccine Candidates and Antimicrobial Resistance Determinants

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.

Keywords: Absolute Quantification; Antibiotics; Bacteria; Drug Resistance; Gonorrhea; Membranes; Neisseria gonorrhoeae; Pathogens; TMT; Vaccine.

Graphical abstract

Fig. 1.

Experimental paradigm of quantitative proteomic profiling of the N. gonorrhoeae 2016 WHO reference strains and the FA6140 strain. All gonococci were cultured concurrently in liquid medium until reaching mid-logarithmic growth. Bacterial cells were harvested, lysed, and subjected to subcellular fractionation to separate the crude cell envelope (CE) and cytoplasmic (C) proteomes. CE proteins were enriched using a sodium carbonate wash and ultracentrifugation. The obtained CE and C protein samples (100 μg) were denatured, reduced, alkylated, trypsinized, and the peptides from each strain were labeled using 10-plex and 6-plex Tandem mass tag (TMT) reagents, as indicated. Finally, samples were pooled, fractionated by strong cation exchange, and analyzed by liquid chromatography electrospray ionization mass spectrometry. Experiments were performed in biological duplicates.

Fig. 2.

Venn diagrams illustrating the distribution of proteins identified in cell envelope and cytoplasmic fractions in two independent proteomic experiments. A, Cell envelope proteomes derived from the 2016 WHO reference strains and FA6140 were analyzed in 10-plex and 6-plex experiments performed in biological duplicates. A total of 1079 and 1081 proteins was identified in Experiments 1 and 2, respectively, and 1010 common proteins were found in both 10-plex experiments. The 6-plex TMT labeling revealed 975 common proteins as well as 197 and 22 unique proteins in Experiments 1 and 2, respectively. Further analyses were applied to 904 proteins mutually identified in both 10-plex and 6-plex experiments. B, The proteomic profiling of cytoplasmic fractions yielded 904 proteins shared among all 10 strains, of which 747 were common in both experiments. The 6-plex TMT identified 904 and 971 proteins in Experiments 1 and 2, respectively; of which 852 were common between replicates. In further analyses solely the 723 proteins shared between both experiments were included. Exp 1 - experiment 1; Exp 2 - experiment 2.

Fig. 3.

Subcellular localization of proteins identified in cell envelope and cytoplasmic subproteomes. Proteins identified in the cell envelope (blue circle) and cytoplasmic (red circle) fractions were subjected to comprehensive assessments of subcellular localization using different prediction algorithms and were allocated into the outer membrane (A), periplasm (B), inner membrane (C), cytoplasm (D), or unknown localization (E).

Fig. 4.

Expression patterns of common proteins identified in the cell envelope fraction. Outer membrane (A), periplasmic (B), inner membrane (C), or proteins with unknown localization (D) are shown. Expression of each protein in each gonococcal strain was compared with the protein level in the reference WHO F isolate. Protein expression is categorized as ubiquitous (green bars); upregulated (red bar); downregulated (blue bar); and variable (gray bar).

Fig. 5.

Expression patterns of common proteins identified in the cytoplasmic proteome. Cytoplasmic (A) and proteins with unknown localization (B) are shown. Protein levels in individual gonococcal strains were compared with the protein level in the reference WHO F isolate. Protein expression is categorized as ubiquitous (green bars); upregulated (red bar); downregulated (blue bar); and variable (gray bar).

Fig. 6.

Decision tree designed for proteomic mining of Neisseria gonorrhoeae vaccine candidates and antimicrobial resistance (AMR) determinants. For vaccine antigen mining, we focused on 904 common proteins identified in all strains in the CE fraction with the overarching goal to discover omnipresent N. gonorrhoeae proteins. Computational predictions were employed to predict protein subcellular localization. Solely 26 OMPs and 121 proteins with unknown localization were included in further analyses. Bioinformatics and literature review yielded six novel vaccine candidates in the latter group of proteins. The OMPs constituted three new antigens and 22 previously proposed gonorrhea vaccine candidates including 13 proteomics-derived. Finally, the 38 identified gonorrhea vaccine candidates were subjected to expression and conservation analyses. To profile AMR signatures, we performed a pairwise comparison of individual strains to the antimicrobial susceptible WHO F strain to enhance the discovery of strain-specific feature(s). The maximum number of identified proteins reached 1100 and 876 for CE and C fractions, respectively. Expression profiling and literature review yielded 398 differentially expressed proteins including 21 verified AMR determinants. Ubiquitous and variably expressed proteins were excluded from the analysis. To link AMR phenotypes with proteomic signatures, COG analysis and phenotypic clustering of gonococcal strains based on their defined antibiograms and the 377 differentially expressed proteins were performed generating seven phenotypic clusters that matched between established and proteome-derived AMR signatures. OMPs - outer membrane proteins; CE - cell envelope; C - cytoplasm; COG - Cluster of Orthologous Genes.