Abstract
Expanding efforts to develop preventive gonorrhea vaccines is critical because of the dire possibility of untreatable gonococcal infections. Reverse vaccinology, which includes genome and proteome mining, has proven very successful in the discovery of vaccine candidates against many pathogenic bacteria. However, progress with this approach for a gonorrhea vaccine remains in its infancy. Accordingly, we applied a comprehensive proteomic platform-isobaric tagging for absolute quantification coupled with two-dimensional liquid chromatography and mass spectrometry-to identify potential gonococcal vaccine antigens. Our previous analyses focused on cell envelopes and naturally released membrane vesicles derived from four different Neisseria gonorrhoeae strains. Here, we extended these studies to identify cell envelope proteins of N. gonorrhoeae that are ubiquitously expressed and specifically induced by physiologically relevant environmental stimuli: oxygen availability, iron deprivation, and the presence of human serum. Together, these studies enabled the identification of numerous potential gonorrhea vaccine targets. Initial characterization of five novel vaccine candidate antigens that were ubiquitously expressed under these different growth conditions demonstrated that homologs of BamA (NGO1801), LptD (NGO1715), and TamA (NGO1956), and two uncharacterized proteins, NGO2054 and NGO2139, were surface exposed, secreted via naturally released membrane vesicles, and elicited bactericidal antibodies that cross-reacted with a panel of temporally and geographically diverse isolates. In addition, analysis of polymorphisms at the nucleotide and amino acid levels showed that these vaccine candidates are highly conserved among N. gonorrhoeae strains. Finally, depletion of BamA caused a loss of N. gonorrhoeae viability, suggesting it may be an essential target. Together, our data strongly support the use of proteomics-driven discovery of potential vaccine targets as a sound approach for identifying promising gonococcal antigens.
Highlights
From the ‡Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon; §Proteomics Facility, Fred Hutchinson Cancer Research Center, Seattle, Washington; ¶Neisseria Reference Laboratory, Department of Global Health, University of Washington, Seattle, Washington; ʈDepartments of Medicine and Global Health, University of Washington, Seattle, Washington; **Department of Microbiology and Immunology, F
Bacterial Strains and Growth Conditions—The following N. gonorrhoeae strains were used in this study: FA1090 [22], MS11 [23], 1291 [24], F62 [25], FA19 [26], the clinical isolates LGB1, LG14, LG20, and LG26, which were collected from two public health clinics in Baltimore from 1991 to 1994 and differ in porB variable region type and pulsed gel electrophoresis patterns [21, 27], 13 isolates from patients attending the Public Health-Seattle & King County Sexually Transmitted Disease clinic from 2011 to 2013, and the WHO 2015 reference strains [10, 28, 29]
Different N. gonorrhoeae clinical isolates were collected from GCB plates, followed by suspension in prewarmed gonococcal base liquid (GCBL), and measurement of the cell density at OD600.Fractions containing either cytoplasmic, cell envelope, membrane vesicles (MVs), or secreted proteins, or whole-cell lysates matched by equivalent OD600 units, were prepared in SDS sample buffer in the presence of 50 mM dithiothreitol and separated in either 10 –20% Criterion Tris-Tricine TGX (BioRad) or 4 –20% Mini-PROTEAN TGX precast gels (Bio-Rad)
Summary
Bacterial Strains and Growth Conditions—The following N. gonorrhoeae strains were used in this study: FA1090 [22], MS11 [23], 1291 [24], F62 [25], FA19 [26], the clinical isolates LGB1, LG14, LG20, and LG26, which were collected from two public health clinics in Baltimore from 1991 to 1994 and differ in porB variable region type and pulsed gel electrophoresis patterns [21, 27], 13 isolates from patients attending the Public Health-Seattle & King County Sexually Transmitted Disease clinic from 2011 to 2013 (supplemental Table S1), and the WHO 2015 reference strains [10, 28, 29]. The following iTRAQ tags were used to label peptides in the cell envelope fractions derived from N. gonorrhoeae cultured on GCB under four growth conditions: for standard aerobic; for growth in the presence of 7.5% normal human serum; for iron-limited; and for anaerobic. Different N. gonorrhoeae clinical isolates were collected from GCB plates, followed by suspension in prewarmed GCBL, and measurement of the cell density at OD600.Fractions containing either cytoplasmic, cell envelope, MVs, or secreted proteins (the same amount of total protein loaded per well), or whole-cell lysates matched by equivalent OD600 units, were prepared in SDS sample buffer in the presence of 50 mM dithiothreitol and separated in either 10 –20% Criterion Tris-Tricine TGX (BioRad) or 4 –20% Mini-PROTEAN TGX precast gels (Bio-Rad). All other experiments were conducted at least in biological triplicates and mean values with corresponding S.E. were presented
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