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. 2016 Jul;15(7):2338-55.
doi: 10.1074/mcp.M116.058800. Epub 2016 May 2.

Proteomics-driven Antigen Discovery for Development of Vaccines Against Gonorrhea

Ryszard A Zielke  1 , Igor H Wierzbicki  1 , Benjamin I Baarda  1 , Philip R Gafken  2 , Olusegun O Soge  3 , King K Holmes  4 , Ann E Jerse  5 , Magnus Unemo  6 , Aleksandra E Sikora  7
Affiliations

Proteomics-driven Antigen Discovery for Development of Vaccines Against Gonorrhea

Ryszard A Zielke et al. Mol Cell Proteomics. 2016 Jul.

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.

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Figures

Fig. 1.
Fig. 1.
Experimental design of quantitative proteomic profiling of the Neisseria gonorrhoeae cell envelopes across different growth conditions. N. gonorrhoeae strain FA1090 was cultured on gonococcal base agar solid medium (GCB) aerobically under standard growth conditions (SGC); on GCB with the addition of 7.5% normal human serum (+NHS); iron limited [GCB without ferric nitrate in Kellogg's Supplements and with deferoxamine mesylate salt at 5 μm final concentration (-Iron)]; and anaerobically on GCB with nitrite as a terminal electron acceptor (-O2). Bacteria were harvested from solid media when the colonies reached approximately the same size. Following lysis, cell envelope proteins were enriched using a sodium carbonate extraction procedure and ultracentrifugation. The same amounts of proteins (100 μg) in each sample were subjected to trypsin digestion. Peptides in different samples were labeled with the following iTRAQ tags: 114 for SGC, 115 for NHS, 116 for -Iron, 117 for -O2. Following labeling, samples were pooled and the peptides were separated by strong cation exchange and reversed-phase chromatography. The Orbitrap Elite was used to collect mass spectra and proteins were identified and quantified using Proteome Discoverer. All experiments described above were repeated on three separate occasions.
Fig. 2.
Fig. 2.
Analysis of identified proteins. A, Venn diagram illustrating the distribution of proteins identified in independent triplicate proteomic experiments. A total of 890, 853, and 956 individual protein species were identified in Experiment 1, 2, and 3; respectively. Further analyses were applied to 751 common proteins identified in all experiments. B, A phylogenetic classification of common proteins was accomplished using the Clusters of Orthologous Groups of proteins (COGs). Letters and digits displayed on the pie chart represent individual COGs and numbers of proteins assigned to each phylogenetic group. C, Heat map illustrating the changes in N. gonorrhoeae proteome upon exposure to the environmental cues relevant to infection. Relative abundance of proteins identified during different growth conditions (NHS, -Iron, and -O2) was compared with the levels of corresponding protein species found in SGC. The color scale covers from fivefold down-regulation (blue), through no change (white), to fivefold up-regulation (red). D, Decision tree to select gonorrhea vaccine candidates from the group of 751 common N. gonorrhoeae cell envelope-associated proteins identified with 1% FDR in three independent experiments via high-throughput proteomic mining. This group contains 259 uniformly expressed and 416 specifically induced proteins. Cytoplasmic proteins were eliminated using bioinformatics predictions and literature searches. Vaccine candidates were ultimately selected from 92 ubiquitously expressed proteins identified by comparing the 168 uniformly expressed cell envelope proteins of this study with the 305 previously identified cell envelopes proteins isolated from FA1090, MS11, F62, and 1291 (21). Subsequently, five of these proteins that were localized to both cell envelopes and MVs fractions (21), and either had pivotal role(s) in other bacterial species (BamA, LptD, TamA) or unknown function (NGO2054 and NGO2139) were subjected to initial assessments as potential gonorrhea vaccine candidates. E, Analysis of anaerobic, iron, and NHS-responsive stimulons of N. gonorrhoeae. Differentially expressed proteins in response to anoxia, iron deprivation, and the presence of NHS were compared with identify the common anaerobic, iron-depleted, and NHS-responsive protein stimulon.
Fig. 3.
Fig. 3.
Generation of polyclonal antibodies and immunoblotting analysis. Recombinant versions of vaccine candidates BamA, LptD, TamA, NGO2054, and NGO2139, as well as proteins employed as controls in the experiments (Laz and BamD) were purified by Ni2+ affinity chromatography and subsequently used to obtain polyclonal rabbit antisera. Wild type, isogenic conditional mutants, or knockouts, as well as complemented variants of FA1090 (as indicated above each immunoblot) were harvested from GCB. Whole-cell lysates matched by equivalent OD600 units were separated in 4–20% Mini-PROTEAN TGX precast gels, transferred onto nitrocellulose membranes and probed with different polyclonal rabbit antibodies.
Fig. 4.
Fig. 4.
Subcellular localization of selected vaccine candidates. Equal amounts of subproteome fractions (C-cytoplasmic, CE-cell envelopes, MVs-naturally released membrane vesicles, SS-soluble fractions of supernatants) derived from FA1090 cultured under standard growth conditions in GCBL were separated in 4–20% gradient gels. The proteins were transferred to nitrocellulose and probed with individual polyclonal rabbit antibodies against tested proteins, as indicated on the left. Controls included protein markers for the periplasmic face of the outer membrane (BamD) and cytoplasm (ObgGC) protein.
Fig. 5.
Fig. 5.
Assessment of surface exposure of candidate antigens BamA, LptD, TamA, NGO2054, and NGO2139. A, Experimental outline of dot blotting of either intact or lysed N. gonorrhoeae FA1090 cells and protease accessibility studies using intact cells. B, Intact cells of FA1090 were spotted on nitrocellulose and probed with different polyclonal antibodies, as shown above individual dot blots. C, Intact and lysed cells were used to detect BamD and Obg, which were utilized as periplasmic and cytoplasmic protein markers, respectively. D, Intact FA1090 cells were incubated with increasing concentrations of trypsin (as indicated), lysed, separated in 4–20% Tris-glycine precast gels and probed with individual antisera. E, Intactness of the cells during trypsin treatment was verified by separation of total-cell lysates by SDS-PAGE and visualization of protein profiles with colloidal Coomassie G-250 staining.
Fig. 6.
Fig. 6.
The purified recombinant variants of BamA, LptD, TamA, NGO2054, and NGO2139 elicit bactericidal antibodies. Serum bactericidal assays were conducted against 5 × 103 CFUs/ml of FA1090 (serum resistant) and MS11 (serum sensitive) N. gonorrhoeae strains using the rabbit anti-BamA, anti-LptD, anti-TamA, anti-NGO2054, and anti-NGO2139 post-immune sera and NHS as the complement source. The rabbit sera were heat-inactivated at 56 °C for 30 min. The bacterial cells were pre-sensitized with dilutions of the antigen-specific heat-inactivated sera for 15 min, followed by the addition of NHS at 10% final concentration, and incubation continued for 30 min. The CFUs were determined by plating bacteria on solid media. The average percent killing was determined from at least four independent experiments and was calculated as the number of CFUs in samples incubated with rabbit post-immune sera and NHS to the number of CFUs recovered from samples treated with rabbit post-immune sera and HI-NHS. N. gonorrhoeae viability was not affected in any of the control samples.
Fig. 7.
Fig. 7.
Expression of potential vaccine targets was evaluated using a panel of 36 temporally and spatially diversified GC isolates. Clinical isolates of N. gonorrhoeae including WHO reference strains (as indicated) were harvested from GCB, matched by equivalent OD600 units and resolved in 4–20% Tris-glycine precast gels. Following transfer onto nitrocellulose membrane and blocking, the proteins were probed with polyclonal rabbit antisera against BamA, LptD, TamA, NGO2054, and NGO2139. An E. coli strain (ER2566) was used as a negative control.
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