Quantitative Proteomics of the Neisseria Gonorrhoeae Cell Envelope and Membrane Vesicles for the Discovery of Potential Therapeutic Targets

Abstract

Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC physiology, which may represent promising targets for designing new therapeutic interventions. Molecular &

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

“…An N. gonorrhoeae FA1090 mutant lacking NGO1985 was the most severely affected, exhibiting increased sensitivity to all of the compounds tested, while the Δngo2121 mutant was sensitive to bile salts and polymyxin B. In contrast, gonococci lacking NGO1344 or NGO2111 were not affected by any of the compounds tested (6).…”

“…S1. In both the previous study (6) and the current one, the Δngo1985 mutant exhibited the most striking phenotypes with three and 46 detrimental and beneficial conditions, respectively (Fig. 4).…”

“…Therefore, we predict that vaccine candidates selected from the FA1090 proteome will provide protection against heterologous strains. In support of our strategy, mice treated with interleukin-12 and immunized with membrane vesicles derived from FA1090 that contain proteins investigated in the current study (6) are protected against infection by both laboratory strains and clinical isolates (15).…”

“…Little is known about the functions of most lipoproteins (1), but those for which information is available have been shown to be involved in nutrient acquisition, cell wall structure and metabolism, cell signaling, antibiotic resistance, attachment to host cells, and ligands for Toll-like receptor 2, which triggers an innate immune response and helps establish adaptive immunity (4). Because of this potential interaction between host and pathogen, we are currently pursuing 20 novel outer membrane-localized proteins, including lipoproteins, as vaccine candidates against Neisseria gonorrhoeae, the gonococcus (GC) (5,6).…”

“…Using proteomics-based reverse vaccinology (16), we have investigated the composition of N. gonorrhoeae cell envelopes and naturally released membrane vesicles across common laboratory strains, including FA1090, MS11, and FA19 (6), as well as FA1090 cell envelopes under four different host-relevant growth conditions (5) to identify vaccine candidate targets. Among the ubiquitously expressed proteins in four examined isolates were novel vaccine candidates NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344 (5,6), with predicted functions associated with cell envelope homeostasis. Herein, we utilized Biolog Phenotype MicroArrays (PMs) to identify the phenotypes of gene knockout mutants in our six vaccine candidates.…”