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.
Highlights
From the ‡Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon 97331; §Proteomics Facility, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024
We propose that largely unexplored proteins localized to bacterial cell envelope and naturally released membrane vesicles are promising as potential novel molecular targets for therapeutic interventions against gonorrhea
Focusing on a homolog of LPS transport protein, LptD (OstA, Imp), which was identified in both the cell envelopes and membrane vesicles (MVs) fractions, and ubiquitously expressed among analyzed strains, we showed that its depletion led to loss of GC viability
Summary
Cells were separated from culture supernatants by low speed centrifugation (6000 ϫ g), and both membrane fractions and MVs were prepared for proteomic analysis as described below. The resulting PCR products were subjected to digestion with appropriate restriction enzymes and cloned into pUC18K-ngo-up These final constructs, pUC18K-⌬ngo, contained kanamycin resistance cassette [54] flanked by homologous regions for recombination and allelic exchange. The PCR product was subjected to digestion with endonuclease FseI and subcloned into ScaI/FseI-pGGC4, to yield pGCC4-Plac ngo1985, which was subsequently used to complement the ⌬ngo1985 strain as described above. Bacterial growth was monitored every hour by both measuring absorbance at OD600 and assessing cell viability by spotting 10 l of 10-fold serial dilutions of GC cultures onto GCB agar plates with or without IPTG. 3 mg (1 mg/ml) of purified, recombinant soluble form of AniA was submitted to Pacific Immunology
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