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

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.Neisseria gonorrhoeae (GC)¹ is an obligate human pathogen and the etiological agent of gonorrhea, a sexually transmitted disease. GC infection remains a significant health and economic burden worldwide (1). It is also the second most commonly reported infectious disease in the United States (2). Gonorrhea ranges from clinically asymptomatic to local genital infections to disseminated bloodstream infections. Asymptomatic infections often have devastating consequences on women''s health including pelvic inflammatory disease, ectopic pregnancy, and infertility (3). Additionally, GC infections facilitate transmission and acquisition of HIV (4). For all of these reasons it is critical to provide effective treatments against gonorrhea. Currently, a dual therapy with cephalosporin and either azithromycin or doxycycline is recommended (5). However, over the past several years treatment failures associated with GC strains displaying decreased susceptibility to extended spectrum cephalosporins have been reported from various parts of the world (6–9). This is especially concerning because no other antibiotics are clinically useful in these cases, and because no appropriate vaccine exists (10). The escalating problem of the spread of antimicrobial resistance in GC, and the importance of the development of new approaches to study, treat, and prevent GC infection, have been recognized by the World Health Organization and by the Centers for Disease Control and Prevention (11, 12).We propose that largely unexplored proteins localized to bacterial cell envelope and naturally released membrane vesicles are particularly promising as potential novel molecular targets for therapeutic interventions against gonorrhea. The small fraction of known components of the GC cell envelope (outer membrane, periplasm, cytoplasmic membrane) plays a fundamental role in establishing infection by enabling the microbes to adhere to and invade host cells, facilitating nutrient acquisition, host tissue destruction, and suppression of immune responses (3, 13–15). Further, GC, like many other Gram-negative bacteria, produces membrane vesicles (MVs), which are nano-sized bilayered proteolipids (16). Naturally produced MVs are potentially an effective way to deliver toxins, enzymes, and other effectors to host tissues. Additionally, evidence from various studies support that MVs participate in intercellular communication and horizontal gene transfer (16–21). In GC, MVs are necessary for biofilm formation, which is thought to play an important role in asymptomatic infection in women, resistance to antimicrobial agents, and suppression of host immune defenses (22–24). MVs may also contribute to the serum resistance by providing an enhanced ability to bind and eliminate bactericidal factors (17).Although the potential importance of proteins localized to the GC cell envelope and MVs has been reported previously (25, 26), only two proteomic studies have been published addressing GC membrane composition (27, 28). Most studies have focused on extensive characterization of factors involved in direct host cell interaction: protruding surface proteins (pili), outer membrane adhesins Opa, porins P.IA and P.IB, lipooligosaccharide, and several iron utilization proteins (3, 4, 15, 29–32). Many of these vital virulence factors undergo phase and/or antigenic variation, making them poor drug or vaccine targets. Therefore, the pursuit for novel and constitutively expressed proteins—therapeutic targets in GC—is of utmost importance.Accordingly, in this study we applied global and unbiased proteomics to compare the composition of both the cell envelopes and MVs isolated from four GC strains: FA1090, F62, MS11, and 1291. Specifically, we used isotope tagging for relative and absolute quantification (iTRAQ) coupled with multidimensional liquid chromatography and tandem mass spectrometry (2D-LC/MS/MS). This approach allowed us to determine a uniformly and differentially expressed repertoire of proteins. Focusing on a homolog of LPS transport protein, LptD (OstA, Imp), which was identified in both the cell envelopes and MVs fractions, and ubiquitously expressed among analyzed strains, we showed that its depletion led to loss of GC viability. Finally, we selected for initial characterization six predicted outer membrane proteins, which were present at similar levels in the GC cell envelopes. We generated Δngo1344, Δngo1955, Δngo1985, Δngo2111, Δngo2121, and Δngo2139 mutant strains and examined their sensitivity toward different cell envelope-perturbing agents as well as chloramphenicol. These studies showed that the lack of NGO1985 resulted in dramatically decreased GC viability, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Overall, these findings further support our hypothesis that the conserved proteins may represent promising targets for designing new therapeutic interventions.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.     

Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication

In vitro infection by human T-cell leukemia virus type 1 and 2 (HTLV-1 and HTLV-2) can result in syncytium formation, facilitating viral entry. Using cell lines that were susceptible to HTLV-2-mediated syncytium formation but were nonfusogenic with HTLV-1, we constructed chimeric envelopes between HTLV-1 and -2 and assayed for the ability to induce syncytia in BJAB cells and HeLa cells. We have identified a fusion domain composed of the first 64 amino acids at the amino terminus of the HTLV-2 transmembrane protein, p21, the retention of which was required for syncytium induction. Construction of replication-competent HTLV genomic clones allowed us to correlate the ability of HTLV-2 to induce syncytia with the ability to replicate in BJAB cells. Differences in the ability to induce syncytia were not due to differences in the levels of total or cell membrane-associated envelope or in the formation of multimers. Therefore, we have localized a fusion domain within the amino terminus of the transmembrane protein of HTLV-2 envelope that is necessary for syncytium induction and viral replication.Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are type C retroviruses that have been associated with a variety of human malignancies. HTLV-1 is the etiological agent of adult T-cell leukemia as well as a degenerative neurological disorder, HTLV-1-associated myelopathy/tropical spastic paraparesis (28, 40, 58, 60, 83). Recent reports have also implicated HTLV-1 infection with arthropathy (42, 65), polymyosis (23, 37), and uveitis (48, 49, 51). HTLV-2 has been associated with a rare form of atypical hairy cell leukemia (62, 63, 68) as well as some cases of neuropathy (33, 39). It is estimated that between 10 million and 20 million individuals worldwide are infected with HTLV, with an overall risk of 5% of disease progression in infected individuals (14). HTLV is endemic in southern Japan, the Caribbean Basin, and Central and South America. In the United States, recent reports have identified a high proportion of HTLV, especially HTLV-2, infection in intravenous-drug abusers (44, 61, 64).Cell-to-cell contact is considered critical for the in vivo and in vitro transmission of HTLV-1 and HTLV-2, as infection by cell-free HTLV virus is inefficient in vitro and in vivo. By analogy with other enveloped viruses, HTLV infection of susceptible cells is likely mediated by the envelope glycoprotein. Antibodies against HTLV envelope are protective against infection in vivo (71, 80), and multiple epitopes that elicit neutralizing antibodies have been identified throughout the protein (31, 34, 56). Initially synthesized as a precursor protein, gp61, HTLV envelope is subsequently modified by glycosylation and cleaved into two subunits, gp46 and p21. The external surface glycoprotein, gp46, is anchored to the cell surface by noncovalent association with the transmembrane envelope glycoprotein, p21. Interaction of envelope with the as yet unidentified cellular receptor leads to cell-to-cell fusion and can result in syncytium formation.We were interested in identifying the molecular determinants of HTLV involved in syncytium formation and viral entry. Our laboratory has several cell lines that are permissive to HTLV-2- but not HTLV-1-mediated cell fusion. Therefore, we constructed recombinants between the HTLV-1 and -2 envelope genes and assayed for the loss of syncytium induction in BJAB cells and HeLa cells. Loss of a 64-amino-acid (aa) domain located at the amino terminus of the HTLV-2 transmembrane protein, p21, correlated with a loss in the ability of the envelope chimera to induce cell fusion. When the chimeric envelopes were expressed in the context of replication-competent genomic clones, there was a good correlation between syncytium induction and the ability to replicate in permissive cells. Present within the identified fusion domain is a hydrophobic region and a heptad repeat resembling a leucine zipper. We examined the contribution of the fusion domain to the structural integrity of the HTLV-2 envelope by using a vaccinia virus expression system. None of the recombinants affected the synthesis, transport, or oligomer formation of the HTLV glycoprotein complex.