|In Silico Biology 9, 0019 (2009); ©2009, Bioinformation Systems e.V.|
1 Centre for Genomics and Applied Gene Technology, Institute of Integrative Omics and Applied Biotechnology (IIOAB), Nonakuri, Purba Medinipur, WB-721172, India
2 School of Biotechnology, Devi Ahilya University, Khandwa Road Campus, Indore, MP-452001, India
* Corresponding author
Edited by H. Michael; received February 05, 2009; revised May 01, 2009; accepted May 05, 2009; published May 10, 2009
Neisseria gonorrhoeae is responsible for causing gonorrhea, one of the most common sexually transmitted diseases prevailing globally. Although extensive researches are in progress in order to control the transmission of the disease and to develop drug(s) against the pathogen, till date no effective vaccine or specific drug could be developed and only antibiotic treatment is in use. Perhaps, due to excess use of antibiotics, several resistant strains have been found. In the present study, metabolic pathways-related candidate drug and vaccine targets have been identified in N. gonorrhoeae virulent strain FA 1090 using an in silico subtractive genomics approach. 106 putative drug targets out of 537 essential genes have been predicted. 67 cytoplasmic and 9 membrane enzymes, along with 10 membrane transporters are found to be the potential drug targets from the host-pathogen common metabolic pathways. Among these targets, competence lipoproteins (NGO0277) and cysW have been identified as candidate vaccine targets. 20 drug targets have been identified from pathogen specific unique metabolic pathways. Out of these, 6 enzymes are involved in dual metabolic pathways and 2 are expressed in cell wall and fimbrium. These gonococci-specific proteins are expected to be better possible drug targets. Screening of the functional inhibitors against these novel targets may result in discovery of novel therapeutic compounds that can be effective against antibiotic resistant strains.
Keywords: drug targets, essential genes, genome analysis, metabolic pathways targets, Neisseria gonorrhoeae, subtractive genomics, candidate vaccine targets
Neisseria gonorrhoeae is a Gram-negative diplococcus and obligate human pathogen that primarily colonizes and invades the reproductive mucosal epithelium and causes one of the most common sexually transmitted diseases known as gonorrhea. Every year about sixty million cases of gonorrhea are reported globally . Nearly 45% of the infected women exhibited ascending gonococcal infection and subsequently pelvic inflammatory disease (PID) [2, 3]. The PID frequently causes infertility and ectopic pregnancy due to permanent blockage of the fallopian tube . The infection in men leads to urethritis, epididymitis, and prostatitis .
Although several virulent factors have been reported in N. gonorrhoeae, and extensive researches are in progress to develop effective therapeutics against the pathogen, still barrier contraceptives and antibiotic therapy are generally prescribed to control transmission and prevalence [2, 5]. Drug resistant strains are also prevalent [6, 7]. There is no vaccine available so far. Therefore, N. gonorrhoeae transmission and infection is a global public health problem . The challenge remains unsolved due to the wide adoptability of the pathogen and the lack of standard experimental animal disease models. However, chimpanzee  and non-primate models viz. mouse, guinea pig, rabbit, rat, hamster, chicken have been developed [10, 11].
In silico subtractive genomics approaches, based on the strategy that an essential survival gene non-homologous to any human host gene is a candidate drug target for a given pathogen [12, 13], have been used to identify putative drug targets in P. aeruginosa [14, 15], H. pylori [16, 17], B. pseudomallei , and A. hydrophila . In the present study, a similar approach has been carried out to screen N. gonorrhoeae virulent strain FA 1090 genome and proteome (2002 proteins) in order to identify its essential genes and subsequent drug and vaccine targets from various metabolic pathways.
N. gonorrhoeae FA 1090 complete genome sequence, BLAST tool and databases primarily NCBI, DEG , and KEGG  were used in the present study. The complete genome and proteome of the pathogen were downloaded from the NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/). A structured database of N. gonorrhoeae strain FA 1090 was also accessed from its location (http://stdgen.northwestern.edu) as reference dataset.
To identify essential genes in N. gonorrhoeae genome, each functional gene and corresponding protein sequence of the bacteria were subjected to standard blastx and blastp, respectively, against DEG (http://tubic.tju.edu.cn/deg). Neisseria homologs that showed significant hits against DEG listed essential genes were selected based on the blastp scores. For short listing these essential genes, cut-off values for bit score, E-value, and percentage of identity at amino acid level were considered >100, <E-10, and >35%, respectively. Genes coding for less than 100 amino acids length were not considered. Selected essential genes of the pathogen were then classified according to Clusters of Orthologous Groups of Proteins (COGs) nomenclature.
Each identified essential gene and corresponding protein sequence of the pathogen were analyzed for sequence homology with the human genome using standard human blastx and blastp in NCBI server and non-homologous essential genes those do not show any similarity with any human sequence were considered as putative drug targets. The function and cellular localization of each protein of identified targets was analyzed with Swiss-Prot protein database (http://us.expasy.org/sprot) and by using sub-cellular localization prediction tools, CELLO , PSLpred , PSORTb , and SOSUI-GramN . Surface and membrane localized proteins were selected for putative candidate vaccine targets. Lists of the selected genes from various pathways were prepared based on the essential non-human homologs that are involved in pathways common in both the host and the pathogen and from pathogen specific metabolic pathways by using the KEGG database. Essential non-human homolog virulent factors were identified by comparing virulence factors of N. gonorrhoeae FA 1090 listed in Virulence Factors of Pathogenic Bacteria Database (VFDB)  with those identified essential genes and drug targets in this study.
We identified 537 genes comprising 26% of the total number of protein coding sequences in N. gonorrhoeae strain FA 1090 genome to be essential and can be grouped into 21 classes according to COGs functional classification (Fig. 1). However, Singh et al.  identified only 223 essential genes using the T-iDT tool supported with DEG version 2.5. The higher number of the essential genes in the present study is due to the increased number of essential genes in the current version of DEG (version 5.2). The identified essential genes are found to be of various functional groups. Genes that are involved in translational machinery constitute the largest group (91 genes) and the RNA processing group represents the smallest number (1 gene). The ratio of the number of essential genes to non-human homologs within a functional group is found to be high (91 to 14) in the translational machinery class of genes, whereas is equal (4 to 4) in the signal transducer group of genes (Fig. 1).
Click on the thumbnail to enlarge the picture
|Figure 1: COGs classification of N. gonorrhoeae essential genes and their comparison with non-human homologs.|
Considering host-pathogen common metabolic pathways, 76 metabolic enzymes are found to be present only in N. gonorrhoeae. Among these enzymes, 67 are cytoplasmic (potential drug targets) (Supplementary Table 1) and 9 are predicted to be localized in the membrane (Supplementary Table 2). However, one is a surface enzyme (competence lipoprotein: NGO0277). From host-pathogen common metabolic pathways, nine integrals to membrane and one cell wall localized gonococcal essential non-human homolog transporter are also identified (Supplementary Table 2). Nine membrane enzymes and ten membrane transporters are found to be potential candidate vaccine targets from common metabolic pathways where sulfate transport permease protein C (cysW) may be one of the best targets (Supplementary Table 2).
On comparison with the list of human metabolic pathways using the KEGG database, 10 pathways are found to be unique in N. gonorrhoeae. Thereafter, each selected pathway was screened for the presence of listed essential enzymes and surface proteins. Four pathogen specific pathways namely, C5-branched dibasic acid metabolism, streptomycin biosynthesis, polyketide sugar unit biosynthesis, and novobiocin biosynthesis did not show involvement of any gonococcal non-human homologous essential enzyme or surface protein.
As shown in Supplementary Table 2, twenty non-human homologous essential proteins viz. 15 enzymes, 3 nitrogen responsive regulators, and 2 pilins are identified from the remaining six pathogen specific pathways. This small group of proteins is required to be further verified for their role(s) in gonococcul survival and virulence by mutagenesis study.
The N. gonorrhoeae outer membrane lipo-oligosaccharides confer the bacterial virulence [28, 29]. Hence, targeting the lipo-polysaccharide biosynthetic pathway may be effective to prevent the infection. The IgA1 proteases , Opa, lipo-oligosaccharides, protein-I, lactoferrin (Lbpl, Lbp2) , and 2C7 oligosaccharide (OS) epitope  are reported to be vaccine candidates. Among eight proteins from lipo-polysaccharide biosynthetic pathway identified, UDP-3-O-acyl N-acetylglucosamine deacetylase (lpxC/envA) and lipopolysaccharide heptosyltransferase-I (rfaC) are also found to be involved in amino acid metabolism (arginine, proline, histidine) and glycan structures - biosynthesis-2 pathways. Therefore, envA and rfaC being involved in dual metabolic pathways may be considered as better targets.
The bacterial two component system is crucial for the growth and survival under extreme conditions. Four essential enzymes and two regulator proteins are found to be potential drug targets in this pathway. Among those identified enzymes, tryptophan synthase subunit a (trpA), indole-3-glycerol-phosphate synthase (trpC), and anthranilate phosphoribosyltransferase (trpD) are also found to be key components in phenylalanine, tyrosine, and tryptophan biosynthesis pathways. Therefore, targeting of these three enzymes may disrupt pathways essential for N. gonorrhoeae survival and virulence and therefore might be a potential antibacterial therapeutic strategy.
In a comparative study, four virulent factors essential for the pathogen are identified from the pool of essential non-human homologous genes. Two virulent factors rfaF and PilF are found to be involved in pathogen specific unique metabolic pathways (lipopolysaccharide biosynthesis and type II secretion system). The other two virulent factors namely, ABC transporter iron-uptake permease inner membrane protein (afuB/fbpB: NGO0216) and transferrin-binding protein-A (TbpA: NGO1495) are identified as membrane transporters (Supplementary Table 2, marked with an asterisk, *).
Type IV pilus assembly proteins are the major components in type II secretion pathway. Type IV pili are associated with bacterial adhesion, aggregation, invasion, host cell signaling, surface motility, and natural transformation . Pilin from gonococcal strain MS11  have been reported to be possible vaccine targets and fragments of purified pili proteins have been patented for preparation of vaccine against N. gonorrhoeae (US Patent: 4443431). In this study, it has been found that type IV pilus assembly protein (PilF) and putative type IV pilin protein (pilV) may be considered for effective anti-gonorrhoeae drug development. The pilV is expressed in fimbrium and therefore, it may be a suitable candidate vaccine target. However, conserved PilE globular domain is found to be non-immunogenic in mouse . Therefore, an effective epitope designing is essential for effective vaccine development with PilE.
In the present study, fructose-1,6-bisphosphate aldolase (alf/tsr) has been identified as a possible drug target due to its involvement in carbon fixation in photosynthetic organisms and host-pathogen common pathways (glycolysis/ pentose phosphate pathway/ fructose and mannose metabolic pathways). Similarly, putative two-components system transcriptional response regulator (ptsN) is found to be an ideal target to block the phosphotransferase system and is unique to the pathogen. Therefore, alf and ptsN may also be taken as good drug targets.
PorB has been reported to be an effective DNA vaccine against the gonococcal infection . Similarly, the outer membrane phospholipase A (pldA)  and transferrin-binding proteins (tbpA and tbpB) have been found to be promising vaccine targets [37-39]. The tspA [T-cell stimulating protein-A (GeneID: 5795626)] and tspB [T-cell stimulating protein-B (GeneID: 904097)] of N. meningitides have been patented as vaccine candidates against pathogenic Neisseria (US patent: 6861507). However, these have not been found much promising in practice. In the present study, screening of the essential genes revealed that tbpA is an essential surface protein that plays a role in inorganic ions transport but is not found to be involved in any metabolic pathway. The identified outer membrane associated proteins viz. competence lipoprotein (nitrogen assimilation pathway) and ddl (D-alanine metabolism) that are essential for vital metabolic, signal transduction, and transport pathways may be considered as alternative options for both targeted drug and vaccine development.
Gonococcal transmission and infection is a global health problem and effective drugs and vaccines are yet to be developed. Here, we have identified six best possible enzyme drug targets and three vaccine targets from various metabolic pathways that are expected to be essential for the pathogen. These are predicted to be superior targets than those identified earlier. However, these targets should be experimentally validated for their role in bacterial survival and virulence. Fold level homology modeling should be carried out for candidate vaccine targets to elucidate the best possible sites and most effective epitopes that can be useful for simulation modeling and vaccine designing. Similarly, better animal models of gonorrhea are needed to study the efficacy of these targets. The functional inhibitors screening against these novel targets might be useful in the discovery of novel therapeutic compounds against drug resistant strains.
We duly acknowledge the motivation and encouragement of all IIOAB members throughout the study.