Despite rapid advances in the diagnosis of bacterial infections and the availability of effective antibiotics, meningococcal disease continues to represent a substantial public health problem for most countries (Tzeng et al., 2000). N. meningitides is a Gram-negative bacterium that colonizes asympto matically the upper nasopharinx tract of about 5-15% of human population. The bacterium can traverse the epithelium and reach the blood stream causing septicemia. From the blood meningococcus is able to cross the blood brain barrier and infect the meninges, causing meningitis potentially leading to shock and death (van et al., 2000; Stephens, 2007). 95% of total cases of meningitis are caused by five major serogroups: A, B, C, Y and W135. Vaccines against serogroups A, C, Y and W135 were developed in the 1960s by using the purified capsular polysaccharide as antigen. In the last 40 years a lot of efforts have been directed to the identification of meningococcus B antigens as basis of new vaccines. However, the high variability of these proteins among the different MenB strains represents a serious obstacle to the production of a globally effective anti-MenB vaccine (Stephens et al., 2007).

Till date the complete genome sequence of about 863 bacteria has been determined and 1653 bacterial genome projects are currently in progress. Availability of genome sequences of pathogens has provided a tremendous amount of information that can be useful in drug target and vaccine target identification. Any gene vital to sustain life and cellular growth of pathogenic organism may be a possible drug target, particularly when the sequence of that gene has little or no similarity to those of the host organism. The differences in the proteins of the host and the pathogen can be effectively used for designing a drug specifically targeting the pathogen. The application of in silico subtractive genomics technology significantly shortens the time required both for identification of antigens and their utility as protective immunogen. Subtractive genomics help enables the subtraction dataset between the host and pathogen genome and provides information for a set of genes that are likely to be essential to the pathogen but absent in the host. This approach has been used successfully to identify essential genes in Pseudomonas aeruginosa (Sakharkar et al., 2004; Perumal et al., 2007). Same methodology was implemented by us for the analysis of the whole proteome sequence of the human pathogen Neisseria meningitides Serogroup B.

The current studies makes use of the subtractive genomics approach,database of essential gene (DEG), differential pathway analysis and sub cellular localization prediction to analyze the complete proteome of N. meningitides serogroup B to search for potential drug and vaccine targets

The essential proteins in N. meningitides serogroup B were identified using subtractive genomics approach and further analyzed for identification of potential drug targets. The flow chart describing the detailed methodology for identifying of pathogen specific essential proteins using subtractive genomics approach is mentioned in Figure 1.

N. meningitides serogroup B proteome was found to contain 2001 proteins. Out of these, 1939 non-paralogs sequences were retrieved by using CD-HIT program at 60% identity. All the proteins of N. meningitides that are non-homologous to the human proteome could not be taken directly as targets as these also include a large number of proteins which are not essential for the viability of the organism. Therefore, out of 1939 proteins, 1413 proteins were identified as non-homologues to the human proteome and were subjected to BLASTP against DEG with E-value cutoff score of 10-10 and 30% identity. 362 proteins comprising 18% of the total number of protein coding sequences in N. meningitides serogroup B found to be essential. Detailed analysis of N. meningitides serogroup B proteome is mentioned in supplementary table 1

Functional classification of the 36 putative uncharacterized essential proteins was performed by using the SVMProt web server based on P value, which is the expected classification accuracy in terms of percentage. 10 proteins were classified as transmembrane proteins , 6 as zinc binding, 4 as lipid-binding, 4 as iron binding, as transferases, 1 as manganese binding, 1 as oxidoreductases, 1 as carbon-carbon lyases, 1 as carbon-sulfur Lyases, 1 as electrochemical potential driven Transporter, 1 as DNA-binding protein (Supplementary Table 2) . The predicted transmembrane proteins and transporter protein may be considered as potential drug targets

The sub cellular localization prediction for the 362 essential proteins of N. meningitides was done by using PA-SUB server to locate the outer membrane proteins which could be probable vaccine targets. 9 outer membrane proteins and 10 periplasmic proteins were identified, which are listed in Supplementary Table 3.

Metabolic pathway analysis of the essential proteins of N. meningitides was done by KEGG Automatic Annotation Server (KAAS). Result of comparative analysis of the metabolic pathways of the host and pathogen by using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database reveals 11 pathways are unique to N.meningitides serogroup B (supplementary table 4). Thereafter, each selected pathway was screened for the presence of pathogen specific essential enzymes. The C5-branched dibasic acid metabolism pathway did not show involvement of any meningococcal essential protein. As shown in supplementary table 4, 33 non-human homologous essential proteins identified from the remaining 10 pathogen specific pathways. This small group of proteins is required to be further verified for their role in meningococcal survival and virulence by mutagenesis study.

In the present study, phosphoenolpyruvate carboxylase (ppc) has been identified as a possible drug target due to its involvement in carbon fixation in photosynthetic organisms, reductive carboxylate cycle and pyruvate metabolism.

Lipopolysaccharides (LPS) and peptidoglycans are the main constituents of the outer cell wall of gram negative bacteria and play an important role in pathogenesis. Out of the thirteen enzymes involved in LPS biosynthetic pathways, eleven enzymes found to be essential for the viability of bacteria, were identified as drug targets as these did not show homology to any human gene. Two proteins (lipid A biosynthesis lauroyl acyl transferase and D-beta-D-heptose 7-phosphate kinase) are not essential meningococcal enzymes

Type II secretion pathway supports the translocation of proteins associated with the virulence factors, across the outer membrane. Five virulent factors i.e type IV pilus assembly protein (pilF), pilus assembly protein (pilG), twitching motility protein1 (pilT-1), twitching motility protein2 (pilT-2), competence protein (comA) are found to be involved in pathogen specific Type II secretion pathway. 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 (Rothbard et al., 1985).In this study, it has been found that type IV pilus assembly protein (PilF) may be considered for effective anti-meningococcal drug development

The bacterial two component system is crucial for the growth and survival under extreme conditions. Eight essential enzymes found to be potential drug targets in this pathway. Among those identified enzymes, tryptophan synthase subunit a (trpA), tryptophan synthase subunit b (trpB), indole-3-glycerol-phosphate synthase (trpC), anthranilate phosphoribosyltransferase (trpD) and anthranilate synthase component I (trpE), are also found to be key components in tyrosine, and tryptophan biosynthesis pathways. Therefore, targeting of these three enzymes may disrupt pathways essential for N. meningitidis survival and virulence and therefore might be a potential antibacterial therapeutic strategy.

Thus, further investigations on the predicted essential proteins are required to verify the reliability of the data. The complete list of the proteins identified via this in silico approach is available as supplementary data

The large scale genome sequencing projects have increased the availability of completely sequenced genomic and proteomic data in public domain. Use of the DEG database is more efficient than conventional methods for identification of essential genes and facilitates the exploratory identification of the most relevant drug targets in the pathogen. The present study has thus led to the identification of several proteins that can be targeted for effective drug design and vaccine development against N.meningitides serogroup B. The drugs developed against these will be specific to the pathogen, and therefore less or not toxic to the host. Since the number of essential genes in the metabolic pathways of N.meningitides serogroup B, identified in the present study, is relatively small (only 33), these can be further characterized and their role in the survival of the bacteria can be verified. Homology modelling of these targets will help identify the best possible sites that can be targeted for drug design by simulation modelling. Virtual screening against these novel targets might be useful in the discovery of novel therapeutic compounds against N. meningitides serogroup B.

The Bioinformatics Facility at our institution, where the work was done, is supported by the Indian Council of Medical Research, New Delhi and the Department of Biotechnology, Government of India, New Delhi