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Increase in emergence of antibiotic resistant and pathogenic microorganisms is one of the biggest challenges for biomedical research and drug development. Bacillus anthracis, the etiological agent of anthrax, is found worldwide and is able to infect virtually all mammals. In the present study, comparative genomics analysis of metabolic pathways of the pathogen B.anthracis and the host Homo sapiens was carried out to identify proteins that are essential for the pathogen but absent in humans. In silico analyses and a customized manual mining identified .. non homologues proteins which could serve as potential drug and vaccine targets. Subcellular localization calculations were performed for each potential drug target. .. genes were predicted to be localized in the cytoplasm, .... were surface proteins. Out of these, .. targets are enzymes and .... are non-enzymes. The .. proteins of B.anthracis were further compared with the experimentally determined essential proteins of different bacterial species listed at DEG database. ... Proteins were found to be essential for survival of pathogen. Furthermore, selected non homologues genes were also evaluated by the drug bank server.
During the past several years, the possibilities of selecting targets using computational approaches with integrated "omics" data such as genomics, proteomics, metabolomics etc have been increasing continuously. Amongst these, two in silico methods; comparative genomics and subtractive genomics are being widely used for the prediction and identification of potential drug targets in numerous pathogenic bacteria and fungal species (Sakharkar et al. 2004; Perumal et al. 2007; Amineni et al. 2010; Abadio et al. 2011). In principle, these approaches rely on search for such genes/proteins which are absent in host but are present in the pathogen. Furthermore, these non-host homologues must be essential for the survival of the pathogen and serve as a critical component in vital physicochemical and metabolic pathways, so that a designed drug or a lead compound specific to such target(s) will only impact on the pathogen's system, without hampering host physiology or any aspect of host biology. A great piece of aid has been provided to this area with the availability of complete genome sequences of several pathogenic microorganisms. Such steps aim to reduce the problem of searching for potential drug targets from a large list to selecting from a chosen few. As we know that the most common mode of mechanism of anti-biotics is to act as inhibitors of targeted bacterial enzymes. Therefore, theoretically all enzymes which are specific to bacteria can be considered as potential drug targets (Galperin and Koonin 1999).
The Bacillus cereus group comprises six species, Bacillus cereus, Bacillus thuringiensis, Bacillus anthracis, Bacillus weihenstephanensis, Bacillus mycoides and Bacillus pseudomycoides (Klee et al. 2010). B.anthracis is a Gram-positive, aerobic, spore-forming bacteria and is the causative agent behind a bacterial disease known as Anthrax. Although primarily a disease of animals, it can also infect human beings with fatal consequences (Baillie 2009; Keim et al. 2009). Isolates of B. anthracis forms a highly monophyletic clade, and are differentiated on the basis of single nucleotide polymorphisms (SNPs) and variable number of tandem repeats (VNTRs). The pathogen is able to cause edema and cell death by a tripartite toxin consisting of the protective antigen, the edema factor, and the lethal factor (Keim et al. 2000; Mock and Mignot 2003; Van Ert et al. 2007). The production of a poly-D-glutamic acid capsule allows it to escape the immune system (Fouet and Mesnage 2002).
The intense focus on the Ames strain during the anthrax letter attack investigation lead to a highly accurate sequence determination that may be completely free of errors. Therefore, it represents best publicly available genome sequence of B.anthracis. The DNA was generated from the frozen stock used for strain distribution to many different laboratories, worldwide. Hence, it is an important reference genome for research purposes. Overall, the ancestral Ames genome is 5,503,926 nucleotides in size with 5,775 protein coding genes identified. In addition, there are 33 ribosomal RNA genes (23S, 16S and 5S) arranged in 11 operons and, along with 95 tRNA genes, found exclusively on the chromosome. The chromosome itself represents about 95% of the genome with the two large plasmids containing the remaining coding capacity (pXO1=181,677 bp; pXO2=94,830 bp) (Read et al. 2003; Ravel et al. 2009).
It is anticipated that the identified common drug targets will expand our understanding of the molecular mechanisms of B.anthracis pathogenesis and facilitate the identification of novel drug candidates.
Results and discussion
Infectious diseases are the second leading cause of death worldwide . Though there is an increasing demand for new antimicrobial agents, their development lacks the much required progress due to requirement of huge investment, less market, short term usage in same patient, and high level of competition with newly developed agents (Spellberg et al. 2004). Developments in Bioinformatics have brought the algorithms, tools, and facilitated the automation of microbial genome sequencing, development of integrated databases over the internet, comparison of genomes, identification of gene product function, and paved the way for development of antimicrobial agents, vaccines, and rational drug design (Bansal 2005).
Here we report a combined comparative and subtractive genomics analysis of different metabolic pathways in B.anthracis for the identification of potential drug and vaccine targets. A systematic workflow was defined which involved use of several bioinformatic tools and databases (Figure ). KEGG contains information about ... pathways of B.anthracis Ames strain. Following our modified approach where we started from analysis of metabolic pathways despite of previous approaches which precede from obtaining complete genome/proteome content of the pathogen and host, initially we identified 28 different metabolic pathways as unique to B.anthracis whereas, .... pathways were common among pathogen and H.sapiens (Table ). Protein sequences from each pathway were obtained and compared using BLASTP against DEG to find out essential genes of B.anthracis (see materials and methods for selection criteria of essential proteins). DEG v6.3 contains information about experimentally determined essential proteins from 16 different Gram-positive and Gram-negative bacteria. Although, at present DEG does not contains information about essential genes of B.anthracis however, it contains essential genes data for B.subtilis and other Gram-positive bacteria. This way we identified ..... essential proteins of B.anthracis. Among them, .... were from unique pathways and from common pathways. In the next step, each essential protein was subjected to NCBI BLASTP search against human proteome (see materials and methods for selection criteria) for the identification of pathogen proteins which show no homology to the host. .....essential proteins were from unique pathways and ...... from common pathways were identified as non homologues proteins. All these essential proteins represent a dataset which could be exploited for drug design and vaccine production against B.anthracis.
As it is known that unique pathways are those which are specific to the pathogen but absent in its host. Proteins in these pathways are therefore also unique to the pathogen and thus can serve as potential drug and vaccine targets. However, not every protein is always a favourable target if for example; it has low accessibility value and/or is not essential for the pathogen. Therefore, there is need to identify proteins which regulate factors such as essential nutrients uptake, virulence and pathogenicity. On the other hand, several unique proteins are also present among common pathways of the pathogen which are non homologues to the host as identified during this study and in several previous studies. The sub cellular localization analysis of all supposed essential and unique enzymes of B.anthracis were evaluated by cello server. As it was suggested that membrane associated protein could be the better target for developing vaccines. After analysis 5 proteins were found to be located in the cytoplasm, 1 as trans membrane and 2 on plasma membrane.
General mechanism of most commonly targeted unique pathways of B.anthracis and other pathogens and potential drug targets identified during this study are discussed below
Phosphotransferase system (PTS)
Firstly discovered in E.coli (Kundig et al. 1964), the phosphoenolpyruvate (PEP) dependent phosphotransferase system (PTS) is now well established as a dynamic transport system among diverse range of bacterial species and is responsible for the uptake and phosphorylation of numerous carbohydrates. The basic composition of the PTS is similar in different bacterial species and consists of two general cytoplasmic components, enzyme 1 (E1) and histidine phosphorcarrier protein (HPr). Membrane bound sugar specific permeases (EII) form the third component of this system and show specificity towards carbohydrates. Therefore, different EIIs are present in bacteria. The PTS uses PEP as a source of energy and phosphoryl donor. Each EII complex consists of one or two integral membrane domains (domains C and D) which are hydrophobic in nature and two hydrophilic domains (domains A and B), which together are responsible for the transport of the carbohydrate across the bacterial membrane as well as its phosphorylation. In a sense, the EII complexes constitute parallel transport pathways connected to a common PEP/EI/HPr phosphoryl transfer pathway. E. coli contains at least 15 different EII complexes. A similar number of PTSs have also been reported in B. subtilis (Reizer et al. 1999; Lindner et al. 2002). A growing body of evidence suggests that the pathogens depend upon their hosts for the uptake of nutrients. Therefore, PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens (Galperin and Koonin 1999; Deutscher et al. 2006). These findings favour the targeting of PTS proteins as potential drug targets. During our in silico comparative analysis, we identified 10 proteins as non homologues to humans from the B.anthracis PTS. These proteins were then checked against DEG database and two proteins, phosphoenolpyruvate-protein phosphotransferase (ptsI) and glucose-specific II ABC component (ptsG) (also present in amino sugar and nucleotide sugar metabolism) were found to be essential for B.anthracis.
Cell wall is one of the major components shared by all bacterial species. Presence of cell wall helps bacteria maintain their morphology as well as to withstand against unfavourable conditions such as in a nonisotonic environment. Typical bacterial cell wall is composed of several components such as peptidoglycan (PG), teichoic acids and proteins (Schleifer KH (1983). Peptidogylcan, which forms more than 70% of the weight of the cell wall, is a large molecule responsible for maintaining the morphology and balance in osmotic pressure. Biosynthesis of PG is a complex two steps process of assembly and polymerization. At the first step, assembly of the peptide moiety of the monomer unit takes place by the successive additions of L-alanine, D-glutamic acid, meso-diaminopimelic acid or L-lysine, and D-alanyl-D-alanine to UDP-N-acetylmuramic acid (UDP-MurNAc). These steps are catalyzed by specific peptide synthetases (ligases), which are designated as MurC, MurD, MurE, and MurF, respectively. The second stage of the PG biosynthesis takes place in the periplasmic space and is catalyzed by the penicillin-binding proteins. This involves transglycosylation and transpeptidation reactions of the disaccharide pentapeptide monomers.
Antibiotics such as Î²-lactams and glycopeptides either inhibit or are poor substrates for the final transpeptidation step of cell wall biosynthesis, resulting in a weakening of the cell wall, followed by rapid lysis and death. These drugs have been a popular mainstay in the treatment of bacterial infections at least in part due to this powerful bactericidal effect. However, there has been a significant increase in the incidence of resistance to these drug classes among important bacterial pathogens. Even so, cell wall biosynthesis remains a valid target for novel antibiotic development, especially for agents that can specifically inhibit any one of the series of essential enzymatic functions involved in assembly of the peptidoglycan.
MurA enzyme which catalyzes has been shown to be inhibited by fosfomycin. However, one potential drawback of MurA as a novel antibiotic target is the presence of two separate genes, murA1 and murA2, that encode for proteins with the same enzymatic activity in gram positive pathogens such as Staphylococcus aureus and Streptococcus pneumoniae. Mutagenic studies have showed that disruption of either murA1 or murA2 had no significant affects on cell growth but cells were unable to survive when both genes were removed (Du et al. 2000). Differences in the active site also make it difficult to develop MurA-specific antibiotic that could effectively inhibit both enzymes because the two homologs of murA share less than 60% identity among gram-positive species. Like other gram-positive bacteria, B.anthracis also contains two murA gene homologs and show high degree of intrinsic fosfomycin resistance. Therefore, there is a need to identify additional drug targets which could effectively block essential metabolic pathways and their respective genes. In our analysis, we compared 17 number of genes from PG biosynthesis pathway of B.anthrax against human genome. A total of 11 enzymes were identified as non homologues to humans. All these enzymes were then further classified following selection criteria (see materials and methods). Subcellular localization were determined for each non homologue and many of the identified proteins
It was identified that UDP-Mur- NAc:L-alanine ligase (MurC) is non homologous to human proteins. Furthermore, MurC is also present in D-glutamine and D-glutamate bacterial metabolic pathways. This makes the MurC, encoded by the murC gene, an interesting candidate for drug development against B.anthracis. Other ligases such as MurD, MurE and MurF from B.anthracis showed homology to.... . Inhibition of cell wall synthesis serves as a primary antibiotic target for Gram-positive and Gram-negative bacteria and will continue to provide new targets for drug development. Therefore,
Two component systems
Since their appearance on earth, bacteria have evolved a variety of functions to respond to environmental changes. One such specific mechanism is the evolution of two-component signal transduction systems (TCS). A typical TCS is composed of a sensor kinase (histidine kinase, HK), which is capable of auto-phosphorylation in response to an environmental signal, and a response regulator (RR) that interacts with the phosphorylated HK (Gao and Stock 2009). A bacterium possesses multiple TCSs to respond appropriately to different environmental changes such as pH, nutrient level, redox state, osmotic pressure, quorum signals, and antibiotics. Some TCSs also control gene clusters that contribute to cell growth, virulence, biofilms, quorum sensing, etc. During the infectious cycle, pathogenic bacteria encounter different microenvironments and their ability to efficiently adapt towards their host organisms is frequently mediated by TCSs, which can, therefore, be considered as an essential prerequisite for their pathogenicity (Beier and Gross 2006). In addition, bacteria also exchange information between different TCSs to form a complex signal transduction network with greater sensitivity (Eguchi and Utsumi 2008; Mitrophanov and Groisman 2008). For example, P.aeruginosa, which inhabits diverse environments, is estimated to have 64 HKs and 72 RRs. Among the TCSs, 19 TCSs are involved in some way with virulence or antibiotic resistance. Even for Gram-positive S. pneumoniae, 10 of its 13 TCSs are involved in pathogenicity (Gooderham and Hancock 2009).
Since signal transduction in mammals occurs by a different mechanism, antimicrobial agents with potential to target enzymes of TCS have attracted much attention in the past years. However, evolution of multi drug resistant microbes is also on the rise therefore, there is need to identify additional drug targets in TCSs. Identification of such targets will be more useful if they found to be non homologue to the host proteins, essential for pathogen survival and common among diverse range of pathogenic bacteria. In case of B.anthracis, we identified 37 TCS proteins which showed no homology to humans. Subsequent sub cellular localization predictions and DEG analysis was performed for each non homologous protein (Table..). Three proteins were identified as essential for the pathogen namely, respiratory nitrate reductase alpha chain narG (also involved in nitrogen metabolism), respiratory nitrate reductase beta chain narH (also involved in nitrogen metabolism), and chromosomal replication initiation protein dnaA.
Bacterial secretion systems
Bacteria form a very wide diversity of biotic associations, ranging from biofilms to mutualistic or pathogenic associations with larger host organisms. Protein secretion pathways plays a key role in modulating all of these interactions including all the known folding and targeting routes of inner and outer membrane proteins as well as of proteins that are secreted by several specific export routes. In Gram-negative bacteria, six general classes of protein secretion systems have been identified. Each class shows considerable diversity and facilitate the process of entry of the secreted proteins inside host cells and modification of host physiology thus promoting colonization. On the other hand, in Gram-positive bacteria, secreted proteins are commonly translocated across the single membrane by the Sec pathway or the two-arginine (Tat) pathway. Although, Gram-positive bacteria share some of the same secretion systems as Gram-negative bacteria, others such as mycobacteria that have a hydrophobic, nearly impermeable cell wall, called the mycomembrane, a specialized type VII secretion system translocates proteins across both the membrane and the cell wall (Sandkvist 2001; Tseng et al. 2009). Therefore, the importance of secretion systems not only for bacterial viability but also for pathogenicity is well established and has a great potential as a target for development of new drugs (Briken 2008). KEGG database contains information about..... proteins of the B.anthracis secretion system. Each protein sequence was subjected to BLAST analysis and four were identified as non homologues to humans. Amongst them three were predicted as membrane localized and one into the cytoplasm. DEG based search for essential proteins suggested....... proteins as essential for B.anthracis namely, .........
Proteins from common pathways
Searching drug targets among homologues proteins
In the current in silico comparative genomics strategy which other authors and we have used, authors follow a workflow in which pathogen's full proteome is compared with human proteome which lead to identification of non homologues proteins. These proteins are then checked against DEG database for the screening of essential but non homologue proteins. However, It is now known that several essential proteins of pathogens show considerable homology to their host proteome. Also, many drugs are in use or proposed whose targets show homology or orthology in human proteome such as parasite cysteine proteases (Engel et al. 1998; Selzer et al. 1999; Abdulla et al. 2007), and the highly conserved Î²-tubulin protein, the target of benzimidazoles (Robinson et al. 2004). has provided a list of currently used drugs and their targets from different pathogens. Interestingly, .... of those drug targets have orthologues in humans. Therefore, when following the current strategy of comparative genomics, there are chances of skipping such potential drug targets which show some level of similarity to humans. Keeping this in context, we retrieved essential yet somewhat homologue proteins of B.anthracis from all of the metabolic pathways and evaluated their potential as drug and vaccine targets. Although, it may be difficult to predict in silico if these homologues proteins can be selected as favourable targets but calculation of certain parameters such as molecular weight, transmembrane domains and availability of experimental determined 3D structures in PDB and Modbase databases may aid towards increased understanding. A list of these proteins along with calculated parameters is presented in table 2.
In this study, we have performed an in silico metabolic pathway analysis of the pathogen B. anthracis. The computational genomic approach has already facilitated the search for potential drug targets against many pathogens. 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 B.anthracis. The drugs developed against identified targets will be specific to the pathogen and may show lesser toxicity to the host. Although the number of essential genes in the metabolic pathways of B.anthracis, identified in the present study, is relatively small (only 8), these can be further characterized and their role in the survival of the bacteria can be verified. Since many of them have been reported to play a role in its virulence, a systematic approach to develop novel drugs against these targets can be adapted for treating B.anthracis infections. Further, homology modeling of these targets will help identify the best possible sites that can be targeted for drug design by simulation modeling.
Materials and methods
Identification of host and pathogen metabolic pathways
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was used as a source (Kanehisa et al. 2006; Kanehisa et al. 2010) of metabolic pathways information. List of metabolic pathways and identification numbers of the host H.sapiens and the pathogen B.anthracis was extracted from the KEGG database and saved locally. A manual comparison was conducted and pathways which do not appear in the host but present in the pathogen according to KEGG database annotations were selected as pathways unique to B.anthracis. Proteins in these unique pathways were identified and amino acid sequences were obtained from Uniprot. Protein sequences were initially filtered on the basis of number of amino acids residues i.e. proteins of less than 100 amino acid residues were excluded.
Identification of potential drug targets
Two step comparison was performed between host and pathogen genomes for the identification of non homologues proteins of B.anthracis. At first, only proteins from pathogen specific pathways were subjected to a BLASTP. Secondly, proteins from common pathways were compared by BLASTP. In each scenario, search was restricted to proteins from H.sapeins only through an option available under BLASTP parameters. Hits were filtered on the basis of e-value inclusion threshold set to 0.005 and minimum bit score of 100. Proteins, which do not have hits below the e-value inclusion threshold of 0.005, were picked out as potential drug targets.
The selected non homologues proteins from the unique and common pathways were then compared against DEG database using BLASTP (Zhang et al. 2004). Expectation value (E-value) cut-off of 10-10 and minimum bit score of 100 was used for screening essential proteins of B.anthracis. Each of the identified essential protein was further submitted to Drug Bank database against approve drug targets and small molecules legends.
Evaluating biological significance of the drug targets
Biological significance and subcellular localization of the non homologues genes were calculated by two methodsâ€¦ The predicted membrane proteins were further analyzed in psortb v3.0  to confirm if the drug targets are identified as membrane proteins irrespective of the subcellular localization prediction methods