Potential therapeutic drug target identification in Community Acquired-Methicillin Resistant Staphylococcus aureus (CA-MRSA) using computational analysis

Abstract

The emergence of multidrug-resistant strain of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strain has highlighted the urgent need for the alternative and effective therapeutic approach to combat the menace of this nosocomial pathogen. In the present work novel potential therapeutic drug targets have been identified through the metabolic pathways analysis. All the gene products involved in different metabolic pathways of CA-MRSA in KEGG database were searched against the proteome of Homo sapiens using the BLASTp program and the threshold of E-value was set to as 0.001. After database searching, 152 putative targets were identified. Among all 152 putative targets, 39 genes encoding for putative targets were identified as the essential genes from the DEG database which are indispensable for the survival of CA-MRSA. After extensive literature review, 7 targets were identified as potential therapeutic drug target. These targets are Fructose-bisphosphate aldolase, Phosphoglyceromutase, Purine nucleoside phosphorylase, Uridylate kinase, Tryptophan synthase subunit beta, Acetate kinase and UDP-N-acetylglucosamine 1-carboxyvinyltransferase. Except Uridylate kinase all the identified targets were involved in more than one metabolic pathways of CA-MRSA which underlines the importance of drug targets. These potential therapeutic drug targets can be exploited for the discovery of novel inhibitors for CA-MRSA using the structure based drug design (SBDD) strategy.

Background

Methicillin resistant Staphylococcus aureus, or MRSA is a grampositive bacterial pathogen which is resistant to methicillin and other beta-lactum antibiotics. It is a major causative agent of skin and soft-tissue infections (SSTIs), endovascular infections, pneumonia, septic arthritis, endocarditis, osteomyelitis, foreignbody infections, and sepsis [1, 2]. The original MRSA infections associated with exposure in the health care setting, particularly in hospitals are referred to as hospital-acquired MRSA (HAMRSA) [3]. In 1990s, a new strain of MRSA emerged in the community setting occurring among young healthy individuals with no exposure to the healthcare setting. The infections caused by these strains are called community-acquired MRSA (CA-MRSA) [4, 5]. Since then, this community-acquired MRSA strain (CA-MRSA) has quickly spread across the globe [6–8]. Outbreaks of CA-MRSA have been reported among children [9], athletes [10], nurseries [11] and obstetrical wards [12]. The CA-MRSA strains have been involved in skin and soft tissue infections including furuncles, abscesses, folliculitis, impetigo, cellulitis, and, more rarely, in cases of severe sepsis, necrotizing fascitis, and necrotizing pneumonia [13]. The CA-MRSA strain is commonly known as the Staphylococcus aureus subsp. aureus MW2. Popovich et al. reported that CA-MRSA may be replacing the traditional hospital-acquired MRSA (HA-MRSA) [14]. The spread of resistant CA-MRSA strains across the globe becoming more common and posing potential threat to the life of community [15]. Because of multidrug resistance, particularly among CA-MRSA, alternative and effective therapeutic options are urgently needed. With the availability of complete genome sequences of CA-MRSA [16], it has now paved the new way for identifying the novel drug targets. Through the complete genome analysis of the pathogen, it is possible to compile a list of potential gene products and their functions which are nonhomologous to the proteome of Homo sapiens. In the present work novel potential theapeutic drug targets have been identified through the metabolic pathways analysis in the community acquired-methicillin resistant Staphylococcus aureus.

Methodology

The entire genome of Staphylococcus aureus subsp. aureus MW2 (CA-MRSA), was sequenced in the year 2002. It is available on the website http://www.genome.jp (Accesion No. NC_003923) which contain 2820462 base pairs and 2624 protein encoding genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database [17] was used http://www.kegg.jp/kegg/pathway.htm for the retrieval of metabolic pathways for the community-acquired methicillin resistant Staphylococcus aureus (Entry no. T00086). The metabolic pathway of CA-MRSA was analyzed which was containing 76 different types of metabolic pathways. All enzymes involved in the different metabolic pathways were listed in a table. The most important criteria for selecting any enzyme or protein as a potential drug target in a pathogen is that it should be nonhomologous to the host i.e. Homo sapiens. The gene products involved in different metabolic pathways of CA-MRSA genome were subjected to the database searching against the proteome of the Homo sapiens using the BLASTp program [18]. The threshold of E-value (expect value) was set to as 0.001. The similar protein sequences which were having less than 30% identity or less than 80% query coverage to the Homo sapiens proteome were considered as the non-homologous to the human. Those enzymes can be considered as the unique potential therapeutic drug targets for the drug designing. After performing the database searching of all metabolic enzymes (gene products) of CA-MRSA against the human proteome, 220 targets were identified as non-homologous to Homo sapiens. These enzymes were involved in 50 different metabolic pathways. Further analysis for all 220 targets was carried out and it was found that some duplicate targets were involved in more than one metabolic pathway. The list of all putative targets was further refined and duplicates were removed. Finally 152 targets were identified as unique putative drug targets. After identifying the novel potential drug targets from metabolic pathways of CA-MRSA, the genes coding for the important enzymes were further searched in the DEG 6.8 database [19] to identify the essentiality or non-essentiality of the genes for the survival of the pathogen. DEG provides the database of essential genes which are indispensable for the survival of an organism (http://www.essentialgene.org/). DEG database has been classified in to two categories prokaryotes and eukaryotes. In the pathogens, essential gene products provide unique potential drug targets for antimicrobial targets. Among all 152 putative drug targets, 39 genes which encode for potential drug targets were identified as essential for the survival of the CA-MRSA.

Discussion

Community acquired-methicillin resistant Staphylococcus aureus (CA-MRSA) strains are now becoming nosocomial pathogen to the human race. In comparison to hospital acquired MRSA, these strains cause infections suddenly, quickly, and with great severity in patients which leads to worse clinical outcome. CAMRSA strains are more virulent than other strains and have very bad impact on conventional therapy particularly with beta-lactum antibiotics which are becoming ineffective for a variety of common staphylococcal infections especially for skin & soft tissue infections [20]. Therefore, we have to find the alternative approach to combat the menace of drug resistance of CA-MRSA. In the present work, post genomic approach has been applied for the identification of potential drug targets for the CA-MRSA. The genes involved in different metabolic pathways of CA-MRSA were analyzed and it was found that total 76 pathways were present in KEGG pathway database. KEGG is the largest database resource consisting 17 different types of databases. For identifying the putative drug targets in the genome of any pathogen, it should be present in the organism and posse’s crucial functional role but absent in the Homo sapiens. Using the BLASTp program, database searching was performed for all the gene products involved in different metabolic pathways of CA-MRSA against the proteome of Homo sapiens. The threshold of E-value was given 0.001 which measures the significance of similarity to the host. Apart from the E-value threshold, the % identity and % query coverage was also considered as the parameter for identifying the putative drug targets non-homologous to the proteome of Homo sapiens. The protein sequences which were having more than 0.001 Evalue and less than 25% sequence identity and/or less than 80% query coverage, were considered as non-homologous drug targets. Total 220 putative drug targets were identified Table 1 (see supplementary material). Out of 220 targets, it was found that some targets (proteins) were involved in more than one metabolic pathway. All the duplicate targets were removed from the list and total 152 unique putative drug targets were identified. The genes encoded for 152 unique putative targets were again searched against the DEG (Database of Essential Genes) database to identify the essentiality of the genes for the survival of CA-MRSA. DEG is the database of essential genes which are indispensable for the survival of any organism. After searching all 152 putative targets, 39 genes were identified as the essential for the survival of CA-MRSA Table 2 (see supplementary material). All 39 essential gene products (targets) were analyzed and it was found that 20 putative drug targets were involved in more than one metabolic pathway. These 20 putative targets can be used as potential therapeutic drug targets for CA-MRSA. Out of 20 putative drug targets, it has been reported in literatures that 7 targets may be used as potential therapeutic drug targets. These targets are Fructosebisphosphate aldolase (EC: 4.1.2.13), Phosphoglyceromutase (EC: 5.4.2.1), Purine nucleoside phosphorylase (EC: 2.4.2.1), Uridylate kinase (EC: 2.7.4.22), Tryptophan synthase subunit beta (EC:4.2.1.20), Acetate kinase (EC:2.7.2.1) and UDP-Nacetylglucosamine 1-carboxyvinyltransferase (EC:2.5.1.7).

Fructose-bisphosphate aldolase (EC: 4.1.2.13):

Fructose-bisphosphate aldolase (FBA) enzyme is encoded by fbaA gene (ID: MW2049). This gene has been found essential in DEG database (DEG10020239) for Staphylococcus aureus N315, Bacillus subtilis, Mycoplasma pulmonis and Escherichia coli. FBA has been reported as potential therapeutic drug target in Mycobacterium tuberculosis and Candida albicans [21, 22]. These FBAs are involved in second reversible step of the glycolytic pathway, which supplies glyceraldehyde 3-phosphate for downstream enzymes in the pathway and fructose 1, 6- bisphosphate (FBP) for gluconeogenesis. Together, the substrates and products of the FBA reaction are crucial for the supply of these precursor molecules to other biochemical pathways essential for the survival of CA-MRSA. This enzyme is also involved in three other metabolic pathways i.e. pentose phosphate pathway, fructose and mannose metabolism & methane metabolism.

Phosphoglyceromutase (EC: 5.4.2.1):

Phosphoglyceromutase (PGM) enzyme is encoded by pgm gene (ID: MW0737). This gene has been found essential in DEG database (DEG10020010) for Bacillus subtilis, Mycoplasma pulmonis, Mycoplasma genitalium and Salmonella enterica. PGM interconvert 2-phosphoglycerate and 3-phosphoglycerate in the glycolytic and gluconeogenic pathways. This enzyme is also involved in glycine, serine, threonine metabolism, and methane metabolism in CA-MRSA. PGM has been reported as important drug target in Wolbachia endosymbiont from the filarial nematode, Brugia malayi (wBm) [23].

Purine nucleoside phosphorylase (EC: 2.4.2.1):

Purine nucleoside phosphorylase (PNP) enzyme is encoded by pnp gene (ID: MW0110). This gene has been found essential in DEG database (DEG10020139) for Staphylococcus aureus N315, E. coli and Acinetobacter baylyi. PNP plays a crucial role in the phosphorolysis of purine nucleosides and deoxynucleosides to generate purine bases. This enzyme is also involved in pyrimidine, nicotinate and nicotinamide metabolism. PNP has been reported as potential therapeutic drug target in M. tuberculosis and Streptococcus mutans [24, 25].

Uridylate kinase (EC: 2.7.4.22):

Uridylate kinase or UMP kinase (UMPK) enzyme is encoded by pyrH gene (ID: MW1141). This gene has been found essential in DEG database (DEG10170157) for Staphylococcus aureus NCTC8325, M. tuberculosis, Mycoplasma pulmonis, Streptococcus pneumoniae, Pseudomona aeruginosa, Salmonella typhimurium, V. cholerae etc. UMP kinase catalyses the phosphorylation of UMP by ATP to yield UDP which is involved in cell wall and RNA biosynthesis. UMPK is conserved in almost all prokaryotic organisms and has been reported as potential therapeutic drug target in Staphylococcus aureus, Streptococcus pneumoniae [26, 27].

Tryptophan synthase subunit beta (EC: 4.2.1.20):

Tryptophan synthase subunit beta (TrpB) enzyme is encoded by trpB gene (ID: MW1259). This gene has been found essential in DEG database (DEG10020152) for Staphylococcus aureus N315, M. tuberculosis, Streptococcus pneumonia, Haemophilus influenzae and Acenetobacter baylayi. TrpB enzyme catalyzes the last step of the tryptophan biosynthetic pathway which is commonly present in almost all prokaryotic organisms but absent in mammals. This enzyme is also involved in the biosynthesis of phenylalanine and tyrosine as well as in the metabolism of glycine, serine and threonine amino acids. TrpB has been reported as potential therapeutic drug target in Mycobacterium tuberculosis and Salmonella typhimurium [28, 29].

Acetate kinase (EC: 2.7.2.1):

Acetate kinase (ACK) enzyme is encoded by ackA gene (ID: MW1654). This gene has been found essential in DEG database (DEG10020202) for Staphylococcus aureus N315, Mycoplasma pulmonis, Mycoplasma genitalium and E. coli. ACK enzyme is involved in the formation of acetate from acetyl-CoA as a metabolic end product. It is involved in many metabolic pathways of CA-MRSA e.g. Taurine & hypotaurine, pyruvate, propanoate and methane metabolism. This enzyme is present in prokaryotic organisms and some eukaryotic organisms e.g. parasites but absent in mammals and it has been reported as attractive drug target for the development of anti-parasitic drugs [30].

UDP-N-acetylglucosamine 1-carboxyvinyltransferase (EC:2.5.1.7):

UDP-N-acetylglucosamine 1-carboxyvinyltransferase enzyme is encoded by murA gene (ID: MW2024). This gene has been found essential in DEG database (DEG10020231) for Staphylococcus aureus N315, Mycobacterium tuberculosis, Bacillus subtilis, Salmonella enterica, Francisella novicida, Helicobacter pylori, E. coli and Acenetobacter baylayi. MurA enzyme catalyses the biosynthesis of peptidoglycan polymer, consisting of Nacetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Peptidoglycan is an integral constituent of bacterial cell wall which is indispensable for the survival of bacteria. UDP-Nacetylglucosamine 1-carboxyvinyltransferase (murA) enzyme catalyses the transfer of the enolpyruvyl group of phosphoenolpyruvate (PEP) to the 3'-hydroxyl group of uridine diphospho-N-acetylglucosamine (UNAG). Furthermore, this enzyme is also involved in amino sugar & nucleotide sugar metabolism. MurA is essential enzyme present in all prokaryotic organism but absent in mammals. It has been reported as potential therapeutic drug target in Haemophilus influenzae, Escherichia coli and Streptococcus pneumonia [31, 32, 33]. Furthermore, except uridylate kinase all above potential therapeutic targets were involved in more than one metabolic pathways of CA-MRSA which underlines the importance of these targets. These drug targets can be used for the discovery of novel drugs which might potentially inhibit the growth of CA-MRSA.

Conclusion

The metabolic pathway of nosocomial community acquiredmethicillin resistant Staphylococcus aureus (CA-MRSA) strain was analyzed from the KEGG database. All the gene products involved in different metabolic pathways of CA-MRSA were searched against the proteome of Homo sapiens and 152 putative targets were identified. 39 genes encoding for important targets were identified as the essential from the DEG database which are indispensable for the survival of CA-MRSA. After extensive literature review, 7 targets were identified as potential therapeutic drug target. These targets are Fructosebisphosphate aldolase (EC: 4.1.2.13), Phosphoglyceromutase (EC: 5.4.2.1), Purine nucleoside phosphorylase (EC: 2.4.2.1), Uridylate kinase (EC: 2.7.4.22), Tryptophan synthase subunit beta (EC:4.2.1.20), Acetate kinase (EC:2.7.2.1) and UDP-Nacetylglucosamine 1-carboxyvinyltransferase (EC:2.5.1.7). Almost all these putative targets were involved in more than one metabolic pathways of CA-MRSA. These potential therapeutic drug targets can be exploited for the discovery of novel inhibitors for CA-MRSA using the structure based drug design (SBDD) strategy.