An experimental study was conducted to evaluate the druggability prioritization targets of salmonella gallinarum in Gallus gallus. Total proteome of pathogen Vs host comparison using comparative genomics was done followed by protein docking of the target proteins of the pathogen and drug target identificationandthen calculation of drug prioritization parameters for therapeutic target. 3965 proteins of S. gallinarum were compared with 24068 proteins of Gallus gallus using BLASTP analysis. The dockingresultsshowedpositiveligandposesforproteins1L50posed with ligands 142, 152, 316 ,477,1114 and atovaquone, ethambutol, olsalazine, protein 1MDZ posed with ligands 152, 388 and ethambutol, olsalazine, protein 1R30 posed with ligands 152,157,273,477,598,701,1039,1114 and atovaquone, ethambutol,olsalazine, lenvatinib, Protein 1XVT posed with ligands 152,157,477,701,1039,1114 and 1197.protein 2KHO posed with ligands 152 and atovaquone, olsalazine, ponatinib, protein 2QVR posed with ligands 142, 152, 273, 316, 388, 477,598, 674, 1114, 1197 and 1213.protein 3E74 posed with ligands 157,477,701 and lenvatinib, Protein 3H8A posed with ligand ponatinib and protein 4ADE posed with ligands ponatinib and 157. The results of the study revealed potential drug targets for developing novel molecules against Salmonella gallinarum and resulted in identification of novel molecules.
Fowl typhoid, an acute septicaemic disease of avian species caused by Salmonella gallinarum (Priyantha, 2012), affects all age groups of chickens. Morbidity is high among all age groups of the birds, whereas mortality may range widely from 10% to 90% (Latife Beyaz et al., 2010). Maintaining a disease free status is a challenging exercise due to the rapid expanding nature of the industry. This is indicated by the fact that a number of Salmonella outbreaks reported in the world are a result of injudicious introduction of infected birds (Meeusen et al., 2007). Thus, poultry industry is facing great setbacks due to frequent outbreaks of salmonellosis (Fatma et al.,2012). Since its discovery, many efforts have been made to control and prevent the occurrence in commercial poultry farming. However, outbreaks of salmonellosis still remain a serious economic problem in countries where control measures are not efficient or in those areas where the climatic conditions favour the environmental spread of these microbes (Barrow and Freitas Neto, 2011). The economic losses are chiefly due to morbidity, mortality, reduced growth rate, reduced feed conversion efficiency, drop in egg production, decreased fertility and hatchability (Mamta Mishra and Deepika Lather, 2010).
Control of fowl typhoid is difficult (Soncine and Back, 2001) due to endemicity of the disease, facultative intracellular nature of causative organism, both vertical (Paiva et al., 2009) and horizontal (Cox et al., 1996) modes of transmission, presence of carrier stage and multiple drug resistance. Fowl typhoid can be controlled by a combination of stringent management procedures and chemotherapy. The widespread and indiscriminate use of antibiotics in the treatment of poultry diseases has lead to antimicrobial resistance of resistant salmonella strains (Enabulele et al., 2010) which is of a global public health concern (Ahmed et al., 2011). However, Enabulele (2010) has reported that Salmonella gallinarum strains are becoming more resistant to antibiotics than other avian salmonellas, which meant that it is more difficult to treat infected flocks successfully. However, this view may change, as its true prevalence becomes known with improved diagnostic tests, and with the likely failure of antibacterial agents to control disease in the future and with the emphasis on curtailing the spread of disease in poultry.Thus, the present study is aimed to evaluate the druggability targets of pathogen viz Gallus gallus.
Materials and Methods
Identification of Host and Pathogen Metabolic Pathways
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was used as a source of metabolic pathways information (Kanehisa et al., 2006; Kanehisa et al., 2010). A list of metabolic pathways and identification numbers of the host and the pathogen Salmonella gallinarum was extracted from the KEGG database and saved locally. Proteins from pathways were identified and the respective amino acid sequences were obtained from the Swiss-Prot database (Boeckmann et al., 2003).
Screening of Non-Homologous and Essential Proteins
Two-step comparisons were performed between host and pathogen proteomes for the identification of non-homologous proteins of Salmonella gallinarum (Altschul et al., 1997). In each scenario, searching was restricted to proteins from broilers only through an option available under BLASTP parameters. Hits were filtered on the basis of expectation value (e-value) inclusion threshold being set to 0.005, and a minimum bit score of 100. Proteins, that did not have hits below the e-value inclusion threshold of 0.005, were picked as non-homologous proteins.
Druggability of Therapeutic Targets
Druggability is another important target prioritization criterion, which is defined as the likelihood of being able to modulate the activity of the protein target with a small-molecule drug (Keller et al., 2006; Cheng et al., 2007). The druggability potential of each of the identified drug targets was calculated by mining Drug Bank contents. The Drug Bank database is a unique bioinformatics and cheminformatics resource that combines detailed drug (i.e. chemical, pharmacological and pharmaceutical) data with comprehensive drug target (i.e. sequence, structure and pathway) information. The database contains 6796 drug entries including 1437 FDA approved small molecule drugs, 134 FDA approved biotech (protein/peptide) drugs, 83 nutraceuticals and 5174 experimental drugs. Additionally,4285 non redundant protein(i.e. drug target/enzyme/transporter/carrier) sequences are linked to these drug entries (Knox et al., 2011). BLASTP with default parameters were used to align the potential drug targets from Salmonella gallinarum against the list of protein targets of compounds found within Drug Bank. The selection criteria for filtering BLAST results were as described previously (Holman et al., 2009), that is, alignments with e-values less significant –25 than 1 x 10 were removed.
Results and Discussion
Identification of Non-Homologous Proteins
3965 proteins of Salmonella gallinarum were compared with 17,227 proteins of Gallus gallususing BLASTP analysis. 1068 non- homologous proteins of SG were found, while 2897 proteins were found homologous. These non-homologous proteins were analyzed using BLAST against PDB and all targets from Drug Bank. Number of proteins found hits against PDB (sequences of proteins with known structure): 35 (Table 1) and against Drug Bank were 29 (Table 2).
Druggability of Therapeutic Targets
Thirty five proteins found with solved PDB structures were analyzed using CLASTLW and that resulted in thirty five proteins having an average alignment of above or minimal 30% (Table 4).The thirty five proteins structures were also determined using PISA software and the structures of the proteins were determined, of which by PISA 5 monomers, 2 dimmers, 1 tetramer and 1 polymer were found (Table 3).
Table 1: List of PDB structures matching with non-homologous proteins of Salmonella gallinarum after BLAST with Gallus gallus proteome
|S. No.||Query Protein ID||Subject Protein ID||%id||Bit Score||Clustalw||PISA|
Thirty five proteins were loaded into Discovery studio version 4.1 for receptor ligand interaction studies with 29 ligands. The docking results showed positive ligand poses for proteins 1L50 posed with ligands DB142, 152, 316, 318, 477, 1114 and atovaquone, ethambutol, olsalazine, protein 1MDZ posed with ligands DB152, 388 and ethambutol, olsalazine, protein 1R30 posed with ligands. DB152, 157, 273, 477, 598, 701, 1039, 1114 and atovaquone, ethambutol, olsalazi ne, lenvatinib, Protein 1XVT posed with ligands DB152, 157, 477, 701, 1039, 1114 and 1197, protein 2KHO posed with ligands DB152 and atovaquone, olsalazi ne, ponatinib, protein 2QVR posed with ligands DB142, 152, 273, 316, 388, 477, 598, 674,1114,1197 and 1213, protein 3E74 posed with ligands DB157, 477, 701 and lenvatinib, protein 3H8A posed with ligand ponatinib and protein 4ADE posed with ligands DB157 and ponatinib (Fig. 1). The functional characters of the above 9 non-homologous proteins are mentioned in Table 4 proteins 1L50
Table 2: List of Drug Targets matching with non-homologous proteins of Salmonella gallinarum after BLAST with Gallus gallus proteome
|SL.No||Query protein ID||Subject protein ID||%id||Bit Score|
posed with ligands 142(L glutamic acid), 152(Thiamine), 316(Acetaminophen) ,477(Chlorepromazine),1114(Chlorephenamine) and atovaquone, ethambutol, olsalazine, protein 1MDZ posed with ligands 152(Thiamine), 388(Phenyleprine) and ethambutol, olsalazine, protein 1R30 posed with ligands 152(Thiamine),157(NADH),273(Topiramate),477(Chlorepromazine),598(Labetolol),701(Amprenavir),1039(Fenofibrate),1114 (Chlorephenamine)and atovaquone, ethambutol,olsalazine, lenvatinib, Protein 1XVT posed with ligands 152(Thiamine),157(NADH),477(Chlorepromazine),701(Amprenavir),1039(Fenofibrate),1114(Chlorephenamine) and 1197(Captopril).protein 2KHO posed with ligands 152(Thiamine) and atovaquone, olsalazine, ponatinib, protein 2QVR posed with ligands 142(L glutamic acid), 152(Thiamine), 273(Topiramate), 316(Acetaminophen), 388(Phenyleprine), 477(Chlorepromazine),598(Labetolol), 674(Galantamine), 1114(Chlorephenamine), 1197(Captopril) and 1213(Fomepizole), protein 3E74 posed with ligands 157(NADH),477(Chlorepromazine),701(Amprenavir) and lenvatinib, Protein 3H8A posed with ligand ponatinib and protein 4ADE posed with ligands ponatinib and 157(NADH). The functional characters of the above 9 non-homologous proteins are mentioned in Table 4.
Table 3: List of Drug Receptor Ligand Interactions with Positive Poses
|Sl.No||Salmonella protein||PDB ID||Ligand Name||Docking Score|
Table 4: List of non-homologous proteins of Salmonella gallinarum after BLASTP with Gallus gallus with their functional characters
|S. No.||Name of the POSSITIVE Salmonella Proteins||POSSITIVE PDB id||Function of Salmonella Protein|
|1||sp|B5R646|ALLB_SALG2||3e74_D||Allantoinase; Catalyzes the conversion of allantoi (5- ureidohydantoin) to allantoic acid by hydrolytic cleavage of the five-member hydantoin ring|
|2||sp|B5RCC2|ARNB_SALG2||1mdz_A||UDP-4-amino-4-deoxy-L-arabinose–oxoglutarate aminotransferase; Catalyzes the conversion of UDP-4-keto-arabinose(UDP-Ara4O) to UDP-4-amino-4-deoxy-L-arabinose (UDP-L-Ara4N)|
|3||sp|B5RB01|ASTC_SALG2||4ade_B||Succinylornithine transaminase;Catalyzes the transamination of N(2)-succinylornithineand alpha-ketoglutarate into N(2)-succinylglutamate semialdehyde and glutamate. Can also act as an acetylornithine aminotransferase.|
|4||sp|B5R761|BIOB_SALG2||1r30_B||Biotin synthase; Catalyzes the conversion of dethiobiotin (DTB) to biotin by the insertion of a sulphur atom into dethiobiotin via a radical-based mechanism.|
|5||sp|B5RGA5|CAIB_SALG2||1xvt_A||L-carnitine CoA-transferase; Catalyzes the reversible transfer of the CoA moiety from gamma-butyrobetainyl-CoA to L-carnitine to generate L-carnitinyl-CoA and gamma-butyrobetaine.|
|6||sp|B5RBK6|COBT_SALG2||1l5o_A||Nicotinate-nucleotide—dimethylbenzimidazole phosphoribosyltransferase; Catalyzes the synthesis of alpha-ribazole-5′- phosphate from nicotinate mononucleotide (NAMN) and 5,6-dimethylbenzimidazole(DMB).|
|7||sp|B5RF08|DNAK_SALG2||2kho_A||Chaperone protein; Heat shock protein 70 ,Acts as a chaperone.|
|8||sp|B5RDS5|ENO_SALG2||3h8a_D||Enolase; Catalyzes the reversible conversion of 2-phosphoglycerate into phosphoenolpyruvate. It is essential for the degradation of carbohydrates via glycolysis.|
|9||sp|B5R9H4|F16PA_SALG2||2qvr_A||Fructose-1,6-bisphosphatase class 1|
Fig. 1: Receptor Ligand Interactions
|Sl.NO||Receptor||Receptor Ligand Interaction|
A variety of compounds which are involved in the management of diseases of non-infectious etiology have shown some antimicrobial activity in vitro, against bacteria and other microorganisms (Tyski, 2003). Such compounds are called “non-antibiotics”. By the end of the nineteenth century, the dyes were known to possess antimicrobial activity, methylene blue (one of phenothiazines compounds) as an antimicrobial agent (Gutman and Ehrlich, 1891). So far, a lot of attention has been focused on thioxanthenes, phenothiazines and other agents with affinities to cellular transport systems which influence the structure of cellular membrane or ions transport etc. (Hendrics et al., 2003). In one study (Kruszewska et al., 2008), it was indicated that some of preparations inhibited growth of at least one of the four examined standard microbial strains. The drugs with the following active substances showed significant antimicrobial activity- amlodipine, acepromazine, butorphanole, cisapride, cisplatine, clomipramine, diltiazem, emadastine, fluvastatine, ketamine, levocabastine, matipranalol. methotrexate, nicergoline, perphenazine, proxymetacaine, sertraline, tegaserole, tetrahydrozoline, ticlopidine and tropicamide. In the present study in silico antimicrobial activity was positive for paracetamol, chlorephenaramine, chlorpromazine, thiamine, labetalol, finofibrate, topiramate, glucosamine, galantamine, L-glutamic acid, ethambutol, phenyleprine. Chen et al., 2002, confirmed that non- antibiotic compounds enhance the in vitro activity of certain antibiotics against specific bacteria. Moreover, the antimicrobial activity of such non antibiotic drugs emphasizes a necessity of the neutralization of their activity during the microbial purity tests of pharmaceutical products (Clonts,1998) This is the reason why any product should be validated towards its possible inhibition against microorganisms. In this study, we examined ligands with pure molecules which gave good scoring in virtual screening and the MIC values of these compounds were ranging from 0.2 to 14.5µg/ml. The less sensitivity of the compounds could be due to variation in the dosages and further dosage studies are required in this study.
Fowl typhoid is prevalent in commercial broiler flocks and is responsible for considerably high morbidity and mortality in affected flocks.In this study, Salmonella gallinarum isolates were found resistant to enrofloxacin, moxifloxacin, azithromycin, erythromycin, chloramphenicol and clindamycin, whereas cefpodoxime showed moderate sensitivity to Salmonella gallinarum. Surveillance, identification and antibiotic sensitivity of the prevalent Salmonella serotypes in the country would help devise suitable prevention and control program for this important poultry pathogen. Since the consumption of poultry products is often associated with salmonellosis, therefore, it becomes necessary to update information about Salmonella resistance to antibiotics used in poultry production.