| dc.description.abstract | Abstract
Increase in resistance pathogens have fade the potential of antibiotics. Thus, alternatives therapies are investigated to conquer the battle of resistance. Antimicrobial peptides (AMPs), the key molecules of innate immunity, are present in all organism and recently gaining attention for its antimicrobial activities as well as antibiotics’ alternative. AMPs exert wide range of activity, have selective nature for pathogenic, lower toxicity, minimal resistance development properties and multiple-targeting mechanism of actions. Despite the knowledge of its multi-targeting activities on cellular membrane and intracellular molecules, only few targets have been identified. In this study, the high-throughput platform of proteome microarrays was utilized for systematical and comprehensive identification of the entire protein targets of AMPs in parallel analysis to the entire proteome of the model organisms: Escherichia coli and Saccharomyces cerevisiae. Intracellular targeting AMPs with antibacterial and antifungal activities were probed on Escherichia coli proteome microarrays and Saccharomyces cerevisiae proteome microarrays to identify their antibacterial and antifungal protein targets, respectively. The bacterial protein targets of AMPs with antibacterial activities (Polyphemusin-I, Sub-5, Penetratin, Histatin-5 and TWF) were systematically identified by individually probing the AMPs on Escherichia coli proteome microarrays. In total of 109, 92, 118, 93 and 48 protein targets were identified for Polyphemusin-I, Sub-5, Penetratin, Histatin-5 and TWF, respectively. The fungal protein targets of AMPs with antifungal activities (Lfcin B, Sub-5, Penetratin and Histatin-5) were systematically identified by individually probing the AMPs on Saccharomyces cerevisiae proteome microarrays. In total of 140, 137, 128 and 123 protein targets were identified for Lfcin B, Sub-5, Penetratin and Histatin-5,respectively. These identified bacterial protein targets of AMPs (Polyphemusin-I, Sub-5, Penetratin, Histatin-5 and TWF) were bioinformatically analyzed together to understand their antibacterial activities. Also, the identified fungal protein targets of AMPs (Lfcin B, Sub-5, Penetratin and Histatin-5) were bioinformatically analyzed together to understand their antifungal activities. In above assays, biotin-streptavidin detection system was used for the signal detection of the biotinylated AMPs bound to the protein targets on proteome microarrays with streptavidin labeled DyLight (fluorescence). As a negative control, only streptavidin labeled DyLight was probing on proteome microarrays. Interesting by probing only streptavidin labeled DyLight on the Escherichia coli proteome microarrays as well as Saccharomyces cerevisiae proteome microarrays, the biotinylated proteins (proteins modification by biotin) of the Escherichia coli and Saccharomyces cerevisiae were identified, respectively. So far, only one biotinylated protein has been identified in Escherichia coli and six biotinylated proteins in Saccharomyces cerevisiae. The essentiality of biotin in living organism cannot be understood by single biotin modified protein. Thus, by probing streptavidin label DyLight on Escherichia coli proteome microarrays and Saccharomyces cerevisiae proteome microarrays, in total of 12 biotinylated proteins for Escherichia coli and 44 biotinylated proteins for Saccharomyces cerevisiae were identified, respectively. Among 44 protein targets of Streptavidin, 30 protein targets overlapped with the protein targets of Anti-biotin labeled Dylight on Saccharomyces cerevisiae proteome microarrays. This anti-biotin probing confirmed the presence of several biotinylated proteins in Saccharomyces cerevisiae. In regard to the targets of AMPs, the entire targets of clinically used antibiotics are also poorly understood. Thus, a novel approach was also used in this study to identify the entire protein targets of antibiotics by utilizing Escherichia coli proteome microarrays and Saccharomyces cerevisiae proteome microarrays, respectively. In total of 93, 81, 87, 65 and 88 protein targets of Sulfamethoxazole, Trimethoprim, Minocycline, Streptomycin and Vancomycin, the commercial antibiotics, were identified from Escherichia coli proteome microarrays. These identified protein targets of antibiotics were bioinformatically analyzed together to understand their antibacterial activities. Whereas, the fungal protein targets of Sulfamethoxazole obtained from Saccharomyces cerevisiae proteome microarrays probing was 33 protein targets in total. Bioinformatics were performed to explore the antifungal activities of Sulfamethoxazole as well as compared the antibacterial and antifungal targets of Sulfamethoxazole. Moreover, the identified protein targets of AMPs from Escherichia coli proteome microarrays as well as Saccharomyces cerevisiae proteome microarrays were further compared to identify the mechanistic difference in their activities against bacterial and fungal pathogens. The comparison results showed completely different mechanism of action of same AMPs and antibiotic in case of bacteria and fungi. Furthermore, the identified protein targets of AMPs and antibiotics from the proteome microarrays approach not only provided the understanding for the mechanism of AMPs and antibiotics but also proved to be a useful tool to study the mechanism of synergistic combinations. The significant higher inhibition effect observed in combination than the sum of individual inhibition is termed as synergistic combination. Synergistic combination has multiple advantages, like enhancing the potential of antibiotics, reducing the doses of individual antibiotics thus lowering their toxicity, prolong in the resistance development as well as exert powerful effect on resistance pathogens. In regard to the limited knowledge of antibiotic target, the mechanism of synergistic combination is unclear. Hence, the entire protein targets identified for AMPs and antibiotics will more clearly explain the mechanism of synergistic combination. The common enrichment in same pathways resulted in the prediction of new synergistic combinations were discovered between AMP and AMP, AMP and antibiotic as well as antibiotic and antibiotic. As well as synthetic lethality approach was used to identify the synthetic lethal pairs between the identified fungal protein targets of AMPs. Based on identified synthetic lethal pairs and their involvement in same protein complex and reversible functions, the synergistic combination was predicted between Lfcin B and Histatin-5 and was experimentally validate in vivo by inhibition growth curve of Saccharomyces cerevisiae in the presence of individual and combination of Lfcin B and Histatin-5. | en_US |