Research Projects

Keratitis, one of the most neglected tropical diseases, often affects farmers, labourers and other field workers. It is highly infectious, inflammatory condition of the cornea, caused by bacteria and fungi. However, bacterial keratitis is more common, generally caused by ESKAPE pathogens like Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. It can destroy the cornea within 24-48 hours if not treated. Since corneal surface has no blood circulation attached to it, most of the existing systemic antibiotics are ineligible for topical application. Besides, the currently available treatment strategies are often challenged by growing rate of antimicrobial resistance (AMR). Thus, current situation necessitates discovery of novel antimicrobials, such as, antimicrobial peptides (AMPs), to which bacteria cannot easily develop resistance. A sub-category of AMPs are ultra-short AMPs, which range from 2-7 amino acids in length and hold capacity to disrupt bacterial membrane without causing much side-effects to the mammalian cells. We plan to design ultra-short peptides against drug-resistant pathogens. Our focus is to harness the strength of natural longer peptides identified from human gut microbiome to derive ultra-short peptides from them. We performed the retrieval of 50 human gut metagenome samples from SRA and predicted AMPs with antibacterial and antibiofilm properties using ML algorithms. 24 such peptides were obtained. 22 were found to be cationic and alpha-helical, out of which 2 also had a positive GRAVY value (AMP19: 0.04, AMP7: 1.23). AMP7 was selected as our parent AMP, using which 4 mutants were generated to increase antibacterial efficacy by replacing leucine (L) with tryptophan (W) at different positions. Using Anti-BP server, we found that our mutants (AMP7_1, AMP7_2, AMP7_3 and AMP7_4) carry a greater number of antibacterial domains (ABPs). AMP7 and its mutants were subjected to Steered MD simulation to study membrane disruption efficacy. The top 3 membrane disrupting peptides identified in silico were AMP7_2, AMP7_3, and AMP7_4 which were taken for wet lab validation. The highest non-cytotoxic concentration for the AMPs was found to be: AMP7_2: 50 µg/ml, AMP7_3:200 µg g/ml, and AMP7_4:200 µg g/ml for 24 h. Further, their MIC was tested against ESKAPE. AMP7_3 demonstrated potent antimicrobial activity, with MIC values of 16 µg/mL or lower against most pathogens, indicating broad-spectrum efficacy. AMP7_2 showed moderate activity, with an MIC of 32 µg/mL against E. coli IMP 4213 (MICs against other pathogens: more than 64 µg/mL). AMP7_4 displayed the weakest activity, with MIC values exceeding 64 µg/mL across all tested strains The above peptides were further subjected to designing for ultra-short peptides, to ensure better efficacy, reduced toxicity, enhanced stability and enhanced cost effectiveness. For each of the 84 ABPs identified within AMP7 and its mutants, we generated all overlapping 4 to 7-mer peptides (ultra-short). Each of these ultra-short peptides was further subjected to sequence scrambling to create shuffled peptides. Additional peptides were generated by shuffling the full parent AMP and re-extracting 4 to 7-mers from the scrambled version. DBAASP antibacterial prediction was carried out on these ultra-short peptides. Peptides that were found to be DBAASP-active were studied for their physiochemical properties. Out of all active peptides, 31 were found to have a charge of +3, and 42 to have a positive GRAVY value. Additionally, 38 were found to have an amphiphilicity index of more than 3.5. Overall, 34 ultra-short peptides were found to fulfil more than 1 criteria of charge, hydrophobicity and amphiphilicity. These have been taken for studying bacterial membrane disruption using steered MD, best of which will be subjected to wet laboratory validation, and testing iv vivo efficacy in SD rat models of keratitis.