Comparative peptidomic profiling of cationic peptides from Solanum lycopersicum under saline stress

Abstract

Antibiotics are frequently and universally common drugs used by humans in advocated and un-prescribed forms. Misuse of commercially available antibiotics has caused emergence of antibiotic-resistant microorganisms in addition to drug toxicity and other health complications. The global crisis of multi-drug resistance necessitates the development of novel antibiotics. Small peptides play a fundamental role in recruiting and promoting agents of innate as well as adaptive immune system. These are relied as good candidates for a new generation of antimicrobial drugs to treat multidrug-resistant microorganisms. The cationic peptides are also known as natural antibiotics and are produced constitutively as well as under the influence of biotic and abiotic stresses. The present project was designed to profile positively charged antimicrobial cationic peptides. Selected glycophytes (Solanum lycopersicum, Silibum marianum and Mentha piperita) and halophyte (Thallungiela halophila) were subjected to salt stress of NaCl at different molar concentrations. The glycophytes were treated with 100 mM salt for 14 days (SLS100-14) and 200 mM of salt for 5 (SLS200-5) and 10 days (SLS200-10) and halophyte was treated with 300 mM NaCl for 3 days (THS300-3). Small proteins and peptides of S. lycopersicum and T. halophila of less than 10 kDa were fractionated by strong cation exchange chromatography. All the obtained fractions were tested for bioactive potential against E. coli and active fractions of tomato plants (control and 100 mM salt stressed). The results showed that tomato plants exhibited salt tolerance at 100 mM of NaCl. The peptides of control and salt treated (SLS100, SLS200-5 and SLS200-10) tomato plants were fractionated on strong cation exchange column and fractions of control and treated with 100 mM NaCl showed significant antibacterial activities against E. coli. The fractions of control and salt-treated (100 mM NaCl) plant samples were run on LC-MS and obtained spectrum peak list were analyzed manually for independent data acquisition (IDA) with a software (Analyst, AB Sciex). The software converted total ionic currents (TIC) of peptide fragments and generated charge to mass ratios for each peak that gave profiles of all the peptides. The LC-MS data files were imported into Progenesis for retention time alignment, peak picking and relative quantitation of unique features. The output from Progenesis were subjected to a python script which removed likely the same features. These features were run through a second python script for filtering to identify x features that were most likely contributed to the bioactivity with their highest abundance in the bioactive region and not elsewhere. Then statistical modeling was employed to further narrow down the list of candidates. Sequence of bioactive peptides were identified by using SwePep database and the obtained peptide sequences were subjected to BLAST search by using bioactive peptide database, “AMPer” for the identification of protein sequence similarities of subject with already reported bioactive plant peptides/ proteins. The statistical modeling listed 12 and 16 bioactive peptides from SLC and SLS100 tomato samples, respectively. The bioinformatics investigation of these bioactive peptides identified antimicrobial cationic peptides of different classes which suggested that these peptides were expressed in tomato plants. Furthermore, bioactive study of crude protein extracts of the tomato seeds and leaves suggested that tomato seeds are rich source of proteins and have significant anti-thrombotic and antioxidant potential. The crude protein extracts of tomato seeds and leaves were found not only DNA friendly and non-mutagenic but also exhibited considerable antibacterial and antifungal activities. Keeping in view the findings of current research project, the Solanum lycopersicum (tomato) plant can be used as potent candidate for cationic peptides with bioactive potential to be used in therapeutics.