The Role Of Toxin-Antitoxin Systems In The Survival Of Multidrug Tolerant Pathogens And Designing Of New Approaches To Treat Them

Background: The chronic infections are often caused by pathogens that are susceptible to antibiotics. Existing antimicrobial therapies are not able to treat or eradicate chronic infections. Recent studies show that treatments for chronic infections are failed due to small subpopulation of microbial cells, called persister cells. Persister cells have ability to survive even at lethal dose of antibiotics known as multidrug tolerance. The molecular mechanism of persister cell formation is unknown and not well understood but it is well established that toxin-antitoxin systems play a key role in the formation of same. Currently available therapeutic suppress, but do not eradicate the infection completely. An effective method for their eradication is great interests to researchers. We found three gene loci in the genome of Pseudomonas aeruginosa codes for toxin-antitoxin proteins. Certain metabolites if given in combination of antibiotics, they can treat persister cells more efficiently. The identification and evaluation of toxin-antitoxin genes can provide a clue to develop new strategies for treating pathogens. The study of various metabolites effects in combination with antibiotics on persister cell can lead to development of new antipersister therapy for treating chronic and latent infections. Methods & Materials: Persister Cell Assay (Antibiotic based): Culture of P. aeruginosa, ampicillin & LB-Agar. Persister Cell Assay (Green fluorescent protein based): Arabinose inducible green fluorescent protein containing vector. Protein-Protein Blast: Putative toxin-antitoxins operon from the genome of P. aeruginosa using NCBI server. (http://blast.ncbi.nlm.nih.gov/Blast.cgi) Phylogenetic Analysis: Different annotated protein sequences were multiple aligned and phylogenetic trees were constructed by using CLC Genomics Workbench (version 5.5 software). Results: Please find attachmentsView Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conclusion: This experiment revealed a small subpopulation of cells that remain alive irrespective of concentration of antimicrobial agents. In exponential phase the number of persister cell was low than stationary growth phase populations. In addition, the persister cells of P. aeruginosa exhibit a high tolerance to the variety of antibiotics, and phenotype was not inherited as tested with four passages of P. aeruginosa populations. In-silico results show the presence of parDE, relBE, and higBA toxin-antitoxin systems among which ParE toxin generates double strand breaks while RelE & HigB toxins induce mRNA cleavage.