Role Of FolP1and FolP2Genes In The Action Of Sulfamethoxazole-trimethoprim Against Mycobacteria

Tuberculosis (TB) is a chronicinfectious disease caused by Mycobacterium tuberculosis (Mtb). The emergence of drug-resistant Mtbis an urgent medical and public health concern as the available anti-TB drugs exhibit limited efficacy. Development of new drugs is not only a herculeantask but expensive. However, if already existing re-purposed drugs could be used for treatment of TB, then faster and cheaper drug development coupled with effective TB management would be attained.Sulfamethoxazole (SMX) and trimethoprim (TMP) are such potential candidates for TB treatment having been used in drug regimens for the treatment ofvarious bacterial infections of the respiratory, urinary, and gastrointestinal tracts. The mechanism of SMX and TMP in these bacteria is by targeting the dihydropteroate synthase (DHPS) and dihydrofolic acid (DHFR) in the folate biosynthesis pathway. However, the mechanism of TMP/SMX drug action in mycobacteria have yet to be unraveled. Therefore, uncovering the mechanism of action of TMP/SMX in Mtb may accelerate the TMP/SMX based anti-TB drug development and use.In mycobacteria, DHPS is encoded by the folPlgene and theprotein encoded by folP2gene has homology to DHPS.This study thus sets out to explore the role of folP1/folP2genes in TMP-SMX resistance in mycobacteria by gene knock out and overexpression. A series of recombinant Mycobacterium smegmatis mc2155(Msm)were constructed as: Msm::p60fp1, Msm::p60fp2, Msm::p60Msfpl and Msm::p60Msfp2and Msm△folP2.The minimum inhibitory concentrations (MIC)of SMX and TMP were determined using the classic agar dilution method. Knocking out of the folP2gene in Msm reduced the MIC of SMX8-fold compared with wild type. Overexpression of the folPl genes from Mtb and Msm increased the MIC by4and2-fold in Msm for SMX and TMP respectively.This suggests that a combination of FolP2inhibitor and SMX could be used for TB treatment with a better outcome.In this study, we also explored an improved technology for gene knock out in mycobacteria. The gene knock out techniques in Mtb have been limited by the slow growth of the organism, the need for biosafety level3(BSL3) containment, and most significantly, the relative inefficiency of existing techniques for targeted gene replacement compared with other model bacteria. The past20years, have seen many advanced gene knock out techniques developed, all but limited by the need for electroporation or recombinant phage to introduce the constructs into the mycobacterial cell, then delete the target gene through allelic replacement. The recombineering used for gene knock out, is still a challenge due to relatively low efficiency of electroporation in the slow-growing mycobacteria. Needless to mention, the construction of recombinant phage is expensive and the size is limited by λphage packaging. In this study, an improved method of introducing the fragment containing the allelic exchange substrates (AESs) into phasmid phAE159generating a specialized transducing phage was used for gene knock out in mycobacteria. Then, the AESs was introduced into mycobacteria through phage improving the efficiency for gene knock out.