Title : Standardization of a pncA Gene Complementation Technique in Mycobacterium tuberculosis pncA-knockout: Tool for the Study of the Relationship Between Mutations in pncA and Phenotypic Parameters
Mutations in the pncA gene are the major cause of resistance to pyrazinamide (PZA), but there are alternative mechanisms that also provide resistance. The objective of the study was to standardize the pncA gene complementation technique in Mycobacterium tuberculosis H37Rv pncA-knockout to evaluate the effect of mutations in the pncA gene, in a scenario controlled by the genetic variability of clinical isolates.
Strains complemented with pncA wild type and D49N, H51R, G78C and F94L mutant genes were generated through the pNIT-1 expression system to evaluate pyrazinoic acid (POA) production by means of the quantitative Wayne test, pncA gene expression level and PZA susceptibility using gradual concentrations of PZA under non-induced and induced conditions.
The pNIT-pncA expression system restored the susceptible phenotype of the pncA-knockout strain. The basal expression levels of the pncA genes were constant and similar to that of the H37Rv strain. The pncA wild type complemented strain showed POA production and Minimum Inhibitory Concentration of PZA similar to H37Rv (0.6 uM and 100 ug/mL, respectively). Although the overexpression of pncA wild type in the complemented strain induced higher production of POA (3 uM), this did not interfere with susceptibility to PZA. However, the mutant pncA strains complemented were resistant in both conditions and the overexpression of the mutant pncA genes, nine times greater than that of the H37Rv strain (P<0.005), made it possible to verify the total or partial loss of the enzymatic function of the mutant pyrazinamidase.
The complementation of the pncA-knockout strain with the pncA wild type gene, by means of the pNIT expression system, allowed the restoration of the susceptible phenotype, and the overexpression of pncA with D49N, H51R, G78C and F94L mutations in the complemented strains really confer resistance to PZA.