FIGURE 5.

Generation and characterization of heterozygous knock-out mutants of LdTyrRS. A, map of LdTyrRS genomic locus and location of the primers used for confirmation by PCR-based analysis along with the expected band sizes. Primer 4 was designed as a forward primer to match the upstream region of the LdTyrRS gene, and primers 8, 3, and 6 were designed internal to TyrRS, HYG, and NEO coding regions, respectively. Primer 2 was designed as a reverse primer to match the downstream region of LdTyrRS gene, and primers 7, 1, and 5 were designed as forward primers, internal to TyrRS, HYG, and NEO coding regions, respectively. B, genomic DNA from heterozygous TyrRS/HYG and TyrRS/NEO mutant parasites was used as a template for PCR analysis. The specific integration of the replacement cassette was checked with HYG, NEO, and LdTyrRS (WT) gene-specific primers. M indicates the molecular size marker in kb. C, Western blotting analysis of equal protein quantities (∼30 μg) from whole cell lysates prepared from WT, TyrRS overexpressors (WT[pTyrRS⁺]), add-back (TyrRS/HYG[pTyrRS⁺]), and heterozygous mutant (TyrRS/HYG) parasites. The loading was normalized with α-tubulin (50 kDa) antibody. D, comparison of the aminoacylation activity of TyrRS in cell lysate of WT, heterozygous (TyrRS/HYG) mutant, and add-back (TyrRS/HYG[pTyrRS⁺]) parasites. E, growth curve of L. donovani WT, add-back (TyrRS/HYG[pTyrRS⁺]), and heterozygous mutant (TyrRS/HYG) promastigotes in M199 media. The results represent mean ± S.D. with n = 3. F, comparison of infectivity of L. donovani WT, heterozygous mutant (TyrRS/HYG), and add-back (TyrRS/HYG[pTyrRS⁺]) parasites in J774A.1 murine macrophage cell line. The murine macrophage cell line J774A.1 was infected with the stationary-phase promastigotes at an multiplicity of infection of 20:1. The cells were stained after 24 h and amastigotes enumerated visually. The results represent mean ± S.D. with n = 3. Student's t test was performed, and the p values are indicated.