Mycobacterial PE_PGRS62 gene is a virulence factor during tuberculosis.

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PE_PGRS62 is thought to be a virulence factor during tuberculosis. To determine whether PE_PGRS62 is a virulence factor of M. tuberculosis, we deleted PE_PGRS62 of M. tuberculosis Erdman (WT) using mycobacterial phage system. We designed the knockout phage of PE_PGRS62/Rv3812 refer to Erdman genomic DNA. A knockout strain, ΔPE_PGRS62 was constructed and was complimented with the integrative plasmid pMV306 into which PE_PGRS62 gene and its promoter are inserted (Δ62Comp). We also transformed an empty pMV306 as a control strain of Δ62 Comp to ΔPE_PGRS62 (Δ62/mock). To confirm these gene disruption and complementation, we extracted genomic DNA from WT, Δ62/mock and Δ62 Comp strains. The fragments treated with SmaI are detected with specific probe. Next, we examined expression of PE_PGRS62 of WT, Δ62/mock and Δ62Comp strains. These strains were cultured in 7H9/ADC medium and their RNAs were extracted. The transcription levels of PE_PGRS62 were determined by qRT-PCR with specific primers. We observed the recovery of PE_PGRS62 expression in Δ62Comp strain. We checked any difference among WT, Δ62/mock and Δ62Comp. Although we confirmed no differences among these strains grown in 7H9/ADC culture medium, Δ62/mock mutant strain showed reduced the survival rate of intracellular mycobacteria in J774 cells. To evaluate the virulence attenuation of ΔPE_PGRS62 in mice, we injected the strains of WT or ΔPE_PGRS62 into tail vein of mice. There was a significant delay in survival infected with the ΔPE_PGRS62 mutant strain relative to WT strain in BALB/c and SCID mice. These results demonstrate that PE_PGRS62 is required for M. tuberculosis virulence.