To construct a lentiviral vector-based short hairpin RNA (shRNA) targeting the gene encoding tryptophan-aspartate containing coat protein (TACO) and to evaluate its inhibitory effect on the expression of TACO, and to further elucidate its effects on the phagocytosing and intracellular killing of Mycobacterium tuberculosis (M.tb) by macrophages and the possible mechanisms.

Three shRNA fragments targeting TACO gene and a scrambling control shRNA fragments were designed and cloned into the lentiviral vector pSicoR. The recombinant lentiviral vectors were identified by sequencing analysis and then packed in 293T cells. Real-time RT-PCR and Western blot assay were performed to evaluate the gene-silencing efficiency of the recombinant lentiviral vectors among RAW264.7 cells transfected with the concentrated lentivirus. The most effective lentivirus was screened out to transfect the RAW264.7 cells for 48 hours, followed by infection those cells with M. tb strains. The entry and intracellular survival of M. tb strains in RAW264.7 cells were determined by bacterial culture at indicated time points. The colocalization of M. tb and lysosomes was detected by immunofluorescence staining. The cyto-ID autophagy kit was used to detect the cellular autophagy and the autophagy-associated protein LC3 was determined by Western blot assay.

The recombinant lentiviral vectors were successfully constructed and confirmed by sequencing analysis. Decreased expression of TACO in RAW264.7 cells was detected after transfecting the cells with the lentiviral vector-based shRNA vectors targeting TACO gene for 48 hours. The most effective lentivirus, LV-pSRT1, decreased the expression of TACO by 85.24% and 69.00% at the mRNA and protein levels, respectively. The bacterial loads in LV-pSRT1 transfected RAW264.7 cells were significantly decreased at the time point of 0 h after M. tb infection as compared with those in the control lentivirus treated cells (5.50×10⁴ vs 8.1×10^4,P<0.05). Compared with the RAW264.7 cells transfected with control lentivirus, the survival rate of intracellular M. tb strains in LV-pSRT1 transfected cells was significantly decreased at the time point of 48 h (134.54% vs 213.58%, P<0.05) and 72 h (148.18% vs 262.96%, P<0.05) considering the bacterial load at the time point of 0 h as the standard. The immunofluorescence staining demonstrated that the colocalization of M. tb strains with lysosomes was significantly enhanced in LV-pSRT1 transfected cells as compared with that in control lentivirus treated cells (75.67% vs 10.66%, P<0.05). Moreover, significantly enhanced autophagy and relative expression of LC3Ⅱ protein were observed in RAW264.7 cells with TACO gene knockdown (16.20% vs 8.50%, P<0.05; 0.51 vs 0.34, P<0.05).

The lentiviral vector-based shRNA targeting TACO gene could effectively knockdown the expression of TACO protein, decrease the entry and increase the intracellular killing of M. tb strains in macrophages. The enhanced intracellular killing of M. tb strains by macrophages was associated with the increased fusion of M. tb-containing phagosome and lysosome.

Tryptophan-aspartate containing coat protein; RNA interference; Lentivirus; Macrophage; Mycobacterium tuberculosis