Mycobacterium tuberculosis PE_PGRS17 promotes the death of host cell and cytokines secretion via Erk kinase accompanying with enhanced survival of recombinant Mycobacterium smegmatis

Abstract

Tuberculosis (TB) remains a serious threat to global public health, largely due to the successful manipulation of the host immunity by its etiological agent Mycobacterium tuberculosis. The PE_PGRS protein family of M. tuberculosis might be a contributing factor. To investigate the roles of PE_PGRS17, the gene of PE_PGRS 17 was expressed in nonpathogenic fast growing Mycobacterium smegmatis. We found that the recombinant strain survives better than the control in macrophage cultures, accompanied by more host cell death and a marked higher secretion of tumor necrosis factor-alpha by a recombinant strain compared with control. Blocking the action of Erk kinase by an inhibitor can abolish the above effects. In brief, our data showed that PE_PGRS 17 might facilitate pathogen survival and disserve the host cell via remodeling the macrophages immune niche largely consisting of inflammatory cytokines. This furnishes a novel insight into the immune role of this mycobacterium unique gene family.

FIG. 1.

Expression of PE_PGRS17 in recombinant Mycobacterium smegmatis revealed by Western Blotting. The 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis exhibited a 36-kDa protein, the sum of a 31-kDa expected protein, and 5-kDa MYC tag expressed by recombinant M. smegmatis strains induced by ɛ-caprolactam. Lane 1: the protein of PE_PGRS 17 expressed in recombinant M. smegmatis strains induced by ɛ-caprolactam for 24 h. Lane 2: recombinant M. smegmatis strains containing the vector only.

FIG. 2.

Survival of recombinant M. smegmatis strains after infection of U937 at an MOI of 10:1. (A) U937 cells were infected with recombinant M. smegmatis strains containing vector only (♦), PE_PGRS 17 (▪) at the MOI of 10. At 24, 48, and 72 h after infection, the macrophages were washed and lysed. Lysates containing the live bacteria were diluted gradually and then plated on 7H10 agar plates to determine CFU. (B) Growth of 2 recombinant M. Smegmatis strains containing vector only (♦), PE_PGRS 17 (▪) at 37°C in 7H9 liquid media were assayed by OD₆₀₀ values. Similar results were obtained in three independent experiments.

FIG. 3.

Death of U937 macrophages infected with recombinant M. smegmatis. U937 macrophages were infected with Msmeg-pNIT (MYC)-Rv0978c (black bars) and M. smeg-pNIT (MYC) (white bars) at an MOI of 10, respectively. At 6-, 24-, and 48-h postinfection, culture supernatants were harvested. The release of LDH was estimated by testing its activity in the culture supernatants. The results showed that recombinant M. smegmatis containing PE_PGRS 17 increased U937 macrophages death at the MOI 10:1 in a time-dependant manner. Each treatment was repeated three times.

FIG. 4.

Secretion of the tumor necrosis factor (TNF)-α and interleukin-10 (IL-10) in U937 infected with recombinant M. smegmatis strains measured by ELISA. (A, B) U937 were infected at an MOI of 10:1 with M. smegmatis containing vector only (□), PE_PGRS 17 (▪) and incubated for different time duration. Culture supernatants were collected at indicated intervals and used to quantify the production of TNF-α and IL-10 by the ELISA kit. (A) The secretion of TNF-α is significantly higher by macrophages infected with M. smegmatis expressing PE_PGRS 17 than the control group at 24 h and 30 h post-infection. (B) The release of IL-10 in macrophages is much higher by the control group at 6 h after infection. Each treatment was repeated three times.

FIG. 5.

The secretion of TNF-α and IL-10 in U937 cells infected with recombinant M. smegmatis strains measured by ELISA and reverse transcription-polymerase chain reaction (RT-PCR). (A) U937 cells were infected at an MOI of 10:1 with M. smegmatis containing vector only (□), PE_PGRS 17 (▪) and incubated for 24 h. Culture sediments were used to extract RNA, followed by semiquantitative RT-PCR to demonstrate the transcriptional levels of TNF-α and IL-10. (B, C) Culture supernatants were collected at 24 h after infection and used to quantify the production of TNF-α and IL-10 by the ELISA kit.