PE11 (Rv1169c) selectively alters fatty acid components of Mycobacterium smegmatis and host cell interleukin-6 level accompanied with cell death

Abstract

PE/PPE family proteins, named after their conserved PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains of N-terminal, are most intriguing aspects of pathologic mycobacterial genome. The roles of most members of this family remain unknown, although selected genes of this family are related to the virulence of Mycobacterium tuberculosis. In order to decipher the role of Rv1169c, the Mycobacterium smegmatis strain heterologous expressed this ORF was constructed and identified that Rv1169c was a cell wall associated protein with a novel function in modifying the cell wall fatty acids. The growth of Rv1169c expressing strain was affected under surface stress, acidic condition and antibiotics treatment. M. smegmatis expressing Rv1169c induced necrotic cell death of macrophage after infection and significantly decreased interlukin-6 production compared to controls. In general, these results underscore a proposing role of Rv1169c in virulence of M. tuberculosis, as it's role in the susceptibility of anti-mycobacteria factors caused by modified cell wall fatty acid, and the induced necrotic cell death by Rv1169c is crucial for M. tuberculosis virulence during infection.

Keywords: Mycobacterium tuberculosis; PE family; Rv1169c; cell death; cell wall integrality; fatty acid components; interleukin-6.

Figure 1

Heterologous expression of M. tuberculosis Rv1169c in M. smegmatis. (A) Ms_Vec and Ms_Rv1169c were grown into an OD₆₀₀ of 0.8 and then were subjected to PCR amplification to detect Rv1169c gene. (B) The cell lysates were prepared from acetamide-induced Ms_Vec and Ms_Rv1169c were subjected to Western blot to determine the expression of His-tagged Rv1169c protein in M. smegmatis by mouse anti-His antibody.

Figure 2

Rv1169c is associated with Mycobacterium cell wall. Cell fractionation experiments were performed to detect detection the expression of Rv1169c protein, and further confirmed by Western blot by specific anti-His antibody. The cytoplasm marker of Mycobacterium GroEL2 served as a control.

Figure 3

The fatty acid component in cell wall of recombinant Ms_Rv1169c (A), Ms_Vec (B), and wild-type M. smegmatis (C).

Figure 4

The survival of Ms_Vec and Ms_Rv1169c after treatment with different antibiotics, including Rif (A), Nor (B), INH (C), and Van (D). Ms_Vec and Ms_Rv1169c were exposed to Rif, Nor, INH and Van, and then ten-fold dilution spotted the bacteria on MB 7H10 supplemented with Hyg, the bacterial number of Ms_Rv1169c and Ms_Vec were counted after 3 days cultivation. ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001.

Figure 5

The sensitivity of Ms_Vec and Ms_1169c strains to acid and surface stress. (A) In vitro growth of recombinant Ms_Vec and Ms_Rv1169c after treatment with different pH gradient for 0, 3, 6, and 9 h. The Ms_Vec and Ms_Rv1169c strains were centrifuged, re-suspended to 5 ml MB 7H9 at an OD₆₀₀ of 0.5, 10-fold serial dilutions of Ms_Vec and Ms_Rv1169c were spotted on MB 7H10 containing Hyg. (B) Survival of Ms_Vec and Ms_Rv1169c after exposure to 0.05% SDS. Re-suspended 5 ml recombinant MS_Vec and MS_Rv1169c (OD₆₀₀ = 0.5) were exposed to 0.05% SDS for 1, 2, 3, and 4 h. And then the recombinant strains were plated onto 7H10 plates by serially ten-fold dilution, the bacterial numbers were counted after 3–4 days of cultivation at 37°C. ^* P < 0.05 and ^** P < 0.01.

Figure 6

Intracellular survival of recombinant Ms_Vec and Ms_Rv1169c within macrophages. PMA-differentiated macrophages were infected with Ms_Vec or Ms_Rv1169c at an MOI of 10. At 6, 24, 48, and 72 h after infection, the macrophages were washed and lysed using 0.01% SDS. Lysates were plated on MB 7H10 medium containing 100 μg/ml Hyg to determine the bacterial number. ^* P < 0.05.

Figure 7

Assay of cell death in macrophages infected with Ms_Vec or Ms_Rv1169c. Macrophages were infected with Ms_Vec (white bars) or Ms_Rv1169c (black bars) at an MOI of 10. After 6, 24, 48, and 72 h infection, culture supernatants were collected and the release of LDH was measured. Data are shown as means ± SEM of triplicate wells. ^* P < 0.05.

Figure 8

The expression of cytokines in macrophage infected by Ms_Vec and Ms_Rv1169c. PMA-differentiated U937 macrophages (2 × 10⁶/well/2 ml) were infected with Ms_Vec and Ms_Rv1169c strains. After 6, 24, 48, and 72 h infection, the infected macrophage were collected and the transcription of IL-6 (A) and IL-1β mRNA (B) was detected by RT-PCR. Culture supernatants were harvested, the secretion of IL-6 (C) and IL-1β (D) were detected by ELISA analysis. ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001.