Mycobacterium tuberculosis Rv0341 Promotes Mycobacterium Survival in In Vitro Hostile Environments and within Macrophages and Induces Cytokines Expression

Abstract

Mycobacterium tuberculosis represents an ancient deadly human pathogen that can survive and multiply within macrophages. The effectors are key players for the successful pathogenesis of this bacterium. M. tuberculosis open reading frame (ORF) Rv0341, a pathogenic mycobacteria-specific gene, was found to be upregulated in macrophages isolated from human tuberculosis granuloma and inside the macrophages during in vitro infection by M. tuberculosis. To understand the exact role of this gene, we expressed the Rv0341 gene in M. smegmatis, which is a non-pathogenic Mycobacterium. We found that Rv0341 expression can alter colony morphology, reduce the sliding capability, and decrease the cell wall permeability of M. smegmatis. Furthermore, Rv0341 remarkably enhanced M. smegmatis survival within macrophages and under multiple in vitro stress conditions when compared with the control strain. Ms_Rv0341 significantly induced expression of TNF-α, IL-1β, and IL-10 compared with M. smegmatis harboring an empty vector. In summary, these data suggest that Rv0341 is one of the M. tuberculosis virulence determinants that can promote bacilli survival in harsh conditions and inside macrophages.

Keywords: Mycobacterium tuberculosis; Rv0341; cytokines; infection; macrophages; stress.

Figure 1

Rv0341 protein was ectopically expressed in M. smegmatis. (A) The 1440 bp Rv0341 gene detected in Ms_Rv0341, Lane M (DNA ladder marker), Lane 1 is the band of Rv0341 gene, and Lane 2 is the negative control (Ms_Vec). (B) Western blot detection of Rv0341 protein expression in M. smegmatis.

Figure 2

Ms_Rv0341 colonial morphology was altered and its sliding motility was reduced. (A) In vitro growth kinetics of Ms_Rv0341 and Ms_Vec strains in liquid MB7H9 medium was determined by measuring growth optical density (OD₆₀₀) every 6 h. (B) The colony of Ms_ Rv0341 and Ms_Vec on MB7H9 agar. (C) Sliding of recombinant M. smegmatis strains in MB7H9 containing 0.03% agarose. The photographs were taken at 5–6 days after incubation at 37 °C.

Figure 3

Rv0341 can enhance the Mycobacterium resistance to multiple stresses. (A) Survival of Ms_Vec and Ms_Rv0341 strain under pH 3. (B) Susceptibility of Ms_Vec and Ms_Rv0341 to the surface stressor (0.05% SDS). (C,D) Survival of Ms_Vec and Ms_Rv0341 in oxidative stress, (C) Survival of recombinant strains in the presence of diamide at a final concentration of 2mM or 7mM. (D) Survival of Ms_Rv0341 and Ms_Vec upon exposure to 7 mM hydrogen peroxide (H₂O₂). Asterisks represent statistical significant difference (* p < 0.05, ** p < 0.01, *** p < 0.001). Experiments were done in triplicate, and similar findings were obtained in three independent experiments.

Figure 4

Rv0341 reduced the cell wall permeability of Mycobacterium. Accumulation of ethidium bromide (EtBr) and Nile Red dyes were used to analyze the cell envelope permeability of recombinant M. smegmatis strains. The ε-caprolactam-induced Ms_Rv0341 and Ms_Vec growth were collected, washed three times, and suspended in sterile phosphate buffer saline (PBS) to an OD600 of ~0.8. Next, the bacterial suspension was treated with 2 μg/mL EtBr (A,B) 20 μM Nile Red dye, and the fluorescence intensity was measured in appointed time using a FLUOstar OPTIMA microplate reader.

Figure 5

Rv0341 expression increased Mycobacterial survival within macrophages. (A) RAW267.4 murine macrophages and (B) phorbol 12-myristate 13-acetate (PAM)-differentiated THP-1 macrophages were infected with Ms_Vec and Ms_Rv0431. The infected macrophages were washed by sterile PBS and then lysed with 1 mL of 0.025% SDS at the indicated time. Next, cell lysate was serially diluted and inoculated on kanamycin MB7H9 agar to enumerate the bacterial colony-forming units (CFU/mL). Asterisks indicated statistical significance; * p < 0.05; ** p < 0.01. The experiments were done in three biological repeats, and similar findings were achieved in three independent experiments.

Figure 6

Ms_Rv0341induced cytokine expression. The cytokine transcription of THP-1 macrophages infected with Ms_Rv0341 or Ms_Vec was examined. Total RNA was extracted at 6 and 48 h of post-infection, and subsequently, cDNA was generated and analyzed by quantitative qRT-PCR. (A) TNF-α, (B) IL-1β, (C) IL-6, and (D) IL-10 expressions were calculated after being normalized with expression levels of beta-actin. Asterisks indicate significant difference as * p < 0.05, ** p < 0.01, *** p < 0.001. The experiments were done in triplicates, and similar findings were obtained in three independent experiments.