Research ArticleMICROBIOLOGY

VapBC22 toxin-antitoxin system from Mycobacterium tuberculosis is required for pathogenesis and modulation of host immune response

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Science Advances  03 Jun 2020:
Vol. 6, no. 23, eaba6944
DOI: 10.1126/sciadv.aba6944
  • Fig. 1 Effect of overexpression of VapC22 on growth and transcriptomics of M. tuberculosis.

    (A and B) The expression of VapC22 and VapC22D8A in M. bovis BCG (A) and M. tuberculosis (B) was induced by the addition of Atc. The growth of various strains was determined by measuring absorbance at 600 nm at regular intervals. The data shown in these panels are representative of three independent experiments. (C to I) Differential gene expression in M. tuberculosis upon VapC22 overexpression. Four hundred forty-seven genes were significantly differentially expressed upon overexpression of VapC22 in M. tuberculosis. (C) The volcano plot displaying gene expression profiles observed 24 hours after VapC22 expression in M. tuberculosis. The y and x axes depict P value and fold change for each gene, respectively. The statistically significant differentially expressed induced and repressed transcripts are highlighted as red and blue dots, respectively. (D) The number of induced and repressed transcripts categorized by functional category is shown. (E) The transcript levels of various differentially expressed genes between the parental and VapC22 overexpression strain at 24 hours after Atc induction were quantified. The data shown in this panel are means ± SE of fold change in the overexpression strain relative to the parental strain obtained from three independent experiments. wrt with respect to (F) Venn diagram showing correlation of differentially expressed genes in VapC22 overexpression strain with nutritionally starved proteome and enduring hypoxia response transcriptome studies. (G to I) Heat maps showing fold change in expression of common genes up-regulated (G) and down-regulated (H) in VapC22 overexpression strain and nutritionally starved bacteria. The common genes up-regulated in our and EHR (enduring hypoxia response) transcriptome are shown in (I). w.r.t., with respect to.

  • Fig. 2 Susceptibility of M. tuberculosis strains under various stress conditions and host tissues.

    (A and B) In vitro growth curves of wild-type, ΔvapC22, and ΔvapC22-complemented strains in liquid cultures. The growth kinetics of parental, ΔvapC22, and ΔvapC22-complemented strains was performed in Middlebrook 7H9 medium by measuring either optical density at 600 nm (OD600nm) (A) or colony-forming unit (CFU) analysis (B) at regular intervals. (C) The effect of deletion of vapC22 on M. tuberculosis susceptibility upon exposure to oxidative and nitrosative stress. Early-log phase cultures of various strains were exposed to either oxidative or nitrosative stress. For bacterial enumeration, 10.0-fold serial dilutions were prepared, and 100 μl was plated on Middlebrook 7H11 medium at 37°C for 3 to 4 weeks. The results shown in these panels are means ± SE of data obtained from three independent experiments. Statistically significant differences were obtained for the indicated groups (paired two-tailed t test, *P < 0.05). (D to J) The effect of deletion of vapC22 on growth of M. tuberculosis in guinea pigs and immunocompetent mice. (D) The representative images of lung and spleen tissue from guinea pigs infected with either wild-type or ΔvapC22 strain via aerosol route are shown. Photo credit: Sakshi Agarwal, Translational Health Science and Technology Institute. (E and F) The bacterial loads were determined by plating lung and spleen homogenates at 4 weeks (E) and 8 weeks (F) after infection. The homogenates were serially diluted and plated on Middlebrook 7H11 medium at 37°C for 3 to 4 weeks. The data shown in these panels are means ± SE of log10 CFU obtained from either six or seven guinea pigs per group per time point. (G) Lung sections of guinea pigs infected with wild-type and ΔvapC22 strain were stained with hematoxylin and eosin (H&E) and viewed at a magnification of ×40. Scale bars, 200 μm. Photo credit: Ashok Mukherjee, National Institute of Pathologist. (H) The total granuloma score in H&E-stained sections from wild-type and ΔvapC22-infected guinea pigs was determined as previously described. The data shown in means ± SE of total granuloma score obtained from six or seven animals per group. (I to J) Female Balb/c mice were infected with parental, ΔvapC22 mutant, and ΔvapC22-complemented strains via a low-dose aerosol infection. At 2 weeks (I), 4 weeks, and 8 weeks (J) after infection, lungs were homogenized, serially diluted, and plated to obtain bacterial loads. The data shown in panels B, C, E, F, H, I, and J are means ± SE of log10 CFU obtained from five animals per group per time point. Statistically significant differences were obtained for the indicated groups (paired two-tailed t test, *P < 0.05, **P < 0.01, and ***P < 0.001).

  • Fig. 3 Effect of VapB22 overexpression on survival of M. tuberculosis under oxidative stress condition and host tissues.

    (A and B) Proteome comparison of parental and ΔvapC22 strains of M. tuberculosis. (A) Volcano plots represent proteome comparisons of the mid-log phase cultures of ΔvapC22 to the parental strain. The number of proteins compared is mentioned in the bottom-right side of the plot. The statistically significant differentially expressed proteins are highlighted as red (increased) and blue (decreased) dots, respectively. The y and x axes depict P value and fold change for each protein, respectively. (B) The number of differentially expressed proteins in ΔvapC22 strain is categorized by their functional category. (C) Proteome comparison of M. tuberculosis strains harboring either vector or VapB22 overexpression construct. Volcano plot representing proteome comparison of mid-log phase cultures from the parental and VapB22 overexpression strains. The number of proteins identified is mentioned in the bottom right side of the panel. The statistically significant differentially expressed proteins are highlighted as red (increased) and blue dots (decreased), respectively. The y and x axes depict P value and fold change for each protein, respectively. (D and E) Overexpression of VapB22 increases susceptibility of both M. tuberculosis and M. bovis BCG upon exposure to oxidative stress. Various M. tuberculosis (D) and M. bovis BCG (E) strains were exposed to oxidative stress, and CFU enumeration was performed. The results shown in these panels are means ± SE of log10 CFU obtained from three independent experiments. Statistically significant differences were observed for the indicated groups (paired-two tailed t test, *P < 0.05 and **P < 0.01). (F to K) The M. tuberculosis VapB22 overexpression strain is attenuated for growth in guinea pigs and immunocompetent mice. (F) Representative image of the lungs and spleens of guinea pigs infected with parental and overexpression strains at 4 and 8 weeks after infection via aerosol route. Photo credit: Sakshi Agarwal, Translational Health Science and Technology Institute. (G and H) The growth of parental and overexpression strains in guinea pigs was assessed by determining lung and splenic bacillary loads at 4 weeks (G) and 8 weeks (H) after infection. The data shown in these panels are means ± SE of log10 CFU obtained from six or seven animals per group per time point. (I) For histopathology analysis, H&E-stained sections of lungs infected with parental and overexpression strain were viewed at a magnification of ×40. Scale bars, 200 μm. Photo credit: Ashok Mukherjee, National Institute of Pathologist. (J) The total granuloma score in H&E-stained sections from guinea pigs infected with various strains was determined. The data shown in this panel are means ± SE of data obtained from six or seven animals per group. (K) Female Balb/c mice were infected with wild-type and VapB22 overexpression strain, and CFU counts were determined in the lungs at 4 and 8 weeks after infection. The data shown in this panel in means ± SE of log10 CFU obtained from five mice per group per time point. Statistically significant differences were obtained for the indicated groups (paired-two tailed t test, *P < 0.05 and **P < 0.01).

  • Fig. 4 Global transcriptome profile and immune response of lung tissues from uninfected mice and from mice infected with either wild-type or ΔvapC22 strain of M. tuberculosis.

    (A) Venn diagram showing correlation of expression profiles obtained from lung tissues of uninfected, wild type (WT)–infected, and ΔvapC22-infected mice. (B) The volcano plot showing comparison of gene expression profiles from mice infected with parental and ΔvapC22 strain of M. tuberculosis. The genes that are significantly up-regulated or down-regulated by more than 2.0-fold have been highlighted as blue and red dots, respectively. The y and x axes depict P value and fold change for each gene, respectively. (C) Heat map showing transcripts differentially expressed by 4.0-fold in lung tissues of mice infected with wild-type and ΔvapC22 strain after 4 weeks of infection. The gene names and pathways are indicated to the right of heat map, and growth conditions are mentioned at the top. The data shown are obtained from three biological replicates. (D) The levels of intracellular cytokines in lung homogenates from 4-week infected mice were assayed by enzyme-linked immunosorbent assay as per the manufacturer’s recommendations. Significant differences were obtained for the indicated groups (paired-two tailed t test, *P < 0.05, **P < 0.01, and ***P < 0.001).

  • Fig. 5 Proposed model for regulation of virulence by VapBC22.

    The imbalance in the relative levels of both VapC22 and VapB22 results in reduced expression of various virulence proteins. The virulence-associated proteins such as ESX-I, ESX-V, KasA, KasB, and AcpM modulate host immune response to enhance replication of intracellular M. tuberculosis. In agreement, the transcripts involved in innate immune responses as evident by induction of apoptosis, recruitment of M2 macrophages, neutrophils, and dendritic cells were enhanced in mice infected with ΔvapC22 strain. We propose that the repression of virulence-associated proteins in ΔvapC22 and VapB22 overexpression strain is associated with their faster clearance by the innate immune response resulting in reduced immunopathology.

Supplementary Materials

  • Supplementary Materials

    VapBC22 toxin-antitoxin system from Mycobacterium tuberculosis is required for pathogenesis and modulation of host immune response

    Sakshi Agarwal, Arun Sharma, Rania Bouzeyen, Amar Deep, Harsh Sharma, Kiran K. Mangalaparthi, Keshava K. Datta, Saqib Kidwai, Harsha Gowda, Raghavan Varadarajan, Ravi Datta Sharma, Krishan Gopal Thakur, Ramandeep Singh

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