Abstract

   Mechanistic understanding of antibiotic persistence is a prerequisite
   in controlling the emergence of MDR cases in Tuberculosis (TB). We have
   reported that the cholesterol-induced activation of VapC12 ribonuclease
   is critical for disease persistence in TB. In this study, we observed
   that relative to the wild type, mice infected with ΔvapC12 induced a
   pro-inflammatory response, had a higher pathogen load, and responded
   better to the anti-TB treatment. In a high-dose infection model, all
   the mice infected with ΔvapC12 succumbed early to the disease. Finally,
   we reported that the above phenotype of ΔvapC12 was dependent on the
   presence of the TLR4 receptor. Overall, the data suggests that failure
   of a timely resolution of the early inflammation by the ΔvapC12
   infected mice led to hyperinflammation, altered T-cell response and
   high bacterial load. In conclusion, our findings suggest the role of
   the VapC12 toxin in modulating the innate immune response of the host
   in ways that favor the long-term survival of the pathogen inside the
   host.

   Keywords: tuberculosis, toxin-antitoxin, virulence, neutrophils,
   persistence, and inflammation

Introduction

   COVID-19 has augmented the tuberculosis (TB) burden by disrupting the
   existing healthcare systems resulting in a delay in diagnosis and
   treatment ([41]1). In spite of the availability of anti-tubercular
   therapy, its eradication still remains obscure ([42]2). In addition,
   non-compliance to a prolonged treatment regimen fuels the development
   of multidrug and extensively drug-resistant TB posing blockades to
   global TB control ([43]3). The virulence of Mycobacterium tuberculosis
   (M. tuberculosis) is highly transmissible primarily through the
   aerosolization of the respiratory secretions from the infected
   individuals. This together with a relatively low virulence has allowed
   this pathogen to successfully co-evolve with humans for centuries
   without causing any symptoms or death ([44]4). We believe that the
   complex network of host-pathogen interaction, thus developed during
   evolution, defines TB in terms of its presentation either as an
   asymptomatic infection or an acute infection presented with clear
   symptoms. Nevertheless, both the pathogen and the host have gradually
   evolved mechanisms that would identify and neutralize any mutually
   directed threats.

   Innate immunity is an indispensable arm of the host immune system
   mediated by phagocytes such as macrophages; bona fide host cells
   harboring the mycobacteria ([45]5). The role of neutrophils which are
   highly motile innate immune cells has been shown in experimental models
   of TB pathogenesis ([46]6–[47]9). Following the M. tuberculosis
   infection, these are the primary cells to percolate the lungs and have
   an extremely intricate role in TB pathology ([48]10). Neutrophils
   mediate their phagocytic activity by producing proteases, reactive
   oxygen and nitrogen intermediates and antimicrobial peptides ([49]11).
   These immune cells are the most common cell type present in the sputum,
   bronchoalveolar fluid and cavities of active TB patients ([50]10,
   [51]12). There is a negative correlation between their abundance in the
   blood of M. tuberculosis-infected patients and disease severity
   ([52]13–[53]16). On the contrary, some neutrophil subpopulations are
   also known to aid in providing a permissive replicating niche and
   nutrient reservoir for M. tuberculosis, indicating their role in
   promoting mycobacterial persistence ([54]17, [55]18). The same has also
   been reported in M. tuberculosis infected rhesus macaques ([56]19) and
   zebrafish ([57]20). Growing evidence suggests the phenotypic and
   functional diversities of neutrophils which extend well beyond their
   conventional role as antimicrobial agent. Timely resolution of
   neutrophils at the site of infection dictates their fate as either
   protective or dysregulated immune cells and is crucial for the
   effective restoration of the excessive inflammatory response to
   homeostasis. The ineffective clearance of neutrophils leads to
   exacerbation of inflammatory response and perpetuation of tissue damage
   ([58]21). Furthermore, the role of mycobacterial genes in shaping an
   appropriate neutrophil-mediated innate immune response and its timely
   resolution that affects the downstream adaptive immunity, remains
   largely unknown.

   Previously, using the guinea pig model, we demonstrated that both lungs
   and spleen isolated from guinea pigs infected with M. tuberculosis
   lacking the VapC12 ribonuclease toxin were presented with higher
   bacterial burden and increased lung pathology relative to the wild type
   (WT) ([59]22). We hypothesize that the VapC12 toxin in M. tuberculosis
   is critical for modulating the host immune response and hence the
   absence of this toxin in M. tuberculosis resulted in a severe form of
   the disease. To further our understanding of the role of the host
   immune system in imparting the above phenotype to the vapC12 deleted
   strain, in the current study, we performed comprehensive
   immune-profiling studies in mice infected with WT, ΔvapC12 and the
   complemented strains to identify host correlates of disease
   persistence. We found that in comparison to WT, the lungs of mice
   infected with the mutant strain were presented with higher bacterial
   load and enhanced pathology. Additionally, these mice showed an excess
   of macrophages, neutrophils and myeloid-derived suppressor cells
   (MDSCs) in the lungs with an alleviated T-cell immune response. In the
   survival study, performed with a high-dose infection model, while all
   the mice infected with WT strain survived none of the mice from the
   ▵vapC12 group survived 18 weeks post-infection. Additionally, lung
   transcriptome analysis revealed that the observed neutrophil
   accumulation was mirrored by increased S100A8 and S100A9 mRNA levels in
   the lungs infected with ΔvapC12 when compared with the WT strain.
   Interestingly, we found that the enhanced pathogenesis observed in the
   mice infected with ΔvapC12 was dependent on the presence of TLR-4
   receptors. These results suggest that vapC12 ribonuclease mediated
   regulation of expression of pathogen-associated molecular patterns
   (PAMPs) in M. tuberculosis modulates host immune response which is
   essential for the long-term survival of the pathogen inside the host. A
   better understanding of the mechanism of generation and maintenance of
   antibiotic persistence in the context of host-pathogen interaction will
   go a long way in designing novel intervention strategies against
   tuberculosis. Host-directed therapies (HDT) targeting such pathways in
   combination with the existing anti-TB treatment can increase the
   efficacy of the current TB regimen.

Methods

Bacterial strains and culture conditions

   All mycobacterial strains were grown and maintained in Middlebrook 7H9
   broth and 7H11 agar (Difco Middlebrook 7H9 broth [catalog no. 271310]
   and 7H11 agar [catalog no. 283810]) and supplemented with 10% OADS
   (bovine serum albumin, oleic acid, and dextrose); 0.05% Tween was added
   in 7H9 broth to enhance bacterial growth. Bacterial enumeration by CFU
   was examined on 7H11 agar plates. The concentrations of antibiotics
   used were as follows: hygromycin B (50 µg/mL) and kanamycin (25 µg/mL).

Mouse infection study

   Animal experiment protocols were reviewed and approved by the animal
   ethics committee of Translational Health Science and Technology
   Institute India [(IAEC/THSTI/183), (IAEC/THSTI/167)], Faridabad, India.
   Animal experiments were performed in accordance with guidelines
   provided by the Committee for the Control and Supervision of
   Experiments on Animals (CCSEA). Pathogen-free C57BL/6 and C3H/HeJ mice
   were obtained from Experimental Animal Facility, THSTI. As described
   earlier, briefly, prior to infection, mid-log phase cultures (O.D
   =0.8-1) of H37Rv (WT), ΔvapC12 and ΔvapC12:vapBC12 M. tuberculosis
   strains were harvested, washed and single-cell suspensions were
   prepared ([60]23). Six to eight-week old mice were infected by aerosol
   exposure in the Glas-Col airborne infection apparatus with
   approximately 50-100 CFUs of different strains. Mice from each group
   (N = 5) were sacrificed at different time points as indicated in the
   experiments (Day 0, 2, 4 and 8 weeks post infection). The bacillary
   load was determined by removing lungs and spleen of infected mice and
   the serial dilution of the organ homogenates was plated on 7H11 agar
   plates supplemented with 10% OADS for bacterial enumeration. The lung
   sections were treated with 10% formalin and hematoxylin and eosin
   stained were used for histopathological analysis. A blind assessment
   strategy was employed for scoring the granulomas. For drug clearance
   study, after establishing bacterial infection till week 4, mice were
   treated with 10mg/kg RIF through oral gavaging till week 8. Also, a
   separate untreated group was kept as a control. Bacillary enumeration
   was done on week 4 and 8 by aseptically removing organs and
   homogenizing them. The homogenized organs were then serially diluted
   and plated on 7H11 agar (containing Polymyxin A, amphotericin B,
   vancomycin, trimethoprim, carbenicillin and cycloheximide mix).

Flow cytometry analysis

   Mice were sacrificed at 2 and 4 week post-infection, and their lungs,
   spleen and LN (draining lymph node) were isolated. Single-cell
   suspension was prepared and stained (1 x 10^6 cells) with fluorescently
   labelled anti-mouse CD11b (eBioscience Invitrogen, USA), anti-mouse Gr1
   (eBioscience Invitrogen, USA), anti-mouse F4/80-FITC (eBioscience
   Invitrogen, USA), anti-mouse CD68 (eBioscience Invitrogen, USA),
   anti-mouse CD206 (eBioscience Invitrogen, USA), anti-mouse CD80
   (eBioscience Invitrogen, USA), anti-mouse NK1.1 (eBioscience
   Invitrogen, USA), anti-mouse CD3 (BD Biosciences USA), anti-mouse CD4
   (BD Biosciences USA), anti-mouse CD8 (eBioscience Invitrogen, USA) and
   anti-mouse γδ-TCR (eBioscience Invitrogen, USA). Briefly, for surface
   staining, cells were stained for 20 minutes at room temperature, and
   were washed twice with PBS. Stained samples were then fixed for 20
   minutes using 4% paraformaldehyde. Data was acquired on FACS Canto (BD
   Biosciences) and analyzed using FlowJo software (Tree Star, Ashland,
   OR, USA) ([61]24, [62]25).

RNA sequencing

   For transcriptome analysis, lung tissues of mice infected with
   different strains (4-week infected WT, ▵vapC12 and ΔvapC12:vapBC12)
   were homogenized by bead beating, and total RNA was isolated using
   RNeasy mini kit according to the manufacturers protocol (Qiagen catalog
   no. 74104). RNA was eluted in nuclease-free water, subjected to DNA
   removal using TURBO DNase. NEB Ultra II directional RNA-sequencing
   Library Prep kit protocol was used to prepare libraries for total
   RNA-Seq (NEB, catalog no. E7760L). An initial concentration of 500ng of
   total RNA was taken for the assay. mRNA molecules are captured using
   magnetic Poly(T) beads (NEB, catalog no. E7490L) following
   purification, the enriched mRNA was fragmented using divalent cations
   under elevated temperatures. The cleaved RNA fragments were copied into
   first-strand cDNA using reverse transcriptase. Second strand cDNA
   synthesis was performed using DNA Polymerase I and RNase H enzyme. The
   cDNA fragments were then subjected to a series of enzymatic steps that
   repair the ends, tails the 3 end with a single ‘A base, followed by
   ligation of the adapters. The adapter-ligated products were then
   purified and enriched using the following thermal conditions: initial
   denaturation 98°C for 30sec; 12 cycles of - 98°C for 10sec, 65°C for
   75sec; final extension of 65°C for 5mins. PCR products are then
   purified and checked for fragment size distribution on Fragment
   Analyzer using HS NGS Fragment Kit (1-6000bp) (Agilent, catalog no.
   DNF-474-1000). Genes with low expression value (mean FPKM value<0.5)
   were treated as background (i.e. ‘noise) ([63]26) and hence excluded
   from further analysis. After the noise removal, the gene expression
   values for each experimental condition were merged, and the final data
   comprised the FPKM value of 13796 genes. To assess the variation in the
   gene expression data, we performed the principal component analysis
   (PCA) using the prcomp package in R, which applies the singular value
   decomposition to the data matrix. For the comparison between gene
   expression profiles from mice infected lungs with WT and ▵vapC12 strain
   of M. tuberculosis, genes with a fold change of 2 and false discovery
   rate (FDR)-adjusted p-value <0.05 were considered differentially
   expressed. The Benjamin-Hochberg multiple hypothesis testing ([64]27)
   was applied to obtain the FDR-adjusted p-value. Subsequently, the
   analyses were performed on the MATLAB and R software. The mafdr
   function in MATLAB was applied to obtain the FDR-adjusted p-value. PCA
   plot, volcano plot and dot plot were generated in R using ggplot2,
   enrichplot and gplots packages. Heatmaps were built using heatmap.2.
   Pathway enrichment analysis was performed in R using the
   clusterProfiler package.

Quantitative RT-PCR

   Total RNAs from lungs were isolated using RNeasy Micro kit (Qiagen),
   and the cDNAs were synthesized by reverse transcriptase (RT) using the
   high capacity cDNA archive random priming kit (Applied Biosystems)
   according to the manufacturers recommended protocol. Real-time PCR was
   performed with 2 × SYBR mix (TaKaRa) in a QuantStudio-6 Flex Real-Time
   PCR System (Applied Biosystems) using the default run program. The
   relative expression of the target genes was calculated using the target
   threshold cycle value (Ct) and the 2^−ΔΔ ^Ct method with GAPDH as the
   internal loading control ([65]28). The primer sequences used for real
   time PCR are listed in [66]Supplementary Table S1 .

Depletion of neutrophils

   Neutrophils were depleted using 200μg anti-mouse Ly6G (Bio X Cell) or
   isotype IgG (Bio X Cell). Briefly, in acute infections (less than 21
   dpi), mice were given 200μg of anti-mouse Ly6G intra- peritoneally
   every other day between 10 and 20 dpi.

Statistical analysis

   Each experiment was repeated at least three times independently and
   three biological replicates were used. Data were plotted as mean ± SEM.
   Students t-test (Mann-Whitney), two-way ANOVA and one-way ANOVA
   (Kruskal-Wallis) were performed wherever applicable by using Graph Pad
   Prism 7.0 software. For the survival curve, Kaplan Meier statistical
   analysis was performed. P-value < 0.05 was considered to be
   statistically significant.

Results

VapC12 ribonuclease toxin modulates growth, virulence and drug tolerance in
M. tuberculosis

   To replicate our earlier findings of the study performed on guinea pigs
   ([67]22), we infected C57BL/6 mice with WT, vapC12 null mutant and
   complemented M. tuberculosis strains. We found a significantly higher
   bacterial load in the lungs and spleen of mice infected with vapC12
   null strain as compared to the WT and complemented strains both at 4
   and 8 weeks post-infection ([68] Figures 1A, B ). In comparison to the
   WT, gross examination of the lungs infected with the mutant strain
   revealed higher inflammation and enhanced tissue damage. Histological
   examination of the lungs infected with the WT strain at 8 weeks
   post-infection revealed a typical necrotic granuloma in the centre of
   the lung (40X) with epithelioid cells and lymphocytes in the granuloma
   (100X) ([69] Figure 1C and [70]Supplementary Figure 1A ). In contrast,
   at a similar time point post infection, lung tissues of mice infected
   with the vapC12 null strain displayed a significantly higher number of
   granulomas ([71] Figure 1D ). Mice infected with complemented strain
   had a phenotype similar to those infected with WT strain ([72]
   Figures 1C, D ). Previously, using kill-curve experiments, we have
   demonstrated that the absence of the vapC12 gene in M. tuberculosis
   reduced the frequency of generation of drug-tolerant population in
   presence of rifampicin under in-vitro growth conditions ([73]22). To
   replicate the above findings under disease conditions, we performed a
   drug clearance study using C57BL/6 mice infection model. We infected a
   group of mice with either WT and ▵vapC12 strain and estimated the rate
   of pathogen clearance by estimating bacterial load in infected organs
   at different time points ([74] Figure 1E ). As expected, 4 week post
   infection, the organs infected with ▵vapC12 had high bacterial load in
   comparison with the organs isolated from the WT infected mice ([75]
   Figures 1F, G ). Importantly, upon treatment with RIF and INH, the rate
   of decline in the bacterial load was higher in mice infected with
   ▵vapC12 strain relative to WT at 8 weeks post infection ([76]
   Figures 1F, G ). To account for the differences in the initial
   bacterial load (week 0), we had to normalize the data relative to week
   0 between H37Rv and vapC12 mutant strains ([77] Figures 1F, G ).
   Importantly, upon treatment with RIF we observed a ~2 fold and ~7 fold
   increase in the killing of the pathogen in the lungs and spleen
   respectively in the mice infected with ▵vapC12 as compared to mice
   infected with the H37Rv ([78] Figures 1F, G ) whereas treatment with
   INH resulted in ~10 fold better killing in the lungs and spleen of the
   mice infected with ▵vapC12 relative of the H37Rv. These results show
   that the absence of vapC12 gene in M. tuberculosis decreased the
   frequency of generation of drug-tolerant population potentiating the
   efficiency of RIF and INH treatment in infected mice ([79] Figures 1F,
   G ). Since vapC12 deleted strain of M. tuberculosis demonstrated
   enhanced pathogenicity, we measured the estimated time of survival of
   WT and ▵vapC12 infected mice in a high dose infection model (500-1000
   bacteria/lung) ([80] Supplementary Figure 1B ). Interestingly, we found
   that 16 weeks post-infection, all the mice (n=10) infected with the WT
   strain survived while none of the mice infected with the ▵vapC12
   survive (n=10) survived ([81] Figure 1H ). Collectively, these data
   suggested that absence of vapC12 imparted hypervirulence phenotype and
   reduces the frequency of generation of persisters in mice infected with
   M. tuberculosis.

Figure 1.

   [82]Figure 1
   [83]Open in a new tab

   vapC12 mutant strain of M. tuberculosis modulates growth and drug
   tolerance. (A, B) Bacterial load in the (A) lungs or (B) spleen of mice
   infected with H37Rv, ΔvapC12, and ΔvapC12:vapBC12 strains of M.
   tuberculosis at designated time points, the lungs were homogenized in 2
   ml of saline, and ten-fold serial dilutions of homogenates were plated
   on 7H11+OADC plates. Each group constituted five mice per time point.
   Data plotted represent the mean ± SEM. Significant differences observed
   between groups are indicated. Data were analyzed using the MannWhitney
   test with **P < 0.01 and *P <0.05. (C) Photomicrographs of H&E-stained
   (40× and 100×) and whole section image of lung sections from mice
   infected with different strains of M. tuberculosis at 8 weeks post
   infection. G=Granuloma, As=Alveolar Space, EC=Epithelioid Cells and
   L=Lymphocytes (D) Granuloma fraction of the lung tissue samples of mice
   infected with different strains of M. tuberculosis, based on the
   semi-quantitative estimation of the fraction of the lung tissue covered
   with granuloma. Data were analyzed using the MannWhitney test with *P <
   0.05 and **P < 0.01. (E) Schematic representation of mice infection
   experiment. Mice (n = 5 per group at each time point) were infected
   with H37Rv and ΔvapC12. After the establishment of infection for
   4 weeks, antibiotics (INH or RIF) were orally administered to the mice
   for the next 4 weeks. A parallel group of mice was left untreated
   (control). Bacterial loads were enumerated in the lungs (F) and spleens
   (G) at different time points post-infection as depicted by scatter
   plot. Each data point represents the log10 CFU of an infected animal in
   individual organs, and the error bar depicts the SEM for each group.
   Bar graph represents the percent killing of the vapC12 mutant strain
   with respect to WT at week 8 post-infection in the (F) lungs and (G)
   spleen. Data was normalized with the untreated group and plotted with
   respect to H37Rv (100%) on the y-axis. Data were analyzed using the
   MannWhitney test with **P < 0.01 and *P <0.05. (H) Percent survival
   plot of mice (n=20) infected with H37Rv and ΔvapC12 strains for 18
   weeks post infection. Data were analyzed using the log-rank test with
   ***P value 0.0005.

VapC12 toxin modulates host immune response during M. tuberculosis infection

   To understand the vapC12 mediated differences in the host immune
   response, we performed detailed immune-profiling studies on organs
   isolated from mice infected with WT, ▵vapC12 and complemented strains
   of M. tuberculosis at week 4 post-infection ([84] Figure 2A ). We
   estimated the innate immune cell population; monocytes, macrophages,
   MDSCs, neutrophils, and Natural Killer-T (NKT) cells in lungs and
   spleen isolated from infected mice ([85] Figures 2B, C and
   [86]Supplementary Figure 2A ). A set of uninfected (UI) mice was
   included as a control group in all the experiments. We found that
   relative to the WT, both lungs and spleen isolated from mice infected
   with ▵vapC12 had a significantly lower percentage (~2%) of monocytes
   population ([87] Figures 2B, C ). In contrast, relative to the WT,
   organs isolated from the mice infected with the vapC12 mutant strain
   had a significantly higher percentages of macrophage, neutrophil and
   MDSCs ([88] Figures 2B, C ). The restoration of the mutant specific
   phenotype to the WT levels in the group of mice infected with the
   complemented strain further confirms the role of VapC12 toxin in
   modulating the host immunity. We also observed that the MDSCs
   population in the lungs was significantly elevated by ~3-fold in the
   mutant strain in comparison to the WT infected group ([89] Figures 2B,
   C ). Our findings corroborated the previous studies as the accumulation
   of MDSCs parallels disease progression in lung parenchyma and
   exacerbate disease by suppressing T-cell functions ([90]29–[91]32).
   Neutrophil infiltration in the lungs; which is a common feature of
   disease severity in TB patients ([92]10) and susceptible mice ([93]18,
   [94]33, [95]34) was found to be ~3-fold higher in the vapC12 mutant
   strain as compared to the WT and complemented strain ([96] Figures 2B,
   C ). Furthermore, the neutrophil population in the spleen isolated from
   vapC12 infected mice was also observed to be ~3 fold higher than in the
   WT ([97] Figure 2C ). Surprisingly, we found that the percentage of NKT
   cells in lungs and spleen isolated from vapC12 mutant infected mice
   were 2-fold and 3-fold elevated as compared to the organs isolated from
   WT infected mice ([98] Figures 2D, E ). It has been reported that
   individuals with pulmonary TB have lower number of NKT cells as
   compared to the healthy controls, indicating that they may have a
   protective role in TB infection ([99]35, [100]36). Together, these data
   indicated that vapC12 mutant induces hyperinflammation by promoting the
   infiltration of myeloid cells in the lungs.

Figure 2.

   [101]Figure 2
   [102]Open in a new tab

   VapC12 toxin modulates host immune response during M. tuberculosis
   infection. (A) Schematic representation of the murine infection
   experiment. Mice (n =5 per group at each time point) were infected with
   H37Rv, ΔvapC12, and ΔvapC12:vapBC12. Immuno-profiling of lung
   infiltrated cells and splenocytes harvested at 4 weeks post infection.
   (B, C) Zebra plots and its representative bar graphs indicating the
   percentage frequency of monocytes (Gr1-CD11b+F4/80-), macrophages
   (Gr1-CD11b+F4/80+), MDSCs (CD11b+ Gr1+^int) and neutrophils
   (CD11b+Gr-1^high) cells harvested from (B) lungs and (C) spleen. Data
   are shown as mean ± SEM (n=5 animals per group). Data were analyzed
   using the one-way ANOVA followed by Tukeys multiple comparison test (*P
   <0.05, **P < 0.01, ***P<0.0005, ****P<0.0001). (D, E) Zebra plots and
   its representative bar graphs showing the percentage frequency of NKT
   (NK1.1+CD3+) cells harvested from (D) lungs and (E) spleen. Data are
   shown as mean ± SEM (n=5 animals per group). Data were analyzed using
   the one-way ANOVA followed by Tukeys multiple comparison test (*P
   <0.05, **P < 0.01, ***P<0.0005, ****P<0.0001).

VapC12 toxin regulates the polarization of macrophages during M. tuberculosis
infection

   The choice of effector mechanism selected by the host to clear
   infection is regulated by the type of cytokine response dictated by the
   initial innate immune response. The fate of Th1 and Th2 polarisation,
   ideal for intracellular and extracellular pathogens respectively, is
   effectively decided by the initial differentiation of the macrophages
   to either classical or alternate types. As an effective host immune
   escape strategy, the pathogen tries to manipulate the macrophage
   differentiation process in its own favor by subverting the host cell
   metabolic pathways ([103]37). To investigate role of the vapC12 gene in
   manipulating macrophage differentiation we used antibodies against CD80
   and CD206, classical markers for M1 and M2 respectively to quantify the
   relative M1/M2 population in the lungs of mice infected with different
   M. tuberculosis strains. We found a ~1.5-fold decrease in the frequency
   of M1 (CD80+) macrophages in lungs isolated from mice infected with
   vapC12 mutant as compared to the WT. On the contrary, we found higher
   frequency of M2 expressing macrophages in the lung tissue infected with
   ▵vapC12 ([104] Figure 3A and [105]Supplementary Figure 3A ).
   Furthermore, the vapC12 mutant strain demonstrated a ~10% increase in
   M1 macrophages and a corresponding ~10% decline in M2 macrophages as
   compared to the WT ([106] Figure 3A ).The same was further validated by
   quantifying the transcripts of markers specific to either M1 or M2
   macrophages. We estimated the transcripts levels of M1 [CD80 and CD86]
   and M2 [Arginase1 (Arg1), YM1 and Fizz1) specific markers by isolating
   RNA from the lungs of WT, vapC12 mutant infected and uninfected (UI)
   mice. As expected, we found ~6-fold (Fizz1), ~10-fold (YM1) and
   ~35-fold (Arg1) increase in the expression of M2 markers in the vapC12
   mutant strain as compared to the WT strain ([107] Figure 3B ). In
   concordance with elevated levels of M2 markers, a clear upregulation in
   the gene expression levels of the Th2 related cytokines; IL 6
   (****P<.0.0001) and IL10 (*P<.0.05) was also observed ([108] Figure 3C
   ). Surprisingly, the transcription signature associated with M1 and Th1
   markers CD80, CD86, IFN-γ and TNF-α showed non-significant changes in
   their gene expression levels and a clear reduction in Th1-induced
   secretion of IL-2 was noted ([109] Figures 3B , [110]C ). In context to
   T cell differentiation, we observed that the vapC12 mutant demonstrated
   an increased percentage of CD4+ T cells producing IFN-γ in both the
   lungs and spleen when compared to the WT. Conversely, there was a
   notable reduction in the IL17-producing T-cell population in the spleen
   of the vapC12 mutant compared to the WT, with no discernible impact
   observed in the lung ([111] Supplementary Figure 3B ). Overall, the
   data convincingly demonstrates that the ratio of M1/M2 (Th1/Th2) is
   significantly lower in the mice infected with ▵vapC12 as compared to
   the WT strain. In sum, these data suggest that levels of VapC12
   ribonuclease help M. tuberculosis control the M1/M2 polarization
   modulating the Th1/Th2 pathway critical for regulating the disease
   outcome.

Figure 3.

   [112]Figure 3
   [113]Open in a new tab

   VapC12 toxin regulates the polarization of macrophages during M.
   tuberculosis infection. (A) Left panel M1: shows the histogram and bar
   graph of CD80 and right panel M2: shows the histogram and bar graph of
   CD206 in Gr1-CD11b+ F4/80+ cells by flow cytometry and bar graph
   represents MFI (Mean fluorescent index) and frequency (%). Data were
   analyzed using FlowJo software (tree star) and statistical calculation
   was presented as mean ± SEM, n = 5. ***P<0.0002, ****P < 0.0001. (B)
   Bar graph for mRNA expression of M1 (CD80 and CD86) and M2 (Arg1, YM1
   and Fizz1) macrophage markers in the lung cells, data representative of
   triplicates *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (C) Bar
   graph for mRNA expression of TH1 (IFN-γ, TNF and IL-2) and TH2 (IL-4,
   IL-10 and IL-6) in the lung cells, data representative of triplicates
   *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

VapC12 is critical for induction of adequate T cell immune response during M.
tuberculosis infection

   In addition to myeloid cells, CD4^+ and CD8^+ T lymphocytes are the key
   players in the establishment of protective immunity against TB in mice.
   The changes in cellular phenotype coincided with the significant
   decrease in the adaptive immune markers i.e. CD4^+ and CD8^+ T
   lymphocytes in the organs isolated from the ▵vapC12 infected mice
   relative to the WT. In lungs harvested from mice infected with vapC12
   mutant strain, we observed a significant 3-fold and 2-fold reduction in
   the percentage of CD4^+ and CD8^+ T-cells respectively, both with
   p<0.05 as compared to the WT infected mice ([114] Figure 4A and
   [115]Supplementary Figure 4A ). Surprisingly, while the CD4^+ T-cell
   profile in the spleen for both groups were identical to the lungs, the
   CD8^+ T-cell remained unaltered ([116] Figure 4B ). As shown in
   [117]Figure 4C , both CD4^+ and CD8^+ T-cell percentages was suppressed
   in the lymph nodes of vapC12 infected mice. Interestingly, in
   comparison to the WT, the secondary lymphoid organs isolated from the
   mice infected with ▵vapC12 harbored significantly higher percentage of
   γδ-T cell 4 weeks post-infection ([118] Figures 4B, C ) with no such
   differences observed in the lungs ([119] Figure 4A ). This data
   corroborates the previous findings which suggest the role of γδ-T cells
   in TB pathogenesis by inducing inflammation and granuloma formation
   ([120]38). Thus, we conclude that the absence of VapC12 toxin in M.
   tuberculosis prevents the induction of adequate T-cell response in the
   lungs and lymphoid organs (lymph nodes and spleen) of infected mice.

Figure 4.

   [121]Figure 4
   [122]Open in a new tab

   vapC12 is critical for induction of adequate T cell immune response
   during M. tuberculosis infection. (A-C) Zebra plots and its
   representative bar graphs indicating percentage frequency of CD4, CD8
   and γδ+T cells in the (A) lung (B) spleen and (C) Lymph nodes. Data
   were analyzed using FlowJo and shown as mean ± SEM (n=5 animals per
   group). Data were analyzed using the one-way Anova test followed by
   Tukeys multiple comparison test (*P <0.05, **P < 0.01, ***p<0.0005,
   ****p<0.0001).

VapC12 is essential for the resolution of neutrophil-mediated inflammation in
tuberculosis

   To further delineate the role of host factors associated with the
   hypervirulent phenotype of the mutant strain, we analysed the RNA
   sequencing data of host genes from lung tissues of mice infected with
   WT, ▵vapC12 and complemented strain. The same was subjected to a
   detailed transcriptome analysis ([123] Figure 5A ). Initially, a
   principal component analysis (PCA) was performed to visualize the
   variations in the obtained expression profiles. Data displayed in the
   first two principal components (PCs) suggest that all three gene
   clusters are well separated from each other ([124] Supplementary
   Figure 5A ). To study the VapC12-mediated differential expression of
   the host genes, we compared the gene expression profiles of lung
   tissues from mice infected with the ▵vapC12 and the WT strains ([125]
   Figure 5B ). In total, 372 differentially expressed genes (DEGs) were
   obtained, out of which 82 and 290 genes were up- and down-regulated,
   respectively ([126] Supplementary Figure 5B and [127]Supplementary File
   S1 ). Pathway enrichment analysis of the upregulated genes revealed an
   enrichment in the processes related to neutrophil functions like
   neutrophil migration and neutrophil-mediated immunity ([128] Figure 5C
   ). We found that in comparison to the WT, infection with the ▵vapC12
   strain enhanced the expression of genes that encode proteins implicated
   in neutrophil-mediated immunity in the host (Cxcl2, Trem1, S100A8/A9
   and Ccl3) ([129] Figure 5D ). S100A8 and S100A9; cytosolic proteins of
   neutrophils are important for the recruitment of neutrophils to the
   granulomas as demonstrated in human and animal models of TB ([130]39).
   A ~2.56 fold (log[10]) increase in the transcript levels of genes
   encoding S100a8 and S100a9 by the host infected with the vapC12 mutant
   strongly suggests an increase in neutrophil recruitment and
   accumulation in the lung tissues. Genes belonging to the
   inflammation-associated markers like matrix metalloproteases (Mmp8 and
   Mmp9), secreted by neutrophils that are known to be critical for the
   formation of granuloma and inducing inflammation during the acute stage
   of infection ([131]7), were found to be significantly induced in the
   lung tissues of mice infected with the mutant strain. We also found an
   increase in the expression of Cxcr2 (22-fold), Cxcl1 (18-fold) and
   Cxcl2 (177-fold) genes which suggests enhanced neutrophil recruitment
   in lungs of mice infected with vapC12 mutant relative to the WT
   ([132]40) ([133] Supplementary File S2 ). Additionally, IL1a (30-fold)
   and IL1b (19-fold), the pro-inflammatory cytokine markers associated
   with TB ([134]41) were also found to be significantly upregulated in
   the tissue isolated from mice infected with mutant strain as compared
   to the WT and complemented strain ([135] Figure 5E ). Interestingly, in
   comparison to the WT, the expression of cystatins such as Cstdc4
   [3.2-fold (log[10])], Stfa2l1 [2.8-fold (log[10])] and Csta2 [2.26-fold
   (log[10])] which are the endogenous inhibitors of the host
   antimicrobial proteins cathepsins ([136]42)were also increased in lungs
   of mutant strain infected animals ([137] Figure 5F ). Simultaneously,
   peptidases which play an important role in pathogen killing by
   activating the antimicrobial peptides were found to be significantly
   reduced in the ▵vapC12 infected mice lungs ([138] Figures 5G, H ),
   setting off an unrestricted growth of M. tuberculosis leading to
   excessive inflammatory responses. Additionally, the observed reduction
   in the transcript level of genes associated with adaptive immune
   response correlates with our earlier findings that the ▵vapC12 infected
   mice fail to induce an effective adaptive immune response ([139]
   Supplementary Figure 5C ). Together, these observations suggest that
   ▵vapC12 proliferates and induces a proinflammatory response by
   modulating the transcript level of proteins that enhances neutrophil
   recruitment, IL-1 production, endopeptidase inhibitors and by
   suppressing the antimicrobial proteases and appropriate adaptive immune
   response inside the host.

Figure 5.

   [140]Figure 5
   [141]Open in a new tab

   VapC12 is essential for the resolution of neutrophil-mediated
   inflammation in tuberculosis. (A) Pictorial representation of the
   process of animal experiment with mice. RNA isolated from lung tissues
   of mice (n=3) infected with WT, ▵vapC12 and complemented strain after 4
   weeks of infection. (B) The volcano plot showing comparison of gene
   expression profiles from mice infected with parental (WT) and ▵vapC12
   strain of M. tuberculosis. Differentially expressed genes (DEGs) were
   selected using the fold change cut-off value of 2 and FDR-adjusted
   p-value < 0.05. The up- and down-regulated genes are highlighted here
   in red and blue, respectively. (C) Dot plot of the gene ontology (GO)
   pathway enriched in upregulated genes. The x-axis represents the number
   of obtained upregulated genes for each pathway. (D) Heatmap
   representing the expression levels of the 21 upregulated genes involved
   in the neutrophil associated function. Data were Z-score transformed
   and the name of the genes are displayed on the right. The names of the
   corresponding infected strains for each sample are mentioned at the
   bottom of the figure. (E) Heatmap representing the expression levels of
   the 9 upregulated genes involved in the IL-1-mediated signaling. (F)
   Heatmap representing the expression levels of the 10 upregulated genes
   involved in the peptidase inhibitor activity. (G) Dot plot of the gene
   ontology (GO) pathway enriched in down-regulated genes. The x-axis
   represents the number of obtained upregulated genes for each pathway.
   (H) Heatmap representing the expression levels of the 15 downregulated
   genes involved in the peptidase regulator activity.

Depletion of neutrophils failed to restore the vapC12 mutant phenotype

   As neutrophils are an abundant component of the innate immune system,
   their highly inflammatory actions have potentially severe consequences
   for host tissues ([142]43). When the regulatory mechanisms responsible
   for clearing apoptotic neutrophils are compromised or if the underlying
   infection cannot be resolved, the actions of neutrophils may turn
   detrimental to the host ([143]44, [144]45). As previously reported,
   neutrophils depletion can be achieved by the administration of 1A8
   antibody ([145]45). To confirm the role of neutrophils in ▵vapC12
   induced hypervirulent phenotype we infected groups of C57BL/6 mice,
   with either 1A8 antibody or their isotype control ([146] Figure 6A ).
   Lungs treated with 1A8 antibody in vapC12 mutant resulted in
   significant decrease in the neutrophils population relative to the
   isotype group ([147] Figure 6B ). Additionally, 1A8 antibody treatment
   resulted in a significant decrease in the monocytes (~4%) and
   macrophages population (~5%) in the lungs ([148] Figure 6B ). Treatment
   with 1A8 antibody also resulted in a significant reduction (5%) in the
   population of CD4^+ and CD8^+ T cells in the lungs and spleen harvested
   from antibody-treated vapC12 mutant mice as compared to WT ([149]
   Figure 6C ). Surprisingly, we did not find any difference in the
   overall lung and spleen bacillary burden in mice infected with vapC12
   mutant strain that were either treated with 1A8 antibody or the isotype
   control ([150] Figure 6D ). These data collectively suggest that the
   ▵vapC12 induced hypervirulence phenotype is independent of the increase
   in the infiltration of neutrophil observed in the lungs of ▵vapC12
   infected mice.

Figure 6.

   [151]Figure 6
   [152]Open in a new tab

   Neutrophil depletion failed to restore the vapC12 mutant phenotype to
   the wild type levels. (A) Schematic representation of antibody
   depletion experiment in mice. ΔvapC12 infected mice (n=5) were either
   treated with neutrophil depleting Anti-Ly6G or IgG control antibody
   every alternative day from day 10 to day 21 post-infection. Mice were
   sacrificed on day 21 for immunophenotyping & bacterial enumeration. (B)
   Zebra plots and its representative bar graph shows the percentage
   frequency of monocytes (Gr1-CD11b+F4/80-), macrophages
   (Gr1-CD11b+F4/80+),neutrophils (CD11b+Gr-1^high) cells in the lungs.
   Data are shown as mean ± SEM (n=5 animals per group). Data were
   analyzed using the one-way ANOVA followed by Tukeys multiple comparison
   test (*P <0.05, **P < 0.01, ***P<0.0005, ****P<0.0001). (C) Zebra plots
   and its representative bar graphs indicating percentage frequency of
   CD4^+ and CD8^+ T cells in the lung and spleen. Data were analyzed
   using FlowJo and shown as mean ± SEM (n=5 animals per group). Data were
   analyzed using the one-way ANOVA test followed by Tukeys multiple
   comparison test (*P <0.05, **P < 0.01, ***p<0.0005, ****p<0.0001). (D)
   Scatter plot showing bacterial load in the lungs and spleen of mice
   infected with ΔvapC12 at designated time points, the lungs were
   homogenized in 2 ml of saline, and ten-fold serial dilutions of
   homogenates were plated on 7H11+OADC plates. Each group constituted
   five mice per time point. Data plotted represent the mean ± SEM.
   Significant differences observed between groups are indicated. Data
   were analyzed using the MannWhitney test with **P < 0.01 and *P <0.05.

VapC12 deletion induced hypervirulence in M. tuberculosis is dependent on
TLR4

   Neutrophil associated proteins, S100A8/A9 function as DAMPs
   (danger-associated molecular pattern) and can act as a ligand of TLR4
   thus activating the innate immune responses ([153]46). Since
   RNA-sequencing data showed that both S100A8/A9 neutrophil-associated
   markers were elevated in the lungs of vapC12 mutant, we hypothesized
   that TLR4 signaling could play an important role in ▵vapC12 mediated
   hypervirulent phenotype. To study the role of TLR4 signaling, we
   infected C3H/HeJ mice (carrying mutated TLR4) with both the WT and
   ▵vapC12 strains ([154] Figure 7A ). In parallel, we included a C57BL/6
   mice group infected with WT and vapC12 mutant as a hypervirulent
   phenotype control. As expected, relative to the WT, we observed an
   increase in the bacterial load in the organs isolated from C57BL/6 mice
   infected with the ▵vapC12 strain ([155] Supplementary Figure 6 ).
   Surprisingly, the above phenotype observed in the C57BL/6 mice was
   completely reversed in C3H/HeJ mice ([156] Figures 7B, C ). In
   comparison to the C57/BL6, C3H/HeJ mice infected with ▵vapC12 were
   presented with a ~10-fold & ~40-fold reduction in bacterial burden in
   the lungs & spleen respectively ([157] Supplementary Figure 6A, B ).
   Interestingly, the reduction in the bacterial burden observed in
   C3H/HeJ mice coincided with a significant reduction in the population
   of neutrophils in these organs (p < 0.05). Additionally, we also
   observed that the percentage of MDSCs and macrophage population from
   the lungs of ▵vapC12 infected TLR4 defective mice were restored to the
   WT levels ([158] Figure 7D ). Surprisingly, in comparison to the lungs,
   restoration of the adaptive immune response in ▵vapC12 infected TLR4
   defective mice were specifically observed in the spleen ([159]
   Figure 7E ). These findings were in line with our bacterial enumeration
   data suggesting that TLR4 signaling pathway could play an important
   role in ▵vapC12 mediated hypervirulent phenotype observed in the mice.

Figure 7.

   [160]Figure 7
   [161]Open in a new tab

   vapC12 deletion induced hypervirulence in M. tuberculosis is dependent
   on TLR4. (A) Schematic representation of the animal experiment. C3H/HeJ
   mice were aerosol infected with H37Rv and ΔvapC12. Mice were sacrificed
   on indicated time point for bacterial enumeration and
   immunophenotyping. (B, C) Scatter plot showing bacterial load in the
   (B) lungs and (C) spleen of mice infected with H37Rv and ΔvapC12 at
   indicated time points, the lungs were homogenized in 2 ml of saline,
   and ten-fold serial dilutions of homogenates were plated on 7H11+OADC
   plates. Each group constituted five mice per time point. Data plotted
   represent the mean ± SEM. Significant differences observed between
   groups are indicated. Data were analyzed using the MannWhitney test
   with **P < 0.01 and *P <0.05. (D) Zebra plot and its representative bar
   graphs showing the percentage frequency of monocytes
   (Gr1-CD11b+F4/80-), macrophages (Gr1-CD11b+F4/80+), MDSCs (CD11b+
   Gr1+^int) and neutrophils (CD11b+Gr-1^high) cells in the lung. Data are
   shown as mean ± SEM (n=5 animals per group). Data were analyzed using
   the one-way ANOVA followed by Tukeys multiple comparison test (*P
   <0.05, **P < 0.01, ***P<0.0005, ****P<0.0001). (E) Zebra plots and its
   representative bar graphs indicating the percentage frequency of CD4^+
   and CD8^+ T cells in the lung and spleen. Data were analyzed using
   FlowJo and shown as mean ± SEM (n=5 animals per group). Data were
   analyzed using the one-way ANOVA test followed by Tukeys multiple
   comparison test (*P <0.05, **P < 0.01, ***p<0.0005, ****p<0.0001).

Discussion

   Earlier we reported that the vapC12 ribonuclease toxin targets proT
   tRNA and modulates the levels of various proline-rich PE-PGRS proteins
   ([162]22). In the same study, we further demonstrated that organs
   isolated from guinea pigs infected with M. tuberculosis strain lacking
   vapC12 gene had higher bacterial load and were found to be more
   pathogenic measured in terms of the degree of inflammation and
   pathological lesions as compared to the WT strain ([163]22). The
   current study was undertaken to further our understanding of the
   hypothesis that vapC12 mediated differential expression of antigenic
   proteins modulates host immune response. Due to easy availability of
   good genetic and immunological toolbox, we in the current study have
   decided to use mice as a surrogate host. Similar to the phenotype
   observed in guinea we found that the vapC12 deficient strain of M.
   tuberculosis demonstrated a hypervirulent phenotype marked by an
   increase in bacterial burden and enhanced pathological lesions.
   Subsequently we have also demonstrated that ▵vapC12 promoted the influx
   of pro-inflammatory myeloid cells at the site of infection resulting in
   robust transcription of S100A8/A9 proteins providing enhanced growth
   benefit to the mutant strain as compared to the WT. By inhibiting the
   S100A8/9-TLR4 axis we were able to mitigate the bacterial growth in
   vapC12 mutant by reducing the detrimental inflammatory immune response.

   The killing of pathogens by macrophages is known to be the primary
   defence mechanism against intracellular pathogens. Like T-lymphocytes,
   macrophages also differentiate into M1 and M2 phenotypes depending on
   their microenvironment and amino acid metabolic shift ([164]37).
   Arginine catabolism via iNOS leads to the production of L-citrulline
   and nitric oxide (NO), playing a predominant role in providing
   resistance against parasitic and bacterial infections. On the contrary,
   use of arginine as a substrate by Arg1 results in the generation of
   L-ornithine and urea and is known to promote disease activity
   ([165]37). During M. tuberculosis infection, Arg1 stimulation in-vitro
   in macrophages through a TLR-dependent and STAT6-independent pathway
   leads to the inhibition of NO production. Conversely, lack of Arg1 was
   connected to elevated NO expression and improved control of
   mycobacterial infection ([166]47). Arginine metabolism has been linked
   to infection as high arginase activity has been reported in lesions of
   leishmaniasis patients and human TB granulomas ([167]48–[168]50). Most
   of the available piece of evidence suggests that pathogens responsible
   for acute as well as chronic infections manipulate STAT6-PPARγ/δ
   pathways to avoid M1 polarization ([169]51–[170]55). While the WT M.
   tuberculosis induced polarization of macrophage towards M1, a skewed
   polarization towards M2 macrophages observed in mice infected with
   vapC12 deficient M. tuberculosis strain suggests that a
   vapC12-dependent protein expression actively influences the macrophage
   polarization in the host. As a consequence, there is an altered T-cell
   response activated in terms of Th1/Th2 by the host. This implies that
   M. tuberculosis actively modulates host immunity by differentially
   expressing its proteins and the resulting spatiotemporal expression of
   M. tuberculosis antigens critically regulate the host immune pathways
   which ultimately affects the disease progression and outcome. Contrary
   to the WT, failure of ▵vapC12 to prevent the accumulation of excessive
   neutrophils at the site of infection results in enhanced inflammation,
   M2 polarization and a Th2 type response which together creates an
   environment conducive for the growth of M. tuberculosis inside the
   host. In agreement with our observations, several reports have
   established that excessive neutrophil infiltration leads to tissue
   damage and exacerbation of inflammation which is detrimental to the
   host ([171]56–[172]59). Additionally, upregulation of the IL-1 cytokine
   signaling genes suggest increased levels of proinflammatory IL-1α and
   IL-1β cytokines in the lungs infected with vapC12 deficient M.
   tuberculosis strain. The dysregulation of the levels of IL-1 and IL-1
   receptor antagonists cannot be ruled out.

   Cathepsins carry out efficient innate and adaptive antibacterial
   functions, either directly by taking part in the death of invasive
   bacteria or by processing and presenting the antigen of infected
   bacteria to lymphocytes, which escalates the removal of the pathogen
   and triggers the development of long-lasting immunity ([173]60).
   However, bacteria have the ability to manipulate cathepsin expression
   and proteolytic activity in order to benefit their own intracellular
   survival in macrophages and escape the host-mediated immune
   surveillance ([174]60). M. tuberculosis avoids contact with cathepsins
   by modulating phagosome maturation, impairing the delivery of lysosomes
   to phagosomes in NF-κB dependent manner and by escaping from the
   phagosomes to the cytosol, thus facilitating its replication and
   survival ([175]61–[176]63). Cystatins, which are the inhibitors of
   cathepsins; the proteolytic enzymes involved in the control of M.
   tuberculosis infection were increased at the transcript level in the
   lungs of ▵vapC12 infected mice ([177]42). In support of increased
   peptidase inhibitor activity, we also observed suppressed levels of
   peptidases in lung homogenates of ▵vapC12 infected mice. Our findings
   are also in concordance with previous reports that cathepsins play a
   central role in the immune system and hence its necessary to regulate
   its activity to maintain homeostasis within cells or tissues ([178]64).
   In recent years, MDSCs which are a heterogeneous population of
   immunocytes from myeloid origin have also been found to be a safe
   harbor for M. tuberculosis proliferation ([179]65, [180]66). As
   reported in recent studies, we have identified that the increase in
   MDSCs population mirrored alleviating protective T-cell response in the
   spleen of mice infected with the vapC12 mutant as compared to the WT
   strain infected mice ([181]30, [182]67, [183]68). These results
   implicate that the influx of neutrophils along with MDSCs, IL-1
   cytokines and endopeptidase inhibitors might be responsible for
   increased bacillary load and lung pathology in the mice infected with
   ▵vapC12 mutant.

   S100A8/A9 proteins, members of the S100 family promote the recruitment
   of myeloid cells, specifically neutrophils, dendritic cells and
   monocytes to the lungs through the induction of inflammatory cytokines
   ([184]39). A recent study has mechanistically demonstrated that the
   expression of these calprotectin is needed for the upregulation of
   CD11b integrin on neutrophils resulting in their accumulation during
   chronic TB infection ([185]69). Indeed, our study revealed that
   S100A8/A9 levels corroborated accumulation of myeloid-derived
   inflammatory cells in the lungs of mice infected with vapC12 strain
   during TB disease progression. The RNA-sequencing data also showed
   increased expression of the marker gene Cxcr2 suggesting increased
   recruitment of myeloid cells at week 4 post-infection in the vapC12
   mutant infected mice. These findings are consistent with the literature
   available on the function of S100A8/A9 in host defence mechanisms
   against bacterial and viral infections ([186]39, [187]70).

   S100A8/A9 plays a decisive role in innate immunity by binding to
   receptors such as TLR4 ([188]46) and receptor for advanced glycation
   end products (RAGE), which activates multiple inflammatory pathways
   mediated by these pattern-recognition receptors (PRRs) ([189]46,
   [190]71, [191]72). Finally, we also investigated the role of TLR4 in
   inducing S100A8/A9 mediated hyper inflammation in vapC12 mutant. By
   blocking the TLR4 signaling (C3He/HeJ mice) the bacillary burden in the
   lungs of vapC12 mice became comparable to that of the WT strain. Based
   on these findings, we believe that the absence of TLR4 in C3He/HeJ
   prevented the induction of PAMP-mediated inflammatory response. This
   resulted in the restoration of the growth of vapC12 mutant to that of
   the WT levels. The exact mechanism of S100A8/A9 overexpression and TLR4
   signal activation induced in vapC12 mutant requires further
   investigation. Characterizing the differentially expressed secretory
   proteins of vapC12 mutant would further give us a better insight into
   mechanistically understanding the downstream processes.

   In conclusion, we have studied how the absence of the ribonuclease
   toxin in M. tuberculosis greatly impacted the ability of the pathogen
   to dampen the early innate immune response. The uninterrupted migration
   of granulocytes and continuous expression of proinflammatory cytokines
   resulted in enhanced pathology. The resulting M2 mediated Th-2
   response, generated in the mice infected with ▵vapC12 favored the
   replication and growth of M. tuberculosis. Overall our data suggest
   that VapC12 ribonuclease toxin-mediated differential expression of M.
   tuberculosis proteins help in the timely resolution of TLR4-mediated
   inflammation which is critical for disease persistence in TB ([192]
   Figure 8 ).

Figure 8.

   [193]Figure 8
   [194]Open in a new tab

   Model depicting the VapC12 toxin-mediated host immunomodulation during
   M. tuberculosis infection. Our findings demonstrate that the absence of
   ribonuclease toxin VapC12 favors unrestricted bacterial growth and
   exacerbated pathology. Additionally, vapC12 mutant hinders the ability
   of M. tuberculosis to control the initial influx of myeloid cells to
   the site of infection leading to continuous inflammation and blunted
   T-cell response. Further, myeloid cells mediated hyper-inflammation
   resulted in over activation of S100A8/A9-TLR4 signaling. Finally,
   vapC12 mediated expression of M. tuberculosis proteins is critical for
   disease persistence in tuberculosis. Illustrated using Biorender
   ([195]www.biorender.com).

Data availability statement

   The datasets presented in this study can be found in online repository
   - India Nucleotide Data Archive (INDA) maintained at Indian Biological
   Data Centre (IBDC), India. The accession number(s) can be found below:
   [196]https://inda.rcb.ac.in/viaaccessionIDPRJEB63988.

Ethics statement

   The animal study was approved by Institutional Animal Ethics Committee
   (IAEC), THSTI. The study was conducted in accordance with the local
   legislation and institutional requirements.

Author contributions

   ST: Data curation, Formal analysis, Investigation, Methodology,
   Software, Validation, Writing – original draft, Writing – review &
   editing. SS: Data curation, Formal analysis, Investigation,
   Methodology, Software, Validation, Writing – review & editing. TS: Data
   curation, Investigation, Methodology, Writing – review & editing. AP:
   Formal analysis, Methodology, Software, Writing – review & editing. MP:
   Investigation, Methodology, Writing – review & editing. VN:
   Investigation, Writing – review & editing. DR: Methodology, Software,
   Writing – review & editing. SC: Methodology, Software, Writing – review
   & editing. AA: Project administration, Resources, Writing – review &
   editing. AKP: Conceptualization, Funding acquisition, Project
   administration, Resources, Supervision, Writing – original draft,
   Writing – review & editing.

Acknowledgments