Mycobacterium tuberculosis Rv1987 protein attenuates inflammatory response and consequently alters microbiota in mouse lung

Abstract

Introduction: Healthy lung microbiota plays an important role in preventing Mycobacterium tuberculosis (Mtb) infections by activating immune cells and stimulating production of T-helper cell type 1 cytokines. The dynamic stability of lung microbiota relies mostly on lung homeostasis. In our previous studies, we found that Mtb virulence factor, Rv1987 protein, can mediate host immune response and enhance mycobacterial survival in host lung. However, the alteration of lung microbiota and the contribution of lung microbiota dysbiosis to mycobacterial evasion in this process are not clear so far.

Methods: M. smegmatis which does not contain the ortholog of Rv1987 protein was selected as a model strain to study the effects of Rv1987 on host lung microbiota. The lung microbiota, immune state and metabolites of mice infected by M. smegmatis overexpressing Rv1987 protein (MS1987) were detected and analyzed.

Results: The results showed that Rv1987 inhibited inflammatory response in mouse lung and anaerobic bacteria and Proteobacteria, Bacteroidota, Actinobacteriota and Acidobacteriota bacteria were enriched in the lung tissues correspondingly. The immune alterations and microbiota dysbiosis affected host metabolic profiles, and some of significantly altered bacteria in MS1987-infected mouse lung, such as Delftia acidovorans, Ralstonia pickettii and Escherichia coli, led to anti-inflammatory responses in mouse lung. The secretory metabolites of these altered bacteria also influenced mycobacterial growth and biofilm formation directly.

Conclusion: All these results suggested that Rv1987 can attenuate inflammatory response and alter microbiota in the lung, which in turn facilitates mycobacterial survival in the host.

Keywords: Mycobacterium tuberculosis; Rv1987; immune response; lung microbiota; metabolites.

Figure 1

The lung microbiota differs between MS1987- and MSVec-infected mice in 16S rRNA sequencing. The mouse was infected by MS1987 or MSVec at 5 × 10⁹ CFU/day for 4 days and the lung tissues were collected at day 9 and day 16 post-infection. (A) The OTUs observed in the lung of MS1987-infected mice, MSVec-infected mice, and uninfected mice. (B) α-diversity analysis of the observed species in each group. (C) β-diversity and PCoA analyses of the observed species in each group. (D) The top ten abundant bacteria at species level in the lung of MS1987-infected mice, MSVec-infected mice and uninfected mice. (E) The heatmap of different bacteria in the lung between MS1987- and MSVec-infected mice. The results were from 5-7 mice of each group. MS1987, M. smegmatis overexpressing Rv1987 protein; MSVec, M. smegmatis carrying empty pVV2 vector; CN, uninfected control. CN-1, MSVec-1 and MS1987-1 represented the samples collected at day 9 post-infection; CN-2, MSVec-2 and MS1987-2 represented the samples collected at day 16 post-infection.

Figure 2

The bacteria in the lung tissues differ between MS1987- and MSVec-infected mice in the cultivation in vitro. Equal quantity of the lung tissues (50 mg) was collected from each mouse for MS1987 and MSVec group and homogenized in 1 mL sterile PBS buffer. Forty microliters of the homogenate from each mouse of one group were mixed together. Then half of the mixture was injected into the aerobic blood enrichment culture, and the other half was injected into the anaerobic blood enrichment culture. (A) The bacteria cultured and enriched in aerobic and anaerobic conditions. (B) The bacteria cultured in various plates. The labeled colonies which were inoculated in the liquid medium and identified by sequencing the V3 region of 16S rRNA. (C) The identified bacteria with different abundance between MS1987 and MSVec groups. The arrows ↑ and ↓ represented the bacteria increased and decreased respectively in the MS1987 group compared to the MSVec group. MS1987, M. smegmatis overexpressing Rv1987 protein; MSVec, M. smegmatis carrying empty pVV2 vector; CN, uninfected control; AE, aerobic culture; AN, anaerobic culture. CN-1, MSVec-1 and MS1987-1 represented the samples collected at day 9 post-infection.

Figure 3

Rv1987 attenuates inflammatory response in mouse lung. (A) The relative mRNA expression of pro-inflammatory and anti-inflammatory cytokines in the lung of MS1987- or MSVec-infected mice at day 9 and 16 post-infection. (B) The protein level of cytokines IL-17 and IL-10 in the lung of MS1987- or MSVec-infected mice at day 9 and 16 post-infection. (C) The cytokine levels in the sera of MS1987- or MSVec-infected mice at day 9 and 16 post-infection. (D) The bacterial CFU in mouse lung. The mice were firstly infected with MS1987 or MSVec and then all re-infected by MSVec at day 9 post-primary-infection. The results were from 5 mice of each group and shown as mean ± SD. In figure (A) and (B), the difference was compared by one-way ANOVA analysis with post-hoc Tukey’s multiple comparisons between MS1987, MSVec and CN groups. In figure (C), the difference was analyzed by unpaired t-test between MS1987 and MSVec groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significant difference. MS1987, M. smegmatis overexpressing Rv1987 protein; MSVec, M. smegmatis carrying empty vector; CN, uninfected control; CFU, colony forming unit.

Figure 4

E. coli, D. acidovorans and R. pickettii affect cytokine production and histopathology in mouse lung. The mouse was infected by E. coli, D. acidovorans or R. pickettii at 5 × 10⁸ CFU/day for 4 days and the sera and lung tissues of mice were collected at day 9 post-infection. (A-C) The mRNA level of cytokines in the mouse lung infected by E. coli (A), D. acidovorans (B) and R. pickettii (C). (D-G) The lung histopathology of the mice without infection (D), or infected by E. coli- (E), D. acidovorans- (F) and R. pickettii (G). The results were shown as mean ± SD from 5 mice and compared between each infected group with CN group by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significant difference. Ec, Escherichia coli AW1.7; Da, Delftia acidovorans; Rp, R. pickettii; CN, uninfected control.

Figure 5

The metabolites of E. coli, D. acidovorans and R. pickettii affect the growth of mycobacterium and its biofilm. MSVec was grown in the glass tubes or the wells in absent or presence of the CFC of E. coli, D. acidovorans and R. pickettii. (A) The absorbance at 600 nm (OD₆₀₀) of bacteria grown in glass tubes. (B) The CFU counting of bacteria in figure (A). (C) The weight of air-liquid layer biofilm grown in the wells. (D) The CV staining of the biofilm formed on the side wall of the wells. The results in bar charts were shown as mean ± SD from 3 independent experiments. The differences were compared between the groups in presence or lack of bacterial CFC by unpaired t-test. **p < 0.01; ns, no significant difference. MSVec, M. smegmatis carrying empty pVV2 vector; Ec, Escherichia coli AW1.7; Da, Delftia acidovorans; Rp, R. pickettii; CFC, cell-free culture; BF, biofilm; CFU, colony forming unit.

Figure 6

Rv1987-induced immune alterations and microbiota dysbiosis affect metabolic profiles of mouse lung. (A) PLS-DA model of the metabolites in the lung of MS1987-infected and MSVec-infected mice. (B) The number of altered metabolites in the lung of MS1987-infected mice compared to MSVec-infected mice. (C) The significantly altered metabolites in the lung of MS1987-infected mice compared to MSVec-infected mice. The results were from 5 mice for each group and the difference was compared by unpaired t-test between MS1987 and MSVec groups. (D) KEGG pathway analysis of different metabolites. (E) The effects of some metabolites on mycobacterial growth. The results were shown as mean ± SD from 3 independent experiments and compared by unpaired t-test between MSVec group and other groups. *p < 0.05; ns, no significant difference. MS1987, M. smegmatis overexpressing Rv1987 protein; MSVec, M. smegmatis carrying empty vector. Ala, L-Alanine; Tau, Taurine; HIAA, 5-Hydroxyindoleacetic acid; Put, Putrescine.