Clindamycin increases MS pulmonary colonization in mice.

(a) The experimental procedures of clindamycin-inducing gut microbiota dysbiosis model in mice. (b) The colonization of MS in the lungs of mice after infection at 72 h. (c) The length of cecum after clindamycin treatment. (d)The level of iFABP in serum. (e) LPS concentration in serum. CL: clindamycin-treatment group, CON: control group. MS: Mycobacterium smegmatis, iFABP: intestinal fatty acid binding protein, LPS: lipopolysaccharides. *P<0.05, **P<0.01, *** P<0.001.

Clindamycin altered the diversity and composition of the gut microbiome in mice.

(a) Chao1 and Inverse Simpson index of gut microbiota. (b) PCoA analysis of gut microbiota based on weighted Unifrac distance. (c) The composition distribution of gut microbiome (bacteria and fungi) at the phylum level. (d) The LEfSe analysis of the differentially abundant gut microbiota between the CL group and CON group at the genera level (LDA>4, P<0.05). CL: clindamycin-treatment group, CON: control group. **P<0.01, **** P<0.0001.

Clindamycin weakens the trans-kingdom network construction of gut bacteria and fungi.

(a) The ratio of ITS2/16S at the genus level. (b) The relative abundant ratio of Firmicutes/Bacteroidota. (c) The relative abundant ratio of Ascomycota/Basidiomycota. (d) The trans-kingdom correlation networks of the CON at the genus level. (e) The trans-kingdom correlation networks of the CL at the genus level. CL: clindamycin-treatment group, CON: control group. *P<0.05; ***P<0.001.

Parameters of the trans-kingdom abundance correlation networks

Gut microbiota dysbiosis enhances the susceptibility of MS pulmonary colonization in mice.

(a) The experimental procedures of the feces microbiota transplantation. (b) The PCoA analysis of gut microbiota based on weighted Unifrac distance. (c) The ITS2/16S diversity ratio at the genus level. (d) The relative abundant ratio of Firmicutes/Bacteroidota. (e) The relative abundant ratio of Ascomycota/Basidiomycota. (f) The trans-kingdom correlation networks of CON-FMT and CL-FMT groups at the genus level. (g) The Venn diagram of gut bacteria and gut fungi in different groups at the genus level. (h) The load of MS in the lungs of mice after infection at 72 h. FMT: fecal microbiota transplantation; CON-FMT: The fecal microbiota of the control group was transplanted, CL-FMT: The fecal microbiota of the clindamycin-treatment group was transplanted. MS: Mycobacterium smegmatis, * P<0.05.

Gut microbiota dysbiosis altered the lung transcriptome of mice.

(a) The Venn diagram of DEGs in the different groups. (b) The top 30 GO enrichment analyses of DEGs. (c) The top 20 KEGG enrichment analyses of DEGs. (d) Interaction network analysis of selected DEGs and significant KEGG pathways related to human diseases. (e) The expression levels of Nos2, Ctsd, Cd74, and Tnf with RT-qPCR and RNA-seq. DEGs: differentially expressed genes, CL: clindamycin-treatment group, CON: control group; CON-FMT: The fecal microbiota of the control group was transplanted, CL-FMT: The fecal microbiota of the clindamycin-treatment group was transplanted. * P<0.05, **P<0.01.

Nos2 regulates the expression of NO, ROS, and Defb1.

(a) The expression levels of the Nos2 in the A549 cells. (b) The load of MS in the A549 cells at different times. (c) The NO concentration in the A549 cells. (d) The concentration of ROS in the A549 cells. (e) The expression levels of Defb1 in A549. A: A549 cells control, AP: Transfected A549 cells with blank pcDNA3.1plasmid, AN: Transfected A549 cells with Nos2-pcDNA3.1 plasmid. * P<0.05, **P<0.01, *** P<0.001.

Mechanisms of the intestinal microbiome dysbiosis effect on the colonization of MS in the mouse lungs.

Gut microbiome dysbiosis and gut permeability-increasing disrupt the lung transcriptome, and increase Nos2 expression through the “gut-lung axis”. Nos2 high expression weakens the intracellular antimicrobial and anti-inflammatory environment by increasing the concentration of NO, decreasing the levels of ROS and Defb1 in the cells, and promoting MS colonization in the lungs of mice. MS: Mycobacterium smegmatis.