K. pneumoniae and M. smegmatis infect epithelial cells via different strategies

Abstract

Background: As the first line of defense, epithelial cells play a vital role in the initiation and control of both innate and adaptive immunity, which participate in the development of disease. Despite its therapeutic significance, little is understood about the specific interaction between pathogenic microorganisms and lung epithelial cells.

Methods: In this study, we performed a head-to-head comparison of the virulence and infection mechanisms of Klebsiella pneumoniae (K. pneumoniae) and Mycobacterium smegmatis (M. smegmatis), which represent Gram-negative/positive respiratory pathogens, respectively, in lung epithelial cell models for the first time.

Results: Through scanning electron microscopy combined with bacterial infection experiments, we confirmed the ability of K. pneumoniae and M. smegmatis strains to form biofilm and cord factor out of the cell wall. M. smegmatis has stronger adhesion and intracellular retention ability, while K. pneumoniae is more likely to induce acute infection. These pathogens could stay and proliferate in lung epithelial cells and stimulate the secretion of specific cytokines and chemokines through a gene transcription regulator. M. smegmatis infection can promote crosstalk among epithelial cells and other immune cells in the lung from a very early stage by prompting the secretion of pro-inflammatory cytokines. Meanwhile, there were significant correlations between K. pneumonia infection and higher levels of interleukin-15 (IL-15), interleukin-1Rα (IL-1Rα), fibroblast growth factor (FGF) basic, and granulocyte colony-stimulating factor (G-CSF). At the same time, K. pneumonia infection also led to changes in the expression of cytoskeletal proteins in epithelial cells.

Conclusions: Our results emphasized the immunoprotection and immunomodulation of lung epithelial cells against exogenous pathogenic microorganisms, indicating that different pathogens damaged the host through different strategies and induced varying innate immune responses. At the same time, they provided important clues and key immune factors for dealing with complicated pulmonary infections.

Keywords: A549; Klebsiella pneumoniae (K. pneumoniae); Mycobacterium smegmatis (M. smegmatis); RNA-seq; cytokines.

Figure 1

Growth curves in vitro and scanning electron microscopy photography of K. pneumoniae and M. smegmatis. (A) Growth curves of K. pneumoniae according to OD₆₀₀ in a 37 ℃ shaker. The experiments were performed in triplicate. The data was expressed as the mean ± SEM at all time points. Scanning electron micrographs showing cell cluster morphology (microcolony), which was strongly suggestive of biofilm formation. Magnification: ×4,000, ×50,000. (B) Growth curves of M. smegmatis according to OD₆₀₀ in a 37 ℃ shaker. The experiments were performed in triplicate. The data was expressed as the mean ± SEM at all time points. Scanning electron micrographs showing cord-like morphology (cord factor). Magnification: ×4,000, ×50,000. OD, optical density; SEM, standard error of mean.

Figure 2

K. pneumoniae and M. smegmatis exhibited endocytosis ability into lung epithelial cells. (A) A549 epithelial cells were infected with K. pneumoniae and M. smegmatis (MOI =1, 5, and 25 for K. pneumonia and MOI =5, 25, and 50 for M. smegmatis) for 3 h. The cells were then washed and lysed, and the intracellular bacteria were enumerated. (B) A549 epithelial cells were infected with K. pneumoniae and M. smegmatis (MOI =1 for K. pneumonia and MOI =25 for M. smegmatis) for 3 h. Then the cells were then washed, and the intracellular bacteria were enumerated at the indicated time points. (C) Lung epithelial cell viability test with a CCK-8 kit. (D) Apoptosis related protein was detected through western blot. Mean ± SEM, two-way ANOVA with Bonferroni comparison test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. The data are representative of three independent experiments. K. pneu, K. pneumoniae; M. smeg, M. smegmatis; E. coil, Escherichia coli; MOI, multiplicity of infection; CCK-8, Cell Counting Kit-8; SEM, standard error of mean; ANOVA, analysis of variance.

Figure 3

Organization structure of F-actin occurs in K. pneumoniae and M. smegmatis-infected A549 lung epithelial cells. (A) F-actins (green) are severed in A549 cells during K. pneumoniae infections. DAPI (blue) shows DNA to identify individual cells. Scale bar =25 µm. (B) F-actins (red) are severed in A549 cells during M. smegmatis infections. DAPI (blue) shows DNA to identify individual cells. Scale bar =50 μm. Ctrl, control group; DAPI, 4,6-diamino-2-phenyl indole; K. pneu, K. pneumoniae; M. smeg, M. smegmatis.

Figure 4

Differentially expressed genes in lung epithelial cells infected by K. pneumoniae and M. smegmatis. (A) Venn diagrams showing the overlap of gene lists between two samples. (B) Correlation heatmap between samples. (C) Heatmap of differentially expressed genes in cells infected by the two strains separately. (D) Volcano diagram of differentially expressed genes in cells infected by the two strains separately. (E) Diagram of the top 20 ranked GO terms among the differentially expressed genes. (F) KEGG pathway enrichment analysis of the differentially expressed genes. MS, M. smegmatis; FC, fold change; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 5

The secretion of cytokines, chemokines, and growth factors from lung epithelial cells infected by K. pneumoniae and M. smegmatis. (A) Cluster analysis of cytokines, chemokines, and growth factors secretion from lung epithelial cells at 24 h post-infection. (B) The secretion of cytokines, chemokines, and growth factors from lung epithelial cells at 3 h post-infection. (C) The secretion of cytokines, chemokines, and growth factors from lung epithelial cells at 24 h post-infection. In this group, there were two infection groups and one control group. (D) The secretion of cytokines, chemokines, and growth factors from lung epithelial cells at 24 h post-infection. In this group, there were only two infection groups to compare. Mean ± SEM, two-way ANOVA with Bonferroni comparison test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. The data are representative of three independent experiments. IL, interleukin; FGF, fibroblast growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte macrophage colony stimulating factor; INF, interferon; IP, interferon gamma induced protein; MCP, monocyte chemoattractant protein; MCAF, monocyte chemotactic and activating factor; MIP, macrophage inflammatory protein; PDGF, platelet-derived growth factor; RANTES, regulated upon activation, normal T cell expressed and presumably secreted; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; M. smeg, M. smegmatis; K. pneu, K. pneumoniae; Ctrl, control group; SEM, standard error of mean; ANOVA, analysis of variance.