Structural Variability of Lipoarabinomannan Modulates Innate Immune Responses within Infected Alveolar Epithelial Cells

Abstract

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen persisting in phagosomes that has the ability to escape host immune surveillance causing tuberculosis (TB). Lipoarabinomannan (LAM), as a glycolipid, is one of the complex outermost components of the mycobacterial cell envelope and plays a critical role in modulating host responses during M. tb infection. Different species within the Mycobacterium genus exhibit distinct LAM structures and elicit diverse innate immune responses. However, little is known about the mechanisms. In this study, we first constructed a LAM-truncated mutant with fewer arabinofuranose (Araf) residues named M. sm-ΔM_6387 (Mycobacterium smegmatis arabinosyltransferase EmbC gene knockout strain). It exhibited some prominent cell wall defects, including tardiness of mycobacterial migration, loss of acid-fast staining, and increased cell wall permeability. Within alveolar epithelial cells (A549) infected by M. sm-ΔM_6387, the uptake rate was lower, phagosomes with bacterial degradation appeared, and microtubule-associated protein light chain 3 (LC3) recruitment was enhanced compared to wild type Mycobacteriumsmegmatis (M. smegmatis). We further confirmed that the variability in the removal capability of M. sm-ΔM_6387 resulted from host cell responses rather than the changes in the mycobacterial cell envelope. Moreover, we found that M. sm-ΔM_6387 or its glycolipid extracts significantly induced expression changes in some genes related to innate immune responses, including Toll-like receptor 2 (TLR2), class A scavenger receptor (SR-A), Rubicon, LC3, tumor necrosis factor alpha (TNF-α), Bcl-2, and Bax. Therefore, our studies suggest that nonpathogenic M. smegmatis can deposit LC3 on phagosomal membranes, and the decrease in the quantity of Araf residues for LAM molecules not only impacts mycobacterial cell wall integrity but also enhances host defense responses against the intracellular pathogens and decreases phagocytosis of host cells.

Keywords: EmbC; LC3-associated phagocytosis; Mycobacterium smegmatis; alveolar epithelial cells; lipoarabinomannan.

Figure 1

Identification of M. smegmatis EmbC gene knockout strain through RT-PCR.

Figure 2

Separation and identification of lipopolysaccharides from the mycobacterial cell wall, including LAM and LM, and the double asterisks (**) represent p < 0.01. (a) PAS staining. (b) Western blotting analysis.

Figure 3

Assessment of the biological characteristics of M. smegmatis and M. sm-ΔM_6387. (a) Growth curves of M. smegmatis and M. sm-ΔM_6387. (b) Measurement of the migration rate through colony diameter on different LB agar plates. (c) Acid-fast staining analysis using the Ziehl–Neelsen method. (d) Detection of cell wall permeability through SDS and crystal violet. (e) Morphology and cell wall structures of M. smegmatis and M. sm-ΔM_6387 were observed by SEM (5000×) and TEM (8000×), respectively. The double asterisks (**) represent p < 0.01.

Figure 3

Assessment of the biological characteristics of M. smegmatis and M. sm-ΔM_6387. (a) Growth curves of M. smegmatis and M. sm-ΔM_6387. (b) Measurement of the migration rate through colony diameter on different LB agar plates. (c) Acid-fast staining analysis using the Ziehl–Neelsen method. (d) Detection of cell wall permeability through SDS and crystal violet. (e) Morphology and cell wall structures of M. smegmatis and M. sm-ΔM_6387 were observed by SEM (5000×) and TEM (8000×), respectively. The double asterisks (**) represent p < 0.01.

Figure 4

Survival and persistence of M. smegmatis and M. sm-ΔM_6387 in A549 cells. (a) CFU over time post-infection. (b) Observations of internalized M. smegmatis-FITC and M. sm-ΔM_6387-FITC in A549 cells under a fluorescence microscope (400×). (c) Reproduction of M. smegmatis::EGFP internalized in A549 cells after PI-LAM/LM and ΔLAM/LM treatment (400×), and the single asterisk (*) represents p < 0.05.

Figure 4

Survival and persistence of M. smegmatis and M. sm-ΔM_6387 in A549 cells. (a) CFU over time post-infection. (b) Observations of internalized M. smegmatis-FITC and M. sm-ΔM_6387-FITC in A549 cells under a fluorescence microscope (400×). (c) Reproduction of M. smegmatis::EGFP internalized in A549 cells after PI-LAM/LM and ΔLAM/LM treatment (400×), and the single asterisk (*) represents p < 0.05.

Figure 5

TEM analysis of A549 cells. (a) A549 cells infected by M. smegmatis at 0 or 12 h post-infection; (b) A549 cells infected by M. sm-ΔM_6387 at 0 or 12 h; (c) A549 cells treated by PI-LAM from M. smegmatis; (d) A549 cells treated by ΔLAM from M. sm-ΔM_6387; (e) The mean sizes of phagosomes and mitochondria in infected A549 were respectively analyzed, and the asterisks (*) represents p < 0.05; (f) The mean sizes of phagosomes and mitochondria in A549 cells pre-treated by LAM mixture were showed, and the double asterisks (**) represent p < 0.01. Red arrows point to all kinds of mitochondria, and the yellow asterisks are exhibited within phagosomes untaked bacteria.

Figure 6

A549 cells were treated by PI-LAM/LM and ΔLAM/LM at 37 °C for 6, 12, or 24 h and loaded with DCFH-DA probes in order to detect the release of ROS by fluorescence microscopy (a) and quantitative detection with a multifunctional microplate reader, and the double asterisks (**) represent p < 0.01 (b).

Figure 7

Confocal images of A549 cells transient transfected by a lentiviral vector with tandem-tagged RFP-GFP-LC3 (A549^+LC3) post-infection. (a) A549^+LC3, respectively, infected by M. smegmatis, M. sm-ΔM_6387, and BCG for 48 h, and control group represent untreated A549 ^{+ LC3} cells (1260×); and white arrows point to LAPsome structures. (b) Dot plots showing yellow and red spot numbers within A549^+LC3 cells treated by different mycobacteria; circles indicate the number of yellow punctates which mean LC3 recruitments, and triangles represent the number of red punctates. (c) A549^+LC3 infected by M. smegmatis or M. smegmatis-EGFP; white arrows indicate LC3-positive recruitment, and red arrows point to intracellular M. smegmatis-EGFP (1260×). The single asterisk (*) represents p < 0.05, and the double asterisks (**) represent p < 0.01.

Figure 8

The identification of the expression levels of proteins or genes related to LAP and inflammatory responses through Western blotting and RT-PCR. (a) A549 cells were infected by M. smegmatis, M. sm-ΔM_6387, or BCG for 6 h, and then were collected 12 h post-infection for the identification of protein expression changes through Western blotting. The proteins analyzed included Bcl-2, Beclin, Rubicon, IFN-γ, and LC3, and the internal reference, β-actin. The significant differences in expression are marked. (b) qRT-PCR was used to detect mRNA expression, and the genes analyzed included TLR2, TLR4, SR-A, Rubicon, IFN-γ, and IL-1β. (c) A549 cells were treated PI-LAM/LM or ΔLAM/LM and were incubated at 37 °C for 6, 12, or 24 h. The expression changes of three genes, Bax, Bcl-2, and TLR2, were identified through Western blotting. (d) The expression changes of four genes, TLR2, SR-A, TNF-α, and IL-1β, were identified through RT-PCR analysis. The single asterisk (*) represents p value < 0.05, and statistical difference, and the double asterisks (**) represent p value < 0.01, and significance difference.

Figure 8

The identification of the expression levels of proteins or genes related to LAP and inflammatory responses through Western blotting and RT-PCR. (a) A549 cells were infected by M. smegmatis, M. sm-ΔM_6387, or BCG for 6 h, and then were collected 12 h post-infection for the identification of protein expression changes through Western blotting. The proteins analyzed included Bcl-2, Beclin, Rubicon, IFN-γ, and LC3, and the internal reference, β-actin. The significant differences in expression are marked. (b) qRT-PCR was used to detect mRNA expression, and the genes analyzed included TLR2, TLR4, SR-A, Rubicon, IFN-γ, and IL-1β. (c) A549 cells were treated PI-LAM/LM or ΔLAM/LM and were incubated at 37 °C for 6, 12, or 24 h. The expression changes of three genes, Bax, Bcl-2, and TLR2, were identified through Western blotting. (d) The expression changes of four genes, TLR2, SR-A, TNF-α, and IL-1β, were identified through RT-PCR analysis. The single asterisk (*) represents p value < 0.05, and statistical difference, and the double asterisks (**) represent p value < 0.01, and significance difference.