Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis

Abstract

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

Fig 1

M. leprae enters the nasal epithelial cell lineage RPMI 2650. Nasal epithelial cells were infected with live M. leprae at an MOI of 10 at 33°C at different time points. The bacteria were labeled with PKH26 (red), and the monolayers were stained with DAPI (blue) and FITC-phalloidin (green). (A) Representative confocal image of 4 independent experiments showing live M. leprae bacilli interacting with RPMI 2650 cells at 24 h of infection. (B, C, and D) Numbers of bacteria that interact with (B), adhere to (C), and enter (D) nasal epithelial cells. (E) Percentages of bacterium-associated cells. Data represent the means ± standard deviations (SD) of the results of 4 experiments performed in duplicate. *, P < 0.05 was considered statistically significant.

Fig 2

M. leprae enters the alveolar epithelial cell lineage A549. Alveolar epithelial cells were infected with live M. leprae at an MOI of 10 at 33°C at increasing incubation times. The bacteria were labeled with PKH26 (red), and the cells were stained with DAPI (blue) and FITC-phalloidin (green). (A) Representative fluorescent image of 4 independent experiments showing live M. leprae bacilli interacting with A549 cells at 24 h of infection. (B, C, and D) Numbers of bacteria that interact with (B), adhere to (C), and enter (D) alveolar epithelial cells. (E) Percentages of bacterium-associated cells. Data represent the means ± standard deviations of the results of 4 experiments performed in duplicate. *, P < 0.05 was considered statistically significant.

Fig 3

Transmission electron micrographs of M. leprae-infected epithelial cells. Epithelial cells were infected with live M. leprae for 24 h at 33°C. RPMI 2650 (A and C) and A549 (B and D) cells were fixed, processed, and visualized by transmission electron microscopy. The arrows point to bacteria; the arrowheads point to the membrane-bound compartments.

Fig 4

M. leprae enters human primary nasal epithelial cells. (A) Primary nasal epithelial cells were isolated from nasal polyps, and culture purity was determined by immunostaining with anti-cytokeratin-19 (CK-19) (red). Cell nuclei were labeled with DAPI (blue). (B) Isotype control. (C and D) Cells were infected with prelabeled PKH67 gamma-irradiated M. leprae at an MOI of 10 at 37°C for 2 h. (C) Representative confocal image of 4 independent experiments showing nasal primary cells infected with M. leprae (green; arrow). (D) The numbers of bacteria that interact with, adhere to, and enter primary nasal epithelial cells were determined. Data represent the means ± standard deviations of the results of 4 experiments performed in duplicate.

Fig 5

M. leprae infects respiratory tract cells in vivo. C57BL/6 mice were intranasally challenged with M. leprae. Airway and lungs were monitored by histological examination for detection of bacteria and lesions. The images show terminal bronchiole and circumjacent lung tissue after 4 h of infection. Acid-fast bacilli (arrows) in luminal macrophages (A) and in shed epithelial cells (B) are shown. Bars, 10 μm (A) and 5 μm (B).

Fig 6

M. leprae enters airway epithelial cells in a passive cytoskeleton-dependent manner. (A to D) Live and heat-killed M. leprae interaction with RPMI 2650 (A and C) and A549 (B and D) cells for 24 h at 33°C. The bacteria were labeled with PKH26, and the numbers of bacteria interacting with the cells and the percentages of bacterium-associated cells were determined by fluorescence microscopy. (E and F) Effect of cytoskeleton inhibitors on M. leprae uptake by RPMI 2650 cells (E) and A549 cells (F). Cells were pretreated with DMSO (drug vehicle), colchicine, or cytochalasin B for 1 h and during the 24 h of the assay at 33°C. Data represent the means ± standard deviations of the results of 3 experiments performed in duplicate. **, P < 0.01.

Fig 7

M. leprae survives inside epithelial cells. Intra- and extracellular M. leprae viability in RPMI 2650 and A549 cells was determined by the use of a Live/Dead kit. (A) RPMI 2650 cells at 33°C. (B) A549 cells at 33°C. (C) A549 cells at 37°C. Intracellular M. leprae bacilli (black bars) were recovered by lysing buffer, while extracellular bacteria (gray bars) were recovered after washing steps. In parallel, M. leprae bacilli were incubated in cell-free medium (white bar). Bacteria were stained by the use of a Live/Dead BacLight Bacterial Viability kit and enumerated by direct counting via fluorescence microscopy at a magnification of ×1,000. The data represent the mean values ± SD of the results of five experiments with duplicate samples from each one. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 8

Functional grouping and intercross of surface-exposed proteins identified in crude and NaOH-treated M. leprae. A Venn diagram illustrating overlap between surface proteins identified in NaOH-treated and crude M. leprae is shown. Proteins identified exclusively in the NaOH-treated bacilli, only in the crude bacilli, or simultaneously found in both samples were functionally classified according to the Leproma database (http://genolist.pasteur.fr/Leproma/). Functional categories are expressed in percentages. seqs, sequences.

Fig 9

M. leprae recombinant Hlp and HBHA mediate the mycobacterial attachment to airway epithelial cells. (A and B) Fluorescent beads coated with Hlp (A), with HBHA (B), or with BSA (negative control) were incubated with RPMI 2650 and A549 cells for 1 h at 37°C. Cells were then washed and fixed. The numbers of beads associated with these cells were counted in a fluorescence microscope in a set of 300 cells using a magnification of ×400. (C) Fluorescent beads coated with Hlp, HBHA, or BSA were incubated simultaneously with A549 cells for 1 h at 37°C. (D) M. smegmatis (MS) was pretreated or not with Hlp (MS+Hlp) or HBHA (MS+HBHA), and an ELISA was performed with anti-Hlp or anti-HBHA, respectively, in order to monitor the binding of these proteins to the bacterial surface. (E) MS and Hlp or MS and HBHA were used to infect epithelial cells for 2 h at 37°C. Internalized bacteria were released with lysis buffer for CFU counting. The data represent the mean values ± SD of the results of at least three experiments performed in duplicate. *, P < 0.05; **, P < 0.01.