Evaluation of the role of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer of epithelial cells

Abstract

This study was undertaken in order to assess the involvement of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer in the M. tuberculosis-epithelial cell interaction. A MTP-deficient strain of M. tuberculosis demonstrated a significant reduction of 69.39% (p=0.047) and 56.20% (p=0.033) in its ability to adhere to and invade A549 pulmonary epithelial cells, respectively, in comparison with the wild-type strain. Complementation of the MTP-deficient mutant restored its adhesion and invasion capacity back to the wild-type levels. Overall, it was found that similar concentrations of IL-1β, IL-4, IL-6, IL-8, G-CSF, IFN-γ, MCP-1, and TNF-α were induced in A549 cells infected with the MTP-proficient and MTP-deficient strains. However, at 48h post-infection, the MTP-deficient mutant induced significantly lower levels of TNF-α than the wild-type strain (p=0.033). Furthermore, at 72h post-infection, the mutant induced significantly higher levels of IL-8 than the wild-type (p=0.005). We conclude that MTP is an adhesin/invasin of epithelial cells and, while playing a role in M. tuberculosis entry, they do not appear to largely influence the epithelial cell cytokine response.

Keywords: Adhesin; Curli pili; Invasin; Mycobacterium tuberculosis.

Fig. 1

The role of M. tuberculosis pili (MTP) in the adhesion (A) to and invasion (B) of epithelial cells. A549 epithelial cells were infected with a wild-type, MTP-deficient mutant, and MTP-overexpressing complemented strains for 1 h (adhesion assay) or 2 h (invasion assay). Amikacin-treated and -untreated infected epithelial cells were washed, lysed, and plated onto agar plates to enumerate viable invaded and adherent bacilli, respectively. Results are expressed as the mean percentage adhesion to or invasion of the mutant and complemented strains, relative to that of the wild-type. Error bars are ±1 × SEM.

Fig. 2

The role of M. tuberculosis pili (MTP) in epithelial cell cytokine production. A549 epithelial cells were exposed to the wild-type, MTP-deficient mutant, and MTP-overexpressing complemented strains for 4 h. Extracellular bacteria were removed by washing, fresh media added, and cell culture supernatants at 24, 48, and 72 h post-infection were assayed for levels of the cytokines IL-1β (A), IL-4 (B), IL-6 (C), IL-8 (D), G-CSF (E), IFN-γ (F), MCP-1 (G), and TNF-α (H), using a Bio-Plex assay (Bio-Rad). Error bars are ± 1 × SEM.