Heparin-binding Hemagglutinin of Mycobacterium tuberculosis Is an Inhibitor of Autophagy.

Qing Zheng,Shan Zhou,Yueyun Ma,Zhi Li,Xiaorui Fu,Liu Yang,Lei Zhou,Qian Zhang,Xiaoke Hao

doi:10.3389/fcimb.2017.00033

Abstract

Airway epithelial cell is often the initial site of attack by pathogens, and cell death is commonly caused by internalization of Mycobacterium tuberculosis (Mtb). However, the mechanism of interaction between epithelial cells and Mtb is not well understood. In this study, we investigated the role of the heparin-binding hemagglutinin (HBHA) protein of Mtb in the function of epithelial cells. In particular, the autophagy of A549 cells was determined based on microtubule-associated protein 1 light chain 3 alpha (LC3) activity. Autophagosome formation was detected by Monodansylcadaverine (MDC) staining and immune fluorescence staining of LC3. Autophagy could be significantly suppressed by HBHA protein. In addition, the LDH assay results showed that HBHA treatment could induce death on A549 cells. To explore the form of cell death, we detected the activity of caspase-3 and LDH release of A549 cells in the presence or absence of caspase inhibitor Z-VAD-FMK. Results demonstrated that HBHA treatment could induce apoptosis of A549 cells. To further confirm these results, we constructed the recombinant Mycobacterium smegmatis (MS) expressing HBHA (rMS-HBHA) and explored the influence of rMS-HBHA on the function of A549 cells. rMS-HBHA infection significantly inhibited LC3 expression and the maturation of autophagosomes in A549 cells. Subsequently, we infected A549 cells with MS and detected the viability of intracellular MS by CFU counts. rMS-HBHA showed higher survival and replication capacity in A549 cells than those of the wild-type MS. Finally, infection of A549 cells with rMS-HBHA caused further apoptosis. These findings suggested that rMS-HBHA could inhibit autophagy, promote its survival and replication within A549 cells, and subsequently induce apoptosis on infected cells to facilitate infection.

Highlights

Tuberculosis (TB) remains a devastating disease with approximately 2 billion people infected worldwide and 1.2 million deaths in 2010 (Hawn et al, 2015)
To evaluate the effect of heparin-binding hemagglutinin (HBHA) on the autophagy of A549 cells, Western blot assay was performed to detect the expression of light chain 3 alpha (LC3) and Beclin-1
When HBHA was added after 90 min, the expression of LC3 and Beclin-1 was suppressed in a dose-dependent manner (Figures 1A–D)

Summary

Introduction

Tuberculosis (TB) remains a devastating disease with approximately 2 billion people infected worldwide and 1.2 million deaths in 2010 (Hawn et al, 2015). The etiological agent of TB, that is, Mycobacterium tuberculosis (Mtb), is the most successful intracellular pathogen that can invade and replicate in many host cell types, including both phagocytic and nonphagocytic cells (Vir et al, 2014). Increasing evidence implicated that alveolar epithelium, type II pneumocyte, plays an important role in both host cell defense and bacterial dissemination (Xiong et al, 2014; Fine-Coulson et al, 2015; Ryndak et al, 2015). Unlike MS, type II pneumocytes could provide a permissive position for Mtb to replicate considerably and help bacterial dissemination (Bermudez and Goodman, 1996; Ryndak et al, 2015). The mechanism of how Mtb destroys the defense system is still unknown

Methods

Results

Conclusion