Heparin-Binding Hemagglutinin Adhesin (HBHA) Is Involved in Intracytosolic Lipid Inclusions Formation in Mycobacteria.

Dominique Raze,Claudie Verwaerde,Carine Rouanet,Elisabeth Werkmeister,Camille Locht,Marc Loyens,Baptiste Coupin,Priscille Brodin,Gaspard Deloison

doi:10.3389/fmicb.2018.02258

Abstract

The heparin-binding hemagglutinin adhesin (HBHA) is an important virulence factor of Mycobacterium tuberculosis. It is a surface-displayed protein that serves as an adhesin for non-phagocytic cells and is involved in extra-pulmonary dissemination of the tubercle bacillus. It is also an important latency antigen useful for the diagnosis of latently M. tuberculosis-infected individuals. Using fluorescence time-lapse microscopy on mycobacteria that produce HBHA-green fluorescent protein chimera, we show here that HBHA can be found at two different locations and dynamically alternates between the mycobacterial surface and the interior of the cell, where it participates in the formation of intracytosolic lipid inclusions (ILI). Compared to HBHA-producing mycobacteria, HBHA-deficient mutants contain significantly lower amounts of ILI when grown in vitro or within macrophages, and the sizes of their ILI are significantly smaller. Lipid-binding assays indicate that HBHA is able to specifically bind to phosphatidylinositol and in particular to 4,5 di-phosphorylated phosphatidylinositol, but not to neutral lipids, the main constituents of ILI. HBHA derivatives lacking the C-terminal methylated, lysine-rich repeat region fail to bind to these lipids and these derivatives also fail to complement the phenotype of HBHA-deficient mutants. These studies indicate that HBHA is a moonlighting protein that serves several functions depending on its location. When surface exposed, HBHA serves as an adhesin, and when intracellularly localized, it participates in the generation of ILI, possibly as a cargo to transport phospholipids from the plasma membrane to the ILI in the process of being formed.

Highlights

Tuberculosis remains one of the major global public health threats
atomic force microscopy (AFM) with a heparin coated tip on living Bacillus Calmette–Guérin (BCG)-HEH revealed that the adhesion events frequencies are similar to that obtained with wild-type BCG (Figures 1A,C), but clearly higher than those obtained with the heparin-binding hemagglutinin adhesin (HBHA)-deleted strain (Figure 1B)
In phenotype A, bright red fluorescence was found within the compartments surrounded by EGFP-HBHA (Figure 2D), indicating that these structures could correspond to ILI for TAG storage

Summary

Introduction

The main causative agent, Mycobacterium tuberculosis, is able to adopt multiple strategies to escape from the immune system, to persist silently within cells and to reactivate (Peddireddy et al, 2017). This life cycle within the host relies on a major step: the dormancy of the bacteria leading to latency of the disease. HBHA and Mycobacterial Lipid Inclusions conditions, including nutrient starvation, acidic stress and oxygen limitation. These conditions often lead to a metabolic switch toward the utilization of fatty acids rather than carbohydrates. Actinobacteria, including mycobacteria, as well as some Gram-negative bacteria and cyanobacteria, usually accumulate triacylglycerol (TAG) or wax esters in the form of granules within the cytoplasm (Alvarez and Steinbuchel, 2002)

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Conclusion