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

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.

Coordinate regulation of virulence and metabolic genes by the transcription factor HbhR in Mycobacterium marinum

“…). The enhanced production of HBHA in Mmar 4.9 was confirmed by immunoblot analyses using monoclonal antibody VF2 (Raze et al , ) (Fig. B).…”

“…Blue bars indicate the position and lengths of qRT-PCR to evaluate the expression levels of each gene. analyses using monoclonal antibody VF2 (Raze et al, 2018) (Fig. 1B).…”

“…It is also a marker of the latency phase of tuberculosis (Hougardy et al , ). Moreover, we recently showed that HBHA is involved in the biogenesis of ILI in M. tuberculosis and M. smegmatis and may function as a moonlighting protein (Raze et al , ). The different functions of HBHA at different steps of the mycobacterial life cycle suggest a coordinated regulation of its gene, as suggested in previous studies (Delogu et al , ; Iona et al , ).…”

Protein scaffold involving MSMEG_1285 maintains cell wall organization and mediates penicillin sensitivity in mycobacteria

“…Total proteins were resolved by SDS/PAGE using 12% acrylamide gels and then electro-transferred onto a nitrocellulose membrane. The membrane was saturated with Trisbuffered saline (TBS) pH 7.5 containing 0.05% Tween-80 and 5% milk and probed overnight at 4°C with anti-HbhA monoclonal antibody VF2 [35], anti-Hsp65/GroL1 monoclonal antibody (Abcam, Paris, France; dilution 1 : 1000), or anti-MBP (NEB Biolabs, Evry, France; dilution 1 : 10 000) in TBS-Tween-3% milk. Finally, the membrane was incubated for 1 h at room temperature with goat anti-mouse horseradish peroxidase-conjugated secondary antibodies (Abcam; diluted 1 : 5000) in TBS-Tween-3% milk.…”

In Vivo Methods to Study Protein–Protein Interactions as Key Players in Mycobacterium Tuberculosis Virulence

“…HbhA is a surface-exposed adhesin that is involved in the binding of mycobacteria to non-phagocytic cells, a necessary process for Mtb dissemination [73], and in the formation of intracellular lipid inclusions [74]. The BACTH system was used to demonstrate that HbhA interacts with Rv0613c and MmpL14 [75].…”