Mycobacterium tuberculosis acquires iron by cell-surface sequestration and internalization of human holo-transferrin

Mycobacterium tuberculosis (M.tb), which requires iron for survival, acquires this element by synthesizing iron-binding molecules known as siderophores and by recruiting a host iron-transport protein, transferrin, to the phagosome. The siderophores extract iron from transferrin and transport it into the bacterium. Here we describe an additional mechanism for iron acquisition, consisting of an M.tb protein that drives transport of human holo-transferrin into M.tb cells. The pathogenic strain M.tb H37Rv expresses several proteins that can bind human holo-transferrin. One of these proteins is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rv1436), which is present on the surface of M.tb and its relative Mycobacterium smegmatis. Overexpression of GAPDH results in increased transferrin binding to M.tb cells and iron uptake. Human transferrin is internalized across the mycobacterial cell wall in a GAPDH-dependent manner within infected macrophages

Mycobacterium tuberculosis H37Ra: a surrogate for the expression of conserved, multimeric proteins of M.tb H37Rv

Additional file 3. Details of primers used, experimental and theoretical molecular weights, pI values and details of post-translational modifications in GAPDH

Reverse overshot water-wheel retroendocytosis of apotransferrin extrudes cellular iron

Iron (Fe), a vital micronutrient for all organisms, must be managed judiciously because both deficiency or excess can trigger severe pathology. Although cellular Fe import is well understood, its export is thought to be limited to transmembrane extrusion through ferroportin (also known as Slc40a1), the only known mammalian Fe exporter. Utilizing primary cells and cell lines (including those with no discernible expression of ferroportin on their surface), we demonstrate that upon Fe loading, the multifunctional enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is recruited to the cell surface, 'treadmills' apotransferrin in and out of the cell. Kinetic analysis utilizing labeled ligand, GAPDH-knockdown cells, (55)Fe-labeled cells and pharmacological inhibitors of endocytosis confirmed GAPDH-dependent apotransferrin internalization as a prerequisite for cellular Fe export. These studies define an unusual rapid recycling process of retroendocytosis for cellular Fe extrusion, a process mirroring receptor mediated internalization that has never before been considered for maintenance of cellular cationic homeostasis. Modulation of this unusual pathway could provide insights for management of Fe overload disorders

Regulation of macrophage cell surface GAPDH alters LL-37 internalization and downstream effects in the cell

Mycobacterium tuberculosis (M.tb), the major causative agent of tuberculosis (TB), has evolved mechanisms to evade host defenses and persist within host cells. Host directed therapies (HDTs) against infected cells are emerging as an effective option. Cationic host defense peptide LL-37 is known to internalize into cells and induce autophagy resulting in intracellular killing of M.tb. This peptide also regulates the immune system and interacts with the multifunctional protein Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), inside macrophages. Our investigations revealed that GAPDH moonlights as a mononuclear cell surface receptor that internalizes LL-37. We confirmed that the surface levels of P2X7, the receptor previously reported for this peptide remained unaltered on M.tb infected macrophages. Upon infection or cellular activation with IFNγ, surface recruited GAPDH bound to and internalized LL-37 into endocytic compartments via a lipid raft dependent process. We also discovered a role for GAPDH in LL-37 mediated autophagy induction and clearance of intracellular pathogen. In infected macrophages wherein GAPDH had been knocked down, we observed an inhibition of LL-37 mediated autophagy which was rescued by GAPDH over expression. This process was dependent on intracellular calcium and P38 MAPK pathways. Our findings reveal a previously unknown process by which macrophages internalize an antimicrobial peptide via cell surface GAPDH and suggests a moonlighting role of GAPDH in regulating cellular phenotypic responses of LL-37 resulting in reduction of M.tb burden