Mycobacterial GAPDH and Iron Uptake
Several previous studies have indicated the existence of some alternate sidero- phore-independent pathways for mycobacterial iron acquisition. Support for this has come from the observation that mutant strains unable to synthesize sidero- phores can continue to acquire transferrin iron and survive both in vivo and in vitro. The siderophore negative strain M. tb H37RvO1A survives within macrophages and acquires transferrin iron during the early stages of infection (Wagner et al. 2005). Similarly, the recombinant siderophore-deficient strain BCG(mbtB)30 continues to acquire transferrin iron and survives in vivo (Tullius et al. 2008). Some researchers have also failed to detect siderophores in M. tb infected tissues, thereby supporting the presence of siderophore-independent iron uptake mechanisms (Lambrecht and Collins 1993).
Biochemical studies combined with proteomic analysis have identified that GAPDH is localized to the cell envelope of M. tb cells (Malen et al. 2007, 2010; Bell et al. 2012). This surface localization has also been associated with its alternate function as an EGF receptor that promotes bacterial growth (Bermudez et al. 1996). In comparison to other species, there is relatively limited information available regarding its other alternate functions.
A preliminary analysis using flow cytometry and transmission electron microscopy revealed that M. tb sequesters holo-transferrin at its surface (Fig. 11.1). Detailed proteomic analysis identified six transferrin-binding proteins, one of which was found to be GAPDH (Table 11.1; Boradia et al. 2014). Sequence analysis revealed significant homology with human GAPDH and S. aureus GAPDH (c. 50 % identity), two organisms where it has previously been identified as a transferrin receptor (Modun and Williams 1999; Raje et al. 2007). GAPDH from cytosol, cell membrane and cell-wall fractions of virulent M. tb H37Rv, and the non-pathogenic strain M.tb H37Ra and M. smegmatis were found to be enzymatically active. The interaction of GAPDH-Tf at the cell surface was confirmed by co-immunopre- cipitation assay, confocal-microscopy-based foster resonance energy transfer studies and transmission-electron-microscopy-based analysis (Fig. 11.2).
To estimate the affinity of interaction with transferrin, recombinant M. tb GAPDH (rGAPDH) was expressed and purified. The KD was determined to be 160 ± 24 nM, which is comparable to the affinity of mammalian GAPDH with transferrin. Transferrin binding to GAPDH on the surface of M. tb was found to be saturable and could be inhibited (up to 80% inhibition could be achieved) with increasing molar concentrations of rGAPDH, a characteristic feature of receptor binding. The total number of transferrin receptors (including GAPDH as well as other identified proteins) was estimated to be approximately 7136 ± 255 receptors per bacterial cell (Boradia et al. 2014).