The Mycobacterium tuberculosis desaturase DesA1 (Rv0824c) is a Ca2+ binding protein.

The hallmark feature of Mycobacterium tuberculosis (M.tb) the causative agent of human tuberculosis, is its complex lipid rich cell wall comprised primarily of mycolic acids, long chain fatty acids that play a key role in structural stability and permeability of the cell wall. In addition, they are involved in inhibiting phagosome-lysosome fusion and aid in granuloma formation during the pathogenic process. M.tb DesA1 is an essential acyl-acyl carrier protein desaturase predicted to catalyze the introduction of position specific double bonds during the biosynthesis of mycolic acids. This protein is one among three annotated desaturases (DesA1-3) in the M.tb genome but is unique in containing a ßγ-crystallin Greek key signature motif, a well-characterized fold known to mediate Ca2+ binding in both prokaryotic and eukaryotic organisms. Using Isothermal Titration Calorimetry and 45CaCl2 overlay, we demonstrate that Ca2+ binds to DesA1. Spectroscopic measurements suggested that this binding induces changes in protein conformation but does not lead to significant alterations in the secondary structure of the protein, a feature common to several ßγ-crystallins. An M. smegmatis strain over-expressing M.tb desA1 showed a Ca2+ dependent variation in surface phenotype, revealing a functional role for Ca2+in DesA1 activity. This study represents the first identification of a Ca2+ binding ßγ-crystallin in M.tb, emphasizing the implicit role of Ca2+ in the pathogenesis of M.tb.

The PE_PGRS Proteins of Mycobacterium tuberculosis Are Ca(2+) Binding Mediators of Host-Pathogen Interaction.

The phenomenal success of Mycobacterium tuberculosis (M.tb) as a pathogen is primarily based on its ability to modulate host immune responses. The genome of M.tb encodes multiple immunomodulatory proteins, including several members of the multigenic PE_PPE family of which the PE_PGRS proteins are a subset. Curiously, 56 of the 61 PE_PGRS proteins contain multiple copies of the glycine-rich sequence motif GGXGXD/NXUX, a nonapeptide sequence predicted to bind Ca(2+), but the functional significance of these motifs remains a mystery. Here we provide evidence via isothermal titration calorimetry, (45)Ca blotting, fluorescence, and circular dichroism spectroscopy that Ca(2+) binds to the PE_PGRS proteins, PE_PGRS33 (Rv1818c) (10 motifs) and PE_PGRS61 (Rv3653) (one motif). Ca(2+) was observed not to bind to PE_PGRS8 (Rv0742), which lacks nonapeptide motifs. Using recombinant Mycobacterium smegmatis strains expressing Rv1818c and Rv3653 and the THP-1 macrophage model of infection, we show that the two proteins mediate Ca(2+)-dependent upregulation of the anti-inflammatory cytokine IL-10, events critical to the pathogenesis of M.tb. Both Rv1818c and Rv3653 interact with TLR2 in a Ca(2+)-dependent manner, providing a novel mechanistic basis for their immunomodulatory effects. Mutations in the nonapeptide motif of Rv3653 led to compromised Ca(2+) binding, validating the functional criticality of this motif. This study demonstrates for the first time not only their Ca(2+) binding properties but also an essential role for Ca(2+) in the functioning of the M.tb PE_PGRS proteins, opening up the possibility of developing novel anti-tuberculosis therapeutics that inhibit Ca(2+)-PE_PGRS binding.

Identification and characterization of a major cell wall-associated iron-regulated envelope protein (Irep-28) in Mycobacterium tuberculosis.

Iron limitation and the expression of mycobactin and carboxymycobactin by Mycobacterium tuberculosis are known. Here, we report how iron regulated the coordinate expression of these two siderophores and a 28-kDa cell wall-associated iron-regulated protein (Irep-28). Irep-28 is identified as the DNA-binding HU homologue HupB protein (hupB [Rv2986c]). Antibodies to this protein were detected in sera from tuberculosis patients. The location of the protein in the cell wall makes it a potential drug target.

Effect of iron concentration on the expression and activity of catalase-peroxidases in mycobacteria.

Mycobacterial catalases are known to exist in different isoforms. We studied the influence of iron concentration on the expression and activity of the different isoforms in Mycobacterium bovis BCG, M. smegmatis, M. fortuitum, M. kansasii and M. vaccae by growing them under iron-sufficient (4 microg Fe/mL) and iron-deficient (0.02 microg Fe/ml) conditions. Upon iron deprivation, significant differences were observed in the catalase/peroxidase activities in both quantitative spectrophotometric assays and in the activity staining in native gels. Notable feature was that the peroxidase activity showed a significant decrease upon iron deprivation in all the mycobacteria, except M. vaccae. Peroxidase activity in all the mycobacteria, irrespective of the iron status was susceptible to heat inactivation. However, the isoforms of catalase showed differences in their heat stability, indicating possible structural differences in these proteins. For example, M. bovis BCG expressed a heat labile catalase under iron-sufficient conditions, while a heat stable catalase band of similar mobility was expressed under iron-deprivation conditions. The study clearly indicates that iron plays an important role in the regulation of expression of the different isoforms of the catalase-peroxidases.