Immunogenicity, Subcellular Localization And Function Of the Eis Protein Of Mycobacterium tuberculosis

by Samuel, Linoj Philip.

The eis gene of M. tuberculosis is believed to play a role in the intracellular survival of this pathogen. Significantly higher levels of antibodies to Eis were detected by ELISA in the sera of patients with tuberculosis as compared to healthy controls. PBMCs from recovered TB donors were also found to demonstrate significantly higher levels of proliferation in response to stimulation with the Eis protein than PBMCs from either active TB cases or healthy controls. Neither the active TB population nor the healthy controls showed significant levels of IFN- or IL-4 secretion in response to stimulation of PBMC with Eis or ESAT-6. Far Western analysis determined that Eis interacts with a ~65 kDa protein that localizes to the cytoplasmic fraction of M. tuberculosis lysate. Realtime PCR analysis of M. tuberculosis infected U-937 macrophages showed that the eis gene is constitutively expressed during infection. Using immunofluorescence microscopy (IF), the Eis protein was detected within the cytoplasm of M. tuberculosis infected macrophages indicating that the protein was being released/secreted from the mycobacterium containing phagosomes. Western blot analysis of the cytoplasm of macrophages infected with M. tuberculosis expressing green fluorescent protein confirmed these results. Western blot analysis also detected the presence of native Eis both in the culture supernatant of infected macrophages and vesicles released from the macrophages. IF also detected the presence of Eis in uninfected macrophages. The Eis protein in the cytoplasm of M. tuberculosis infected macrophages was also found to colocalize with EEA1, an endosomal marker, indicating a possible association of the protein with early endosomes. Eis was also shown to elicit higher levels of IL-10 16 secretion than PPD in human monocytes. Infection of monocytes from healthy tuberculin reactors with M. tuberculosis wild type and eis mutant demonstrated that eis plays a role in modulation of IL-10/TNF- secretion in response to infection. Bioinformatic analysis of the amino acid sequence of Eis indicates that Eis is an acetyltransferase of the GCN5 related family of N-acetyltransferases. Further work is required to determine the role Eis plays in the survival of M. tuberculosis within the macrophage. 17