Thermostable Hexameric Form of Eis (Rv2416c) Protein of M. tuberculosis Plays an Important Role for Enhanced Intracellular Survival within Macrophages

Abstract

Eis protein is reported to enhance the intracellular survival of Mycobacterium tuberculosis in human macrophages. Eis protein is not only known to skew away the immunity by disturbing the protective TH1 response, but aminoglycoside acetyltransferase activity of Eis is reported to regulate autophagy, inflammation and cell death. Here we have gained insight into the structure-function properties of Eis. Eis protein is a hexameric αβ protein. Although urea and guanidinium hydrochloride (GdmCl) was found to induce one-step unfolding of Eis but size exclusion chromatography showed that GdmCl treated Eis maintained its hexameric form. SDS-PAGE assay confirmed that hexameric form of Eis is partially stable to SDS and converts into trimers and monomers. Out of these three forms, aminoglycoside acetyltransferase activity is found to be associated only with hexamers. The Tm of Eis was found to be ∼75°C. Aminoglycoside acetyltransferase Eis demonstrated remarkable heat stability retaining >80% of their activity at 70°C which falls down to ∼50% at 75°C and is completely inactive at 80°C. Further, intracellular survival assay with heated samples of M. smegmatis harboring eis gene of M. tuberculosis H37Rv demonstrated a possible role for the thermostability associated with Eis protein in the enhanced intracellular survival within macrophages. In sum, these data reveal that only hexameric form of Eis has a thermostable aminoglycoside acetyltransferase activity. This is the first report showing the thermostability associated with aminoglycoside acetyltransferase activity of Eis protein being one of the essential features for the execution of its biological role.

Interaction of Erp Protein of Mycobacterium tuberculosis with Rv2212 Enhances Intracellular Survival of Mycobacterium smegmatis

“…This result also indicates the delayed clearance of M. smegmatis harboring either RvErp or Rv2212. Similar results have also been reported earlier for Eis protein, a thermostable aminoglycoside acetyltransferase, which enhances the survival of Mycobacterium within macrophages (13,31). A recent report by Pedroza-Roldan et al (17) has also shown that the overexpression of Rv2212 caused the enhanced replication and survival of Mycobacterium bovis BCG Pasteur 1173P2.…”

“…M. smegmatis transformed with vector alone was included in every experiment as a negative control. The intracellular survival assay was performed as described earlier (13).…”

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

“…(56) The escape mechanism is unknown, but efficient intracellular survival of Mt requires a strong thermodynamic stability of proteins. (57) We recently showed in vitro that isolated sub-hexameric assemblies of Mt-NDPK are inactive, complete assembly being essential for function. (53) By combining crystallography and biochemistry, we also demonstrated that hexamer's high thermostability (76°C) is dependent on six inter-subunit R80-D93 salt bridges.…”

Autophagy in Tuberculosis

“…An M. tuberculosis protein termed Eis (enhanced intracellular survival) (Shin et al 2010a;Ganaie et al 2011;Kim et al 2012b) may interact with specific autophagy factors or affect upstream signaling regulators. Eis is an 1-aminoacetyltransferase that acetylates Lys-55 in a JNK-specific phosphatase (Kim et al 2012b).…”

“…Eis is an 1-aminoacetyltransferase that acetylates Lys-55 in a JNK-specific phosphatase (Kim et al 2012b). JNK action is important for activation of the key autophagy regulator Beclin 1, and thus Eis may modulate autophagy (Shin et al 2010a;Ganaie et al 2011;Kim et al 2012b). A mycobacterial glycolipid lipoarabinomannan has also been reported as inhibiting autophagy (Shui et al 2011).…”