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

Ravi K. Lella,Laura Ellen Via,Rahul Solanki,Charu Sharma,Arsheed A. Ganaie

doi:10.1371/journal.pone.0027590

Ravi K. Lella, Laura Ellen Via + Show 3 more

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https://doi.org/10.1371/journal.pone.0027590

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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.

Highlights

Mycobacterium tuberculosis can successfully survive inside the host macrophages in spite of the antimicrobial effector functions of the macrophages [1,2,3,4]
Eis protein is hexameric in nature We amplified the eis gene from the M. tuberculosis H37Rv genomic DNA and cloned, over expressed and purified using NiNTA chromatography as described previously [13]
The results of the subunit mass, as determined by SDS-Polyacrylamide Gel Electrophoresis (PAGE), along with size exclusion chromatography (SEC) studies demonstrate that Eis protein of M. tuberculosis exists as a homo-hexamer under experimental conditions

Summary

Introduction

Mycobacterium tuberculosis can successfully survive inside the host macrophages in spite of the antimicrobial effector functions of the macrophages [1,2,3,4]. A variety of mechanisms have been suggested to contribute to the survival of M. tuberculosis within macrophages [5,6]. Eis protein (Rv2416c of M. tuberculosis) is a secretory protein of 42.0 kDa which provides enhanced intracellular survival to M. tuberculosis in the human macrophage cell line U-937 [7]. Eis protein has the aminoglycoside acetyltransferase activity and belongs to GCN5-related family of N-acetyltransferases (GNAT) [8,9]. Members of the GNAT family of proteins are involved in a variety of activities, ranging from transcriptional activation to antibiotic resistance [10]. The over-expression of Eis aminoglycoside acetyltransferase due to the mutations in the -10 and -35 promoter region was found to confer kanamycin resistance [8]. Kanamycin is one of the important second-line anti-TB drugs which inhibit protein synthesis [11]

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