Recombinant preparation and functional studies of EspI ATP binding domain from Mycobacterium tuberculosis

https://doi.org/10.1016/j.pep.2016.03.009Get rights and content

Highlights

  • The MtEspI ATP binding domain was solubly expressed in Escherichia coli.

  • The purity of MtEspI415–493 was 96% after purified by GST- and His-dual-tag.

  • Recombinant MtEspI415–493 possessed the ATP binding activity.

Abstract

The ESX-1 secretion system of Mycobacterium tuberculosis is required for the virulence of tubercle bacillus. EspI, the ESX-1 secretion-associated protein in Mycobacterium tuberculosis (MtEspI), is involved in repressing the activity of ESX-1-mediated secretion when the cellular ATP level is low. The ATP binding domain of MtEspI plays a crucial role in this regulatory process. However, further structural and functional studies of MtEspI are hindered due to the bottleneck of obtaining stable and pure recombinant protein. In this study, we systematically analyzed the structure and function of MtEspI using bioinformatics tools and tried various expression constructs to recombinantly express full-length and truncated MtEspI ATP binding domain. Finally, we prepared pure and stable MtEspI ATP binding domain, MtEspI415–493, in Escherichia coli by fusion expression and purification with dual tag, Glutathione S-transferase (GST) tag and (His)6 tag. 31P NMR titration assay indicated that MtEspI415–493 possessed a moderate affinity (∼μM) for ATP and the residue K425 was located at the binding site. The protocol described here may provide a train of thought for recombinant preparation of other ESX-1 secretion-associated proteins.

Introduction

Tuberculosis kills millions of people annually, which is caused by Mycobacterium tuberculosis (Mt) [1], [2]. The ESX-1 secretion-associated proteins in Mycobacterium tuberculosis are encoded by 16 genes in the esx-1 locus [3], which play very important roles in the viability and virulence performance of tubercle bacillus. The deletion of the esx-1 locus causes Mycobacterium tuberculosis attenuated survival in macrophages [4], [5], [6]. Up to date, there are only a few ESX-1 secretion-associated proteins whose function and structure are well studied. For example, the ESAT-6/CFP-10 pair is responsible for the tuberculosis pathogenesis [7], [8], [9]. The secreted transcription factor, EspR, controls Mycobacterium tuberculosis virulence [10], [11]. The heptameric PE/PPE-like secreted substrate, EspB, might be involved in ESX-1-mediated macrophage killing during Mycobacterium tuberculosis disease progression [12], [13], [14]. However, the structures and functions of most proteins involved in the ESX-1 system are still unclear, due to the obstacles in obtaining high-quality recombinant proteins.

MtEspI is a component of ESX-1 system, which is encoded by Rv3876 (espI gene) located in the esx-1 locus. The orthologs of MtEspI merely exist in the pathogenic mycobacteria with ESX-1 system [15], [16]. Recently, Zhang et al. [16] demonstrated that MtEspI could block the ESX-1-mediated secretion in response to low ATP level in Mycobacterium tuberculosis. What's more, the negative regulation of MtEspI might play a vital role in chronic infection by maintaining ATP homeostasis and virulence in Mycobacterium tuberculosis [16], [17]. Besides, it was reported that the mutant of conserved lysine K425 in the MtEspI ATP binding domain would lose its regulation capability [15], [16], [18]. However, the detailed mechanism of the MtEspI negative regulation remains unclear. To further understand MtEspI regulation mechanism, we need to prepare recombinant MtEspI protein, and explore its structure and function at molecular level. In this work, we constructed various recombinants containing different MtEspI fragments, and tried extensive expression and purification conditions. Ultimately, we obtained the pure MtEspI ATP binding domain, MtEspI415–493, by fusion expression and two-step affinity purification with N-terminal Glutathione S-transferase (GST) tag and C-terminal (His)6 tag. 31P NMR titration experiments indicated that MtEspI415–493 had the capacity for binding with ATP. Our purification protocol documented herein provides a clue for recombinant preparation of other ESX-1 secretion-associated proteins in Mycobacterium tuberculosis.

Section snippets

Materials

The full-length MtEspI gene was a generous gift from Associate Professor Xuelian Zhang (State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 200433, China). Vectors pGEX-6p-1, pET-28a, modified pMAL-c2X (A PreScission Protease recognition sequence was inserted following Maltose Binding Protein (MBP) tag.), modified pET-16b (Dual fusion partner (His)10-MBP tag and Tobacco Etch Virus (TEV) protease recognition sequence were cloned into the vector.) and

Bioinformatics analysis and truncation selection of MtEspI

In our preparatory work, the full-length MtEspI was not expressed at all although we tried many different vectors with various expression conditions (Data not shown). The bioinformatics analysis suggested that the protein expression propensity of the full-length MtEspI was low, only reaching 0.065 (Table 3). Previous researches suggested that MtEspI contained a conserved FlhG domain (411–447) which was involved in the negative regulation of flagellar biogenesis in Vibrio cholera [15], [26], [27]

Conclusions

The ESX-1 secretion system is vital for Mycobacterium tuberculosis virulence and MtEspI regulates ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis. In our works, we successfully prepared MtEspI ATP binding domain, MtEspI415–493, after laborious works for trying various expression and purification conditions. We further confirmed that the recombinant MtEspI415–493 possessed a moderate affinity for ATP. And K425 was verified as a key residue for MtEspI415–493-ATP

Acknowledgements

This work was supported by the Natural Science Foundation of China (No. 31470034) and the Fundamental Research Funds for the Central Universities (No. 20720160033). We thank Associate Professor Xuelian Zhang for providing the full-length MtEspI gene (State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 200433, China). We also thank Yunbao Zhi and Associate Professor Kejiang Lin for their assistance in predicting the 3D structure of MtEspI (Department

References (40)

  • F.X. Berthet et al.

    Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10)

    Microbiology

    (1998)
  • A.L. Sorensen et al.

    Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis

    Infect. Immun.

    (1995)
  • P.S. Renshaw et al.

    Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6

    EMBO J.

    (2005)
  • S. Raghavan et al.

    Secreted transcription factor controls Mycobacterium tuberculosis virulence

    Nature

    (2008)
  • O.S. Rosenberg et al.

    EspR, a key regulator of Mycobacterium tuberculosis virulence, adopts a unique dimeric structure among helix-turn-helix proteins

    Proc. Natl. Acad. Sci. U. S. A.

    (2011)
  • B. McLaughlin et al.

    A mycobacterium ESX-1-secreted virulence factor with unique requirements for export

    PLoS Pathog.

    (2007)
  • C. Das et al.

    Computational analysis of the ESX-1 region of Mycobacterium tuberculosis: insights into the mechanism of type VII secretion system

    PloS One

    (2011)
  • M. Zhang et al.

    EspI regulates the ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis

    Mol. Microbiol.

    (2014)
  • N.A. Kruh et al.

    Portrait of a pathogen: the Mycobacterium tuberculosis proteome in vivo

    PloS One

    (2010)
  • N. Dasgupta et al.

    Interaction of the antiactivator FleN with the transcriptional activator FleQ regulates flagellar number in Pseudomonas aeruginosa

    J. Bacteriol.

    (2001)
  • View full text