Significance of a histone-like protein with its native structure for the diagnosis of asymptomatic tuberculosis

Yukiko Ohara,Ichiro Nakagawa,Tsukasa Mashima,Yukihiro Kaneko,Sohkichi Matsumoto,Saburo Yamamoto,Yutaka Yoshida,Fumio Arisaka,Yoshie Fujiwara,Masato Katahira,Haruka Kobayashi,Yoshitaka Tateishi,Yuriko Ozeki,Yasuo Tsunaka,Akihito Nishiyama,Kengo Kitadokoro,Ryoji Maekura,Riccardo Manganelli

doi:10.1371/journal.pone.0204160

Abstract

Tuberculosis causes the highest mortality among all single infections. Asymptomatic tuberculosis, afflicting one third of the global human population, is the major source as 5–10% of asymptomatic cases develop active tuberculosis during their lifetime. Thus it is one of important issues to develop diagnostic tools for accurately detecting asymptomatic infection. Mycobacterial DNA-binding protein 1 (MDP1) is a major protein in persistent Mycobacterium tuberculosis and has potential for diagnostic use in detecting asymptomatic infection. However, a previous ELISA-based study revealed a specificity problem; IgGs against MDP1 were detected in both M. tuberculosis-infected and uninfected individuals. Although the tertiary structures of an antigen are known to influence antibody recognition, the MDP1 structural details have not yet been investigated. The N-terminal half of MDP1, homologous to bacterial histone-like protein HU, is predicted to be responsible for DNA-binding, while the C-terminal half is assumed as totally intrinsically disordered regions. To clarify the relationship between the MDP1 tertiary structure and IgG recognition, we refined the purification method, which allow us to obtain a recombinant protein with the predicted structure. Furthermore, we showed that an IgG-ELISA using MDP1 purified by our refined method is indeed useful in the detection of asymptomatic tuberculosis.

Highlights

Tuberculosis remains a serious threat to human health
The pellet was washed with ice-cold phosphate-buffered saline (PBS), and the bacteria were subsequently collected by centrifugation
The results revealed that the IgG responses to culture filtrate protein 10 kDa (CFP10), early secretary antigen target with 6 kDa (ESAT6), Antigen 85B (Ag85), and purified protein derivatives (PPD) are significantly higher in active tuberculosis patients

Summary

Introduction

Tuberculosis remains a serious threat to human health. The most recent report by the World Health Organization announced that, in 2016, 10.4 million people newly developed tuberculosis and 1.7 million people died from this disease[1]. Mycobacterium tuberculosis, an acid-fast Gram-positive bacillus, is an etiologic agent of tuberculosis. This intracellular pathogen can escape from the bactericidal mechanisms of its hosts. It is estimated that more than 90% of M. tuberculosis-infected individuals do not initially develop the disease but instead remain in an asymptomatic state without eradicating the pathogen[2, 3]. Asymptomatic infections are the major silent source of tuberculosis. The establishment of a precise diagnosis of asymptomatic tuberculosis is crucial for disease control[3, 4]. The sensitivity of currently available tools, such as the interferongamma release assay (IGRA), is limited, probably because they detect immune responses to proteins produced from growing rather than from persistent M. tuberculosis[5, 6]

Methods

Results

Conclusion