Mycobacterium tuberculosis Prolyl Oligopeptidase Induces In vitro Secretion of Proinflammatory Cytokines by Peritoneal Macrophages.

Brina Portugal,Izabela M D Bastos,Diego O Nolasco,Francisco D Vieira,Flávia N Motta,Flávia N Motta,Jaime M Santana,Andre F Correa,Andre F Correa,Ana L V Atta,Hugo De Almeida,Kelly G Magalhães

doi:10.3389/fmicb.2017.00155

Abstract

Tuberculosis (TB) is a disease that leads to death over 1 million people per year worldwide and the biological mediators of this pathology are poorly established, preventing the implementation of effective therapies to improve outcomes in TB. Host–bacterium interaction is a key step to TB establishment and the proteases produced by these microorganisms seem to facilitate bacteria invasion, migration and host immune response evasion. We presented, for the first time, the identification, biochemical characterization, molecular dynamics (MDs) and immunomodulatory properties of a prolyl oligopeptidase (POP) from Mycobacterium tuberculosis (POPMt). POP is a serine protease that hydrolyzes substrates with high specificity for proline residues and has already been characterized as virulence factor in infectious diseases. POPMt reveals catalytic activity upon N-Suc-Gly-Pro-Leu-Gly-Pro-AMC, a recognized POP substrate, with optimal activity at pH 7.5 and 37°C. The enzyme presents KM and Kcat/KM values of 108 μM and 21.838 mM-1 s-1, respectively. MDs showed that POPMt structure is similar to that of others POPs, which consists of a cylindrical architecture divided into an α/β hydrolase catalytic domain and a β-propeller domain. Finally, POPMt was capable of triggering in vitro secretion of proinflammatory cytokines by peritoneal macrophages, an event dependent on POPMt intact structure. Our data suggests that POPMt may contribute to an inflammatory response during M. tuberculosis infection.

Highlights

Despite global effort to stop tuberculosis (TB), it remains the second-deadliest infectious disease worldwide causing over 1 million deaths per year and an additional 0.4 million death resulting from TB disease among HIV-positive patients (WHO, 2015)
POPMt was expressed in E. coli BL21(DE3) as a soluble and active enzyme which allowed us to proceed to its purification (Figure 1A) and biochemical characterization
prolyl oligopeptidase (POP) have already been described in bacteria like Flavobacterium meningosepticum (Yoshimoto et al, 1980, 1991), Sphingomonas capsulate (Kabashima et al, 1998), Aeromonas hydrophila (Kanatani et al, 1993), and M. xanthus (Shan et al, 2004)

Summary

Introduction

Despite global effort to stop tuberculosis (TB), it remains the second-deadliest infectious disease worldwide causing over 1 million deaths per year and an additional 0.4 million death resulting from TB disease among HIV-positive patients (WHO, 2015). The pathology results from a highly evolved and multifactorial ability of Mycobacterium tuberculosis, an intracellular bacterium, to Mycobacterium tuberculosis Prolyl Oligopeptidase prevent or evade effective host responses (Goldberg et al, 2014). Even though M. tuberculosis can infect a variety of cell types, alveolar macrophages are its main niche. M. tuberculosis spread and dissemination is deeply correlated with its ability to infect and immunomodulate macrophages. In reaction to M. tuberculosis infection, macrophages upregulate effectors and signaling pathways to both prevent bacilli replication and recruit other immune cells into the site of infection (Cooper and Torrado, 2012; Sia et al, 2015). M. tuberculosis has an arsenal of potent mechanisms for evading those antimicrobial reactions, thereby changing the host immune response toward a pathological rather than a protective one. Pulmonary TB is the most typical presentation of the disease, M. tuberculosis may disseminate into a variety of organs causing extrapulmonary TB (Galimi, 2011)

Methods

Results

Conclusion