Molecular characterization of Histomonas meleagridis exoproteome with emphasis on protease secretion and parasite-bacteria interaction.

Rounik Mazumdar,Michael Hess,Katharina Nöbauer,Karin Hummel,Ivana Bilic,John Parkinson

doi:10.1371/journal.pone.0212429

Abstract

The exoproteome of parasitic protists constitutes extracellular proteins that play a fundamental role in host-parasite interactions. Lytic factors, especially secreted proteases, are capable of modulating tissue invasion, thereby aggravating host susceptibility. Despite the important role of exoproteins during infection, the exoproteomic data on Histomonas meleagridis are non-existent. The present study employed traditional 1D-in-gel-zymography (1D-IGZ) and micro-LC-ESI-MS/MS (shotgun proteomics), to investigate H. meleagridis exoproteomes, obtained from a clonal virulent and an attenuated strain. Both strains were maintained as mono-eukaryotic monoxenic cultures with Escherichia coli. We demonstrated active in vitro secretion kinetics of proteases by both parasite strains, with a widespread proteolytic activity ranging from 17 kDa to 120 kDa. Based on protease inhibitor susceptibility assay, the majority of proteases present in both exoproteomes belonged to the family of cysteine proteases and showed stronger activity in the exoproteome of a virulent H. meleagridis. Shotgun proteomics, aided by customized database search, identified 176 proteins including actin, potential moonlighting glycolytic enzymes, lytic molecules such as pore-forming proteins (PFPs) and proteases like cathepsin-L like cysteine protease. To quantify the exoproteomic differences between the virulent and the attenuated H. meleagridis cultures, a sequential window acquisition of all theoretical spectra mass spectrometric (SWATH-MS) approach was applied. Surprisingly, results showed most of the exoproteomic differences to be of bacterial origin, especially targeting metabolism and locomotion. By deciphering such molecular signatures, novel insights into a complex in vitro protozoan- bacteria relationship were elucidated.

Highlights

Histomonas meleagridis is a unicellular microaerophilic flagellate pathogen causing histomonosis in gallinaceous birds with a worldwide prevalence
The attenuated H. meleagridis displayed a slight increase in cell number within first 2 hours of incubation, which was followed by a plateau until 6 hours and steady decline in cell count after 6 hours in medium without serum (Fig 1)
In contrast to virulent histomonads, the cell count of attenuated parasites slightly increased within first two hours of incubation. This could be due to the fact that at the start of the serum-free experiment, cells were taken from the mid logarithmic phase and just continued their programmed cell division

Summary

Introduction

Histomonas meleagridis is a unicellular microaerophilic flagellate pathogen causing histomonosis (blackhead disease) in gallinaceous birds with a worldwide prevalence. Molecular investigations of H. meleagridis predominantly relay on in vitro culture, in which the pathogen is isolated from the intestinal content of naturally infected birds and propagated in cell culture media Such cultures often constitute an accumulation of diverse poultry-specific caecal microbes including protozoa and bacteria, hampering histomonad specific analysis. The establishment of the mono-eukaryotic culture facilitated protozoan specific analysis, the background of coexisting ill-defined xenic bacterial flora complicated advanced molecular studies on H. meleagridis, those encompassing ‘-omics’ approaches This necessitated the development of monoxenic culture, in which the xenic bacterial background of the mono-eukaryotic culture was exchanged with that of a single bacterial strain, the Escherichia coli DH5α, thereby generating a ‘monoxenic-mono-eukaryotic’ culture [12]. Comparative proteome study performed on H. meleagridis unravelled the difference between the native virulent strain and its’ mitigated attenuated strain [9]

Methods

Results

Discussion

Conclusion