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

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.

Fig 1. Growth curves of H. meleagridis virulent and attenuated strains under serum-free conditions.

The cell viability was monitored by the trypan blue exclusion under light microscope. At any given time-point no dead cells were detected. The cell count/mL for both virulent and attenuated H. meleagridis it is shown for each time point in the growth curve and represents both total cell number and the number of viable cells.

Fig 2. In vitro secretion kinetics assay demonstrating the time-dependent accumulation of proteins and proteases in the H. meleagridis exoproteome.

(A) 1D-IGZ with the virulent H. meleagridis exoproteome. (B) Conventional 8% 1D SDS-PAGE gel with the virulent H. meleagridis exoproteome. (C) 1D-IGZ with the attenuated H. meleagridis exoproteome. (D) Conventional 8% 1D SDS-PAGE gel with the attenuated H. meleagridis exoproteome. E. coli 24h exoproteome serves as a control due to its’ presence in monoxenic co-cultivation.

Fig 3. Dose dependent inhibitory effects of TLCK, E-64, PMSF and EDTA on the H. meleagridis exoproteomes.

(A) 1D-IGZ demonstrating the inhibitory effect of TLCK (B), E-64 (C), PMSF and (D) EDTA. E. coli 24h exoproteome serves as a control due to its’ presence in monoxenic co-cultivation.

Fig 4. Pie-chart displaying the proteins identified in the H. meleagridis exoproteome sorted according to their biological process.

The proteins were categorized into 12 groups termed as: anti-oxidative stress, cell signalling, cytoskeletal, hypothetical protein, kinase activity, metabolism, nucleotide activity, other processes, oxidation-reduction activity, protein binding, protein degradation and ribosomal protein.

Fig 5. Protein function network of up-regulated proteins detected by the SWATH-MS approach (≥ 2-fold and p-value < 0.05).

(A) H. meleagridis virulent exoproteome, (B) H. meleagridis attenuated exoproteome. The source nodes are categorized according to biological process of the proteins. The colour-coding is based on the n-fold over-expression data associated with each significantly differentially expressed protein (Tables 1 and 2). The network layout was manually determined. Over expressed proteins in virulent histomonads (A) are displayed in small inserted figure.