An Alliance of Gel-Based and Gel-Free Proteomic Techniques Displays Substantial Insight Into the Proteome of a Virulent and an Attenuated Histomonas meleagridis Strain

Abstract

The unicellular protozoan Histomonas meleagridis is notorious for being the causative agent of histomonosis, which can cause high mortality in turkeys and substantial production losses in chickens. The complete absence of commercially available curative strategies against the disease renders the devising of novel approaches a necessity. A fundamental step toward this objective is to understand the flagellate's virulence and attenuation mechanisms. For this purpose we have previously conducted a comparative proteomic analysis of an in vitro cultivated virulent and attenuated histomonad parasite using two-dimensional electrophoresis and MALDI-TOF/TOF. The current work aimed to substantially extend the knowledge of the flagellate's proteome by applying 2D-DIGE and sequential window acquisition of all theoretical mass spectra (SWATH) MS as tools on the two well-defined strains. In the gel-based experiments, 49 identified protein spots were found to be differentially expressed, of which 37 belonged to the in vitro cultivated virulent strain and 12 to the attenuated one. The most frequently identified proteins in the virulent strain take part in cytoskeleton formation, carbohydrate metabolism and adaptation to stress. However, post-translationally modified or truncated ubiquitous cellular proteins such as actin and GAPDH were identified as upregulated in multiple gel positions. This indicated their contribution to processes not related to cytoskeleton and carbohydrate metabolism, such as fibronectin or plasminogen binding. Proteins involved in cell division and cytoskeleton organization were frequently observed in the attenuated strain. The findings of the gel-based studies were supplemented by the gel-free SWATH MS analysis, which identified and quantified 42 significantly differentially regulated proteins. In this case proteins with peptidase activity, metabolic proteins and actin-regulating proteins were the most frequent findings in the virulent strain, while proteins involved in hydrogenosomal carbohydrate metabolism dominated the results in the attenuated one.

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

“…meleagridis strains and the SWATH MS approach identified 42 differentially expressed H . meleagridis proteins [41]. However, beside the fact that in this study the medium for cultivation of parasites contained serum, the analysed protein fraction was different and contained endogenous proteins.…”

“…The recent comparative proteome studies of H . meleagridis , identified actin, enolase, FBA and GAPDH as up regulated by virulent histomonads [9, 41]. However, this only reflected spots localized on positions that differed to the bulk of both proteins, indicating possible post-translational modification(s) and/or truncation(s).…”

Comparative Surfaceome Analysis of Clonal Histomonas meleagridis Strains with Different Pathogenicity Reveals Strain-Dependent Profiles

“…Raw data of previously published experiments [ 10 , 12 ] were re-analyzed with the appropriate software packages for SWATH data: ProteinPilot Software 5.0.2, Sciex (Framingham, USA), PeakView 2.2, Sciex (Framingham, USA), and MarkerView, 1.3.1.1, Sciex (Framingham, USA), as stated in the original publications using the combination of the new H. meleagridis proteome database, the UniProt database for E. coli (taxonomy 83333, , accessed on 25 June 2019) and a common contaminant database ( , accessed on 25 June 2019) as described above. Exported abundance values were used for further statistical evaluation with the DEP package in R as mentioned above.…”

“…Most of the initial molecular investigations focused on the phylogeny of H. meleagridis , and just recently the “omics”-based research has started [ 7 ]. Proteome and exoproteome studies identified differentially expressed proteins in virulent and attenuated strains using mass spectrometry combined with gel-based and gel-free methods, supported by sequences from a transcriptome database [ 8 , 9 , 10 , 11 , 12 ]. In addition, the recently reported full genome sequences of a virulent H. meleagridis strain and an attenuated H. meleagridis strain now provide the underlying genomic data [ 13 ].…”

Proteomic analysis to unravel the biochemical mechanisms triggered by Bacillus toyonensis SFC 500-1E under chromium(VI) and phenol stress

“…The gel-based technologies include one or bi-dimensional electrophoresis strongly established (Abdallah et al 2012), while shotgun technologies, among gel-free, are taking over by utilizing the rapid expansion and advancement of mass spectrometry (Heyer et al 2015; Aebersold and Mann 2016). Current, both technologies have used as complementary approaches given the limitations and advantages that present each one (Bonilla et al, 2020;Monoyios et al, 2018). There has been considerable proteomic research devoted to understanding the mechanisms adopted by microorganisms to remove a particular contaminant such as heavy metals (Dekker et Gang et al 2019).…”