Unravelling the differences: comparative proteomic analysis of a clonal virulent and an attenuated Histomonas meleagridis strain

Abstract

The current study focused on Histomonas meleagridis, a unicellular protozoan, responsible for histomonosis in poultry. Recently, the occurrence of the disease increased due to the ban of effective chemotherapeutic drugs. Basic questions regarding the molecular biology, virulence mechanisms or even life cycle of the flagellate are still puzzling. In order to address some of these issues, we conducted a comparative proteomic analysis of a virulent and an attenuated H. meleagridis strain traced back to a single cell and propagated in vitro as monoxenic mono-eukaryotic cultures. Using two-dimensional electrophoresis (2-DE) for proteome visualization with computational 2-DE gel image and statistical analysis, upregulated proteins in either of the two H. meleagridis strains were detected. Statistical analysis fulfilling two criteria (≥threefold upregulation and P < 0.05) revealed 119 differentially expressed protein spots out of which 62 spots were noticed in gels with proteins from the virulent and 57 spots in gels with proteins from the attenuated culture. Mass spectrometric analysis of 32 protein spots upregulated in gels of the virulent strain identified 17 as H. meleagridis-specific. The identification revealed that these spots belonged to eight different proteins, with the majority related to cellular stress management. Two ubiquitous cellular proteins, actin and enolase, were upregulated in multiple gel positions in this strain, indicating either post-translational modification or truncation, or even both. Additionally, a known virulence factor named legumain cysteine peptidase was also detected. In contrast to this, mass spectrometric analysis of 49 protein spots, upregulated in gels of the attenuated strain, singled out 32 spots as specific for the flagellate. These spots were shown to correspond to 24 different proteins that reflect the increased metabolism, in vitro adaptation of the parasite, and amoeboid morphology. In addition to H. meleagridis proteins, the analysis identified differential expression of Escherichia coli DH5α proteins that could have been influenced by the co-cultivated H. meleagridis strain, indicating a reciprocal interaction of these two organisms during monoxenic cultivation.

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

“…Despite substantial differences between 2-DE and 2D-DIGE, such as in the experimental design, the applied amount of protein, influence of technical variation and protein spot detection, the protein patterns obtained by the two techniques showed a high level of similarity. The observed distribution of protein spots in the 2D-DIGE-α and 2D-DIGE-β experiments was in agreement with previously published conventional 2-DE gels of H. meleagridis cultures (Monoyios et al, 2018 ). In the conventional 2-DE study as well as in the 2D-DIGE, a noticeable difference in the M r distribution of proteins between virulent and attenuated culture samples was revealed (Monoyios et al, 2018 ).…”

“…Protein spots were distributed within a pH range of 5–7 and a M r of 15–140 kDa (Figures S3, S4). The landmark spot, which was distinctive at pI 5 and a M r of ~50 kDa (Figures S3, S4), was known to be actin of H. meleagridis origin based upon a previous study (Monoyios et al, 2018 ). The visualization of this actin spot with yellow color was an indication that it was not differentially expressed between the in vitro cultivated virulent and attenuated parasites (Figure S3 ).…”

“…Subsequently, the virulent low-passaged parasites were harvested at passage number 25 and the attenuated high-passaged parasites at passage number 303. A purification protocol consisting of consecutive washing and centrifugation steps was used to collect H. meleagridis cells and to remove most of the bacteria as previously described (Monoyios et al, 2018 ). Pellets of collected parasites were stored at −80°C until further use.…”

“…A non-comparative two-dimensional electrophoresis (2-DE) study on H. meleagridis proteome revealed 17 out of 19 identified proteins as actin (Pham et al, 2016 ). A recent comparative 2-DE proteome analysis of the virulent and attenuated H. meleagridis culture, identified 49 significantly differentially expressed protein spots between the two strains, corresponding to 31 unique H. meleagridis proteins (Monoyios et al, 2018 ). In that study, mass spectrometric identification of significant protein spots was supported by the recent de novo transcriptome sequencing of the virulent and attenuated parasite, which was the latest contribution toward the enrichment of H. meleagridis sequence data (Mazumdar et al, 2017 ).…”

Unravelling the Immunity of Poultry Against the Extracellular Protozoan Parasite Histomonas meleagridis Is a Cornerstone for Vaccine Development: A Review

“…In addition, the study elucidated potential pathogenic mechanisms in respect to cytoadherence and host cell membrane disruption, together with the possible regulation of such processes. Monoyios et al ( 41 ) addressed differences between in vivo cultivated virulent and attenuated H. meleagridis parasites on protein expression level. Based on mass spectrometry data it could be shown that eight different proteins, with the majority related to cellular stress management, have been found up-regulated in virulent histomonads compared to the attenuated strain which potentially affect the host-pathogen interaction between the two strains.…”