A Comparison of Bottom-Up Proteomic Sample Preparation Methods for the Human Parasite Trichomonas vaginalis
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ABSTRACT:
SUBMITTER: Mayr A
PROVIDER: S-EPMC10905575 | biostudies-literature | 2024 Feb
REPOSITORIES: biostudies-literature
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Project description:T. vaginalis, a human-infective parasite, causes the most common nonviral sexually transmitted infection (STI) worldwide and contributes to adverse inflammatory disorders. The immune response to T. vaginalis is poorly understood. Neutrophils (polymorphonuclear cells [PMNs]) are the major immune cell present at the T. vaginalis-host interface and are thought to clear T. vaginalis. However, the mechanism of PMN clearance of T. vaginalis has not been characterized. We demonstrate that human PMNs rapidly kill T. vaginalis in a dose-dependent, contact-dependent, and neutrophil extracellular trap (NET)-independent manner. In contrast to phagocytosis, we observed that PMN killing of T. vaginalis involves taking "bites" of T. vaginalis prior to parasite death, using trogocytosis to achieve pathogen killing. Both trogocytosis and parasite killing are dependent on the presence of PMN serine proteases and human serum factors. Our analyses provide the first demonstration, to our knowledge, of a mammalian phagocyte using trogocytosis for pathogen clearance and reveal a novel mechanism used by PMNs to kill a large, highly motile target.
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Project description:Bottom-up proteomic strategies rely on efficient digestion of proteins into peptides for mass spectrometry analysis. In-solution and filter-based strategies are commonly used for proteomic analysis. In recent years, filter-aided sample preparation (FASP) has become the dominant filter-based method due to its ability to remove SDS prior to mass spectrometry analysis. However, the time-consuming nature of FASP protocols have led to the development of new filter-based strategies. Suspension traps (S-Traps) were recently reported as an alternative to FASP and in-solution strategies as they allow for high concentrations of SDS in a fraction of the time of a typical FASP protocol. In this study, we compare the yields from in-solution, FASP, and S-Trap based digestions of proteins extracted in SDS and urea-based lysis buffers. We performed label-free quantification to analyze the differences in the portions of the proteome identified using each method. Overall, our results show that each digestion method had a high degree of reproducibility within the method type. However, S-Traps outperformed FASP and in-solution digestions by providing the most efficient digestion with the greatest number of unique protein identifications. This is the first work to provide a direct quantitative comparison of two filter-based digestion methods and a traditional in-solution approach to provide information regarding the most efficient proteomic preparation.
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Project description:Trichomonas vaginalis, the most common non-viral sexually transmitted parasite, causes more than 270 million infections annually. The infection's outcome varies greatly depending on different factors that include variation in human immune responses, the vaginal microbiome, and the inherent virulence of the strain. Although the pathogenicity of the different strains depends, at least partially, on differential gene expression of virulence genes; the regulatory mechanisms governing this transcriptional control remain incompletely understood. While many studies have reported a positive correlation between gene expression and chromatin accessibility in other cells, this relationship has not been analyzed in T. vaginalis. To address these questions, we selected two contrasting T. vaginalis strains based on their interactions with host cells: B7268 strain, a highly adherent one and resistant to metronidazole, and NYH209 strain, a poorly adherent one and sensitive to metronidazole. Next, we combined the assay for transposase-accessible chromatin using sequencing (ATAC-seq) with RNA sequencing (RNA-seq), to delve into the relationship between chromatin accessibility and gene expression in these distinct T. vaginalis strains. Our findings demonstrate a correlation between chromatin accessibility and gene expression across both strains. Moreover, we found that chromatin accessibility plays a pivotal role in modulating mRNA expression levels of several established genes linked to parasite pathogenesis and drug resistance. We also identified several open chromatin peaks residing at intergenic regions, revealing possible distal regulatory elements that may control gene expression. These results highlight the importance of chromatin accessibility in modulating gene expression in the parasite T. vaginalis, with possible consequences in pathogenesis and/or drug treatment.
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