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Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a
valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple,
comparable, and reproducible analytical methods must be applied. Although progress is being
made in EV separation methods for human biofluids, the implementation of EV assays for clinical
diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of
established EV separation techniques from human serum and plasma, including ultracentrifugation
and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation,
(b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number,
protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo.
Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant
difference in miRNA content was observed for the different separation methods, ultracentrifugation
was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for
subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of
EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure
and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose
this combination as the preferred EV isolation method for biomarker studies from human serum
or plasma.
Exploring Virulence Factors and Alternative Therapies against Staphylococcus aureus Pneumonia
(2020)
Abstract
Proteome analyses are often hampered by the low amount of available starting material like a low bacterial cell number obtained from in vivo settings. Here, the single pot solid‐phase enhanced sample preparation (SP3) protocol is adapted and combined with effective cell disruption using detergents for the proteome analysis of bacteria available in limited numbers only. Using this optimized protocol, identification of peptides and proteins for different Gram‐positive and Gram‐negative species can be dramatically increased and, reliable quantification can also be ensured. This adapted method is compared to already established strain‐specific sample processing protocols for Staphylococcus aureus, Streptococcus suis, and Legionella pneumophila. The highest species‐specific increase in identifications is observed using the adapted method with L. pneumophila samples by increasing protein and peptide identifications up to 300% and 620%, respectively. This increase is accompanied by an improvement in reproducibility of protein quantification and data completeness between replicates. Thus, this protocol is of interest for performing comprehensive proteomics analyses of low bacterial cell numbers from different settings ranging from infection assays to environmental samples.
Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 106 bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.