Quantitative Proteomics of Extracellular Vesicles Released from Human Monocyte-Derived Macrophages upon β‑Glucan Stimulation

Fungal infections (mycoses) are common diseases of varying severity that cause problems, especially to immunologically compromised people. Fungi express a variety of pathogen-associated molecular patterns on their surface including β-glucans, which are important immunostimulatory components of fungal cell walls. During stimulatory conditions of infection and colonization, besides intensive intracellular response, human cells actively communicate on the intercellular level by secreting proteins and other biomolecules with several mechanisms. Vesicular secretion remains one of the most important paths for the proteins to exit the cell. Here, we have used high-throughput quantitative proteomics combined with bioinformatics to characterize and quantify vesicle-mediated protein release from β-glucan-stimulated human macrophages differentiated in vitro from primary blood monocytes. We show that β-glucan stimulation induces vesicle-mediated protein secretion. Proteomic study identified 540 distinct proteins from the vesicles, and the identified proteins show a proteomic signature characteristic for their cellular origin. Importantly, we identified several receptors, including cation-dependent mannose-6-phosphate receptor, macrophage scavenger receptor, and P2X7 receptor, that have not been identified from vesicles before. Proteomic data together with detailed pathway and network analysis showed that integrins and their cytoplasmic cargo proteins are highly abundant in extracellular vesicles released upon β-glucan stimulation. In conclusion, the present data provides a solid basis for further studies on the functional role of vesicular protein secretion upon fungal infection

Comparative Exoprotein Profiling of Different <i>Staphylococcus epidermidis</i> Strains Reveals Potential Link between Nonclassical Protein Export and Virulence

Staphylococcus epidermidis (SE) includes commensal and pathogenic strains capable of infecting humans and animals. This study reports global exoproteome profiling of bovine mastitis strain PM221 and two human strains, commensal-type ATCC12228 and sepsis-associated RP62A. We identified 451, 395, and 518 proteins from culture supernatants of PM221, ATCC12228, and RP62A, respectively. Comparison of the identified exoproteomes revealed several strain-specific differences related to secreted antigens and adhesins, higher virulence capability for RP62A, and similarities between the PM221 and RP62A exoproteomes. The majority of the identified proteins (∼80%) were predicted to be cytoplasmic, including proteins known to be associated in membrane vesicles (MVs) in Staphylococcus aureus and immunogenic/adhesive moonlighting proteins. Enrichment of MV fractions from culture supernatants and analysis of their protein composition indicated that this nonclassical protein secretion pathway was being exploited under the conditions used and that there are strain-specific differences in nonclassical protein export. In addition, several predicted cell-surface proteins were identified in the culture media. In summary, the present study is the first in-depth exoproteome analysis of SE highlighting strain-specific factors able to contribute to virulence and adaptation

Molecular Mechanisms of Selective Estrogen Receptor Modulator Activity in Human Breast Cancer Cells: Identification of Novel Nuclear Cofactors of Antiestrogen–ERα Complexes by Interaction Proteomics

Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that controls key cellular pathways <i>via</i> protein–protein interactions involving multiple components of transcriptional coregulator and signal transduction complexes. Natural and synthetic ERα ligands are classified as agonists (17β-estradiol/E₂), selective estrogen receptor modulators (SERMs: Tamoxifen/Tam and Raloxifene/Ral), and pure antagonists (ICI 182,780-Fulvestrant/ICI), according to the response they elicit in hormone-responsive cells. Crystallographic analyses reveal ligand-dependent ERα conformations, characterized by specific surface docking sites for functional protein–protein interactions, whose identification is needed to understand antiestrogen effects on estrogen target tissues, in particular breast cancer (BC). Tandem affinity purification (TAP) coupled to mass spectrometry was applied here to map nuclear ERα interactomes dependent upon different classes of ligands in hormone-responsive BC cells. Comparative analyses of agonist (E₂)- vs antagonist (Tam, Ral or ICI)-bound ERα interacting proteins reveal significant differences among ER ligands that relate with their biological activity, identifying novel functional partners of antiestrogen–ERα complexes in human BC cell nuclei. In particular, the E₂-dependent nuclear ERα interactome is different and more complex than those elicited by Tam, Ral, or ICI, which, in turn, are significantly divergent from each other, a result that provides clues to explain the pharmacological specificities of these compounds

Molecular Mechanisms of Selective Estrogen Receptor Modulator Activity in Human Breast Cancer Cells: Identification of Novel Nuclear Cofactors of Antiestrogen–ERα Complexes by Interaction Proteomics

Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that controls key cellular pathways <i>via</i> protein–protein interactions involving multiple components of transcriptional coregulator and signal transduction complexes. Natural and synthetic ERα ligands are classified as agonists (17β-estradiol/E₂), selective estrogen receptor modulators (SERMs: Tamoxifen/Tam and Raloxifene/Ral), and pure antagonists (ICI 182,780-Fulvestrant/ICI), according to the response they elicit in hormone-responsive cells. Crystallographic analyses reveal ligand-dependent ERα conformations, characterized by specific surface docking sites for functional protein–protein interactions, whose identification is needed to understand antiestrogen effects on estrogen target tissues, in particular breast cancer (BC). Tandem affinity purification (TAP) coupled to mass spectrometry was applied here to map nuclear ERα interactomes dependent upon different classes of ligands in hormone-responsive BC cells. Comparative analyses of agonist (E₂)- vs antagonist (Tam, Ral or ICI)-bound ERα interacting proteins reveal significant differences among ER ligands that relate with their biological activity, identifying novel functional partners of antiestrogen–ERα complexes in human BC cell nuclei. In particular, the E₂-dependent nuclear ERα interactome is different and more complex than those elicited by Tam, Ral, or ICI, which, in turn, are significantly divergent from each other, a result that provides clues to explain the pharmacological specificities of these compounds

New Insights into <i>Staphylococcus aureus</i> Stress Tolerance and Virulence Regulation from an Analysis of the Role of the ClpP Protease in the Strains Newman, COL, and SA564

In <i>Staphylococcus aureus</i>, ClpP proteases were previously shown to be essential for virulence and stress tolerance in strains derived from NCTC8325. Because these strains exhibit a severely reduced activity of the alternative sigma factor, SigB, we here reassessed the role of ClpP in SigB-proficient clinical strains. To this end, <i>clpP</i> was deleted in strains COL, Newman, and SA564, and the strains were characterized phenotypically. The proteomic changes accomplished by the <i>clpP</i> deletion in the different strains were analyzed using the 2-D DIGE technique. The proteomic analyses revealed mostly conserved changes in the protein profiles of the ClpP-deficient strains. Among the strain-specific changes were the up-regulation of prophage proteins that coincided with an increased spontaneous release of prophages and the relatively poorer growth of the <i>clpP</i> mutants in some strain backgrounds. Interestingly, the effect of ClpP on the expression of selected virulence genes was strain-dependent despite the fact that the expression of the global virulence regulators RNAIII, <i>mgrA, sarZ</i>, <i>sarR</i>, and <i>arlRS</i> was similarly changed in all <i>clpP</i> mutants. ClpP affected the expression of <i>sarS</i> in a strain-dependent manner, and we propose that the differential expression of <i>sarS</i> is central to the strain-dependent effect of ClpP on the expression of virulence genes

Genomics and Proteomics Provide New Insight into the Commensal and Pathogenic Lifestyles of Bovine- and Human-Associated <i>Staphylococcus epidermidis</i> Strains

The present study reports comparative genomics and proteomics of Staphylococcus epidermidis (SE) strains isolated from bovine intramammary infection (PM221) and human hosts (ATCC12228 and RP62A). Genome-level profiling and protein expression analyses revealed that the bovine strain and the mildly infectious ATCC12228 strain are highly similar. Their genomes share high sequence identity and synteny, and both were predicted to encode the commensal-associated <i>fdr</i> marker gene. In contrast, PM221 was judged to differ from the sepsis-associated virulent human RP62A strain on the basis of distinct protein expression patterns and overall lack of genome synteny. The 2D DIGE and phenotypic analyses suggest that PM221 and ATCC12228 coordinate the TCA cycle activity and the formation of small colony variants in a way that could result in increased viability. Pilot experimental infection studies indicated that although ATCC12228 was able to infect a bovine host, the PM221 strain caused more severe clinical signs. Further investigation revealed strain- and condition-specific differences among surface bound proteins with likely roles in adhesion, biofilm formation, and immunomodulatory functions. Thus, our findings revealed a close link between the bovine and commensal-type human strains and suggest that humans could act as a reservoir of bovine mastitis-causing SE strains