Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase

Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence

Development of novel technologies for effective phosphorylation analysis

Protein phosphorylation plays a critical role in the regulation of many cellular functions. To assist in phosphoproteomic analyses, in the Tao lab, we have been developing new, more effective techniques based on soluble nanopolymers, which demonstrate excellent solubility, compact spherical shape and chemical homogeneity. Functionalization of such molecules with appropriate groups has demonstrated that the techniques are highly versatile and allow for highly efficient analyses of phosphorylation and signaling pathways. The first dendrimer-based method discussed is PolyMAC (Polymer-based Metal ion Affinity Capture), a phosphopeptide enrichment reagent. Such a reagent is useful because the analysis of phosphoproteomes by mass spectrometry depends on an efficient method to enrich phosphopeptides from complex mixtures. The soluble nature of the novel reagent makes it a competent choice for complete phosphopeptide binding and isolation, which was demonstrated through comprehensive comparisons with existing techniques. The two PolyMAC-based technologies developed were further utilized for biologically-relevant studies of Syk kinase-dependant changes in the breast cancer phosphoproteome and B cell receptor (BCR) stimulated changes in human B cell phosphoproteome. Another approach utilizing nanopolymers we have developed in our lab is designed for effective blotting/detection of phosphoproteins on a membrane or in a 96-well plate. We named this phospho-imaging reagent pIMAGO. Unlike antibodies, the new reagent is capable of selectively binding a phosphorylated residue independent of amino acid microenvironment. Currently, it shows no preference for any of the phosphosites, and thus holds great promise in the biological analyses where the site of phosphorylation is not known or its specific antibody is not available. Using this technique, the phosphorylation levels of proteins of interest under physiological conditions can be easily detected as part of standard Western blot or ELISA formats without the need for radioactivity or expensive phosphosite-specific antibodies. The utility of the approach has been demonstrated using standard mixtures of proteins, as well as by in vitro kinase and phosphatase assays. Overall, dendrimer-based reagents designed for phosphoproteomics have demonstrated outstanding selectivity toward phosphorylated residues, great reproducibility and high recovery yield. They have the potential to become powerful tools for a number of biochemical analyses

Global Phosphoproteomics of Activated B Cells Using Complementary Metal Ion Functionalized Soluble Nanopolymers

Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events. Most studies to date have focused on the BCR and protein tyrosine phosphorylation. Because spleen tyrosine kinase, Syk, is an upstream kinase in multiple BCR-regulated signaling pathways, it also affects many downstream events that are modulated through the phosphorylation of proteins on serine and threonine residues. Here, we report a novel phosphopeptide enrichment strategy and its application to a comprehensive quantitative phosphoproteomics analysis of Syk-dependent downstream signaling events in B cells, focusing on serine and threonine phosphorylation. Using a combination of the Syk inhibitor piceatannol, SILAC quantification, peptide fractionation, and complementary PolyMAC-Ti and PolyMAC-Zr enrichment techniques, we analyzed changes in BCR-stimulated protein phosphorylation that were dependent on the activity of Syk. We identified and quantified over 13 000 unique phosphopeptides, with a large percentage dependent on Syk activity in BCR-stimulated B cells. Our results not only confirmed many known functions of Syk, but more importantly, suggested many novel roles, including in the ubiquitin proteasome pathway, that warrant further exploration

Data-Independent Acquisition Phosphoproteomics of Urinary Extracellular Vesicles Enables Renal Cell Carcinoma Grade Differentiation

Translating the research capability and knowledge in cancer signaling into clinical settings has been slow and ineffective. Recently, extracellular vesicles (EVs) have emerged as a promising source for developing disease phosphoprotein markers to monitor disease status. This study focuses on the development of a robust data-independent acquisition (DIA) using mass spectrometry to profile urinary EV phosphoproteomics for renal cell cancer (RCC) grades differentiation. We examined gas-phase fractionated library, direct DIA (library-free), forbidden zones, and several different windowing schemes. After the development of a DIA mass spectrometry method for EV phosphoproteomics, we applied the strategy to identify and quantify urinary EV phosphoproteomes from 57 individuals representing low-grade clear cell RCC, high-grade clear cell RCC, chronic kidney disease, and healthy control individuals. Urinary EVs were efficiently isolated by functional magnetic beads, and EV phosphopeptides were subsequently enriched by PolyMAC. We quantified 2584 unique phosphosites and observed that multiple prominent cancer-related pathways, such as ErbB signaling, renal cell carcinoma, and regulation of actin cytoskeleton, were only upregulated in high-grade clear cell RCC. These results show that EV phosphoproteome analysis utilizing our optimized procedure of EV isolation, phosphopeptide enrichment, and DIA method provides a powerful tool for future clinical applications

Analytical Pipeline for Discovery and Verification of Glycoproteins from Plasma-Derived Extracellular Vesicles as Breast Cancer Biomarkers

Glycoproteins comprise more than half of current FDA-approved protein cancer markers, but the development of new glycoproteins as disease biomarkers has been stagnant. Here we present a pipeline to develop glycoproteins from extracellular vesicles (EVs) through integrating quantitative glycoproteomics with a novel reverse phase glycoprotein array and then apply it to identify novel biomarkers for breast cancer. EV glycoproteomics show promise in circumventing the problems plaguing current serum/plasma glycoproteomics and allowed us to identify hundreds of glycoproteins that have not been identified in blood. We identified 1,453 unique glycopeptides representing 556 glycoproteins in EVs, among which 20 were verified significantly higher in individual breast cancer patients. We further applied a novel glyco-specific reverse phase protein array to quantify a subset of the candidates. Together, this study demonstrates the great potential of this integrated pipeline for biomarker discovery

Analytical Pipeline for Discovery and Verification of Glycoproteins from Plasma-Derived Extracellular Vesicles as Breast Cancer Biomarkers

Glycoproteins comprise more than half of current FDA-approved protein cancer markers, but the development of new glycoproteins as disease biomarkers has been stagnant. Here we present a pipeline to develop glycoproteins from extracellular vesicles (EVs) through integrating quantitative glycoproteomics with a novel reverse phase glycoprotein array and then apply it to identify novel biomarkers for breast cancer. EV glycoproteomics show promise in circumventing the problems plaguing current serum/plasma glycoproteomics and allowed us to identify hundreds of glycoproteins that have not been identified in blood. We identified 1,453 unique glycopeptides representing 556 glycoproteins in EVs, among which 20 were verified significantly higher in individual breast cancer patients. We further applied a novel glyco-specific reverse phase protein array to quantify a subset of the candidates. Together, this study demonstrates the great potential of this integrated pipeline for biomarker discovery