Dataset Information

Quantitative analysis of the exoproteome from a Staphylococcus aureus LAC sarA mutant

ABSTRACT: The staphylococcal accessory regulator A (sarA) impacts the extracellular accumulation of Staphylococcus aureus virulence factors at the level of intracellular production and extracellular protease-mediated degradation. To assess the relative impact of these two functions, we previously used a proteomics approach that measures protein abundance as a function of all proteoforms to demonstrate that mutation of sarA results in increased levels of extracellular proteases and assess the impact of this on the accumulation of S. aureus exoproteins1. While this approach confirmed that protease-mediated degradation has a significant impact on the S. aureus exoproteome, it was potentially limited in that it did not take into account the possibility that large, stable proteolytic products from a given protein could result in false negatives when quantified by total proteoforms. Here, we present an expanded proteomics approach that utilizes a dual quantitative method for measuring abundance at both the total proteoform and full-length exoprotein levels. Specifically, proteins present in conditioned medium from overnight, stationary phase cultures of the USA300 strain LAC, an isogenic sarA mutant, and a sarA mutant unable to produce any of the known extracellular proteases (sarA/protease) were resolved using one-dimensional gel electrophoresis. Using methods that focus on total proteoforms vs. methods that focus specifically on full-length proteins, quantitative proteomic comparisons of sarA vs sarA/protease mutants identified proteins that were degraded in a protease dependent manner owing to mutation of sarA, while comparisons of a sarA/protease mutant vs the LAC parent strain identified proteins in which abundance was altered in a sarA mutant in a protease-independent manner. Furthermore, the proteins uniquely identified by the full-length data analysis approach eliminated false negatives observed in the total proteoform analysis. This approach provided for a more comprehensive and robust analysis of the impact of mutating sarA and protease-mediated degradation on the S. aureus exoproteome.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Staphylococcus Aureus

TISSUE(S): Cell Culture

DISEASE(S): Osteomyelitis

SUBMITTER: Stephanie Byrum

LAB HEAD: Alan J. Tackett

PROVIDER: PXD009614 | Pride | 2018-11-21

REPOSITORIES: Pride

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Publications

Label-Free Proteomic Approach to Characterize Protease-Dependent and -Independent Effects of sarA Inactivation on the Staphylococcus aureus Exoproteome.

Byrum Stephanie D SD Loughran Allister J AJ Beenken Karen E KE Orr Lisa M LM Storey Aaron J AJ Mackintosh Samuel G SG Edmondson Ricky D RD Tackett Alan J AJ Smeltzer Mark S MS

Journal of proteome research 20180927 10

The staphylococcal accessory regulator A ( sarA) impacts the extracellular accumulation of Staphylococcus aureus virulence factors at the level of intracellular production and extracellular protease-mediated degradation. We previously used a proteomics approach that measures protein abundance of all proteoforms to demonstrate that mutation of sarA results in increased levels of extracellular proteases and assesses the impact of this on the accumulation of S. aureus exoproteins. Our previous appr ...[more]

PMID: 30209945

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Project description:Binding of transcription factors (TFs) to regulatory sequences is a pivotal step in the control of gene expression. Despite many advances in the characterization of sequence motifs recognized by TFs, our ability to quantitatively predict TF binding to different regulatory sequences is still limited. Here, we present a novel experimental assay termed BunDLE-seq that provides quantitative measurements of TF binding to thousands of fully designed sequences of 200 bp in length within a single experiment. Applying this binding assay to two yeast TFs we demonstrate that sequences outside the core TF binding site profoundly affect TF binding. We show that TF-specific models based on the sequence or DNA shape of the regions flanking the core binding site are highly predictive of the measured differential TF binding. We further characterize the dependence of TF binding, accounting for measurements of single and co-occurring binding events, on the number and location of binding sites and on the TF concentration. Finally, by coupling our in vitro TF binding measurements, and another application of our method probing nucleosome formation, to in vivo expression measurements carried out with the same template sequences now serving and promoters, we offer insights into mechanisms that may determine the different expression outcomes observed. Our assay thus paves the way to a more comprehensive understanding of TF binding to regulatory sequences, and allows the characterization of TF binding determinants within and outside of core binding sites. Binding experiments were carried out with one of two input libraries; Lib1 (also referred to as “main lib”) includes designed sequence variants of length 150bp, and Lib2 (also referred to as “constant flanks lib”) includes designed sequence variants of length 200bp. The sequences of the variants of interest can be found in the second column of the processed data file (BunDLE-seq_data, see 3 excel sheets). EXP1, EXP2 and EXP3 were carried out using Lib1, and EXP4 was carried out using Lib2. Binding experiments were carried out with different proteins (in different concentrations) – DNA was extracted from bound bands that were formed on the gel and amplified with a primer with a unique 5bp upstream tail specifying the identity of the originating band (also referred to as “band barcode”). Data from the following bands was used in the analysis: EXP1_band1, no-protein band – with band barcode “AACGA”, EXP1_band2, single Gcn4 (conc 1:8) bound band* – with band barcode “TCGTA” , EXP1_band3, histones bound band – with band barcode “AGATA”, EXP2_band1, no-protein band – with band barcode “AACGA”, EXP2_band2, single Gcn4 (conc 1:8) bound band* – with band barcode “AGATA”, EXP2_band2, single Gcn4 (conc 1:7) bound band – with band barcode “CACTT”, EXP2_band2, single Gcn4 (conc 1:6) bound band – with band barcode “TCGTA”, EXP2_band2, single Gcn4 (conc 1:5) bound band – with band barcode “CATCA”, EXP2_band2, single Gcn4 (conc 1:4) bound band – with band barcode “TATTA”, EXP2_band2, single Gcn4 (conc 1:3) bound band – with band barcode “AGTCA”, EXP2_band2, single Gcn4 (conc 1:2) bound band – with band barcode “ACGAA”, EXP2_band2, single Gcn4 (conc 1:1) bound band – with band barcode “ACAGA”, EXP2_band2, two Gcn4 (conc 1:1) bound band – barcoded with “GTAGA”, EXP3_band1, no-protein band – with band barcode “GCATA”, EXP3_band2, single Gal4 (conc 1:1) bound band – with band barcode “GATGT”, EXP3_band3, single Gal4 (conc 3:1) bound band – with band barcode “GTTCA”, EXP3_band4, two Gal4 (conc 3:1) bound band – with band barcode “CTCAA”, EXP4_band1, no-protein band – with band barcode “GAGTA”, EXP4_band2, single Gcn4 (conc 1:3) bound band – “TACCA”, EXP4_band3, no-protein band – with band barcode “TCGTT”, EXP4_band3, single Gal4 (conc 3:1) bound band – with band barcode “ACATC”, *biological replicates

Dataset Information

Quantitative analysis of the exoproteome from a Staphylococcus aureus LAC sarA mutant

Publications

Label-Free Proteomic Approach to Characterize Protease-Dependent and -Independent Effects of sarA Inactivation on the Staphylococcus aureus Exoproteome.

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