Aureolib - a proteome signature library: towards an understanding of staphylococcus aureus pathophysiology

Abstract

Gel-based proteomics is a powerful approach to study the physiology of Staphylococcus aureus under various growth restricting conditions. We analyzed 679 protein spots from a reference 2-dimensional gel of cytosolic proteins of S. aureus COL by mass spectrometry resulting in 521 different proteins. 4,692 time dependent protein synthesis profiles were generated by exposing S. aureus to nine infection-related stress and starvation stimuli (H2O2, diamide, paraquat, NO, fermentation, nitrate respiration, heat shock, puromycin, mupirocin). These expression profiles are stored in an online resource called Aureolib (http://www.aureolib.de). Moreover, information on target genes of 75 regulators and regulatory elements were included in the database. Cross-comparisons of this extensive data collection of protein synthesis profiles using the tools implemented in Aureolib lead to the identification of stress and starvation specific marker proteins. Altogether, 226 protein synthesis profiles showed induction ratios of 2.5-fold or higher under at least one of the tested conditions with 157 protein synthesis profiles specifically induced in response to a single stimulus. The respective proteins might serve as marker proteins for the corresponding stimulus. By contrast, proteins whose synthesis was increased or repressed in response to more than four stimuli are rather exceptional. The only protein that was induced by six stimuli is the universal stress protein SACOL1759. Most strikingly, cluster analyses of synthesis profiles of proteins differentially synthesized under at least one condition revealed only in rare cases a grouping that correlated with known regulon structures. The most prominent examples are the GapR, Rex, and CtsR regulon. In contrast, protein synthesis profiles of proteins belonging to the CodY and σ(B) regulon are widely distributed. In summary, Aureolib is by far the most comprehensive protein expression database for S. aureus and provides an essential tool to decipher more complex adaptation processes in S. aureus during host pathogen interaction.

Figure 1. The relational database model.

Aureolib uses a MySQL database to store expression data and relevant information on protein annotation, statistical analyses, and experimental setups.

Figure 2. Functions and graphical user interface of Aureolib.

(A) Aureolib consists of three different levels for data management, data processing and visualization of data. (B) Combined with an intuitive user interface accessible by generic web browsers Aureolib provides helpful tools for expression data analyses and data visualization.

Figure 3. Intra-experimental comparison of protein synthesis patterns in response to different stimuli.

Quantification data for all detected protein spots were normalized (total) and standardized (z-score). Significant changes of spot intensities were determined for each experiment (ANOVA, α = 0.1, distribution based on 1000 permutations) and the corresponding expression values were used for hierarchical sample clustering (HCL, Euclidean distance, complete linkage). Accordingly, the stimuli can be divided into two classes: (A) Stimuli causing continuous changes of the protein synthesis pattern and (B) stimuli transiently affecting the protein synthesis pattern.

Figure 4. General aspects of S. aureus stress response.

Inter-experimental analyses of stress induced protein synthesis patterns revealed general and specific effects on global protein synthesis. (A) The majority of the synthesis profiles showed a significant (ANOVA, α = 0.1, p-values based on 1000 permutations, absolute threshold of 2.5) induction or repression rate in response to one or two stimuli. (B) To identify more general effects a pairwise comparison of all experiments was performed using protein synthesis profiles significantly changed at least one time point following exposure to the different stimuli (t-test, α = 0.1; p-values based on all possible permutations, adjusted Bonferroni correction). The number of induced (orange) and repressed (blue) protein synthesis profiles shared by the respective stimuli is shown. The total number of synthesis profiles significantly induced or repressed by the respective stimulus are given on the right side. (C) Overlap of marker proteins induced in response to diamide, puromycin, and heat as well as to nitric oxide, fermentation, and nitrate respiration are shown.

Figure 5. Inter-experimental expression profiles of selected proteins.

Synthesis ratios (stress vs. control) are shown as log₂ values. Fold changes above 2.5 and below 0.4 are colored in orange and blue, respectively. Significant changes are marked by an asterisk (*). Synthesis profiles of proteins belonging to the Rex (A) and Ctsr/HrcA (B) regulon as well as proteins involved in leucine biosynthesis (C) are shown in more detail.

Figure 6. Differential expression of protein isoforms in response to diamide (A) and nitrosative stress (B).

Sectors of 2D gels covering the regions where CysK, GuaB, Upp, SACOL0618 and SACOL1895 are located. Proteins represented by multiple spots indicate post translational modifications. In the present approach the synthesis of protein isoforms differing in pI and/or molecular weight was separately analyzed.

Figure 7. Expression clusters of σ^B-dependent proteins.

(A) Hierarchical clustering (Euclidian distance/complete linkage) of synthesis profiles of proteins for which transcription of the respective genes has been shown to be influenced by σ^B , , . (B) Expression profiles of solely σ^B-dependent proteins. Abbreviations for stress conditions are: O (H₂O₂), D (diamide), P (paraquat), N (nitrogen monoxide), F (fermentation), A (nitrate respiration), H (heat stress), U (puromycin), and M (mupirocin). ¹⁾ Support for a σ^B-dependent transcriptional start point: experimental (black square), predicted (gray square), not identified yet (white square) , , . ²⁾ Impact of σ^B: solely σ^B-dependent/major effect on expression (black square), co-regulated by additional factors (gray squares), or not determined (white square) , . Csb (controlled by sigma B) nomenclature based on Gertz et al. .

Figure 8. Response of S. aureus to H₂O₂.

(A) S. aureus COL was cultivated in synthetic medium at 37°C and treated with 10 mM H₂O₂ at OD₅₀₀ 0.5 (0 min). (B) Protein synthesis profiles were normalized using total normalization and standardized (z-score). Significantly changed synthesis profiles were determined by ANOVA (α = 0.1, distribution based on all permutations, absolute threshold of 2.5). Based on their expression kinetics in response to H₂O₂, proteins can be allocated to three groups: proteins which are induced during the acute phase immediately after imposition of stress (red lines), proteins induced during resumption of growth (blue lines) and proteins highly expressed in the tolerance phase (green lines).