Label-Free Quantitative Proteomics Analysis of Antibiotic Response

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Label-free Quantitative Proteomics Analysis of Antibiotic Response in Staphylococcus aureus to Oxacillin Xiaofen Liu, Yingwei Hu, Pei-Jing Pai, Daijie Chen, and Henry Lam J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 24 Oct 2013 Downloaded from http://pubs.acs.org on November 2, 2013

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Journal of Proteome Research

Label-free Quantitative Proteomics Analysis of Antibiotic Response in Staphylococcus aureus to Oxacillin Xiaofen Liu1, Yingwei Hu1, Pei-Jing Pai1, Daijie Chen2,3,*, Henry Lam1,4,* 1

Department of Chemical and Biomolecular Engineering, the Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

2

China State Institute of Pharmaceutical Industry, Shanghai Institute of Pharmaceutical Industry, Shanghai, China

3

School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China

4

Division of Biomedical Engineering, the Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

* Corresponding authors: Prof. Henry Lam Department of Chemical and Biomolecular Engineering The Hong Kong University of Science and Technology Clear Water Bay, Kowloon, Hong Kong Email: [email protected] Phone: +852-2358-7133 Fax: +852-2358-0054

Prof. Daijie Chen China State Institute of Pharmaceutical Industry, Shanghai Institute of Pharmaceutical Industry 1320 West Beijing Road Shanghai, China Email: [email protected] Phone: +86-137-0175-6930 Fax: +021-6247-0851

Key words: beta-lactam antibiotics, oxacillin, Staphylococcus aureus; label-free quantitative proteomics 1

ACS Paragon Plus Environment


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Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of fatal bacterial infections in hospitals and has become a global health threat. Although the resistance mechanisms of β-lactam antibiotics have been studied for decades, there are few attempts at systems-wide investigations into how the bacteria respond to antibiotic stress. In this paper, spectral counting-based label-free quantitative proteomics has been applied to study global responses in MRSA and methicillin-susceptible S. aureus (MSSA) treated with sub-inhibitory doses of oxacillin, a model beta-lactam antibiotic. We developed a simple and easily repeated sample preparation procedure that is effective for extracting surface-associated proteins. On average, 1025 and 1013 proteins were identified at a false discovery rate threshold of 0.01, for the untreated group of MRSA and MSSA. Upon treatment with oxacillin, 81 proteins (65 upregulated, 16 down-regulated) were shown differentially expressed in MRSA (p 4.3) and the fold changes are higher/ lower than ±1.5 fold (log2(fold change) > ±0.585), i.e., the rectangular regions labeled in Figure 4. The filter parameters were used based on the PepC results from the technical replicates of the control samples. Under these stringent filter parameters, only 13 (1.0 %) out of 1025 proteins were found to have significant changes between technical replicates of MRSA control samples, and only 10 (0.9 %) out of 1013 have significantly changes between technical replicates of MSSA control samples. Since reproducibility is an important issue for label-free quantitative proteomics, the reproducibility of our approach was evaluated using technical replicates obtained from the same biological samples and same preparation procedures but prepared separately and injected in a randomized order in LC-MS/MS. Spectral counts for the same protein in corresponding technical replicates were compared using PepC. For all groups of samples, a good agreement was found with R2 > 0.91, slope around 1 and intercept around 0 between two replicates on each biological replicates (Supplementary Figure S3). This shows that our experimental workflow exhibits good reproducibility. 16


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In our results, 81 proteins (16 proteins were down-regulated and 65 proteins were up-regulated) were differentially expressed in oxacillin-treated MRSA compared to untreated MRSA, and 225 proteins (63 proteins were down-regulated and 162 proteins were up-regulated) were differentially

expressed

in

oxacillin-treated

MSSA

compared

to

untreated

MSSA

(Supplementary Table S2). There are more differentially expressed proteins uniquely in MSSA than MRSA. It might be because MRSA has evolved specific resistance mechanism to oxacillin, and therefore is well equipped to neutralize the antibiotics stress. In contrast, the MSSA strain undergoes more widespread changes in its proteome in response to oxacillin. These differentially expressed proteins were subjected to further bioinformatic studies: protein localization by SELP and PSORTb v3.0, pathway enrichment by DAVID and protein-protein interaction network by STRING. Differentially expressed proteins in oxacillin-treated MRSA and MSSA were compared for the localization distribution predicted by SELP and PSORTb v3.0. Figure 5 shows that both strains have similar localization distribution, except for the cytoplasmic membrane/cell wall compartment, which has a higher percentage of differentially expressed proteins in oxacillintreated MRSA. More specifically, penicillin binding protein 2a (PBP2a), β-lactamase, and βlactamase regulatory protein are uniquely found in this compartment only in oxacillin-treated MRSA, but not in MSSA. PBP2a was the most significantly changed protein (up-regulated about 168.5 folds) while PBP1, PBP2, PBP3 and PBP4 did not show significant changes. Betalactamase and β-lactamase regulatory protein were not detected in control sample, but exhibited a high abundance in oxacillin-treated MRSA. These results are consistent with the known βlactam antibiotics resistance mechanisms in MRSA.9

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Even though similar localization distributions for the differentially expressed proteins in oxacillin treated MRSA and MSSA were found, two totally different interaction networks were obtained (Figure 6, predicted by STRING). There appears to be substantial differences in the cellular responses to oxacillin stress between MRSA and MSSA. For the interaction network of the oxacillin-treated MRSA, the most prominent sub-networks of perturbed proteins (Figure 6A, labeled by red rectangle) are involved in the β-lactam resistance pathway and the peptidoglycan biosynthesis pathway. For oxacillin-treated MSSA, a much more complicated network of differential expressed proteins emerged, among them proteins involved in RNA degradation, the ribosome, and oxidative phosphorylation (Figure 6B). The enriched pathways predicted by DAVID given the set of differentially expressed proteins in oxacillin-treated MRSA and MSSA were summarized in Figure 7. These results also indicate vastly different responses between MRSA and MSSA to oxacillin stress. In this study, sub-inhibitory doses of oxacillin were used to challenge both MRSA and MSSA. Under these conditions, oxacillin-treated MRSA is expected to activate the known resistance mechanism, in addition to potentially other pathways to tolerate the antibiotic. On the other hand, oxacillin-treated MSSA does not possess the resistance genes, but would still turn on tolerance mechanisms that allow the cells to continue growing.60-62 Proteins which are differentially expressed in both oxacillin-treated MRSA and MSSA are potentially important as a generic tolerance response to the antibiotic. We found that 31 proteins were differentially expressed in both oxacillin-treated MRSA and MSSA. Two up-regulated pathways were identified by DAVID for these 31 proteins: the peptidoglycan biosynthesis pathway and the pantothenate and Coenzyme A (CoA) biosynthesis pathway.

18


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The peptidoglycan biosynthesis pathway is well known for its importance in antibiotic targeting and resistance.63 In this pathway, two enzymes were shown up-regulated in both oxacillin-treated MRSA and MSSA: Aminoacyltransferase FemB and UDP-N-acetylmuramoyltripeptide-D-alanyl-D-alanine ligase (EC 6.3.2.10), encoded by the genes femB and murF, respectively. In the same pathway, two enzymes were observed up-regulated only in oxacillin treated-MRSA, including UDP-N-acetylglucosamine 1-carboxyvinyltransferase (EC 2.5.1.7) and UDP-N-acetylmuramoylalanine-D-glutamate ligase, encoded by murA and murD, respectively. In addition, three enzymes in the same pathway were found up-regulated only in oxacillin-treated MSSA: methicillin resistance factor FemA (EC 2.3.2.-), UDP-N-acetylmuramate--L-alanine ligase (EC 6.3.2.8) and glutamine-fructose-6-phosphate aminotransferase [isomerizing] (EC 2.6.1.16), encoded by femA, murC and glmS, respectively. These enzymes are known to be presented in both MRSA and MSSA strains, and studies has shown that disruption of these genes reduces the level of resistance in MRSA.64, 65 This seems plausible that the cells up-regulated these proteins to increase antibiotic tolerance in both MRSA and MSSA. In the pantothenate and CoA biosynthesis pathway, four enzymes were involved (Supplementary Figure S4). In our results, two of them were shown up-regulated in both oxacillin-treated MRSA and MSSA. They were bifunctional phospho-pantothenoylcysteine decarboxylase/ phosphopantothenate-cysteine ligase (EC 4.1.1.36), de-phospho-CoA kinase (EC 2.7.1.24) encoded by coaBC, and coaE. CoA is an important and essential metabolite for all organisms; it participates in the tricarboxylic acid cycle (TCA cycle), fatty acid metabolism, and a host of other biological processes performing diverse biochemical functions.66, 67 In E. coli, reduction of CoA levels leads to growth stasis.68 Knockout of the CoA biosynthetic genes leads to massive, and most often lethal, disruption of cell development and regulation.69, 70 In S. aureus, 19



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CoA and its metabolites are believed to help maintain the reducing environment in the cell.71,72 Recent studies have suggested that antibiotics cause oxidative stress by inducing the production of hydroxyl radicals, which ultimately leads to cell death.11 It follows that an increased production of CoA may be a response to fight the oxidative stress caused by oxacillin in both MRSA and MSSA. Therefore, our results are consistent with the conjecture that oxacillin imposes oxidative stress to S. aureus, and the up-regulation of the pantothenate and CoA biosynthesis pathway is a key mechanism used by the cell to tolerate the antibiotic. Other than these two pathways, several efflux pump proteins were detected up-regulated in both oxacillin-treated MRSA and MSSA. For example, multidrug resistance protein A (encoded by emrA) was found up-regulated in both oxacillin-treated MRSA and MSSA. Several studies have suggested that the increase of the efflux pump protein expression is an early stage of developing antibiotic resistance. It is surmised that efflux proteins actively transport antibiotics out to the intracellular space to help cell survive the antibiotic treatment.73,74 The up-regulated pathways unique to oxacillin-treated MRSA are the β-lactam resistance pathway, which is well expected from known resistance mechanisms of MRSA. Oxacillintreated MSSA exhibits a large number of uniquely up-regulated pathways, such as TCA cycle, two component system, oxidation phosphorylation and purine metabolism (Figure 7). These results are consistent with the literature75 that suggests that changes in energy metabolism, TCA cycle, and purine/pyrimidine metabolism are common bacterial responses against antibiotics. Down-regulated pathway unique in oxacillin-treated MRSA is the alanine, aspartate and glutamate metabolism pathway. Several pathways are uniquely down-regulated in oxacillintreated MSSA, such as the ABC transporters pathway, the porphyrin and cholorophyll metabolism pathway, the pentose and glucuronate interconversions pathway and the aminoacyl20


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tRNA biosynthesis pathway. We were not able to find literature that discusses these downregulated pathways related to the antibiotics response, but they may be useful targets for followup studies. Two more sets of experiments were carried out using the same methodology to verify the effectiveness of this approach in revealing resistance mechanisms in MRSA. In the first experiment, the same MRSA strain was dosed with a sub-inhibitory concentration of cefoxitin, another beta-lactam antibiotic against which MRSA also has resistance. The results showed that PBP2a, beta-lactamase and beta-lactamase regulatory protein are the most up-regulated proteins relative to the untreated control (Supplementary Table S3). In the second experiment, erythromycin, a macrolide antibiotic against which MRSA is also resistant, was applied instead. It is well-established that macrolide antibiotics target the ribosome and interfere with protein synthesis, and a mutation in the binding site renders the antibiotic ineffective.76,77 In erythromycin-treated MRSA, the most up-regulated protein is rRNA (Adenine-N(6)-)methyltransferase (encoded by ermA) (Supplementary Table S4) which is consistent with the known resistance mechanism of MRSA against macrolide antibiotics. These results have again demonstrated that our experimental strategy is capable of revealing antibiotic resistance mechanisms in bacteria.

Conclusion

In our study, a sub-inhibitory dose of oxacillin was applied to two strains of S. aureus, ATCC 43300 (MRSA) and ATCC 25923 (MSSA), to reveal the cellular responses to β-lactam antibiotics, oxacillin, through spectral counting-based label-free quantitative proteomics analysis. 21



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Firstly, we developed an effective and easily repeatable sample preparation method for shotgun proteomics analysis. On average, 1025 and 1013 proteins were identified for the untreated group of MRSA and MSSA, with more than 200 cell surface associated proteins among them, surpassing previous methods. Secondly, using spectral counting, 81 proteins and 225 proteins were found differentially expressed in oxacillin treated MRSA and MSSA, respectively. The known resistance mechanisms for MRSA involving PBP2a and β-lactamase were detected. The peptidoglycan biosynthesis pathway and the pantothenate and CoA biosynthesis pathway were identified in both oxacillin-treated MRSA and MSSA as potential tolerance mechanisms. Overall, more pathways are disrupted in MSSA than in MRSA. It might be because MRSA has its own mechanisms to overcome antibiotics stress, but MSSA is forced to respond instead by regulating its energy metabolism, the efflux pump system and the oxidase and reductase system. These new data offer a more complete view of the proteome changes in bacteria in response to the antibiotic. This report is the first in using label-free quantitative proteomics to study antibiotic responses in S. aureus.

Supporting information This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgement This research is supported by the University Grant Council of the Hong Kong Special Administrative Region Government, China (Grant No. HKUST DAG08/09.EG02), and the National Natural Science Foundation of China (Grant No. 81273413). We also thank the 22


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Tables and Figures

Figure 1. Schematic diagrams of (A) the sample preparation and (B) the data analysis procedures.

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Figure 2. Venn diagrams for proteome comparison on (A) control samples of ATCC 43300 (MRSA) and ATCC 25923 (MSSA), and (B) oxacillin treated MRSA and MSSA

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Figure 3. Venn diagram of cell surface associated proteins identified in Hempel et al, 2011 on MRSA compared with MRSA (ATCC 43300) and MSSA (ATCC25923) in our study

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Figure 4. PepC result for oxacillin-treated MRSA compared with control; and oxacillin-treated MSSA compared with control. Only proteins with average spectral counts of at least 5 are shown as data points. Differentially expressed proteins are deemed significant for p < 0.05 (absolute (log2 p) > 4.3) and fold change higher/lower than ±1.5, corresponding to the rectangular regions.

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Figure 5. . Localization distribution of differentially expressed proteins, with localization predicted by SLEP and PSORTb v3.0.

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(A)

34


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(B)

Figure 6: Differentially expressed proteins of oxacillin-treated MRSA compared to controls (Figure 6A), and oxacillin-treated MSSA compared to controls (Figure 6B) as depicted in their interaction networks by STRING50 and visualized by Medusa.51 Each dot represents a protein found to be differentially expressed in the presence of oxacillin by quantitative proteomics, and the lines represent putative protein interactions recorded or predicted by STRING. Dots in different color represent different ranges of fold changes. Proteins in the red rectangular (Figure 6A) are involved in beta-lactam resistance and peptidoglycan biosynthesis pathways. 35



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Figure 7. Pathway enrichment study by DAVID of the differentially expressed proteins in oxacillin treated MRSA and MSSA, compared to their untreated controls.

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Table 1: Comparison of six independent researches with our method in Surface associated proteins identification

Predicted sub-cellular location Cytoplasmic membrane

Glowalla et al.49

Hempel et al. (2010)50

Solis et al.51

Venture et al.53

Dreisbach et al.54

Hempel et al. (2011)52 *

5

38

18

11

11

9

11

15

14

5

5

Cell wall Extracellular

8

Lipoprotein

ATCC

ATCC

43300

25923

130

165

152

9

30

16

18

11

11

62

33

42

3

2

3

22

24

26

Cytoplasmic

22

160

47

55

39

771

672

669

Unknown

4

43

11

19

23

112

115

106

Total

39

269

95

113

96

1127

1025

1013

Note: * For Hempel et al.52, the reported protein identifiers are first transformed to UniProt accession number by UniProt, and then the protein names are matched according to the BLASTclust results of our custom-made sequence database. In total 1127 out of 1129 proteins listed in Hempel et al. were matched for subsequent localization prediction and comparison.

37



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Graphical Abstract

38


Label-Free Quantitative Proteomics Analysis of Antibiotic Response

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