A comprehensive mass spectrometric survey of Streptococcus

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A comprehensive mass spectrometric survey of Streptococcus pyogenes subcellular proteomes Laura Wilk, Lotta Happonen, Johan Malmström, and Heiko Herwald J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00701 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 22, 2017

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Journal of Proteome Research

A comprehensive mass spectrometric survey of Streptococcus pyogenes subcellular proteomes Laura Wilk+*, Lotta Happonen+, Johan Malmström+ and Heiko Herwald+

+

Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden. *

to whom correspondence should be addressed: BMC, Floor B14, Lund University, Tornavägen 10, 221 84 Lund, Sweden. Phone: +46 46 222 68 07 Email: [email protected], [email protected]

Keywords Streptococcus pyogenes, bacterial proteome, cellular fractionation, secretome, cell wall, membrane, vaccine, drug target, virulence factor, mass spectrometry

1 ACS Paragon Plus Environment

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Abstract

Streptococcus pyogenes is a major global health burden causing a wide variety of diseases. As a vaccine against this bacterium is still lacking, vaccine candidates or antimicrobial therapies are urgently needed. Here, we use an invasive and clinical relevant streptococcal M1 serotype in order to characterise the bacterial proteome in-depth. An elaborate fractionation technique is employed to separate the different cell fractions followed by shotgun mass spectrometry analysis, allowing us to confirm the expression of nearly two thirds (1022) of the 1690 open reading frames predicted for the streptococcal M1 reference proteome. In contrast to other studies, we present the entire isolated membrane proteome, which opens up a whole new source for drug targets. We show both the unique and most prevalent proteins for each cellular fraction, and analyse the presence of predicted cell wall-anchored proteins and lipoproteins. With our approach, we also identify a variety of novel proteins whose presence has not been reported in previous proteome studies. Proteins of interest, potential virulence factors and drug or vaccine targets are discussed for each cellular fraction. Overall, the results of this work represent the so far widest proteomic approach to characterise the protein composition and localisation in S. pyogenes.


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Introduction Streptococcus pyogenes, a Gram-positive bacterium, is an important human pathogen accounting for more than 700 million infections annually1. Also known as group A streptococcus (GAS), the microorganism mainly evokes harmless throat and skin infections that can be treated with penicillin or macrolides. However, under invasive and more severe conditions, such as streptococcal toxic shock syndrome and necrotising fasciitis, antibiotic treatment can fail2 and infections are then associated with high morbidity and mortality rates1. Moreover, due to emerging antibiotic resistances and a steady drop in discovery of new antibiotics, the development of new antimicrobial therapies is urgently required. The best option to cope with GAS infections would be the development of a vaccine, but unfortunately all attempts have failed so far. Following the early days of vaccine research using the whole microorganisms themselves, the genomic era opened up the possibility to select vaccine candidates based solely on the genetic information. However, it soon became apparent that identification of the gene sequences alone does not necessarily provide information about the actual expression of the targeted proteins. Therefore, proteomic profiles of pathogens provide a much more detailed picture of protein amounts. With the introduction of proteomic tools, streptococcal vaccine research has experienced a revival. Previous proteomic studies on GAS strains have mainly focused on surface exposed proteins, with analyses being performed by 2D-PAGE and mass spectrometry (for a recent overview of conducted studies see3). Though mass spectrometric technologies are constantly advancing and can identify a high number of proteins, an extensive characterisation of all subcellular proteomes from a streptococcal serotype has not been conducted yet. While proteomic analysis of whole cells yields an average of the protein composition, subcellular proteome analysis is able to not only confirm the presence of a specific vaccine or



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drug target within a cellular fraction but also to provide information about its abundance. Moreover, the development of new strategies to cope with GAS infections requires not only knowledge about surface-exposed and secreted proteins, which are commonly targeted in vaccine research, but also about drug targets in other cell fractions. Of particular interest is the bacterial membrane, as it determines the boundary between the cytoplasm and the environment. Within most sequenced genomes, 20-30% of all genes are predicted to encode membrane proteins4. However, not much is known about the membrane proteome of GAS. Since membrane proteins are involved in important cellular processes, such as signaling and transport, this protein class provides promising targets for attacking the pathogen. Improved subcellular annotation extends beyond vaccine and drug development. This type of information can also be used to explain fundamental microbiology phenomena, for instance cell adherence and host colonisation. As outlined in Figure 1, GAS harbors three different cellular compartments, i.e. the cytoplasm, enclosed by the plasma membrane, and a rather thick cell wall surrounding the plasma membrane5. While capsular polysaccharides can form an additional capsule around the cell of Gram-positive bacteria, protection and stability provided by an outer membrane as seen in Gram-negative bacteria is missing. Therefore, the cell wall of Gram-positive bacteria is in general much thicker, but as such open to the environment6. However, there are extracellular proteins present in Gram-positive bacteria, which are either secreted or remain associated with the cell surface by mechanisms adhering them either to the cell wall or the plasma membrane6, 7

.


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Figure 1: Schematic depiction of cell structure and subcellular proteomes of S. pyogenes. Secreted proteins are released into the environment. A hyaluronic acid capsule surrounds the bacterium. The cell wall consists of peptidoglycan decorated with various proteins (M protein) and glycopolymers. Two types of anionic cell wall glycopolymers can be distinguished in GAS, one attached to peptidoglycan (wall teichoic acid) and one to membrane lipids (lipoteichoic acid, LTA)5. Additional proteins (X) could interact with the cell wall components. The presence of a periplasmic space is proposed, with residing lipoproteins and other soluble proteins (Y) that could be retained by the membrane/membrane proteins. Sortase A is located at the membrane and mediates peptidoglycan-anchoring of respective proteins (M protein). Cytoplasmic proteins might not only diffuse freely in the cytoplasm but also interact with the membrane/membrane proteins.



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Extracellular proteins are synthesised in the cytoplasm and have to be translocated across the plasma membrane. Proteins destined for the secretory pathway harbor an N-terminal signal peptide and their translocation is facilitated by a membrane complex known as SecYEG, along with the ATPase SecA6, 7. These precursor proteins are cleaved by signal peptidases and the mature proteins are released into the environment or retained at the cell surface. Both covalent and non-covalent attachment of proteins to the cell wall has been observed in Gram-positive bacteria6, 7. A well-characterised covalent anchoring mechanism is based on the presence of a C-terminal region, containing the so-called LPXTG motif8. This motif is recognised by a sortase, SrtA, (Figure 1) which couples the protein to the peptidoglycan layer8. Prominent representatives of such surface proteins in S. pyogenes include M- and M-like proteins (Figure 1) and the C5a peptidase5, 9. In several Gram-positive organisms proteins can also noncovalently interact with the peptidoglycan or the teichoic acids (Figure 1)6, 7. In more recent studies, several proteins have also been detected on the cell surface of S. pyogenes that lack both a signal and an anchor sequence7, 9. These proteins, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were thought to be confined to the cytoplasm as members of the glycolytic pathway and their translocation mechanism still remains unknown. Most likely, they are retained at the surface by charge and/or hydrophobic interactions9. It becomes increasingly clear now that these proteins exhibit more than one relevant function7, 9. Beneath the cell wall, a periplasmic space is located in Gram-negative bacteria. Although a similar space has already been suggested earlier for Gram-positive bacteria5, only recent studies employing cryo-electron microscopy support this notion10. The presence of such a periplasmic space appears plausible, at the least to harbor the lipoproteins, which are tethered to the outer leaflet of the cytoplasmic membrane (Figure 1)11. Lipoproteins have an N-terminal signal sequence that targets the protein for translocation via the Sec machinery. The N-terminal sequence also contains the lipobox motif, which anchors it to the plasma membrane11. Due to


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the absence of an outer membrane, lipoproteins are proposed to serve as anchored equivalents to their periplasmic counterparts in Gram-negative bacteria, as in the case of substrate-binding proteins of ABC transporters7, 11. It is possible that additional soluble proteins are present in the putative periplasmic space that could be retained e.g. by interactions with membrane proteins (Figure 1). Such protein interactions might also occur at the cytoplasmic side of the plasma membrane (Figure 1). Integral membrane proteins are synthesised in the cytoplasm and can be targeted to the SecYEG complex for membrane insertion12. In addition, the insertase YidC mediates membrane protein insertion independently from SecYEG12. Different techniques to analyse the protein composition of GAS have been employed previously. A common approach for secretome analysis, also adopted in this work, employs cell growth of GAS in protein-reduced medium to reduce the presence of non-streptococcal proteins with subsequent analysis of the harvested medium3, 13 (Figure 2).



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Figure 2: Workflow for cellular fractionation of S. pyogenes. Bacterial growth conditions and subsequent preparation steps (light blue) of cellular fractions (dark blue; analysed by shotgun mass spectrometry in this study) are shown. Secreted extracellular proteins were obtained from bacteria grown in protein-reduced medium, while the remaining cellular fractions were prepared from bacteria grown in protein-rich medium. P=pellet, SN=supernatant; obtained after centrifugation.

Techniques for surface proteome analysis include cell wall digest with mutanolysin or lysozyme3 (Figure 2) and ‘surface shaving’ by means of proteases14. In the latter approach peptides originating not only from cell wall-associated proteins are obtained, but also from other surface-exposed proteins such as lipoproteins or protruding regions of membrane proteins14. Though studies on whole GAS cells have been performed by others15,

16

,


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fractionation following cell breakage only yielded mixtures of the cellular compartments, i.e. combined cytoplasmic and cell wall15 or cell wall and membrane proteins16. In contrast to these reports, we present here a complete GAS proteome analysis by means of separated cell fractions: secretome, cell wall, plasma membrane and cytoplasm (Figure 2). Moreover, we describe peripheral proteins associated with the cell membrane. Therefore, our approach allows for a more detailed localisation and characterisation of putative drug and vaccine targets in GAS.

Experimental Section Bacterial strain and culture conditions - S. pyogenes strain AP1 (40/58; covS truncated) of the M1 serotype was obtained from the WHO Collaborating Centre for Reference and Research on Streptococci, Prague, Czech Republic. Bacteria were grown at 37 °C and 5 % CO2 for 16-18 h from a single hemolytic colony to stationary phase (OD620nm~0.8) in Todd-Hewitt (TH) broth (Becton Dickinson) supplemented with 0.3 % (w/v) yeast extract (Becton Dickinson). 100 ml of this pre-culture were added to 2 l TH with 0.3 % (w/v) yeast extract. Cells from this culture were used for later subcellular fractionation. In addition, 5 ml pre-culture were added to 100 ml protein-reduced TH broth13 to enable subsequent analysis of secreted proteins. Both cultures were grown to midlogarithmic phase (OD620nm~0.4) at 37 °C and 5 % CO2.

Preparation of secreted extracellular proteins - Cells were harvested at 12,000 x g for 15 min at 4 °C. The culture supernatant was filtered through a 0.22 µm pore size filter unit (Millipore). A 15 ml aliquot was further concentrated by ultrafiltration at 4,000 x g for 15 min at 4 °C with an Amicon Ultracel 10 kDa molecular weight cutoff centrifugal filtration unit (Millipore) and



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washed three times with 5 ml ice-cold PBS. Concentrated supernatant proteins were collected at a final volume of 1 ml and stored at -80 °C until sample preparation for mass spectrometry.

Subcellular fractionation - Cells were harvested at 12,000 x g for 15 min at 4 °C and washed with ice-cold PBS. The pellet was stored at -20 °C until further use. Isolation of cell wallassociated proteins and generation of protoplasts were essentially performed as described previously17 with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) added to buffers. Isolated cell wall-associated proteins were stored at -80 °C until sample preparation for mass spectrometry. All further steps were performed on ice and with ice-cold buffers. Remaining protoplasts were washed twice with 50 mM Tris-HCl, pH 8.0, 1 mM EDTA (TEbuffer), 20 % (w/v) sucrose, 1 mM AEBSF (AppliChem) by centrifugation at 14,000 x g for 15 min at 4 °C. The pellet was resuspended in 20 ml TE-buffer including Protease and Phosphatase Inhibitor Mini Tablets (Thermo Scientific). Protoplasts were disrupted by sonication (Branson Sonifier 150) with 6 x 10 s cycles at output 6, followed by 6 x 10 s cycles at output 10, with 1.5 min of cooling on ice in between cycles. The suspension was centrifuged at 10,000 x g for 15 min at 4 °C in order to remove cellular debris and unbroken protoplasts. The supernatant was then centrifuged at 200,000 x g for 1 h at 4 °C. The supernatant containing the cytosolic proteins was removed from the membrane pellet. The pellet was rinsed three times with 2 ml TE buffer, 1 mM AEBSF, and taken up in 0.5 ml of the same buffer. Both the cytosolic proteins and the crude membrane pellet were stored at -80 °C until sample preparation for mass spectrometry.

Isolation of membrane-associated peripheral proteins – 700 µl of TE-buffer, 1 M NaCl were added to 300 µl crude membrane suspension. The suspension was homogenised with a handheld glass Dounce homogeniser and incubated on ice for 30 min. Membrane-associated 10 ACS Paragon Plus Environment

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peripheral proteins were retrieved in the supernatant after centrifugation at 200,000 x g for 1 h at 4°C. Prior to further analyses, the supernatant was washed with TE-buffer, 1 mM AEBSF and concentrated to 200 µl by ultrafiltration at 14,000 x g at 4 °C with an Amicon Ultracel 3 kDa molecular weight cutoff centrifugal filtration unit (Millipore). The remaining purified membrane pellet was rinsed three times with TE-buffer, 1 mM AEBSF and taken up in 300 µl of the same buffer. Samples were stored at -80 °C until sample preparation for mass spectrometry.

Sample preparation for mass spectrometry - Membrane fractions were solubilised with 0.05 % ProteaseMAX (Promega) for 20 min. ProteaseMAX treated fractions were subsequently centrifuged (16000 x g for 10 min at 4 °C) and supernatants containing the solubilised membrane proteins were harvested. The ProteaseMAX reagent was inactivated at 95 °C for 5 min. The protein concentration in all of the cellular fractions was measured using the BCA kit (Pierce) and additionally estimated by SDS-PAGE on a Criterion TGX 4-20% gel (Bio-Rad). Based on this, an estimated 20 µg of each fraction was used for trypsin digestion. Samples were mixed with 8 M urea and 100 mM ammonium bicarbonate, and the cysteine bonds were reduced with 5 mM tris(2-carboxyethyl)phosphine (TCEP) (37 °C for 50 min) and alkylated with 10 mM iodoacetamide (22 °C for 90 min). Samples were diluted with 100 mM ammonium bicarbonate to a final urea concentration of 1.5 M, and sequencing grade trypsin (Promega) was added for protein digestion (37 °C for 18 h). Samples were acidified (to a final pH 3.0) with 10 % formic acid, and the peptides subsequently purified with C18 reverse phase spin columns according to the manufacturer's instructions (Macrospin columns, Harvard Apparatus). Peptides were dried in a speedvac and reconstituted in 2 % acetonitrile, 0.2 % formic acid prior to mass spectrometric analyses.



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Liquid chromatography mass spectrometry and protein identification - Liquid chromatography mass spectrometry was essentially performed as described earlier18. Peptides were analysed on a Q Exactive Plus mass spectrometer (Thermo Scientific) connected to an EASY-nLC 1,000 ultra-high-performance liquid chromatography system (Thermo Scientific). Peptides were separated on an EASY-Spray column (Thermo Scientific; ID 75 μm × 25 cm, column temperature at 45 °C). Column equilibration and sample load were performed using a constant pressure of 600 bar. A linear gradient from 5 to 35 % acetonitrile in aqueous 0.1 % formic acid was run for 90 min at a flow rate of 300 nl min−1. One full MS scan (resolution 70,000 at 200 m/z; mass range 400–1,600 m/z) was followed by MS/MS scans (resolution 17,500 at 200 m/z) of the 15 most abundant ion signals. Precursor ions were isolated with 2 m/z isolation width and fragmented using higher-energy collisional-induced dissociation at a normalised collision energy of 30. Charge state screening was enabled, and precursors with an unknown charge state and singly charged ions were rejected. The automatic gain control was set to 1e6 for both MS and MS/MS with ion accumulation times of 100 ms and 60 ms, respectively. The intensity threshold for precursor ion selection was set to 1.7 e4. The mass spectrometry data have been deposited to the ProteomeXchange19 consortium via the MassIVE partner repository (University of California San Diego, CA, USA, http://massive.ucsd.edu) with

the

dataset

identifier

PXD006345

(http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD006345).

Mass spectrometry data analysis – MS raw data were converted to gzipped and Numpressed20 mzML using the tool msconvert from the ProteoWizard, v3.0.5930 suite21. Acquired spectra were analysed using the search engine X! Tandem (2013.06.15.1-LabKey, Insilicos, ISB)22 against an in-house compiled database containing the Homo sapiens and Streptococcus pyogenes serotype M1 reference proteomes (UniProt proteome IDs


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UP000005640 and UP000000750, respectively23), with the S. pyogenes Protein H added (UniProt ID P50470), yielding a total of 72241 protein entries and an equal amount of reverse decoy sequences. Fully tryptic digestion was used allowing 1 missed cleavage. Carbamidomethylation (C) was set to static and oxidation (M) to variable modifications, respectively. Mass tolerance for precursor ions was set to 20 ppm, and for fragment ions to 50 ppm. Identified peptides were processed and analysed through the Trans-Proteomic Pipeline (TPP v4.7 POLAR VORTEX rev 0, Build 201403121010) using PeptideProphet24 and ProteinProphet25 scoring. The protein false discovery rate (FDR) was set to 1 % in ProteinProphet at a probability of 0.85. The peptide FDR was equally 1 %. Abacus (version 2.5) was used for label-free spectral counting26. Identified peptide sequences, precursor charge and m/z for each assignment, all modifications observed, peptide identification score, peptides assigned for each protein, and protein accession numbers are presented in Table S-1. The percent coverage of each protein assigned is presented in Table S-2.

Experimental design and statistical rationale – In total, three biological replicates were performed for culture growth originating from different clones picked from agar plates. Mass spectrometry was performed on each cellular fraction in two technical duplicates. GraphPad Prism 7.0 software (GraphPad software) was used for initial processing of Abacus results. Only proteins with a mean average spectral count of at least 1 were considered for further analysis.

Results Mass spectrometric characterisation of the S. pyogenes M1 strain AP1 reveals prevalent and unique proteins in subcellular proteomes Throughout this study, we used cells of the clinically relevant invasive M1 serotype, and the cells were grown to mid-exponential phase. After cellular fractionation, secreted extracellular



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proteins, the cell wall, plasma membrane, and cytoplasm were obtained (Figure 2). In order to release membrane-associated peripheral proteins, crude membranes were treated with high ionic strength buffer27. The resulting six fractions (Figure 2) were analysed by shotgun mass spectrometry and 1132 different proteins were identified in total using a 1% false-discovery rate (FDR) (Table S-3). In order to further reduce false positives, only those proteins with a mean average spectral count of at least 1 (n = 1023) were included in subsequent analyses, thus omitting 109 proteins. Proteins identified via this selection process cover 60.5 % of predicted ORFs (n = 1690) in the M1 SF370 reference genome28 (Figure 3). All 1690 proteins belonging to the M1 SF370 reference genome were analysed by the subcellular prediction tool PSORTb v3.029. The percentages of proteins assigned to each cellular fraction by PSORTb as well as percentages for proteins included and excluded in data analysis are given in Figure 3. Within the SF370 proteome, 33 % of predicted ORFs have currently no known putative function and are listed as uncharacterised and unreviewed in the UniProt database23. With our data, we can confirm the expression of 235 (42 %) of these 556 uncharacterised proteins in S. pyogenes (Table S-4).

Figure 3: Predicted sub-cellular protein localisation for the S. pyogenes proteome. The S. pyogenes SF370 (M1 serotype) reference proteome28 was obtained from the UniProt database23 (Proteome ID: UP000000750) and all 1690 predicted ORFs were analysed by PSORTb v3.029 in order to predict the sub-cellular localisation. The doughnut chart shows the percentage distribution of proteins in each of the five cellular fractions based on PSORTb prediction. In 14 ACS Paragon Plus Environment

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addition, percentages for included proteins identified via our mass spectrometry analysis with a mean average spectral count (m.a.s.c.) of ³ 1 and excluded proteins with a m.a.s.c. < 1 are listed for each cellular fraction. Note, that Protein H is present in S. pyogenes AP1 M1 but not listed in the reference proteome and therefore not included in this analysis.

Analysis of the protein composition of each of the cellular fractions revealed a number of (i) n = 98 extracellular secreted proteins in the growth medium, (ii) n = 552 proteins in the cell wall, (iii) n = 627 proteins in the cytoplasm, and (iv) n = 806 proteins in the crude membrane preparation (Figure 2, Table S-3). Following extraction of membrane-associated peripheral proteins (n = 342), the purified membranes displayed the highest number of proteins of all fractions investigated (n = 900). A high number of so far uncharacterised proteins was detected especially within the membrane fraction (n = 14 for extracellular proteins, n = 102 for cell wall, n = 97 for cytoplasm, n = 174 for crude membranes, n = 49 for membrane-associated peripheral proteins and n = 202 for purified membranes; Table S-3). The 10 most prevalent proteins within each cellular fraction are listed in Table 1. For each fraction, the gene ontology (GO) biological function of all proteins was obtained from the UniProt database and the 20 most frequent functions are presented as pie charts in Figure S-1. While the biological function of the majority of the identified proteins in these fractions is still unknown, the membrane-associated peripheral protein fraction constitutes an exception, since most proteins are involved in translation and transcription processes. Based on the protein composition of the analysed cellular fractions, it is apparent that many proteins are present in more than one fraction (Figure S-2 and Table S-3). However, some proteins are detected only in a given cellular fraction (Table 2), and these unique proteins can serve as markers for the various subproteoms. It should be noted that selection of these proteins is unique due to the set cut-off for data analysis. The vast bulk of unique proteins are present 15 ACS Paragon Plus Environment


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in the purified membrane fraction (n = 76). Proteins for which PSORTb could not predict a localisation but which were exclusively proven in one cellular fraction in this work are presented in Figure 4.

Figure 4: Proteins of previously unknown cellular localisation. Proteins that were detected only in a particular cellular fraction (see Table 2) but had no localisation site prediction available from PSORTb analysis (“unknown”; see also Figure 3), are listed as UniProt IDs with the respective fraction.

The S. pyogenes M1 subcellular proteomes

Secreted extracellular proteins (growth medium) First, we focused on the secretome, since many extracellular proteins play an important role in streptococcal pathogenesis, can counteract host immune responses, or are involved in nutrient acquisition30. Therefore, these proteins are interesting targets for drug development. We identified a total of n = 98 proteins in the growth medium (Table S-3 and Figure S-2). In order to compare these data with previous secretome studies on S. pyogenes M1, we converted 16 ACS Paragon Plus Environment

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protein identifiers from two recent publications16, 31 to available UniProt IDs and compared them with our results (Table S-5). The most abundant secreted proteins are the previously detected16, 31 and well characterised Streptokinase A, GAPDH and M protein (Table 1)30. We also found further glycolytic enzymes such as phosphoglycerate kinase and fructose-bisphosphate aldolase (Table 1) in the growth medium, in line with previous results16. Moreover, we detected 37 proteins (Table S-5), including enolase, which have not been described as extracellular proteins previously. Both GAPDH and enolase are known to be associated with the streptococcal cell surface where they display other functions than in the cytoplasm, e.g. the binding to fibronectin or plasminogen, respectively9. Interestingly, we also detect C5a peptidase and Protein H extracellularly (Table 1). Both proteins are usually cell wall-anchored just like the M protein, which is likewise prevalent in the growth medium. The genes of these three proteins are located in the vir regulon and it has been shown that all three proteins can be released from the streptococcal surface by the cysteine protease SpeB (Streptopain; P0C0J1)32. Since SpeB is also detectable in the growth medium (Table 2)16, the release of these proteins also in vivo appears plausible. Our data further confirm the presence of Prts, also known as SpyCEP, in the extracellular space (Table S-3). In contrast to SpeB, Prts is a serine proteinase, which can process and inactivate a number of pro-inflammatory chemokines30. Two additional identified enzymes are the immunogenic secreted protein (Q99XU7) and the previously reported immunogenic secreted-like protein (Q99Y99)16 (Table S-3). While the latter protein has no ascribed function, Q99XU7 has been shown to elicit an antibody response in humans33. Both proteins share a high sequence identity and harbor a CHAP domain23, which is often present in enzymes involved in peptidoglycan hydrolysis. This putative function has also been suggested for protein Q9A0A8, which was exclusively found in the extracellular medium herein (Table 2).



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DNAses, including for instance mitogenic factor (Q7DAK9; Table 2), constitute another important class of secreted GAS proteins, though their roles as potential extracellular virulence factors are not yet fully understood30. Apart from Q7DAK9, we found another putative DNAse, Q99Z26, which is exclusively secreted into the growth medium (Table 2). The precise function of this protein has also not been elucidated yet. While we detected the reported secreted virulence factors Streptolysin O (P0C0I3), the cotranscribed NAD-glycohydrolase (Q7DAN2) and hyaluronat lyase (Q99ZX4), we did not find superoxide dismutase SodA (P0C0I1) among secreted proteins30. SodA was identified in the cell wall fraction instead (Table S-3). Finally, among the novel identified secretome proteins herein (Table S-5), we observed a so far uncharacterised protein (Q9A173) to be relatively abundant (Table S-3).

Cell wall Proteins present at the cell wall of S. pyogenes are not only important for host- pathogen interactions but have also been in the focus of proteomic studies as they could serve as potential vaccine candidates. It is well known that certain proteins, such as M protein, are anchored to the peptidoglycan layer (Figure 1). These proteins are synthesised in the cytoplasm and targeted for translocation by a discrete N-terminal signal peptide8. The presence of a C-terminal sorting signal, consisting of the LPXTG motif, a hydrophobic stretch and a charged tail, enables anchoring via Sortase A8. Based on these characteristics, it is possible to predict cell wall anchoring of proteins in S. pyogenes. Therefore, the S. pyogenes (serotype M1) proteome was searched for cell wall-anchored proteins with PSORTb and in addition a list with predictions from CW-Pred234 was retrieved. Maximally 20 proteins are predicted to be anchored via this mechanism to peptidoglycan (note that Protein H is missing in the M1 SF370 reference genome and was analysed separately in here). Table 3 lists predicted cell wall-anchored proteins


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together with experimental evidence for their presence and abundance in the purified cell wall fraction. A total of n = 14 proteins was detected in the cell wall, n = 2 only at < 1 mean average spectral count value (Q9A0A2 and Q9A0K5), n = 1 only in the membrane fraction (Q99YV5) and n = 4 proteins (Q7DAL7, Q99YX9, Q99YT0, and Q99Z76) were not found in any fraction at all. Protein GRAB (Q7DAL7) belongs to the latter group and its expression is generally not observed in the AP1 strain of S. pyogenes35. Likewise, expression of the other three proteins might also be strain-specific or require different growth conditions. Among those 14 cell wall located proteins, 5 are so far uncharacterised and 1 (Q99ZV7) harbours an LPXTG motif but no obvious signal peptide. The cell division protein DivIB (Q99YV5) was assigned to multiple locations (cell wall and membrane) by PSORTb with a preference for the cell wall, although lacking both a signal peptide and an LPXTG motif. Our data now confirm DivIB to be located at the cell membrane (Table S-3), most likely by spanning the membrane once23. It is surprising that out of 1690 ORFs in S. pyogenes only 20 proteins were predicted to be anchored to the cell wall by PSORTb and only 14 were identified herein. However, previous surface analyses have revealed that also anchorless proteins cover the surface of S. pyogenes9. Indeed, our analysis yields a total of n = 552 different proteins in the cell wall fraction (Table S-3 and Figure S-2). In an attempt to put our results into context with previous proteomic studies on GAS M1, we extracted published proteomic data on surface proteins and converted all identifiers, where applicable, to UniProt IDs. One proteome study employed mutanolysin treatment36, three studies applied surface shaving14, 31, 37 and one study yielded a mixed cell wall/membrane preparation16. As summarised in Table S-6, we find n = 531 more proteins than reported for the mutanolysin extraction study36. In order to compare our results with those from the latter four studies, in which the fractions were mixed, we combined our data sets for proteins detected in cell wall, membrane-associated peripheral proteins and both crude and purified membrane fractions (Table S-6). This analysis yields a total of n = 820 proteins that



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are newly reported in our study and of which n = 399 are present in the cell wall preparation (Table S-6). However, it should be noted though that cross-contamination of the cell wall fraction with cytosolic proteins due to protoplast lysis after mutanolysin treatment has been described previously3 . Therefore, we calculated the ratio of spectral counts between the cell wall and the cytoplasm for all newly detected proteins (Table S-6), applying a cut-off of 1.0 as a criterion for cross-contamination. We base this cut-off value on calculated ratios from well-known cell wall proteins such as enolase (ratio = 1.2). This analysis yields 213 novel proteins for the cell wall fraction, with 18 proteins even having a ratio of 5.0 or above. Among the most abundant cell wall proteins (Table 2; Table S-3) are previously well characterised proteins such as enolase, phosphoglycerate kinase, GAPDH and triosephosphate isomerase, which are part of the glycolytic pathway and usually located in the cytoplasm9. In addition, we detect 5 more glycolytic enzymes in the cell wall (Table S-3): the previously described fructose-bisphosphate aldolase (P68905); three enzymes not unambiguously assigned to the cell wall before: glucose-6-phosphate isomerase (Q9A1L1), ATP-dependent6-phosphofructokinase (Q99ZD0) and pyruvate kinase (Q99ZD1); and phosphoglycerate mutase (Q99Z29), so far not detected on GAS M114, 16, 31, 36, 37. Other novel proteins identified in the cell wall fraction of GAS M1, that are present at relatively high abundance as compared to the other proteins in the fraction, include 3 peptidases (PepN, PepO and PepF) as well as 2 NADH oxidases (Q99XR6 and Q99ZN6) (Table S-3 and S-6). NADH oxidases (Noxs) catalyse the reduction of molecular oxygen to either hydrogen peroxide or water. Nox has been implied in coping with oxidative stress in aerobic environments38 and might therefore contribute to virulence. As mentioned in the section above, we also find superoxide dismutase SodA (P0C0I1) located in the cell wall. Superoxide dismutases play an important role in directly protecting the


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cell from reactive oxygen species (ROS), namely by converting superoxide anions to molecular oxygen and hydrogen peroxide. In addition to enzymes that scavenge ROS, others are involved in repairing oxidised proteins. Among all amino acid residues, methionine (and cysteine) residues are particularly susceptible to oxidation by ROS. Therefore, protective methionine sulfoxide reductases (Msrs) are found in many aerobic organisms and their role in oxidative stress defense has been particularly studied in the cytoplasm39. Here, we find two Msrs, MsrA (Q9A149) and MsrB (Q99ZV6). Interestingly, both enzymes are only present in the cell wall but absent in the cytoplasm (Table 2). This is in agreement with recent reports on extracytoplasmic Msrs that have only been observed in very few bacteria so far, including, for instance, S. pneumoniae39. It has been proposed that the presence of such reducing enzymes in the cell envelope aids the bacteria not only in correct protein folding and repair, but also in withstanding killing by oxidative burst from macrophages and thereby contributing to virulence39. The presence of elongation factors G and Tu (Table 1) on the cell surface has been reported prior to this work14, 16, 31, 37. In our study, we find EF-Ts as an additional prevalent elongation factor (Table S-3). Another class of proteins at first glance present unexpectedly on the cell surface are tRNA ligases, also known as aminoacyl-tRNA synthetases, that specifically attach an amino acid to its tRNA. The 3 predominant ones found herein are alanine-, valine- and arginine tRNA ligase (Table S-3). It should be noted that only the latter enzyme has been reported earlier to be associated with the cell surface16. Interestingly, recent reports have highlighted the usage of aminoacyl-tRNAs not only in protein synthesis but also in nonribosomal biosynthetic pathways in bacteria, such as peptidoglycan cross-linkage or membrane phospholipid modification40. Both features have been linked to antibiotic resistance in respective strains40.



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Lastly, we identified further proteins so far unreported for the cell wall, including an ATPbinding protein (Q99XH2), a putative phosphotransacetylase (Q99ZQ5), phosphoglucomutase (Q99ZH8) and transketolase (Q99YJ3), a GMP synthase (P64299) and an adenylosuccinate synthetase (Q9A1P8).

Cytoplasm We detected a total of n = 627 proteins in the cytoplasm (Table S-3 and Figure S-2) of which the most prevalent proteins are listed in Table 1. In agreement with previous reports on GAS, enolase and GAPDH are present in high abundance3, and we also find high levels of Elongation factor G (Table 1). Other enzymes involved in the glycolytic pathway are also predominant in the cytoplasm, as well as elongation factors, ribosomal proteins, chaperones and proteins destined for surface anchoring, such as M protein (Table S-3). We next sought to compare the high number of cytoplasmic proteins with another proteome study15 in order to identify putative differences (Table S-7). It must be pointed out though that the previous study employed whole cell breakage, which resulted in a mixture of cytoplasmic, cell wall and membrane(-associated) proteins. Based on this comparison, we find n = 451 identical and n = 176 proteins that have so far not been ascribed to the cytoplasm (Table S-7). Among these novel proteins are n = 34 so far uncharacterised proteins. Other proteins include DNA helicase PcrA (Q99ZE1), for which gene disruption was found to be lethal in Gram-positive bacteria41, and low levels of DNA helicase RuvA (P66751). Interestingly, we only find one DNA polymerase, PolC (P0C0B8), in the cytoplasm, while we do not detect DNA polymerase DnaE (P0C0F3; alpha subunit of DNA polymerase III holoenzyme) at all (Table S-3). Instead, DnaE as well as subunits HolA (Q7DAL5; delta subunit) and DnaX (Q7DAL6; gamma/tau subunits) are associated with the membrane and remain so even after purification (Table S-3). On the contrary, subunit DnaN (Q9A209; beta subunit) is not only located at the membrane but also


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in the cytoplasm (Table S-3). Other proteins detected at elevated levels in the cytoplasm are DNA ligase (Q9A0J5), DNA gyrase subunit B (Q9A0L0) and a putative toxic anion resistance protein (Q9A1Q0), belonging to the TelA like protein family associated with tellurite resistance42, see also Table S-3.

Plasma membrane and membrane-associated peripheral proteins Starting from a crude membrane preparation, the number of detectable proteins increased from n = 806 to n = 900 in the purified membrane fraction following release of membraneassociated peripheral proteins (n = 342) (Table S-3 and Figure S-2). This can probably be explained by a better amenability of more proteins in the purified membrane fraction to tryptic digest. Proteins detected either in both membrane fractions (n = 777) or only in one fraction (n =29 for crude membrane and n = 123 for purified membrane) are presented in Table S-8. Table 2 lists the 10 most prominent proteins in both membrane fractions as well as in the membraneassociated peripheral proteins. It can be noticed that also cell wall-anchored proteins, such as M protein and C5a peptidase are present in these fractions. This is not surprising, since both proteins belong to the most common proteins displayed on the cell surface and have to be translocated via the SecYEG translocase. The presence of these proteins is therefore most likely explainable by them being “trapped” in the translocon. Among peripheral proteins extracted from crude membranes by high ionic strength buffer are many so far uncharacterised proteins (n = 22) as well as a number of ribosomal proteins of the large 50S and small 30S subunit (n = 50) (Table S-3). Interestingly, one 50S ribosomal protein (Q48WA5) was only detectable in the membrane-associated peripheral protein pool (Table 2). F-ATP synthase subunits alpha and beta are also very prevalent in the extract as they belong to the soluble catalytic F1 complex (Table I).



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The purified membrane fraction not only contains the highest number of proteins of all fractions (Table S-3) but also the highest number of unique proteins (Table 2). We list the 20 most prominent proteins that are also assigned to the membrane by PSORTb in Table 4. In order to specify the protein localisation better, we also provide available information from gene ontology and for predicted lipoproteins in Table 4, with lipoproteins described in more detail in the next section. In order to characterise detected integral membrane proteins, their predicted biological functions were retrieved via UniProt and are depicted in Figure S-3. A total of n = 40 biological functions are present, with the largest fraction (60.55 %) constituted by a so far unknown function. The second largest fraction (6.42 %) consists of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS), which is essential for sugar uptake from the environment. Proteins involved in transport function represent the third most important fraction (3.67%). Among integral membrane proteins, protein Q99ZA4 is the highest prevalent (Table 4) - both in crude and purified membranes. This protein is predicted to belong to the class of ionotropic glutamate receptors, a highly conserved family of ligand-gated ion channels that regulate ion passage over the plasma membrane42. Another abundant protein is Q99Y37 (Table 4), which belongs to the Stomatin family and harbors a Band 7 domain42. Proteins in this family often occur as oligomers, may serve as scaffolding proteins, and are frequently involved in modulation of ion channel activity42. Another large portion of membrane proteins found herein is encoded by the opp operon, such as the oligopeptide permease Opp, which has been suggested to be important for GAS virulence in mice43. OppB (Q9A1F9) and OppC (Q9A1F8) constitute the transmembrane domain of this ABC transporter, while the two accessory nucleotide-binding domains are constituted by OppD (Q9A1F7) and OppF (P0A2V6) (Table 4). In our study, we detected lower levels of OppC than OppB in membranes (Table S-3).


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In an attempt to identify novel proteins in GAS membranes, we compared our data to proteomic data obtained from surface shaving14, 31, 37, mixed cell wall/membrane preparation16 as well as whole cell extraction15, since the methods employed in these studies also yield membrane proteins to a certain extent. Therefore, we combined our data on proteins detected in the cell wall, the cytoplasm, both membrane preparations and on membrane-associated proteins - as we do not compare pure fractions - and used this for comparison with the other aforementioned studies (Table S-9). This resulted in a total of n = 486 proteins that had not been reported in these studies before, of which n = 435 were detected in the membrane or membrane-associated peripheral protein fractions presented in this study (see Table S-9 for distribution). Among these proteins, especially hyaluron synthase HasA (P0C0H1), the putative PTS enzyme II component D (Q99ZV1), penicillin-binding protein 2a (Q99XS5), and a putative heavy metal-transporting ATPase (Q99Z27) were prevalent in purified membranes (Table S-3). Notably, HasA synthesises and translocates hyaluronan chains for capsule formation although the precise mechanism is still not fully understood44. We also detect elevated levels compared to the other cellular fractions of UDP-glucose 6-dehydrogenase HasB (P0C0F5) from the hasABC operon in the membrane fractions, which might indicate a strong interaction with HasA (Table S-3). Metal-ATPases belong to the P-type superfamily of transport ATPases and have been shown to function both in metal homeostasis and extracellular metalloprotein assembly by driving metal efflux, with both functions contributing to virulence in bacteria45. In addition to the above-mentioned proteins, our data further confirm the presence of two putative transferases, one sugar transferase (Q9A117) and one glycosyl transferase (Q9A0F8) (Table S-3).

Lipoproteins



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Putative lipoproteins are not recognised by PSORTb and therefore we retrieved predicted lipoproteins (n = 31) from the Pred-Lipo database46. The predictions are based on the characteristic secretory signal peptides of bacterial lipoproteins. These sequences contain the “lipobox” motif, which harbors an essential cysteine residue for membrane anchoring after export11, 46. We searched for the presence of predicted lipoproteins in both crude and purified membrane fractions and found a total of n = 27 lipoproteins (Table 5). Out of these lipoproteins, 24 were present in both membrane preparations. Two lipoproteins were only detectable after the purification step (Q9A0V0 and Q99YQ3), while one lipoprotein (Q9A1I9) was only identified in the crude membrane fraction. By comparing our data to previous proteomic studies employing surface shaving14, 31, 37, mixed cell wall/membrane preparations16, and whole cell extraction15, we can confirm the presence of 9 novel predicted lipoproteins in GAS M1 (P65631, Q99YA2, Q9A0H2, Q9A198, Q99XV3, Q99ZB1, Q99ZC4, and the aforementioned Q9A1I9 and Q99YQ3) (Table 5; Table S-10). From these 9 lipoproteins, 6 are substratebinding proteins of ABC transporters and therefore embody the principle of tethering these proteins to the membrane in Gram-positive bacteria as opposed to their periplasmic soluble counterparts in Gram-negative bacteria11. We also detect the membrane protein insertase YidC1 (P65631), a paralog of YidC2 (Q9A1C3), since two YidC proteins are found in most Gram-positive bacteria. The other 2 observed proteins are a laminin-binding protein (Q99XV3), previously shown to be immunogenic in humans47, and a protein of unknown function (Q99ZC4), which belongs to a protein family with shared structural similarity to betalactamase inhibitory proteins42. Lastly, another noteworthy finding is that one of the predicted lipoproteins (Q9A0A8) was exclusively found in the growth medium (Table 2 and V), in agreement with the study conducted by Sharma et al.16.

Subcellular localisation of putative new drug targets in S. pyogenes


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In a recent study from 2016, Singh et al. performed an in silico analysis in order to identify protein targets that are essential for GAS survival but share no homology with human proteins48. The authors found a total of 28 essential proteins. In our study, we searched our proteome data for these drug target candidates. Table 6 lists both their cellular localisation and amount in respect to the other proteins in the cellular fraction. Seven of these proteins were undetectable in any cellular fraction in our study, including, for instance, the beta subunit of citrate lyase (Q99ZK8). Both malonyl-CoA-acyl carrier protein transacylase (Q99YD5) and glucose-6-phosphate isomerase (Q9A1L1) were the only two proteins detectable in the growth medium albeit at lower levels. However, the latter enzyme was found in elevated levels on the cell surface rendering it an interesting drug target. The same applies to putative phoshoglucomutase (Q99ZH8) and adenylosuccinate synthase (Q9A1P8). In their study, Singh et al. especially pointed to chorismate synthase, glutamate racemase and ATP synthase subunit a as previously reported candidate drug targets and these proteins were indeed found among five proteins solely present in the membrane fractions. Proteins Q99YK1 and P65781 were also confined to the membranes, but could be partially extracted by high ionic strength buffer.

Discussion Surface-exposed proteins are obvious candidates for vaccine development and thus we give a detailed overview of streptococcal cell wall components and lipoproteins in this study. Strikingly, only very few proteins appear to be directly cell wall-anchored while many proteins are retained by other mechanisms. This observation also supports the notion of a periplasmiclike space in GAS5. The display of cytoplasmic proteins on the GAS surface is well documented and often these proteins display an additional function performed “at nighttime”, which is why they have been termed “moonlighting” proteins49. For example, surface-exposed GAPDH has a binding affinity for fibronectin. It has been speculated that molecule binding to surface



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proteins induces functional conformational changes, since these proteins do not harbor cytoplasmic domains partaking in cell signaling9. In addition, GAPDH could be involved in extracellular ATP formation in concert with the other present glycolytic enzymes9,

49

.

Interestingly, the aforementioned formation of aminoacyl-tRNAs by tRNA-ligases is an ATPdependent process. In this study, we detect several additional glycolytic enzymes extracellularly. This supports the hypothesis that sequestering the glycolytic pathway - maybe as a macromolecular complex - on the bacterial surface can be advantageous for the bacteria during colonisation/infection and also could increase its activity as compared with the cytoplasm49. Therefore, it appears highly likely that the elevated number of surface proteins serves a purpose and is not due to cross contamination, opening perspectives in the discovery of new functions and drug targets. Another class of surface-exposed proteins is constituted by lipoproteins that have been brought out as putative vaccine candidates. The family of bacterial lipoproteins is involved in a variety of processes such as nutrient acquisition, adhesion, and immune modulation9, 50. Previous proteomic approaches have targeted several lipoproteins in GAS and here we identified nine additional predicted members of this family in M1 GAS. However, some predicted lipoproteins were not found, which might be due to a wrong prediction strategy, strain specificity, or growth conditions. In 2004, Beia et al. reported that active immunisation of mice with recombinantly expressed lipoproteins resulted in growth-inhibitory activity50. This substantiates that lipoproteins are promising vaccine candidates. In recent years, the bacterial membrane and its associated proteins were moved into the spotlight as a novel target for drug development. The cell membrane is involved in many crucial processes, such as transport of waste products or nutrients, cell-cell communication, and respiration, all of which are essential for bacterial survival. Therefore, one therapeutic approach being pursued is the direct targeting and disruption of the bacterial membrane by


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means of antimicrobial peptides and related molecules. Moreover, some membrane proteins protrude into the extracellular space and hence constitute putative vaccine candidates. One especially interesting target is bacterial energy metabolism and related respiratory and metabolic enzymes located at the membrane51. These targets need to be sufficiently distinct from their human counterparts. Two interesting candidates without human homologue that were detected in the membrane herein are the putative penicillin-binding protein 2X and the FATPase subunit a 48. Targeting another class of ATPases, the P-type ATPases, could also prove a worthwhile approach.

Conclusions S. pyogenes is one of the most important human pathogens and an alarming increase in resistance rates against macrolide antibiotics is being observed52. Resistance to clindamycin is of special concern, as it is often used in combination with penicillin to treat GAS infections52. So far, safe and effective vaccines against GAS infections are not available and while the search for protective antigen candidates needs to be pursued, it is also worthwhile to explore new therapeutic strategies in coping with GAS infections. In order to select and study novel drug targets, it is crucial to gain a better understanding of the proteins produced by the bacteria, their cellular location, and their relative amounts. The present work describes the most extensive approach to date that characterises the entire S. pyogenes proteome of an invasive M1 serotype. Here, we analyse for the first time the protein composition of all isolated cellular fractions, including the plasma membrane, by means of state of the art mass spectrometry. We also aimed for a systematic terminology approach in this work, including the use of UniProt identifiers, gene names (when available) and loci, since we believe that utilisation of as well as comparison between proteomic studies is often hampered by usage of different/outdated identifiers. Therefore, our data analysis is based on up-to-date identifiers from the M1 SF370 reference



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proteome. Since growth conditions impact protein expression levels and distribution, we decided to compare our results only with proteome studies employing M1 serotype GAS grown at comparable conditions. Nearly two thirds of predicted ORFs of the M1 SF370 genome were found to be present in GAS grown to mid-exponential phase. With this approach we sought not only to map the cellular location of interesting drug target candidates, but also to identify novel, previously undetected, proteins and uncharacterised proteins, whose cellular functions are yet to be determined. We believe that this study will enable other researchers to better understand GAS cellular organisation and its interaction with the human host. Moreover, our work will also be beneficial in finding new drug targets for development of antimicrobial therapies.

Associated content SUPPORTING INFORMATION: The following files are available free of charge at ACS website http://pubs.acs.org:

Figure S-1 Functional classification of identified proteins. Figure S-2 Visualisation of protein abundance within and distribution between subcellular proteomes as shown by heat maps. Figure S-3 Functional classification of integral membrane proteins. Table legends SI Legends for all supplementary tables and references. Table S-1 Peptide and protein identification. (Excel file) Table S-2 Protein coverage for S. pyogenes proteins identified via mass spectrometry. (Excel file)


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Table S-3 Proteins detected in S. pyogenes subcellular proteomes. (Excel file) Table S-4 Uncharacterised proteins expressed in S. pyogenes. (Excel file) Table S-5 Novel proteins detected in the S. pyogenes secretome. (Excel file) Table S-6 Novel proteins detected in the S. pyogenes cell wall. (Excel file) Table S-7 Novel proteins detected in the S. pyogenes cytoplasm. (Excel file) Table S-8 Comparison of protein content between the crude and purified membrane fraction. (Excel file) Table S-9 Novel proteins detected in or associated with the S. pyogenes plasma membrane. (Excel file) Table S-10 Novel predicted lipoproteins detected in S. pyogenes. (Excel file)

Corresponding Author To whom correspondence should be addressed: Laura Wilk, BMC, Floor B14, Lund University, Tornavägen 10, 221 84 Lund, Sweden. Phone: +46 46 222 68 07 Email: [email protected], [email protected]

Author contributions



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L.W. designed the experiments and performed the cell fractionation. L.H. performed the MSanalysis. LW and LH performed the bioinformatics analysis and analysed the data. L.W., L.H., J.M. and H.H. wrote the paper.

Funding Sources To Johan Malmström: Swedish Research Council (project 621-2012-3559), the Swedish Foundation for Strategic Research (grant FFL4), the Crafoord Foundation (grant 20100892), Stiftelsen Olle Engkvist Byggmästare, the Wallenberg Academy Fellow KAW (2012.0178), European research council starting grant (ERC-2012-StG-309831) and the Medical Faculty, Lund University. To Heiko Herwald: Alfred Österlund, Knut and Alice Wallenberg Foundation (2011.0037), Ragnar Söderberg Foundation (M121/11), the Swedish Foundation for Strategic Research SB12-0019, Swedish Research Council.2016-01104

ACKNOWLEDGMENT This work was supported in part by the foundations of Alfred Österlund, Crafoord, European research council starting grant, the Knut and Alice Wallenberg Foundation, the Medical Faculty at Lund University, the Ragnar Söderberg Foundation, Stiftelsen Olle Engkvist Byggmästare, the Swedish Foundation for Strategic Research, the Swedish Research Council, and the Wallenberg Academy Fellow KAW.

Figure Legends 32 ACS Paragon Plus Environment

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Figure 1: Schematic depiction of cell structure and subcellular proteomes of S. pyogenes. Secreted proteins are released into the environment. A hyaluronic acid capsule surrounds the bacterium. The cell wall consists of peptidoglycan decorated with various proteins (M protein) and glycopolymers. Two types of anionic cell wall glycopolymers can be distinguished in GAS, one attached to peptidoglycan (wall teichoic acid) and one to membrane lipids (lipoteichoic acid, LTA)5. Additional proteins (X) could interact with the cell wall components. The presence of a periplasmic space is proposed, with residing lipoproteins and other soluble proteins (Y) that could be retained by the membrane/membrane proteins. Sortase A is located at the membrane and mediates peptidoglycan-anchoring of respective proteins (M protein). Cytoplasmic proteins might not only diffuse freely in the cytoplasm but also interact with the membrane/membrane proteins.

Figure 2: Workflow for cellular fractionation of S. pyogenes. Bacterial growth conditions and subsequent preparation steps (light blue) of cellular fractions (dark blue; analysed by shotgun mass spectrometry in this study) are shown. Secreted extracellular proteins were obtained from bacteria grown in protein-reduced medium, while the remaining cellular fractions were prepared from bacteria grown in protein-rich medium. P=pellet, SN=supernatant; obtained after centrifugation.

Figure 3: Predicted sub-cellular protein localisation for the S. pyogenes proteome. The S. pyogenes SF370 (M1 serotype) reference proteome28 was obtained from the UniProt database23 (Proteome ID: UP000000750) and all 1690 predicted ORFs were analysed by PSORTb v3.029 in order to predict the sub-cellular localisation. The doughnut chart shows the percentage distribution of proteins in each of the five cellular fractions based on PSORTb prediction. In addition, percentages for included proteins identified via our mass spectrometry



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analysis with a mean average spectral count (m.a.s.c.) of ³ 1 and excluded proteins with a m.a.s.c. < 1 are listed for each cellular fraction. Note, that Protein H is present in S. pyogenes AP1 M1 but not listed in the reference proteome and therefore not included in this analysis.

Figure 4: Proteins of previously unknown cellular localisation. Proteins that were detected only in a particular cellular fraction (see Table 2) but had no localisation site prediction available from PSORTb analysis (“unknown”; see also Figure 3), are listed as UniProt IDs with the respective fraction.


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Tables Table 1. Most abundant proteins identified in Streptococcus pyogenes subcellular proteomes

Subcellular localisation

Protein annotation

UniProt ID

SPy_1979 ska

Streptokinase A

P10520

SPy_0274 gap

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

P0C0G7

Locus tag

Gene name

Secreted extracellular proteins

(Plasmin receptor; Plasminogenbinding protein) SPy_2018 emm1

M protein type 1

Q99XV0

SPy_2010 scpA

C5a peptidase (SCP)

P58099

SPy_1801 isp2

Immunogenic secreted-like protein

Q99Y99

SPy_1760 dnaK

Chaperone protein DnaK (HSP70)

P0C0C6

SPy_0136

Uncharacterised protein

Q9A1R6

SPy_1881 pgk

Phosphoglycerate kinase

P68897

SPy_0611 tuf

Elongation factor Tu (EF-Tu)

P69952

SPy_1889 fba

Fructose-bisphosphate (FBPA)

SPy_0731 eno

Enolase

P69949

SPy_0274 gap


P0C0G7

aldolase P68905

Cell wall

(Plasmin receptor; Plasminogenbinding protein) SPy_1881 pgk


P68897

SPy_0273 fus

Elongation factor G (EF-G)

P69946

SPy_1282 pyk

Pyruvate kinase

Q99ZD1




Protein annotation

UniProt ID

SPy_0611 tuf


P69952

SPy_1371 gapN

Putative NADP-dependent Q99Z67 glyceraldehyde-3-phosphate dehydrogenase

SPy_1760 dnaK


P0C0C6

SPy_2010 scpA

C5a peptidase (SCP)

P58099

SPy_0613 tpiA

Triosephosphate isomerase (TIM; P69887 TPI)

SPy_0731 eno

Enolase

P69949

SPy_0273 fus

Elongation factor G (EF-G)

P69946

SPy_0274 gap


P0C0G7

Locus tag

Gene name

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Cytoplasm

(Plasmin receptor; Plasminogenbinding protein) SPy_1282 pyk

Pyruvate kinase

Q99ZD1

SPy_0099 rpoC

DNA-directed RNA polymerase P0C0E0 subunit beta' (RNAP subunit beta')

SPy_1881 pgk


P68897

SPy_0611 tuf


P69952

SPy_2070 groL

60 kDa chaperonin (GroEL protein) P69883 (Protein Cpn60)

SPy_1760 dnaK


SPy_1371 gapN

Putative NADP-dependent Q99Z67 glyceraldehyde-3-phosphate dehydrogenase

SPy_2010 scpA

C5a peptidase (SCP)

P0C0C6

Crude membrane P58099


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Protein annotation

UniProt ID

SPy_2018 emm1

M protein type 1

Q99XV0

SPy_0416 prtS

Putative cell envelope proteinase

Q9A180

SPy_1315


Q99ZA4

SPy_0611 tuf


P69952

SPy_1228

Putative lipoprotein

Q99ZH4

SPy_2000 dppA

Surface lipoprotein

Q99XV9

SPy_0760 atpD

ATP synthase subunit beta

Q9A0I7

SPy_0913

Putative ribosomal protein S1-like Q9A066 DNA-binding protein

SPy_1294

Putative maltose/maltodextrin- Q99ZC0 binding protein

SPy_0913

Putative ribosomal protein S1-like Q9A066 DNA-binding protein

SPy_0760 atpD


Q9A0I7

SPy_0758 atpA

ATP synthase subunit alpha

Q9A0I9

SPy_0731 eno

Enolase

P69949

SPy_0611 tuf


P69952

SPy_0274 gap


P0C0G7

Locus tag

Gene name

Membraneassociated peripheral proteins

(Plasmin receptor; Plasminogenbinding protein) SPy_2010 scpA

C5a peptidase (SCP)

P58099

SPy_2018 emm1

M protein type 1

Q99XV0

SPy_0724 rplS

50S ribosomal protein L19

P66084

SPy_0061 rplN

50S ribosomal protein L14

Q9A1W4




Protein annotation

UniProt ID

SPy_2010 scpA

C5a peptidase (SCP)

P58099

SPy_2018 emm1

M protein type 1

Q99XV0

SPy_0416 prtS

Putative cell envelope proteinase

Q9A180

SPy_1228


Q99ZH4

SPy_1315


Q99ZA4

SPy_0611 tuf


P69952

SPy_2000 dppA

Surface lipoprotein

Q99XV9

SPy_1884

Eukaryotic hypersensitive-induced Q99Y37 response-like protein

SPy_1316

Putative ABC transporter (ATP- Q99ZA3 binding protein)

SPy_0760 atpD


Locus tag

Gene name

Page 38 of 59

Purified membrane

Q9A0I7

The ten predominant proteins for each cellular fraction are listed in descending order according to their mean average spectral counts as given in Table S-3. Proteins were identified by shotgun mass spectrometry and are listed with their respective annotation and UniProt identifier as well as gene name (when available) and locus. Spy-numbers refer to the S. pyogenes SF370 (serotype M1) genome.

Table 2. Unique proteins in each Streptococcus pyogenes sub-proteome


Locus tag

Gene name

Protein annotation

UniProt ID

SPy_2043

mf

Mitogenic factor

Q7DAK9

SPy_1436

mf3

Putative deoxyribonuclease

Q99Z26


Q7DAM2

Secreted extracellular proteins

SPy_0861


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Locus tag

Gene name

Protein annotation

UniProt ID

SPy_0857

mur1.2

Putative peptidoglycan hydrolase

Q9A0A8

SPy_2039

speB

Streptopain (SPP)

P0C0J1

SPy_0167

slo

Streptolysin O

P0C0I3

SPy_1352

aroA

3-phosphoshikimate carboxyvinyltransferase

1- Q99Z83

Uncharacterized protein

Q99XL4

Cell wall

SPy_2152 SPy_0877

mvaD

Mevalonate decarboxylase

pyrophosphate Q9A097

SPy_1217

Putative glycine cleavage system H Q99ZI5 protein

SPy_0186


Q9A1M7

SPy_0577


Q9A0W9

SPy_1055

msrB

Peptide methionine reductase MsrB

sulfoxide Q99ZV6

SPy_1710

Putative PTS system, enzyme IIB Q99YG9 component

SPy_0377


Q9A1A5

SPy_1604


Q99YP5

SPy_0265


Q9A1H7

SPy_2045

Protein low temperature requirement Q99XT6 C

SPy_0121

Putative deoxyguanosine

Q9A1S5

SPy_1840

Hypothetical protein

Q99Y71

SPy_1218


Q99ZI4

SPy_0747


Q9A0J7

SPy_0352

Acylphosphatase

Q9A1C2

SPy_1600

Putative hyaluronidase

Q99YP8




Protein annotation

UniProt ID

SPy_1581


Q99YR1

SPy_1144


Q99ZP2

Locus tag

Gene name

Page 40 of 59

SPy_1707

lacB1

Galactose-6-phosphate subunit LacB 1

isomerase Q99YH2

SPy_0466

msrA

Peptide methionine reductase MsrA

sulfoxide Q9A149

SPy_0900

pyrF

Orotidine 5'-phosphate decarboxylase Q9A077 OMPdecase)

SPy_1811

scrK

Putative fructokinase

Q99Y93

SPy_1905

hsdS

Putative type I deoxyribonuclease

site-specific Q99Y25

SPy_1189

citF

Putative citrate lyase, alpha subunit

Q99ZK7

Putative arsenate reductase

Q9A183


Q99ZM1


Q99XJ2

Cytoplasm

SPy_0410 SPy_1171

satD

SPy_2181 SPy_1186

citD

Citrate lyase acyl carrier protein Q99ZK9 (Citrate lyase gamma chain)

SPy_1201

UPF0122 protein

P67253

SPy_0850

Ferredoxin--NADP reductase (FNR)

Q9A0B5

SPy_1857

Putative protein

SPy_1552


Q99YT3

SPy_2104

UPF0246 protein

Q99XQ1

SPy_1042


Q99ZW5 Q99ZQ1

transcriptional

regulatory Q99Y59

SPy_1135

guaC

GMP reductase

SPy_1638

atoD.1

Putative Acetyl-CoA:acetoacetyl- Q99YM1 CoA transferase A subunit


Page 41 of 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Protein annotation

UniProt ID

SPy_1927


Q99Y09

SPy_0686


Q9A0N9

SPy_1971


Q99XX9

SPy_0004


Q9A208

SPy_0117


Q9A1S6

SPy_0252

Putative sugar transporter binding lipoprotein

Locus tag

Gene name

Crude membrane

sugar Q9A1I9

SPy_0209

tadA

tRNA-specific adenosine deaminase

P68999

SPy_0497

mutM

Formamidopyrimidine-DNA Q9A131 glycosylase, (DNA-(apurinic or apyrimidinic site) lyase MutM) (AP lyase MutM) (EC 4.2.99.18)

Membraneassociated peripheral proteins

SPy_2058.1 rpmG2

50S ribosomal protein L33 2

Q48WA5

SPy_0467

UPF0346 protein

Q9A148

SPy_0599

Cys-tRNA(Pro)/Cys-tRNA(Cys) deacylase

Q9A0V4

Purified membrane




Page 42 of 59

Locus tag

Gene name

SPy_1103

potB

Putative spermidine / putrescine ABC Q99ZS7 transporter (Permease protein)

SPy_1900

thiD

Putative kinase

SPy_2026

UniProt ID

Protein annotation

phosphomethylpyrimidine Q99Y29

Histidine kinase

Q99XU6 helicase/nuclease Q9A0H3

SPy_0777

addA

ATP-dependent subunit A

SPy_2148

mutS

DNA mismatch repair protein MutS

Q99XL8

Putative histidine kinase protein

Q99ZG9

SPy_1236 SPy_0755

atpB

ATP synthase subunit a (F-ATPase Q9A0J2 subunit 6)

SPy_1247

Putative myo-inositol-1(Or monophosphatase

SPy_2211


SPy_1062

Putative two-component regulator

4)- Q99ZF9 Q99XH1

response Q99ZU9

SPy_1964

cdsA

Phosphatidate cytidylyltransferase

Q99XY2

SPy_1233

coaA

Pantothenate kinase

Q99ZH1

Putative amino acid permease

Q9A1Z9

Undecaprenyl-diphosphatase

P67392

SPy_0016 SPy_0280

uppP

(Bacitracin resistance protein) SPy_1592

Putative ABC transporter substrate Q99YQ3 binding lipoprotein

SPy_0045


Q9A1X6

SPy_1056

UPF0324 membrane protein

Q99ZV5

SPy_1085

srtF

Lantibiotic transport protein SrtF

ATP-binding P0C0E3

SPy_1149

Putative ABC transporter binding protein)

SPy_0808


(ATP- Q99ZN7 Q9A0E6


Page 43 of 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Locus tag

Gene name

Protein annotation

UniProt ID

SPy_0021

recO

DNA repair protein RecO

P65987

SPy_1215


Q99ZI7

SPy_1791


SPy_0797


Q9A0F6

Glutamate 5-kinase

Q99YJ7

SPy_1174

Putative transcarboxylase subunit

Q99ZL8

SPy_0397

UPF0298 protein

P60419

SPy_0874

Putative two-component regulator

SPy_1385


Q99Z59

SPy_0887


Q9A087

Ribonucleoside-diphosphate reductase

Q9A175

SPy_0363

Phosphoesterase

Q9A1B5

SPy_0440

Putative dehydrogenease oxidoreductase

SPy_1672

SPy_0427

proB

nrdE.1

(ATP- Q99YA5

response Q9A0A0

/ Q9A166

SPy_1665

ftsL

Cell division protein FtsL

Q99YK0

SPy_1841

rnhC

Ribonuclease HIII (RNase HIII)

P66677

SPy_1785

recG

ATP-dependent DNA helicase RecG

Q99YB0

SPy_0530

vicX


Q9A106

SPy_0498

coaE

Dephospho-CoA kinase

P58102

SPy_0229

Putative ABC binding protein

SPy_1844

Putative exodeoxyribonuclease (Alpha subunit)

SPy_2105

nrdG

Anaerobic triphosphate protein

transporter,

ATP- Q9A1K5 V Q99Y68

ribonucleoside- Q99XQ0 reductase-activating




Protein annotation

UniProt ID

SPy_1937


Q99Y03

SPy_1736


Q99YE8

SPy_0622


Q9A0U0

SPy_0237

Carbonic anhydrase

Q9A1K0

SPy_1246

Putative nucleolar protein

Q99ZG0

SPy_0201


Q9A1L7

SPy_0570

Putative drug resistance protein

Q9A0X6

SPy_1061

Putative two-component histidine kinase

Locus tag

SPy_0327

Gene name

Page 44 of 59

ntpJ

sensor Q99ZV0

Putative V-type Na+-ATPase subunit Q9A1D8 J

SPy_1790


SPy_0527


Q9A109

SPy_0604


Q9A0V0

SPy_2099

Putative transcriptional (GntR family)

SPy_0400

Putative DNA polymerase III delta' Q9A189 subunit

SPy_1549

argR1

(ATP- Q99YA6

regulator Q99XQ3

Arginine regulator

Q99YT5

SPy_1507

Putative amino acid ABC transporter Q99YW8 (Permease protein)

SPy_0233


Q9A1K3

SPy_0357


Q9A1C0

SPy_0390


Q9A195

SPy_0475

dgk

Diacylglycerol kinase

Q9A142

SPy_1648

recU

Holliday junction resolvase RecU

Q99YL2


Q99Z72

Streptolysin S associated ORF

Q9A0K4

SPy_1366 SPy_0740

sagC


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Locus tag

Gene name

SPy_0507 SPy_0856

mur1.1

Protein annotation

UniProt ID


Q9A124

Putative peptidoglycan hydrolase

Q9A0A9

SPy_0242

Putative histidine kinase, possibly Q9A1J7 involved in competence

SPy_1255


Q99ZF1

SPy_0437


Q9A168

SPy_1257


(ATP- Q99ZF0

SPy_0876

mvaK1

Mevalonate kinase

Q9A098

SPy_2182

holB

Replicative DNA helicase

Q99XJ1


Q9A174

Putative mutator protein

Q99YW5

SPy_1879


Q99Y39

SPy_0685


Q9A0P0

SPy_0428 SPy_1510

mutT

Unique candidates were identified by Venn analysis (http://bioinformatics.psb.ugent.be/webtools/Venn/) of proteins detected by shotgun mass spectrometry of cellular fractions, using a cut-off of 1 for the mean average spectral count. Number of unique proteins detected in supernatant was n = 6, in cell wall n = 24, in cytoplasm n = 15, in crude membrane n = 7, in membrane associated proteins n = 3 and in purified membrane n = 76. Proteins are listed in descending order in relation to relative abundance within each fraction, together with their respective annotation, UniProt identifier, gene name (when available) and locus. Spy-numbers refer to the S. pyogenes SF370 (serotype M1) genome.

Table 3. Cell wall assigned proteins detected in Streptococcus pyogenes

Locus tag

Gene name

SPy_2010 scpA

Protein annotation

UniProt ID

Prediction tool

MS analysis of cell wall fraction

C5a peptidase (SCP)

P58099

CW-Pred2;

detected (114±5)

PSORTb SPy_0416 prtS

Putative cell envelope Q9A180 proteinase

CW-Pred2;

detected (104±22)



Locus tag

Gene name

Protein annotation

UniProt ID

Prediction tool

Page 46 of 59


PSORTb SPy_2018 emm1

M protein type 1

Q99XV0

CW-Pred2;

detected (103±12)

PSORTb Immunoglobulin G- P50470 binding protein H

CW-Pred2;

Collagen-like surface Q7DAL3 protein

CW-Pred2;

SPy_2000 dppA

Surface lipoprotein

Q99XV9

PSORTb

detected (17±13)

SPy_2009


Q99XV2

CW-Pred2;

detected (7±8)


Q9A0C0


Q9A1S0

SPy_1798


Q99YA0

SPy_1054

SPy_1983 scl

SPy_0843

SPy_0130

SPy_1972 pulA

detected (44±13)

PSORTb detected (24±5)

PSORTb

PSORTb CW-Pred2;

detected (6±2)

PSORTb CW-Pred2;

detected (5±3)

PSORTb PSORTb

detected (5±6)

Putative collagen-like Q99ZV7 protein

CW-Pred2;

detected (4±2)

Putative pullulanase

CW-Pred2;

Q99XX8

PSORTb detected (3±2)

PSORTb SPy_1801 isp2

Immunogenic secreted-like protein

Q99Y99

PSORTb

detected (2±2)

SPy_0747


Q9A0J7

PSORTb

detected (2±1)*

SPy_1357 grab

Protein (Protein

CW-Pred2;

not present in AP1

GRAB Q7DAL7 G-related


Page 47 of 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Locus tag

Gene name

Protein annotation alpha protein)

SPy_1494

UniProt ID

2M-binding

Prediction tool


PSORTb

S. pyogenes

CW-Pred2;

not detected (in any cellular fraction)


Q99YX9

SPy_1558


Q99YT0

PSORTb


SPy_1361 inlA

Putative internalin A

Q99Z76

PSORTb


SPy_1523 divIB

Cell division protein Q99YV5 DivIB

PSORTb

not detected in cell wall;

PSORTb

only detected in membrane

SPy_0872

SPy_0737 epf

Putative secreted 5'- Q9A0A2 nucleotidase

CW-Pred2;

Putative extracellular Q9A0K5 matrix binding protein

CW-Pred2;

PSORTb

PSORTb

only detected if cut-off < 1 for mean average spectral count is used not detected in cell wall; only detected in membrane if cutoff < 1 for mean average count is used

The SF370 (serotype M1) proteome (Proteome ID: UP000000750) was searched for putative cell wall proteins with PSORTb v3.0.229 (n = 20). In addition, predicted cell wall proteins (n = 14) were retrieved from the CW-Pred2 database34 for S. pyogenes M1 GAS. Protein H was also analysed by CW-Pred2 and PSORTb and added to the list. Predicted proteins detected in the cell wall fraction by shotgun mass spectrometry are listed in descending order (mean average spectral count and standard deviation), applying a cut-off of 1 for the mean average spectral count. Proteins labeled with an asterisk (*) are solely detected in the cell wall fraction. UniProt identifier, gene name (when available) and locus are given. Spy-numbers refer to the S. pyogenes SF370 (serotype M1) genome.



Page 48 of 59

Table 4. Prevalent proteins in the Streptococcus pyogenes plasma membrane Locus tag Gene name

Protein annotation

UniProt ID

Protein localisation

MS analysis of purified membrane fraction

SPy_1315


Q99ZA4

Integral

194±27

SPy_1884

Eukaryotic hypersensitive-induced response-like protein

Q99Y37

Integral

104±35

SPy_1316

Putative ABC transporter Q99ZA3 (ATP-binding protein)

-

104±42

SPy_0760 atpD

ATP synthase subunit Q9A0I7 beta

Membrane

103±33

Lipoprotein

96±10

(F-ATPase subunit beta) SPy_1390 prsA1

Foldase protein PrsA 1

P60811

SPy_1740 manN

Putative mannose- Q99YE4 specific phosphotransferase system component IID

Integral

90±25

SPy_0453 mtsA

Metal ABC transporter P0A4G4 substrate-binding lipoprotein

Lipoprotein

84±20

SPy_0015 ftsH

ATP-dependent zinc Q9A200 metalloprotease FtsH

Integral

77±29

SPy_2037 prsA2


Lipoprotein

71±7

SPy_1227

Putative sugar ABC Q99ZH5 transporter (ATP-binding protein)

-

70±8

SPy_0855

Putative fructose-specific Q9A0B0 enzyme II, PTS system BC component

Integral

67±16

SPy_1625


Q99YN0

Integral

66±6

SPy_0097 pbp1b

Putative penicillin- Q9A1U2 binding protein 1b

Integral

63±4

SPy_0296 oppD

Oligopeptidepermease (ATP-binding protein)

-

61±16

P60812

Q9A1F7


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Locus tag Gene name

Protein annotation

UniProt ID

Protein localisation


SPy_1748 fabF

3-oxoacyl-[acyl-carrierprotein] synthase 2

Q99YD7

-

58±23

SPy_0294 oppB

Oligopeptidepermease

Q9A1F9

Integral

56±16

SPy_0623 pacL

Putative calcium- Q9A0T9 transporting ATPase

Integral

56±27

SPy_0297 oppF

Oligopeptide ATP-binding OppF

Membrane

50±14

transport P0A2V6 protein

SPy_0184 opuABC Putative glycine-betaine Q9A1M9 Integral binding permease protein

49±16

SPy_1963

49±10

Putative metalloprotease

zinc Q99XY3

Integral

The 20 most prominent proteins both assigned to the membrane by PSORTb and detected in the purified membrane fraction by shotgun mass spectrometry are listed in descending order (mean average spectral count and standard deviation). A cut-off of 1 for the mean average spectral count was applied. Note that PSORTb does not detect lipoprotein motifs and thus predicted lipoproteins from Table 5 are listed under the section protein localisation. Proteins predicted to be an integral component of membrane (gene ontology cellular compartment, GO:0016021) or to be located at the membrane (gene ontology cellular compartment, GO:0005886) are listed there as well. Protein annotation, UniProt identifier, gene name (when available) and locus are given. Spy-numbers refer to the S. pyogenes SF370 (serotype M1) genome.

Table 5. Predicted lipoproteins detected in Streptococcus pyogenes

Protein annotation

UniProt ID


SPy_1228


Q99ZH4

detected (213±53)

SPy_2000 dppA

Surface lipoprotein

Q99XV9

detected (121±28)

SPy_1294

Putative maltose/maltodextrinbinding protein

Q99ZC0

detected (101±2)

SPy_1390 prsA1


P60811

detected (96±10)

Locus tag

Gene name



Locus tag

Gene name

Protein annotation

UniProt ID

Page 50 of 59


SPy_0453 mtsA

Metal ABC transporter P0A4G4 substrate-binding lipoprotein

detected (84±20)

SPy_2037 prsA2


detected (71±7)

SPy_0903

Putative ABC transporter Q9A074 (Binding protein)

detected (56±11)

SPy_0317


Q9A1E5

detected (50±3)

SPy_0163

Putative ABC transporter Q9A1P7 (Lipoprotein)

detected (45±4)

SPy_0351 yidC2

Membrane protein insertase Q9A1C3 YidC 2 (Foldase YidC 2)

detected (33±2)

SPy_1306 malX

Maltose/maltodextrinbinding protein

Q99ZB1

detected (24±9) ‡

SPy_1882 lppC

Putative acid phosphatase

Q99Y38

detected (19±24)

SPy_1274

Putative amino acid ABC Q99ZD7 transporter, periplasmic amino acid-binding protein

detected (17±9)

SPy_1245 pstS

Putative phosphate ABC Q99ZG1 transporter, periplasmic phosphate-binding protein

detected (16±2)

SPy_0457

Peptidyl-prolyl cis-trans Q9A156 isomerase (PPIase)

detected (11±15)

SPy_0385 fhuD

Ferrichrome ABC Q9A198 transporter (Ferrichromebinding protein)

detected (10±5) ‡

SPy_0247 yidC1

Membrane protein insertase P65631 YidC 1 (Foldase YidC 1)

detected (8±1) *‡

SPy_0210


Q9A1L2

detected (7±7) *

SPy_0319

Lipoprotein

Q9A1E4

detected (5±2)

SPy_2033


Q99XU1

detected (5±7)

SPy_2007 lmb

Putative laminin adhesion

Q99XV3


SPy_1795

Putative ABC transporter Q99YA2 (Periplasmic binding protein)


P60812


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Locus tag

Gene name

Protein annotation

UniProt ID


Putative ABC transporter Q99YQ3 substrate binding lipoprotein

detected (2±1) ‡

SPy_0778

Putative ABC transporter Q9A0H2 (Substrate-binding protein)


SPy_1290


Q99ZC4


SPy_0604


Q9A0V0

detected (1±1)

SPy_1592

(only detectable purified membrane)

(only detectable purified membrane)

peptidoglycan Q9A0A8

in

in

SPy_0857 mur1.2

Putative hydrolase

not detected (only detectable in supernatant)

SPy_0252

Putative sugar transporter Q9A1I9 sugar binding lipoprotein

not detected (only detected in purified membrane if cut-off < 1 for mean average count is used; but detectable in crude membrane) ‡

SPy_1558


Q99YT0

not detected (in cellular fraction)

any

SPy_1405


Q99Z45


any

SPy_1361 inlA

Putative internalin A

Q99Z76


any

Predicted lipoproteins (n = 31) were retrieved from the Pred-Lipo database for S. pyogenes M1 GAS46. Proteins are given with annotation, UniProt identifier, gene name (when available) and locus. Spy-numbers refer to the S. pyogenes SF370 (serotype M1) genome. A total of n = 27 lipoproteins were detected. Lipoproteins detected in the purified membrane fraction (n = 26) by shotgun mass spectrometry are listed in descending order (mean average spectral count and standard deviation). A cut-off of 1 for the mean average spectral count was used. Proteins labeled with an asterisk (*) were solely detected in both crude and purified membrane fractions. Lipoproteins newly confirmed in this study (n = 9) are labeled with a double cross (‡).

Table 6. Subcellular localisation of putative new drug targets in Streptococcus pyogenes 51 ACS Paragon Plus Environment


Page 52 of 59

Locus tag Gene name

Protein annotation

UniProt ID

SPy_1188 citE

Putative citrate lyase, beta subunit

Q99ZK8

SPy_0215 pgi

Glucose-6-phosphate isomerase (GPI)

Q9A1L1

SPy_0890 deoB

Phosphopentomutase

P63927

SPy_1224 pgmA

Putative phosphoglucomutase

Q99ZH8

SPy_1750 fabD

Malonyl CoA-acyl carrier protein transacylase

Q99YD5

SPy_0755 atpB

ATP synthase subunit a (F-ATPase subunit 6)

Q9A0J2

SPy_0027 purM

Phosphoribosylformylglycinamidine cyclo-ligase Q9A1Z0

SPy_0362

Non-canonical purine NTP pyrophosphatase Q9A1B6 (NTPase)

SPy_0003 dnaN

DNA polymerase III subunit beta

SPy_0160 purA

Adenylosuccinate synthetase (AMPSase) (AdSS) Q9A1P8

SPy_0462 pyrH

Uridylate kinase (UK)

P65938

SPy_2190 sdhA

Putative L-serine dehydratase alpha subunit

Q99XI5

SPy_0911 bcaT

Branched-chain-amino-acid aminotransferase

Q9A068

SPy_1576

Putative chorismate mutase

Q99YR4

SPy_0810 aroC

Chorismate synthase (CS)

Q9A0E4

SPy_0361 murI

Glutamate racemase

Q9A1B7

SPy_0251 nanE

Putative N-acetylmannosamine-6-phosphate 2- P65522 epimerase

SPy_1101 murB

UDP-N-acetylenolpyruvoylglucosamine reductase

Q99ZS9

SPy_0280 uppP

Undecaprenyl-diphosphatase

P67392

SPy_1664 pbpX

Putative penicillin binding protein 2X

Q99YK1

SPy_1684 glpK

Glycerol kinase (GK)

Q99YI7

SPy_0226 gpsA

Glycerol-3-phosphate dehydrogenase [NAD(P)+] P68887

SPy_1110

Putative malic enzyme ((S)-malate:NAD+ Q99ZS1 oxidoreductase (Decarboxylating))

SPy_1219

Putative trimethylamine dehydrogenase

Q9A209

Q99ZI3


Page 53 of 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


SPy_1126 nadK

NAD kinase

SPy_0197 nadC

Putative nicotinate-nucleotide pyrophosphorylase Q9A1L9

SPy_1362 birA

Putative biotin operon repressor

Q99Z75

SPy_1098 folP

Dihydropteroate synthase (DHPS)

P0C0G1

Uniprot ID

Cell wall

Secreted extracellular

P65781

Cytoplasm Crude Membrane- Purified membrane associated membrane peripheral

proteins

proteins

Q99ZK8

-

-

Q9A1L1

3±6

P63927

-

-

-

-

49±25 56±6

6±3

4±4

9±0

-

26±7

6±8

1±2

8±6

Q99ZH8

-

54±36 38±6

19±17

2±2

28±12

Q99YD5

2±3

29±19 26±9

8±6

2±1

11±6

Q9A0J2

-

-

-

-

-

3±1

Q9A1Z0

-

-

-

-

-

-

Q9A1B6

-

5±2

4±0

1±2

-

2±1

Q9A209

-

19±15 17±2

7±3

-

9±2

Q9A1P8

-

58±19 40±10

13±4

3±2

20±3

P65938

-

2±3

3±2

6±5

1±1

8±5

Q99XI5

-

9±8

9±4

7±7

1±2

10±7

Q9A068

-

24±12 16±5

3±3

-

4±3

Q99YR4

-

-

5±5

-

9±4

12±5

-



Page 54 of 59

Q9A0E4

-

-

-

2±1

-

2±2

Q9A1B7

-

-

-

2±1

-

4±1

P65522

-

-

-

-

-

-

Q99ZS9

-

5±1

6±5

2±1

-

5±4

P67392

-

-

-

-

-

2±0

Q99YK1

-

-

-

9±10

4±4

11±8

Q99YI7

-

-

-

-

-

-

P68887

-

17±13 14±3

12±7

2±0

19±4

Q99ZS1

-

-

-

-

-

-

Q99ZI3

-

-

-

-

-

-

P65781

-

-

-

9±5

4±4

6±5

Q9A1L9

-

-

-

-

-

-

Q99Z75

-

-

3±2

5±5

-

8±6

P0C0G1

-

4±4

7±3

4±3

-

6±4

The cellular distribution of 28 novel drug target proteins, identified by Singh et al48, was analysed in proteome data obtained by shotgun mass spectrometry. A cut-off of 1 for the mean average spectral count was applied to subcellular proteome data and then drug targets were localised. Searched drug targets are given in the top part of the table with respective annotation, UniProt identifier, gene name (when available) and locus. Spy-numbers refer to the S. pyogenes SF370 (serotype M1) genome. Protein localisation and mean average spectral count with standard deviation for each subproteom are given in the lower part of the table. - = not detected

References

1. Carapetis, J. R.; Steer, A. C.; Mulholland, E. K.; Weber, M., The global burden of group A streptococcal diseases. Lancet Infect Dis 2005, 5, (11), 685-94. 2. Cunningham, M. W., Pathogenesis of group A streptococcal infections and their sequelae. Adv Exp Med Biol 2008, 609, 29-42. 3. Callegari, E. A.; Chaussee, M. S., The Streptococcal Proteome. In Streptococcus pyogenes: Basic Biology to Clinical Manifestations [Online]; Ferretti, J. J.; Stevens, D. L.; Fischetti, V. A., Eds.; Oklahoma City (OK): University of Oklahoma Health Sciences Center, 2016; https://www.ncbi.nlm.nih.gov/books/NBK333416/ (accessed June 1, 2017). 4. Krogh, A.; Larsson, B.; von Heijne, G.; Sonnhammer, E. L., Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001, 305, (3), 567-80. 54 ACS Paragon Plus Environment

Page 55 of 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5. Navarre, W. W.; Schneewind, O., Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol Mol Biol Rev 1999, 63, (1), 174-229. 6. Silhavy, T. J.; Kahne, D.; Walker, S., The bacterial cell envelope. Cold Spring Harb Perspect Biol 2010, 2, (5), a000414. 7. Scott, J. R.; Barnett, T. C., Surface proteins of gram-positive bacteria and how they get there. Annu Rev Microbiol 2006, 60, 397-423. 8. Schneewind, O.; Missiakas, D., Sec-secretion and sortase-mediated anchoring of proteins in Gram-positive bacteria. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2014, 1843, (8), 1687-1697. 9. Fischetti, V. A., M Protein and Other Surface Proteins on Streptococci. In Streptococcus pyogenes: Basic Biology to Clinical Manifestations [Online]; Ferretti, J. J.; Stevens, D. L.; Fischetti, V. A., Eds.; Oklahoma City (OK): University of Oklahoma Health Sciences Center, 2016; https://www.ncbi.nlm.nih.gov/books/NBK333431/ (accessed June 1, 2017). 10. Matias, V. R.; Beveridge, T. J., Cryo-electron microscopy reveals native polymeric cell wall structure in Bacillus subtilis 168 and the existence of a periplasmic space. Mol Microbiol 2005, 56, (1), 240-51. 11. Hutchings, M. I.; Palmer, T.; Harrington, D. J.; Sutcliffe, I. C., Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold 'em, knowing when to fold 'em. Trends Microbiol 2009, 17, (1), 13-21. 12. Xie, K.; Dalbey, R. E., Inserting proteins into the bacterial cytoplasmic membrane using the Sec and YidC translocases. Nature Reviews Microbiology 2008, 6, (3), 234-244. 13. Lei, B.; Mackie, S.; Lukomski, S.; Musser, J. M., Identification and immunogenicity of group A Streptococcus culture supernatant proteins. Infect Immun 2000, 68, (12), 6807-18. 14. Rodriguez-Ortega, M. J.; Norais, N.; Bensi, G.; Liberatori, S.; Capo, S.; Mora, M.; Scarselli, M.; Doro, F.; Ferrari, G.; Garaguso, I.; Maggi, T.; Neumann, A.; Covre, A.; Telford, J. L.; Grandi, G., Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome. Nat Biotechnol 2006, 24, (2), 191-7. 15. Malmström, J.; Karlsson, C.; Nordenfelt, P.; Ossola, R.; Weisser, H.; Quandt, A.; Hansson, K.; Aebersold, R.; Malmström, L.; Björck, L., Streptococcus pyogenes in Human Plasma ADAPTIVE MECHANISMS ANALYZED BY MASS SPECTROMETRY-BASED PROTEOMICS. Journal of Biological Chemistry 2012, 287, (2), 1415-1425. 16. Sharma, A.; Arya, D. K.; Sagar, V.; Bergmann, R.; Chhatwal, G. S.; Johri, A. K., Identification of potential universal vaccine candidates against group A Streptococcus by using high throughput in silico and proteomics approach. J Proteome Res 2013, 12, (1), 33646. 17. Cole, J. N.; Djordjevic, S. P.; Walker, M. J., Isolation and solubilization of gram-positive bacterial cell wall-associated proteins. Methods Mol Biol 2008, 425, 295-311. 18. Malmström, E.; Kilsgård, O.; Hauri, S.; Smeds, E.; Herwald, H.; Malmström, L.; Malmström, J., Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics. Nature Communications 2016, 7, 10261. 19. Vizcaino, J. A.; Deutsch, E. W.; Wang, R.; Csordas, A.; Reisinger, F.; Rios, D.; Dianes, J. A.; Sun, Z.; Farrah, T.; Bandeira, N.; Binz, P. A.; Xenarios, I.; Eisenacher, M.; Mayer, G.; Gatto, L.; Campos, A.; Chalkley, R. J.; Kraus, H. J.; Albar, J. P.; Martinez-Bartolome, S.; Apweiler, R.; Omenn, G. S.; Martens, L.; Jones, A. R.; Hermjakob, H., ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat Biotechnol 2014, 32, (3), 223-6. 55 ACS Paragon Plus Environment


Page 56 of 59

20. Teleman, J.; Dowsey, A. W.; Gonzalez-Galarza, F. F.; Perkins, S.; Pratt, B.; Rost, H. L.; Malmstrom, L.; Malmstrom, J.; Jones, A. R.; Deutsch, E. W.; Levander, F., Numerical compression schemes for proteomics mass spectrometry data. Mol Cell Proteomics 2014, 13, (6), 1537-42. 21. Chambers, M. C.; Maclean, B.; Burke, R.; Amodei, D.; Ruderman, D. L.; Neumann, S.; Gatto, L.; Fischer, B.; Pratt, B.; Egertson, J.; Hoff, K.; Kessner, D.; Tasman, N.; Shulman, N.; Frewen, B.; Baker, T. A.; Brusniak, M. Y.; Paulse, C.; Creasy, D.; Flashner, L.; Kani, K.; Moulding, C.; Seymour, S. L.; Nuwaysir, L. M.; Lefebvre, B.; Kuhlmann, F.; Roark, J.; Rainer, P.; Detlev, S.; Hemenway, T.; Huhmer, A.; Langridge, J.; Connolly, B.; Chadick, T.; Holly, K.; Eckels, J.; Deutsch, E. W.; Moritz, R. L.; Katz, J. E.; Agus, D. B.; MacCoss, M.; Tabb, D. L.; Mallick, P., A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotechnol 2012, 30, (10), 918-20. 22. Craig, R.; Beavis, R. C., A method for reducing the time required to match protein sequences with tandem mass spectra. Rapid Commun Mass Spectrom 2003, 17, (20), 2310-6. 23. UniProt, C., UniProt: a hub for protein information. Nucleic Acids Res 2015, 43, (Database issue), D204-12. 24. Keller, A.; Nesvizhskii, A. I.; Kolker, E.; Aebersold, R., Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Analytical Chemistry 2002, 74, (20), 5383-5392. 25. Nesvizhskii, A. I.; Keller, A.; Kolker, E.; Aebersold, R., A statistical model for identifying proteins by tandem mass spectrometry. Analytical Chemistry 2003, 75, (17), 4646-4658. 26. Fermin, D.; Basrur, V.; Yocum, A. K.; Nesvizhskii, A. I., Abacus: A computational tool for extracting and pre-processing spectral count data for label-free quantitative proteomic analysis. Proteomics 2011, 11, (7), 1340-1345. 27. Smith, S. M., Strategies for the purification of membrane proteins. Methods Mol Biol 2011, 681, 485-96. 28. Ferretti, J. J.; McShan, W. M.; Ajdic, D.; Savic, D. J.; Savic, G.; Lyon, K.; Primeaux, C.; Sezate, S.; Suvorov, A. N.; Kenton, S.; Lai, H. S.; Lin, S. P.; Qian, Y.; Jia, H. G.; Najar, F. Z.; Ren, Q.; Zhu, H.; Song, L.; White, J.; Yuan, X.; Clifton, S. W.; Roe, B. A.; McLaughlin, R., Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc Natl Acad Sci U S A 2001, 98, (8), 4658-63. 29. Yu, N. Y.; Wagner, J. R.; Laird, M. R.; Melli, G.; Rey, S.; Lo, R.; Dao, P.; Sahinalp, S. C.; Ester, M.; Foster, L. J.; Brinkman, F. S. L., PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 2010, 26, (13), 1608-1615. 30. Hynes, W.; Sloan, M., Secreted Extracellular Virulence Factors. In Streptococcus pyogenes: Basic Biology to Clinical Manifestations [Online]; Ferretti, J. J.; Stevens, D. L.; Fischetti, V. A., Eds.; Oklahoma City (OK): University of Oklahoma Health Sciences Center, 2016; https://www.ncbi.nlm.nih.gov/books/NBK333411/ (accessed June 1, 2017). 31. Bensi, G.; Mora, M.; Tuscano, G.; Biagini, M.; Chiarot, E.; Bombaci, M.; Capo, S.; Falugi, F.; Manetti, A. G.; Donato, P.; Swennen, E.; Gallotta, M.; Garibaldi, M.; Pinto, V.; Chiappini, N.; Musser, J. M.; Janulczyk, R.; Mariani, M.; Scarselli, M.; Telford, J. L.; Grifantini, R.; Norais, N.; Margarit, I.; Grandi, G., Multi high-throughput approach for highly selective identification of vaccine candidates: the Group A Streptococcus case. Mol Cell Proteomics 2012, 11, (6), M111 015693. 32. Berge, A.; Björck, L., Streptococcal cysteine proteinase releases biologically active fragments of streptococcal surface proteins. J Biol Chem 1995, 270, (17), 9862-7. 56 ACS Paragon Plus Environment

Page 57 of 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


33. McIver, K. S.; Subbarao, S.; Kellner, E. M.; Heath, A. S.; Scott, J. R., Identification of isp, a locus encoding an immunogenic secreted protein conserved among group A streptococci. Infect Immun 1996, 64, (7), 2548-55. 34. Fimereli, D. K.; Tsirigos, K. D.; Litou, Z. I.; Liakopoulos, T. D.; Bagos, P. G.; Hamodrakas, S. J., CW-PRED: A HMM-Based Method for the Classification of Cell Wall-Anchored Proteins of Gram-Positive Bacteria. In Artificial Intelligence: Theories and Applications: 7th Hellenic Conference on AI, SETN 2012, Lamia, Greece, May 28-31, 2012. Proceedings, Maglogiannis, I.; Plagianakos, V.; Vlahavas, I., Eds. Springer Berlin Heidelberg: Berlin, Heidelberg, 2012; pp 285290. 35. Rasmussen, M.; Müller, H.-P.; Björck, L., Protein GRAB of Streptococcus pyogenes Regulates Proteolysis at the Bacterial Surface by Binding α2-Macroglobulin. Journal of Biological Chemistry 1999, 274, (22), 15336-15344. 36. Henningham, A.; Chiarot, E.; Gillen, C. M.; Cole, J. N.; Rohde, M.; Fulde, M.; Ramachandran, V.; Cork, A. J.; Hartas, J.; Magor, G.; Djordjevic, S. P.; Cordwell, S. J.; Kobe, B.; Sriprakash, K. S.; Nizet, V.; Chhatwal, G. S.; Margarit, I. Y.; Batzloff, M. R.; Walker, M. J., Conserved anchorless surface proteins as group A streptococcal vaccine candidates. J Mol Med (Berl) 2012, 90, (10), 1197-207. 37. Severin, A.; Nickbarg, E.; Wooters, J.; Quazi, S. A.; Matsuka, Y. V.; Murphy, E.; Moutsatsos, I. K.; Zagursky, R. J.; Olmsted, S. B., Proteomic analysis and identification of Streptococcus pyogenes surface-associated proteins. J Bacteriol 2007, 189, (5), 1514-22. 38. Henningham, A.; Dohrmann, S.; Nizet, V.; Cole, J. N., Mechanisms of group A Streptococcus resistance to reactive oxygen species. FEMS Microbiol Rev 2015, 39, (4), 488508. 39. Arts, I. S.; Gennaris, A.; Collet, J. F., Reducing systems protecting the bacterial cell envelope from oxidative damage. FEBS Lett 2015, 589, (14), 1559-68. 40. Shepherd, J.; Ibba, M., Direction of aminoacylated transfer RNAs into antibiotic synthesis and peptidoglycan-mediated antibiotic resistance. Febs Letters 2013, 587, (18), 2895-2904. 41. Anand, S. P.; Khan, S. A., Structure-specific DNA binding and bipolar helicase activities of PcrA. Nucleic Acids Res 2004, 32, (10), 3190-7. 42. Mitchell, A.; Chang, H. Y.; Daugherty, L.; Fraser, M.; Hunter, S.; Lopez, R.; McAnulla, C.; McMenamin, C.; Nuka, G.; Pesseat, S.; Sangrador-Vegas, A.; Scheremetjew, M.; Rato, C.; Yong, S. Y.; Bateman, A.; Punta, M.; Attwood, T. K.; Sigrist, C. J.; Redaschi, N.; Rivoire, C.; Xenarios, I.; Kahn, D.; Guyot, D.; Bork, P.; Letunic, I.; Gough, J.; Oates, M.; Haft, D.; Huang, H.; Natale, D. A.; Wu, C. H.; Orengo, C.; Sillitoe, I.; Mi, H.; Thomas, P. D.; Finn, R. D., The InterPro protein families database: the classification resource after 15 years. Nucleic Acids Res 2015, 43, (Database issue), D213-21. 43. Wang, C. H.; Lin, C. Y.; Luo, Y. H.; Tsai, P. J.; Lin, Y. S.; Lin, M. T.; Chuang, W. J.; Liu, C. C.; Wu, J. J., Effects of oligopeptide permease in group a streptococcal infection. Infect Immun 2005, 73, (5), 2881-90. 44. Weigel, P. H., Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior. Int J Cell Biol 2015, 2015, 367579. 45. Argüello, J. M.; González-Guerrero, M.; Raimunda, D., Bacterial transition metal P(1B)ATPases: transport mechanism and roles in virulence. Biochemistry 2011, 50, (46), 9940-9.



Page 58 of 59

46. Bagos, P. G.; Tsirigos, K. D.; Liakopoulos, T. D.; Hamodrakas, S. J., Prediction of lipoprotein signal peptides in Gram-positive bacteria with a Hidden Markov Model. J Proteome Res 2008, 7, (12), 5082-93. 47. Wahid, R. M.; Yoshinaga, M.; Nishi, J.; Maeno, N.; Sarantuya, J.; Ohkawa, T.; Jalil, A. M.; Kobayashi, K.; Miyata, K., Immune response to a laminin-binding protein (Lmb) in group A streptococcal infection. Pediatr Int 2005, 47, (2), 196-202. 48. Singh, S.; Singh, D. B.; Singh, A.; Gautam, B.; Ram, G.; Dwivedi, S.; Ramteke, P. W., An Approach for Identification of Novel Drug Targets in Streptococcus pyogenes SF370 Through Pathway Analysis. Interdiscip Sci 2016, 8, (4), 388-94. 49. Henderson, B.; Martin, A., Bacterial virulence in the moonlight: multitasking bacterial moonlighting proteins are virulence determinants in infectious disease. Infect Immun 2011, 79, (9), 3476-91. 50. Lei, B.; Liu, M.; Chesney, G. L.; Musser, J. M., Identification of new candidate vaccine antigens made by Streptococcus pyogenes: purification and characterization of 16 putative extracellular lipoproteins. J Infect Dis 2004, 189, (1), 79-89. 51. Hurdle, J. G.; O'Neill, A. J.; Chopra, I.; Lee, R. E., Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections. Nat Rev Micro 2011, 9, (1), 62-75. 52. Antibiotic resistance threats in the United States, 2013. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention Web site. https://www.cdc.gov/drugresistance/threat-report-2013/ (accessed June 1, 2017).


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“for TOC only”


A comprehensive mass spectrometric survey of Streptococcus

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