Proteomic Characterization of a Natural HostâPathogen Interaction

Article pubs.acs.org/jpr

Proteomic Characterization of a Natural Host−Pathogen Interaction: Repertoire of in Vivo Expressed Bacterial and Host SurfaceAssociated Proteins Megan A. Rees,†,‡,§ Oded Kleifeld,§ Paul K. Crellin,†,‡ Bosco Ho,‡ Timothy P. Stinear,‡,∥ A. Ian Smith,*,†,§ and Ross L. Coppel†,‡ †

Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, ‡Department of Microbiology, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia ∥ Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia §

S Supporting Information *

ABSTRACT: Interactions between a host and a bacterial pathogen are mediated by cross-talk between molecules present on, or secreted by, pathogens and host binding-molecules. Identifying proteins involved at this interface would provide substantial insights into this interaction. Although numerous studies have examined in vitro models of infection at the level of transcriptional change and proteomic profiling, there is virtually no information available on naturally occurring host−pathogen interactions in vivo. We employed membrane shaving to identify peptide fragments cleaved from surface-expressed bacterial proteins and also detected proteins originating from the infected host. We optimized this technique for media-cultured Corynebacterium pseudotuberculosis, a sheep pathogen, revealing a set of 247 surface proteins. We then studied a natural host− pathogen interaction by performing membrane shaving on C. pseudotuberculosis harvested directly from naturally infected sheep lymph nodes. Thirty-one bacterial surface proteins were identified, including 13 not identified in culture media, suggesting that a different surface protein repertoire is expressed in this hostile environment. Forty-nine host proteins were identified, including immune mediators and antimicrobial peptides such as cathelicidin. This novel application of proteolytic shaving has documented sets of host and pathogen proteins present at the bacterial surface in an infection of the native host. KEYWORDS: Proteomics, Corynebacterium pseudotuberculosis, Mycobacterium, surface proteins

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INTRODUCTION Proteins either secreted from, or located on, the outer surface of a bacterial pathogen may be involved in interactions with infected hosts. Some surface proteins also mediate a bacterium’s interaction with the environment by sensing the chemical and physical conditions of the external milieu and sending the appropriate signals to the cytoplasm to trigger responses.1 Surface-expressed proteins also transport nutrients, waste products, secreted virulence factors, and other compounds, which are important for bacterial growth and competition with other microbes.2 During infection, membrane proteins have important roles in mediating interactions with the host, including adherence, internalization, toxin synthesis, and escape from the host immune system.3 In addition, it has been proposed that cytoplasmic proteins released from lysed cells into the extracellular environment can bind to the surface of intact cells,4 where they may perform an alternate function.5 The myriad roles of surface proteins has led to great interest in the determination of the surfaceomes of bacterial pathogens as an initial step toward identifying all interactions between pathogen and host and to narrow the search for protective antigens.6 © XXXX American Chemical Society

When developing proteomic strategies to identify membrane proteins,7 it is important to acknowledge that membraneassociated proteins can include proteins embedded in the membrane via one or multiple transmembrane helices as well as peripheral proteins that have a looser association with the membrane, usually via some form of protein−protein interaction.8 In contrast to genome sequencing studies, proteomics can provide information about the expressed repertoire of proteins: their degradation or modification and location.9 However, the low abundance and hydrophobicity of many membrane proteins have been significant impediments to surfaceome determination, especially in some of the earlier proteomic efforts that used 2D gel electrophoresis for protein separation and quantitation.10 The surface or membrane shaving technique aims to identify the in vivo expressed surface proteins of a microorganism (Figure 1). This proteomic strategy was initially developed in Gram-positive bacteria,11 was later adapted to Gram-negative Special Issue: Environmental Impact on Health Received: September 28, 2014

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dx.doi.org/10.1021/pr5010086 | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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Figure 1. Membrane shaving as a tool to identify proteins on the surface of bacteria. In this approach, live bacteria are exposed to the protease trypsin, which cleaves peptide fragments from exposed surface proteins. These peptides remain in the supernatant after centrifugation and filtration to remove the cells. Peptides in the supernatant are then desalted and concentrated prior to identification by mass spectrometry.

organisms,3,12 and has recently been used to characterize proteins in some mycobacterial species1314 and some eukaryotes.15 The technique is based on the proteolytic cleavage of surface-exposed proteins to generate peptide fragments that are separated and identified by mass spectrometry and then matched to whole proteins predicted from an annotated genome. The membrane shaving strategy may also identify adherent or membraneassociated host proteins in an in vitro setting.16 Although numerous studies have examined laboratory inducible proteins in various in vitro models of infection, both at the level of transcriptome17 and proteome profiles,18 there is virtually no information available on naturally occurring host−pathogen interactions in vivo. Here, we have documented sets of host and pathogen proteins in an infection of the native host. In this study, we have applied the membrane shaving technique to bacteria harvested directly from naturally infected sheep lymph nodes as an example of mycobacterial-like infection. Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CLA), which occurs globally, primarily affecting ruminant animals19 and occasionally humans.20 CLA is clinically and histologically similar to caseous lymphadenitis, caused by Mycobacterium tuberculosis in humans,21 which is the most common form of extra-pulmonary tuberculosis. Mycobacteria and Corynebacteria are closely genetically related and share many cell wall attributes.22 Accordingly, identifying proteins involved in this host−pathogen interaction may provide valuable insight into a significant mycobacterial disease of humans. Resistance to infection with C. pseudotuberculosis involves both humoral and cell-mediated immunity.23 CLA is a florid inflammatory process including granuloma formation and necrosis that is macroscopically confined to the thickened

capsule of the lymph node. This complex process is likely to involve both surface and secreted bacterial proteins as well as multiple components of the host immune system. Studies characterizing surface-expressed proteins of C. pseudotuberculosis have not been reported. Predictions of the potential surface localization24 of proteins have been made,25 resulting in an in silico surface proteome for C. pseudotuberculosis, but experimental validation is lacking. Proteomics has been used to identify the repertoire of exported proteins,26 and this preparation was able to elicit a humoral response but not substantial protection from a bacterial challenge in goats.27 Secreted proteins are important in C. pseudotuberculosis, with phospholipase D and CP40 being recognized as virulence factors that can elicit a specific immune response.28 A greater interferon-γ response in vitro has been documented to secreted antigens than to whole cell lysates.29 Additional virulence factors have been postulated, 30 including by genomic comparison studies.31 In M. tuberculosis infection, individual secreted proteins, such as ESAT 6, although important, are not the only agents responsible for driving the host immune response.32 Therefore, it is likely there are as yet unidentified host and bacterial components involved in this complex immune response. For this reason, capturing surface protein expression in vivo in a natural infection with an unbiased high-throughput approach such as proteomics appears to offer particular advantages. Here, we present a novel application of the membrane shaving technique to characterize surface bacterial and host proteins present in vivo in a natural infection. These data will aid the understanding of host−pathogen interactions in other mycobacterial diseases, including tuberculosis. B



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EXPERIMENTAL METHODS

genome (Ovis aires Oar_ v3.1, NCBI 9940, downloaded 19.08.2013) and an annotated reference strain of C. pseudotuberculosis C231 (NCBI Genbank CpC231:CP001829) as well as a database of common reagents and contaminants (common Repository of Adventitious Proteins, ftp://ftp.thegpm.org/fasta/ cRAP). The following search parameters were used: missed cleavages, 2; peptide mass tolerance, 20 ppm; peptide fragment tolerance, ±0.02 Da; peptide charge, 2+, 3+, and 4+; variable modifications included deamination NQ, methionine oxidation, acetylation protein N-terminal, and an isotope error (carbon 13) of 2. A decoy search was performed simultaneously using a Mascot built in option, and results were adjusted to a false detection rate for protein identity of less than 1%. Significance was determined at the peptide level, with a peptide ion score cut off set at p < 0.01. Triplicate biological samples were prepared, significance was determined at the peptide level, and only proteins identified in more than one biological replicate were reported in the results.

Bacterial Culture

All C. pseudotuberculosis strains were cultured in brain−heart infusion (BHI CM1135, pH 7.4) broth or agar (Oxoid, Hampshire England) for 2−3 days at 37 °C. C. pseudotuberculosis C231 (Cptb_C231)25 was obtained from Dr. Rob Moore, Commonwealth Scientific and Industrial Research Organization (CSIRO). Bacteria were harvested at early exponential phase by centrifugation at 2500g and washed three times with Sorenson’s buffer at 4 °C for membrane shaving studies. Membrane Shaving

Membrane shaving was performed as described11 with some modifications. After washing with Sorenson’s buffer, harvested cells, approximately 107, were resuspended in 1/100th volume of the same buffer (pH 7.2) containing 20% sorbitol to minimize osmotic stress. Digestion was performed with 5 μg of trypsin (Roche trypsin sequencing grade from bovine pancreas, cat. no. 11047841001) for 15 min at 37 °C in the presence of 5 mM DTT to maximize the availability of trypsin cleavage sites. After incubation, the sample was centrifuged at 2500g at 4 °C for 10 min to pellet bacterial cells, with peptide fragments cleaved by trypsin remaining in the supernatant. The supernatant was filtered with 22 μm filters (Millex-GP (25 mm) sterile filter cat. no SLGP R25, Millipore, Billerica, MA, USA) to ensure removal of cellular material. A further digestion was performed with the addition of 5 μg of fresh trypsin and incubation overnight at 37 °C. The reaction was stopped with formic acid to pH 2, and then samples were frozen at −80 °C prior to proteomic analysis.

Bioinformatics Analysis

Subcellular localization was predicted by the Inmembrane program,33 which includes a transmembrane helix predictor (TMMOD), a secretion signal predictor (SignalP), a lipoprotein signal predictor (LipoP), and a sequence alignment for protein profiles (HMMER). The Inparanoid program,34 which is based on reciprocal BLAST, was used for prediction of homology with M. tuberculosis. Likely protein functions were assigned by the COG database;35 proteins sequence data was searched against the COG target HMM database/bio/db/hmmer3/COG.hmm a database of hidden Markov Models using the hmmpfam program from the HMMER software suite (http://selab.janelia.org/software.html) with predicted orthologous functional group from COG and NCBI. Mammalian protein subcellular location and likely function were derived from UniProt database (http://www.uniprot.org).

Mass Spectrometry Analysis

For proteomic analysis, samples were concentrated and purified with benchtop C18 columns according to the manufacturer’s instructions (TopTip TM, Glygen Corp, Columbia, MD, USA). Peptides were eluted in 60% acetonitrile with 0.01% formic acid, concentrated under vacuum, and resuspended in 0.01% formic acid. Tryptic digests were analyzed by LC−MS/MS using a QExactive mass spectrometer (Thermo Scientific, Bremen, Germany) coupled online with a RSLC nano HPLC (Ultimate 3000, Thermo Scientific, Bremen, Germany). Samples were injected onto a Thermo RSLC pepmap100, 75 μm i.d., 100 Å pore size, 15 cm reversed-phase nano column with 95% buffer A (0.1% formic acid) at a flow rate of 300 nL/min. The peptides were eluted over a 60 min gradient to 40% buffer B (80% acetonitrile, 0.1% formic acid). The elutant was nebulized and ionized using the Thermo nano electrospray source stainless steel emitter with a capillary voltage of 1800 V. The resolving power was 70 000 at 200 m/z for MS1 and was 17 500 at 200 m/z for MS2. Peptides were selected for MS/MS analysis in full MS/dd-MS2 (TopN) mode with the following parameter settings: TopN 10, MSMS AGC target 5 × 104, 120 ms Max IT, NCE 27, and 3 m/z isolation window. Dynamic exclusion was set to 30 s. Data from LC−MS/MS runs was exported in Mascot generic file format (*.mgf) using ProteoWizard 3.0.3631 (open source software, http://proteowizard.sourceforge.net). MS convert settings selected included MS level for filter, peak picking 1−2, output format.mgf, 32bit, write index, and zlib compression. Peptides were then identified by a Mascot (version 2.4.01) search of a database constructed in house that contained 8890 sequences. The database contained the fully annotated sheep

Membrane Shaving of C. pseudotuberculosis Reference Strain (Cptb_C231)

Cptb_C231 was cultured in BHI medium to the early exponential phase of growth, generally 18 to 24 h, and triplicate samples were subjected to membrane shaving (Figure 1). Peptide fragments were identified by spectra generated by MS/MS and matched to the database that included the predicted proteome of Cptb_C231. Triplicate biological samples were prepared, significance was determined at the peptide level, and only proteins identified in more than one biological replicate were included in the results. In parallel, trypsin-free controls were performed to identify proteins shed into the buffer by viable cells. Viability of bacteria was not significantly affected by the trypsin treatment, as determined by the absence of significant differences in the numbers of colony forming units between shaven and unshaven bacteria as determined by the plate dilution method (data not shown). Processing of Infected Lymph Nodes

Specimens of macroscopically infected lymph nodes were obtained from a local abattoir (Herd Abattoir, Geelong) shortly after humane slaughter for human consumption. The selection of infected lymph nodes was based on observation and palpation of the carcass. Three animals with CLA and three animals without apparent disease but from the same flock were studied. Groups of lymph nodes and surrounding connective tissues were retrieved. Lymph nodes were processed individually and classified to one of three groups: (1) diseased nodes, (2) normal nodes from diseased animals, and (3) normal nodes from animals without any apparent C



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as part of the processes of infection and resistance to host responses. C. pseudotuberculosis Cptb_C231, a sheep isolate, was selected for this analysis due to the availability of a full genome sequence.25 A total of 137 proteins were recurrently identified following membrane shaving of Cptb_C231, and 57 proteins were identified in the trypsin-free controls for shed proteins, of which 54 were seen in both data sets (Figure 2 and Supporting Information Data 1). Using a bioinformatics algorithm to predict likely subcellular location, we found that 30% of trypsincleaved proteins had a likely surface location, with proteins with predicted cell wall features such as a transmembrane domains accounting for almost half (19/41) of these. Within the data set obtained from the trypsin-free controls, 44% of these likely shed proteins had a predicted surface location, more than half of which (15/25) were predicted to be secreted. These secreted proteins identified in both samples included the three characterized virulence factors of phospholipase D, CP40, and lysozyme M128,38 as well as several secreted proteins with roles in cell wall synthesis39 such as trehalose corynomycolyl transferase C, two penicillin binding proteins, and a surface layer A protein. These bacteria were cultured in BHI media, which is derived from animal products, predominately sheep and calf, and despite washing of the bacterial cells, it is possible for proteins from this media to be carried over from the media. The database search included the whole proteome of sheep, and several mammalian proteins were recurrently identified, which are listed in Supporting Information Table 1; however, these contaminants only composed 4−6% of the proteins identified in any replicate. Importantly, no mammalian peptides sets were inadvertently ascribed to a bacterial protein or vice versa, as such a misattribution would compromise later studies examining both host and bacterial proteins.

disease. Lymph nodes were processed and the bacteria were isolated for species identification and subsequent membrane shaving experiments. Purification of Bacteria from Infected Tissues

Specimens of intact macroscopically infected lymph nodes were frozen immediately after collection at −20 °C. Lymph node specimens were defrosted at 4 °C and dissected back to the capsule, and connective tissue was discarded. Lymph node capsules were opened, and contents were liberated by blunt tweezer dissection into a buffer of PBS (pH 7.4) and 4 mM EDTA. Samples were then suspended in twice the volume of the same buffer in sterile Falcon tubes and then centrifuged twice at 300g for 10 min at 4 °C to remove mammalian cellular material. The supernatant was collected and centrifuged at 2500g for 10 min at 4 °C. Pelleted bacterial cells were then washed twice with Sorenson’s buffer (pH 7.2, 0.15 mol/L monopotassium phosphate, 0.15 mol/L disodium phosphate) and collected following centrifugation at 2500g for 10 min at 4 °C. Three aliquots of wet pellet were taken from each sheep sample, and each underwent separate membrane shaving. Starting amounts of bacteria were equilibrated by weight of wet pellet. Membrane Shaving of Bacteria from Infected Material

Membrane shaving was performed separately for each of the nine isolated tissues, comprising tissues from three overtly infected lymph nodes and six sets of lymph node tissue from control settings described earlier, in which each sample underwent trypsin incubation to cleave peptide fragments from surface-exposed proteins. Shaving was performed in triplicate on each tissue sample, resulting in three technical repeats for each tissue sample, and underwent mass spectrometry analysis individually, following which spectral data files (.mgf) were merged for database searching. Identified peptides were paired to the predicted proteome of both host (Ovis aries) and bacteria (Cptb_C231). Comparison of these protein repertoires was then made between the diseased and normal tissues, with the comparison between host proteins and bacterial proteins considered separately.

Species and Strain Identification of Bacteria Isolated from Caseous Material

A sample of the bacterial pellet isolated from a diseased node was taken for direct Gram stain, which revealed Gram-positive rods on a background of occasional mammalian cells and cellular debris (data not shown). A sample of bacterial pellet was also streaked onto BHI agar, from which uniform pale opaque colonies grew. A single colony was selected for subculture and was taken as a representative of this field strain in subsequent experiments. The identity of this strain designated Cptb_RLC001 was initially confirmed as C. pseudotuberculosis by sequencing the rpoB gene and comparison to other corynebacterial rpoB orthologues, as described by Khamis et al.37 This field-isolated strain (Cptb_RLC001) also underwent ion torrent genome sequencing to yield 3 507 459 reads with a mean length of 183 bp. These reads were mapped at 200× coverage to the 16 publically available C. pseudotuberculosis genomes. This analysis revealed that Cptb_RLC001 was most closely related to Cptb_C231, separated by only 62 SNPs; however, the sequencing coverage for Cptb_RLC001 was not adequate for use as a proteome template. The Cptb_C231 genome was thus chosen as a suitable reference sequence for peptide matching in subsequent experiments.

Genome Sequencing and Analysis

Genomic DNA was extracted from strain Cptb_RLC001 using a Nucleon kit according to the manufacturer’s instructions (Amersham Biosciences), and high-throughput sequencing was performed using the Ion Torrent Personal Genome Machine (Life Technologies, Guilford, CT, USA) with a 316 chip and 200 bp sequencing chemistry. The sequence reads were mapped to the C. pseudotuberculosis C231 reference genome using SHRiMP 2.2.36 SNPs were identified using Nesoni, v0.70, to construct a tally of putative differences at each position that included substitution mutations only (www.vicbioinformatics.com). The species identity of Cptb_RLC001 was confirmed by sequencing of the rpoB gene37 with Sanger sequencing (Applied Biosystems 3730 DNA Analyzer, Life Technologies, Guilford, CT, USA).

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RESULTS

Membrane Shaving of C. pseudotuberculosis Reference Strain (Cptb_C231)

Membrane Shaving of a Field Isolate of C. pseudotuberculosis (Cptb_RLC001)

Prior to an analysis of proteins on the surface of bacteria isolated from infected lymph nodes, we performed an experiment with liquid culture grown C. pseudotuberculosis to derive an initial set of surface-exposed proteins. This would aid subsequent identification of additional proteins that were induced

Cptb_RLC001 was grown in BHI broth media and also underwent membrane shaving using the approach and controls described above for Cptb_C231. Overall, 225 proteins were recurrently identified following membrane shaving, whereas 55 D



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Figure 2. A comparison of the membrane shaving protein repertoires of two strains of C. pseudotuberculosis grown in liquid media. Proteins identified in the reference strain (Cptb_C231) by membrane shaving (green) and those shed by trypsin-free control (yellow) and compared to proteins identified in the field isolate (Cptb_RLC001) by membrane shaving (red) and those shed by trypsin free control (blue). Numbers represent unique proteins identified in three biological replicates, after significance of match was determined at the peptide level.

Although this centrifugation process enriched the sample for bacteria, host cells and cellular debris were not completely removed, as observed by direct microscopy. Rather than subject the bacteria to a more rigorous purification process such as filtration, which may have resulted in cell lysis and potentially alter the surface, we elected to include controls that could account for background host proteins present in quiescent and uninfected lymph nodes. Thus, the same series of centrifugation steps was performed with lymph node material from two control settings: (1) normal lymph nodes from the same three animals that did not have any macroscopic disease and (2) normal lymph nodes from three other animals without any clinical evidence of disease, which were from the same herd as the animals with CLA. Membrane shaving was performed separately for each of the isolated tissues. Identified peptides were matched to the proteomes of host (Ovis aries) and pathogen (Cptb_C231). Comparison of these protein repertoires was then made between the diseased and normal tissues, with the data sets of host proteins listed in Supporting Information Table 4 and bacterial proteins presented in Supporting Information Tables 2 and 3.

were identified in the trypsin-free controls, 51 of which were seen in both procedures, suggesting that a quarter of the proteins identified by membrane shaving were actually shed into the suspension buffer (Figure 2 and Supporting Information Data 1). Similar to the results of the Cptb_C231 shaving, several known secreted virulence factors,30,38 such as phospholipase D, lysozyme M1, and the culture filtrate protein CP40, were identified both by membrane shaving and in the shed set of proteins detected in the trypsin-free control. The predicted subcellular location of the proteins within these two groups was determined by the Inmembrane program.33 The trypsin-free control, designed to reveal spontaneously shed proteins, had 56% of proteins with a predicted surface feature, of which 55% (17/31) contained a predicted signal sequence for export. Within the surface shaving set, 42% had a predicted surface feature such as a transmembrane region or lipoprotein motif. If the shed proteins were excluded from the analysis of surface proteins, then 35% still had a predicted surface feature. The data sets generated with Cptb_C231 and Cptb_RLC001 showed considerable overlap, with 114 of the 137 proteins identified in Cptb_C231 also identified within the Cptb_RLC001 repertoire (Figure 2 and Supporting Information Table 1). A greater total number of proteins was identified in Cptb_RLC001; this is unlikely to be due to increased cell lysis, as the field strain had fewer predicted cytoplasmic proteins as a proportion of the total number. Indeed, this may imply a slightly more robust cell wall in this strain in the face of trypsin exposure.

Bacterial Proteins Identified in Caseous Nodes

Thirty-one bacterial proteins were recurrently identified in caseous material from more than one diseased animal out of a total of 89 bacterial proteins individually identified (Table 1). When we examined the individual peptides identified in each animal (Supporting Information Data 2 and 3), many identical peptides were detected in more than one replicate, suggesting that the same portion of the protein was exposed to trypsin cleavage. The bacterial proteins identified in the infected lymph nodes included some putative virulence factors such as largeconductance mechanosensitive channel (locus tag CpC231_0664 and gene mscL) and Antigen 84 (locus tag CpC231_1383), both of which are putative virulence factors of C. pseudotuberculosis and related species.24 Three of the identified proteins (FadF, large-conductance mechanosensitive channel, and ascorbate-specific permease IIC

Membrane Shaving of Bacteria Isolated Directly from Caseous Material

Having successfully liberated and identified the surface proteins of pure cultures of Cptb_C231 and Cptb_RLC001, we utilized membrane shaving to characterize infected lymph node tissue from three animals with CLA (Supporting Information Data 1). Animal tissues were enriched for bacteria by a series of centrifugation steps, with the concentration of bacteria increasing from 5.4 × 106 to 2.4 × 107 colony forming units per milliliter, as determined by the plate dilution method. E



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Table 1. Bacterial Proteins Identified in Diseased Nodes of More than One Animal in Vivoa name

locus

no. of peptides identfied

Proteins Identified in Vivo and in Vitro 30S ribosomal protein S16 CpC231_1323 2,3-Bisphosphoglycerate-dependent phosphoglycerate CpC231_0261 mutase 60 kDa chaperonin CpC231_1799 60 kDa chaperonin 10 kDa chaperonin Alkyl hydroperoxide reductase subunit C Uncharacterized protein Uncharacterized protein Large-conductance mechanosensitive channel Antigen 84 30S ribosomal protein S2 Acetate kinase Malate dehydrogenase Transketolase Chaperone protein DnaK ATP-dependent chaperone protein ClpB Dihydroxyacetone kinase family protein Phosphoenolpyruvate carboxykinase [GTP] Proteins Identified Only in Vivo Chromosome partitioning protein ParB Regulatory protein RecX Protein fadF Enolase Fumarate hydratase class II Argininosuccinate synthase Sulfurtransferase Glycerol-3-phosphate dehydrogenase Aspartate-semialdehyde dehydrogenase Lipid A biosynthesis lauroyl acyltransferase Ascorbate-specific permease IIC component ulaA Purine nucleoside phosphorylase DeoD-type Methylmalonyl-CoA carboxyltransferase 12S subunit

M. tuberculosis M. tuberculosis gene homologue (synonyms)

presence in other tb membrane proteomics studies (ref)

8 10

Rv2909c Rv0489

rpsP gpm1 (gpm)

43, 44 43

22

Rv0440

42−44

CpC231_0423 CpC231_0422 CpC231_1218 CpC231_0348 CpC231_2052 CpC231_0664 CpC231_1383 CpC231_1308 CpC231_1824 CpC231_1596 CpC231_1096 CpC231_1890 CpC231_1868 CpC231_1056 CpC231_1936

5 9 28 4 3 10 8 9 4 18 5 66 18 7 13

Rv3417c Rv3418c Rv2428 #N/A

groEL2 (groL2 groEL-2 hsp65) groEL1 groES (mpt57) aphC

Rv0985c Rv2145c Rv2890c Rv0409 Rv1240 Rv1449c Rv0350 Rv0384c #N/A Rv0211

mscL wag31 (ag84) rpsB

42−44 43, 44 32, 42−44 44 43, 44 43

pckA (pckG pck1)

42, 43

CpC231_2097 CpC231_1245 CpC231_1903 CpC231_0713 CpC231_0737 CpC231_0954 CpC231_0481 CpC231_1859 CpC231_0174 CpC231_1182 CpC231_0082 CpC231_0163 CpC231_0572

8 8 2 12 12 15 9 27 10 5 41 4 10

Rv3917c Rv2736c Rv0338c Rv1023 Rv1098c Rv1658 Rv3283 Rv3302c Rv3708c Rv2611c N/A N/A N/A

parB (parA) recX

44

mdh tkt dnaK clpB (htpM)

eno f um argG sseA glpD2 asd

42−44 43, 44 43, 44

43, 44

42−44 32, 43, 44 42−44 43, 44 43 42−44 43, 44 44

a Proteins also identified in media-grown shaving experiments (in vitro) are grouped together. TMHMM is transmembrane region, as predicted by a bioinformatics algorithm.80 M. tuberculosis homologue is the closest homologous protein, as predicted by a bioinformatics algorithm.34

reductase subunit C.45 In addition, three proteins (Wag31, ParB, and MscL) have roles in cell wall synthesis and therefore a possible surface location. So, our method appears to enrich for proteins with M. tuberculosis homologues that are involved in virulence and cell wall synthesis. The likely function for each of the proteins was predicted using the Clusters of Orthologous Groups (COG) algorithm.46 This list was subsequently compared to the predicted functions of all gene products of C. pseudotuberculosis and also to the surface proteome derived by membrane shaving of the same strain grown in liquid media (Figure 3). Compared to shaved proteins identified from bacteria grown in media, those shaved from bacteria isolated from lymph nodes had an increased proportion of signaling, metabolite production, and transport proteins and a reduction in proteins involved in cell wall synthesis. Two bacterial proteins were identified in lymph nodes of normal appearance from animals with disease elsewhere. These were the cytoplasmic protein D9QA50 N-acetyl-gammaglutamyl-phosphate reductase (CpC231_0948) and a membrane protein with two predicted trans-membrane regions, D9QCG4 ATP-dependent zinc metalloprotease FtsH (CpC231_1784).

component UlaA) have a transmembrane region predicted by the Inmembrane bioinformatics program,33 suggesting bacterial surface expression. All other proteins were predicted by this algorithm to be cytoplasmic, as they did not possess a surface feature motif; however, six of these have previously been identified in the culture filtrate of C. pseudotuberculosis,26,40 so they may actually be surface or secreted proteins. This data set was then analyzed to detect close homology with proteins identified in the human pathogen M. tuberculosis, as there is a larger body of scientific research characterizing proteins in this organism and because proteins involved in the disease process are likely to be shared with this exclusive pathogen. We used a reciprocal blast match of greater than 50% of the larger protein and then a gating algorithm to exclude paralogues or gene duplication events.34 Twenty six of the 31 proteins (84%) had a close homologue in M. tuberculosis; of these, 18 are essential for growth of M. tuberculosis41 (Table 1). Most of these had been identified in other proteomic studies that characterized proteins in the membrane fraction of M. tuberculosis.32,42−44 Furthermore, six are thought to have roles in virulence, including several chaperone proteins, endopeptidase ATP binding protein, and alkyl hydroperoxide F



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Figure 3. Distribution of predicted function of identified proteins, as determined by Clusters of Orthologous Groups functional attribution of individual proteins, as a proportion of each protein data set. Innermost circle represents proteins identified in bacteria harvested directly from lymph nodes (in vivo). Middle circle represents the surface protein repertoire from bacteria cultivated in liquid media (in vitro). Outer circle represents all proteins predicted to be transcribed from the sequenced genome (in silico).

which was included to identify spontaneously shed proteins. Fourteen proteins were identified only in the membrane shaving of bacteria harvested directly from lymph nodes (Table 1). These included two proteins with predicted transmembrane regions as well as enolase, which lacks a predicted surface feature but has been identified as surface expressed in other studies.47

The presence of these bacterial proteins in tissue distant from caseous lymphadenitis suggests the possibility of either subclinical infection or transport of bacterial proteins from a distant site to other nodes. Comparison of Protein Repertoires between Bacteria from Caseous Nodes and Media-Grown Bacteria

Host Proteins

A greater number of bacterial proteins (225) was identified when membrane shaving was performed with Cptb_RLC001 grown in BHI media. Interestingly, only 18 of the 31 proteins recurrently identified in the tissue sample were also seen in the surface shaving of media grown organisms (Table 1). This suggests that different surface proteins are expressed in the hostile environment of the host lymph node, although, alternately, it may reflect significant release of cytoplasmic proteins in the tissue samples. Many of these proteins lacked a predicted surface feature, including three chaperone proteins, 30S ribosomal protein S16, alkyl hydroperoxide reductase subunit C, and 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase, and two uncharacterized proteins. Interestingly, proteins with a likely surface localization such as the virulence factor Antigen 84 and the membrane protein large-conductance mechanosensitive channel were identified in both settings but not in the trypsin-free control,

Host Proteins Identified in Caseous Nodes. Proteins of host origin were abundant in all samples prepared from caseous material. One-hundred twenty unique host proteins were identified in more than one animal. We anticipated that many of these proteins may be usually present in lymph nodes, and we were interested to identify those proteins that were detectable only in the presence of overt bacterial infection. Therefore, we compared the host protein repertoire from caseous nodes to that of proteins identified from lymph node material from three animals without any apparent C. pseudotuberculosis infection. Forty-nine host proteins were identified only in the caseous lymph nodes (Table 2), of which nine are proteins with known roles in immunity and 10 have known roles in the inflammatory response. The remaining 30 may also have as yet undefined roles in mediating the inflammatory response to this bacterial infection. G



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Identified proteins known to be involved in the immune response included complement components, MHC class 1 antigens, and receptors for IgG. The surface marker CD11b is of particular interest in mycobacterial infections as it has a crucial role in bacterial phagocytosis by macrophages.48 Antimicrobial peptides were also identified in the caseous material including cathelicidin-2 and regakine 1-like protein. Cathelicidin-2 is recognized as a vitamin D-dependent mediator of the innate immune response.49 Ten proteins involved in the inflammatory response were also identified in the caseous nodes but not in the nodes of normal appearance. Several of these proteins are the direct result of inflammation such as amyloid and heat shock protein. However, there were also several proteins identified, such as serpin and alpha-1-antitrypsin, whose roles are to modulate or control inflammation, which would be in keeping with CLA in which the inflammatory process, while florid, is contained within the boundary of the lymph node, with little apparent systemic effect. Proteins Identified in Lymph Nodes of Normal Appearance. Immune and inflammatory proteins were also present in normal lymph nodes, but the specific proteins identified differed (Figure 4A). Seventy-one host proteins were identified both in diseased and normal lymph nodes. These included two bacteriocidal peptides, cathelicidin-1 and myeloid antimicrobial protein (Supporting Information Data 4). Onehundred three host proteins were recurrently identified in normal tissue but not diseased lymph nodes (Figure 4A), including several immune mediators including CD3 receptor components, mannose C-type lectin receptor, interferoninduced GTP-binding protein Mx1, and CD81 antigen. To investigate whether the host protein response to C. pseudotuberculosis is systemic and generalized or just contained within the caseous nodes, we compared our results to lymph nodes of normal appearance obtained from the same animals that had overtly caseous nodes elsewhere (Supporting Information Data 4). These data revealed a protein repertoire similar to that of the uninfected animals (Figure 4B). Overall, 75% (130/174) of the proteins identified in normal nodes of apparently uninfected animals were also seen in membrane shaving of nodes of normal appearance from animals with known infection elsewhere. Six proteins were identified in the both the caseous and normal appearing nodes of infected animals but were absent from disease-free animals (indicated with an asterisk in Table 2). These included heat shock protein and alpha-2-HS glycoproteins, which are usually associated with inflammation in the acute phase response. None of these proteins are specifically recognized as immune mediators. The combined protein repertoire of nodes which appeared normal, from both diseased and normal animals, are likely to represent some of the proteome of the quiescent lymph node in uninfected or subclinically infected animals.

Table 2. Host Proteins Identified Only in Diseased Lymph Nodesa UniProt ID Role in Immune Response CTHL2_SHEEP O46544_SHEEP A6N7G0_SHEEP A2TJJ2_SHEEP Q4LBE1_SHEEP Q4LBE3_SHEEP B6C7C8_SHEEP OSTP_SHEEP Q5MJE8_SHEEP Role in Inflammation FETUA_SHEEP* B0LRN2_SHEEP B6UV62_SHEEP D8 × 187_SHEEP Q9TR77_SHEEP H2DGR2_SHEEP* I1WXR3_SHEEP CH3L1_SHEEP B0LRN4_SHEEP Q30DU3_SHEEP Other Functions F16P1_SHEEP Q683L9_SHEEP PDXK_SHEEP C8BKD1_SHEEP PADI3_SHEEP C7BDV7_SHEEP Q7YS10_SHEEP CERU_SHEEP Q9N109_SHEEP H2AZ_SHEEP* B5AN56_SHEEP GTR3_SHEEP Q9N113_SHEEP C5IJ84_SHEEP 1433Z_SHEEP A3FFK9_SHEEP ALBU_SHEEP* CAH2_SHEEP ANGT_SHEEP RL40_SHEEP B0LRN6_SHEEP F5CC79_SHEEP Q7YQJ6_SHEEP C5ISA8_SHEEP B6E3I8_SHEEP C8BKE6_SHEEP* Q7M331_SHEEP C5IJ99_SHEEP B0LRP3_SHEEP* A4ZYA6_SHEEP

protein name Cathelicidin-2 Complement component C3 (Fragment) Regakine 1-like protein CD11b MHC class I antigen MHC class I antigen Fc gamma 2 receptor Osteopontin Lactoferrin Alpha-2-HS-glycoprotein Serum amyloid A protein (fragment) SERPINF1 Serpin peptidase inhibitor clade B ovalbumin member 1 175 antigen (Fragment) Heat shock protein 70 Alpha-1-antitrypsin transcript variant 1 Chitinase-3-like protein 1 Cytochrome b-245 alpha polypeptide (fragment) Cytochrome b-245 beta polypeptide (fragment) Fructose-1,6-bisphosphatase 1 Putative H-ATPase subunit B (Fragment) Pyridoxal kinase Coagulation factor II Protein-arginine deiminase type-3 SRI LDHA protein (fragment) Ceruloplasmin Lactate dehydrogenase A (fragment) Histone H2A.Z Insulin-like growth factor-binding protein-6 Solute carrier family 2, facilitated glucose transporter member 3 Enolase (Fragment) RAB10 14-3-3 protein zeta/delta NADPH oxidase heavy chain subunit (fragment) Serum albumin Carbonic anhydrase 2 Angiotensinogen Ubiquitin-60S ribosomal protein L40 Asparaginase-like 1 protein (fragment) Beta-1,4-galactosyltransferase I Carnitine Copine I Enolase 1 (fragment) Eukaryotic translation initiation factor 5A Protein kinase C inhibitor KCIP-1 isoform eta (fragment) RHOA Seryl-tRNA synthetase (fragment) Vimentin (fragment)

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DISCUSSION Multiple proteins act in concert in infection, both to cause damage by a pathogen and in the host’s attempts to control the infection.50 Therefore, the simultaneous unbiased characterization of an extended repertoire of proteins in a system, as presented here, has distinct advantages for defining and understanding the disease process. Strategies previously applied to identify features of pathogenicity include comparing virulent and avirulent bacterial strains,51 identifying proteins produced under disease mimicking,52 or identifying proteins expressed in

a

Only proteins identified in more than one biological replicate are listed. Proteins are grouped according to proposed role. Proteins also identified in normal lymph nodes from animals with disease elsewhere are indicated with an asterisk. H



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Figure 4. (A) Venn diagram of host proteins identified in overtly caseous nodes (red) and nodes obtained from normal animals (blue). Proteins were identified in more than one animal in each group. Numbers represent numbers of individual unique proteins identified. (B) Venn diagram of host protein repertoire of overtly caseous nodes (red) and nodes obtained from normal animals (blue) and nodes of normal appearance from an animal with overtly caseous nodes elsewhere in the carcass (green). Proteins were identified in more than one animal in each group. Numbers represent numbers of individual unique proteins identified.

an in vivo culture system.53 By contrast, the approach described here samples a natural field infection directly. To our knowledge, this is the first time such characterization has been performed, and we anticipate that the technique would lend itself well to tissue samples acquired from human disease states. The relative paucity of bacterial proteins compared to host proteins is a particular challenge for the proteomic characterization of pathogens in the in vivo state.18 Several strategies have been applied to enrich for bacteria including centrifugation, immune labeling with magnetic beads, or flow cytometry.50 The proportion of bacterial proteins identified in published studies range from just 0.4% of all spectra in a whole tissue digest53 up to 30% of proteins identified after enrichment of GFP labeled bacteria with flow cytometry.50 In this study, we were able to enrich for bacterial proteins during sample preparation with several centrifugation steps, so that for infected sheep nodes 29% of all proteins identified were of bacterial origin. Our technique, aided by the high bacillary load of C. pseudotuberculosis in infected nodes,54 has produced substantial enrichment with minimal processing of the tissues, allowing adequate detection of the naturally occurring in vivo protein repertoire. The bacterial proteins identified in the overtly infected lymph nodes contained some putative virulence factors (Table 1). Antigen 84, encoded by CpC231_1383, is a close orthologue of

the M. tuberculosis protein Wag31. In other mycobacteria, Wag31 is surface-expressed,55 and in M. tuberculosis, it has been shown to be involved in cell wall synthesis56 and resistance to oxidative stress.57 The protein encoded by CpC231_0664, described as large-conductance mechanosensitive channel (mscL), was also identified. This is a member of a family that protects against osmotic stress58 and has been proposed as a drug target in C. pseudotuberculosis24,59 and M. tuberculosis.60 The presence of these proteins in addition to FadF and UlaA, which have predicted transmembrane regions, validates the use of this method to target proteins on the cell surface. Interestingly, enolase and protein FadF were also identified in the in vivo proteomic study of a mouse model of M. tuberculosis infection,53 suggesting that these proteins are more likely to be expressed in the host than during growth in media. Although the functions of many of the identified proteins have yet to be determined, the large proportion of these with close homology to essential M. tuberculosis proteins (58%) suggests that this method may reveal proteins with important roles in mycobacterial infection. Surprisingly, several anticipated proteins were not detected in our analysis of caseous nodes, including the well-characterized virulence factor phospholipase D (PLD)23,30,61 and protein CP40.62 PLD has been readily identified during growth in I



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media in other studies.26,63 These proteins may be expressed only during tissue invasion and not in an established infection such as that examined here. Alternatively, these secreted proteins may have been removed during the initial washing of bacterial cells. Similarly, many exported proteins known to be important in virulence and immune stimulation were not detected in an in vivo animal model of tuberculosis.53 Exported proteins may not be identified if they have been trafficked to another location or if they are no longer present at a detectable level after sample processing. Many of the bacterial proteins identified in this work as surface expressed lacked any predicted surface feature. The presence of predicted cytoplasmic proteins following membrane shaving is commonly reported in the literature. In other studies, the proportion of predicted cytoplasmic proteins identified varies from 0 to 80%;64 indeed, even in the secretome of carefully cultured mammalian cell lines, only 12% of identified proteins possessed a classical secretion feature.65 Explanations66 include imprecise bioinformatic predictions,67 proteins with more than one function and/or location,5,68 truly cytoplasmic proteins present on the surface either due to active transport or via noncovalent association with cell wall proteins,69 and cytoplasmic proteins released from membrane vesicles, which have been demonstrated in some mycobacterial species.70 These theories are not mutually exclusive, and, additionally, some degree of bacterial cell lysis may be present in the tissues analyzed here. Indeed, any cytoplasmic protein released into the necrotic tissue may have a significant role in this host−pathogen interaction, and confirmation of this would require further targeted studies. Proteins lacking a predicted surface feature have been documented as surface exposed in M. avium hominissuis using a biotinylation method.71 Among 24 surface proteins identified by biotinylation were malate dehydrogenase, 30S ribosomal S2, and phosphoenolpyruvate carboxykinase, also identified in this study. The finding of predicted cytoplasmic proteins at the surface by a different experimental approach supports the proposition that a number of the cytoplasmic proteins that we found in this study are in fact surface associated. Bacterial proteins were identified sporadically in apparently normal lymph node tissue, and there are several possible explanations. First, it is possible that subclinical infection is present in these apparently uninfected nodes; indeed, not all lymph nodes of an animal are uniformly infected.72 We have shown significant similarity in the protein repertoire of both bacterial and host proteins seen in apparently uninfected nodes from animals with infection elsewhere as well as in those animals assessed as normal by abattoir workers (Figure 4B). A second possibility is that bacterial secreted proteins have been trafficked from a distal location and taken up by these lymph nodes. The membrane-shaving approach has previously been used to define host proteins interacting with the bacterial surface by incubating cultured bacteria with its usual host body fluid. These include Streptococcus gallolyticus bacteria incubated with human intestinal epithelial cell secretions73 and Staphylococcus aureus incubated in human sera.16 In the current study, we have been able to apply the membrane shaving technique to a natural infection to identify host proteins that occur in the presence of bacterial proteins in vivo. In addition to bacterial proteins, 49 host proteins were recurrently identified only in the infected tissues, suggesting that they are enriched by their association with bacteria and may be adherent to the bacterial surface. These include several

proteins already demonstrated to have a direct antibacterial function such as cathelicidin49,74 or an immune role by binding to the pathogen and signaling to other cells, for example, regakine 1 like protein.75 CD11b, identified in this study, has been proposed to bind to LAM in the cell wall of mycobacteria76 and mediate both opsonic77 and nonopsonic78 uptake of M. tuberculosis by macrophages. The presence of host proteins known to bind to the mycobacterial surface is proof of concept that our membrane shaving approach described here is targeted to protein identifications at the level of the host− pathogen interface. Whole proteome analysis of both host and pathogen proteins has been made with experimentally inoculated in vivo infections but not naturally occurring infections. A proteomic analysis of whole murine lung after inoculation with M. tuberculosis has been performed.53 Interestingly, the protein products of Rv0338c and Rv1023 were observed and are close homologues of proteins FadF and enolase, respectively, which were also identified in our study. Several proteins that had increased expression in an in vivo bovine model of infection with M. avium paratuberculosis79 were also identified in caseous nodes in our study, including the bacterial protein chaperonin GroEL and host proteins cathelicidin, actin, myosin, and tubulin. Interestingly, 103 host proteins were present only in uninfected tissue. Even when this tissue was harvested from an animal with a known infected lymph node elsewhere, 64% of these proteins had also been identified in uninfected animals. This included CD3 receptor components, CD81 antigen, mannose C-type lectin receptor, and interferon induced Mx1, some of which may defend against the establishment of a bacterial infection. It is likely that within this group there will be some host proteins that provide a degree of local protection against the establishment of a florid caseous infection. An advantage of this work is that the broad milieu of host proteins surrounding the infecting pathogen can be captured simultaneously without any adulteration of the system that may occur if an attempt was made to separate proteins prior to protease digestion. We believe that this augments the strengths of the membrane shaving technique as applied to intact replicating bacteria. A necessary consequence of this approach is that specific interacting pairs of host and bacterial proteins are not identified. This will require more targeted approaches such as pull-down assays or the use of cross-linking agents prior to shaving. However, broad proteomic studies such as ours provide a useful set of candidates for future studies.

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CONCLUSIONS We have applied a surface shaving proteomic technique to bacterial cells harvested directly from a pathological specimen and have, to our knowledge, obtained the first characterization of surface-expressed bacterial and adherent host proteins in a natural infection. Identified C. pseudotuberculosis proteins included several established and putative virulence factors as well as some uncharacterized proteins that may be responsible for pathogenic processes and thus potential therapeutic targets. The host proteins identified included proteins with previously described bacterial interactions as well as several immune mediators. In addition, host proteins identified in apparently uninfected material may represent agents that are important in resistance to infection. Further work will be required to unravel the specific interactions between pairs of bacterial and host proteins, but our data maps, for the first time, the in vivo protein milieu of a natural infection. J



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ASSOCIATED CONTENT

S Supporting Information *

ABBREVIATIONS

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REFERENCES

BHI, brain−heart infusion; BLAST, Basic Local Alignment Search Tool; CFU, colony forming units; CLA, caseating lymphadenitis; MS/MS, tandem mass spectrometry; SNP, single nucleotide polymorphism

Tables documenting mass spectrometry results and details of proteins identified. Data Table 1: Tables of proteins identified following membrane shaving of the reference (Cptb_C231) and field strains (Cptb_RLC001) of C. pseudotuberculosis grown in BHI liquid media. (Tab 1) Proteins identified in both Cptb_C231 and Cptb_RLC001 as illustrated in the venn diagram of Figure 2. (Tab 2) Proteins identified following membrane shaving of Cptb_C231 grown in BHI media. (Tab 3) Proteins identified in trypsin free control (shed proteins) with Cptb_C231 incubated in BHI media without trypsin. (Tab 4) Proteins identified following membrane shaving of Cptb_RLC001 grown in BHI media. (Tab 5) Proteins identified in trypsin free control (shed proteins) with Cptb_RLC001 incubated in BHI media without trypsin. (Tabs 6−16) All peptides identified following Mascot search per biological replicate. Data Table 2: All peptides identified following Mascot search per animal. (Tab 1) Diseased sheep A. (Tab 2) Diseased sheep B. (Tab 3) Diseased sheep C. (Tab 4) Normal sheep D. (Tab 5) Normal sheep E. (Tab 6) Normal sheep F. (Tab 7) Normal node tissue from diseased sheep A. (Tab 8) Normal node tissue from diseased sheep B. (Tab 9) Normal node tissue from diseased sheep C. Data Table 3: All peptides detected listed for each bacterial protein identified in caseous lymph nodes of diseased. These proteins correspond to the proteins listed in Table 1. Data Table 4: All host (sheep) proteins identified in tissues from sheep lymph nodes following membrane shaving. (Tab 1) Sheep proteins identified only in diseased nodes grouped by function. (Tab 2) Sheep proteins identified only in diseased nodes listed alphabetically. (Tab 3) Sheep proteins identified in nodes of normal appearance, corresponding to Figure 4A,B. (Tab 4) Sheep proteins identified in both diseased and nondiseased nodes listed by mascot score. (Tab 5) Sheep proteins identified in both diseased and nondiseased nodes listed by number of peptides identified. (Tab 6) Sheep proteins identified in both diseased and nondiseased nodes listed by relative abundance as calculated by emPAI (exponentially modified protein abundance index). For results tables in Tabs 4−6, all values are additionally color coded according to their relative distribution within the data set: red indicates the lowest values, yellow indicates the 50% percentile values, and green indicates the highest values. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: (61 3) 99024050. Fax: (61 3) 99029222. E-mail: ian.smith@ monash.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Australian Research Council Centre of Excellence in Structural and Functional Genomics (COE562063) and the National Health and Medical Research Council of Australia (project grants ID1007676 and ID1064466). We acknowledge the assistance of M. C. Herd, Pty Ltd, Geelong, and CSIRO, Australian Animal Health Laboratory, Geelong. We are grateful for technical assistance in sequencing the rpoB gene from Rajini Brammananth. K



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