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Evaluating Common Humoral Responses Against Fungal Infections With Yeast Protein Microarrays Paulo Sergio Rodrigues Coelho, Hogune Im, Karl V. Clemons, Michael P. Snyder, and David A. Stevens J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00365 • Publication Date (Web): 10 Aug 2015 Downloaded from http://pubs.acs.org on August 15, 2015

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Journal of Proteome Research

Evaluating Common Humoral Responses Against Fungal Infections With Yeast Protein Microarrays Paulo S. R. Coelho1,2,4, Hogune Im3,*, Karl V. Clemons1,2, Michael P. Snyder3, David A. Stevens1,2 1 Stanford University, Division of Infectious Diseases and Geographic Medicine 300 Pasteur Drive, Lane L-134 Stanford, CA, USA 94305-5107 2 California Institute for Medical Research 2260 Clove Dr San Jose, CA, USA 95128 3 Stanford University, Department of Genetics 300 Pasteur Drive, Alway Stanford, CA, USA 94305-5120 4 Universidade de São Paulo Departamento de Biologia Celular, Molecular e Bioagentes Patogênicos Faculdade de Medicina de Ribeirão Preto Avenida dos Bandeirantes, 3900, Ribeirão Preto, SP, Brasil, CEP:14049-900 * Current address: Samsung SDS, Healthcare division 123 Olympic-ro 35-gil, Songpa-gu, Seoul, Korea Running title: Antibody profiling to fungal infections with protein microarray Key words: Fungal infection, Serum profiling, Saccharomyces cerevisae, Protein microarray

Corresponding author: Paulo Sergio Rodrigues Coelho Departamento de Biologia Celular, Molecular e Bioagentes Patogênicos Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo Avenida dos Bandeirantes, 3900 Ribeirão Preto, SP, CEP:14049-900 Phone: 55-16-3602-3257 Email [email protected]

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Abstract In this study we profiled the global immunoglobulin response against fungal infection by using yeast protein microarrays. Groups of CD-1 mice were infected systemically with human fungal pathogens (Coccidioides posadasii, Candida albicans or Paracoccidioides brasiliensis) or inoculated with PBS as a control. Another group was inoculated with heat-killed yeast (HKY) of Saccharomyces cerevisiae. After 30 days, serum from mice in the groups were collected and used to probe S. cerevisiae protein microarrays containing 4,800 full-length glutathione S-transferase (GST)-fusion proteins. Anti-mouse IgG conjugated with Alexafluor 555 and anti-GST antibody conjugated with Alexafluor 647 was used to detect antibody-antigen interactions and presence of GST-fusion proteins, respectively. Serum after infection with C. albicans reacted with 121 proteins; C. posadasii, 81; P. brasiliensis, 67; and after HKY, 63 proteins on the yeast protein microarray, respectively. We identified a set of 16 antigenic proteins that were shared across the three fungal pathogens. These include retrotransposon capsid proteins, heat shock proteins, and mitochondrial proteins. Five of these proteins were identified in our previous study of fungal cell wall by mass spectrometry (Ann N Y Acad Sci. 1273:44-51). The results obtained give a comprehensive view of the immunological responses to fungal infections at the proteomic level. They also offer insight into immunoreactive protein commonality amongst several fungal pathogens, and provide a basis for a panfungal vaccine.


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Introduction Human fungal infections may occur as a self-limiting cutaneous disease, an acute pneumonia or widely disseminated systemic infections. Although a number of fungal infections can manifest in a healthy individual, especially the endemic diseases, they have become increasingly important as causes of infection in immune compromised patient populations, particularly in a nosocomial setting. Immunosuppression caused by cancer treatment, organ transplant or autoimmune disease are important risk factors for increased susceptibility to fungal disease. Other factors important to increased susceptibility include invasive procedures such prosthetic surgery or application of indwelling catheters; imbalance of normal microbiota after treatment with broad spectrum antibiotics and immunosuppressive diseases (HIV for ex.) (1). The development of vaccines against fungal infections remains a great scientific and technological challenge, in part because of a lack of complete understanding of the mechanisms that underlie protective immunity. For example, we have recently shown that heat-killed yeast (HKY) used as a vaccine immunogen protects against five different fungal infections (2-9). Among the possible antigens responsible for the cross-protection are those present in the cell wall moieties (glucans and proteins). These molecular components may elicit a broad antibody response, with memory, that may contribute to protecting the host against subsequent fungal infections. The antibodies may provide immunity by the activation of complement, with neutralization of virulence traits, or opsonization, affecting the direction and vigor of cell-mediated immunity to directly inhibit fungal growth, or inhibit adherence and germination (6). To obtain a global overview of the antibody response against the HKY vaccine, and compare that to the response to other fungal infections, we have used a yeast protein microarray, which covers most of the yeast proteome, to identify antigens that elicit an antibody response and are common to S. cerevisiae and the HKY vaccine, as well as other fungal pathogens. We identified a set of common proteins (e.g., heat shock proteins), and also a novel



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set of antigens. These findings will help to understand the mechanism and development of a pan-fungal vaccine.


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Material and Methods Infection and serum collection Five-week-old male and female CD-1 mice were purchased from Charles River Laboratories (Hollister, CA). Mice were housed five per cage under conventional conditions and provided food and water ad libitum. After one week of acclimatization, groups of 5 mice were infected with one of the fungal pathogens. Experimental systemic infections with Coccidioides posadasii, strain Silveira were done using intravenous inoculation of 250 arthroconidia; pulmonary infection with Paracoccidioides brasiliensis, strain Garcia, by instilling 30 µl of yeast suspension (6 x107 yeast) intranasally; and systemic infection with Candida albicans, strain #5 by intravenous inoculation of 3x105 yeast, as previously described (10-12). In addition, groups of mice were immunized subcutaneously with heat-killed Saccharomyces cerevisiae (HKY) using three doses of 6 x107 killed yeasts as described previously by Capilla et al.(9). PBS-treatment was used as a control. After 30 days of infection/immunization, mice were anesthetized with isoflurane and exsanguinated. The blood was allowed to clot and serum collected for use. For each experimental group, serum samples (from 750 µl blood) were pooled for use in the protein microarray probing. All in vivo experiments were done under an approved animal care and use protocol from the California Institute for Medical Research. Protein microarray screen and data analysis Purified recombinant yeast proteins were printed on the nitrocellulose coated glass slides as previously described (13). Each microarray contains 4,800 fulllength GST fusion proteins spotted in duplicate (14, 15). The array probing was performed as described in the Invitrogen protocol for Protoarray Human Protein Microarray for Immune Response Biomarker Profiling (15). Collected sera were diluted (1:100) in washing buffer and loaded into the microarray chamber (4 ml). Two arrays were probed for each sample. After 1 h incubation at 4C and washing, the arrays were probed with anti-mouse IgG conjugated with Alexafluor 555 (1:100) and anti-GST antibody conjugated with Alexafluor 647



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(1:1000) to detect antibody-antigen interactions and presence of GST-fusion proteins, respectively. After washing, the protein–antibody interactions were detected using a 4200AL microarray scanner (Molecular Devices, Sunnyvale, CA). The image file was processed using Genepix Pro 7 software (Molecular Devices) to determine the signal intensity of the immune reaction. A local background subtraction was used to reduce the noise. The Alexa 555/ Alexa 647 mean ratios greater than 0.050 was considered as positive signals (Supplementary Tables 1-4). Amino acid (aa) sequences of the identified proteins were retrieved from the Saccharomyces cerevisiae Genome database (SGD) (http://yeastgenome.org/) and used as queries in BLASTP searches for sequence similarity in Candida albicans, Coccidioides posadasii and Paracoccidioides brasiliensis genomes. The resulting aa sequences were analyzed for sequence identity by using ClustalW. For protein functional classification and probability of enrichment of the categories, SGD and Funspec analysis tools were used (http://funspec.med.utoronto.ca/)


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Results In these studies, our goal was to determine whether cross-reactive immunogenic antigens could be found among several fungal pathogens and with HKY of S. cerevisiae. We did this by infecting mice with the fungal pathogens C. posadasii, C. albicans or P. brasiliensis or vaccinating mice with HKY; mice inoculated with PBS served as a control. Sera from these groups were used to probe S. cerevisiae protein microarrays as described in the Methods section (Fig. 1). Serum from mice infected with C. albicans reacted with 121 proteins; C. posadasii, 81; and P. brasiliensis, 67. After HKY vaccination, 63 proteins were detected on the yeast protein microarray (Fig. 2, and Suppl Tables 1-4). Table 1 shows the proteins that were detected by two or more of the sera tested, thus showing cross-reactivity among the different fungi with S. cerevisiae proteins. It should be noted that fewer proteins were identified by the serum from HKY vaccinated mice, which is likely due to the heat killing of the yeast and degradation of proteins and epitopes. By comparative analysis of the proteins detected by each serum, we identified a set of 16 antigenic proteins that were cross-reactive with sera from mice infected with each of the three fungal pathogens (Fig 2 and Table 2). These proteins are: SSE2/HSP70 (YBR169C), PET117 (YER058W), UBP3 (YER151C), TOM71 (YHR117W), ENO2 (YHR174W), TRZ1 (YKR079C), HBS1 (YKR084C), YDR161W, TRR1 (YDR353W), ARC1 (YGL105W), CUS2 (YNL286W), SOL1 (YNR034W), SUA7 (YPR086W) and the gag proteins (YPL257W-A, YPR158W-A, YGR038C-A). The degree of amino acid sequence conservation among the different fungal organisms is shown in table 2. In total, we identified in all assays 207 unique proteins that were recognized as antigens and elicited IgG antibody formation (Supplementary Tables 1-4). We observed 18 unique antigens between P. brasiliensis and C. posadasii, two phylogenetically closely related organisms and 2 to 4 unique proteins are shared within other pairs (Venn diagram in Figure 2). Bioinformatic analysis (using Funspec and GO functional analysis) showed a significant enrichment of common reactive proteins involved in protein folding (SSE2, CCT6, SNL1, HSP60, HSC82, SIS1, YDJ1, APJ1 STI1; p- value



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of 3.714e-07, ) translational elongation (TEF2, HBS1, MEF1, STM1, CAM1; pvalue of 1.976e-06) and tRNA aminoacylation for protein translation (GRS1 ARC1 GUS1 THS1 DPS1; p-value of 2.203e-05). Table 3 shows a general functional classification of the identified proteins.


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Discussion A goal of our work over the last several years has been that of vaccine development for fungal infection, since at present there are no commercially approved vaccines for human use. Our studies showing that HKY could induce protection against five different fungal pathogens is suggestive that a panfungal vaccine is a real possibility. This is further supported by studies using laminarin conjugated with diphtheria toxoid that showed protection against candidosis and aspergillosis (16, 17) and the use of a multivalent vaccine enhanced protection against coccidioidomycosis (18). However, one difficulty in the development of a panfungal vaccine is that of choosing the best immunogenic antigens for inclusion. The availability of a 4800 element ordered protein microarray from S. cerevisiae provides a useful tool for not only the study of comparative proteomics among various fungi, but for the study of comparative antigenic profiles. By probing the array with serum from infected animals, we are able to detect antibodies reactive against known proteins. Thus, the yeast proteins spotted onto the array present sufficient amino acid sequence conservation of epitopes to permit recognition by antibodies generated against other ascomycetous fungal species. This result then allows us to compare antigenic profiles of different fungi and determine what immunogenic proteins these organisms have in common. In the current study we have identified a large set of antigenic proteins in S. cerevisiae that react with sera from mice infected with one of three fungal pathogens or from those inoculated with heat-killed S. cerevisiae itself. This is the most comprehensive antibody profiling of fungal infection study to date using protein microarrays. One published study generated an array of cell surface proteins from C. albicans representing 363 different proteins (19). Those authors used their array to screen patient sera from different stages of candidal infection to generate biomarker profiles that might be useful for diagnostic or prognostic purposes. The identification of many of the proteins in our study support previously reported findings. Five of the reactive proteins were identified by our group in a proteomic study of C. posadasii and Aspergillus fumigatus extracts, which were



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determined by immunoblotting and mass spectroscopy (20): enolase (YHR174W), elongation factor 1 alpha (YBR118W), heat shock 70 kDa protein (SSE2/YBR169C) and aconitate hydratase mitochondrial (YDR234W) (20) . SSE2, a member of heat shock 70 protein family, is among the group of proteins that were detected by each of the experimental sera from the fungal pathogens we tested. Hsp70 has been observed in C. posadasii spherules by nano-high-performance liquid chromatography-tandem mass spectrometry (21). Furthermore, this protein has been shown to be immunogeneic in P. brasiliensis, H. capsulatum and C. albicans, even though it does not induce protection against experimental infections (22-27). Hsp70 is immunogenic in vivo in pulmonary cryptococcosis caused by the basidiomycete Cryptococcus neoformans (28, 29). We also observed that other HSPs commonly reacted with the sera: HSP40/DnaJ family members (YNL064C, YNL077W) for sera after C. posadasii, C. albicans and HKY; HSP42 after C. albicans; HSP60 after C. albicans or P. brasiliensis; and HSP90 after C. posadasii or P. brasiliensis. The HSP90 result is of interest, since it was not detected in the serum of mice infected with C. albicans, and antibody against this protein has been proposed as a therapeutic for treatment of candidiasis (30). It is known that many proteins including heat shock proteins are exported by unconventional secretory mechanisms (22). Antibodies may react with these proteins residing transiently in the cell surface or secreted to the external milieu. These results validate our approach to identify common antigens that elicit antibody responses in fungal infections. We noted the absence of covalently linked cell wall proteins including GPI-proteins in the list of common reactive proteins in Table 1, (for example, ECM33). We have also noted the absence of reactivity for many isoforms present in the array, such as Hsp70 family members (SSE1 paralog) and reactivity to HSP90 in C. albicans for example (31). One explanation would be that the antibody titers elicited by these proteins or the amount of each protein in the spot present on the array were not high enough to allow detection above the baseline.


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The identification of 91 additional unique proteins with the C. albicansinfected serum was not unexpected as C. albicans and S. cerevisiae are the most closely related among the organisms tested. Furthermore, it is not surprising that the serum from the mice vaccinated with heat-killed yeast did not induce more antibodies, since it is very likely that many epitopes were altered conformationally because of the heating used to kill the yeast. It is also possible that the amount of some proteins present in the killed yeast was insufficient to induce a strong antibody response with IgG at a titer high enough to detect proteins on the array. We believe that is difficult to statistically estimate the degree of proteome sequence divergence from its mostly non-pathogenic relative S. cerevisiae sufficient to elicit a pathogen-specific antibody repertoire during an infection. However, we certainly know that many pathogen-specific proteins are not present in the S. cerevisiae array because they were acquired by gene duplication or gene family expansion during lineage-specific evolution. In C. albicans, antibodies for a number of immunogenic cell wall proteins (e.g., ALS), proteins involved in nutrient acquisition (secreted aspartyl proteinases and lipases), and nutrient uptake (ferric reductases, iron transporters, and oligopeptide and amino acid permeases) are necessarily impossible to show reactivity in our study. A newly evolved class of proline-rich proteins, known to be highly immunogenic in C. posadasii, is under-represented in the S. cerevisiae genome as well. Another aspect that makes it difficult to estimate differences in immune reactivity in fungal species is that the most relevant homology among the various protein orthologs relates to the epitopes in the proteins. Although many are likely conformational epitopes, homologous linear epitopes likely also contribute to the demonstration of the cross-reactivities. Thus, it is possible that while the overall percent homology is low, that the homology of some epitopes, particularly the linear epitopes, is very high. Interestingly, we identified a set of antigens not previously known to react with sera of infected animals. As the most intriguing example, several retrotransposon gag proteins reacted with all experimental sera (table 1 and 2).



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One transcriptomic study has observed the expression of Gag RNAs, during the dimorphic transition after in vitro heat induction in P. brasiliensis (32). It is plausible that these proteins are induced during infection by the host temperature. Additional studies are required to evaluate the role of gag proteins in the infection process and the nature of the humoral response against these proteins. We conclude that protein microarrays represent a comprehensive approach to verify the entire antibody specificity repertoire induced during a specific fungal infection. The data presented in this study further represent a resource for evaluating candidates for vaccine development. The analysis of the antibody profile may be useful to guide the selection of vaccine components in future studies.


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Table 1. Yeast proteins identified on protein microarrays that react with serum from mice infected with different fungal pathogens (Cp, Pb, Ca) or inoculated heat-killed Yeast (Sc). Gray shading indicates reactivity. Proteins in bold are discussed in the paper. Sc: Saccharomyces cerevisiae, Ca: Candida albicans; Pb: Paracoccidioides brasiliensis, Cp: Coccidioides posadasii ORF YDR353W YER151C YGL105W YGR038C-A YPL257W-A YPR158W-A YHR117W

Gene TRR1 UBP3 ARC1 "" "" "" TOM71

YKR079C YKR084C YNL286W YNR034W YDR161W YHR174W YBR169C YER058W YPR086W YER101C YER125W YGL245W YGR256W YHL033C YHR214C-C YIL078W YLL018C YLR003C YLR192C YLR244C YNL272C YOR195W YOR198C YOR239W YOR335C YPL048W YPR008W YDL070W YNL007C YNL077W YNL292W YNL217W YIL016W YBR118W

TRZ1 HBS1 CUS2 SOL1 "" ENO2 SSE2 PET117 SUA7 AST2 RSP5 GUS1 GND2 RPL8A "" THS1 DPS1 CMS1 HCR1 MAP1 SEC2 SLK19 BFR1 ABP140 ALA1 CAM1 HAA1 BDF2 SIS1 APJ1 PUS4

YBR121C YDL002C YDR234W YDR257C YDR358W

GRS1 NHP10 LYS4 RKM4 GGA1

YFR010W YJL218W YLR069C YML045W-A YMR004W YMR186W YNL001W YOL038W YOR027W YOR054C YPL100W YPL192C YDR109C YDR188W YNL054W-A YPR074C YER036C

UBP6 "" MEF1 "" MVP1 HSC82 DOM34 PRE6 STI1 VHS3 ATG21 PRM3 "" CCT6 TKL1" ARB1

YIL075C YLR150W YPL266W YER067W YLR259C YER120W YGR126W YJL084C YIL072W YNL064C

RPN2 STM1 DIM1 RGI1 HSP60 SCS2 "" ALY2 HOP1 YDJ1

SNL1 TEF2

Function ThioRedoxin Reductase UBiquitin-specific Protease Acyl-RNA-Complex gag protein gag protein gag protein Translocase of the Outer Mitochondrial membrane tRNase Z Hsp70 subfamily B Suppressor Cold sensitive U2 snRNA Suppressor Suppressor Of Los1-1 (DNA repl. Stress) Unknown ENOlase HSP70 PETite colonies Transcription initiation Lipid raft associated protein E3 ubiquitin ligase GlUtamyl-tRNA Synthetase 6-phosphogluconate dehydrogenase Ribosomal Protein of the Large subunit gag protein THreonyl tRNA Synthetase Aspartyl-tRNA synthetase 90S preribosome processome (DNA stress) eIF3 Methionine AminoPeptidase GEF for Sec4 Synthetic Lethal Kar3p Component of mRNP complexes Actin Binding Protein ALAnyl-tRNA synthetase Calcium And Membrane-binding protein Transcriptional activator (DNA repl. Stress) Transcription initiation (DNA repl. Stress) HSP40 co-chaperone Hsp40/DnaJ family PseudoUridine Synthase unknown Ribosome-associated protein Translational elongation factor EF-1 alpha Glycyl-tRNA Synthase INO80 chromatin remodeling complex lysine biosynthesis pathway Ribosomal lysine (K) Methyltransferase Golgi-localized, Gamma-adaptin ear homology, Arf-binding protein UBiquitin-specific Protease Putative acetyltransferase Mitochondrial Elongation Factor gag protein membrane traffic to the vacuole Hsp90 family ribosomal subunit dissociation (DNA stress) PRoteinase yscE Hsp90 cochaperone Subunit of the 20S proteasome AuTophaGy related Pheromone-Regulated Membrane protein Putative kinase Chaperonin Containing TCP-1 gag protein TransKetoLase ATP-binding cassette protein involved in Ribosome Biogenesis 26S proteasome Suppressor of ToM1 pre-ribosomal RNA processing Respiratory Growth Induced Heat Shock Protein Suppressor of Choline Sensitivity DNA damage response Arrestin-Like Yeast protein HOmolog Pairing Yeast dnaJ

Cp


Pb

Ca

Sc


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Table 2. Percentage amino acid sequence identity among protein orthologs

Sc

Ca

Pb

Cp

TRR1

79.6

69.0

67.7

ENO2

76.0

73.0

73.0

SOL1

62.3

42.5

40.9

SSE2/HSP70

59.3

48.6

46.3

SUA7

51.5

36.8

37.4

ARC1

48.5

34.0

35.6

PET117

45.4

32.3

34.8

TOM71

39.0

25.8

25.8

HBS1

36.0

29.8

32.1

UBP3

34.7

19.9

27.9

TRZ1

32.5

25.2

23.2

YDR161W

31.5

22.5

23.5

CUS2

26.5

24.6

23.5

Sc: Saccharomyces cerevisiae, Ca: Candida albicans; Pb:Paracoccidioides brasiliensis, Cp: Coccidioides posadasii



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Table 3. Functional classification of proteins reacting with sera. Classs/ORF

Gene

Function

Category

Glycolysis YHR174W

ENO2

Enolase

Glycolysis

Chaperones YBR169C YNL007C YNL077W YMR186W YOR027W YLR259C YNL064C YDR188W

SSE2 SIS1 APJ1 HSC82 STI1 HSP60 YDJ1 CCT6

HSP70 HSP40 co-chaperone Hsp40/DnaJ family Hsp90 family Hsp90 cochaperone Hsp60 Yeast dnaJ Chaperonin Containing TCP-1

Stress; chaperone; protein folding Chaperone Chaperone chaperone chaperone chaperone chaperone Chaperonin

tRNA modification YGL105W YKR079C YNR034W YIL078W YLL018C YGL245W YNL292W YOR335C YOR239W YBR121C

ARC1 TRZ1 SOL1 THS1 DPS1 GUS1 PUS4 ALA1 ABP140 GRS1

Acyl-RNA-Complex tRNase Z Suppressor Of Los1-1 (DNA repl. Stress) THreonyl tRNA Synthetase Aspartyl-tRNA synthetase GlUtamyl-tRNA Synthetase PseudoUridine Synthase ALAnyl-tRNA synthetase Actin Binding Protein Glycyl-tRNA Synthase

tRNA aminoacylation; protein translation; tRNA metabolism tRNA processing; export tRNA aminoacylation tRNA aminoacylation tRNA aminoacylation tRNA modification tRNA aminoacylation Actin cytoskeleton and tRNA methylation tRNA aminoacylation

"" "" "" "" ""

gag protein gag protein gag protein gag protein gag protein gag protein

Protein synthesis YHL033C YBR118W YLR192C YIL016W YKR084C YNL001W YLR069C YPL048W

RPL8A TEF2 HCR1 SNL1 HBS1 DOM34 MEF1 CAM1

Ribosomal Protein of the Large subunit Translational elongation factor EF-1 alpha eIF3 Ribosome-associated protein Hsp70 subfamily B Suppressor ribosomal subunit dissociation (DNA stress) Mitochondrial Elongation Factor Calcium And Membrane-binding protein

Protein synthesis Protein synthesis Protein synthesis; component of translation initiation factor 3 Protein synthesis; protein folding translation translation translation;mitochondria Protein translation; transcription initiation

Proteasome YOL038W YOR054C YIL075C YFR010W

PRE6 VHS3 RPN2 UBP6

PRoteinase yscE Subunit of the 20S proteasome 26S proteasome UBiquitin-specific Protease

Protease; 20S proteasome Protease; 20 S proteasome Protease: proteasome 26S UBiquitin-specific Protease

Stress YDR353W YER151C YLR150W

TRR1 UBP3 STM1

ThioRedoxin Reductase UBiquitin-specific Protease Suppressor of ToM1

Oxidative stress Osmotic stress; proteolysis Regulation of translational initiation in response to stress

Unknown YNL217W YJL218W YLR003C YDR109C YDR161W YGR126W YER067W

"" CMS1 "" "" "" RGI1

unknown Putative acetyltransferase 90S preribosome processome (DNA stress) Putative kinase unknown DNA damage response Respiratory Growth Induced

unknown unknown unknown unknown

Others YHR117W

TOM71

YNL286W YOR198C YER058W YPR086W YER101C YER125W

CUS2 BFR1 PET117 SUA7 AST2 RSP5

YGR256W YLR244C YNL272C YOR195W YPR008W YDL070W YDL002C YDR234W YPR074C YDR257C YDR358W

GND2 MAP1 SEC2 SLK19 HAA1 BDF2 NHP10 LYS4 TKL1" RKM4 GGA1

YMR004W YPL100W YPL192C YER036C

MVP1 ATG21 PRM3 ARB1

YPL266W YER120W YJL084C YIL072W

DIM1 SCS2 ALY2 HOP1

Gag proteins YGR038C-A YPL257W-A YPR158W-A YNL054W-A YHR214C-C YML045W-A

Translocase of the Outer Mitochondrial membrane Cold sensitive U2 snRNA Suppressor Component of mRNP complexes PETite colonies Transcription initiation Lipid raft associated protein E3 ubiquitin ligase 6-phosphogluconate dehydrogenase Methionine AminoPeptidase GEF for Sec4 Synthetic Lethal Kar3p Transcriptional activator (DNA repl. Stress) Transcription initiation (DNA repl. Stress) INO80 chromatin remodeling complex lysine biosynthesis pathway TransKetoLase Ribosomal lysine (K) Methyltransferase Golgi-localized, Gamma-adaptin ear homology, Arf-binding protein membrane traffic to the vacuole AuTophaGy related Pheromone-Regulated Membrane protein ATP-binding cassette protein involved in Ribosome Biogenesis pre-ribosomal RNA processing Suppressor of Choline Sensitivity Arrestin-Like Yeast protein HOmolog Pairing

unknown Unknown; stress related

Mitochondrial outer membrane protein mRNA processing; splicing mRNA metabolic process mitochondria Transcription initiation protein targeting to membrane E3 ubiquitin ligase of NEDD4 family; regulates many cellular processes oxidative branch, pentose pathway Methionine removal; aminopeptidase exocytosis Chromosome segregation; Kinetochore protein Transcriptional activator (DNA repl. Stress) Transcription initiation (DNA repl. Stress) chromatin remodeling complex Aminoacid metabolism Aminoacid metabolism peptidyl-lysine monomethylation Golgi to endosome transport protein targeting to vacuole autophagy Karyogamy; membrane protein Ribosome biogenesis rRNA modification ER membrane protein; phospholipid metabolism regulation of intracellular transport meiosis


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Supporting Information This material is available free of charge via http://pubs.acs.org/. Supplementary table: Supplementary Tables_JPR.docx



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Acknowlegment This work was supported in part by funding from the Foundation for Research in Infectious Diseases (grant 424 to D.A.S.), the NIH (R01GM076102 to M.P.S) and Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (08/558316 to P.S.R.C.). P.S.R.C. received a scholarship from Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (CsF 246793/2012-0).


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Figure Legends

Fig 1. Protein microarrays probed with sera from uninfected mice (negative control) and from Coccidioides posadasii-infected mice. 4,800 GST-tagged yeast proteins were spotted in duplicate (see Material and Methods). After probing with primary sera (sera from infected or vaccinate mice), the arrays were probed simultaneously with secondary sera consisting of anti-mouse IgG conjugated with Alexafluor-555 (green signal) and anti-GST antibody conjugated with Alexafluor-647 (red signal). Block 27 from both arrays is shown after scanning and image processing. (16 X 16 spots representing a total of 128 yeast proteins and controls printed in duplicate). Two representative antibody– protein interactions (Hsp70/SSE2 and Gag proteins) are indicated by green boxes in the array probed with sera from C. posadasii-infected mice. The yellowish spots in the green boxes represent a pseudocolor ratio signal revealed after analysis with Genepix software. These proteins are indicated in red boxes in the negative control. Fig 2. Interactions detected on the protein microarray after probing with sera from mice infected with Candida albicans (Ca), Coccidioides posadasii (Cp), Paracoccidioides brasiliensis (Pb) or immunized with heat-killed yeast (HKY). Each number represents the number of unique proteins that were visualized by the serum from the mice.



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Figure 1 Protein microarrays probed with sera from uninfected mice (negative control) and from Coccidioides posadasii-infected mice 80x48mm (300 x 300 DPI)



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Figure 2 Interactions detected on the protein microarray after probing with sera from mice infected with Candida albicans (Ca), Coccidioides posadasii (Cp), Paracoccidioides brasiliensis (Pb) or immunized with heat-killed yeast (HKY) 54x44mm (200 x 200 DPI)


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