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Accepted Manuscript Characterization of extracellular proteins in members of the Paracoccidioides complex Amanda Rodrigues de Oliveira, Lucas Nojosa Oliveira, Edilânia Gomes Araújo Chaves, Simone Schneider Weber, Alexandre Melo Bailão, Juliana Alves ParenteRocha, Lilian Cristiane Baeza, Célia Maria de Almeida Soares, Clayton Luiz Borges PII:

S1878-6146(18)30062-X

DOI:

10.1016/j.funbio.2018.04.001

Reference:

FUNBIO 916

To appear in:

Fungal Biology

Received Date: 24 November 2017 Revised Date:

11 March 2018

Accepted Date: 3 April 2018

Please cite this article as: Rodrigues de Oliveira, A., Oliveira, L.N., Araújo Chaves, E.G., Weber, S.S., Bailão, A.M., Parente-Rocha, J.A., Baeza, L.C., Almeida Soares, C.M.d., Borges, C.L., Characterization of extracellular proteins in members of the Paracoccidioides complex, Fungal Biology (2018), doi: 10.1016/j.funbio.2018.04.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Characterization of extracellular proteins in members of the Paracoccidioides complex

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Amanda Rodrigues de Oliveira1,ǂ, Lucas Nojosa Oliveira1,2,ǂ, Edilânia Gomes Araújo

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Chaves1, Simone Schneider Weber3,4, Alexandre Melo Bailão1, Juliana Alves Parente-

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Rocha1, Lilian Cristiane Baeza1, Célia Maria de Almeida Soares1, Clayton Luiz Borges1*

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Federal de Goiás, Goiânia, Goiás, Brazil

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Laboratório de Biologia Molecular; Instituto de Ciências Biológicas; Universidade

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Universidade de Brasília, Brasília, Distrito Federal, Brazil

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Itacoatiara, Amazonas, Brazil

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Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul, Brazil

Instituto de Ciências Exatas e Tecnologia; Universidade Federal do Amazonas,

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Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição; Universidade Federal do

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Programa de Pós-graduação em Patologia Molecular; Faculdade de Medicina;

These authors have contributed equally to this work.

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*

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Clayton Luiz Borges

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Laboratório de Biologia Molecular

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Instituto de Ciências Biológicas II

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Campus II

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Universidade Federal de Goiás

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790-900, Goiânia, Goiás, Brazil.

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Tel/Fax: +55 62 3521-1110

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E-mail address: [email protected] (C. L. Borges)

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Corresponding author

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Abstract Paracoccidioides is a thermodimorphic fungus that causes Paracoccidioidomycosis

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(PCM) – an endemic systemic mycosis in Latin America. The genus comprises several

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phylogenetic species which present some genetic and serological differences.

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diversity presented among isolates of the same genus has been explored in several

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microorganisms. There have also been attempts to clarify differences that might be related

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to virulence existing in isolates that cause the same disease. In this work, we analyzed the

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secretome of two isolates in the Paracoccidioides genus, isolates Pb01 and PbEpm83, and

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performed infection assays in macrophages to evaluate the influence of the secretomes of

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those isolates upon an in vitro model of infection. The use of a label-free proteomics

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approach (LC-MSE) allowed us to identify 92 proteins that are secreted by those strains. Of

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those proteins, 35 were differentially secreted in Pb01, and 36 in PbEpm83. According to

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the functional annotation, most of the identified proteins are related to adhesion and

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virulence processes. These results provide evidence that different members of the

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Paracoccidioides complex can quantitatively secrete different proteins, which may

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influence the characteristics of virulence, as well as host-related processes.

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Keywords: Paracoccidioides species; macrophage infection; secretome; label-free

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proteomics; virulence factors.

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1. Introduction Fungi

of

the

Paracoccidioides

genus

are

the

causative

agents

of

paracoccidioidomycosis (PCM) – a prevalent systemic mycosis in Latin America (Restrepo

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& Tobon 2005). These fungi present thermal dimorphism, growing in the environment as

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mycelium, the saprobiotic form, in temperatures below 28°C, and as yeast cells at 36°C in

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human host tissues (Restrepo 1985). The mycelia form produces conidia that act as

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infectious propagules, which, when inhaled, suffer dimorphic transition in the lungs,

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resulting in the pathogenic yeast form (Brummer et al. 1993; Borges-Walmsley et al.

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2002). The disease evolution and the manifestation of clinical forms depend on

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immunological factors (Franco et al. 1987), the virulence levels of the fungus isolates

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(San-Blas & Niño-Vega 2001), and the host gender (Restrepo et al. 1984).

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The Paracoccidioides genus is currently subdivided into five species, namely P.

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lutzii, P. brasiliensis, P. americana, P. restrepiensis and P. venezuelensis (Turissini et al.

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2017), based on genotypic studies and geographic distribution. Diversity presented among

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isolates of the same genus has been explored with regard to various microorganisms,

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including Paracoccidioides. These studies have used various approaches in an attempt to

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characterize biochemical and molecular differences that can reflect on the virulence of

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isolates that cause the same disease (Kurokawa et al. 2005; Carvalho et al. 2005; Pigosso

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et al. 2013; Siqueira et al. 2016).

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Many of the virulence factors of medically-important pathogenic fungi are related to

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extracellular proteins. These act on the adverse environment of the host, combatting the

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immune response, and leading to successful infection (Kniemeyer & Brakage 2008;

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Ranganathan & Garg 2009; Holbrook et al. 2011). It is clear that extracellular proteins play

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an essential role in fungal-infection strategies, acting on the molecular dialogue with the

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host cells, enabling survival, multiplication, pathogen dissemination, as well as in

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ACCEPTED MANUSCRIPT modulation of host defenses (Ranganathan & Garg 2009; Silva et al. 2012). Secretome

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characterization of microorganisms has been widely explored (Sorgo et al. 2010;

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Rampitsch et al. 2013; Campbell et al. 2015), and through this characterization it is

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possible to identify new fungal virulence factors. In Paracoccidioides, proteins such as

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heat shock proteins, thioredoxin, superoxide dismutase, catalases, TCTP family proteins,

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disulfide isomerase, secreted serine proteinase - are all involved in cell rescue, defense, and

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virulence, and have been described in this milieu (Vallejo et al. 2012; Weber et al. 2012;

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Pigosso et al. 2017).

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Some Paracoccidioides spp. proteins that play an important role in the virulence of

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the fungus against the host have been identified in extracellular environments. In this

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context, adhesion can mediates host-fungi interactions during infection. Enolase, 14-3-3

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protein, fructose-1,6-bisphosphate aldolase, triose phosphate isomerase, glyceraldeyde-3-

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phosphate dehydrogenase, and glycoprotein gp43 have been described as Paracoccidioides

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adhesins (Barbosa et al. 2006; Pereira et al. 2007; Donofrio et al. 2009; Nogueira et al.

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2010; Chaves et al. 2015; Oliveira et al. 2015). A recent study has provided molecular

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details of the interaction between P. brasiliensis and extracellular matrix (ECM)

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components, using in vitro cellular models and adherence assays. It has shown that the

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peptide 1 (NLGRDAKRHL) from gp43 contributes to P. brasiliensis’ adhesion to Vero

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cells (Mendes-Giannini et al. 2006). Using antisense RNA (aRNA) technology, Torres and

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collaborators correlated low gp43 expression with lower P. brasiliensis pathogenicity in

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the mice model of infection, noting the protein involvement in fungal virulence (Torres et

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al. 2013). The role of the 30 kDa protein of P. brasiliensis as adhesin was also

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demonstrated in the ex vivo model of infection (Andreotti et al. 2005). In addition,

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Paracoccidioides secretes a serine proteinase during the murine model of infection

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(Pigosso et al. 2017). However, a comparison between members of the genus has not yet been established. The majority of information about the eco-epidemiology, infection, and

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Paracoccidioidomycosis has been obtained in studies that did not distinguish between the

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species involved (Martinez 2017). Taking this into account, we performed the

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characterization of the extracellular proteome profile between two members of the genus

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Paracoccidioides, namely Pb01 (P. lutzii) and PbEpm83 (P. restrepiensis). In addition, we

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performed infection assays in macrophages, which allowed us to suggest that the fungus

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can modify their secretome, influencing their behavior in infection. This is the first

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comparative study between Paracoccidioides members of the extracellular proteome, and

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it will provide a significant set of data for future research. Finally, our data brings an

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important dataset of putative virulence/antigenic molecules from the Paracoccidioides

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genus. These data serve to highlight molecules that need to be explored further in order to

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better understand Paracoccidioides pathobiology.

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2. Material and methods

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2.1. Microorganisms and culturing conditions

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Paracoccidioides, isolates Pb01 (ATCC MYA-826) (Carrero et al. 2008) and

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PbEpm83 (Theodoro et al. 2008; Machado et al. 2013) were used in this study.

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Paracoccidioides isolates were both from chronic PCM. Pb01 was isolated from a patient

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from the state of Goiás, Brazil (Central-Western region), and PbEpm83 from a patient

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from Bogota (Colombia). The conidia production also differs between these two species.

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Paracoccidioides lutzii isolates produced an intermediate to high number of conidia, while

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P. restrepiensis isolates were unable to generate conidia, when incubated in the media

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containing potato dextrose agar and Soil Extract Agar. This may explain the delimited

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distribution of this species (Theodoro et al. 2012). The yeast cells were maintained in BHI solid medium [Brain Heart Infusion] (pH

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7.0), plus 4% glucose (w/v), 1% agar (w/v) in weekly samplings at 36 ºC for 5 days. The

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viability, cell growth and vitality assays were performed with a Trypan Blue (Sigma

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Aldrich), cell counting on hemacytometer and quantification of glucose consumption

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(Doles), respectively.

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2.2. Preparation of extracellular extracts

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The yeast extracellular proteins from Paracoccidioides isolates Pb01 and PbEpm83

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were prepared as previously described (Weber et al. 2012). In brief, the yeast cells from

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the isolates were obtained by inoculating 50 µg/mL of wet weight cells in BHI liquid

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medium. They were then maintained at 36ºC for 24 hours under shaking (150 rpm).

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Afterwards, the cells were removed by centrifugation at 10,000 x g at 4°C for 30 minutes.

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The culture supernatants were sequentially filtered through 0.45 and 0.22 µm-pore-size

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membrane filters. The filtrates were concentrated and then washed three times with

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ultrapure water by centrifugation through a 10 kDa molecular weight cut-off in Ultracel®

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regenerated membrane (Amicon Ultra centrifugal filter, Millipore, Bedford, MA, USA).

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The absence of cell lysis was performed as previously described (Weber et al. 2012) (data

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not shown). Protein concentrations were determined by the Bradford method (Bradford

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1976). For the proteomic analysis, three biological replicates of protein samples from three

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independent experiments were obtained for each Paracoccidioides isolate.

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2.3. Mass spectrometry analysis by nanoUPLC-MSE

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ACCEPTED MANUSCRIPT The yeast extracellular proteins from Paracoccidioides isolates were analyzed by

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using the label-free nanoUPLC-MSE approach. An equal amount of each sample (150 µg)

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was prepared for nanoUPLC-MSE, as previously described (Murad et al. 2011; Lima et al.

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2014; Bailão et al. 2014). Samples were subjected to the action of 0.2% (v/v) of RapiGest

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SF™ surfactant (Waters Corporation, Milford, MA) at 80°C for 15 minutes. In order to

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make the protein more accessible to alkylation and digestion, 2.5 µL of 100 mM DTT

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(Dithiotreitol, GE Healthcare) was added at 60°C for 15 minutes. Next, 2.5 µL of 300 mM

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iodoacetamide (GE Healthcare) was added, and the samples were incubated at room

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temperature for 30 minutes for cysteines alkylation, while being protected from the light.

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The protein samples were digested with 40 µL of 50 ng/µL trypsin (Promega, Madison,

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WI, USA) at 37°C for 16 hours. After digestion, 30 µL of trifluoroacetic acid (TFA)

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(Sigma-Aldrich) 5% (v/v) was added to the samples. The mixture was left at 37°C for 90

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minutes in order to precipitate the surfactant. Afterwards, the samples were submitted to

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centrifugation at 18,000 x g, at 6°C for 30 minutes. The supernatant was transferred to new

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tubes and dried under vacuum. The precipitate was resuspended in 30 µL of ultrapure

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water and submitted to further purification with ZipTips® Pipette Tips (ZipTips® Pipette

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Tips C18, Millipore, Bedford, MA, USA). Peptides were dried again under vacuum and

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resuspended in ammonium formate (20 mM). In order to quantify the proteins, rabbit

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phosphorylase B (PHB) (MassPREPTM Digestion Standard) was used. The final

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concentration of PHB was 200 fmol/µL to Pb01 sample and 250 fmol/µL to PbEpm83

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sample.

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The digested peptides were further analyzed via nanoUPLC-MSE by using a

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nanoACQUITYTM system coupled to Synapt G1 MS™ mass spectrometer (Waters

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Corporation, Manchester, UK). The [GLU1]-Fibrinopeptide B (GFB) was used as a lock

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mass for calibration during the sample’s analysis. The UPLC-MSE spectra that was

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obtained was then processed and examined using the ProteinLynx Global Server (PLGS)

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version 2.4 (Waters Corporation, Manchester, UK). The raw data were searched against the

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Paracoccidioides

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(http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHo

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me.html) using algorithms previously described in correct and reverse sequences (Li et al.

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2009). The tables generated by PLGS were processed as previously described (Murad &

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Rech 2012; Lima et al. 2014; Bailão et al. 2014). Graphics indicating the quality of the

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proteomic data (dynamic detection range, type of peptides fragmentation and peptide mass

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identification accuracy) were generated using the FBAT software (Laird and Horvath,

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2000), MassPivot (kindly provided by Dr. Andrew M. Murad) Spotfire® (TIBCO Software

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Inc. ©) and Microsoft Office Excel (Microsoft ©) programs. Furthermore, only those

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proteins that presented 1.3-fold differences in expression values were considered to be

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regulated.

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spp.

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2.4. Bioinformatics Analysis

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The identified proteins were functionally classified according to the MIPS Functional

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Catalogue Database (http://mips.helmholtz-muenchen.de/funcatDB/) using query tools

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available

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(http://pedant.gsf.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_r48325_Pa

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r_lutzi), UniProt (http://www.uniprot.org/) and the National Center for Biotechnology

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Information (NCBI) (https://www.ncbi.nlm.nih.gov/).

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proteins were submitted to the BlastP tool (Basic Local Alignment Search Tool;

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https://blast.ncbi.nlm.nih.gov/Blast.cgi) from NCBI to search for sequence similarities and

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conserved domains in order to find some function related to non-classified proteins. Venn

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online,

such

as

Pedant

Hypothetical and conserved

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diagrams

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(http://bioinfogp.cnb.csic.es/tools/venny/index.html).

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were

constructed

by

using

the

Venny

2.1

tool

With regard to the secretion pathways prediction, the detected proteins were also subjected

to

analysis

in

the

online

algorithm

SignalP

3.0

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(http://www.cbs.dtu.dk/services/SignalP) in order to determine the presence of the signal

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peptide. Proteins with a signal peptide sequence were considered as being secreted by a

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classical pathway, and the standard cut-offs of the algorithm were adopted. In addition, we

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used the SecretomeP 2.0 algorithm (http://www.cbs.dtu.dk/services/SecretomeP) to predict

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proteins secreted by non-classical or alternative pathways, adopting the cut-off suggested

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by the software. In addition, TargetP 1.0 (http://www.cbs.dtu.dk/services/TargetP-1.0/),

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TMHMM

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(http://gpcr.biocomp.unibo.it/predgpi/pred.htm),

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(http://wolfpsort.seq.cbrc.jp/) predictive servers were also used in order to evaluate the

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subcellular location, contributing to discard false positive, as employed previously by

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Beys-da-Silva et al. (2014).

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(http://www.cbs.dtu.dk/services/TMHMM/), and

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software (http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::antigenic), which searched for

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potential antigenic epitopes within the protein sequences. The adhesin prediction analysis

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was also performed by using the FaaPred server (Fungal adhesins and Adhesin-like

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Proteins Prediction; http://bioinfo.icgeb.res.in/faap/) (Ramana & Gupta 2010) by adopting

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software default settings.

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2.5. Immunoblotting analysis

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Extracellular extracts (40 µg) were fractionated by 12% SDS-polyacrylamide gel

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electrophoresis (SDS-PAGE). After gel electrophoresis, proteins were stained with

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ACCEPTED MANUSCRIPT Coomassie Blue R or transferred to a Hybond™ ECL™ Membrane (GE Healthcare Life

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Sciences). Membranes were blocked with blocking buffer [1X PBS, 10% (w/v) skim milk,

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0.1% (v/v) Tween 20] for 2 hours at room temperature. After blocking, the membranes

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were washed with wash buffer [1X PBS, 0.1% (v/v) Tween 20] and incubated under

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agitation with specific primary antibodies for 1 hour at room temperature. Afterwards, the

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membranes were washed three times, followed by incubation with secondary antibodies

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conjugated with alkaline phosphatase for 1 hour at room temperature. Labelled bands

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detection was carried out using a 5-bromo-4-chloro-3-indolylphosphate/nitroblue

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tetrazolium (BCIP/NBT) protocol. The antibodies used for this analysis were: anti-enolase

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(1:40,000) (Nogueira et al. 2010); anti-triosephosphate isomerase (1:1,000) (Pereira et al.

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2007), anti-glyceraldehyde 3-phosphate dehydrogenase (1:5,000) (Barbosa et al. 2006),

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anti-aldolase (1:5,000) (Chaves et al. 2015), and anti-formamidase (1:1,000) (Borges et al.

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2010). Raw Tiff images were analyzed by densitometry of immunoblotting bands using the

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ImageJ 1.51 software. The pixel intensity of the analyzed bands was generated and

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expressed as arbitrary units.

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2.6. Measurement of formamidase activity

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Formamidase activity (FMD) was assessed by the ammonia formation from the

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formamide hydrolysis (Borges et al. 2005). The test was performed with 1µg of total

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protein extract. The volume was adjusted to 250 µL with formamide + PEB buffer (100

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mM formamide, 100 mM sodium phosphate and 10 mM EDTA, pH 7.4). The samples

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were incubated at 37°C for 30 minutes to allow formamidase enzymatic activity. After

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that, 400 µL of sodium phenol-nitroprusside and 400 µL of alkaline solution (Sigma

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Aldrich, Co.) were added. The samples were incubated at 50°C for 6 minutes and the

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absorbance read at 625nm. The amount of ammonia formed was determined by

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ACCEPTED MANUSCRIPT comparison with a standard curve. One unit (U) of formamidase-specific activity

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corresponds to the amount of enzyme required to hydrolyze 1 mmol of formamide

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(corresponding to the formation of 1 mmol of ammonia)/min/mg of total protein (Bury-

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Moné et al. 2003).

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2.7. Macrophage model of infection

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The macrophage infection assay was conducted as previously described (Lima et al.

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2014; Bailão et al. 2014), with some modifications. Macrophages, cell line J774 A.1 (Rio

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de Janeiro Cell Bank – BCRJ/UFRJ, accession number: 0121), were maintained in RPMI

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medium (RPMI 1640, Vitrocell, Brazil), with 10% (v/v) FBS at 37°C in a 5% CO2

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incubator chamber. The assay in J774 macrophage cells was conducted in the proportion of

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1:5 (macrophages:fungi cells) in order to favor the fungal internalization. A total of 1 x 106

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macrophages cells was distributed in 12 well culture plates, with RPMI medium, plus 100

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U/mL IFN-γ (PeproTech, Rocky Hill, NJ, USA) and incubated at 37°C, 5% CO2 for 16

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hours. For infection, 5 x 106 Paracoccidioides yeast cells were added to plate wells

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presenting adherent macrophages. The experimental infection had a course of 12 and 24

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hours, incubated at 5% CO2 and at 37°C. The recovered Paracoccidioides cells inside

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macrophages were plated on BHI solid medium (4% glucose and 4% FBS), and the growth

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of colonies was evaluated on the 12th day after infection.

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To evaluate the secretome influence on the Paracoccidioides-macrophages

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adhesion/interaction, a phagocytosis assay was conducted as described above by adding

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100 µg of secreted protein extract together with Paracoccidioides yeast cells, followed by

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incubation at 37°C and 5% CO2, for 12 hours and 24 hours. After incubation, the cells were

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washed 3 times with 1X PBS to remove Paracoccidioides non-adhered cells. Then, the

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macrophage cells were lysed with cold ultrapure water 3 times for 5 minutes. Fungal cells

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ACCEPTED MANUSCRIPT were recovered in 500 µL of saline solution (NaCl 0.9%), and 100 µL were plated on BHI

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solid medium supplemented with 4% FBS, which was incubated at 37°C. The growth of

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the colonies could be observed from the 5th day, and the counting of colony-forming units

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(CFUs) was performed on the 12th day. All infection assays were performed in biological

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and experimental triplicates and the data were presented as the mean ± standard deviation

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of replicates. Statistical analysis was carried out using the Student’s t-test, and p-values ≤

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0.05 were considered statistically significant.

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3. Results

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3.1. Label-free Proteomic analysis of Paracoccidioides isolates

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In order to verify the in vitro cellular behavior between strains, viability, growth, and

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vitality assays were performed. As shown in Supplementary Figure S1, the strains behave

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similarly. These data demonstrate that the in vitro conditions do not show any experimental

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bias on the analyzes. In addition, the quality of the extracellular protein extracts was

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evaluated by using SDS-PAGE assay. Supplementary Figure S2 shows good protein

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extract integrity, as well as it was possible to observe a distinct protein pattern between the

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samples of isolates. The dynamic range chart depicted measure abundance of 3 to 3.5

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orders of magnitude, indicating a satisfactory detection distribution of a high and low

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proteins concentrations levels (Supplementary Figure S3 panel A). Most of the peptides

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identified in Pb01 and PbEmp83 secretomes presented less than 15 ppm of error, which

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can be seen in Supplementary Figure S3 panel B. Supplementary Figure S3 panel C depicts

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that 44 and 50% of those peptides were obtained from peptide match type data in the first

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pass (PepFrag 1) to Pb01 and PbEpm83, respectively; a total of 6% of the peptides were

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obtained for both isolates in the second pass (PepFrag 2); 16 and 15% were identified by a

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missed trypsin cleavage, whereas insource fragmentation rates of 17 and 13% were related

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to Pb01 and PbEpm83, respectively. Altogether, these results show that all proteomic data

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is of a good quality.

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3.2. An overview of the extracellular proteome profiles

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Proteomic data from the Paracoccidioides spp. extracellular extract acquired by

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nanoUPLC-MSE resulted in the identification of 92 proteins (Supplementary Table S1).

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From the 92 proteins identified, 35 were upregulated in Pb01 (Fig. 1 panel A, Table 1)

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and 36 were upregulated in PbEpm83 (Fig. 1 panel A, Table 2). The remaining 21

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proteins, which showed no statistical differences in the detected amounts between the

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isolates, were grouped as constitutive proteins (Fig. 1 panel A, Supplementary Table S2).

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Additionally, all identified protein sequences were subjected to prediction software

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of protein secretion pathways and subcellular location, revealing concordant results. Of 35

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proteins differentially secreted in Pb01, 15 (42.8%) possess a signal peptide predicted by

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SecretomeP (Fig. 1 panel B, Table 1). Of 36 extracellular proteins differentially secreted

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in PbEpm83, 2 (5.5%) proteins possess a signal peptide predicted by SignalP, whereas 13

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(36.1%) proteins were predicted by SecretomeP (Fig. 1 panel B, Table 2). These data

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were compared with the Fungal Secretome Database (FSD), which is a platform of several

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secretomes of fungal species that provides information on protein secretion pathways in

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these microorganisms. The FSD describes that 58% of the genome-encoded proteins of

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Pb01 are secreted by presumably non-classical routes and 14% by the conventional route,

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being 4.4% predicted by SignalP. This highlights the fact that the vast majority of proteins

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secreted by this species use alternatives routes of secretion, which is in accordance with

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our data.

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The eight most abundant extracellular proteins in each isolate, which correspond to

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30% of the total number of secreted proteins, include: enolase (7%), fructose-1,6-

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ACCEPTED MANUSCRIPT bisphosphate aldolase (5.5%), nucleoside diphosphate kinase (5%), 2-methylcitrate

322

synthase (3.5%), phosphoglycerate kinase (2.5%), heat shock protein SSC1 (2.4%),

323

thioredoxin (2.2%), and transaldolase (2%) in PbEpm83; while hsp70-like protein (10%),

324

enolase (5%), 2-methylcitrate dehydratase (3%), 2-methylcitrate synthase (2.7%),

325

nucleoside diphosphate kinase (2.5%), heat shock protein SSC1 (2.6%), malate

326

dehydrogenase (2.4%), and phosphoglycerate kinase (2.3%) were abundant in Pb01.

RI PT

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The extracellular proteins were also classified into functional categories according to

328

the MIPS Functional Catalogue database (FunCatDB) (Fig. 1 panel C). The main classes

329

identified were related to metabolism, energy, protein synthesis, protein fate, and cell

330

rescue/defense/virulence. Among the identified proteins are many defense proteins which

331

function as chaperones (heat shock proteins). They form an important part of the cellular

332

machinery to make the correct protein folding and help to protect cells against stress. Pb01

333

isolate was shown to differ from PbEpm83 with regard to two functions, namely transport

334

and proteins with binding functions. While biogenesis of cellular compounds and

335

differentiation of cell types was detected only in PbEpm83. Proteins with unusual

336

functional categories in the extracellular milieu were described in other Paracoccidioides

337

extracellular proteomes. This indicates that these proteins may have a secondary function

338

outside of the fungal cells.

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SC

327

Next, we compared the identified extracellular proteins of Pb01 and PbEpm83 with

340

orthologous proteins described in Pb18 (Vallejo et al. 2012) and H. capsulatum

341

(Albuquerque et al. 2008), as depicted in Figure 2. The comparison demonstrated that 14

342

secreted proteins were identified in the four strains analyzed. On the other hand, some

343

extracellular proteins were only detected in Pb01 secretome (decarboxylase family protein

344

- PAAG_03537; homogentisate 1,2-dioxygenase - PAAG_08164; nuclear transcription

345

factor Y subunit C - PAAG_08441, suaprga1 - PAAG_03309), while others 17

14

ACCEPTED MANUSCRIPT extracellular proteins were detected only in PbEpm83 (isochorismatase family hydrolase -

347

PAAG_04083; ssDNA binding protein- PAAG_07296; 12-oxophytodienoate reductase -

348

PAAG_03631; aromatic-L-amino-acid decarboxylase - PAAG_01563; cysteine synthase -

349

PAAG_07813; glutathione S-transferase Gst3 - PAAG_03931; heat shock protein -

350

PAAG_05679; HET-C domain containing protein HetC - PAAG_03921; hexokinase -

351

PAAG_01015;

352

phosphoribosyltransferase - PAAG_06643; UTP-glucose-1-phosphate uridylyltransferase -

353

PAAG_06817; two conserved hypothetical protein - PAAG_00340/PAAG_07921 and

354

three hypothetical proteins – PAAG_02985/PAAG_05550/PAAG_07158). This analysis

355

demonstrates that isolates of Paracoccidioides genus present many common proteins when

356

compared with the characterized Histoplasma extracellular proteome. The data reinforce

357

the sharing of extracellular proteins between Paracoccidioides species and the pathogenic

358

fungus H. capsulatum (Xavier et al. 2009).

adenylyltransferase

-

PAAG_05929;

uracil

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SC

sulfate

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346

For the purpose of validate the proteomic data, we performed immunoblot and

360

enzymatic activity assays. The proteomic data revealed the enzymes formamidase and

361

fructose-1,6-bisphosphate aldolase as differentially secreted in PbEpm83. Triosephosphate

362

isomerase and glyceraldehyde-3-phosphate dehydrogenase were found as differentially

363

secreted in Pb01. These results can be confirmed by the fact that the protein species reacts

364

against to its respective antibodies with a high intensity, as depicted in the immunoblotting

365

analysis (Fig. 3). Enolase, which is a very abundant protein in Paracoccidioides, was

366

identified as constitutive in our analyses. It reacted strongly and similarly in

367

immunoblotting, corroborating its constitutive expression between the two isolates (Fig.

368

3). In addition, the specific activity of enzyme formamidase was measured in extracellular

369

extracts of Pb01 and PbEpm83 by enzymatic assay. The test showed that this enzyme is

370

functionally active in both extracts. A higher activity was detected in the PbEpm83

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15

ACCEPTED MANUSCRIPT 371

secreted sample, indicating increased enzyme activity in this condition compared with the

372

Pb01 isolate, corroborating the proteomic results (Fig. 4).

373

3.4. Proteins involved in adherence and virulence

375

In order to list possible proteins involved in adherence, the proteins were submitted

376

to FaaPred software (Fungal adhesins and adhesin-like proteins prediction tool) which is

377

designed to predict adhesins from human pathogenic fungi. The analysis showed 17

378

adhesin-like proteins, representing almost 20% of the total of identified proteins, amongst

379

them 3 hypothetical proteins, 1 thioredoxin, and 1 heat shock protein. Eight proteins were

380

differentially secreted in Pb01, 6 differentially secreted in PbEpm83, and 3 with no

381

differential expression between the isolates (Table 3).

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374

In addition, the search for proteins related to antigenicity and virulence was carried

383

out using Antigenics Pasteur software. It was possible to verify that all identified proteins

384

present antigenic hits that may be important in host–fungus interaction. Also, the proteins

385

identified in Paracoccidioides isolates were searched in digital databases such as PubMed

386

of the National Center for Biotechnology Information (NCBI) for the identification of

387

virulence factors already described in other pathogenic fungi in literature. We found that

388

18 proteins detected in our analysis are described as virulence factors, such as: mannitol-1-

389

phosphate 5-dehydrogenase, dipeptidyl peptidase, alcohol dehydrogenase, mitochondrial

390

peroxiredoxin PRX1, fructose-1,6-bisphosphate aldolase, enolase, and heat shock protein

391

Hsp88. These proteins are involved in many processes related to virulence, such as

392

antioxidant activities, protection against reactive oxygen species (ROS), decreased

393

susceptibility to phagocytes, adhesive properties, as well as interaction with components of

394

the extracellular matrix (ECM) (Table 4).

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ACCEPTED MANUSCRIPT 3.5. Macrophage Infection Assay

397

It is known that species from the same genus can present morphological and

398

biochemical differences that may reflect in different behaviors and skills with regard to

399

infection. As a result, we sought to evaluate the behavior of two members of different

400

phylogenetic species of Paracoccidioides, Pb01 and PbEpm83, with reference to

401

macrophage infection. The results show that Paracoccidioides species display similar

402

behaviors within 12 hours of infection with regard to the number of recovered cells. It is

403

similar in both species (360 and 362 CFU/mL from Pb01 and PbEpm83 respectively) (Fig.

404

5 panel A). However, with the course of infection, Pb01 was shown to be more resistant to

405

the macrophage environment compared with PbEpm83, and had a higher number of

406

recovered cells after 24 hours of infection (436 and 252 CFU/mL from Pb01 and

407

PbEpm83, respectively) (Fig. 5 panel B). Taking into account that the secretome of

408

pathogenic microorganisms can be directly involved in the host cell interaction, it is

409

evident that the secretome is very important in the early stages of infection. To evaluate the

410

effect of the secretome, the extracellular protein extract from both isolates was added to

411

macrophage infection assay (Fig. 5 panels A, B, C and D). The results showed that the

412

CFU increased slightly considering both evaluated times of infection. This data reinforces

413

the influence of secreted proteins in Paracoccidioides adhesion/internalization/survival in

414

activated macrophages.

SC

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396

In order to assess the secretome influence between the species, the secretome protein

416

extracts were cross-tested between species to check the effect of secretomes in this in vitro

417

model of infection. After 12 hours of phagocytosis assay of Pb01 + SPb01 (secretome

418

from Pb01), and Pb01 + SPbEpm83 (secretome from PbEpm83), it was possible to

419

visualize a survival increase of 30 and 7 %, respectively (Fig. 5 panel C). In addition, we

420

tested PbEpm83 + SPbEpm83 and PbEpm83 + SPb01, which presented an increasing rate

17

ACCEPTED MANUSCRIPT of 26 and 16%, respectively (Fig. 5 panel D). After 24 hours of assay we detected an

422

increasing rate of 60 and 29% to Pb01 + SPb01 and Pb01 + SPbEpm83 (Fig.5 panel C)

423

and 45 and 13% to PbEpm83 + SPbEpm83 and PbEpm83 + SPb01, respectively (Fig. 5

424

panel D). Statistical differences between the confronted conditions are presented in bars.

425

Both secretomes also increased the number of CFU recovered, but this effect was less

426

pronounced than when exposed to the additional secretome of their own species.

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421

428

SC

427

5. Discussion

Several factors may influence the isolate’s virulence at the time of infection, and

430

these factors may act differently in members of a defined genus. A recent work has shown

431

that P. brasiliensis presents a faster conversion of yeast to mycelium, larger colonies and

432

greater conidia production. In addition, P. brasiliensis and P. lutzzi presented different

433

abilities to grow after long periods under stress conditions, being P. brasiliensis more

434

tolerant in these conditions, suggesting increased chances of causing infection (Hrycyk et

435

al. 2017). In the current study, the secretome profile comparison between two

436

Paracoccidioides complex members was described to search for secreted molecules that

437

may be related to the mechanisms of virulence released by different species from the same

438

genus. Using a label-free MSE proteomics approach, we identified a total of 92 proteins, 71

439

of which were differentially expressed between the strains, while 21 were described as

440

constitutive (considering a cut-off of 1.3-fold change). Among the proteins identified in

441

this study, there are proteins related to various biological functions, such as carbohydrate

442

metabolism, energy processes, synthesis and protein fate, oxidation/reduction, transport,

443

cell signaling, defense and virulence, which is in accordance with descriptions of

444

secretome profiles in previous works (Holbrook et al. 2011; Vallejo et al. 2012; Weber et

445

al. 2012; Gil-Bona et al. 2015).

AC C

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429

18

ACCEPTED MANUSCRIPT Many of the proteins found in our work, such as heat shock proteins, glyceraldehyde-

447

3-phosphate dehydrogenase, mannitol-1-phosphate 5-dehydrogenase, triose phosphate

448

isomerase, fructose-1,6-bisphosphate aldolase, peptidyl-prolyl cis-trans isomerase, and

449

disulfide isomerase, were also found in vesicles secreted by Histoplasma capsulatum

450

(Albuquerque et al. 2008). Characterization studies of extracellular vesicles have also been

451

widely explored in Cryptococcus neoformans (Rodrigues et al. 2008, 2014; Oliveira et al.

452

2010); proteomic analysis of vesicles revealed a further 76 proteins and various others

453

related to virulence and protection against oxidative stress, such as capsule synthesis,

454

urease, laccase, heat shock proteins, superoxide dismutase, thioredoxin reductase, and

455

catalase A (Rodrigues et al. 2008). Some of them were also found in our secretome

456

analysis. These findings reinforce the idea that fungi have mechanisms for removing some

457

classical cytoplasmic proteins from their cells in order to help the microorganisms in non-

458

classical functions in this milieu.

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446

The classical mechanism of secretion involves release via endoplasmic reticulum

460

(ER) and Golgi apparatus, guided by the signal peptide (Blobel & Dobberstein 1975).

461

However, it has been shown that numerous proteins are secreted without the signal

462

sequence, suggesting the existence of non-classical transport routes (Nombela et al. 2006;

463

Nickel & Rabouille 2009). Our prediction analysis shows that most extracellular proteins

464

identified in this work use alternative mechanisms of secretion (Fig. 1 panel B), which is

465

consistent with the literature (Cuervo et al. 2009). In Paracoccidioides, glyceraldehyde-3-

466

phosphate dehydrogenase – GAPDH (Barbosa et al. 2006), triose phosphate isomerase –

467

TPI (Pereira et al. 2007), and enolase - ENO (Nogueira et al. 2010), have been described

468

in the extracellular environment, where their behavior is supposed to differ from the

469

classical behaviors.

AC C

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19

ACCEPTED MANUSCRIPT A variety of studies have addressed secreted proteins and their functions in the

471

extracellular environment (Tsang et al. 2009; Sorgo et al. 2010; Wang et al. 2011; Fekkar

472

et al. 2012; Rampitsch et al. 2013; Campbell et al. 2015) including in Paracoccidioides

473

(Vallejo et al. 2012; Weber et al. 2012; Chaves et al. 2015). A vesicle and vesicle-free

474

protein study in Paracoccidioides brasiliensis isolated Pb18, and revealed some secreted

475

proteins performing functions related to infection, immune response modulation, as well as

476

adhesion to the extracellular matrix components (Vallejo et al. 2012).

SC

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470

Adhesion is an important pathogenicity factor in a variety of pathogenic

478

microorganisms. A study in Paracoccidioides demonstrated that 14-3-3 and enolase are the

479

most highly expressed adhesins during pathogen-host interaction and identified higher

480

levels of adhesin expression in P. brasiliensis when compared with P. lutzii, and correlated

481

this with the increased in vivo virulence of the fungus (Oliveira et al. 2015). Among the

482

most abundant extracellular proteins that we identified in the Paracoccidioides species, the

483

adhesion skill is the most relevant of them. Our findings showed a similar amount of

484

adhesin expressed for both Pb01 and PbEpm83 isolates.

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Secretome analysis of P. lutzii showed that approximately 21% of identified proteins

486

were predicted as adhesin-like proteins. Also, eighteen extracellular proteins were found in

487

the cytoplasm of macrophages experimentally infected by P. lutzii, suggesting that the

488

fungus uses secreted proteins as one of the strategies to gain a successful infection (Weber

489

et al. 2012). The proteins ATPase alpha, elongation factor 1-alpha subunit, and malate

490

dehydrogenase were found as being up-regulated in PbEpm83. The proteins DNA damage

491

checkpoint rad24, glyceraldehyde-3-phosphate dehydrogenase, nucleoside diphosphate

492

kinase, and peptidyl-prolyl cis-trans isomerase D, were found as up-regulated in Pb01.

493

These findings reinforce the hypothesis that these secreted proteins can help fungal

494

survival within macrophages/host tissues.

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20

ACCEPTED MANUSCRIPT The infectious process involves the adhesion of fungus to host cells and theirs

496

components. In this context, pathogens express molecules that can interact, for example,

497

with human plasminogen and end up favoring the pathogen spread to deeper tissues.

498

Studies show fructose-1,6-bisphosphate aldolase, 2-methylcytrate synthase, malate

499

dehydrogenase

500

Paracoccidioides secretome (Chaves et al. 2015); phosphoglycerate kinase, fructose-1,6-

501

bisphosphate aldolase and thioredoxin, also as a plasminogen ligant, found on Candida cell

502

wall (Crowe et al. 2003). The fructose-1,6-bisphosphate aldolase, enolase, heat shock

503

SSC1, and nucleoside diphosphate kinase, were described as adhesin-like molecules,

504

identified during copper-deprivation conditions in Paracoccidioides lutzii (Pb01) in the

505

presence of extracellular matrix components (Oliveira et al. 2014). Our group identified

506

fifteen extracellular proteins with plasminogen binding ability. Of these, fructose-1,6-

507

bisphosphate aldolase (FBA) was able to increase the fungus-macrophages interaction and

508

was found in vesicles in the releasing process (Chaves et al. 2015). In this work FBA was

509

identified suggesting its participation in the invasiveness capacity of fungal.

phosphoglycerate

kinase

as

plasminogen

binders

in

the

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and

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495

Many extracellular identified proteins related to defense and virulence were

511

identified in our analysis. Among the virulence functions described in other fungi (Hogan

512

et al. 1996; Crowe et al. 2003; Brown et al. 2007; Karkowska-kuleta et al. 2009; Oh et al.

513

2010; Viefhues A, Heller J, Temme N 2014) there is the ability of secreted proteins to act

514

as adhesins, allowing pathogen invasion of the host tissues, such as glucose-6-phosphate

515

isomerase in Cryptococcus neoformans, which interacts with plasminogen (Stie et al.

516

2009) or cellular detoxification with GST in Candida albicans (Garcerá et al. 2010). In the

517

category of cell rescue, defense and virulence (RDV) protection proteins such as

518

chaperones and foldases are included. They comprise part of the machinery of cells against

519

stress, and assist in the correct folding of nascent secretory proteins. Peptidyl-prolyl

AC C

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510

21

ACCEPTED MANUSCRIPT isomerases are foldases which catalyze the cis - trans isomerization of peptide bonds N-

521

terminal proline residues in polypeptide chains. They play a role in correct folding of

522

newly synthesized proteins, which is an important step in maintaining the structure and

523

function of proteins (Shaw 2002). In our analysis many chaperones (six heat shock

524

proteins) and foldases (three peptidyl-prolyl cis-trans isomerase and one disulfide

525

isomerase) were identified. Of these, peptidyl-prolyl cis-trans isomerase D was also found

526

as secreted protein in Pb01 yeast cells infecting macrophages (Weber et al. 2012),

527

suggesting that its appearance in the secretome may be related to the maintenance and/or

528

acceleration of the formation of the final and correct structure of proteins in this

529

environment.

M AN U

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520

During the adaptation of Paracoccidioides cells to changes in temperature and stress

531

conditions in general, there is a significant increase in expression of heat shock proteins

532

(HSPs). In Paracoccidioides, a study using antisense RNA technology described the

533

protective role of heat shock protein HSP90 during adaptation to hostile environments; it

534

promotes the survival of the fungus during host-pathogen interactions (Tamayo et al.

535

2013). The Hsp90 co-chaperone AHA1 and the Hsp90 binding co-chaperone Sba1 were

536

found in this work to be up-regulated in Pb01 isolate. In addition, HSP70 was the most

537

abundant protein in Pb01 secretome. HSP70 was identified as a major target of antibodies

538

in cryptococcose patients (Kakeya et al. 1999), and was previously identified serving as a

539

plasminogen-binding receptor in C. albicans and C. neoformans (Crowe et al. 2003; Stie et

540

al. 2009).

AC C

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530

541

In our findings we also found some proteins that present the classical localization on

542

cytosol compartment. Many cytoplasmic proteins have been described in other

543

compartments as having non classical functions; they are known as moonlighting proteins

544

(Karkowska-Kuleta & Kozik 2014; Gil-Bona et al. 2015). A study of virulent and hypo-

22

ACCEPTED MANUSCRIPT virulent Cryptococcus strains detected that the secretome of virulent strains has largely

546

been limited to proteolytic and hydrolytic enzymes, while the hypo-virulent strains have a

547

diverse secretome, including non-conventionally secreted cytosolic canonical and

548

immunogenic proteins that have been implicated in virulence (Campbell et al. 2015). In

549

our work, both isolates presented a similar number of identified virulence factors. Until

550

now, it hasn’t been possible to correlate, whether quantitatively or qualitatively, the

551

influence of secretome content on the fitness of the fungus. However, during infection the

552

secretome can act differently for each isolate. In addition, it can also be affected when

553

combined with others factors present in the host milieu. These changes assist the fungus in

554

combating the host defenses, and enables it to achieve infection successfully.

M AN U

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545

Paracoccidioides-secreted proteins such as aconitase, aldolase, glyceraldehyde-3-

556

phosphate dehydrogenase, isocitrate lyase, malate synthase, triose phosphate isomerase,

557

fumarase and enolase have also been described as not performing classic functions

558

(Marcos et al. 2014). Of these, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose

559

phosphate isomerase and enolase were found in our analysis. Besides the glycolytic

560

activities performed by Paracoccidioides enolase, this enzyme presents an affinity for

561

extracellular matrix (ECM) proteins such as fibronectin and laminin. In addition, enolase

562

has the capacity to bind plasminogen, thus facilitating fungal invasion and distribution

563

(Nogueira et al. 2010). Triose phosphate isomerase, which also belongs to the glycolytic

564

pathway, interacts with the ECM components fibronectin and laminin (Pereira et al. 2007;

565

Ikeda & Ichikawa 2014). This enzyme was found up-regulated in Pb01 secretome. Despite

566

not being detected as an adhesin by FaaPred software, it has previously been shown that

567

fructose bisphosphate aldolase interacts with human plasminogen. This interaction

568

promotes an increase in the fibrinolytic ability of the fungus (Chaves et al. 2015), which

569

can play an important role in the establishment of the fungus in host tissues. In addition to

AC C

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555

23

ACCEPTED MANUSCRIPT its role in the metabolism of carbohydrates and other cellular functions, the GAPDH

571

enzyme also binds to ECM proteins and is associated with Paracoccidioides infection

572

establishment (Barbosa et al. 2006). The GAPDH enzyme was identified in this work as

573

being up-regulated in Pb01 isolate.

RI PT

570

In addition, it has been assumed that oxidative stress in response to pathogens

575

includes the production of antioxidants, which are related to pathogenicity. The glutathione

576

S-transferase enzyme found in this work as differentially secreted in PbEpm83 is involved

577

in cellular detoxification processes and protection against oxidative stress produced by the

578

host cell’s defenses (Garcerá et al. 2010). The identification of proteins involved in the

579

oxidative stress response in Paracoccidioides’ extracellular proteome contributes to the

580

possibility that pathogenic fungi release defense molecules that possibly act as virulence

581

factors. DNA damage checkpoint protein rad24, glutathione S-transferase, thioredoxin,

582

alcohol dehydrogenase, thimet oligopeptidase, heat shock proteins and disulfide isomerase,

583

which were identified in this work, have already been described as upregulated expression

584

in Paracoccidioides, after exposure to oxidative stress (Grossklaus et al. 2013). It

585

demonstrates that both isolates studied present mechanisms for reacting against oxidative

586

stress, and that these mechanisms can be produced by the host.

EP

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M AN U

SC

574

Formamidase (FMD), an enzyme involved in nitrogen metabolism, was our target

588

chosen for confirmation of proteomic data by enzymatic assay. The findings show that the

589

enzyme is active in the extracellular environment and also prove that it is differentially

590

expressed in PbEpm83 secretome. This enzyme may be associated with fungal

591

pathogenesis once is reactive with antibodies present in sera from patients with

592

Paracoccidioidomycosis (Borges et al. 2005). In addition, as a functional enzyme that

593

produces ammonia, this enzyme can be related to invasion processes through ammonia-

594

mediated tissue injury.

AC C

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24

ACCEPTED MANUSCRIPT By searching the literature, we were able to point to various proteins related to

596

processes associated with infection establishment and considered as virulence factors. The

597

proteins induced in PbEpm83 secretome, compared with those in Pb01, were: heat shock

598

protein, elongment translation factor 1-alpha, alcohol dehydrogenase, mitochondrial

599

peroxiredoxin PRX 1, glutathione S-transferase-3, mitochondrial ATP synthase D chain,

600

fructose-1,6-bisphosphate aldolase and dipeptidyl peptidase. These, in general, relate to

601

motility processes, adhesion and colonization, as well as antioxidant activity. Among the

602

proteins up-regulated in Pb01 were: glyceraldehyde-3-phosphate dehydrogenase, triose

603

phosphate isomerase, thioredoxin, TCTP family protein, glucose-6-phosphate isomerase,

604

and three heat shock proteins. Its relations with the virulence are based on interactions

605

reports with plasminogen, cellular redox homeostasis and adhesive properties. Therefore,

606

our findings showed that PbEpm83 and Pb01 use several proteins related to adhesion and

607

virulence at the time of infection. Even though they share some expressed proteins, each

608

species was shown to adopt different expression levels of these.

TE D

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SC

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595

It is clear that secretome plays an essential role in fungal infection strategy, working

610

in the molecular dialogue with the host cells, enabling survival, multiplication and

611

pathogen dissemination (Silva et al. 2012). Therefore, the importance of fungal

612

secretome’s characterization as a tool for host-pathogen interaction studies has been

613

demonstrated (Kniemeyer & Brakage 2008; Holbrook et al. 2011; Weber et al. 2012;

614

Girard et al. 2013). This is the first comparative extracellular proteome study between

615

members of Paracoccidioides complex, and it will provide a significant set of data for

616

future research regarding the importance of secreted proteins in host-pathogen interactions.

617

Our proteomic analysis showed a range of secreted proteins expressed by the isolates, with

618

most of them being related to adhesion and virulence. In this sense, we can speculate that

619

the secretome has an important role regarding infection, and that, together with the protein

AC C

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609

25

ACCEPTED MANUSCRIPT expression of the other compartments, it comprises the necessary set adopted by each

621

isolate to develop effective infection strategies. However, the progression of the disease

622

depends on multiplex parameters, not only the fungal strain, and therefore it is still not

623

enough to infer which specie studied here is really more virulent and/or which molecules

624

are crucial for that. Additional studies are needed to explore our findings regarding the

625

fractionation of secretomes, including functional studies using genetics tools, as well as

626

production and evaluation of secreted proteins and its influence in Paracoccidioides

627

biology and pathogenesis.

SC

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620

629

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628

Acknowledgments

This work at Universidade Federal de Goiás was supported by grants from Conselho

631

Nacional de Desenvolvimento Científico e Tecnológico (CNPq - Processo 477962/2010-

632

6), and Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG) (INCT-IPH). ARO

633

has a fellowship from Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG).

634

LNO and EGAC have fellowships from Coordenação de Aperfeiçoamento de Pessoal de

635

Nível Superior (CAPES).

638 639

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637

Conflict of interest

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The authors declare that they have no competing interests.

640 641 642 643

26

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ACCEPTED MANUSCRIPT References

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Albuquerque PC, Nakayasu ES, Rodrigues ML, Frases S, Casadevall A, Zancope-Oliveira RM, Almeida IC, Nosanchuk JD, 2008. Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes. Cell Microbiol. 10: 1695–1710. https://doi.org/10.1111/j.14625822.2008.01160.x.Vesicular

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ACCEPTED MANUSCRIPT Figures legends

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Figure 1. Graphic summary of bioinformatic analysis. A) Venn diagram showing the

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number of proteins up regulated in Pb01 and PbEpm83 isolates; at intersection showing the

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number of proteins without statistical differences. B) Number of proteins predicted to be

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secreted by classical and non-classical secretory pathways. SP: SignalP tool; ScP:

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SecretomeP tool; NP: no prediction. C) Functional categories of the proteins differentially

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secreted with significant statistical alteration in Pb01 (left) and PbEpm83 (right) identified

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protein by nanoUPLC-MSE classified into functional categories according to the database

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MIPS Functional Catalogue (FunCatDB).

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Figure 2: Analysis of orthologous proteins found in fungal extracellular proteome.

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The Venn diagram shows the number of proteins that overlap and not between Pb01 (this

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work), PbEpm83 (this work), Pb18 (Vallejo et al. 2012) and H. capsulatum (Albuquerque

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et al. 2008).

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Figure 3. Immunoblotting analysis. Immunoblotting analysis of Pb01 and PbEpm83

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secreted extracts. The transferred membrane was incubated with polyclonal antibodies

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against proteins tested. ENO - PbEnolase; FMD - PbFormamidase; ALD - PbAldolase;

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GAPDH - PbGlyceraldehyde-3-phosphate dehydrogenase; TPI - PbTriose phosphate

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isomerase. Raw Tiff images were analyzed by densitometry of immunoblotting bands

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using the ImageJ 1.51 software. Pixel intensity for the analyzed bands was generated and

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expressed as arbitrary units. ‘*’ indicates statistically significant difference. The

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densitometry p-values are: ENO: p=0.2033; FMD: p=0.0004; ALD: p=0.0001; GAPDH:

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p=0.0073; TPI: p=0.0005.

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ACCEPTED MANUSCRIPT Figure 4. Formamidase activity assay. The FMD activity was determined by the

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measuring the amount of ammonia formation at 37°C. Data was expressed as the mean ±

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standard deviation of the biological triplicates of independent experiments. Student’s t-test

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was used. ‘*’ differences statistically significant (p ≤ 0.05).

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Figure 5. Macrophage Infection Assay. Fungal cells of Pb01 and PbEpm83 were

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incubated for 12h and 24h with the J774 macrophage lineage for fungal

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adhesion/internalization. After this period, the infected macrophages were lysed and the

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lysis products were plated on BHI medium to recover fungi. The colony forming units

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(CFUs) were counted after 12 days of growth and subjected to analysis of the standard

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deviation of the triplicates. A) Results presented comparing both isolates in 12h infection.

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B) Results presented comparing both isolates in 24 h infection. C) Results presented in

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function of time of infection (12 and 24 h) to Pb01 isolate. D) Results presented in

975

function of time of infection (12 and 24 h) to PbEpm83 isolate. ‘S’ indicates addition of

976

50µL of secreted extracts of Paracoccidioides yeast cells (2 mg/mL) to the time of

977

infection (S01 to Pb01 and S83 to PbEmp83).

M AN U

TE D

EP

978

SC

967

Supplementary Figure S1. Cell viability, growth and vitality assay. Yeast cells of Pb01

980

and PbEpm83 were grown in identical experimental conditions and obtained in biological

981

triplicates. For all experiments the timepoints of 0, 12, 24 and 36 hours were evaluated. A)

982

The cells were stained with Trypan Blue and counted in hemocytometer. B) Equivalent

983

amounts of cells were initially inoculated and at time-points were counted in

984

hemocytometer. C) The amount of glucose was measured by the enzymatic action of

985

Glucose Oxidase method.

AC C

979

36

ACCEPTED MANUSCRIPT Supplementary Figure S2. Extracellular extract quality analysis. Protein extract of the

987

secretome of Pb01 and PbEpm83 using 12% gel electrophoresis (SDS-PAGE). Molecular

988

mass protein standards (kDa) are indicated on the left of the gels. MW: molecular weight

989

marker.

RI PT

986

990

Supplementary Figure S3. Global analysis of NanoUPLC-MSE proteomic data. A)

992

Dynamic detection range of proteins according to their abundance and molecular weight of

993

Pb01 (left) and PbEpm83 (right). Identified proteins regularly (open square) and reverse

994

form (closed circle) and endogenous default PHB (phosphorylase B, yellow triangle). B)

995

The graphics show accuracy mass analysis in the detection of Pb01 (left) and PbEpm83

996

(right) proteins. C) The pie charts show the percentages of the type of detection of peptides

997

in of Pb01 (left) and PbEpm83 (right). PepFrag1 and PepFrag2 indicates the type of

998

identification using the database of Paracoccidioides by PLGS applying the algorithms

999

described (Geromanos et al. 2009; Li et al. 2009); VarMod, variable modifications;

1000

InSource corresponds fragmentation occurred in the ionization source; MissedCleavage,

1001

loss of cleavage by trypsin; NeutralLoss H2O, NH3 and H3PO4, corresponding to loss of

1002

water precursors, ammonia and phosphoric acid, respectively.

AC C

EP

TE D

M AN U

SC

991

37

ACCEPTED MANUSCRIPT

Log2

FunCat2

Metabolism PAAG_05019

HIT domain protein

3079.07

-0.62

1.03

PAAG_02869

phosphoglycerate kinase

6335.24

-0.9

1.05

PAAG_06526

glucose-6-phosphate isomerase

2453.99

-0.66

1.05

PAAG_08313

43258.32

-1.12

PAAG_00433

L-PSP endoribonuclease family protein Hmf1 adenosine kinase

1719.2

-0.62

01.01.11. 02.01 01.03.01

PAAG_02859

adenosylhomocysteinase

8659.62

-1.37

01.07.01

PAAG_00588

fumarate hydratase

1100.07

Pb01

1.05

PAAG_00889

phosphomannomutase

676.36

Pb01

1.05

PAAG_02585

triosephosphate isomerase

Pb01

1.05

PAAG_03537

decarboxylase family protein

2795.32

Pb01

PAAG_08164

homogentisate 1,2-dioxygenase

2550.28

Pb01

01.01.06. 06.02 01.01.09. 05.02

glyceraldehyde-3-phosphate dehydrogenase

1412.95

-0.67

Energy PAAG_08468

TE D

AC C

3332.27

Protein Function

nucleotide/nucleoside/nucleoba se metabolism C-compound and carbohydrate metabolism C-compound and carbohydrate metabolism biosynthesis of isoleucine

SC

Score

M AN U

Protein description

EP

Access numbers1

RI PT

Table 1. Protein preferentially secreted in Pb01, compared with PbEpm83 secretome identified by UPLC-MSE.

2.01

Signal P3

Secretome P4

Antigenics Pasteur5

-

-

17

-

0.66268

17

-

-

22

-

-

6

-

-

16

-

-

25

-

-

17

-

-

12

purin nucleotide/nucleoside/nucleoba se metabolism biosynthesis of vitamins, cofactors, and prosthetic groups C-compound and carbohydrate metabolism C-compound and carbohydrate metabolism C-compound and carbohydrate metabolism biosynthesis of lysine

-

-

13

-

-

10

degradation of tyrosine

-

0.908391

19

glycolysis and gluconeogenesis

-

0.912034

18

ACCEPTED MANUSCRIPT

1400.53

Pb01

02.13.03

aerobic respiration

Cell Cycle and DNA Processing PAAG_01986 nucleosome binding protein

3314.72

Pb01

PAAG_00773

4186.77

Pb01

10.01.09. 05 10.03.01. 03

DNA conformation modification (e.g. chromatin) cell cycle checkpoints (checkpoints of morphogenesis, DNA-damage,-replication, mitotic phase and spindle)

967.29

Pb01

11.02.03. 04

8074.72 1020.18 13221.49 9916.27 1799.46

-0.9 -1.99 -1.26 -0.66 Pb01

2707.78 4356.69

0.662756

12

-

0.918277

4

-

-

13

transcriptional control

-

0.901699

12

12.01 12.01.01 12.01.01 12.01.01 12.01

ribosome biogenesis ribosomal proteins ribosomal proteins ribosomal proteins ribosome biogenesis

-

0.651432 0.718675 0.65348 -

5 6 4 3 7

-1.68 -0.96

14.01 14.01

protein folding and stabilization protein folding and stabilization

-

0.94122 0.864046

15 16

5744.41

-0.93

14.01

protein folding and stabilization

-

0.871257

8

3920.94 3431.9 8273.84

-0.83 -0.8 -0.63

14.01 14.01 14.04

-

0.936126

6 26 42

5951.87

-0.75

14.07.03

protein folding and stabilization protein folding and stabilization protein targeting, sorting and translocation modification by phosphorylation, dephosphorylation,

-

-

9

Transcription PAAG_08441 nuclear transcription factor Y subunit C Protein Synthesis PAAG_04425 60S ribosomal protein L22 PAAG_06536 ubiquitin PAAG_07841 60S acidic ribosomal protein P1 PAAG_09096 40S ribosomal protein S28 PAAG_09083 TCTP family protein

AC C

EP

Protein Fate (folding, modification and destination) PAAG_02686 Hsp90 co chaperone AHA1 PAAG_03334 peptidyl-prolyl cis-trans isomerase D PAAG_06168 peptidyl-prolyl cis-trans isomerase cypE PAAG_05226 Hsp90 binding co-chaperone Sba1 PAAG_08059 heat shock protein PAAG_05643 nuclear protein localization protein 4 PAAG_04291 nucleoside diphosphate kinase

M AN U

SC

DNA damage checkpoint protein rad24

-

RI PT

suaprga1

TE D

PAAG_03309

ACCEPTED MANUSCRIPT

Protein with ligation function PAAG_07750 heat shock protein Hsp88 PAAG_05518 cell division cycle protein

6682.68 1751.52

Pb01 -0.59

16.01 16.19.03

Regulation of Protein Activity PAAG_06344 rab GDP-dissociation inhibitor

3475.8

Pb01

18.01.07

Cellular Transport and Transport Routes PAAG_02364 thioredoxin PAAG_02515 cytochrome P450 55A1

40461.62 2581.77

-1.34 Pb01

20.01.15 20.01.15

Cell Rescue, Defense and Virulence PAAG_08003 hsp70 like protein

44260.87

-0.67

Unclassified PAAG_03243

7366.56

-1.48

-

-

27 32

regulation by binding / dissociation

-

-

18

electron transport electron transport

-

0.933168 -

5 17

32.01

stress response

-

-

26

-

-

-

0.924308

11

SC

protein binding ATP binding

M AN U

TE D

1

conserved hypothetical protein

RI PT

autophosphorylation

AC C

EP

Number of general information in the Broad Institute Database (http://archive.broadinstitute.org/ftp/pub/annotation/fungi/paracoccidioides/genomes/). Number of functional category according to the MIPS Functional Catalogue (FunCat2). 3 Prediction secretion according to SignalP 3.0. The number represents the probability of a signal peptide (http://www.cbs.dtu.dk/services/SignalP/). 4 Prediction secretion according to SecretomeP 2.0. The number corresponds to the neural network that exceeds the value 0.5 (http://www.cbs.dtu.dk/services/SecretomeP/). 5 Number of antigenic hits found by the Antigenics Software (http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::antigenic). 2

ACCEPTED MANUSCRIPT

Access numbers1

Log2

FunCat2

Metabolism PAAG_00053 malate dehydrogenase

1772.66

0.82

1.05

PAAG_03333 formamidase

1774.38

1.54

1.02

PAAG_03279 aminopeptidase PAAG_05929 sulfate adenylyltransferase PAAG_01015 hexokinase

976.46 4689.2 1413.37

PbEpm83 PbEpm83 PbEpm83

1.01 1.01 1.05

PAAG_04541 alcohol dehydrogenase

660.42

PbEpm83

1.05

PAAG_06817 UTP-glucose-1-phosphate uridylyltransferase PAAG_07813 cysteine synthase PAAG_01563 aromatic-L-amino-acid decarboxylase PAAG_06643 uracil phosphoribosyltransferase

798.05

PbEpm83

1.05

Energy PAAG_03330 dihydrolipoyl dehydrogenase

M AN U

TE D

PbEpm83 01.01.09.03.01 PbEpm83 01.01.09.04.01

908.32

PbEpm83

01.03.04

PbEpm83

01.05.02.07

704.74

PbEpm83

01.05.25

1607.68

PbEpm83

01.20.15.03

1824.05

PbEpm83

2.1

EP

1505.8 1930.43

8664.16

AC C

PAAG_01995 fructose-1,6-bisphosphate aldolase 1 PAAG_09004 puromycin sensitive aminopeptidase PAAG_04083 isochorismatase family hydrolase

Protein Function

Signal Secretome Antigenics P3 P4 Pasteur5

C-compound and carbohydrate metabolism nitrogen, sulfur and selenium metabolism amino acid metabolism amino acid metabolism C-compound and carbohydrate metabolism C-compound and carbohydrate metabolism C-compound and carbohydrate metabolism biosynthesis of cysteine biosynthesis of phenylalanine

-

0.816364

17

-

-

17

-

-

35 26 18

-

0.837883

11

-

-

22

-

0.507512 -

15 27

pyrimidine nucleotide/nucleoside/nucleobase metabolism sugar, glucoside, polyol and carboxylate catabolism regulation of C-compound and carbohydrate metabolism metabolism of ubiquinone

-

-

10

-

0.66289

11

-

-

35

-

-

9

-

-

26

SC

Score

Protein description

RI PT

Table 2. Protein preferentially secreted in PbEpm83, compared with Pb01 secretome identified by UPLC-MSE.

tricarboxylic-acid pathway (citrate cycle, Krebs cycle, TCA

ACCEPTED MANUSCRIPT

PbEpm83

2.1

PAAG_04820 ATPase alpha subunit

905.42

PbEpm83

2.11

PAAG_04570 ATP synthase D chain mitochondrial PAAG_03631 12-oxophytodienoate reductase

1673.84

PbEpm83

2.13

7223.48

PbEpm83

2.45

Cell Cycle and DNA processing PAAG_07296 ssDNA binding protein

7652.94

PbEpm83

1946.27

PbEpm83

Protein Fate PAAG_00986 PAAG_05679 PAAG_03719 PAAG_07467

disulfide isomerase Pdi1 heat shock protein thimet oligopeptidase dipeptidyl peptidase

Cell Rescue, Defense and Virulence PAAG_03216 mitochondrial peroxiredoxin PRX1

-

-

20

-

-

20

-

-

7

energy conversion and regeneration

-

0.739896

15

10.01.03

DNA synthesis and replication

-

0.542166

5

11.02.03.04

transcriptional control

-

0.903215

6

M AN U

TE D

590.47 1506.82

PbEpm83 PbEpm83

12.04 12.01.01

translation ribosomal proteins

-

0.683646 -

19 11

5899.09 600.64

PbEpm83 PbEpm83

12.01.01 12.01.01

ribosomal proteins ribosomal proteins

0,386 -

-

4 10

2079.57 693.39 561.54 954.06

PbEpm83 PbEpm83 PbEpm83 PbEpm83

14.01 14.01 14.13 14.13.01

protein folding and stabilization protein folding and stabilization protein/peptide degradation cytoplasmic and nuclear protein degradation

0,772 -

-

20 24 35 34

2950.48

PbEpm83

32.01.01

oxidative stress response

-

-

10

AC C

Protein Synthesis PAAG_02024 elongation factor 1-alpha PAAG_00801 60S acidic ribosomal protein P0 lyase PAAG_04691 60S acidic ribosomal protein P2 PAAG_07707 60S ribosomal protein L10a

EP

Transcription PAAG_04496 nascent polypeptide associated complex subunit beta

cycle) tricarboxylic-acid pathway (citrate cycle, Krebs cycle, TCA cycle) electron transport and membraneassociated energy conservation respiration

RI PT

907.93

SC

PAAG_08075 citrate synthase

ACCEPTED MANUSCRIPT

42.01

10

PbEpm83

43.01.03

1154.2 1186.76 1026.17 5971.67 1765.91

PbEpm83 PbEpm83 PbEpm83 PbEpm83 PbEpm83

1

cell wall

-

-

17

fungal and other eukaryotic cell type differentiation

-

0.941415

32

-

0.883262 0.936902

6 8 5 5 2

RI PT

PbEpm83

SC

Differentiation of cell types PAAG_03921 HET-C domain containing protein HetC Unclassified PAAG_00340 conserved hypothetical protein PAAG_02985 hypothetical protein PAAG_05550 hypothetical protein PAAG_07158 hypothetical protein PAAG_07921 conserved hypothetical protein

1956.35

-

M AN U

Biogenesis of cellular compounds PAAG_03931 glutathione S-transferase Gst3

-

Number of general information in the Broad Institute Database (http://archive.broadinstitute.org/ftp/pub/annotation/fungi/paracoccidioides/genomes/). Number of functional category according to the MIPS Functional Catalogue (FunCat2). 3 Prediction secretion according to SignalP 3.0. The number represents the probability of a signal peptide (http://www.cbs.dtu.dk/services/SignalP/). 4 Prediction secretion according to SecretomeP 2.0. The number corresponds to the neural network that exceeds the value 0.5 (http://www.cbs.dtu.dk/services/SecretomeP/). 5 Number of antigenic hits found by the Antigenics Software (http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::antigenic).

AC C

EP

TE D

2

0.518989 0.952615

ACCEPTED MANUSCRIPT Table 3. Proteins of Paracoccidioides Pb01 and PbEpm83 related to adherence. Access numbers1 PAAG_08313

Protein description

FAApred2

Isolate3

L-PSP endoribonuclease family protein Hmf1

0.32413393

Pb01

triosephosphate isomerase

0.475649

Pb01

PAAG_08468

glyceraldehyde-3-phosphate dehydrogenase

0.6656069

Pb01

PAAG_09096

40S ribosomal protein S28

0.45667675

Pb01

PAAG_02686

Hsp90 co-chaperone AHA1

0.49974454

Pb01

PAAG_08059

0.75891651

Pb01

0.64670755

Pb01

PAAG_02364

heat shock protein endoplasmic reticulum and nuclear membrane protein Npl4 thioredoxin

0.72402533

Pb01

PAAG_07813

cysteine synthase

PAAG_06643

uracil phosphoribosyltransferase

PAAG_07296

ssDNA binding protein

PAAG_02985

hypothetical protein

PAAG_05550

hypothetical protein

PAAG_07921

conserved hypothetical protein

PAAG_00771

enolase nascent polypeptide associated complex subunit alpha RNP domain protein

PAAG_04571

1

SC

PbEpm83

0.54851004

PbEpm83

0.41982781

PbEpm83

0.74640492

PbEpm83

0.64984866

PbEpm83

0.29603461

PbEpm83

NO*

C

0.34804424

C

0.67499902

C

TE D

PAAG_04913

0.49329971

M AN U

PAAG_05643

RI PT

PAAG_02585

Number of general information on the Broad Institute database. (http://archive.broadinstitute.org/ftp/pub/annotation/fungi/paracoccidioides/genomes/). 2 Prediction of the adhesins by FAApred software (http://bioinfo.icgeb.res.in/faap/query.html). 3

Isolate of Paracoccidioides in that the protein was found. C = refers to the constituent proteins (no

differences in expression levels for the parameters adopted).

EP

*Protein predicted to be non-adhesin by the FAApred software, but predicted to be adhesin in the FungalRV software -Adhesin Prediction and Immunoinformatics portal for human fungal pathogens

AC C

(http://fungalrv.igib.res.in/query.php) and supported by literature.

ACCEPTED MANUSCRIPT

Table 4. Identification of proteins secreted in Paracoccidioides Pb01 and PbEpm83, putatively related to virulence. Access numbers1

Expression2

Microorganism3

PAAG_04559

2-metilcitratre dehydratase

C

Aspergillus spp

PAAG_06473

mannitol-1-phosphate 5-dehydrogenase

C

Cryptococcus neoformans

PAAG_08059 PAAG_06811 PAAG_05679

heat shock proteins-Hsp

Pb01 C PbEpm83

Candida albicans

Adaptive response to stress.

PAAG_07467

dipeptidyl peptidase

PbEpm83

Aspergillus fumigatus

It facilitates the colonization of the lungs and other tissues

PAAG_08468

glyceraldehyde-3-phosphate dehydrogenase

Pb01

Paracoccidioides

Binding to fibronectin and laminin

PAAG_00771

enolase

C

Paracoccidioides

Fibronectin binding protein

PAAG_02585

triose phosphate isomerase

Pb01

Paracoccidioides

PAAG_01995

fructose-1,6-bisphosphate aldolase 1

PAAG_02364

SC

M AN U

TE D EP

Important findings4

References

Reduction in pathogenicity (Biosynthesis Lysine) Increased susceptibility to human neutrophils with low strains in vitro production mannitol;

(Hogan et al. 1996)

RI PT

Protein description

Adhesive properties; Connection epithelial cells cultured in vitro Motility and adhesion (interactions with plasminogen)

Candida albicans

thioredoxin

Pb01

Botrytis cinerea

Cellular redox homeostasis

PAAG_03216

mitochondrial peroxiredoxin PRX1

PbEpm83

Candida albicans

Antioxidant activity;

PAAG_03931

glutathione S-transferase 3 (GST)

PbEpm83

Candida albicans

Cellular detoxification;

PAAG_04541

alcohol dehydrogenase

PbEpm83

Candida albicans

Interactions with plasminogen

AC C

PbEpm83

(Hogan et al. 1996) (Brown et al. 2007) (Karkowskakuleta et al. 2009) (Barbosa et al. 2006; Seidler 2013) (Donofrio et al. 2009; Nogueira et al. 2010) (Pereira et al. 2007) (Crowe et al. 2003) (Viefhues A, Heller J, Temme N 2014) (Srinivasa et al. 2012) (Garcerá et al. 2010) (Crowe et al. 2003)

ACCEPTED MANUSCRIPT

elongation factor 1-alpha

PbEpm83

Candida albicans

PAAG_08003 PAAG_01262

Hsp70-like protein

Pb01 C

PAAG_06526

glucose-6-phosphate isomerase

Pb01

PAAG_04570

mitochondrial ATP synthase D chain

PbEpm83

Cryptococcus neoformans Cryptococcus neoformans Cryptococcus neoformans

PAAG_09083

TCTP family protein

Pb01

Aspergillus nidulans

PAAG_07750

heat shock protein Hsp 88

Pb01

Aspergillus nidulans

SC

M AN U

1

Interactions with plasminogen

(Crowe et al. 2003)

Interactions with plasminogen

(Stie et al. 2009)

Interactions with plasminogen

(Stie et al. 2009)

Interactions with plasminogen

(Stie et al. 2009)

RI PT

PAAG_02024

Induced in early stages of germination of conidia Induced in early stages of germination of conidia

(Oh et al. 2010) (Oh et al. 2010)

Number of general information on the Broad Institute database (http://archive.broadinstitute.org/ftp/pub/annotation/fungi/paracoccidioides/genomes/). Isolate of Paracoccidioides in that the protein was found. C - refers to the constituent proteins (no differences in expression levels for the parameters adopted). 3 Microorganism in which the virulence factor was already described. 4 Description in the literature of the protein action as a virulence factor.

AC C

EP

TE D

2

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights

Paracoccidioides species exhibit different extracellular protein profiles



P. lutzii shown to be more resistant to macrophage infection



Secreted proteins are request to Paracoccidioides survival in activated macrophages

AC C

EP

TE D

M AN U

SC

RI PT