Proteomic analysis of Paracoccidioides brasiliensis complex isolates: Correlation of the levels of differentially expressed proteins with in vivo virulence

RESEARCH ARTICLE Proteomic analysis of Paracoccidioides brasiliensis complex isolates: Correlation of the levels of differentially expressed proteins with in vivo virulence Cristiane Candida do Amaral1☯, Geisa Ferreira Fernandes2☯, Anderson Messias Rodrigues2☯, Eva Burger3☯, Zoilo Pires de Camargo ID2☯* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: do Amaral CC, Fernandes GF, Rodrigues AM, Burger E, de Camargo ZP (2019) Proteomic analysis of Paracoccidioides brasiliensis complex isolates: Correlation of the levels of differentially expressed proteins with in vivo virulence. PLoS ONE 14(7): e0218013. https://doi.org/10.1371/ journal.pone.0218013 Editor: Monde Ntwasa, University of South Africa, SOUTH AFRICA 1 Department of Medicine, Discipline of Infectious Diseases, Federal University of São Paulo (UNIFESP), São Paulo, Brazil, 2 Department of Microbiology, Immunology and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo (UNIFESP), São Paulo, Brazil, 3 Department of Microbiology and Immunology, Federal University of Alfenas (UNIFAL), Alfenas, Brazil ☯ These authors contributed equally to this work. * Abstract Background Paracoccidioidomycosis (PCM) is a systemic mycosis commonly found in Latin America that is caused by distinct species of Paracoccidioides genus: Paracoccidioides brasiliensis complex (S1, PS2, PS3 and PS4) and Paracoccidioides lutzii. Its pathobiology has been recently explored by different approaches to clarify the mechanisms of host-pathogen interactions underpinning PCM. The diversity of clinical forms of this disease has been attributed to both host- and fungus-related factors. Received: October 18, 2018 Methodology/Principal findings Accepted: May 23, 2019 For better understanding of the molecular underpinnings of host-fungus interactions, we evaluated in vivo virulence of nine Paracoccidioides brasiliensis complex isolates and correlated it to protein expression profiles obtained by two-dimensional gel electrophoresis. Based on the recovery of viable fungi from mouse organs, the isolates were classified as those having low, moderate, or high virulence. Highly virulent isolates overexpressed proteins related to adhesion process and stress response, probably indicating important roles of those fungal proteins in regulating the colonization capacity, survival, and ability to escape host immune system reaction. Moreover, highly virulent isolates exhibited enhanced expression of glycolytic pathway enzymes concomitantly with repressed expression of succinylCoA ligase beta chain, a protein related to the tricarboxylic acid cycle. Published: July 2, 2019 Copyright: © 2019 do Amaral et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was supported, in part, by grants from São Paulo Research Foundation (FAPESP), the National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). ZPdC acknowledges the financial support of FAPESP (2009/54024-2) Conclusions/Significance Our findings may point to the mechanisms used by highly virulent P. brasiliensis isolates to withstand host immune reactions and to adapt to transient iron availability as strategies to survive and overcome stress conditions inside the host. PLOS ONE | https://doi.org/10.1371/journal.pone.0218013 July 2, 2019 1 / 24 Proteomic analysis of P. brasiliensis isolates and CNPq (429594/2018-6). AMR acknowledges the financial support of FAPESP (2017/27265-5) and CAPES (88887.177846/2018-00). GFF is a fellow and acknowledges the financial support of CNPq (150605/2015-3). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Introduction Paracoccidioidomycosis (PCM) is the most frequent endemic systemic mycosis in Latin America with high incidence in Brazil, Argentina, Colombia, and Venezuela [1, 2]. It is caused by the thermally dimorphic species of the genus Paracoccidioides. Until recently, Paracoccidioides was considered a monotypic taxon typified by Paracoccidioides brasiliensis [3]. However, the introduction of molecular phylogenetics shed light on the taxonomy of Paracoccidioides, leading to the description of new cryptic entities. To date, four phylogenetic species are recognized inside the P. brasiliensis complex: S1, PS2, PS3 and PS4 [4, 5]. P. brasiliensis sensu stricto (s. str.), formerly known as S1, is the most widely distributed agent of PCM, occurring in Brazil, Argentina, Paraguay, Uruguay, Peru and Venezuela [6]. Paracoccidioides americana (formerly known as PS2) occurs in Venezuela and Brazil, in sympatry with P. brasiliensis s. str. [6]. Paracoccidioides restrepiensis (PS3) and P. venezuelensis (PS4) are geographically restricted to Colombia and Venezuela, respectively [6]. Finally, P. lutzii, an ancient divergent of the P. brasiliensis complex occurs in Brazil with its epicenter in the Central-West region [7, 8]. A recent speciation event is assumed for species embedded in the P. brasiliensis complex (especially PS3 and PS4), whereas it seems that P. brasiliensis sensu lato (s.l.) and P. lutzii are reproductively isolated in nature [6]. The ecological niche or exact habitat of these species remains poorly understood [9]. Paracoccidioides brasiliensis s. str. and P. americana has been described in armadillos, but not P. lutzii. The nine-banded armadillo (Dasypus novemcinctus) is a natural reservoir of the fungus and animal infection due to Paracoccidioides spp. has been observed repeatedly in several endemic areas of Brazil and Colombia [10]. Also, culture-independent surveys based on DNA detection techniques revealed that P. brasiliensis complex and P. lutzii are present in the soil [11]. It is accepted that conidia present in nature are inhaled by patients, transformed into budding yeast cells in the lungs, and then, these cells spread to different organs [12]. PCM may manifest itself in a variety of clinical forms, ranging from a benign and localized condition to a more severe and disseminated disease, depending on the extent of the depression of cellular immunity [12–14]. Paracoccidioides spp. are able to cause disease symptoms in the murine model. However, different isolates are not homogeneous in their virulence characteristics, a fact that could explain different clinical forms of PCM. The virulence profile of Paracoccidioides spp. isolat (...truncated)