Proteomic analysis of a Candida albicans pir32 null strain reveals proteins involved in adhesion, filamentation and virulence

RESEARCH ARTICLE Proteomic analysis of a Candida albicans pir32 null strain reveals proteins involved in adhesion, filamentation and virulence Pamela El Khoury☯, Andy Awad☯, Brigitte Wex, Roy A. Khalaf* Department of Natural Sciences Lebanese American University, Byblos, Lebanon ☯ These authors contributed equally to this work. * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: El Khoury P, Awad A, Wex B, Khalaf RA (2018) Proteomic analysis of a Candida albicans pir32 null strain reveals proteins involved in adhesion, filamentation and virulence. PLoS ONE 13(3): e0194403. https://doi.org/10.1371/journal. pone.0194403 Editor: Yong-Sun Bahn, Yonsei University, REPUBLIC OF KOREA Abstract We have previously characterized Pir32, a Candia albicans cell wall protein that we found to be involved in filamentation, virulence, chitin deposition, and resistance to oxidative stress. Other than defining the cell shape, the cell wall is critical for the interaction with the surrounding environment and the point of contact and interaction with the host surface. In this study, we applied tandem mass spectrometry combined with bioinformatics to investigate cell wall proteome changes in a pir32 null strain. A total of 16 and 25 proteins were identified exclusively in the null mutant strains grown under non-filamentous and filamentous conditions. These proteins included members of the PGA family with various functions, lipase and the protease involved in virulence, superoxide dismutases required for resisting oxidative stress, alongside proteins required for cell wall remodeling and synthesis such as Ssr1, Xog1, Dfg5 and Dcw1. In addition proteins needed for filamentation like Cdc42, Ssu81 and Ucf1, and other virulence proteins such as Als3, Rbt5, and Csa2 were also detected. The detection of these proteins in the mutant and their lack of detection in the wild type can explain the differential phenotypes previously observed. Received: November 1, 2017 Accepted: March 4, 2018 Published: March 19, 2018 Copyright: © 2018 El Khoury 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: Data are available from figshare, doi: https://doi.org/10.6084/m9. figshare.5743212.v1. Funding: The Department of Natural Sciences has contributed to funding this project. Competing interests: The authors have declared that no competing interests exist. 1. Introduction Candida albicans is normally found in many homeotherms as a benign commensal organism residing asymptomatically on mucosal surfaces and on the skin [1]. However, following an ecological shift or disturbance in the microbial flora, C. albicans becomes an opportunistic pathogen. Factors causing such disturbance include the uptake of broad-spectrum antibiotics, hormonal imbalances, malnutrition, and excessive carbohydrates intake [2]. Patients of radiotherapy, chemotherapy, xerostomia, organ transplantation, endocrine disorders, or HIV infection are at risk of developing a C. albicans infection ranging from local to systemic candidiasis [1, 3]. Statistically, C. albicans is among the most frequently identified agents causing nosocomial infections and the third most frequently isolated pathogen from the bloodstream as per the rankings of the Center for Disease Control [4]. C. albicans has been reported to cause 250 PLOS ONE | https://doi.org/10.1371/journal.pone.0194403 March 19, 2018 1 / 16 pir32 null reveals adhesion, filamentation, virulence genes to 400 thousand deaths per year worldwide [5]. Several factors contribute to the transition of C. albicans between commensalism and pathogenicity. This transition is governed in large parts by the ability of C. albicans to interchange between 2 morphologies: yeast and hyphal forms. Both forms are crucial for the development and maintainance of C. albicans pathogenicity (Jacobsen et al., 2012). The first form is required for the attachment to host cells and subsequent dissemination and the latter form is required for tissue invasion and biofilm formation [6]. Yeast-to-hyphae switching is triggered by various environmental conditions including hypoxia, temperature of 37˚C, serum availability, neutral to basic pH, glucose depletion, and contact with a host cell [7]. In both morphologies, the cells are bound by a cell wall that is continuously modified upon morphogenesis and growth. The cell wall is formed of an inner chitin layer, a β-1,3-glucan layer, a β-1,6-glucan layer, and an outer mannan layer [8]. The fungal cell wall comprises 30% of the overall cell dry weight, where polysaccharides correspond to almost 90% of which, and the remainder is represented by proteins [9]. These proteins have various functions. Proteins attached to the outer surface are related to host cell adhesion such as lectins and adhesins. Some cell wall proteins, like lipases and proteases, degrade the external structures of the host cells and tissues thus aiding in invasion. Others, such as chitinases and mannosidases, are engaged in the synthesis and remodeling of the cell wall itself thus affecting its rigidity and resistance to stresses. Additionally, some proteins are needed to escape the host defenses such as superoxide dismutases [10]. One group of wall proteins, called the Pir proteins standing for proteins with internal repeats, are attached to the β-1,3-glucan layer by alkali-labile bonds and are highly O-glycosylated. These proteins lack a glycosyl phosphatidyl inositol (GPI) anchor motif, but include a signal peptide, internal repeats, a sensitive site for Kex2, and 4 Cys residues at the C-terminal sequence [11]. In C. albicans, there are two Pir proteins: Pir1 and Pir32. Pir1 was found to be an essential protein, required for the stability and rigidity of the cell wall [12]. Pir32 is a 422 amino acid long protein previously characterized in our lab by homologous recombination, a pir32 null strain [13]. Pir32 was found to be involved in virulence, chitin deposition, stress response, and filamentation. Since this protein is located at the cell surface, we hypothesized that the lack of Pir32 would be reflected in various defects in the cell wall proteome and associated pathways. When compared to the wild type strain, the pir32 null strain exhibited hyperfilamentation, enhanced virulence, doubled chitin content, and elevated response to stresses [13]. Accordingly, the aim of this study was to analyze the cell wall proteome of the pir32 null strain in order to explain the observed mutant phenotypes. As such, cell walls from the wild type and mutant strains, under filamentous and non-filamentous growth conditions, were isolated and various chemicals and enzymes were added to fractionate the proteins on the basis of their cell wall anchorage. Tandem-MS, following try (...truncated)