ORIGINAL RESEARCH published: 01 July 2021 doi: 10.3389/fcimb.2021.642658 Comparison of Cryptococcus gattii/ neoformans Species Complex to Related Genera (Papiliotrema and Naganishia) Reveal Variances in Virulence Associated Factors and Antifungal Susceptibility Lana Sarita de Souza Oliveira 1, Luciana Magalhães Pinto 1, Mariana Arau´ jo Paulo de Medeiros 1, Dena L. Toffaletti 2, Jennifer L. Tenor 2, Edited by: Taˆ nia Fraga Barros 3, Rejane Pereira Neves 4, Reginaldo Gonc¸ alves de Lima Neto 5, Brian Wickes, Eveline Pipolo Milan 6, Ana Carolina Barbosa Padovan 7, Walicyranison Plinio da Silva Rocha 8, The University of Texas Health Science John R. Perfect 2 and Guilherme Maranhão Chaves 1* Center at San Antonio, United States Reviewed by: 1 Laboratory of Medical and Molecular Mycology, Department of Clinical and Toxicological Analyses, Federal University of Rio Sudha Chaturvedi, Grande do Norte, Natal, Brazil, 2 Division of Infectious Disease, Department of Medicine, Duke University School of Medicine, Wadsworth Center, United States Durham, NC, United States, 3 Department of Clinical and Toxicological Analyses, Federal University of Bahia, Salvador, Brazil, Jianping Xu, 4 Department of Mycology, Federal University of Pernambuco, Recife, Brazil, 5 Department of Tropical Medicine, Federal University McMaster University, Canada of Pernambuco, Recife, Brazil, 6 Department of Infectology, Federal University of Rio Grande do Norte, Natal, Brazil, 7 Department of Microbiology and Immunology, Federal University of Alfenas, Alfenas, Brazil, 8 Department of Pharmaceutical Sciences, Federal *Correspondence: University of Paraiba, João Pessoa, Brazil Guilherme Maranhão Chaves [email protected] Cryptococcosis is an infectious disease of worldwide distribution, caused by Specialty section: encapsulated yeasts belonging to the phylum Basidiomycota. The genus Cryptococcus This article was submitted to Fungal Pathogenesis, includes several species distributed around the world. The C. gattii/neoformans species a section of the journal complex is largely responsible for most cases of cryptococcosis. However, clinical series Frontiers in Cellular and have been published of infections caused by Papiliotrema (Cryptococcus) laurentii and Infection Microbiology Naganishia albida (Cryptococcus albidus), among other related genera. Here, we Received: 21 January 2021 Accepted: 10 June 2021 examined the pathogenic potential and antifungal susceptibility of C. gattii/neoformans Published: 01 July 2021 species complex (clades I and II) and related genera (Papiliotrema and Naganishia) Citation: isolated from environmental and clinical samples. P. laurentii (clade III), N. liquefasciens/ Oliveira LSdS, Pinto LM, de Medeiros MAP, Toffaletti DL, N. albidosimilis (clade IV); and N. adeliensis/N. albida (clade V) strains produced higher Tenor JL, Barros TF, Neves RP, levels of phospholipase and hemolysins, whereas the C. gattii/neoformans species Neto RGdL, Milan EP, Padovan ACB, complex strains (clades I and II) had markedly thicker capsules, produced more biofilm Rocha WPdS, Perfect JR and Chaves GM (2021) Comparison of biomass and melanin, which are known virulence attributes. Interestingly, 40% of C. Cryptococcus gattii/neoformans neoformans strains (clade II) had MICs above the ECV established for this species to Species Complex to Related Genera (Papiliotrema and Naganishia) Reveal amphotericin B. Several non-C. gattii/neoformans species complex (clades III to V) had Variances in Virulence Associated MICs equal to or above the ECVs established for C. deuterogattii and C. neoformans for all Factors and Antifungal Susceptibility. the three antifungal drugs tested. Finally, all the non-C. gattii/neoformans clinical isolates Front. Cell. Infect. Microbiol. 11:642658. doi: 10.3389/fcimb.2021.642658 (clades III to V) produced more melanin than the environmental isolates might reflect their Frontiers in Cellular and Infection Microbiology | www.frontiersin.org1 July 2021 | Volume 11 | Article 642658 Oliveira et al. Cryptococcus spp. Reveal Phenotypic Differences particularly enhanced need for melanin during in vivo protection. It is very clear that C. gattii/neoformans species complex (clades I and II) strains, in general, show more similar virulence phenotypes between each other when compared to non-C. gattii/neoformans species complex (clades III to V) isolates. These observations together with the fact that P. laurentii and Naganishia spp. (clades III to V) strains were collected from the outside of a University Hospital, identify features of these yeasts important for environmental and patient colonization and furthermore, define mechanisms for infections with these uncommon pathogens. Keywords: Cryptococcus spp., Papiliotrema laurentii, Naganishia spp., virulence factors, antifungal drug susceptibility INTRODUCTION responsible for 80% of the cases of infection caused by non- Cryptococcus gattii/neoformans species complex yeasts (Johnson Cryptococcosis is an infectious disease of worldwide distribution, et al., 1998; Khawcharoenporn et al., 2007; Smith et al., 2017; caused by encapsulated yeasts belonging to the phylum Castro-Lainez et al., 2019; Zhang et al., 2019). Based on DNA- Basidiomycota (Kwon-Chung and Bennett, 1992; Maziarz and sequencing of seven different genes, the class Tremellomycetes Perfect, 2016). Cryptococcus spp., such as the Cryptococcus gattii/ (Agaricomicotina lineage, Phylum Basidiomycota) including the neoformans species complex, are able to enter mammalian hosts and genus Cryptococcus was recently reclassified. Therefore, the cause damage to the central nervous system (CNS) (Kwon-Chung former species C. laurentii is now a synonym of Papiliotrema et al., 2014; Lakoh et al., 2020) and respiratory tract (Lakoh et al., laurentii and a few other Cryptococcus species of medical interest 2020). In the last few decades, cryptococcal meningoencephalitis has have been currently included in the genus Naganishia as follows: become an infection of global relevance, with up to 1 million new Naganishia (Cryptococcus) diffluens, Naganishia (Cryptococcus) infections annually and significant attributable morbidity and liquefaciens, Naganishia (Cryptococcus) albidosimilis, Naganishia mortality. Even with widespread effective antiretroviral therapy, albida (Cryptococcus albidus) and Naganishia (Cryptococcus) there is still approximately 200,000 deaths per year especially adeliensis (Liu et al., 2015). among patients with human immunodeficiency virus (HIV) Several virulence factors have classically been carefully infection and AIDS (Park et al., 2009; Park et al., 2011). Besides characterized and validated for C. gattii/neoformans complex, HIV/AIDS infection, other risk factors for cryptococcosis include such as the presence of a polysaccharide capsule that protects corticosteroids and other immunosuppressive therapies, solid organ yeast cells against phagocytosis, the ability to grow at 37°C, transplantation, malignant and rheumatological diseases (Maziarz biofilm formation, and the production of additional factors and Perfect, 2016). including melanin, phospholipase, metalloprotease, DNase, The genus Cryptococcus includes several species distributed urease, superoxide dismutase and other antioxidant enzymes around the world and found in different environments (Chee and (Mancianti et al., 2002; Sanchez et al., 2008; Peng et al., 2018; Lee, 2005; Loperena-Alvarez et al., 2010), such as bird droppings Zaragoza, 2019). However, only a few studies have investigated the (mainly pigeons) (Irokanulo et al., 1997; Soltani et al., 2013; Kassi potential role of these and other virulence factors in the non- et al., 2018), soil, rivers, decomposing vegetation and tree hollows Cryptococcus gattii/neoformans species complex (Ikeda et al., 2002; (Lazera et al., 1998; Kidd et al., 2007; Alves et al., 2016; Santos Andrade-Silva et al., 2010; Araujo Gde et al., 2012; Ferreira-Paim Bentes et al., 2019). Recently, Hagen et al. (2015) recognized et al., 2012; Araujo et al., 2017). multiple species within the Cryptococcus gattii/neoformans Because of the variations of virulence factors and clinical species complex, including the existence of four C. neoformans resistance to azole monotherapy, it is important to know and genotypes: VNI and VNII (var. grubii, serotype A), VNIII compare the relevant pathogenic potential and antifungal (hybrid serotype AD), VNIV (var. neoformans, serotype D); susceptibility profiling of Cryptococcus spp. collected from clinical and four genotypes of C. gattii: VGI, VGII, VGIII and VGIV and environmental sources. Therefore, the primary objectives of (serotypes B and C). Others have also reported the presence of this study were to evaluate the pathogenic features and antifungal VNBI and VNBII genotypes within the taxon previously susceptibility of Cryptococcus-related genera isolated from pigeon considered C. neoformans var. grubii (Desjardins et al., 2017). droppings (environmental), compared to the Cryptococcus gattii/ More recently, a new C. gattii molecular type has been discovered neoformans species complex strains obtained from clinical samples in the African environments of the Central Miombo Woodlands of patients belonging to 3 different Brazilian states (Bahia, (VGV) (Farrer et al., 2019). Pernambuco and Rio Grande do Norte) in the Northeast region. Cryptococcus gattii/neoformans species complex is primarily This is the first study to demonstrate that non-Cryptococcus gattii/ related to the vast majority of cases of cryptococcosis