Characterization of a Paracoccidioides spp. strain from southeastern Brazil genotyped as Paracoccidioides restrepiensis (PS3) and review of this phylogenetic species

Abstract

Phylogenetic species of Paracoccidioides brasiliensis complex (S1a and S1b, PS2, PS3, and PS4) and Paracoccidioides lutzii are agents of paracoccidioidomycosis, an endemic fungal disease in Latin America. P. restrepiensis (PS3 genotype) was classified as monophyletic and geographically restricted to Colombia and neighboring territories. BAT (or Pb-327B) was isolated from a patient living in the southeast region of Brazil but with genotype similar to Colombian Paracoccidioides spp. strains. This study aimed to define the phylogenetic species of BAT isolate by using additional genotyping methods, as well as reviewing the epidemiological and clinical studies related to P. restrepiensis isolates. Genomic DNA of BAT isolate and reference strains of P. brasiliensis sensu stricto (S1b), P. americana (PS2), P. restrepiensis (PS3), and P. lutzii were analyzed by conventional polymerase chain reaction (PCR) of partial gp43 exon 2 loci, by PCR-RFLP technique of tub1 gene, and by sequencing of the whole gp43 exon 2 loci. Here, we show that BAT isolate belongs to P. restrepiensis species, which is an unusual identification in southeastern Brazil, where P. brasiliensis sensu stricto is the prevalent genotype. This identification has relevance for geographical distribution and propagation of the genus Paracoccidioides in South America.

Keywords:
Paracoccidioides restrepiensis; Paracoccidioides brasiliensis PS3; phylogenetic species; evolution; paracoccidioidomycosis epidemiology

Introduction

Paracoccidioidomycosis (PCM) is a systemic fungal infection endemic and restricted to Latin American countries such as Brazil, Argentina, Colombia, and Venezuela (Martinez, 2017). Pathogens that cause the acute and chronic forms of PCM are thermodimorphic fungi belonging to the genus Paracoccidioides, family Ajellomycetaceae, order Onygenales, class Eurotiomycetes, and species Paracoccidioides brasiliensis and Paracoccidioides lutzii (Gonzalez and Hernandez, 2016). P. brasiliensis clade is composed of five phylogenetic species, in which S1a and S1b belong to the paraphyletic group distributed in Brazil, Argentina, Paraguay, Peru, and Venezuela; PS2 belongs to the monophyletic group distributed in Brazil and Venezuela; PS3 belongs to the monophyletic group found mainly in Colombia; and the PS4 monophyletic group is found exclusively in Venezuela (Matute et al., 2006; Carrero et al., 2008; Teixeira et al., 2009; Teixeira et al., 2014; Muñoz et al., 2016). Turissini et al. (2017) analyzed microsatellites, mitochondrial and nuclear genes, proposing four new species belonging to the genus Paracoccidioides: P. brasiliensis sensu stricto (S1a and S1b), P. americana (PS2), P. restrepiensis (PS3), and P. venezuelensis (PS4). These species show among them genotypic and micromorphological divergences (Turissini et al., 2017). The P. lutzii clade contains exclusively P. lutzii (Teixeira et al., 2009).

Phylogenetic species 3 (PS3), now P. restrepiensis, was characterized by Matute et al. (2006) and classified as monophyletic, geographically restricted to Colombia, and considered an evolutionary lineage independent of other phylogenetic species of Paracoccidioides spp. complex. The same authors described the phylogenetic relationship of P. restrepiensis (PS3) with other species of P. brasiliensis complex, showing ancestral proximity to P. brasiliensis sensu stricto (S1a and S1b), but having a greater genetic distance from P. americana (PS2). Munõz et al. (2016), when analyzing genotypic divergences among the phylogenetic species, verified the ancestral proximity of Colombian P. restrepiensis (PS3) isolates with Venezuelan isolates of P. venezuelensis (PS4) and Argentinian and Brazilian isolates of P. brasiliensis sensu stricto (S1a and S1b). Besides the genetic proximity of P. restrepiensis (PS3) to other phylogenetic species of P. brasiliensis complex, Roberto et al. (2016) characterized two strains (human isolate chronic form PCM, and soil isolate) obtained in the Venezuelan territory as PS3 (now P. restrepiensis), suggesting its regional dissemination in South America.

This study aimed to characterize a clinical isolate from southeastern Brazil as P. restrepiensis (PS3), an unusual finding in such geographical area. Additionally, a review has been presented with studies on human and environmental isolates of the same genotype.

Material and Methods

Paracoccidioides spp. isolates and culture conditions

BAT (also known as Pb-327-B) clinical strain was isolated in 1985 from a suppurated lymph node of a patient resident in a city belonging to the metropolitan region of Ribeirão Preto, São Paulo State, Brazil (21º10’13.44” S and 47º48’37.17” W). The patient was a 33-year-old male rural worker who had the subacute form of PCM manifested by generalized lymphadenomegaly, hepatosplenomegaly, disseminated cutaneous lesion, fungal lesions in duodenal and colonic mucosa, and jaundice. The patient denied previous disease history or travel to other Brazilian states and South American countries. PCM diagnosis was supported by Paracoccidioides spp. isolation in culture, histopathological examination of intestinal lesions, and a 1:1024 serum titer in the counterimmunoelectrophoresis for anti-Paracoccidioides spp. antibodies. The patient obtained clinical cure after two years of treatment with sulfa drugs.

The following reference strains, whose genotypes were determined in other studies, were employed for BAT clinical isolate comparison: Pb 18 – representative of P. brasiliensis sensu stricto (S1b) species (Matute et al., 2006); Pb dog-EPM 194-representative of P. americana (PS2) species and T2-EPM 54-representative of P. restrepiensis (PS3) species (Roberto et al., 2016); and Pb 01 representative of P. lutzii (Teixeira et al., 2009). All the strains are maintained by successive subcultures on Sabouraud Agar Dextrose medium (Oxoid) plus 0.15 g l^-1 chloramphenicol sodium succinate (Blau Farmacêutica), and incubated at 25 °C. The study was approved by the Research Ethics Committee of the Hospital das Clínicas of Ribeirão Preto Medical School, University of São Paulo (Protocol HCRP nº 4456/2017).

Genomic DNA extraction of Paracoccidioides spp. strains

The genomic DNA of Paracoccidioides spp. strains were obtained from the fungal mycelia, which were grown in a synthetic modified McVeigh-Morton liquid medium for 35 days at 25 °C in an orbital shaker at 130 rpm (Infors HT-Ecotron) (Restrepo and Jimenez, 1980). The mycelia were subjected to extraction of genomic DNA according to the method I (treated glass beads and phenol-chloroform-isoamyl alcohol), with minimal modifications (van Burik et al., 1998). The genomic DNA was treated with 300 ng ml^-1 RNase A^® (Thermo Fisher Scientific) at 37 °C for one hour. The concentration of genomic DNA was determined by using NanoDrop 2000^® (Thermo Fisher Scientific) and its integrity checked in 1% agarose gel using SYBR^® Safe DNA gel stain (Thermo Fisher Scientific) and visualized using the ChemiDoc XRS+ imager with Image Lab software (Bio-Rad).

Partial gp43 exon 2 loci PCR amplification

To identify and classify BAT clinical isolate into the genus Paracoccidioides, the genomic DNA of Paracoccidioides spp. reference strains and BAT isolate were submitted to partial amplification of the gp43 exon 2 loci by using the primers gp43-E2F: (5- CCA GGA GGC GTG CAG GTG TCC C – 3) and gp43-E2R: (5- GCC CCC TCC GTC TTC CAT GTC C – 3) (Cisalpino et al., 1996; Roberto et al., 2016) at 10 mM concentration, and annealing temperature at 58 °C. PCR reaction was performed with Taq polymerase enzyme-GoTaq® Green Master Mix (Promega) according to the manufacturer’s instructions. The final volume of PCR reaction was 25 μl, containing 500 ng genomic DNA. Thermocycling was performed in the Vapo Protect^® thermocycler (Eppendorf). PCR products, approximately 533 bp, had their integrity verified in 2% agarose gel by using SYBR® Safe DNA gel stain (Thermo Fisher Scientific). Its molecular weight was determined by 100-bp Ladder marker, Ready-To-Use (Sinapse), and visualized and photographed on the ChemiDoc XRS+ imager with Image Lab software (Bio-Rad).

Polymerase Chain Reaction – Restriction Fragment Length Polymorphism of tub1 gene – PCR-RFLP

Phylogenetic species identification of BAT clinical isolate was made according to Roberto et al. (2016). Briefly, PCR-RFLP of alpha-tubulin (tub1) gene was performed with Taq polymerase-GoTaq® Green Master Mix enzyme (Promega). The final volume of PCR reaction was 25 μl, containing 500 ng genomic DNA and the primers tub1F: (5-CTG GGA GGT ATG ATA ACA CTG C-3) and tub1R: (5- CGT CGG GCT ATT CAG ATT TAA G -3) (Kasuga et al., 2002; Roberto et al., 2016) at a concentration of 10 mM, and annealing temperature of 58 °C. PCR tub1 products (263 bp) were cleaved with BclI and MspI endonucleases (Thermo Fisher Scientific) at a concentration of 10 U μL^-1 each at 37 °C per 16 hours, according to manufacturer’s instructions. Cleaved DNA fragments were visualized in 2.5% agarose gel at 70 V for 140 minutes in presence of SYBR® Safe DNA gel stain (Thermo Fisher Scientific) and 50 bp DNA ladder molecular marker (Sinapse) and compared according to the method described by Roberto et al. (2016).

Sequencing of gp43 exon 2 loci

To validate the tub1 gene PCR-RFLP method, gp43 exon 2 loci was sequenced using the primers Pbgp43-E2F: (5-CTA GAA TAT CTC ACT CCC AG-3) and Pb_gp43-E2R: (5-GCC CCC TCC GTC TTC CAT GTC C-3) (Cisalpino et al., 1996; Hrycyk et al., 2018) at a concentration of 20 mM and annealing temperature of 58 °C. PCR product of gp43 exon 2 loci, approximately 722 bp, had nonspecific amplification and/or integrity verified in 2% agarose gel. Then PCR amplicons were purified using Wizard^® SV Gel and PCR Clean-Up System kit (Promega), as instructed by the manufacturer. DNA sequences were determined with an ABI3730^® DNA Analyzer (Applied Biosystems), using the BigDye^® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). Chromatograms were analyzed with ChromasPro^® software (ChromasPro 2.6.5). The DNA sequences were compared to nucleotide database using the Basic Local Alignment Search Tool (blastn): https://blast.ncbi.nlm.nih.gov/Blast.cgi (Altschul et al., 1990). The sequences of gp43 exon 2 loci determined in this study were submitted to alignment and analysis of similar and conserved regions, using Clustal Omega software https://www.ebi.ac.uk/Tools/msa/clustalo/ (Sievers et al., 2011).

BAT clinical isolate sequence of gp43 exon 2 loci was deposited at GenBank: (https://www.ncbi.nlm.nih.gov/genbank/) under accession number MH484614.

Results

BAT clinical isolate belongs to the genus Paracoccidioides

Genomic DNA from Paracoccidioides spp. reference strains and BAT isolate were subjected to standard PCR to partially amplify gp43 exon 2 loci. A PCR product of approximately 533 bp (Figure 1A) was observed, confirming that all study samples, including BAT clinical isolate, belong to the genus Paracoccidioides.

Figure 1
(A) Partial amplification of the gp43 exon 2 loci of Paracoccidioides spp. by conventional PCR of BAT clinical isolate and reference isolates. M: 100 bp DNA ladder molecular marker, Ready-To-Use (Sinapse® Inc., United States). (B) Amplification of the tub1 gene by PCR-RFLP. M: 50 bp DNA ladder molecular marker (Sinapse® Inc., United States). (C) PCR-RFLP DNA fragment patterns obtained after cleavage with BclI and MspI endonucleases, showing similarity of BAT clinical isolate with T2-EPM54 (P. restrepiensis – PS3 reference strain). M: 50 bp DNA ladder molecular marker (Sinapse® Inc., United States). Pb 18: P. brasiliensis sensu stricto (S1b); Pb dog-EPM 194: P. americana (PS2); T2-EPM 54: P. restrepiensis (PS3); and Pb 01: P. lutzii; BAT: Clinical isolate under study.

Molecular characterization of BAT clinical isolate as P. restrepiensis (PS3)

The tub1 gene of Paracoccidioides spp. reference strains and BAT isolate were PCR amplified, and 263 bp amplification products were observed (Figure 1B). BAT clinical isolate was identified as P. restrepiensis (PS3) (Figure 1C) since the 263 bp fragment of the tub1 gene does not have cleavage sites for BclI and MspI endonucleases; thus, it was maintained in its complete integrity (263 bp). The reference strains Pb 18, Pb dog–EPM194, T2 – EPM54, and Pb 01 had DNA fragment patterns produced by the endonucleases, as described by Roberto et al. (2016), validating the molecular identification of BAT isolate species by PCR-RFLP as P. restrepiensis (PS3) genotype (Figure 1C). The whole gp43 exon 2 loci of BAT clinical isolate was sequenced to confirm the result obtained by PCR-RFLP. The gp43 exon 2 loci DNA sequence showed 100% identity for nucleotide sequence of the reference strain T2-EPM54 P. restrepiensis (PS3). Alignment of nucleotide sequences of the gp43 exon 2 loci of BAT clinical isolate and the reference strains also showed genetic proximity with Pb 18-P. brasiliensis sensu stricto (S1b), but greater phylogenetic distance from Pb dog EPM194 – P. americana (PS2) and from Pb01 (P. lutzii) (Figure 2).

Figure 2
Alignment of gp43 exon 2 loci nucleotide sequences from Paracoccidioides spp. reference strains and BAT clinical isolate. The identity between BAT isolate and T2-EPM54 (P. restrepiensis – PS3 reference strain) was observed, as well as genetic proximity with Pb 18 (P. brasiliensis sensu stricto – S1b) but with greater phylogenetic distance from Pb dog-EPM194 (P. americana – PS2) and Pb 01 (P. lutzii). *: represents similar nucleotides in the analyzed sequences.

The geographical origin of BAT clinical isolate and of the other Paracoccidioides spp. isolates classified as P. restrepiensis (PS3 genotype) are shown in Figure 3.

Figure 3
Geographic distribution of human and environmental isolates of P. restrepiensis (PS3 genotype) in South America. Each circle represents the identified isolates and their respective regions (Colombia and Venezuela) according to reports in the literature (Matute et al., 2006; Muñoz et al., 2016; Roberto et al., 2016). In Brazil: BAT clinical isolate (PCM-subacute form) isolated from a patient in the macro-region of Ribeirão Preto, São Paulo State, Brazil, and B18 or Pb339 was isolated from a patient in the state of São Paulo, Brazil.

Discussion

BAT clinical strain was isolated in 1985 and maintained at the Ribeirão Preto Medical School-USP, southeastern Brazil. It was classified as P. restrepiensis (PS3) by sequencing of gp43 exon 2 loci and PCR–RFLP of tub1 gene. This result was unexpected since phylogenetic species of PS3 (P. restrepiensis) have so far been isolated in Colombia and Venezuela, countries in northwestern South America. A sample of this strain was sent to Venezuela in the 1990s and included in two investigations on genetic diversity of Paracoccidioides spp. These studies showed a genotypic difference between BAT clinical isolate and other Brazilian isolates. First, BAT isolate (named Pb 327-B) was evaluated together with 32 P. brasiliensis clinical and environmental isolates from different South American countries. By employing randomly amplified polymorphic DNA (RAPD), BAT isolate was grouped near P. brasiliensis isolates from Colombia, which was later classified as PS3 genotype (Calcagno et al., 1998). All 33 P. brasiliensis isolates but one had their DNA analyzed by restriction fragment length polymorphism (RFLP), using HinfI and HincII endonucleases. The dendrogram showed a great relationship of BAT clinical isolate with Colombian P. brasiliensis strains (Nino-Vega et al., 2000).

Some phenotypic characteristics of BAT isolate were evaluated. Micromorphological aspects, virulence for guinea pig, and serological reaction of BAT-isolate exoantigen with rabbit anti GP43 serum were typical traits of P. brasiliensis strains (Lacaz CS et al., 1999). Compared to other P. brasiliensis isolates, BAT isolate had high exoantigen production, which was recognized by sera from patients with PCM in southeastern Brazil (Silva-Vergara et al., 1998; Panunto-Castelo et al., 2003). BAT clinical isolate has been used as a source of antigens of Paracoccidioides spp. in serological tests for PCM diagnosis, since it, together with antigens of other strains, formed a pool with high reactivity against sera from PCM patients from São Paulo state, Brazil (Vidal et al., 2014). Sera from patients infected by P. brasiliensis S1 genotype reacted with BAT isolate exoantigen in immunodiffusion test (data not shown). Paracoccin, a 160 kDa Glc-NAc-binding lectin of the BAT isolate yeast wall, adhered to laminin and induced TNFα and NO production by macrophage cells (Coltri et al., 2006).

To date, 31 P. restrepiensis (PS3) strains (clinical and environmental isolates) have been identified and reported in the literature. The studies encompassed phylogeny, molecular characterization, morphology, serology, and/or epidemiology (Table 1). The geographic distribution of isolates characterized as P. restrepiensis (PS3), including BAT clinical strain, is predominant in Colombia, totaling 87.2% of the 31 isolates, followed by 6.4% in Venezuela, and 6.4% in Brazil (Matute et al., 2006; Muñoz et al., 2016; Roberto et al., 2016). The occurrence of P. restrepiensis (PS3) in other South American countries (Brazil and Venezuela) suggests that its geographical distribution is not restricted to the Colombian territory as initially presumed. It is believed that the phylogeographic characteristics of P. restrepiensis (PS3) are due to a possible and relatively recent biogeographic expansion of P. brasiliensis sensu stricto (S1a and S1b) to Colombia, associated to events of geographic barriers represented by the uplifting of the Andes mountain range and submersion of the Colombian territory by formation of the Pebas-Solimões lake (Wesselingh and Salo, 2006; Teixeira et al., 2014). The emergence of P. restrepiensis (PS3) in Brazil was not clarified within the process of speciation of these phylogenies, due to absence of a geographical barrier in the territory (Teixeira et al., 2014).

Table 1
Paracoccidioides spp. isolates genotypically characterized as P. restrepiensis (PS3): origin, source, molecular identification, and study type (1967 – 2018).

Strain		Origin	Source	Identification method	Study Type	Reference
Original ID	Other ID
C1	P149	Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C2	P159	Antioquia, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C3	P163	Antioquia, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C4	ATCC 60855	Antioquia, Colombia	Chronic /PCM	MLST	Phenotypic	(Bustamante-Simon et al., 1985)
					Phenotypic	(Villar et al., 1988)
					Virulence	(Gomez et al., 2001)
					Phylogenetic	(Matute et al., 2006)
C5	P141	Antioquia, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C6	P196 / Higuita	Antioquia, Colombia	Chronic /PCM	MLST	Molecular Identification	(Corredor et al., 2005)
					Phylogenetic	(Matute et al., 2006)
C7	P204	Antioquia, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C8	P292	Antioquia, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C9	P68	Antioquia, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C10	P72	Cordoba, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C11	P46	Antioquia, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C12	P161	Antioquia, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C13	76533	Antioquia, Colombia	Chronic /PCM	MLST	Susceptibility	(Hoyos et al., 1984)
					Phylogenetic	(Matute et al., 2006)
C14	H31	Boyaca, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C15	H45	Cundinamarca, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C16	H47	Arauca, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C17	P206	Antioquia, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C18	P151	Antioquia, Colombia	Chronic /PCM	MLST	Phylogenetic	(Matute et al., 2006)
C19	CIB44197	Caldas, Colombia	Armadillo	MLST	Molecular Identification	(Corredor et al., 2005)
					Phylogenetic	(Matute et al., 2006)
C20	Pb73/ ATCC 32071	Antioquia, Colombia	Unknown	MLST	Serological tests	(Restrepo-Moreno 1967)
					Phylogenetic	(Matute et al., 2006)
C21	CIB40392	Caldas, Colombia	Armadillo	MLST	Epidemiological	(Corredor et al., 1999)
					Phylogenetic	(Matute et al., 2006)
T2	EPM54	Caracas, Venezuela	Soil	MLST, PCR – RFLP	Phylogenetic	(Roberto et al., 2016)
5598	EPM62	Caracas, Venezuela	Acute/PCM	MLST, PCR – RFLP	Phylogenetic	(Roberto et al., 2016)
JDA – 80	EPM77	Medellin, Colombia	Unknown	MLST, PCR – RFLP	Phylogenetic	(Roberto et al., 2016)
MSCol	EPM81	Medellin, Colombia	Chronic /PCM	MLST, PCR – RFLP	Phylogenetic	(Roberto et al., 2016)
I9	EPM83	Bogotá, Colombia	Chronic /PCM	MLST, PCR – RFLP	Phylogenetic	(Theodoro et al., 2008)
					Serological	(Machado et al., 2013)
					Proteome	(Pigosso et al., 2013)
					Secretome	(de Oliveira et al., 2018)
*B18	Pb339 or	Brazil	Unknown	MLST, PCR - RFLP	Serological tests	(Restrepo-Moreno 1967)
	ATCC 32069				Virulence	(Gomez et al.
	EPM01 or B339				Serological	(Camargo et al., 2003)
	or ATCC 200273				Phylogenetic	(Matute et al., 2006)
					Mitochondrial DNA	(Salgado – Salazar et al., 2010)
					Phylogenetic	(Roberto et al., 2016)
					Phylogenetic	(Muñoz et al., 2016)
PbBac	-	Colombia	PCM	Genomic sequencing	Phylogenetic	(Muñoz et al., 2016)
PbCnh	-	Colombia	Chronic /PCM	Genomic sequencing	Phylogenetic	(Muñoz et al., 2016)
PbJam	-	Colombia	Chronic /PCM	Genomic sequencing	Phylogenetic	(Muñoz et al., 2016)
BAT	Pb327 - B	Ribeirão Preto – São Paulo, Brazil	Subacute/PCM	MLST, PCR – RFLP	Serological	(Silva – Vergara et al., 1998)
					Mycological	(Lacaz Cda, S et al., 1999)
					Phylogenetic	(Calcagno et al., 1998)
					Phylogenetic	(Niño – Vega et al., 2000)
					Serological	(Panuto – Castelo et al., 2003)
					Immunologic	(Coltri et al., 2006)
					Molecular Identification	This Study

Besides the geographical origin of P. restrepiensis (PS3), the respective clinical manifestation of PCM is one of the important aspects for understanding the pathogenicity of this phylogenetic species. Among the 31 P. restrepiensis (PS3) isolates described in the literature, including BAT isolate, 25 (76%) strains were isolated from patients. The chronic form of PCM was more prevalent in patients infected with P. restrepiensis (PS3), representing 88% of the cases. Acute/subacute forms of PCM caused by P. restrepiensis (PS3) were only reported in two patients, including that with a disseminated disease from which the strain evaluated in this study was isolated (Matute et al., 2006; Muñoz et al., 2016; Roberto et al., 2016). In a comparative study of PCM cases caused by P. brasiliensis sensu stricto (S1a and S1b) and P. americana (PS2) in Rio de Janeiro state-Brazil, the prevalence of chronic form was observed for both species (de Macedo et al., 2019). The same was observed in a clinical and epidemiological study of PCM caused by P. lutzii in the state of Mato Grosso-Brazil, wherein all patients were diagnosed with the chronic form (Hahn et al., 2019). In general, chronic form predominance is common in PCM, so it does not distinguish P. restrepiensis (PS3) genotype in this regard.

Some of the Paracoccidioides spp. isolates characterized as P. restrepiensis (PS3) have already had their biology studied (Matute et al., 2006; Theodoro et al., 2008). Genotypic and phenotypic studies for P. restrepiensis (PS3) isolates were fungal antigenicity (Restrepo-Moreno and Schneidau, 1967), ketoconazole susceptibility (Hoyos et al., 1984), murine immune response to PCM, conidia morphology and sporulation at different culture media (Bustamante-Simon et al., 1985), dimorphism (Villar et al., 1988), morphological analysis and molecular identification of armadillo isolates (Corredor et al., 1999), and melanin production (Gomez et al., 2001). Other studies compared P. restrepiensis (PS3) isolates with different species belonging to Paracoccidioides spp. complex (P. brasiliensis sensu stricto (S1), P. americana (PS2)) to evaluate genotypic and phenotypic differences. Corredor et al. (2005) studied polymorphic genes (gp43 exon 2 loci, ITS_1, and ITS_4) from clinical and armadillo strains isolated in the Colombian territory, comparing them with strains isolated in other South American countries. Polymorphic differences were found among genes when compared to strains identified as P. brasiliensis sensu stricto (S1); P. restrepiensis (PS3) showed high differentiation from other species (Corredor et al., 2005; Matute et al., 2006). PRP8 intein protein gene sequences from species of the Paracoccidioides spp. complex, including P. restrepiensis EPM83, were analyzed in a phylogenetic study. This gene can be used as a molecular marker since its polymorphism can separate species from the P. brasiliensis complex and P. lutzii (Theodoro et al., 2008).

Some studies were directed to a phenotypic comparison of P. restrepiensis (PS3) isolates and other species belonging to the Paracoccidioides spp. (Table 2). Turissini et al. (2017) carried out a phenotypic study on yeast cells of cryptic species of P. brasiliensis complex (S1, PS2, PS3, and PS4) and P. lutzii. These authors observed that P. restrepiensis (PS3) has yeast cells larger than P. brasiliensis sensu stricto (S1) and P. americana (PS2) ones but no cell size differences with P. venezuelensis (PS4) and P. lutzii strains. A comparative study to evaluate PCM immunodiagnosis using species of the P. brasiliensis complex and P. lutzii showed higher GP43 production and best antigenic reactivity in an immunodiffusion assay with the EPM83 strain (P. restrepiensis – PS3) when tested with sera from patients living in a geographic area where P. brasiliensis sensu stricto (S1a e S1b) and P. americana (PS2) are prevalent (Machado et al., 2013). Another comparative study of proteomes by disrupting yeast cells of Paracoccidioides species representative isolates showed that EPM83 (P. restrepiensis – PS3) preferentially expressed 38 proteins, including heat shock proteins (HSP) and a higher level of GP43 production (Pigosso et al., 2013). An analysis of secretomes of two Paracoccidioides spp strains identified 95 extracellular proteins, 35 specific of P. lutzii and 36 specific of P. restrepiensis (PS3), including several ones related to fungal virulence factors and adhesion (de Oliveira et al., 2018).

Table 2
Phenotypic characteristics of P. restrepiensis (PS3) isolates compared to other species of the P. brasiliensis complex and P. lutzii

P. restrepiensis isolate	P. brasiliensis complex or P. lutzii isolate	Evaluated trait	Observed differences	Study
C4, BAC, CNH	Pb18, T15LN1, T4B14 - P. brasiliensis sensu stricto (S1)	Yeast cell size	P. restrepiensis (PS3) shows larger yeast area than P. brasiliensis sensu stricto (S1) and P. americana (PS2) isolates but similar to P. venezuelensis (PS4)	(Turissini et al., 2017)
	Pb3 and T10B1 - P. americana (PS2)
	Pb300 and Pb305 - P. venezuelensis (PS4)
	Pb66, Pb01, EE - P. lutzii
BAT	Pb18 - P. brasiliensis sensu stricto (S1)	Exoantigen production	Higher number of BAT exoantigens recognized by patient sera	(Panuto-Castelo et al., 2003)
	DGO and C – 9 - Unknown Genotype
	B_339 – P. brasiliensis sensu stricto (S1)
EPM83	Pb265 - P. brasiliensis sensu stricto (S1)	GP43 production and antigenicity	EPM83 showed higher GP43 production and antigenicity in the immunodiffusion test with PCM sera	(Machado et al., 2013)
	Pb01, Pb8334, Pb66 - P. lutzii
EPM83	Pb339 – P. brasiliensis sensu stricto (S1)	Proteome	EPM83 presented higher expression of 38 proteins, including HSP and GP43	(Pigosso et al., 2013)
	Pb2 – P.americana (PS2)
	Pb01 – P. lutzii
EPM83	Pb01 - P. lutzii	Secretome	EPM83 production of 36 specific proteins; 21 proteins shared with Pb01	(de Oliveira et al., 2018)

B18 or Pb339 (ATCC32069) is a strain that has been reported in the literature to have divergent results regarding its classification as P. restrepiensis (PS3). It is originally from the state of São Paulo (Brazil) but from an unknown source. This strain was obtained from the National Communicable Disease Center (Atlanta, USA) and first studied by Restrepo-Moreno and Scheneidau JD in 1967. Matute et al. (2006) classified B18 as P. brasiliensis sensu stricto (S1b) by analyses of polymorphisms in nuclear genes, chitin synthase (CHS) 2, glucan synthase (FKS), tub1 (TUB), adenyl ribosylation factor (ARF), and gp43 exon 2 loci. On the other hand, Salgado-Salazar et al. (2010) studied polymorphism of five mitochondrial genes used as markers in molecular characterization of Paracoccidioides species. The findings enabled reclassification of B18 strain as P. restrepiensis (PS3) from Paracoccidioides spp. complex (Salgado-Salazar et al., 2010). Later, Roberto et al. (2016) evaluated B18 strain, termed in that study as B339 and /or EPM01 (ATCC200273) (Camargo et al., 2003), and classified B18 strain as P. restrepiensis (PS3) by PCR-RFLP of tub1 gene and sequencing of CHS2, FKS, TUB, ARF, and GP43 nuclear genes, the same ones studied by Matute et al. (2016). A study conducted by Turissini et al. (2017) classified B18 strain as an independent genotype due to its phylogenetic and micromorphological differences already observed in previous studies, suggesting a hybrid species belonging to the genus Paracoccidioides.

Identification of an unusual genotypic variant in southeastern Brazil contributes to understanding speciation and propagation involving PCM agents and may help in knowing P. restrepiensis characteristics (PS3 genotype) such as morphology, virulence, and serological reactivity.

Acknowledgments

T. A. Cocio thanks Prof. Dr Zoilo Pires de Camargo, Department of Microbiology, Immunology, and Parasitology, Federal University of São Paulo – SP (Brazil) for kindly ceding Pb dog–EPM194 and T2–EPM54 strains used in this study, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – (CAPES Brazil - Finance Code 001) for by granting doctoral and postdoctoral scholarships [Grant numbers: 88882.180015/2018-01 (T.A.C.) and 88882.317609/2019-1 (E.N.)], and the Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FAEPA) for financial support.

References

• Altschul SF, Gish W, Miller W, Myers EW and Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403-410.
• Bustamante-Simon B, McEwen JG, Tabares AM, Arango M and Restrepo-Moreno A (1985) Characteristics of the conidia produced by the mycelial form of Paracoccidioides brasiliensis Sabouraudia 23:407-414.
• Calcagno AM, Nino-Vega G, San-Blas F and San-Blas G (1998) Geographic discrimination of Paracoccidioides brasiliensis strains by randomly amplified polymorphic DNA analysis. J Clin Microbiol 36:1733-1736.
• Camargo ZP, Berzaghi R, Amaral CC and Silva SH (2003) Simplified method for producing Paracoccidioides brasiliensis exoantigens for use in immunodiffusion tests. Med Mycol 41:539-542.
• Carrero LL, Nino-Vega G, Teixeira MM, Carvalho MJ, Soares CM, Pereira M, Jesuino RS, McEwen JG, Mendoza L, Taylor JW et al. (2008) New Paracoccidioides brasiliensis isolate reveals unexpected genomic variability in this human pathogen. Fungal Genet Biol 45:605-612.
• Cisalpino PS, Puccia R, Yamauchi LM, Cano MI, da Silveira JF and Travassos LR (1996) Cloning, characterization, and epitope expression of the major diagnostic antigen of Paracoccidioides brasiliensis J Biol Chem 271:4553-4560.
• Coltri KC, Casabona-Fortunato AS, Gennari-Cardoso ML, Pinzan CF, Ruas LP, Mariano VS, Martinez R, Rosa JC, Panunto-Castelo A and Roque-Barreira MC (2006) Paracoccin, a GlcNAc-binding lectin from Paracoccidioides brasiliensis binds to laminin and induces TNF-alpha production by macrophages. Microbes Infect 8:704-713.
• Corredor GG, Castano JH, Peralta LA, Diez S, Arango M, McEwen J and Restrepo A (1999) Isolation of Paracoccidioides brasiliensis from the nine-banded armadillo Dasypus novemcinctus in an endemic area for paracoccidioidomycosis in Colombia. Rev Iberoam Micol 16:216-220.
• Corredor GG, Peralta LA, Castano JH, Zuluaga JS, Henao B, Arango M, Tabares AM, Matute DR, McEwen JG and Restrepo A (2005) The naked-tailed armadillo Cabassous centralis (Miller 1899): a new host to Paracoccidioides brasiliensis Molecular identification of the isolate. Med Mycol 43:275-280.
• de Macedo PM, Teixeira MM, Barker BM, Zancope-Oliveira RM, Almeida-Paes R and Francesconi do Valle AC (2019) Clinical features and genetic background of the sympatric species Paracoccidioides brasiliensis and Paracoccidioides americana PLoS Negl Trop Dis 13:e0007309.
• de Oliveira AR, Oliveira LN, Chaves EGA, Weber SS, Bailao AM, Parente-Rocha JA, Baeza LC, de Almeida Soares CM and Borges CL (2018) Characterization of extracellular proteins in members of the Paracoccidioides complex. Fungal Biol 122:738-751.
• Gomez BL, Nosanchuk JD, Diez S, Youngchim S, Aisen P, Cano LE, Restrepo A, Casadevall A and Hamilton AJ (2001) Detection of melanin-like pigments in the dimorphic fungal pathogen Paracoccidioides brasiliensisin vitro and during infection. Infect Immun 69:5760-5767.
• Gonzalez A and Hernandez O (2016) New insights into a complex fungal pathogen: the case of Paracoccidioides spp. Yeast 33:113-128.
• Hahn RC, Rodrigues AM, Della Terra PP, Nery AF, Hoffmann-Santos HD, Gois HM, Fontes CJF and de Camargo ZP (2019) Clinical and epidemiological features of paracoccidioidomycosis due to Paracoccidioides lutzii PLoS Negl Trop Dis 13:e0007437.
• Hoyos GL, McEwen JG, Brummer E, Castaneda E, Restrepo A and Stevens DA (1984) Chronic murine paracoccidioidomycosis: effect of ketoconazole on clearance of Paracoccidioides brasiliensis and immune response. Sabouraudia 22:419-426.
• Hrycyk MF, Garcia Garces H, Bosco SMG, de Oliveira SL, Marques SA and Bagagli E (2018) Ecology of Paracoccidioides brasiliensisP. lutzii and related species: infection in armadillos, soil occurrence and mycological aspects. Med Mycol 56:950-962.
• Kasuga T, White TJ and Taylor JW (2002) Estimation of nucleotide substitution rates in Eurotiomycete fungi. Mol Biol Evol 19:2318-2324.
• Lacaz CS, Vidal MS, Heins-Vaccari EM, de Melo NT, Del Negro GM, Arriagada GL and Freitas RS (1999) Paracoccidioides brasiliensis A mycologic and immunochemical study of two strains. Rev Inst Med Trop Sao Paulo 41:79-86.
• Machado GC, Moris DV, Arantes TD, Silva LR, Theodoro RC, Mendes RP, Vicentini AP and Bagagli E (2013) Cryptic species of Paracoccidioides brasiliensis: impact on paracoccidioidomycosis immunodiagnosis. Mem Inst Oswaldo Cruz 108:637-643.
• Martinez R (2017) New Trends trends in paracoccidioidomycosis epidemiology. J Fungi 3:e1.
• Matute DR, McEwen JG, Puccia R, Montes BA, San-Blas G, Bagagli E, Rauscher JT, Restrepo A, Morais F, Nino-Vega G et al. (2006) Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies. Mol Biol Evol 23:65-73.
• Muñoz JF, Farrer RA, Desjardins CA, Gallo JE, Sykes S, Sakthikumar S, Misas E, Whiston EA, Bagagli E, Soares CM et al. (2016) Genome diversity, recombination, and virulence across the major lineages of Paracoccidioides mSphere 1:e00213-00216.
• Nino-Vega GA, Calcagno AM, San-Blas G, San-Blas F, Gooday GW and Gow NA (2000) RFLP analysis reveals marked geographical isolation between strains of Paracoccidioides brasiliensis Med Mycol 38:437-441.
• Panunto-Castelo A, Freitas-da-Silva G, Bragheto IC, Martinez R and Roque-Barreira MC (2003) Paracoccidioides brasiliensis exoantigens: recognition by IgG from patients with different clinical forms of paracoccidioidomycosis. Microbes Infect 5:1205-1211.
• Pigosso LL, Parente AF, Coelho AS, Silva LP, Borges CL, Bailao AM and Soares CM (2013) Comparative proteomics in the genus Paracoccidioides Fungal Genet Biol 60:87-100.
• Restrepo-Moreno A and Schneidau JD (1967) Nature of the skin-reactive principle in culture filtrates prepared from Paracoccidioides brasiliensis J Bacteriol 93:1741-1748.
• Restrepo A and Jimenez BE (1980) Growth of Paracoccidioides brasiliensis yeast phase in a chemically defined culture medium. J Clin Microbiol 12:279-281.
• Roberto TN, Rodrigues AM, Hahn RC and de Camargo ZP (2016) Identifying Paracoccidioides phylogenetic species by PCR-RFLP of the alpha-tubulin gene. Med Mycol 54:240-247.
• Salgado-Salazar C, Jones LR, Restrepo A and McEwen JG (2010) The human fungal pathogen Paracoccidioides brasiliensis (Onygenales: Ajellomycetaceae) is a complex of two species: phylogenetic evidence from five mitochondrial markers. Cladistics 26:613-624.
• Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Soding J et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539.
• Silva-Vergara ML, Martinez R, Chadu A, Madeira M, Freitas-Silva G and Leite Maffei CM (1998) Isolation of a Paracoccidioides brasiliensis strain from the soil of a coffee plantation in Ibia, State of Minas Gerais, Brazil. Med Mycol 36:37-42.
• Teixeira MM, Theodoro RC, de Carvalho MJ, Fernandes L, Paes HC, Hahn RC, Mendoza L, Bagagli E, San-Blas G and Felipe MS (2009) Phylogenetic analysis reveals a high level of speciation in the Paracoccidioides genus. Mol Phylogenet Evol 52:273-283.
• Teixeira MM, Theodoro RC, Nino-Vega G, Bagagli E and Felipe MS (2014) Paracoccidioides species complex: ecology, phylogeny, sexual reproduction, and virulence. PLoS Pathog 10:e1004397.
• Theodoro RC, Bagagli E and Oliveira C (2008) Phylogenetic analysis of PRP8 intein in Paracoccidioides brasiliensis species complex. Fungal Genet Biol 45:1284-1291.
• Turissini DA, Gomez OM, Teixeira MM, McEwen JG and Matute DR (2017) Species boundaries in the human pathogen Paracoccidioides Fungal Genet Biol 106:9-25.
• van Burik JA, Schreckhise RW, White TC, Bowden RA and Myerson D (1998) Comparison of six extraction techniques for isolation of DNA from filamentous fungi. Med Mycol 36:299-303.
• Vidal MS, Del Negro GM, Vicentini AP, Svidzinski TI, Mendes-Giannini MJ, Almeida AM, Martinez R, de Camargo ZP, Taborda CP and Benard G (2014) Serological diagnosis of paracoccidioidomycosis: high rate of inter-laboratorial variability among medical mycology reference centers. PLoS Negl Trop Dis 8:e3174.
• Villar LA, Salazar ME and Restrepo A (1988) Morphological study of a variant of Paracoccidioides brasiliensis that exists in the yeast form at room temperature. J Med Vet Myco 26:269-276.
• Wesselingh F and Salo J (2006) A Miocene perspective on the evolution of the Amazonian biota. Scr Geol 133:439-458.

• Associate Editor: Célia Maria de Almeida Soares

Publication Dates

• Publication in this collection
  29 May 2020
• Date of issue
  2020

History

• Received
  20 June 2019
• Accepted
  09 Mar 2020