By using nested PCR, we have successfully been able to amplify a 196-bp fragment of the PbGP43 exon 2 from 23 formalin-fixed, paraffin-embedded (FFPE) samples out of 31 obtained from 20 PCM patients. The human β-globulin control gene was also amplified in these samples and also in two others (25 total), which probably lacked fungal DNA. We identified phylogenetic lineages of the P. brasiliensis complex from the sequencing of the nested PCR products. These 23 sequenced samples were collected in four Brazilian states located in the Southeast and South regions, which are recognized as the mainly endemic areas of PCM in the country [3]. The majority of the samples (91.3%) were obtained from male patients. It is in agreement with the previously described in a study that analyzed 584 PCM patients (575 of them from the Southeast region of Brazil), in which 84.2% of the samples were from male patients [46]. A predominance of male patients is characteristic of PCM, irrespective of phylogenetic species [47, 48], and the reasons include the fact that circulating estrogens in women can inhibit the transformation of the inhaled conidia into yeast cells, and the greater proportion of men involved in agricultural activities in endemic areas [3, 49].
The alignment of our sequences with other PbGP43 sequences available in the GenBank database showed the presence of five parsimony-informative sites in the gene region analyzed. We analyzed a fragment of the second exon of PbGP43 because it is the most polymorphic region among P. brasiliensis genes already studied [20, 27, 50]. A highly polymorphic gene region (more informative) is critical in FFPE samples, since the extracted DNA is more prone to be fragmented, challenging the amplification of long PCR products [51]. The limitations in getting DNA with good quality from FFPE tissue biopsies are formalin fixation and paraffin embedding procedures, resulting in DNA damage. The difficulty of amplifying DNA from FFPE samples is well discussed and explored in the literature [30, 52, 53], but despite this difficulty, only six samples did not show any amplification products, which indicates that for most of the samples, we obtained a good quality DNA from the FFPE blocks. Indeed, the successful use of FFPE blocks for PCM diagnosis has been demonstrated in several clinical investigations to detect the fungus DNA as a marker of infection, highlighting the value of these samples for molecular diagnosis [17, 31-35].
Sequences were assigned to seven haplotypes, six of them belonging to the P. brasiliensis complex. The other haplotype comprises P. lutzii, which was first considered as P. brasiliensis, but later proposed as a new species in the genus Paracoccidioides [8]. Our results also indicated the existence of an unsampled or extinct haplotype in the P. brasiliensis complex, as represented by the median vector in the haplotype network. We found a relatively high value of haplotype diversity and a low value of nucleotide diversity, a pattern commonly observed in recently diverged species. Indeed, that is the case for the P. brasiliensis complex, since it has been demonstrated that the divergence times between species pairs range between 0.127 and 0.714 million years ago [19].
Each recognized species of the P. brasiliensis complex was assigned to a distinct haplotype (H1, H2, H3, and H6), but sequences of two haplotypes (H4 and H5) could not be assigned to any species of the complex. The ML phylogenetic tree supported the haplotype network results and indicated that P. americana was the most basal species of the complex, in agreement with previous studies [19, 21, 54]. The expected phylogeny for the other taxa in the complex is P. restrepiensis and P. venezuelensis as sister species, with P. brasiliensis sensu stricto as the sister group of these dyad [19]. However, we found H5 located between the P. americana clade and a polytomy involving the other haplotypes. The isolates that comprise H5 were obtained from human patients in Costa Rica, and as far as we know, only Takayama et al. [55] reported a phylogeny considering these isolates. They analyzed a different region of the PbGP43 gene and also obtained an exclusive clade for these isolates, which prevented the assignment of a phylogenetic species. We, therefore, considered these isolates as an indeterminate species of the P. brasiliensis complex.
FFPE samples were collected in an area of sympatry between P. brasiliensis sensu stricto and P. americana. The former is usually reported as the most frequent species, associated with most PCM cases, while the latter is found at a considerably lower frequency [10, 20, 54, 56, 57]. Our results showed that nine out of 23 FFPE samples (39.1%) were assigned to P. americana, a higher proportion than expected. In comparison, a recent study analyzing samples collected in the southwest region of Brazil reported six out of 39 (15.4%) samples assigned to P. americana [57]. The other 14 FFPE sequences were grouped in H4 and could not be assigned to any species of the complex, as observed for H5. Unexpectedly, no FFPE samples were assigned to the species P. brasiliensis sensu stricto. Since H4 comprises only FFPE samples, we named the clade that contains these sequences as FFPE clade.
Our findings indicated genotypes not covered by the four species currently recognized, suggesting a higher genetic diversity in the P. brasiliensis complex than that described so far. Similar findings had already been described for the genus Paracoccidioides in a study that reported isolates obtained from environmental samples clustering apart from the clinical referenced strains of P. lutzii and P. brasiliensis [58]. By analyzing the rRNA universal fungal region ITS1-5.8S-ITS2, the authors found two new genotypes in soil samples and one in aerosol samples that indicated a higher genetic variation than the previously reported for the ITS in Paracoccidioides. Our study expands the knowledge regarding the genetic diversity in Paracoccidioides by finding new genotypes inside the P. brasiliensis complex and considering FFPE human tissue samples. Moreover, the higher genetic diversity found in FFPE and environmental samples, compared with isolates maintained in culture collections, may suggest that some lineages of Paracoccidioides are not successful in culture adaptation. Further studies are needed to evaluate this issue.
We obtained four PbGP43 sequences from the same patient (named Patient 3), an initially healthy male, 22 years-old, ex-rural worker living in Botucatu (São Paulo State, Brazil). He was hospitalized with a diagnosis of pneumonia, whose symptoms lasted for over one month and aggravated in the last days before hospitalization. He showed multiple cutaneous lesions, fever, pneumonia, and was later diagnosed with the acute juvenile form of PCM, established by both the microscopic findings and skin test. Serology was negative for HIV and he died a week after being admitted to the hospital. It is noteworthy that we detected a coinfection in Patient 3, for which three samples were clustered in H4, and one sample was clustered with P. americana. Coinfection by Paracoccidioides has already been reported in armadillos [59], and a recent study reported a case of coinfection in humans, showing P. brasiliensis sensu stricto and P. lutzii in the same patient [35]. Here, for the first time, we report a coinfection involving two different lineages of the P. brasiliensis complex. Pinheiro et al.[35] hypothesize that the use of non-selective media most likely favors the isolation of a faster-growing or a major representative genotype in Paracoccidioides, while FFPE samples have a higher potential to detect coinfections. Likewise, our results reinforce the high potential of FFPE samples in Paracoccidioides coinfection detection.
The PbGP43 gene region analyzed includes the inner core of the P10 T-cell epitope. P10 can induce protective immune responses in vitro and in vivo, promising therapeutic alternatives to combat PCM [60]. The inner core is formed by the conserved amino acid sequence HTLAIR, which was identical among the six haplotypes comprising the P. brasiliensis complex, including H4 and H5 that could not be assigned to any recognized species. Our results corroborate the high conservation level of the P10 inner core among species in the complex and reinforce its potential to be used in the PCM treatment and possible vaccines. The gene region analyzed also includes the N-glycosylation site of this glycoprotein [27], which showed no nucleotide substitutions among the species of the complex, but showed one non-synonymous substitution in P. lutzii. This substitution prevents glycosylation, as already proven experimentally [61].
Interestingly, the five variable parsimony-informative sites identified among the haplotypes consisted of non-synonymous changes in one or more species of the P. brasiliensis complex, in relation to P. lutzii. Genes encoding pathogenesis-related proteins, as PbGP43, are likely to evolve in response to selective pressure from the host’s immune system, which explains why non-synonymous substitutions are common and have been maintained by positive selection in genes that code for virulence factors [28]. Three out of five sites showed radical non-synonymous changes, i.e., amino acid changes that can promote structural and/or functional changes in the protein.
We highlight the change from lysine (basic amino acid) to glutamic acid (acidic amino acid) observed in H1, H2, H3, and H4, associated with changes in the isoelectric point (pI). Changes related to this property are essential because the electrical charge of the protein affects its solubility. That is especially relevant in the gp43 isoforms since they are primarily secreted to the extracellular environment and bound to major histocompatibility complex (MHC) molecules [62]. Indeed, a variety of pIs have been reported in gp43 [25, 27, 50, 63, 64], which have the potential to influence immunogenicity, i.e., the ability of an antigen to induce an adaptive immune response. We also found substitutions associated with alpha-helical tendencies (in H1 and H3), power to be at the N-terminal, and turn tendencies (both in H3), affecting the protein tertiary structure.
Our data shed additional light to the genetic diversity existing in the P. brasiliensis complex and contribute to the knowledge of the geographic distribution of lineages. In addition, we confirmed PbGP43 as a remarkable informative gene for further species identification in the complex, even considering a short gene fragment. It is also important to point out the correlation between molecular and phylogenetic analyzes of Paracoccidioides found in FFPE human biopsies, which is rare in the literature and shows a high potential to detect cases of coinfection, an interesting aspect that is worth exploring in the future.