Genome-wide mapping using new AFLP markers to explore intraspecific variation among pathogenic Sporothrix species

Carvalho, Jamile Ambrósio de; Hagen, Ferry; Camargo, Zoilo Pires de; Rodrigues, Anderson Messias

doi:10.1371/journal.pntd.0008330

“…The mating‐type characterisation in both species revealed a skewed ratio between the MAT1‐1 : MAT1‐2 idiomorphs, with a predominance of MAT1‐2 isolates. As asexual amplification can skew mating frequencies, our results suggest that S brasiliensis genotypes during outbreaks in the feline population are likely to be predominantly clonal, which does not necessarily imply the absence of sex but indicates the emergence of a successful genotype 29 …”

Section: Discussionmentioning

confidence: 82%

“…isolates. As asexual amplification can skew mating frequencies, our results suggest that S brasiliensis genotypes during outbreaks in the feline population are likely to be predominantly clonal, which does not necessarily imply the absence of sex but indicates the emergence of a successful genotype 29. The distribution pattern of the clinical Sporothrix isolates evaluated suggests how the mating-type between Sporothrix species is widespread in Espírito Santo, proposed two distinct scenarios of the disease in the state.…”

mentioning

confidence: 74%

Molecular‐based assessment of diversity and population structure of Sporothrix spp. clinical isolates from Espírito Santo‐Brazil

Rocha

¹

,

Terra

²

,

Oliveira

³

et al. 2020

Mycoses

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Background Sporotrichosis is a subcutaneous mycosis caused by Sporothrix species that affects humans and animals. Little information on the genetic diversity and population structure of the pathogen is available for Brazil, which is needed to design effective strategies to tackle the advance of sporotrichosis in endemic areas. Objectives We assessed the genetic diversity and mating‐type distribution of Sporothrix isolates recovered from human and feline cases of sporotrichosis in Espírito Santo‐Brazil to better understand the population structure, epidemiology and diversification of this pathogen, as well as to explore the possible routes of transmission involved in the ongoing outbreaks. Methods In all, 75 Sporothrix isolates were identified with phenotypic characteristics. Then, fungal DNA extraction was performed, and the species‐specific PCR technique was applied, using markers directed to the calmodulin gene. The mating‐type idiomorph of species was identified by PCR using primers targeting the MAT1‐1 and MAT1‐2 loci. Results Among the 75 Sporothrix isolates, 76% were confirmed as S brasiliensis and 24% as S schenckii sensu stricto. S brasiliensis was more prevalent in the metropolitan area and S schenckii s. str. in the mountainous region of the state. In both species, the presence of the two sexual idiomorphs was detected, suggesting that they are heterothallic species. Conclusions Our data suggest that sporotrichosis takes on an epidemic‐urban character involving S brasiliensis. This species in Espírito Santo is likely to originate from Rio de Janeiro, as most isolates harbour the same MAT 1‐2 locus. We confirm that S brasiliensis has significantly broadened its area of occurrence, an essential feature of emerging pathogens.

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“…Our analysis showed that the fragments observed following AFLP with EcoRI-AC / MseI-CT or EcoRI-AT / MseI-CT primers represent the optimal combinations to explore genetic diversity in Paracoccidioides. A similar in silico framework has been successfully applied for medically relevant Sporothrix species ( de Carvalho et al. 2020 , 2021a ), supporting that combining bioinformatics tools and whole-genome sequences can make the AFLP method more predictable instead of, rather, using random combinations of suboptimal endonuclease-combinations ( Rombauts et al.…”

Section: Discussionmentioning

confidence: 91%

“…All oligonucleotides were provided by Applied Biosystems in the AFLP Microbial Fingerprinting kit. AFLP fragments were determined by capillary electrophoresis with an ABI3730 Genetic Analyzer alongside a GeneScan 500 ROX internal size standard (35–500 bp; Applied Biosystems) at the Human Genome and Stem Cell Research Centre Core Facility (University of São Paulo, São Paulo, Brazil) under previously described conditions ( de Carvalho et al. 2020 ).…”

Section: Methodsmentioning

confidence: 99%

Trends in the molecular epidemiology and population genetics of emergingSporothrixspecies

Roberto¹,

Carvalho²,

Beale³

et al. 2021

Studies in Mycology

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Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection acquired after inhalation of Paracoccidioides propagules from the environment. The main agents include members of the P. brasiliensis complex (phylogenetically-defined species S1, PS2, PS3, and PS4) and P. lutzii . DNA-sequencing of protein-coding loci (e.g., GP43 , ARF , and TUB1 ) is the reference method for recognizing Paracoccidioides species due to a lack of robust phenotypic markers. Thus, developing new molecular markers that are informative and cost-effective is key to providing quality information to explore genetic diversity within Paracoccidioides . We report using new amplified fragment length polymorphism (AFLP) markers and mating-type analysis for genotyping Paracoccidioides species. The bioinformatic analysis generated 144 in silico AFLP profiles, highlighting two discriminatory primer pairs combinations (#1 EcoRI-AC/MseI-CT and #2 EcoRI-AT/MseI-CT). The combinations #1 and #2 were used in vitro to genotype 165 Paracoccidioides isolates recovered from across a vast area of South America. Considering the overall scored AFLP markers in vitro (67–87 fragments), the values of polymorphism information content ( PIC = 0.3345–0.3456), marker index ( MI = 0.0018), effective multiplex ratio ( E = 44.6788–60.3818), resolving power ( Rp = 22.3152–34.3152), discriminating power ( D = 0.5183–0.5553), expected heterozygosity ( H = 0.4247–0.4443), and mean heterozygosity ( H avp = 0.00002–0.00004), demonstrated the utility of AFLP markers to speciate Paracoccidioides and to dissect both deep and fine-scale genetic structures. Analysis of molecular variance (AMOVA) revealed that the total genetic variance (65-66 %) was due to variability among P. brasiliensis complex and P. lutzii (PhiPT = 0.651–0.658, P < 0.0001), supporting a highly structured population. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for P. brasiliensis s. str. (χ 2 = 1.025; P = 0.3113), P. venezuelensis (χ 2 = 0.692; P = 0.4054), and P. lutzii (χ 2 = 0.027; P = 0.8694), supporting random mating within each species. In contrast, skewed distributions were found ...

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“…To perform the AFLP, we followed the protocol of Vos et al. 1995 with modifications described by de Carvalho et al. 2020 .…”

Section: Methodsmentioning

confidence: 99%

Exploring genetic diversity, population structure, and phylogeography in Paracoccidioides species using AFLP markers

Carvalho¹,

Beale²,

Hagen³

et al. 2021

Studies in Mycology

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Sporothrix ( Ophiostomatales ) comprises species that are pathogenic to humans and other mammals as well as environmental fungi. Developments in molecular phylogeny have changed our perceptions about the epidemiology, host-association, and virulence of Sporothrix . The classical agent of sporotrichosis, Sporothrix schenckii , now comprises several species nested in a clinical clade with S. brasiliensis , S. globosa , and S. luriei . To gain a more precise view of outbreaks dynamics, structure, and origin of genetic variation within and among populations of Sporothrix , we applied three sets of discriminatory AFLP markers (#3 EcoRI-GA/MseI-TT, #5 EcoRI-GA/MseI-AG, and #6 EcoRI-TA/MseI-AA) and mating-type analysis to a large collection of human, animal and environmental isolates spanning the major endemic areas. A total of 451 polymorphic loci were amplified in vitro from 188 samples, and revealed high polymorphism information content ( PIC = 0.1765–0.2253), marker index ( MI = 0.0001–0.0002), effective multiplex ratio ( E = 15.1720–23.5591), resolving power ( Rp = 26.1075–40.2795), discriminating power ( D = 0.9766–0.9879), expected heterozygosity ( H = 0.1957–0.2588), and mean heterozygosity ( H avp = 0.000007–0.000009), demonstrating the effectiveness of AFLP markers to speciate Sporothrix . Analysis using the program structure indicated three genetic clusters matching S. brasiliensis (population 1), S. schenckii (population 2), and S. globosa (population 3), with the presence of patterns of admixture amongst all populations. AMOVA revealed highly structured clusters (PhiPT = 0.458–0.484, P < 0.0001), with roughly equivalent genetic variability within (46–48 %) and between (52–54 %) populations. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for S. schenckii (χ 2 = 2.522; P = 0.1122), supporting random mating. In contrast, skewed distributions were found for S. globosa (χ 2 = 9.529; P = 0.0020) with a predominance of MAT1-1 isolates, and regional differences were highlighted for S. brasiliensis with the overwhelming occurrence of MAT1-2 in Rio de Janeiro (χ 2 = 14.222; P = 0.0002) and Pernambuco (χ 2 ...

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Genome-wide mapping using new AFLP markers to explore intraspecific variation among pathogenic Sporothrix species

Cited by 25 publications

References 91 publications

Molecular‐based assessment of diversity and population structure of Sporothrix spp. clinical isolates from Espírito Santo‐Brazil

Molecular‐based assessment of diversity and population structure of Sporothrix spp. clinical isolates from Espírito Santo‐Brazil

Trends in the molecular epidemiology and population genetics of emergingSporothrixspecies

Exploring genetic diversity, population structure, and phylogeography in Paracoccidioides species using AFLP markers

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