xploring genetic diversity, population structure, and phylogeography in Paracoccidioides species using AFLP marker

Author(s)

Roberto, T. N. ^[1] ; de Carvalho, J. A. ^{[1, 2]} ; Beale, M. A. ^[3] ; Hagen, F. ^{[4, 5, 6]} ; Fisher, M. C. ^[7] ; Hahn, R. C. ^{[8, 9]} ; de Camargo, Z. P. ^{[1, 2]} ; Rodrigues, A. M. ^{[1, 2]}

Affiliation

^[1] Fed Univ Sao Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, Lab Emerging Fungal Pathogens, Discipline Cellular Biol, BR-04023062 Sao Paulo - Brazil

^[2] Fed Univ Sao Paulo UNIFESP, Dept Med, Discipline Infect Dis, BR-04023062 Sao Paulo - Brazil

^[3] Wellcome Sanger Inst, Parasites & Microbes Programme, Wellcome Genome Campus, Cambridge CB10 1SA - England

^[4] Jining 1 Peoples Hosp, Lab Med Mycol, Jining, Shandong - Peoples R China

^[5] Westerdijk Fungal Biodivers Inst, Dept Med Mycol, Uppsalalaan 8, NL-3584 CT Utrecht - Netherlands

^[6] Univ Med Ctr Utrecht, Dept Med Microbiol, Heidelberglaan 100, NL-3584 CX Utrecht - Netherlands

^[7] Imperial Coll London, MRC Ctr Global Infect Dis Anal, Sch Publ Hlth, London W2 1PG - England

^[8] Univ Fed Mato Grosso, Fac Med, Lab Mycol Res, BR-78060900 Cuiaba - Brazil

^[9] Univ Fed Mato Grosso, Julio Muller Univ Hosp, BR-78048902 Cuiaba - Brazil

• Document type: Journal article
• Source: STUDIES IN MYCOLOGY; n. 100 SEP 2021.
• Web of Science Citations: 2

Abstract

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 (Havp = 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. (chi(2) = 1.025; P = 0.3113), P. venezuelensis (chi(2) = 0.692; P = 0.4054), and P. lutzii (chi(2) = 0.027; P = 0.8694), supporting random mating within each species. In contrast, skewed distributions were found for P. americana (chi(2) = 8.909; P = 0.0028) and P. restrepiensis (chi(2) = 4.571; P = 0.0325) with a preponderance of MAT1-1. Geographical distributions confirmed that P. americana, P. restrepiensis, and P. lutzii are more widespread than previously thought. P. brasiliensis s. str. is by far the most widely occurring lineage in Latin America countries, occurring in all regions of Brazil. Our new DNA fingerprint assay proved to be rapid, reproducible, and highly discriminatory, to give insights into the taxonomy, ecology, and epidemiology of Paracoccidioides species, guiding disease control strategies to mitigate PCM. (AU)

• FAPESP's process: 13/05405-9 - Analysis of genetic diversity in agents of paracoccidioidomycosis using AFLP (Amplified fragment length polymorphism)
• Grantee: Thiago Nunes Roberto
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 17/27265-5 - Molecular epidemiology and genomic perspectives on the evolution and spread of emerging fungal pathogens
• Grantee: Anderson Messias Rodrigues
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 18/21460-3 - Study of different antigenic preparations of Paracoccidioides lutzii for the standardization of the ELISA test as an aid in the diagnosis of paracoccidioidomycosis due to P. lutzii
• Grantee: Zoilo Pires de Camargo
• Support Opportunities: Regular Research Grants