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

Author(s)

de Carvalho, Jamile Ambrosio ; Hagen, Ferry ; Fisher, Matthew C. ; de Camargo, Zoilo Pires ; Rodrigues, Anderson Messias

• Document type: Journal article
• Source: PLoS Neglected Tropical Diseases; v. 14, n. 7, p. 26-pg., 2020-07-01.

Abstract

Author summary Sporotrichosis is a subacute or chronic infection characterized by nodular lesions of the (sub)cutaneous tissues and adjacent lymphatics.Sporothrix brasiliensis,S.schenckii, andS.globosaare the main agents of sporotrichosis in humans and other mammals.Sporothrixpropagules gain entrance by traumatic implantation in the skin following two main routes of infection, which include animal transmission (e.g., cat-cat and cat-human) and plant origin. In recent years there has been a significant increase in the number of atypical and more severe cases of sporotrichosis, along with the expansion of the area of occurrence ofSporothrixspecies, such as the highly virulentS.brasiliensis. We investigated the usefulness of the AFLP technology, a DNA fingerprinting technique, which is based on the selective amplification of genomic restriction fragments by PCR to explore genetic diversity and population structure inSporothrixduring ongoing outbreaks. We report six highly effective sets of AFLP markers to discriminateSporothrixat species and strain level, thus allowing tracking the spread of sporotrichosis. Adding molecular data in an outbreak response context can reveal better ways to improve public policies to contain the advance of sporotrichosis, by early detection, response, intervention, and follow-up. Sporotrichosis is a chronic subcutaneous mycosis caused bySporothrixspecies, of which the main aetiological agents areS.brasiliensis,S.schenckii, andS.globosa. Infection occurs after a traumatic inoculation ofSporothrixpropagules in mammals' skin and can follow either a classic route through traumatic inoculation by plant debris (e.g.,S.schenckiiandS.globosa) or an alternative route through zoonotic transmission from animals (e.g.,S.brasiliensis). Epizootics followed by a zoonotic route occur in Brazil, with Rio de Janeiro as the epicenter of a recent cat-transmitted epidemic. DNA-based markers are needed to explore the epidemiology of theseSporothrixexpansions using molecular methods. This paper reports the use of amplified-fragment-length polymorphisms (AFLP) to assess the degree of intraspecific variability amongSporothrixspecies. We used whole-genome sequences fromSporothrixspecies to generate 2,304 virtual AFLP fingerprints.In silicoscreening highlighted 6 primer pair combinations to be testedin vitro. The protocol was used to genotype 27 medically relevantSporothrix. Based on the overall scored AFLP markers (97-137 fragments), the values of polymorphism information content (PIC= 0.2552-0.3113), marker index (MI= 0.002-0.0039), effective multiplex ratio (E= 17.8519-35.2222), resolving power (Rp= 33.6296-63.1852), discriminating power (D= 0.9291-0.9662), expected heterozygosity (H= 0.3003-0.3857), and mean heterozygosity (H-avp= 0.0001) demonstrated the utility of these primer combinations for discriminatingSporothrix. AFLP markers revealed cryptic diversity in species previously thought to be the most prevalent clonal type, such asS.brasiliensis, responsible for cat-transmitted sporotrichosis, andS.globosaresponsible for large sapronosis outbreaks in Asia. Three combinations (#3 EcoRI-FAM-GA/MseI-TT, #5 EcoRI-FAM-GA/MseI-AG, and #6 EcoRI-FAM-TA/MseI-AA) provide the best diversity indices and lowest error rates. These methods make it easier to track routes of disease transmission during epizooties and zoonosis, and our DNA fingerprint assay can be further transferred between laboratories to give insights into the ecology and evolution of pathogenicSporothrixspecies and to inform management and mitigation strategies to tackle the advance of sporotrichosis. (AU)

• 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
• 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