Sporotrichosis is a fungal (sub)cutaneous infection of humans and felines caused by a group of dimorphic pathogens of the Sporothrix genus embedded in the so-called pathogenic clade (Sporothrix schenckii, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei). However, the genus also contains occasional opportunists, such as Sporothrix pallida and Sporothrix mexicana (environmental clade) which have the potential to cause sporotrichosis in susceptible hosts.
During infection, pathogenic fungi encounter oxidative stress that can be overcome by expressing an antioxidant defense system, including the production of catalase enzymes (CATs).
Among the different Sporothrix spp., there is a high degree of variability in terms of virulence, pathogenicity and ability to survive in oxidative stress conditions. Therefore, in the light of these differences, we decided to test the susceptibility of all members of the pathogenic and environmental clades to hydrogen peroxide (H2O2) under specific experimental conditions. Moreover, we also performed an in-silico analysis of the genes encoding catalases, by using the recently sequenced Sporothrix genomes, in order to understand if genetic differences exist among pathogenic and environmental Sporothrix species.

Methods
A total of 55 Sporothrix isolates were examined in this study. The susceptibility to H2O2 was assayed using the spot-assay on YEPD-agar containing 6mM H2O2 and by microdilution broth method. Bioinformatics analysis were performed to identify and classify the catalase-encoding genes in published genomes. CAT-genes were also experimentally validated in a sub-panel of different Sporothrix strains using standard Sanger sequencing.

Results
All S. pallida and S. mexicana isolates, including the unique pathogenic S. luriei isolate, grew under oxidative stress conditions used whereas all the examined S. schenckii and S. chilensis strains resulted unable to grow in the same experimental conditions. S. brasiliensis and S. globosa strains were variably resistant to H2O2 suggesting the occurrence of a high intraspecific variability within the populations of these two pathogens. Bioinformatics analysis of the whole-proteome of Sporothrix species revealed a total of 15 proteins containing the “Catalase Core Domain”, classified as “monofunctional catalases”. Species belonging to the pathogenic clade (S. schenckii, S. brasiliensis, S. globosa), contained 4 different catalase-encoding genes while only 3 genes were found within the environmental species S. pallida.



Conclusions
This study showed that Sporothrix species differ in their response to H2O2 and that the environmental species, S. pallida and S. mexicana, appear to be naturally resistant to this compound. However, differences were also found among members of the pathogenic clade and S. schenckii seems to be the most susceptible species to H2O2. 
Based on the obtained data, it seems that the degree of susceptibility to H2O2 among examined Sporothrix species can be defined as S. pallida or S. mexicana > S. brasiliensis or S. globosa > S. schenckii. We also identified a different content of CAT-isoforms in the sequenced genomes but it was not possible to know their individual contribution to the different phenotypes observed. Therefore future gene expression studies will be needed in order to expand our knowledge about the molecular mechanisms associated with oxidative stress mediated by H2O2 in Sporothrix species.

6 / Susceptibility to hydrogen peroxide and molecular characterization of catalase-encoding genes in different Sporothrix species