Gene Function Analysis in the Ubiquitous Human Commensal and Pathogen Malassezia Genus

Abstract

The genus Malassezia includes 14 species that are found on the skin of humans and animals and are associated with a number of diseases. Recent genome sequencing projects have defined the gene content of all 14 species; however, to date, genetic manipulation has not been possible for any species within this genus. Here, we develop and then optimize molecular tools for the transformation of Malassezia furfur and Malassezia sympodialis using Agrobacterium tumefaciens delivery of transfer DNA (T-DNA) molecules. These T-DNAs can insert randomly into the genome. In the case of M. furfur, targeted gene replacements were also achieved via homologous recombination, enabling deletion of the ADE2 gene for purine biosynthesis and of the LAC2 gene predicted to be involved in melanin biosynthesis. Hence, the introduction of exogenous DNA and direct gene manipulation are feasible in Malassezia species.

Horizontal gene transfer in the human and skin commensal Malassezia: a bacterially-derived flavohemoglobin is required for NO resistance and host interaction

“…Moreover, most of the HGT candidate genes identified in Malassezia operate as a self-contained metabolic units, which has been proposed to facilitate HGT 13 . Intriguingly, the high number of HGT events suggests also a predisposition of Malassezia to bacterial conjugation, in line with our previous findings that A. tumefaciens -mediated transformation is the only effective technique for molecular manipulation of Malassezia 18 . There are a number of identified HGT that are predicted to be important for Malassezia pathophysiology and that can be characterized using the methodologies reported in the present study.…”

“…Flavohemoglobins are critical for nitric oxide (NO) detoxification and counteract nitrosative stress 9 . To assess whether this HGT event in Malassezia resulted in a gain of function, we deleted the YHB1 ORF (MSYG_3741) of M. sympodialis ATCC42132 through targeted mutagenesis using our recently developed transformation protocol based on transconjugation mediated by Agrobacterium tumefaciens 18,19 (Fig. S4A).…”

“…The M. sympodialis flavohemoglobin-encoding gene YHB1 was identified in the genome assembly though BLASTp analysis with the Saccharomyces cerevisiae Yhb1 protein sequence. The plasmid for targeted mutagenesis of M. sympodialis YHB1 was generated by cloning regions of 1.5 kb flanking the YHB1 gene fused with the NAT marker within the T-DNA of the binary plasmid pGI3 as previously reported 18,59 . For yhb1 Δ functional complementation, three different GFP-fusion plasmids were generated.…”

Expression of aMalasseziacodon optimized mCherry fluorescent protein in a bicistronic vector

“…Transformations were carried out as previously described (Ianiri et al 2016;Celis et al 2017), and 113 randomly selected NAT-resistant colonies were analyzed by PCR to detect the presence of NAT and 114 mCherry ORFs. Predicted amplicons of 576 bp and 708 bp for NAT and mCherry respectively, were 115 observed in 4 out of 23 transformants but absent in wild type (Figure 2A).…”

Advancing functional genetics throughAgrobacterium-mediated insertional mutagenesis and CRISPR/Cas9 in the commensal and pathogenic yeastMalassezia furfur

“…Although these models represent an important advance in understanding the 154 mechanisms of host responses to Malassezia, a lack of technologies for functional genetic 155 studies has hampered the identification and characterization of the fungal components 156 that promote inflammation and induce host responses. We were the first group to develop 157 a transformation system based on transconjugation-mediated by Agrobacterium 158 tumefaciens (AtMT, A. tumefaciens-mediated transformation) that is effective for both 159 insertional and targeted mutagenesis and enabled the first genetic manipulation of M. 160 furfur and M. sympodialis (IANIRI et al 2016;IANIRI et al 2017). Subsequently, M. 161 pachydermatis has also been transformed (CELIS et al 2017).…”