HGT in the human and skin commensal Malassezia : A bacterially derived flavohemoglobin is required for NO resistance and host interaction

Abstract

<jats:p>The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions.<jats:italic>Malassezia</jats:italic>are the most abundant fungal skin inhabitant of warm-blooded animals and have been implicated in skin diseases and systemic disorders, including Crohn’s disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within the<jats:italic>Malassezia</jats:italic>genus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation in<jats:italic>Malassezia sympodialis</jats:italic>, we demonstrated that bacterially derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNA-sequencing analysis revealed that endogenous accumulation of nitric oxide resulted in up-regulation of genes involved in stress response and down-regulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure of<jats:italic>Malassezia</jats:italic>flavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent of<jats:italic>Malassezia</jats:italic>flavohemoglobin. Lastly, we identified an additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin.</jats:p>

Approaches for Genetic Discoveries in the Skin Commensal and Pathogenic Malassezia Yeasts

“…Another characteristic of Malassezia genomes is the presence of bacterial genes acquired through horizontal gene transfer (HGT) events. While the number of these events is usually limited, in Malassezia more than 30 HGT have been identified (Wu et al, 2015 ; Ianiri et al, 2020 ). HGT candidates found in the majority of the Malassezia species include genes involved in broad stress resistance, such as flavohemoglobin, catalase, and oxidoreductases, found in some cases in multiple copies.…”

“…This gene is absent in all Malassezia species phylogenetically related to M. sympodialis , and is present in five copies in M. globosa . Other acquired genes encode a variety of proteins with different functions, such as hydrolysis, protein transport and folding, and detoxification of xenobiotics (Ianiri et al, 2020 ).…”

“…Using molecular techniques described in the section Agrobacterium tumefaciens -Mediated Transformation Enables Insertional Mutagenesis and Targeted Gene Deletion in Malassezia , we demonstrated that the HGT of the bacterial flavohemoglobin in Malassezia resulted in a gain of function critical for resistance to nitrosative stress and nitric oxide (NO) detoxification (Ianiri et al, 2020 ). Analysis of the available Malassezia genomes revealed additional HGT of another flavohemoglobin-encoding gene that originated from different donor bacteria.…”

“…Another vector that encodes a fluorescent marker includes a Malassezia codon-optimized mCherry gene fused with the NAT marker, as in plasmid pAIM2, through a P2A sequence (Goh et al, 2020 ); P2A is a 2A self-cleaving peptide derived from the porcine teschovirus-1 that was used to guarantee high expression of both the NAT and mCherry genes. These gene markers were also modified and reassembled to perform protein localization and chromatin immunoprecipitation (ChIP) through the generation of both N-terminal and C-terminal GFP fusion proteins, and 3xFLAG-tagged proteins (Ianiri et al, 2020 ; Sankaranarayanan et al, 2020 ).…”

Advances in understanding the evolution of fungal genome architecture

“…However, advances in whole-genome sequencing have, by increasing efficiency and reducing costs, expanded the number of fungal genome sequences available and allowed the identification of numerous HGT events across the fungal kingdom; many instances of HGT events in fungi have been extensively characterized and reviewed 16 – 19 . HGT events appear to be more frequent in organisms that have undergone significant niche adaptation such as commensal organisms like the saprophytic skin commensal Malassezia species, which have acquired a bacterial flavohemoglobin as well as many other bacterially derived genes 93 . Similarly, obligate intracellular pathogens belonging to the Microsporidia and Cryptomycota phyla have experienced significant HGT, in which up to 2% of genes are estimated to be derived from HGT events 85 .…”