Identification of small RNAs in extracellular vesicles from the commensal yeast Malassezia sympodialis

Abstract

Malassezia is the dominant fungus in the human skin mycobiome and is associated with common skin disorders including atopic eczema (AE)/dermatitis. Recently, it was found that Malassezia sympodialis secretes nanosized exosome-like vesicles, designated MalaEx, that carry allergens and can induce inflammatory cytokine responses. Extracellular vesicles from different cell-types including fungi have been found to deliver functional RNAs to recipient cells. In this study we assessed the presence of small RNAs in MalaEx and addressed if the levels of these RNAs differ when M. sympodialis is cultured at normal human skin pH versus the elevated pH present on the skin of patients with AE. The total number and the protein concentration of the released MalaEx harvested after 48 h culture did not differ significantly between the two pH conditions nor did the size of the vesicles. From small RNA sequence data, we identified a set of reads with well-defined start and stop positions, in a length range of 16 to 22 nucleotides consistently present in the MalaEx. The levels of small RNAs were not significantly differentially expressed between the two different pH conditions indicating that they are not influenced by the elevated pH level observed on the AE skin.Extracellular vesicles (EV) are released not only from different mammalian cell-types but also from microorganisms and parasites and have the capacity to transfer complex biological information [1][2][3][4][5] . Various types of EV ranging in size from 20 nm to 1,000 nm in diameter have been described and are classified mainly on their mechanisms of biogenesis and their physiological functions 1,6 . Those designated exosomes are nanosized vesicles of 50-100 nm which are released extracellularly after fusion of multicellular endosomes with the cell membrane, whereas microvesicles (MV) are larger vesicles (100-1,000 nm) generated through outward budding of the plasma membrane 1,5 . Gram-negative bacteria produce MV by outward budding of the outer membrane and these vesicles are therefore referred to as outer membrane vesicles (OMV) with a diameter in the range of 20-500 nm 6 . Exosomes can be detected in body fluids such as urine, bronchoalveolar lavage fluid (BAL), breast milk and serum 7 . The functions of exosomes include immunoregulatory mechanisms such as modulation of antigen presentation, immune activation, immune suppression, immune surveillance and intercellular communication 6 . EV from microorganisms with thick cell walls, such as Gram-positive bacteria and fungi, have been associated with cytotoxicity, the invasion of host cells, and the transfer of virulence factors 2 . As seen with exosomes 1,8 , fungal EV have been observed to deliver functional messenger (m)RNAs and micro (mi)RNA-like RNAs to recipient cells 9,10 . miRNAs are small non-coding RNAs with a length between 20 and 22 nucleotides (nt) 11 . They are spliced from precursor sequences that form the stable hairpin necessary for transportation from the nucleus to the cytoplasm. After the miRNA has been c...

Movement of small RNAs in and between plants and fungi

“…Extracellular vesicles play critical roles in fungal growth and the ability to derive nutrients from their environment (including invading potential hosts). Recently, a number of different types of noncoding small RNAs have been characterized inside EVs from fungi as described above (Garcia‐Silva et al , ; Peres da Silva et al , ; Rayner et al , ).…”

“…However, the discovery of diverse small RNA species in Arabidopsis extracellular vesicles is consistent with this pathway where small RNAs could be loaded into vesicles and secreted to the apoplast(Baldrich et al, 2019).Extracellular vesicles play critical roles in fungal growth and the ability to derive nutrients from their environment (including invading potential hosts). Recently, a number of different types of noncoding small RNAs have been characterized inside EVs from fungi as described above(Garcia-Silva et al, 2014;Peres da Silva et al, 2015;Rayner et al, 2017).…”

Fungal Extracellular Vesicles with a Focus on Proteomic Analysis

“…The commensal skin microflora yeast M. sympodialis , which is often associated with skin disorders including atopic eczema, produces EVs that carry allergens that can induce cytokine responses in patients with atopic eczema . These EVs also carry small RNA (sRNA) but the function of the sRNA remains uncharacterized . Proteomic analysis of M. sympodialis EVs identified the most proteins of any fungal EV studies with a total of 2439 proteins across four replicates.…”

A two‐way road: novel roles for fungal extracellular vesicles

“…EV sRNAs were identified in M. sympodialis , S. cerevisiae , C. albicans , P. brasiliensis and C. neoformans (Peres da Silva et al, ; Rayner et al, ), suggesting that mechanisms similar to those observed in the trans‐kingdom communication between fungi and plants could be functional for the transfer of biological information within fungal populations. In this context, a recent study has demonstrated that RNA‐containing fungal EVs mediate virulence in the human pathogen C. gattii (Bielska et al, ).…”