*1.3. ABC Multidrug Transporters in Fungal Kingdoms*

Invasive fungal diseases account for ~1.5 million deaths per year worldwide [220,221]. The increasing numbers of immunosuppressed people, including the elderly, transplant recipients, cancer and HIV/AIDS patients will most likely increase future cases of infections by opportunistic pathogens like *Candida* species (spp.) [222]. Candida spp. are commensal colonizers and part of the microflora present on mucosal and epithelial barriers such as the gastrointestinal and urogenital tracts. *Candida* spp. are a major part of the physiological mycobiome species [223], along with several thousand bacterial species constituting tissuespecific microbiomes [224–229]. Several *Candida* spp. can cause life-threatening invasive systemic disease in severely immunocompromised individuals [230]. The small number of chemical entities in antifungal drugs have been very problematic in the past, especially after prolonged use or after extensive prophylaxis, as was the case for pronounced azole resistance in HIV patients. Hence, the propensity to develop MDR is lower when larger drug arsenals against different targets are available [231]. For example, *C. auris* is a newly emerging pan-resistant fungal pathogen first reported from an ear infection in Japan in 2009 [232]. Within a decade, *C. auris* appeared in more than 40 countries around the globe, causing hospital outbreaks of invasive candidemia [233–240]. Importantly, *C. auris* is also causing severe superinfections with viruses, as seen in a recent co-infection of COVID-19 patients in a Mexican hospital ICU, leading to a dramatic overall mortality of 83%. In fact, intrinsic MDR in other *Candida* spp. such as *C. glabrata* and *C. kruzei* have also been increasing over the last decade [230,241,242]. Importantly, among other mechanisms, such as drug target gene mutations [243,244], efflux-based MDR/PDR has been recognized as a major cause of fungal anti-infective drug resistance [219,235,244–247].

Fungal ABC proteins are also quite diverse and are implicated in many biological functions, contributing to pivotal cellular processes, including cellular detoxification and stress adaptation [214,248,249]. Owing to space constraints, we will limit our discussion to paradigm fungal PDR family members implicated in drug resistance phenomena, but refer the reader to numerous recent reviews discussing fungal ABC proteins at large [249,250]. Fungal ABC proteins of the PDR subfamily are the closest eukaryotic orthologues of human ABCG family exporters [245,251]. For instance, *C. albicans* and *C. glabrata* harbor 27 and 18 ABC proteins in total, respectively [250]. Interestingly, *Cryptococcus neoformans* and *Aspergillus fumigatus* harbor even larger numbers, with 54 and 49 ABC transporters, respectively [252,253]. While we are not discussing transporters which are not linked to MDR phenomena, we still provide a comprehensive list of all known ABC transporters in non-pathogenic yeasts (Table 1) and their phylogenetic relationships (Figure 1A). MRP-like yeast ABC transporters including Yor1, Ycf1, Ybt1, Vmr1 and Bpt1 mediate vacuolar detoxification and heavy metal resistance [214]. Of note, Ycf1, which also has a rudimentary R-domain motif as present in human CFTR [50], was the first yeast homologue of mammalian MRP [254], while pathogenic fungi such as *C. albicans* harbor only Yor1 and Mlt1 (Table 2).

**Figure 1.** Phylogeny and structural organization of ABC transporters in mammals and yeast. (**A**) The phylogenetic tree shows the evolutionary relationships of ABC transporter subfamilies in yeast and mammals. Some 29 ABC transporters were subjected to amino acid sequence alignments. Branch length was analyzed using MEGA-X, and represents the evolutionary distance in the units of the number of amino acid substitutions per site. Names are given in the UniProt code, protein name and organism, respectively. The analysis reveals two major exporters subfamilies referred to as type I and II. Type I was sub-classified into ABCB/MDR (blue) and ABCC/MRP (green) subgroups. The type II family represents ABCG/PDR subgroups with the fungal (red) and mammalian transporters (orange). Ca: *Candida albicans*, Cg: *Candida glabrata*, Caur: *Candida auris*, Cd: *Candida dubliniensis*, Ct: *Candida tropicalis*, Cn: *Cryptococcus neoformans*, Sc: *Saccharomyces cerevisiae*, Sp: *Schizosaccharomyces pombe*. (**B**) Predicted membrane topologies of three MDR ABC exporter families. The transporters hold several diagnostic hallmark domains, including two NBDs (NBD1: light green; NBD2: green), two TMD regions usually with 6 putative membrane-spanning helices each (TMD1: light yellow; TMD2: bright orange), elbow helix (pink), re-entry helix (blue) and TMD0 (purple), respectively.

The fungal PDR (ABCG) or pleiotropic drug resistance family is the largest subfamily of ABC transporters of the type II exporter class [251]. PDR transporters are the closest structural orthologues of all mammalian ABCG subfamily transporters (Figure 1A),

sharing the same topological orientation and domain arrangements with mammalian ABCG5/ABCG8 [255] and ABCG2 [256–259] (Supplementary Figure S1). PDR transporters in fungal pathogens implicated in clinical antifungal resistance in *C. albicans* include Cdr1, Cdr2, Cdr3, Cdr6 and Cdr11, in *C. glabrata* (Cdr1, Pdh1, Snq2 and Aus1) [260], in *C. auris* (Cdr1), in *C. tropicalis* (Cdr1), in *C. dubliniensis* (Cdr1 and Cdr2) and in *Cryptococcus neoformans* (Afr1) (Table 2). All are highly conserved in non-pathogenic yeasts (Table 1). Their overexpression in pathogens causes hallmark MDR phenotypes seen for mammalian or bacterial ABC transporters [111,141,217,218,235,261–264].

Diversity of fungal PDR transporters and evolutionary relationships to mammalian ABC transporters. Most ABC proteins from non-pathogenic baker's yeasts (Table 1) are also found in various numbers and functions in pathogenic fungi (Table 2). A phylogenetic tree analysis suggests two major groups, referred to as exporter type I and type II [4,248,265,266], contain three MDR, MRP and PDR subfamilies (Figure 1A). While type I exporters (MDR/ABCB and MRP/ABCC) hold a TMD1–NBD1–TMD2–NBD2 configuration, all PDR/ABCG type II exporters adopt a "reverse" architecture, such as NBD1– TMD1–NBD2–TMD2 (Figure 1B). By contrast with the mammalian ABCG subfamily, all fungal PDR proteins are full-size transporters (Figure 1B).

**Table 2.** ABC efflux transporters mediating MDR in pathogenic fungi.


\* All transporters located in the plasma membrane, except for Atm1 and Mlt1, residing in the mitochondrial and vacuolar membrane, respectively. GS: glutathione, PC: phosphatidylcholine, PE: phosphatidylethanolamine, PS: phosphatidylserine.
