Previous studies of laboratory mutants of S. cerevisiae
and clinical isolates of diverse Candida
species have localized RES-conferring mutations to hot spot 1 or, less commonly, hot spot 2 of Fks1 or its paralog Fks2 (27
). With few exceptions, these mutations confer relatively uniform cross-RES to CSP, MCF, and ANF. This cross-RES may reflect interaction of the mutated Fks residue with a structural element common to all three echinocandins; e.g., the cyclic hexapeptide or the alkyl portion of the lipid side chain (). Alternatively, it could reflect an indirect effect of the mutation on echinocandin susceptibility. Indeed, to date there has been no evidence that echinocandins directly interact with Fks. By analogy, the majority of mutations conferring azole resistance in C. glabrata
confer fluconazole-itraconazole cross-resistance and localize to transcription factor Pdr1, clearly distinct from the azole target, which is 14α-sterol demethylase (9
). Mutations that confer differential fluconazole versus itraconazole resistance have, however, been characterized in other fungi and, as expected, localize to the target enzyme (33
). This rationale led us to screen S. cerevisiae
mutants selected on CSP- or MCF-containing plates for differential RES. Both of the mutants identified (in an fks1
Δ strain) exhibited an Fks2 mutation, confirming Fks as an echinocandin target. However, both involved residues not previously implicated in RES, specifically, S1380P, 4 residues downstream of the previously characterized hot spot 2, and W714L, about 50 residues downstream of hot spot 1. The latter mutation defines the new hot spot 3, the focus of this study.
Site-directed mutagenesis of Fks1 confirmed a role in RES for residue W695, the equivalent of Fks2 W714. This mutation did not exhibit the same MCF/ANF RES and CSP susceptibility but, rather, a distinct pattern of differential RES. This suggests that the hot spot 3 environment differs somewhat for Fks1 (in the WT background) and Fks2 (in a Δfks1 background) in terms of its interaction with other Fks regions, other proteins, or perhaps, the lipid membrane. Further site-directed mutagenesis implicated several adjacent residues in RES or, intriguingly, echinocandin hypersusceptibility. The latter result suggests that echinocandin-Fks interaction even in susceptible organisms, such as S. cerevisiae, is not fully optimized; i.e., echinocandin modifications that further enhance activity are feasible.
Despite the expanded clinical use of echinocandins, acquired RES remains rare. A more pressing concern surrounding echinocandin use is the intrinsic RES of many emerging fungal pathogens. Our central hypothesis is that specific substitutions in otherwise conserved Fks1 hot spot residues contribute to intrinsic RES. In support of this, evidence has been presented that hot spot 1 substitutions P647A and F639Y contribute to the low- and high-level RES of C. parapsilosis
and F. solani
, respectively (10
). Here, we present evidence for a similar role for the W695F substitutions within hot spot 3 of both S. prolificans
and S. apiospermum
, as this mutation in S. cerevisiae
decreased MCF and ANF susceptibilities by 8- and 128-fold, respectively. On the other hand, this single mutation does not fully account for the RES profile of Scedosporium
species; substitutions elsewhere within Fks1 (e.g., F639Y) (14
) and other factors, such as relatively low reliance on β-1,3-glucan versus α-1,3-glucan, most likely play a role. Our S. cerevisiae
data also predict that the W696C substitution in B. dermatitidis
would confer CSP-specific RES to the mold phase of this dimorphic fungus. The MCF susceptibility of this phase (23
), in contrast to the intrinsic RES of the yeast phase, further supports a role for other factors in echinocandin susceptibility.
Mutations conferring differential RES are potentially powerful reagents for modeling Fks-echinocandin interaction. However, since Fks is an integral membrane protein and since echinocandins have lipid side chains that are likely to be membrane embedded (analogous to the antibacterial agent daptomycin) (1
), these models will need to incorporate Fks topology. For example, the hot spot 3-defining residue W695 is robustly predicted by the widely used topology algorithm TMHMM (18
) to fall within a transmembrane helix (data not shown), where it could interact with the echinocandin lipid chains. If this model is correct, the differential RES conferred by W695 replacement with L, F, or C would reflect their differential binding to the alkyl (CSP) versus aryl (MCF and ANF) components of these lipid chains. Topological analysis of Fks to more rigorously model Fks-echinocandin-membrane interaction is in progress.
Following completion of this work, Martins et al. (21
), in their studies of the Fks1 homolog Bgs4 from fission yeast Schizosaccharomyces pombe
, described a mutation in residue W760, which is equivalent to the S. cerevisiae
hot spot 3-defining residues Fks1 W695 and Fks2 W714. The mutation, W760S, conferred resistance to CSP but not aculeacin A, an echinocandin structurally most similar to ANF; this is analogous to the differential RES effects of the Fks1 W695C mutation (). These data provide further, independent support for the role of hot spot 3 in echinocandin activity. Since this S. pombe
mutant was selected on papulacandin B, which shares the lipid but not the peptide moieties of the echinocandins, it also supports the model above in which hot spot 3 is membrane embedded.