Efflux pump proteins in antifungal resistance

Abstract

It is now well-known that the enhanced expression of ATP binding cassette (ABC) and major facilitator superfamily (MFS) proteins contribute to the development of tolerance to antifungals in yeasts. For example, the azole resistant clinical isolates of the opportunistic human fungal pathogen Candida albicans show an overexpression of Cdr1p and/or CaMdr1p belonging to ABC and MFS superfamilies, respectively. Hence, azole resistant isolates display reduced accumulation of therapeutic drug due to its rapid extrusion and that facilitates its survival. Considering the importance of major antifungal transporters, the focus of recent research has been to understand the structure and function of these proteins to design inhibitors/modulators to block the pump protein activity so that the drug already in use could again sensitize resistant yeast cells. The review focuses on the structure and function of ABC and MFS transporters of Candida to highlight the recent advancement in the field.

Keywords: ABC transporters; Candida; MFS transporters; azoles; efflux pumps; multidrug resistance.

Figure 1

Venn diagram showing substrates which are common and distinct for Cdr1p and CaMdr1p.

Figure 2

Schematic representation of the predicted topology of Cdr1p. Cdr1p is predicted to have two transmembrane domains (TMD1 and TMD2) and two NBDs, (N- and C-terminal NBDs) organized in reverse (NBD-TMS6)₂ topology. The amino and carboxyl terminals of protein are indicated. Each TMD consists of six TMSs, which are numbered 1–6 in TMD1 and 7–12 in TMD2. Six extracellular loops (ECL1-6) and four intracellular loops (ICL1-4) are indicated.

Figure 3

A list of residues from Cdr1p that upon substitution gave a phenotype. The color gradient of individual TMS in the central Cdr1p cartoon shows the relative transmembrane conservation based on conservation scores obtained from JALVIEW2.4.0.b2 (Rawal et al., 2013). A red color indicates the highest conservation score while yellow indicates the lowest score. Abbreviations: MUT (mutants), P (phenotype), TS (susceptible to all drugs), SS (selectively susceptible), ECL (extracellular loop), NBD (nucleotide-binding domain), TMS (transmembrane segments). Below each table are the pepwheels showing LIPS (LIPid-facing Surface) residues. Helical wheel projections of each TMS sequence were constructed using http://rzlab.ucr.edu/scripts/wheel/wheel.cgi (Rawal et al., 2013). The sequences are displayed in a helical representation as if looking down the axis of the helix. The mutations that affected drug resistance are circled pink. The red curve on each wheel marks the location of the LIPS surface.

Figure 4

(A) Schematic representation of Cdr1p, wherein N-NBD showing degeneracy (red typeface) in conserved Walker A motif, Walker B motif, Q loop and H loop while C-NBD showing degeneracy in signature C. The sequences shown for each motif is from Cdr1p. Sequences of 85 Fungal PDR transporters having topology similar to Cdr1p (Rawal et al., 2013) have been used for the consensus panels below the motif sequences. Sequence conservation of NBD motifs among fungal PDR transporters is individually depicted by Sequence logos, which are a graphical representation of an amino acid multiple sequence alignment. Each logo consists of stacks of symbols, one stack for each position in the sequence. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino or nucleic acid at that position. (B) A highly conserved PDR subfamily specific motif in Cdr1p which is presenting adjacent to Walker A in both the N-NBD (M1 motif) and C-NBD (M2 motif). Sequence conservation of both the motifs among fungal PDR transporters is individually depicted by Sequence logos.

Figure 5

(A) Predicted topology of the CaMdr1p with 12 transmembrane segments. The amino and carboxyl terminals of protein are indicated. Six extracellular loops (ECL1-6) and five intracellular loops (ICL1-5) are indicated. Two homologous halves are connected by a central cytoplasmic loop (CCL) or ICL3. The TMS5 is colored dark and magnified to show the amino acid residues of TMS5. (B) Alignment of the protein sequences of the C. albicans antiporter CaMdr1p TMS5 with the other fungal and bacterial drug antiporters, showing the presence of the unique and conserved antiporter motif or motif C. The amino acid sequence of TMS5 of CaMdr1p is boxed. The sequence of the antiporter motif is written for comparison, where X can be any amino acid. Residues conserved in all the MFS transporters that are part of the motif are highlighted in gray, whereas residues conserved only in fungal MFS that were found critical for the activity are highlighted in black.

Figure 6

A list of residues from CaMdr1p that upon substitution gave a phenotype. TS, susceptible to all drugs; SS, selectively susceptible; ECL, extracellular loop; TMS, transmembrane segments; ICL, intracellular loops; CCL, central cytoplasmic loop.