Investigation of the MFS drug transporter family in Candida parapsilosis using CRISPR-Cas9

The function of specific transporters is a key feature underlying drug resistance in Candida species. Drug transporters fall into two main classes – ATP-binding cassette (ABC) transporters, and the major facilitator superfamily (MFS) transporters. Some members of the drug/H (+) antiporter (DHA1) of the MFS superfamily have been shown to function as multidrug transporters. We targeted 16 genes belonging to five families that compose one branch of the DHA1 transporter group. These include MDR1/FLR1, associated with multidrug resistance in C. albicans (3 members); TPO4, associated with polyamine transport (1 member); NAG3/4, associated with transport of N-acetyl glucosamine (2 members); TPO2/3, associated with polyamine transport (1 member); and TPO1/FLU1, possibly associated with fluconazole resistance (9 members). We used CRISPR-Ca9 based gene editing to explore the function of of the five families in C. parapsilosis. All 16 members were individually disrupted by introducing stop codons in the first third of the open reading frames (editing), or by deleting the whole gene. In addition, members of each family were disrupted together, including all 9 members of the TPO1/FLU1 family. CPAR2_603010, CPAR2_207540, and CPAR2_301760 all belonged to the MDR1 family. Editing CPAR2_603010 conferred sensitivity to fluconazole and voriconazole, though disrupting the other two genes had no effect. The azole sensitivity of the CPAR2_603010 edited strain was reverted by introducing the wild type sequence. Disrupting CPAR2_603010 or CPAR2_301760 individually did not affect sensitivity to 4-nitroquinoline 1-oxide. However, the double disruptant was sensitive. Disrupting CPAR2_300760, a member of the TPO1/FLU1 family, resulted in sensitivity to mycophenolic acid. Whole genome sequencing analysis of a strain in which all nine TPO1 genes were disrupted revealed that few off-target effects introduced by the CRISPR-Cas9 system, as few unexpected changes were found after eight rounds of transformation.

Interactions of cytosolic termini of the Jen1 monocarboxylate transporter are critical for trafficking, transport activity and endocytosis

Plasma membrane (PM) transporters of the major facilitator superfamily (MFS) are essential for cell metabolism and growth, as well as for survival in response to stress or cytotoxic drugs, in both prokaryotes and eukaryotes. In the yeast Saccharomyces cerevisiae, Jen1 is a monocarboxylate/H+ symporter that has been used to dissect the molecular details underlying control of cellular expression, transport mechanism and turnover of MFS transporters. Here, we present evidence supporting previously non-described roles of the cytosolic N- and C- termini in Jen1 biogenesis, PM stability and activity, through functional analyses of rationally designed truncations and chimeric constructs with UapA, a S. cerevisiae endocytosis-insensitive purine transporter from Aspergillus nidulans. Our results reveal a cryptic role of the N-terminal region and thus show that both cytosolic N- and C-termini are critical for Jen1 trafficking to the PM, transport activity and endocytosis. In particular, we provide evidence that the N- and the C-cytosolic termini of Jen1 undergo transport-dependent dynamic intra-molecular interactions, which critically affect the mechanism of transport and turnover of Jen1. Our results support an emerging concept where the cytosolic tails of PM transporters control transporter expression and function, through flexible intra-molecular interactions with each other and the transmembrane core of the protein. This idea may be extended to other MFS members providing a deeper understanding of conserved, but also evolving, mechanisms underlying MFS transporter structure-function relationships.

X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.

Hunting monolignol transporters: membrane proteomics and biochemical transport assays with membrane vesicles of Norway spruce

Both the mechanisms of monolignol transport and the transported form of monolignols in developing xylem of trees are unknown. We tested the hypothesis of an active, plasma membrane-localized transport of monolignol monomers, dimers, and/or glucosidic forms with membrane vesicles prepared from developing xylem and lignin-forming tissue-cultured cells of Norway spruce (Picea abies L. Karst.), as well as from control materials, comprising non-lignifying Norway spruce phloem and tobacco (Nicotiana tabacum L.) BY-2 cells. Xylem and BY-2 vesicles transported both coniferin and p-coumaryl alcohol glucoside, but inhibitor assays suggested that this transport was through the tonoplast. Membrane vesicles prepared from lignin-forming spruce cells showed coniferin transport, but the Km value for coniferin was much higher than those of xylem and BY-2 cells. Liquid chromatography-mass spectrometry analysis of membrane proteins isolated from spruce developing xylem, phloem, and lignin-forming cultured cells revealed multiple transporters. These were compared with a transporter gene set obtained by a correlation analysis with a selected set of spruce monolignol biosynthesis genes. Biochemical membrane vesicle assays showed no support for ABC-transporter-mediated monolignol transport but point to a role for secondary active transporters (such as MFS or MATE transporters). In contrast, proteomic and co-expression analyses suggested a role for ABC transporters and MFS transporters.

A Genomic and Transcriptomic Overview of MATE, ABC, and MFS Transporters in Citrus sinensis Interaction with Xanthomonas citri subsp. citri

The multi-antimicrobial extrusion (MATE), ATP-binding cassette (ABC), and major facilitator superfamily (MFS) are the main plant transporters families, playing an essential role in the membrane-trafficking network and plant-defense mechanism. The citrus canker type A (CC), is a devastating disease caused by Xanthomonas citri subsp. citri (Xac), affecting all citrus species. In this work, we performed an in silico analysis of genes and transcripts from MATE, ABC, and MFS families to infer the role of membrane transporters in Citrus-Xac interaction. Using as reference, the available Citrus sinensis genome and the citrus reference transcriptome from CitrusKB database, 67 MATE, 91 MFS, and 143 ABC genes and 82 MATE, 139 MFS, and 226 ABC transcripts were identified and classified into subfamilies. Duplications, alternative-splicing, and potentially non-transcribed transporters’ genes were revealed. Interestingly, MATE I and ABC G subfamilies appear differently regulated during Xac infection. Furthermore, Citrus spp. showing distinct levels of CC susceptibility exhibited different sets of transporters transcripts, supporting dissimilar molecular patterns of membrane transporters in Citrus-Xac interaction. According to our findings, 4 MATE, 10 ABC, and 3 MFS are potentially related to plant-defense mechanisms. Overall, this work provides an extensive analysis of MATE, ABC, and MFS transporters’ in Citrus-Xac interaction, bringing new insights on membrane transporters in plant-pathogen interactions.

Trichophyton rubrum Azole Resistance Mediated by a New ABC Transporter, TruMDR3

ABSTRACT The mechanisms of terbinafine resistance in a set of clinical isolates of Trichophyton rubrum have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of T. rubrum to develop resistance through multidrug efflux transporters. The introduction of a T. rubrum cDNA library into Saccharomyces cerevisiae allowed the isolation of one transporter of the major facilitator superfamily (MFS) conferring resistance to azoles (TruMFS1). To identify more azole efflux pumps among 39 ABC and 170 MFS transporters present within the T. rubrum genome, we performed a BLASTp analysis of Aspergillus fumigatus, Candida albicans, and Candida glabrata on transporters that were previously shown to confer azole resistance. The identified candidates were further tested by heterologous gene expression in S. cerevisiae. Four ABC transporters (TruMDR1, TruMDR2, TruMDR3, and TruMDR5) and a second MFS transporter (TruMFS2) proved to be able to operate as azole efflux pumps. Milbemycin oxime inhibited only TruMDR3. Expression analysis showed that both TruMDR3 and TruMDR2 were significantly upregulated in TIMM20092. TruMDR3 transports voriconazole (VRC) and itraconazole (ITC), while TruMDR2 transports only ITC. Disruption of TruMDR3 in TIMM20092 abolished its resistance to VRC and reduced its resistance to ITC. Our study highlights TruMDR3, a newly identified transporter of the ABC family in T. rubrum, which can confer azole resistance if overexpressed. Finally, inhibition of TruMDR3 by milbemycin suggests that milbemycin analogs could be interesting compounds to treat dermatophyte infections in cases of azole resistance.

Conformational Transition Pathways in Major Facilitator Superfamily Transporters

Major facilitator superfamily (MFS) of transporters consists of three classes of membrane transporters: symporters, uniporters, and antiporters. Despite such diverse functions, MFS transporters are believed to undergo similar conformational changes within their distinct transport cycles. While the similarities between conformational changes are noteworthy, the differences are also important since they could potentially explain the distinct functions of symporters, uniporters, and antiporters of MFS superfamily. We have performed a variety of equilibrium, non-equilibrium, biased, and unbiased all-atom molecular dynamics (MD) simulations of bacterial proton-coupled oligopeptide transporter GkPOT, glucose transporter 1 (GluT1), and glycerol-3-phosphate transporter (GlpT) to compare the similarities and differences of the conformational dynamics of three different classes of transporters. Here we have simulated the apo protein in an explicit membrane environment. Our results suggest a very similar conformational transition involving interbundle salt-bridge formation/disruption coupled with the orientation changes of transmembrane (TM) helices, specifically H1/H7 and H5/H11, resulting in an alternation in the accessibility of water at the cyto- and periplasmic gates.