Plasma membrane transporters Alp1 and Nrt1 mediate the uptake of benzamidine conjugates in fungi

Abstract

Efflux pumps confer antifungal resistance. Benzamidine (BM) conjugates overcome resistance by enhancing intracellular accumulation, but their cellular uptake mechanism remains unclear. A screen of membrane transport protein mutants revealed that plasma membrane transporters Alp1 and Nrt1 are importers of BM conjugates and are essential for their antifungal activity.

Invasive candidiasis is a leading cause of morbidity and mortality in immunocompromised and critically ill patients (Kullberg and Arendrup, 2015; Pappas et al., 2018). The limited repertoire of antifungal drug classes, coupled with the escalating prevalence of antifungal resistance, further highlights the urgent need for the development of novel antifungal agents (Lee et al., 2021; Revie et al., 2018). While the FDA approved the first echinocandin-class antifungal agent in 2001 (Aguilar-Zapata et al., 2015), the subsequent development of antifungal therapeutics has progressed relatively slowly, with few agents harboring novel mechanisms of action approved for the treatment of invasive fungal infections to date (Ahmad et al., 2016; Denning and Bromley, 2015). The major challenges in antifungal drug development are largely attributable to the pivotal role of the fungal efflux system, which protects fungal cells by non-selectively extruding potential toxicants (Cannon et al., 2009; Zhu et al., 2023). Some efflux substrates, known as expelled potential active agents (EPAAs), may exhibit antifungal activity when accumulated within fungal cells (Chang et al., 2018; Wang et al., 2023). However, research into the conversion of EPAAs into functional antifungal agents remains largely underexplored.

We previously developed a benzamidine (BM)-based mitochondrial targeting strategy that leverages the BM moiety as a targeting warhead to convert EPAAs into potent, broad-spectrum antifungal agents with a minimal risk of resistance development, representing a safe and broadly applicable platform for developing novel treatments against drug-resistant fungal pathogens (Wang et al., 2023). However, the underlying molecular mechanisms that mediate the cellular uptake of BM conjugates remain incompletely elucidated. The fungal plasma membrane harbors a large number of membrane transport proteins. Previous studies have shown that chemotherapeutic agents such as oxaliplatin and doxorubicin are transported across cellular membranes via solute carrier (SLC) transporters (Galetin et al., 2024), which led us to hypothesize that BM and its conjugates may also enter fungal cells through specific membrane transport proteins. Identifying the membrane transport proteins mediating BM conjugates uptake will extend our insights into the cell surface transport mechanisms and is critical for the development and clinical application of these conjugates as antifungal agents.

A yeast knockout strain collection purchased from Horizon Discovery Ltd. was used to select 142 mutant strains with disruptions in genes encoding membrane transport proteins, thus constructing a customized mutant library (Table S1). Subsequently, we employed a coumarin-benzamidine (DC-BM) fluorescent probe (Fig. 1A) (Wang et al., 2023), which accumulates rapidly in fungal cells and emits blue fluorescence, to screen this library. Specifically, overnight cultures of strains from the library were washed once with PBS buffer and subsequently diluted to a concentration of 1 × 10⁶ CFU/mL in PBS buffer. Each strain was assayed in both a blank control group and a DC-BM treatment group, with three biological replicates per condition, using the wild-type strain BY4743 as a control. The DC-BM treatment group was incubated in the dark at 30 °C and 200 rpm with 10 μg/mL of DC-BM for 30 min. After incubation, the cells were centrifuged, washed three times with PBS buffer, and resuspended in 200 μL of PBS buffer. Intracellular probe accumulation in different strains was quantified by flow cytometry-based fluorescence intensity analysis. Compared with the parental BY4743 strain, alp1Δ/Δ and nrt1Δ/Δ exhibited a significant reduction in intracellular DC-BM accumulation (Fig. 1B and C), suggesting that these two membrane transport proteins may play a key role in the cellular uptake of BM conjugates.

Fig. 1.

Characterization of benzamidine uptake channels in fungi via fluorescent probe screening. (A) Fluorescent probe used for screening. (B) Comparison of the relative intracellular levels of DC-BM in 142 channel protein-related gene knockout strains against the BY4743 strain. (C) Different S. cerevisiae cells were cultured in PBS with DC-BM for 30 min, and the fluorescence intensity was measured by flow cytometry. The bars represent means ± SD from three independent experiments. ***P < 0.001.

To further confirm that these two membrane transport proteins regulate the antifungal activity of BM conjugates by mediating their cellular uptake, we assessed the antifungal susceptibility of the corresponding mutant strains to IS-2-BM (Wang et al., 2023) (Fig. 2A), a previously reported BM conjugate with potent antifungal activity. Specifically, overnight-cultured yeast cells were diluted in YPD medium to a concentration of 1 × 10⁶ CFU/mL. A 100 μL aliquot of the cell suspension was dispensed into a 96-well plate. Serial concentrations of IS-2-BM were added to the cultures and incubated at 30 °C for 16 h. Cells that were not treated with IS-2-BM served as a control. Growth curves were plotted based on the values recorded by a microplate reader at 600 nm. Our results demonstrated that both mutant strains exhibited varying degrees of resistance to IS-2-BM compared with the parental strain BY4743 (Fig. 2B), confirming that Alp1 and Nrt1 are indeed responsible for facilitating the cellular uptake of BM conjugates and consequently modulate their antifungal activity.

Fig. 2.

Antifungal activity of IS-2-BM against different S. cerevisiae. (A) Structure of compound IS-2-BM. (B–D) Different S. cerevisiae cells were incubated with IS-2-BM. Growth was measured by a microplate reader at 600 nm. Cells that were not treated with IS-2-BM served as the control. Each point represents the mean of three replicated measurements.

To our knowledge, Alp1 acts as an arginine transporter (Sychrova and Chevallier, 1994), whereas Nrt1 is a plasma membrane-localized nicotinamide riboside transporter (Belenky et al., 2008). These two proteins exhibit substantial differences in both structure and physiological function, and the precise molecular mechanisms by which they facilitate benzamidine uptake into fungal cells remain to be elucidated. Current investigations into small-molecule drug internalization often rely on structural biology approaches such as cryo-electron microscopy, whereas computational methods including molecular docking are generally insufficient to precisely define the dynamic transport processes underlying drug entry (Chen et al., 2024; Zhang et al., 2025). At present, however, we cannot determine whether Alp1 and Nrt1 are directly involved in the cellular uptake of BM conjugates. Nevertheless, despite this uncertainty, our current findings demonstrate that Alp1 and Nrt1 cooperatively promote the cellular entry of benzamidine conjugates and thus regulate their antifungal activity, which extends our understanding of the cell surface transport mechanisms underlying benzamidine conjugate uptake.

Berberine (BBR) exerts antifungal activity by inhibiting the activity of mitochondrial complex I (Tong et al., 2021). Thus, enhancing the mitochondrial targeting of BBR via benzamidine conjugation is expected to markedly boost its antifungal activity, and the resulting conjugates can be employed to validate the role of Alp1 and Nrt1 in mediating the cellular uptake and antifungal activity of benzamidine conjugates. Subsequently, we synthesized a berberine-benzamidine conjugate (BBR-BM) (Fig. 3A and Scheme S1). The colocalization experiment was conducted using Candida albicans TOM70-GFP-CAI4 cells. Tom70 is a mitochondrial outer membrane protein, and its fusion with GFP allows for the visualization of mitochondria within fungal cells. Specifically, overnight-cultured TOM70-GFP-CAI4 cells were diluted in RPMI 1640 medium to a concentration of 1 × 10⁶ CFU/mL, followed by the addition of varying concentrations of BBR and BBR-BM. The cells were incubated at 30 °C for 6 h. The cells were then collected and washed twice with PBS. Colocalization of Tom70-GFP with BBR or BBR-BM was observed using confocal microscopy. The results indicated that BBR-BM accumulated intracellularly and was predominantly localized in the mitochondria (Fig. 3B), which is consistent with our previous observations (Wang et al., 2023).

Fig. 3.

Alteration of antifungal activity of berberine following the conjugation with benzamidine. (A) Structure of compound BBR-BM. (B) Subcellular distribution of BBR or BBR-BM in C. albicans TOM70-GFP-CAI4 cells were treated with BBR or BBR-BM for CLSM observation. Scale bar, 10 μm. (C) Different S. cerevisiae cells were incubated with BBR-BM. Growth was measured by a microplate reader at 600 nm. Cells that were not treated with BBR-BM served as the control. Each point represents the mean of three replicated measurements. (D) Survival curve of infected G. mellonella by C. albicans Gu5 (n = 20 per group). The number of surviving larvae was recorded each day. The Mantel-Cox test was used to compare differences between the groups. *P < 0.05, **P < 0.01; ns, not significant.

Given that BBR-BM exhibits mitochondrial localization, we hypothesize that it possesses stronger antifungal activity than BBR. In accordance with the CLSI document M27-A3 (Wayne, 2008), the minimum inhibitory concentrations (MICs) of BBR-BM and BBR against C. albicans were determined. Specifically, overnight cultures of fungal cells were diluted in RPMI 1640 medium to 1 × 10³ CFU/ml, and 100 μl of the suspension was added to each well of a 96-well plate. Each test compound was two-fold serially diluted from 0.25 to 128 μg/mL. After incubation at 35 °C for 24 h, the optical density (OD) at 600 nm was measured using a microplate reader. The MIC was defined as the lowest concentration that reduced the OD by 80% relative to untreated control wells. As expected, BBR-BM displayed enhanced antifungal activity against a panel of Candida species, including azole-resistant strains, relative to BBR (Table 1). We next assessed the antifungal activity of BBR-BM against the alp1Δ/Δ and nrt1Δ/Δ mutant strains. The results showed a significant reduction in susceptibility to BBR-BM in both mutant strains (Fig. 3C), which was consistent with the susceptibility phenotype observed with IS-2-BM. These findings further confirm that these two membrane transporters mediate the uptake of benzamidine conjugates into fungal cells and deepen our understanding of the cell surface transport mechanisms.

Table 1.

Antifungal activity of BBR-BM against different Candida species.

Strainsa	MIC80 (μg/mL)
	BBR-BM	BBR	FLC
SC5314	4	16	2
Gu5	8	64	>128
CA632	8	32	>128
CP1	8	32	2
CT2	4	32	2

^aSC5314 is a wild-type reference strain of C. albicans; Gu5 is a FLC-resistant C. albicans clinical isolate from an AIDS patient, which harbors a gain-of-function mutation in the TAC1 and thereby causes the overexpression of CDR1 and CDR2. CA632 is a clinical pan-azole-resistant C. albicans clinical isolate from a cancer patient; CP1 is a clinical isolate of Candida parapsilosis；CT2 is a clinical isolate of Candida tropicalis.

We further employed Galleria mellonella as an in vivo infection model to evaluate the therapeutic potential of BBR-BM against C. albicans Gu5, an azole-resistant strain with CDR1/CDR2 overexpression. The use of Gu5 can further demonstrate the potential of BM conjugation strategy in overcoming azole resistance. Moreover, C. albicans Gu5 displayed the strongest intrinsic resistance to BBR and an approximately 8-fold susceptibility enhancement toward BBR-BM, ideal for verifying the potency of this conjugation strategy against pump-mediated resistance. G. mellonella larvae were randomly assigned to four groups: blank group (injected with an equivalent volume of PBS), control group (infected with fungi without drug treatment), fluconazole (5 μg/larva) group, BBR (5 μg/larva) group, and BBR-BM (5 μg/larva) group, with 20 larvae in each group. All compounds were dissolved in DMSO as 10 mg/mL stock solutions. Each stock was diluted 1:20 in PBS to yield a 0.5 mg/mL working solution. Each G. mellonella larva received an injection of 10 μL, providing a final dose of 5 μg/larva. The Gu5 strain was grown to the logarithmic phase, centrifuged to collect the cells, washed once with PBS, and subsequently diluted to a concentration of 2.5 × 10⁷ CFU/mL in PBS. A volume of 10 μL of this suspension was injected into the abdominal legs of the larvae using a microsyringe. Infection was determined to be successful when the larvae exhibited a black coloration or a distinct black line along their backs, accompanied by a noticeable decline in vitality. The test compounds were diluted in PBS to the required concentrations, and the resultant working solutions were injected into the opposite abdominal legs of the larvae, also using a microsyringe at a volume of 10 μL per injection. The untreated control group received an equivalent volume of PBS. Following treatment, the larvae were cultured at 30 °C and survival was recorded daily for 8 days. Consistently, BBR-BM exerted a more potent therapeutic effect than BBR in a C. albicans-infected larval infection model (Fig. 3D), further confirming the potential of BM conjugation strategy for antifungal drug discovery.

In conclusion, we identified two plasma membrane transporters, Alp1 and Nrt1, as key mediators of BM conjugates uptake and antifungal activity. These findings clarify the uncharacterized transmembrane transport mechanism of BM conjugates. Together, these results provide a solid experimental basis for developing and translating benzamidine conjugates as novel therapeutics against drug-resistant fungal infections.

CRediT authorship contribution statement

Xueyang Jin: Writing – original draft, Validation, Methodology, Investigation, Funding acquisition, Data curation. Yunfan Yang: Validation, Investigation, Data curation. Xue Wang: Methodology, Investigation, Funding acquisition, Conceptualization. Ming Yuan: Supervision, Investigation, Data curation. Shaohua Zhao: Writing – review & editing, Supervision. Guoyun Wang: Writing – review & editing, Supervision. Wenqiang Chang: Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Funding acquisition, Data curation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material 1

Utilized Strains and Synthetic Procedure of BBR‑BM.

Supplementary material 2

142 Genes Encoding Membrane Transport Proteins.

Data availability

Data will be made available on request.