Plasma Membrane Proteolipid 3 Protein Modulates Amphotericin B Resistance through Sphingolipid Biosynthetic Pathway

Abstract

Invasive opportunistic fungal infections of humans are common among those suffering from impaired immunity, and are difficult to treat resulting in high mortality. Amphotericin B (AmB) is one of the few antifungals available to treat such infections. The AmB resistance mechanisms reported so far mainly involve decrease in ergosterol content or alterations in cell wall. In contrast, depletion of sphingolipids sensitizes cells to AmB. Recently, overexpression of PMP3 gene, encoding plasma membrane proteolipid 3 protein, was shown to increase and its deletion to decrease, AmB resistance. Here we have explored the mechanistic basis of PMP3 effect on AmB resistance. It was found that ergosterol content and cell wall integrity are not related to modulation of AmB resistance by PMP3. A few prominent phenotypes of PMP3 delete strain, namely, defective actin polarity, impaired salt tolerance, and reduced rate of endocytosis are also not related to its AmB-sensitivity. However, PMP3 overexpression mediated increase in AmB resistance requires a functional sphingolipid pathway. Moreover, AmB sensitivity of strains deleted in PMP3 can be suppressed by the addition of phytosphingosine, a sphingolipid pathway intermediate, confirming the importance of this pathway in modulation of AmB resistance by PMP3.

Figure 1. S. cerevisiae PMP3 and its homologs from C. albicans and C. glabrata modulate AmB resistance.

(a) Multicopy overexpression of S. cerevisiae PMP3 (ScPMP3) and its homologs from C. glabrata (CgPMP3) and C. albicans (CaPMP3-O: PMP3 ortholog, orf19.1655.3; CaPMP3-B: PMP3 best hit, orf19.2959.1) in pmp3Δ strain of S. cerevisiae enhance AmB resistance by about 4-fold with respect to wild-type strain (BY4741) and about 32-fold with respect to pmp3Δ strain. The relative growth of the strains on 0.1 μg/ml AmB (not shown) was comparable to that of respective strains on 0.2 μg/ml AmB. (b) AmB sensitivity of C. glabrata strain deleted in PMP3 ortholog (Cgpmp3Δ) and C. albicans strains deleted in both alleles of PMP3 ortholog (Capmp3-OΔ/Δ) and PMP3 best hit (Capmp3-BΔ/Δ), with respect to their respective parent strains CG462 and SN95. Five μl of 10-fold serial dilutions of cells were spotted starting from about 10⁵ cells/spot, as described in Methods.

Figure 2. Slow rate of endocytosis of pmp3Δ strain is restored to normal level by overexpression of ScSUR7.

(a) Wild type strain 3818 (SEY6210-Mup1pHluorin) and pmp3Δ strain (3818 pmp3Δ::HIS3) transformed with either vector or ScSUR7, were grown without methionine to promote accumulation of Mup1-pHluorin in the plasma membrane. After addition of 20 μg/ml methionine, random fields of cells were imaged at different time intervals. All images were obtained at identical exposure conditions. (b) After addition of methionine, Mup1-pHluorin fluorescence was measured at indicated time intervals in a flow cytometer, as described in Methods. The values shown are average of two replicates from one representative experiment. Experiments were repeated thrice with comparable results.

Figure 3. Actin polarization defect of pmp3Δ strain is suppressed by multicopy SUR7 overexpression.

Cells were grown to log phase and actin was visualized by rhodamine phalloidin staining. About 200 cells with small buds were scored according to their polarization state. Cells with actin patches concentrated in the small bud, with fewer than four patches in the mother cell, were classified as polarized cells. Other cells with more actin patches in the mother cell than in the small bud were classified as depolarized cells. Representative images are shown in Figure S6. Mean values of two independent experiments are given. The error bars indicate the range.

Figure 4

SUR7 overexpression can suppress salt sensitivity (a), but not AmB sensitivity (b) of strains deleted in PMP3, RVS161 or RVS167. Wild-type (BY4741) and PMP3 overexpression strains are included as controls.

Figure 5. PMP3 modulates AmB resistance through sphingolipid biosynthetic pathway.

(a) Sphingolipid biosynthetic pathway genes FEN1 and SUR4 and regulatory genes YPK1 and SAC1 are important for PMP3 mediated increase in AmB resistance. Wild-type (BY4741) and pmp3Δ strains overexpressing ScPMP3 serve as positive controls. (b) PMP3 modulates tolerance to myriocin, a sphingolipid biosynthetic pathway inhibitor. While strains overexpressing PMP3 are about 4-fold more tolerant, the strain deleted in PMP3 is about 2-fold more sensitive to myriocin, compared to the wild-type strain BY4741.

Figure 6. Phytosphingosine (PHS), a sphingolipid pathway intermediate, modulates AmB resistance.

(a) Growth of wild-type (BY4741), PMP3 deletion and overexpression strains of S. cerevisiae on indicated concentrations of AmB alone or in combination with 5 μM phytosphingosine (PHS). Relative growth of strains at 0.8 μg/ml AmB (not shown) was comparable to their growth at 1.6 μg/ml. (b) Growth of wild-type (CG462) and PMP3 delete (Cgpmp3Δ) strains of C. glabrata on indicated concentrations of AmB alone or in combination with 5 μM PHS. (c) Growth of C. albicans strains deleted in both alleles of PMP3 ortholog (Capmp3-OΔ/Δ) or PMP3 best hit (Capmp3-BΔ/Δ), with respect to their parent SN95 on indicated concentrations of AmB alone or in combination with 10 μM PHS.