Neomycin Interferes with Phosphatidylinositol-4,5-Bisphosphate at the Yeast Plasma Membrane and Activates the Cell Wall Integrity Pathway

Abstract

The cell wall integrity pathway (CWI) is a MAPK-mediated signaling route essential for yeast cell response to cell wall damage, regulating distinct aspects of fungal physiology. We have recently proven that the incorporation of a genetic circuit that operates as a signal amplifier into this pathway allows for the identification of novel elements involved in CWI signaling. Here, we show that the strong growth inhibition triggered by pathway hyperactivation in cells carrying the "Integrity Pathway Activation Circuit" (IPAC) also allows the easy identification of new stimuli. By using the IPAC, we have found various chemical agents that activate the CWI pathway, including the aminoglycoside neomycin. Cells lacking key components of this pathway are sensitive to this antibiotic, due to the disruption of signaling upon neomycin stimulation. Neomycin reduces both phosphatidylinositol-4,5-bisphosphate (PIP₂) availability at the plasma membrane and myriocin-induced TORC2-dependent Ypk1 phosphorylation, suggesting a strong interference with plasma membrane homeostasis, specifically with PIP₂. The neomycin-induced transcriptional profile involves not only genes related to stress and cell wall biogenesis, but also to amino acid metabolism, reflecting the action of this antibiotic on the yeast ribosome.

Keywords: MAPK; PIP2; Saccharomyces cerevisiae; Slt2; cell wall integrity; neomycin.

Figure 1

Integration of IPAC results in greater CWI pathway hyperactivation than the IPAC-bearing plasmid. (a) Sensitivity to different stresses of BY4741 strain bearing either the empty vector YCplac111 or the plasmid YCplac111-IPAC, and control wild type with the integrated vector (YSTH1) or with integrated IPAC (YSTH2). Ten-fold serial dilutions of cell suspensions were spotted onto YPD plates in the absence (no stress) or presence of 5 μg/mL CR, 10 U/mL (100 μg/mL) zymolyase 100T, or 100 μg/mL SDS. A representative assay from three different experiments with distinct transformants is shown. (b) Western blotting analysis of extracts from BY4741 strain transformed with plasmid YCplac111-IPAC or the YSTH2 strain (integrated IPAC) treated with 30 μg/mL CR for the indicated times (min). Dually phosphorylated Slt2 and G6PDH (as a loading control) were detected with anti-phospho-p44/42 and anti-G6PDH antibodies, respectively. The numbers below correspond to the amount of phosphorylated Slt2, normalized with respect to the loading control for each sample and expressed as a fold increase relative to the basal phosphorylation levels in the absence of stress of BY4741 cells bearing the YCplac111-IPAC plasmid. A representative blot from three independent experiments is shown. (c) β-galactosidase activity of cell extracts from the same strains as in (a), all bearing the MLP1p-lacZ plasmid. Cells were either left untreated (no stress) or treated for 4 h with 30 μg/mL CR. Data represent the mean of β-galactosidase activity of three independent transformants. Error bars indicate standard deviation, and an asterisk indicates statistical significance relative to plasmidic IPAC according to Student’s t-test (* = p-value < 0.05). (d) Multiwell plate sensitivity assay of the YSTH1 strain (integrated vector) and the YSTH2 strain (integrated IPAC) to the indicated concentrations of zymolyase 100T. Data represent the mean of three independent transformants. Error bars indicate standard deviation. (e) Halo sensitivity assay of the same strains as in (d). Disks were impregnated with 10 mg/mL CR. A representative assay from three different experiments is displayed.

Figure 2

Neomycin, lithium chloride (LiCl), zinc chloride (ZnCl2), ethylenediaminetetraacetic acid (EDTA), and diphenhydramine chlorhydrate (DPH) activate the IPAC circuit. (a) Growth inhibition halo assay of vector (WT) or IPAC-integrated Y3656 strains. Six-millimeter-diameter disks were impregnated with 50 mg/mL neomycin sulfate, 424 mg/mL LiCl, 136 mg/mL ZnCl₂, 11 mg/mL EDTA, or 50 mg/mL DPH. Mean and standard deviations of halo diameters from three different experiments are shown. (b) Western blotting analysis of extracts from BY4741 RLM1-6MYC strain transformed with YCplac111 (−) or Ycplac111-IPAC (+) and treated or not (no stress) for 3 h with 30 μg/mL CR, 10 mg/mL neomycin (Neo), 8.5 mg/mL LiCl, 681.4 μg/mL ZnCl₂, 11.2 mg/mL EDTA, or 1 mg/mL DPH. Rlm1-Myc, dually phosphorylated Slt2, and G6PDH (as a loading control) were detected with anti-Myc, anti-phospho-p44/42, and anti-G6PDH antibodies, respectively. A representative blot from two independent experiments is shown.

Figure 3

Neomycin is the only protein synthesis inhibitor that activates the CWI pathway. (a) Halo sensitivity assay of vector (WT) or Y3656 strains with integrated IPAC. Disks were impregnated with 100 mg/mL of neomycin sulfate, 1 mg/mL of cycloheximide, 50 mg/mL of G418, 50 mg/mL of hygromycin B, or 50 mg/mL of gentamycin. Mean and standard deviations of halo diameters from three different experiments are shown. (b) Western blotting analysis of extracts from Y3656 strain without (−) or with IPAC (+) and treated or not (no stress) for 4 h with 10 mg/mL neomycin, 25 μg/mL cycloheximide, 31.3 μg/mL G418, 62.5 μg/mL hygromycin B, or 10 mg/mL gentamycin sulfate. Dually phosphorylated Slt2, total Slt2 protein, and G6PDH (as a loading control) were detected with anti-phospho-p44/42, anti-Slt2, and anti-G6PDH antibodies, respectively. A representative blot from three independent experiments is shown.

Figure 4

The CWI pathway is essential for yeast cells to survive in the presence of neomycin. (a) Scheme depicting the main components of the CWI pathway. (b) Sensitivity to neomycin of isogenic Y3656 mutants lacking the indicated CWI pathway components was assayed by spotting 10-fold serial dilutions of cell suspensions onto YPD plates in the absence (no stress) or presence of either 2 or 5 mg/mL neomycin or 10 μg/mL of CR. Red letters indicate higher sensitivity to neomycin than the wild-type strain. A representative assay from three different experiments is shown. (c) Neomycin-elicited signaling to Slt2 is mediated by core components of the CWI pathway. Western blotting analysis of extracts of isogenic Y3656 mutants lacking the indicated CWI pathway components treated (+) or not (−) for 4 h with 10 mg/mL neomycin. (d) Western blotting analysis of CML128 (WT) and pkc1Δ mutant isogenic strains in the absence (−) or presence of 30 μg/mL CR or 10 mg/mL neomycin (N) for 4 h. Dually phosphorylated Slt2 and G6PDH (as a loading control) were detected with anti-phospho-p44/42 and anti-G6PDH antibodies, respectively. A representative blot from three independent experiments is displayed. (e) Neomycin induces Rlm1-mediated gene transcription. Transcriptional induction analysis of the MLP1 gene by RT-qPCR in response to neomycin compared to CR in the Y3656 strain. Cells were cultured in the absence of stress (−) or with 10 mg/mL neomycin or 30 μg/mL CR for 1 or 4 h. The graph shows the mean and standard deviation of the expression level from three independent experiments. Asterisks indicate statistical significance relative to the absence of stress according to a Student’s t-test (* = p-value < 0.05; ** = p-value < 0.01; *** = p-value < 0.001).

Figure 5

(a) Fluorescence microscopy images of the wild-type YPH499 strain co-expressing the PIP₂ marker GFP-2XPH (PLCδ) and p110α-KD from the plasmids pJMCS-DM23 and YCpLG-p110αKD, respectively, treated or not with neomycin 5 mg/mL and cultured in SR-G for 5 h. (b). Fluorescence microscopy images of YPH499 strain expressing the phosphatidylserine (PS) marker RFP-LactC2 from the plasmid pRS416- RFP-LactC2 treated or not with neomycin 5 mg/mL cultured in SD for 5 h. (c) Growth assays of YPH499 strain transformed with plasmids YcpLG-p110α or YcpLG-p110αKD on SD and SG plates containing no stress, 2 mg/mL of neomycin, or 5 µg/mL of CFW (upper panel). Growth assays of YPH499 cells transformed with plasmids BG1805-PLC1 or pYES2 as control on SD/SG plates containing no stress, 5 mg/mL of neomycin, or 10 µg/mL of CR (lower panel). A representative assay from three independent experiments is shown. (d) Fluorescence microscopy images of the YPH499 strain expressing the PIP₂ marker GFP-2X-PH(PLCδ1) with either an inactive version of p110α (control) or Myr-p110α from plasmids pJMCS-DM23, YcpLG-p110αKD and YcpLG-Myr-p110α, respectively, treated or not with 5 mg/mL of neomycin for 5 h and cultured in SR-G. Scale bars correspond to 5 μm. The graph shows the mean percentage of cells with PM labeling with GFP-2XPH (PLCδ1) from three independent experiments and error bars indicate the standard deviation. An asterisk indicates statistical significance relative to the control strain according to a Student’s t-test (* = p-value < 0.05).

Figure 5

(a) Fluorescence microscopy images of the wild-type YPH499 strain co-expressing the PIP₂ marker GFP-2XPH (PLCδ) and p110α-KD from the plasmids pJMCS-DM23 and YCpLG-p110αKD, respectively, treated or not with neomycin 5 mg/mL and cultured in SR-G for 5 h. (b). Fluorescence microscopy images of YPH499 strain expressing the phosphatidylserine (PS) marker RFP-LactC2 from the plasmid pRS416- RFP-LactC2 treated or not with neomycin 5 mg/mL cultured in SD for 5 h. (c) Growth assays of YPH499 strain transformed with plasmids YcpLG-p110α or YcpLG-p110αKD on SD and SG plates containing no stress, 2 mg/mL of neomycin, or 5 µg/mL of CFW (upper panel). Growth assays of YPH499 cells transformed with plasmids BG1805-PLC1 or pYES2 as control on SD/SG plates containing no stress, 5 mg/mL of neomycin, or 10 µg/mL of CR (lower panel). A representative assay from three independent experiments is shown. (d) Fluorescence microscopy images of the YPH499 strain expressing the PIP₂ marker GFP-2X-PH(PLCδ1) with either an inactive version of p110α (control) or Myr-p110α from plasmids pJMCS-DM23, YcpLG-p110αKD and YcpLG-Myr-p110α, respectively, treated or not with 5 mg/mL of neomycin for 5 h and cultured in SR-G. Scale bars correspond to 5 μm. The graph shows the mean percentage of cells with PM labeling with GFP-2XPH (PLCδ1) from three independent experiments and error bars indicate the standard deviation. An asterisk indicates statistical significance relative to the control strain according to a Student’s t-test (* = p-value < 0.05).

Figure 6

Western blotting of cell lysates of Y3656 and the isogenic slt2∆ strains transformed with plasmid pLB215 (Ypk1-HA) from untreated cultures (control) or treated with 0.8 mg/mL (2 µM) myriocin for 2 h (Myrio), the combination of 0.8 mg/mL (2 µM) and 5 mg/mL neomycin for 2 h (Myrio+Neo), or 5 mg/mL neomycin for 4 h (Neo), all of them cultured in YPD. Membranes were probed with anti-phospho T662-Ypk1 (upper panel), anti-HA (middle panel), and anti-G6PDH (lower panel), as a loading control.

Figure 7

Genome-wide expression profile of wild-type cells following neomycin treatment. (a) Gene Ontology term enrichment within the genes up- or downregulated upon neomycin exposure. The graph indicates the percentage of genes clustered into the indicated GO terms for the differentially expressed genes upon neomycin treatment (dark blue) compared to that of the overall genome (light blue). According to a χ2 test, only p-values ≤ 0.05 were considered. (b) Comparison of the neomycin transcriptional induction determined by DNA microarrays and RT-qPCR. The graphic shows the level of induction of six genes selected to confirm by RT-qPCR (dark blue) the initial values obtained in the DNA microarray assays (light blue). The columns represent the mean, and the error bars indicate the standard deviation from three independent experiments.

Figure 8

Comparison of the transcriptional profiles in response to neomycin, gentamycin (GEO Accession Number GDS2999), RPL22 deletion [39], Congo red (CR) exposure [23] and p110α-CAAX overexpression [34]. Red colors are assigned to upregulated genes in each condition while green indicates downregulation. Hierarchical clustering using Euclidean distance was generated using the software Multi-Experiment Viewer (MeV).