Suppressed Protein Translation Caused by MSP-8 Deficiency Determines Fungal Multidrug Resistance with Fitness Cost

Abstract

Antifungal resistance, particularly the rise of multidrug-resistance strains, poses a significant public health threat. In this study, the study identifies a novel multidrug-resistance gene, msp-8, encoding a helicase, through experimental evolution with Neurospora crassa as a model. Deletion of msp-8 conferred multidrug resistance in N. crassa, Aspergillus fumigatus, and Fusarium verticillioides. However, the transcript levels of genes encoding known drug targets or efflux pumps remain unaltered with msp-8 deletion. Interestingly, MSP-8 interacted with ribosomal proteins, and this mutant displays compromised ribosomal function, causing translational disturbance. Notably, inhibition of protein translation enhances resistance to azoles, amphotericin B, and polyoxin B. Furthermore, MSP-8 deficiency or inhibition of translation reduces intracellular ketoconazole accumulation and membrane-bound amphotericin B content, directly causing antifungal resistance. Additionaly, MSP-8 deficiency induces cell wall remodeling, and decreases intracellular ROS levels, further contributing to resistance. The findings reveal a novel multidrug resistance mechanism independent of changes in drug target or efflux pump, while MSP-8 deficiency suppresses protein translation, thereby facilitating the development of resistance with fitness cost. This study provides the first evidence that MSP-8 participates in protein translation and that translation suppression can cause multidrug resistance in fungi, offering new insights into resistance mechanisms in clinical and environmental fungal strains.

Keywords: Aspergillus fumigatus; Fusarium verticilioides; Neurospora crassa; antifungal drug; helicase; multidrug resistance; protein translation.

Figure 1

The multidrug resistance phenotype of 2k‐6 is associated with the MSP‐8 point mutation (E749K). A) Spider web diagram depicting changes in antifungal susceptibility profiles of the indicated strains. B) Schematic illustrating the inheritance stability of multidrug resistance in strain 2k‐6 via backcrossing. The multidrug resistance phenotype is determined by drug susceptibility testing at designated concentrations of different kinds of antifungals. Plates are incubated at 28 °C for the indicated time points. The antifungal drugs used in this study are as follows: KTC (ketoconazole), VRC (voriconazole), PoxB (polyoxin B), AmB (amphotericin B). C) CAPS assay performed with 18 markers as described by Randy Lambreghts et al.(2009) spaced across LGV. CAPS makers are named by their physical location given as ‐) on the X‐axis. Map ratios estimated from band intensities for SNPs are shown on the Y‐axis. Map ratios below 0.01 are indicated red numbers. The mapping methodology is detailed in the Experimental Section. D) SNP information for the mapped mutation between ilv‐2 (1828369) and poi‐1 (3397430) on LGV, highlighted by a red line. The colors represent the frequency of SNPs. Source data are provided in File S1 (Supporting Information). The specific SNPs (dashed box) in the 2k‐6 strain are shown in the chart below.

Figure 2

Identification of E749K mutation in MSP‐8 associated with multidrug resistance phenotype in the strain 2k‐6. A) Drug susceptibility test of the indicated N. crassa strains: the msp‐8 knockout mutant (cdr4 ^KO::msp‐8 ^KO), the point mutant (cdr4 ^KO::MSP‐8^E749K), the background strain cdr4 ^KO mutant, the evolved mutant 2k‐6 and the msp‐8 complementary strain in 2k‐6 (2k‐6::msp‐8 ^com). B) Drug susceptibility test of the msp‐8 knockout mutant (msp‐8 ^KO), the point mutant (MSP‐8^E749K), the msp‐8 complementary strain (msp‐8 ^KO::msp‐8 ^com), and the point mutated msp‐8 complementary strain (msp‐8 ^KO::msp‐8 ^E749K), against designated concentrations of different antifungals. Two microliter aliquots of conidial suspension (2 × 10⁶ conidia/mL) were inoculated in the center of plates (ϕ90‐mm) with or without the antifungals. The plates were then incubated at 28 °C for the indicated time. C) Drug susceptibility test of the msp‐8 homologous gene knockout mutant (Fvmsp‐8 ^KO) in F. verticillioides and the wild‐type strain (Fv7600). Two microliter aliquots of conidial suspension with gradient dilution (10⁷, 10⁶, 10⁵, 10⁴ conidia /mL) were inoculated onto PDA plates (ϕ150‐mm) with or without the antifungals. The plates were then incubated at 28 °C for the indicated time. D) Drug susceptibility test of the msp‐8 homologous gene knockout mutant (Afmsp‐8 ^KO) in A. fumigatus and the wild‐type strain (CEA17). E) Drug susceptibility test of the clinical isolated strain 2707 (containing point mutation of F367Y in Afmsp‐8), the point mutant (Afmsp‐8^F367Y), and the reference strain (ATCC), against designated concentrations of different antifungals. Two microliter aliquots of conidial suspension with gradient dilution (10⁷, 10⁶, 10⁵ conidia/mL) were inoculated in CM plates (ϕ90‐mm) with or without the antifungals. The plates were then incubated at 37 °C for the indicated time. All experiments were independently repeated at least three times. Abbreviations: TDF (Triadimefon), ITC (Itraconazole).

Figure 3

Deletion of MSP‐8 disrupts protein translation. A) Laser confocal microscopic images showing the distribution of MSP‐8‐GFP in N. crassa cells under control condition. DAPI staining is used to visualize the nucleus (blue). The merged 3D image of GFP and DAPI staining demonstrates non‐nuclear localization of MSP‐8‐GFP (Pearson's Correlation Value for co‐localization = 0.2813). Mitochondria are visualized using Red‐MitoTracker dye. B) Gene ontology (GO) analysis of the MSP‐8‐interacting proteins identified by Y2H, with the Sankey diagram on the left illustrating the connections between the interacting proteins and the enriched GO items. Source data are provided in File S3 (Supporting Information). C) Heat map comparison of the proteomic data showing the expression levels of RQC system proteins in WT and msp‐8 ^KO strains. Colors represent the log₂‐transformed fold changes in protein levels detected in the indicated strains. D,E) Representative polysome profiles (D) from WT and msp‐8 ^KO strains, and quantification of the 80S monosome to non‐polysome (80S/Non‐P) ratios (E). Data are presented as mean ± SD. Statistical significance between the WT strain and msp‐8 ^KO mutant is assessed using a two‐tailed t‐test. Values with 0.01 < p < 0.05 are marked with*. p = 0.0296, n = 3.

Figure 4

Deletion of MSP‐8 and translation inhibitors reduce both intracellular KTC accumulation and the membrane‐bound AmB content, leading to fungal multidrug resistance. A,B) Drug susceptibility test of the WT and msp‐8 ^KO strains to designated concentration of different antifungals, either untreated or treated with 0.02 µM CHX (A), or 6.25 µg mL⁻¹ ASM (B). Both CHX and ASM are protein translation inhibitors. C) Analysis of KTC accumulation in WT and msp‐8 ^KO strains under KTC stress, and under KTC treatment combined with either CHX or ASC. D) Analysis of the membrane‐bound‐AmB content in WT and msp‐8 ^KO strains under AmB stress, and under AmB treatment combined with either CHX or ASC. All the data are presented as mean ± SD. Statistical significance between each pair of groups was determined using two‐way ANOVA. Values with p < 0.0001 and p > 0.05 are marked with **** and n.s, respectively. Statistical values are as follows: p_{msp‐8 KO(KTC, KTC+CHX)} = 0.7528, n = 3; p_{msp‐8 KO(KTC, KTC+ASM)} = 0.9996, n = 3; p_{msp‐8 KO(AmB, AmB+CHX)} = 0.9988, n = 3; p_{msp‐8 KO(AmB, AmB+ASM)} = 0.9885, n = 3.

Figure 5

MSP‐8 deficiency leads to down‐regulation of transport proteins, remodeling of cell wall, and reduced ROS levels A) Relative expression of PUP‐6 protein in WT and msp‐8 ^KO strains, with or without KTC treatment. The data are presented as mean ± SD with n = 3. Statistical significance between groups is determined by two‐way ANOVA. B) Drug susceptibility test of WT and pup‐6 ^KO strains to KTC. C) Relative expression of oligopeptide/dipeptide transporters NCU08397, NCU09874, and MFS‐9 in WT and msp‐8 ^KO strains. The data are presented as mean ± SD with n_NCU08397 = 2 and n_NCU09874 = 3. Statistical significance between groups is determined by the two‐tailed t‐test. D–F) Relative amounts of ergosterol (D,E) and chtin (F) in msp‐8 ^KO and WT strains under control conditions and with KTC (D), AmB (E), or PoxB (F) treatment. These data are presented as mean ± SD with n = 3. Statistical significance between groups is determined by the two‐way ANOVA. G) Accmulation of ROS in the indicated strains under the untreated (control) or after treatment with KTC, AmB, and PoxB. The data are presented as mean ± SD with n = 3. Statistical significance between groups is determined by the two‐tailed t‐test. H) Relative expression of oxidoreductases CEM‐5, CAT‐3, and CAT‐4 in WT and msp‐8 ^KO strains. The data are presented as mean ± SD with n_CEM‐5 = 3, n_CAT‐3 = 3, and n_CAT‐4 = 2. Statistical significance between groups is determined by the two‐tailed t‐test. I) Drug susceptibility test of WT and cem‐5 ^KO strains to KTC, AmB, and PoxB.