Rufomycin Exhibits Dual Effects Against Mycobacterium abscessus Infection by Inducing Host Defense and Antimicrobial Activities

Abstract

Nontuberculous mycobacterial pulmonary infection is often aggravated due to antibiotic resistance issues. There is a need for development of new drugs inducing both host immune responses and antimicrobial activities. This study shows that the rufomycins 4/5/6/7 (Rufomycin 4-7), which targets ClpC1 as a subunit of caseinolytic protein complex ClpC1/ClpP1/ClpP2 of mycobacteria, exhibits a dual effect in host innate defense and in vivo antimicrobial activities against a rough morphotype of Mycobacterium abscessus (Mabs-R), a clinically severe morphotype that causes hyperinflammation. Rufomycin 4-7 treatment showed antimicrobial effects against Mabs pulmonary infection in vivo and in macrophages. In addition, Rufomycin 4-7 significantly decreased inflammation, but enhanced the autophagy/lysosomal genes through upregulation of the nuclear translocation of transcription factor EB (TFEB). Furthermore, Rufomycin 4-7 treatment effectively inhibited mitochondrial damage and oxidative stresses in macrophages during Mabs-R infection. Collectively, Rufomycin 4-7-mediated dual effects inducing both antimicrobial activities and host immune defense might confer an advantage to treatment against Mabs-R infection.

Keywords: Mycobacterium abscessus; antimicrobial activity; host immune defense; inflammation; rufomycin.

FIGURE 1

Rufomycin 4–7 treatment increases antimicrobial effects against mycobacterial infection in vivo and in vitro. (A) Mice were infected with Mabs-R (1 × 10⁶ CFU) intranasally, followed by administration of Rufomycin 4–7 (200 mg/kg, three times per week; by gavage) and sacrificed at 21 dpi. Bacterial loads in lung tissues for infected mice (n = 5; each group) were determined by CFU assay. (B) The sectioned lung tissues of infected mice (n = 3; each group) from panel (A) were H&E stained (scale bar: 300 μm), and the inflamed area of each group was quantified. (C) The sectioned lung tissues from panel (A) were stained with anti-Ly6G (red) and DAPI (blue) (scale bar: 25 μm), and the relative fluorescence intensity of Ly6G-stained neutrophils were quantified (n = 9 for each group; at least 200 cells per image). (D) BMDMs were infected with Mabs-R (MOI = 1 or 3) for 2 h and then incubated with solvent control (sc) or Rufomycin 4–7 (10 μM) in the freshly changed media. Intracellular survival of Mabs-R was determined by CFU assay at 0 and 1 dpi. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Statistical analysis was determined with unpaired t-test (A–C) or two-way ANOVA (D). Data are presented as means ± SEM (A–C), or ± SD from at least three independent experiments performed in triplicate (D). Images are representative of three independent experiments (B,C). MOI, multiplicity of infection; dpi, days post-infection.

FIGURE 2

Rufomycin 4–7 regulates the expression of inflammatory cytokines during Mabs-R infection. (A) BMDMs were infected with Mabs-R (MOI = 3) for 2 h and then incubated with sc or Rufomycin 4–7 (10 μM) in the freshly changed media. The cells were harvested at the indicated times and subjected to qRT-PCR analysis to measure the expression of inflammatory cytokine/chemokine genes. (B) The supernatants from the BMDMs prepared as in (A) were collected at 6 and 18 hpi and subjected to ELISA to measure the cytokine level of TNF-α, IL-1β, and IL-6. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Statistical analysis was determined with unpaired t-test and presented as means ± SD from at least three independent experiments performed in triplicate. ns, not significant; UI, uninfected; hpi, hours post-infection.

FIGURE 3

Rufomycin 4–7 decreases Mabs-R-induced p38-MAPK inflammatory signaling in macrophages. (A) BMDMs were pretreated with Rufomycin 4–7 (10 μM) or vehicle and infected with Mabs-R (MOI = 3) for the indicated times. Cells were lysed and subjected to immunoblot analysis with antibodies of p-NF-κB, p-ERK, p-JNK, p-p38, p-Akt, and β-actin. (B) Densitometric analysis was performed and normalized to β-actin with the p-p38 band. ***p < 0.001. Statistical analysis was determined with one-way ANOVA and presented as means ± SD (B). Western blot images are representative of three independent experiments (A). UI, uninfected.

FIGURE 4

Rufomycin 4–7 enhances TFEB-lysosomal gene expression during Mabs-R infection. BMDMs were infected with Mabs-R (MOI = 3) for 2 h and then incubated with sc or Rufomycin 4–7 (10 μM) in the freshly changed media for the designated time. (A) The mRNA expression level of TFEB was determined by qRT-PCR. (B) At 12 hpi, the cells were fixed, permed, and stained with anti-TFEB antibodies (green) and DAPI (blue) to visualize fluorescent images using confocal microscopy (scale bar: 12.5 μm). The cells with TFEB translocated into the nucleus was manually calculated from the confocal images (n = 8 for each group; at least 80 cells per image). (C) Cells were lysed and total RNAs were extracted, followed by qRT-PCR analysis with primers of TFEB-downstream autophagic/lysosomal genes including Uvrag, Beclin1, Gabarap, Rab7, Lamp1, and Lamp2. ^∗∗∗p < 0.001. Statistical analysis was determined with one-way ANOVA (A,C) or unpaired t-test (B). Data are presented as means ± SD from at least three independent experiments performed in triplicate. ns, not significant; UI, uninfected; hpi, hours post-infection.

FIGURE 5

Rufomycin 4–7 increases the colocalization of phagosomes and lysosomes against GFP-Mabs infection. BMDMs were infected with GFP-Mabs (MOI = 3) for 2 h and incubated with Rufomycin 4–7 (10 μM) for 18 h in the fresh media. Cells were then stained with LAMP1 (red) and DAPI (blue) and subjected to confocal microscopy. Representative confocal images from each group (scale bar: 5 μm) and the quantitative data of colocalization of LAMP1 and GFP-Mabs were presented (n = 10 for each group; at least 80 cells per image). ^∗∗p < 0.01. Statistical analysis was determined with unpaired t-test and presented as means ± SD from at least three independent experiments performed in triplicate.

FIGURE 6

Rufomycin 4–7 ameliorates mitochondrial damage and oxidative stress during Mabs-R infection. (A) BMDMs were infected with Mabs-R (MOI = 3) for 2 h and further cultured with Rufomycin 4–7 (1, 5, or 10 μM) for 2 h in the fresh media. The cells were stained with MitoSOX and visualized with confocal images (scale bar: 12.5 μm). (B) The relative intensity level of mtROS of infected BMDMs was calculated relative to UI (n = 9 for each group; at least 80 cells per image). (C) BMDMs were infected with Mabs-R (MOI = 3) for 2 h and cultured with Rufomycin 4–7 (10 μM) for 18 h. Cells were harvested and then subjected to TEM analysis. Representative images and their magnified images (a–c) from each group are shown (scale bar: 500 nm or 1 μm). (D) The ratio of damaged/total mitochondria was manually calculated from at least nine EM images. ^∗∗∗p < 0.001. Statistical analysis was determined with one-way ANOVA (B,D) and presented as means ± SD from at least three independent experiments performed in triplicate. UI, uninfected.