A small volatile bacterial molecule triggers mitochondrial dysfunction in murine skeletal muscle

Abstract

Mitochondria integrate distinct signals that reflect specific threats to the host, including infection, tissue damage, and metabolic dysfunction; and play a key role in insulin resistance. We have found that the Pseudomonas aeruginosa quorum sensing infochemical, 2-amino acetophenone (2-AA), produced during acute and chronic infection in human tissues, including in the lungs of cystic fibrosis (CF) patients, acts as an interkingdom immunomodulatory signal that facilitates pathogen persistence, and host tolerance to infection. Transcriptome results have led to the hypothesis that 2-AA causes further harm to the host by triggering mitochondrial dysfunction in skeletal muscle. As normal skeletal muscle function is essential to survival, and is compromised in many chronic illnesses, including infections and CF-associated muscle wasting, we here determine the global effects of 2-AA on skeletal muscle using high-resolution magic-angle-spinning (HRMAS), proton ((1)H) nuclear magnetic resonance (NMR) metabolomics, in vivo (31)P NMR, whole-genome expression analysis and functional studies. Our results show that 2-AA when injected into mice, induced a biological signature of insulin resistance as determined by (1)H NMR analysis-, and dramatically altered insulin signaling, glucose transport, and mitochondrial function. Genes including Glut4, IRS1, PPAR-γ, PGC1 and Sirt1 were downregulated, whereas uncoupling protein UCP3 was up-regulated, in accordance with mitochondrial dysfunction. Although 2-AA did not alter high-energy phosphates or pH by in vivo (31)P NMR analysis, it significantly reduced the rate of ATP synthesis. This affect was corroborated by results demonstrating down-regulation of the expression of genes involved in energy production and muscle function, and was further validated by muscle function studies. Together, these results further demonstrate that 2-AA, acts as a mediator of interkingdom modulation, and likely effects insulin resistance associated with a molecular signature of mitochondrial dysfunction in skeletal muscle. Reduced energy production and mitochondrial dysfunctional may further favor infection, and be an important step in the establishment of chronic and persistent infections.

Figure 1. NMR spectra of in vivo ³¹P NMR saturation-transfer on mouse hind limb skeletal muscle.

Representative summed ³¹P-NMR spectra acquired from control and 2-AA treated mice at day 4, before (A) and after (B) saturation of the γ-ATP resonance, with the difference spectrum between the two shown below (A–B). The arrow on γ-ATP indicates the position of saturation (sat) by rf irradiation (−2.4 ppm). ppm, chemical shift in parts per million.

Figure 2. 2-AA treatment differentially modulates the genes involved in energy production and intermediate metabolism in skeletal muscle.

Differentially expressed genes involved in energy production (A) and intermediate metabolism (B) in response to 2-AA treatment. Grey boxes represent up–egulation, and black boxes represent down-regulation, of the respective gene in muscle from 2-AA treated versus control mice. The negative log10 of p-values represented by gray triangles are shown in the right vertical axis. The expression of certain key genes is down-regulated, consistent with the in vivo ³¹P-NMR data (Figure 1 and Table 2).

Figure 3. NMR spectra from ¹H-NMR HRMAS analysis of gastrocnemius skeletal muscle from 2-AA treated versus control mice.

The spectra were acquired from 2-AA treated mice at 4 d versus control mice, and scaled to the phosphocreatine plus creatine peak (3.02 ppm). Resonance signals of lipids correspond to: 1) terminal methyl CH₃ protons (0.9 ppm); 2) acyl chain methylene protons (CH₂)_n of intramyocellular lipids (IMCLs) (1.3 ppm); 3) methylene protons CH₂C-CO (1.6 ppm); 4) allylic methylene protons C = C-CH₂-C of monounsaturated fatty acyl moieties (MUFAs) (2.05 ppm); 5) α methylene protons CH₂CO (2.25 ppm); 6) diallylic methylene protons  = C-CH₂-C =  of polyunsaturated fatty acyl moieties (PUFAs); and 7) N-methyl protons of phosphocreatine and creatine (3.0 ppm), respectively. The NMR spectra demonstrate increased biomarkers of insulin resistance IMCLs.

Figure 4. 2-AA treatment dampens key metabolic protein levels in mouse skeletal muscle cells.

Western blotting of cellular extracts with specific antibodies of PGC-1β, PPAR-γ and Sirt-1 in 2-AA treated cells at the indicated time points. One representative experiment (out of three) is shown. Loading was normalized relative to mouse α-tubulin. Densitometric data are the average of three replicate experiments and are expressed as mean ± SD (vertical bars).*p<0.05 vs. naïve. DU, densitometric units.

Figure 5. Effects of 2-AA on viability of mouse muscle cells.

MTT assay measuring cell viability in mouse skeletal muscle cells (C2C12) after treatment with 0.8 mM 2-AA over time, as indicated in the figure. SDs (vertical bars) were calculated from three replicate experiments.

Figure 6. 2-AA treatment down-regulates muscle function.

Black bars indicate the number of down-regulated genes; gray bars indicate the number of up-regulated genes, in the skeletal muscle of mice 4 days post 2-AA treatment versus control mice (left vertical axis). The negative log10 of p-values represented by gray triangles are indicated on the right vertical axis.

Figure 7. Schematic model for 2-AA mediated molecular mechanisms of mitochondrial dysfunction in skeletal muscle.

2-AA triggers mitochondrial dysfunction via the down-regulation of peroxisome proliferative activated receptor, gamma, coactivator (PGC)-1β, peroxisome proliferator activated receptor (PPAR)-γ, sirtuin (Sirt)-1, the insulin signaling pathway, overall energy metabolism, and increased lipid accumulation.