Comparison of Macrophage Immune Responses and Metabolic Reprogramming in Smooth and Rough Variant Infections of Mycobacterium mucogenicum

Abstract

Mycobacterium mucogenicum (Mmuc), a rapidly growing nontuberculous mycobacterium (NTM), can infect humans (posttraumatic wound infections and catheter-related sepsis). Similar to other NTM species, Mmuc exhibits colony morphologies of rough (Mmuc-R) and smooth (Mmuc-S) types. Although there are several case reports on Mmuc infection, no experimental evidence supports that the R-type is more virulent. In addition, the immune response and metabolic reprogramming of Mmuc have not been studied on the basis of morphological characteristics. Thus, a standard ATCC Mmuc strain and two clinical strains were analyzed, and macrophages were generated from mouse bone marrow. Cytokines and cell death were measured by ELISA and FACS, respectively. Mitochondrial respiration and glycolytic changes were measured by XF seahorse. Higher numbers of intracellular bacteria were found in Mmuc-R-infected macrophages than in Mmuc-S-infected macrophages. Additionally, Mmuc-R induced higher levels of the cytokines TNF-α, IL-6, IL-12p40, and IL-10 and induced more BMDM necrotic death. Furthermore, our metabolic data showed marked glycolytic and respiratory differences between the control and each type of Mmuc infection, and changes in these parameters significantly promoted glucose metabolism, extracellular acidification, and oxygen consumption in BMDMs. In conclusion, at least in the strains we tested, Mmuc-R is more virulent, induces a stronger immune response, and shifts bioenergetic metabolism more extensively than the S-type. This study is the first to report differential immune responses and metabolic reprogramming after Mmuc infection and might provide a fundamental basis for additional studies on Mmuc pathogenesis.

Keywords: Mycobacterium mucogenicum; TLR2; glycolysis; immune response; metabolism; mitochondrial respiration.

Figure 1

M. mucogenicum morphologies. (A) Colony characteristics of M. mucogenicum in 7H10 media after 7 days. (B) Scanning electron microscopy images of the Mmuc-ATCC (ATCC 49650) strain and other clinical strains. (C) The purified GPLs and the acetone-precipitated pellet (APP) from total lipids extracted from Mmuc-ATCC (lane l), Mmuc-S (lane 2), and Mmuc-R (lane 3) were analyzed by thin-layer chromatography (TLC) using chloroform/methanol (9:1, v/v) as the mobile phase. GPL: glycopeptidolipids.

Figure 2

M. mucogenicum is an intracellular bacteria. (A) Intracellular staining of CFSE-coupled Mmuc in BMDMs infected at an MOI of 10 bacteria per cell for 4 h. Cortical F-actin was stained using phalloidin-Texas red, and nuclei were stained with DAPI. Scale bar = 5 μm. (B) Infection kinetics of M. mucogenicum in BMDMs during the first 4 h postinfection. BMDMs were infected with Mmuc at an MOI of 10. At each indicated time point, cells were washed three times and lysed with 0.05% Triton X-100 containing distilled water to release intracellular bacteria. This experiment was repeated three times with similar results. (C) Growth profiles of Mmuc within BMDMs over a 5-day period after infection. BMDMs were infected at an MOI of 10. Noninternalized bacteria were washed off after 4 h. The number of bacterial colony-forming units (CFUs) was determined at the indicated time point after removing the supernatant. Values are the means of triplicate samples, and error bars represent standard deviations (SD) (** p < 0.01). This experiment was repeated three times with similar results.

Figure 3

Quantitative analyses of the necrosis and apoptosis of BMDMs infected with M. mucogenicum. Representative results of Annexin V–PI staining (A) and quantitative analysis (B). BMDMs were infected with Mmuc at an MOI of 10 for 24 h. After incubation, cells were collected, and FITC-Annexin V and PI were added. Samples were analyzed by flow cytometry. Values are mean ± SD of three samples (* p < 0.05, ** p < 0.01, *** p < 0.001 versus control group; ^### p < 0.001 versus Mmuc-ATCC group).

Figure 4

Comparison of inflammatory cytokine production by BMDMs infected with M. mucogenicum strains. BMDMs were infected with Mmuc strains at an MOI of 1 or 10 for 24 h. The results are presented as the mean pg/mL ± SD for all the experiments performed. Supernatants were collected, and the levels of TNF-α (A), IL-12p40 (B), IL-6 (C), and IL-10 (D) were determined by the enzyme-linked immunosorbent assay. Values are mean ± SD of three samples (* p < 0.05, ** p < 0.01, or *** p < 0.001). Un: uninfected.

Figure 5

M. mucogenicum-induced activation of MAPKs and NF-kB. (A) BMDMs were infected with the indicated Mmuc strains at an MOI of 10, and protein expression was detected at 30 min. Cell lysates were subjected to SDS–PAGE, and immunoblot analysis was performed using specific antibodies against phospho-p38 (p-p38), phospho-ERK1/2, phospho-JNK, and β-actin. (B–D) Each protein bands in A were scanned, and relative band intensities were normalized for the β-actin band. The column diagrams represent average relative band intensity with standard error from three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 versus control; ^# p ≤ 0.05, ^## p ≤ 0.01 for Mmuc-ATCC versus Mmuc-S or Mmuc-ATCC versus Mmuc-R or Mmuc-S versus Mmuc-R). ns: nonsignificant.

Figure 6

TLR2 plays an essential role in M. mucogenicum-induced inflammatory cytokine production by BMDMs. BMDMs from wild-type, TLR2^−/−, and TLR4^−/− mice were infected with Mmuc for 24 h at an MOI of 10 and then screened for the secretion of the cytokines TNF-α, IL-12p40, and IL-6 by the enzyme-linked immunosorbent assay. Values are mean ± SD of three samples (*** p < 0.001 for WT versus tlr2^−/−). UI: uninfected, I: infected.

Figure 7

Oxygen consumption rate of Mmuc-infected BMDMs. BMDMs were infected with Mmuc-ATCC, Mmuc-S, or Mmuc-R at an MOI of 10 for 24 h. (A) Mitochondrial respiration was determined while monitoring oxygen consumption rates (OCRs) with a Seahorse XFp analyzer using a Cell Mito stress test kit. The sequential injection of oligomycin (Oli, 1.0 μM), cyanide-4-[trifluoromethoxy] phenylhydrazone (FCCP, 2.0 μM), and rotenone/antimycin A (R/A, 0.5 μM) is indicated. (B–G) Representative nonmitochondrial oxygen consumption, basal respiration, maximum respiration, H⁺ (proton) leakage, ATP production, and spare respiratory capacity values were determined using a Seahorse XF Cell Mito stress report generator. The data are normalized to the protein concentration. All data are presented as the mean ± SD (n = 3). (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 versus control). ns: nonsignificant.

Figure 8

Extracellular acidification profiles and glycolytic parameters of Mmuc-infected BMDMs. BMDMs were infected with Mmuc-ATCC, Mmuc-S, or Mmuc-R at an MOI of 10 for 24 h. (A) Representative measurements of the extracellular acidification rates (ECARs) in Mmuc-infected BMDMs were acquired using the XF Glycolysis Stress Test kit. The sequential injection of glucose (Glu, 10 mM), oligomycin (Oli, 1.0 μM), and 2-deoxyglucose (2-DG, 50 mM) is indicated. (B–F) Representative glycolysis, glycolytic capacity, glycolytic reserve, nonglycolytic acidification, and glycolytic reserve as a percentage values were determined using the Seahorse XF Cell ECAR report generator. The data are normalized to the protein concentration. All data are presented as the mean ± SD (n = 3). (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 versus control; # p ≤ 0.05 for Mmuc-ATCC versus Mmuc-R). ns: nonsignificant.