Journal: bioRxiv

Article Title: Glucose selectively drives a rapid oxidative burst and immunometabolic reprogramming in human neutrophils during Mycobacterium tuberculosis infection

doi: 10.64898/2026.05.05.722986

Figure Lengend Snippet: | Intact Mtb triggers an immediate, glucose-dependent oxidative burst in human neutrophils . a , Workflow for isolating neutrophils from healthy donors and measuring neutrophil oxidative burst and other bioenergetic parameters during Mtb infection using the Agilent Seahorse XFe96 Extracellular Flux Analyzer. All XF data were normalized to 10,000 cells using Gen5 software on an Agilent BioTek Cytation 5 Multimode Reader. Serum-free RPMI 1640 medium was used in all assays. b , Representations of XF assay design, including injection strategies for measuring extracellular acidification rate (ECAR), oxygen consumption rate (OCR), and oxidative burst. The magnitude of oxidative burst is determined by calculating the area under the OCR curve (AUC) and is plotted as column graphs. c , Oxidative burst of neutrophils initially cultured on glucose (10 mM; Glu and Glu + PMA groups) or other nutrients (10 mM) following stimulation with PMA. After calibration and three baseline measurements, PMA (100 ng/mL final concentration) was injected via the first injection port and OCR was measured. Approximately 100 min later, glucose (10 mM final concentration) was injected via the second port (final glucose concentration for Glu and Glu + PMA groups was 20 mM, all others were 10 mM) and OCR measured. The magnitude of the initial oxidative burst (UAC 1 st injection, PMA) was determined from the area between the first and second injections. The second oxidative burst (AUC 2nd injection, glucose) was determined from the AUC following the second injection. The overall oxidative burst (AUC Overall) is the sum of the two bursts. d , Neutrophil oxidative burst following Mtb infection (MOI of 5). Neutrophils cultured in medium containing 10 mM glucose were exposed to Mtb for 30 minutes, washed three times in glucose-free medium to remove extracellular bacilli, then cultured in medium containing 0-10 mM glucose. Uninfected controls were maintained in 10 mM glucose. AUC was calculated beginning with the first OCR measurement. e , Infection of neutrophils by injecting Mtb bacilli through XF injection port B in medium containing 10 mM glucose. After calibration and baseline measurements, rotenone (1.25 μM) and antimycin A (2.5 μM) were injected via port A to inhibit mitochondrial respiration. Mtb -GFP bacilli were injected through port B at an MOI of 1 or 5. After the completion of the XF run, Mtb -GFP distribution in the wells was visualized using the Agilent BioTek Cytation 5 Cell Imaging Multimode Reader to confirm uniform bacterial distribution. f , Oxidative burst of neutrophils cultured on glucose (0-10 mM) following injection of Mtb bacilli (MOI of 5) via port B. g, Oxidative burst of neutrophils cultured on glucose (10 mM) in response to live Mtb (MOI of 5), irradiated (irr.) Mtb , Mtb whole-cell lysate, or Mtb cellular fractions containing cell wall, cell membrane, or total lipids. h , Oxidative burst of neutrophils in medium containing various nutrients, with rotenone (1.25 μM) and antimycin A (2.5 μM) injected via port A prior to Mtb injection (MOI 1:5) via Port B. Glucose (10 mM final concentration) was later injected into all wells. i , Oxidative burst of neutrophils cultured in 10 mM glucose following infection with Mtb , M. avium , M. abscessus , S. aureus , or L. monocytogenes (all MOI of 5), via injection from port B. c–i, column graphs represent the magnitude of the oxidative burst and are shown as the mean ± SEM of the areas under the OCR curve for each group (n = 6-8 technical replicates per group). Statistical significance was determined using ordinary two-way ANOVA with Tukey’s multiple comparisons test (* P < 0.03, ** P < 0.002, *** P < 0.0002, **** P < 0.0001). All assays were performed independently three or more times.

Article Snippet: Liquid cultures of Mtb H37Rv and H37Rv-GFP, M. avium 2285 Rough (BEI Resources, NR-44264) and M. abscessus (Moore and Frerichs) Kusunoki and Ezaki (ATCC, cat # 19977) were grown at 37 °C with shaking in Middlebrook 7H9 broth (Thermo Fisher cat # DF0713-17-9) supplemented with 0.2% glycerol, ADS (albumin [Millipore Sigma cat # 3116956001], dextrose [Thermo Fisher cat # DF0155-17-4], and sodium chloride), and 0.02 % tyloxapol (Sigma-Aldrich cat # T8761).

Techniques: Infection, Software, XF Assay, Injection, Cell Culture, Concentration Assay, Imaging, Irradiation, Membrane

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: ETH improves the transformation efficiency of Mycobacterium abscessus . (A, B) Susceptibility of the indicated M. abscessus to the drug ETH (A) and EMB (B). The MIC 50 was defined as the concentration of the compound that inhibited bacterial growth by 50% and was analyzed by a nonlinear fit model in GraphPad Prism 8.0. Data are expressed as the mean ± SD of triplicate samples and are representative of two independent experiments. (C) Transformation efficiency of M. abscessus when different concentrations of ETH or EMB were present in the culture medium used for competent cell preparation. ETH (10, 20, and 40 μg/ml) or EMB (200, 400, and 800 μg/ml) was added to the M. abscessus culture. The transformation efficiency was defined as the total number of CFU generated per transformation. Statistically significant differences were determined using multiple paired t ‐tests. EMB, ethambutol; ETH, ethionamide. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Initially, M. abscessus ATCC 19977 was recovered on 7H10 solid medium.

Techniques: Transformation Assay, Concentration Assay, Generated

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: Genome editing using CRISPR‐Cas9‐assisted recombineering in M. abscessus. (A) Schematic showing the lacZ ‐targeting sgRNA and oligonucleotides used for recombineering. The oligonucleotides lacZ .lag (79 nt) and lacZ .lead (79 nt, sequences reverse and complementary to lacZ .lag) targeting the lagging strand and the leading strand of DNA replication were designed to introduce a continuous stop codon into the lacZ open reading frame, respectively. (B) Number of CFU per transformation and percentage of white transformant colonies after electroporation of the pSG‐ lacZ ‐1 plasmids and oligonucleotides into M. abscessus ATCC 19977 harboring the recombinase‐expression plasmid. lacZ‐1 and lacZ‐2 denote sgRNAs targeting two different sites in the lacZ gene. The transformants were plated on X‐gal plates for blue‐white screening. The black bar corresponds to the left Y axis, indicating the number of transformants, and the gray bar corresponds to the right Y axis, indicating the percentage of white colonies. Results represent the average of two independent experiments. (C) Transformation and recombination efficiency of the dual‐reporter detection system resulting from electroporation of the shear plasmid expressing gfp and the oligonucleotides targeting lacZ . Recombination efficiency was defined as the percentage of white colonies on the X‐gal plate. GFP positivity was calculated based on the proportion of GFP‐positive colonies. lacZ‐1 and lacZ‐2 denote sgRNAs targeting two different sites in the lacZ gene. (D) Transformation efficiency and recombination efficiency of gene deletion using CRISPR‐Cas9‐assisted dsDNA recombineering. Deletions of 620 bp were introduced into M. abscessus chromosomal DNA using approximately 1 kb dsDNA fragments. X‐gal, 5‐bromo‐4‐chloro‐3‐indolyl β‐ d ‐galactopyranoside.

Article Snippet: Initially, M. abscessus ATCC 19977 was recovered on 7H10 solid medium.

Techniques: CRISPR, Introduce, Transformation Assay, Electroporation, Expressing, Plasmid Preparation, Shear

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: MAB_2806–2807 is required for antibiotic resistance of Mycobacterium abscessus. (A) Disk diffusion assay of the MAB_2806‐2807 operon to different antibiotics. The zone of inhibition for FOX, LZD, and CLR was measured for Δ MAB_2806 , Δ MAB_2807 , and Δ MAB_2806–2807 , as well as their single‐ or double‐gene complementary strains. Statistically significant differences were determined using a paired two‐tailed t ‐test. All p values were calculated by comparison with the wild type. ** p < 0.01; *** p < 0.001; and **** p < 0.0001. (B) Uptake of EtBr by wild type, Δ MAB_2806‐2807 , and the complementary strain. CLR, clarithromycin; EtBr, ethidium bromide; FOX, cefoxitin; LZD, linezolid.

Article Snippet: Initially, M. abscessus ATCC 19977 was recovered on 7H10 solid medium.

Techniques: Diffusion-based Assay, Inhibition, Two Tailed Test, Comparison

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: Screening of efflux pump genes affecting the survival of M. abscessus in Galleria mellonella larvae. (A) Screening of an M.abscessus efflux pump mutant library in G. mellonella larvae. Efflux pump mutants and the wild‐type ATCC 19977 strain of M. abscessus harboring barcodes were mixed and used to infect G. mellonella larvae. At 3 and 5 days postinfection, the larvae were homogenized and plated. The plated bacteria were collected, and the barcode sequences were amplified for next‐generation sequencing analysis. Created in BioRender. Duan, D. (2025) https://BioRender.com/bi8qu91 . (B) Volcano plots depict the log 2 FC values and p values of each mutant from the library at 3 days (left) and 5 (right) days postinjection of G. mellonella larvae. Blue dots represent mutants depleted from G. mellonella larvae, while red dots represent those that are enriched. Gray dots indicate mutants showing no significant difference. The horizontal dashed line indicates a p ‐value of 0.05, while the left and right vertical dashed lines indicate log 2 FC of −1 and 1, respectively.

Article Snippet: Initially, M. abscessus ATCC 19977 was recovered on 7H10 solid medium.

Techniques: Mutagenesis, Bacteria, Amplification, Next-Generation Sequencing

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: ETH improves the transformation efficiency of Mycobacterium abscessus . (A, B) Susceptibility of the indicated M. abscessus to the drug ETH (A) and EMB (B). The MIC 50 was defined as the concentration of the compound that inhibited bacterial growth by 50% and was analyzed by a nonlinear fit model in GraphPad Prism 8.0. Data are expressed as the mean ± SD of triplicate samples and are representative of two independent experiments. (C) Transformation efficiency of M. abscessus when different concentrations of ETH or EMB were present in the culture medium used for competent cell preparation. ETH (10, 20, and 40 μg/ml) or EMB (200, 400, and 800 μg/ml) was added to the M. abscessus culture. The transformation efficiency was defined as the total number of CFU generated per transformation. Statistically significant differences were determined using multiple paired t ‐tests. EMB, ethambutol; ETH, ethionamide. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: The M. abscessus ATCC 19977 WT and efflux pump mutant strains were cultured in 7H9 without OADC until the OD 600 reached ~1.0, followed by dilution to an OD 600 of ~0.7.

Techniques: Transformation Assay, Concentration Assay, Generated

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: Genome editing using CRISPR‐Cas9‐assisted recombineering in M. abscessus. (A) Schematic showing the lacZ ‐targeting sgRNA and oligonucleotides used for recombineering. The oligonucleotides lacZ .lag (79 nt) and lacZ .lead (79 nt, sequences reverse and complementary to lacZ .lag) targeting the lagging strand and the leading strand of DNA replication were designed to introduce a continuous stop codon into the lacZ open reading frame, respectively. (B) Number of CFU per transformation and percentage of white transformant colonies after electroporation of the pSG‐ lacZ ‐1 plasmids and oligonucleotides into M. abscessus ATCC 19977 harboring the recombinase‐expression plasmid. lacZ‐1 and lacZ‐2 denote sgRNAs targeting two different sites in the lacZ gene. The transformants were plated on X‐gal plates for blue‐white screening. The black bar corresponds to the left Y axis, indicating the number of transformants, and the gray bar corresponds to the right Y axis, indicating the percentage of white colonies. Results represent the average of two independent experiments. (C) Transformation and recombination efficiency of the dual‐reporter detection system resulting from electroporation of the shear plasmid expressing gfp and the oligonucleotides targeting lacZ . Recombination efficiency was defined as the percentage of white colonies on the X‐gal plate. GFP positivity was calculated based on the proportion of GFP‐positive colonies. lacZ‐1 and lacZ‐2 denote sgRNAs targeting two different sites in the lacZ gene. (D) Transformation efficiency and recombination efficiency of gene deletion using CRISPR‐Cas9‐assisted dsDNA recombineering. Deletions of 620 bp were introduced into M. abscessus chromosomal DNA using approximately 1 kb dsDNA fragments. X‐gal, 5‐bromo‐4‐chloro‐3‐indolyl β‐ d ‐galactopyranoside.

Article Snippet: The M. abscessus ATCC 19977 WT and efflux pump mutant strains were cultured in 7H9 without OADC until the OD 600 reached ~1.0, followed by dilution to an OD 600 of ~0.7.

Techniques: CRISPR, Introduce, Transformation Assay, Electroporation, Expressing, Plasmid Preparation, Shear

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: MAB_2806–2807 is required for antibiotic resistance of Mycobacterium abscessus. (A) Disk diffusion assay of the MAB_2806‐2807 operon to different antibiotics. The zone of inhibition for FOX, LZD, and CLR was measured for Δ MAB_2806 , Δ MAB_2807 , and Δ MAB_2806–2807 , as well as their single‐ or double‐gene complementary strains. Statistically significant differences were determined using a paired two‐tailed t ‐test. All p values were calculated by comparison with the wild type. ** p < 0.01; *** p < 0.001; and **** p < 0.0001. (B) Uptake of EtBr by wild type, Δ MAB_2806‐2807 , and the complementary strain. CLR, clarithromycin; EtBr, ethidium bromide; FOX, cefoxitin; LZD, linezolid.

Article Snippet: The M. abscessus ATCC 19977 WT and efflux pump mutant strains were cultured in 7H9 without OADC until the OD 600 reached ~1.0, followed by dilution to an OD 600 of ~0.7.

Techniques: Diffusion-based Assay, Inhibition, Two Tailed Test, Comparison

Journal: mLife

Article Title: CRISPR‐based genome editing reveals the roles of efflux pumps in Mycobacterium abscessus

doi: 10.1002/mlf2.70048

Figure Lengend Snippet: Screening of efflux pump genes affecting the survival of M. abscessus in Galleria mellonella larvae. (A) Screening of an M.abscessus efflux pump mutant library in G. mellonella larvae. Efflux pump mutants and the wild‐type ATCC 19977 strain of M. abscessus harboring barcodes were mixed and used to infect G. mellonella larvae. At 3 and 5 days postinfection, the larvae were homogenized and plated. The plated bacteria were collected, and the barcode sequences were amplified for next‐generation sequencing analysis. Created in BioRender. Duan, D. (2025) https://BioRender.com/bi8qu91 . (B) Volcano plots depict the log 2 FC values and p values of each mutant from the library at 3 days (left) and 5 (right) days postinjection of G. mellonella larvae. Blue dots represent mutants depleted from G. mellonella larvae, while red dots represent those that are enriched. Gray dots indicate mutants showing no significant difference. The horizontal dashed line indicates a p ‐value of 0.05, while the left and right vertical dashed lines indicate log 2 FC of −1 and 1, respectively.

Article Snippet: The M. abscessus ATCC 19977 WT and efflux pump mutant strains were cultured in 7H9 without OADC until the OD 600 reached ~1.0, followed by dilution to an OD 600 of ~0.7.

Techniques: Mutagenesis, Bacteria, Amplification, Next-Generation Sequencing