Table 1.
List of NTM isolates used in this study.
Fig 1.
Itaconic acid inhibits the growth of ATCC M. abscessus.
Smooth and rough ATCC M. abscessus was grown in 7H9 minimal media supplemented with 10 mM SCFAs: Acetate; propionate; or butyrate. IA was added at the indicated concentrations and an OD600 reading was taken each day to track bacterial growth. Statistical tests were performed using Two-Way ANOVA; Tukey’s multiple comparison test, on log transformed data. Error bars indicate standard deviation and significance indicates where the SCFA control was first found to be significantly different from 10 mM Itaconic acid samples. * p ≤ 0.05. ** p ≤ 0.005, ** p ≤ 0.0005, **** p ≤ 0.0001. n = 3.
Fig 2.
Itaconic acid inhibits the growth of ATCC M. avium.
ATCC M. avium was grown in 7H9 minimal media supplemented with 10 mM SCFAs: Acetate; propionate; or butyrate. IA was added at the indicated concentrations and an OD600 reading was taken each day to track bacterial growth. Statistical tests were performed using Two-Way ANOVA; Tukey’s multiple comparison test, on log transformed data. Error bars indicate standard deviation and significance indicates where the SCFA control was first found to be significantly different from 10 mM Itaconic acid samples. * p ≤ 0.05. ** p ≤ 0.005, ** p ≤ 0.0005, **** p ≤ 0.0001. n = 2–5.
Fig 3.
Itaconic acid inhibits the growth of clinically isolated M. abscessus.
Smooth and rough clinical isolates of M. abscessus (A) 253, (B) 711, (C) 1243, (D) 493, (E) 1238, were grown in 7H9 minimal media supplemented with 10 mM of the indicated SCFAs. IA was added at the indicated concentrations and an OD600 reading was taken each day to track bacterial growth. Statistical tests were performed using Two-Way ANOVA; Tukey’s multiple comparison test, on log transformed data. Error bars indicate standard deviation and significance indicates where the SCFA control was first found to be significantly different from 10 mM Itaconic acid samples. * p ≤ 0.05. ** p ≤ 0.005, ** p ≤ 0.0005, **** p ≤ 0.0001. n = 2–5.
Fig 4.
Itaconic acid inhibits the growth of clinically isolated MAC.
Clinical isolates of MAC (A) 622, (B) 813, (C) 1071, (D) 1151, were grown in 7H9 minimal media supplemented with 10 mM of the indicated SCFAs. IA was added at the indicated concentrations and an OD600 reading was taken each day to track bacterial growth. Statistical tests were performed using Two-Way ANOVA; Tukey’s multiple comparison test, on log transformed data. Error bars indicate standard deviation and significance indicates where the SCFA control was first found to be significantly different from 10 mM Itaconic acid samples. * p ≤ 0.05. ** p ≤ 0.005, ** p ≤ 0.0005, **** p ≤ 0.0001. n = 2–3.
Fig 5.
pH neutral itaconic acid does not inhibit the growth of M. abscessus and M. avium.
Clinically isolated (A) M. abscessus 253 and (B) M. avium 813 was grown in 7H9 minimal media supplemented with 10 mM SCFAs: Acetate, propionate, and butyrate. IA (pH≈7) was added at the indicated concentrations and an OD600 reading was taken each day to track bacterial growth. Statistical tests were performed using Two-Way ANOVA; Tukey’s multiple comparison test, on log transformed data. Error bars indicate standard deviation and significance indicates where the SCFA control was first found to be significantly different from 10 mM Itaconic acid samples. * p ≤ 0.05. ** p ≤ 0.005, ** p ≤ 0.0005, **** p ≤ 0.0001. n = 3–5.
Fig 6.
Increased acidity enhances the inhibitory effects of itaconic acid on M. abscessus and M. avium.
Clinical isolates of (A) smooth and rough M. abscessus 253 and (B) M. avium 813 was grown in 7H9 minimal media adjusted to pH = 6.1 supplemented with 10 mM SCFAs: Acetate, propionate, and butyrate. IA was added at the indicated concentrations and an OD600 reading was taken each day to track bacterial growth. Statistical tests were performed using Two-Way ANOVA; Tukey’s multiple comparison test, on log transformed data. Error bars indicate standard deviation and significance indicates where the SCFA control was first found to be significantly different from 10 mM Itaconic acid samples. * p ≤ 0.05. ** p ≤ 0.005, ** p ≤ 0.0005, **** p ≤ 0.0001. n = 3–6.
Fig 7.
Itaconic acid reduces the enzymatic activity of ICL in M. abscessus.
ATCC isolates of smooth and rough M. abscessus 253 grown in 7H9 media to stationary phase, collected, and lysed to acquire the bacterial ICL enzymes. IA (10 mM) was added to the cell lysate solutions and an OD340 reading was taken 20 minutes post addition to track enzymatic activity compared to the untreated controls. Statistical tests were performed using a paired t-test on log transformed data. Error bars indicate median with interquartile range and significance indicates where the SCFA control was first found to be significantly different from 10 mM Itaconic acid samples.* p ≤ 0.05. n = 3.
Fig 8.
4-OI enhances the clearance of MABC by THP-1 cells.
Clinically isolated M. abscessus 253 was added to differentiated THP-1 cells at an MOI of (A) 5:1 Smooth; (B) 5:1 Rough; and (C) 15:1 Smooth. 4-OI was added at the indicated concentrations (5 mM 4-OI for panel A) and cells were incubated at 37°C at 5% CO2 for the indicated lengths of time. Control refers to THP-1 cells exposed to NTM without the addition of any 4-OI. Statistical tests were performed using Two-Way ANOVA; Sidak’s multiple comparisons test, on log transformed data. Error bars indicate median with interquartile range and asterisks indicates a significant difference between the compared samples. **** p ≤ 0.0001. n = 2–4.
Fig 9.
4-OI enhances the clearance of MAC by THP-1 cells.
Clinically isolated M. avium 813 was added to differentiated THP-1 cells at an MOI of 15:1. 4-OI was added at the indicated concentrations and THP-1 cells with NTM were incubated at 37°C at 5% CO2 for the indicated lengths of time while the samples with NTM alone were incubated at 37°C. Control refers to THP-1 cells exposed to NTM without the addition of any 4-OI. Statistical tests were performed using Two-Way ANOVA; Sidak’s multiple comparisons test, on log transformed data. Error bars indicate median with interquartile range and asterisks indicates a significant difference between the compared samples. **** p ≤ 0.0001. n = 1–4.