In Mycobacterium abscessus, the Stringent Factor Rel Regulates Metabolism but Is Not the Only (p)ppGpp Synthase

Abstract

The stringent response is a broadly conserved stress response system that exhibits functional variability across bacterial clades. Here, we characterize the role of the stringent factor Rel in the nontuberculous mycobacterial pathogen, Mycobacterium abscessus (Mab). We found that deletion of rel does not ablate (p)ppGpp synthesis and that rel does not provide a survival advantage in several stress conditions or in antibiotic treatment. Transcriptional data show that Rel_Mab is involved in regulating expression of anabolism and growth genes in the stationary phase. However, it does not activate transcription of stress response or antibiotic resistance genes and actually represses transcription of many antibiotic resistance genes. This work shows that there is an unannotated (p)ppGpp synthetase in Mab. IMPORTANCE In this study, we examined the functional roles of the stringent factor Rel in Mycobacterium abscessus (Mab). In most species, stringent factors synthesize the alarmone (p)ppGpp, which globally alters transcription to promote growth arrest and survival under stress and in antibiotic treatment. Our work shows that in Mab, an emerging pathogen that is resistant to many antibiotics, the stringent factor Rel is not solely responsible for synthesizing (p)ppGpp. We find that Rel_Mab downregulates many metabolic genes under stress but does not upregulate stress response genes and does not promote antibiotic tolerance. This study implies that there is another critical but unannotated (p)ppGpp synthetase in Mab and suggests that Rel_Mab inhibitors are unlikely to sensitize Mab infections to antibiotic treatment.

Keywords: (p)ppGpp; Mab; Mycobacterium abscessus; RNA-seq; RelA; antibiotic tolerance; ppGpp; pppGpp; stress adaptation; stress response.

FIG 1

Thin-layer chromatography (TLC) of guanine nucleotides extracted from ³²P-labeled M. abscessus strains from logarithmic phase and starvation.

FIG 2

Contribution of rel_Mab to survival in various stresses. (A) CFU of wild-type M. abscessus strain ATCC 19977 (WT), the Δrel_Mab strain, and the complemented Δrel_Mab L5::rel_Mab strain in Hartman’s du Bont medium with no glycerol and tyloxapol as a detergent. (B) CFU in 7H9 Middlebrook medium with a pH of 4. (C) CFU in Lennox LB with 1 M NaCl. (D) CFU in Hartmans du Bont medium with 5, 25, or 60 mM tert-butyl peroxide after 24 h. Relative CFU is calculated by taking the ratio between each CFU value and the initial CFU value at time zero. All data points are averages from three biological replicates. Error bars represent standard deviation. There are no significant differences in any of these data by a two-tailed Student’s t test.

FIG 3

Growth and stationary-phase survival of Δrel_Mab. (A) CFU in the stationary phase in 7H9 medium. The 2-day time point is 48 h after diluting logarithmic phase cultures to optical density at 600 nm (OD₆₀₀) = 0.05. (B) CFU during logarithmic phase growth in 7H9 medium. The graph is set at a log₂ scale. Doubling times ± 95% confidence interval are as follows: wild type (WT), 2.15 h ± 0.08; Δrel_Mab strain, 4.67 h ± 4.26; Δrel_Mab L5::rel_Mab, 2.84 ± 1.85. The P values are for the wild type compared to Δrel_Mab. are as follows: for t = 4, P = 0.0415; for t = 7, P = 0.00015; for t = 10, P = 0.006; and for t = 12, P = 0.00029. The P values between wild type and the complemented strain were not significant. The P values between strains in the stationary phase were not significant (data not shown). Asterisks represent significance as measured by the two-tailed Student’s t test: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; NS, P > 0.05.

FIG 4

Contribution of rel_Mab to survival in antibiotic treatment. Relative CFU of strains treated with either 200 μg/mL of clarithromycin (Clar), 150 μg/mL of amikacin (Amk), or 80 μg/mL of cefoxitin (Fox) for either (A) 48 h in logarithmic phase or (B) 72 h in the stationary phase. Relative CFU were calculated by taking the ratio between the CFU value after treatment and the initial CFU value at time zero. The bars represent the mean of six to nine biological replicates; the individual values are shown by the dots. Error bars represent standard deviation. The logarithmic phase P values were as follows: (Amk150) WT versus Δrel_Mab strain, 0.005; WT versus Δrel_Mab L5::rel_Mab strain, 0.01; Δrel_Mab versus Δrel_Mab L5::rel_Mab strain, 0.004. The stationary phase P values were as follows: (AMK150) WT versus Δrel_Mab strain, 0.00017; WT versus Δrel_Mab L5::rel_Mab strain, 0.0217; Δrel_Mab versus Δrel_Mab L5::rel_Mab strain, 0.0017. Asterisks represent significance as measured by the two-tailed Student’s t test: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; NS, P > 0.05.

FIG 5

Repression of central metabolic genes by Rel_Mab during stationary phase. Genes in red are downregulated by Rel_Mab at least 3-fold in the stationary phase, P < 0.05. Genes in black are not significantly regulated by Rel_Mab in the stationary phase. See Table S4 for data. TCA, tricarboxylic acid.