Altered serine metabolism promotes drug tolerance in Mycobacterium abscessus via a WhiB7-mediated adaptive stress response

Abstract

Mycobacterium abscessus is an emerging opportunistic pathogen responsible for chronic lung diseases, especially in patients with cystic fibrosis. Treatment failure of M. abscessus infections is primarily associated with intrinsic or acquired antibiotic resistance. However, there is growing evidence that antibiotic tolerance, i.e., the ability of bacteria to transiently survive exposure to bactericidal antibiotics through physiological adaptations, contributes to the relapse of chronic infections and the emergence of acquired drug resistance. Yet, our understanding of the molecular mechanisms that underlie antibiotic tolerance in M. abscessus remains limited. In the present work, a mutant with increased cross-tolerance to the first- and second-line antibiotics cefoxitin and moxifloxacin, respectively, has been isolated by experimental evolution. This mutant harbors a mutation in serB2, a gene involved in L-serine biosynthesis. Metabolic changes caused by this mutation alter the intracellular redox balance to a more reduced state that induces overexpression of the transcriptional regulator WhiB7 during the stationary phase, promoting tolerance through activation of a WhiB7-dependant adaptive stress response. These findings suggest that alteration of amino acid metabolism and, more generally, conditions that trigger whiB7 overexpression, makes M. abscessus more tolerant to antibiotic treatment.

Keywords: Mycobacterium abscessus; WhiB7; antibiotic tolerance; mycobacteria; serine metabolism; β-lactam.

Fig 1

Isolation of M. abscessus cefoxitin-tolerant mutants by experimental evolution. (a) Schematic representation of the experimental evolution protocol. (b) MIC determination of cefoxitin for M. abscessus WT and MABserB2. (c) Evolution of the survival rate over the experimental evolution.

Fig 2

Genomic characterization of cefoxitin-tolerant mutants. (a) The phosphorylated pathway of L-serine biosynthesis and its connection with other metabolic pathways. (b) Mapping of the frame-shift mutation in the last codon of serB2. The frameshift mutation, along with amino acid changes, is shown in red.

Fig 3

Antibiotic cross-tolerance of tolerant mutant MABserB2. (a) Bactericidal activity of cefoxitin on stationary-phase cultures diluted 1:100 in the fresh medium of M. abscessus WT, MABserB2, and MABserB2 complemented with the WT allele of serB2 (MABserB2-rev; n = 6). (b) Bactericidal activity of cefoxitin on log-phase cultures of M. abscessus WT and MABserB2 (n = 3). (c) Bactericidal activity of imipenem (n = 6), moxifloxacin (n = 8), amikacin (n = 3), and rifabutin (n = 3) on stationary phase cultures diluted 1:100 in the fresh medium of M. abscessus WT and MABserB2. Statistical analyses were performed using Mann–Whitney U test. Error bars represent SD. P-value: *: P < 0.0332, **: P < 0.0021, ***: P < 0.0002, and ****: P < 0.0001.

Fig 4

Similar growth rate and lag time of M. abscessus WT and MABserB2. (a) Growth curves of the WT and MABserB2 strains in 7H9 (upper; n = 3) or M9 minimum medium (lower; n = 2). (b) Determination of the lag time by dilution of stationary-phase bacteria into fresh media and CFU counting (n = 3). (c) Determination of the lag time using the fluorescence dilution dual reporter plasmid pTIGc. Flow cytometric detection of TurboFP635 fluorescence is shown at time points 2, 4, and 8 hours for M. abscessus WT:pTIGc (dashed black lines) and MABserB2:pTIGc (dashed red lines). Gray and red plain-colored histograms correspond to the TurboFP635 fluorescence at time point 0 hour for the WT:pTIGc and MABserB2:pTIGc strains, respectively. (d) Percentage of TurboFP635 fluorescence intensity (FI) over time compared to fluorescence at t0 in the WT:pTIGc and MABserB2:pTIGc strains. Error bars represent SD.

Fig 5

Altered serine metabolism is responsible for the cefoxitin tolerance of MABserB2. (a) Supplementation with either serine or glycine suppresses the tolerant phenotype. Bactericidal activity of cefoxitin on stationary phase cultures diluted 1:100 in the fresh medium of M. abscessus WT and MABserB2, cultivated in the presence or absence of serine (left; n = 4) or glycine (right; n = 6). Statistical analyses were performed using Mann–Whitney U test. Error bars represent SD. P-value: *: P < 0.0332, **: P < 0.0021, ***: P < 0.0002, and ****: P < 0.0001. (b) Down-regulating serB2 expression by CRISPRi confers auxotrophy to serine. Growth of the strain WT transformed with pLJR965 (vector control) (1), p965MAB3117 (targeting essential control gene MAB_3117 c) (2), or p965MAB3388 (targeting serB2, two independent clones) (3 and 4) on 7H10 solid medium containing various combinations of ATc and serine. Seven microliters of serial dilutions (10⁻¹ to 10⁻⁶) of cultures of these strains grown in the absence of ATc were spotted on plates.

Fig 6

Metabolomics analysis of MABserB2. (a) Relative abundance of selected metabolites in the MABserB2 and WT strains, grown either in stationary phase without (stat.) and with serine (stat./serine) or in exponential phase (expo.). Each point represents the ratio of the mean abundance of the metabolite in the MABserB2 strain to that in the WT strain within one experiment, calculated from three biological replicates of each strain, each performed in duplicate. (b) α-ketoglutarate/glutamate ratio in the MABserB2 and WT strains, grown in stationary phase in the absence (stat.) or presence of serine (stat./serine) or in exponential phase (expo.). Each point represents the ratio of the mean abundance of α-ketoglutarate and glutamate for each strain within one experiment, calculated from three biological replicates, each performed in duplicate.

Fig 7

Tolerance of MABserB2 to cefoxitin is mediated by WhiB7. (a) RT-qPCR analysis of the expression of whiB7 in MABserB2 compared to the WT strain. Analyses were carried out on bacteria cultured in the exponential phase (expo.; n = 4) or in the stationary phase without (stat.; n = 6) or with serine (stat. + serine; n = 4). (b) Bactericidal activity of cefoxitin on stationary-phase cultures diluted 1:100 in the fresh medium of the WT and MABserB2 strains transformed with pLJR965 (control vector targeting no gene) and of MABserB2 transformed with p965MAB3508 (targeting whiB7; left; n = 5). RT-qPCR analysis of the expression of whiB7 in MABserB2 transformed with either pLJR965 or p965MAB3508 compared to the WT strain harboring pLJR965 (right; n = 2). Analyses were done on bacteria grown to stationary phase in the presence of ATc to induce the production of the dCAS9 protein and expression the sgRNA. (c) Bactericidal activity of cefoxitin on stationary phase cultures diluted 1:100 in the fresh medium of the WT, MABserB2, WTΔwhiB7, and MABserB2ΔwhiB7 strains and of the MABserB2ΔwhiB7 mutant complemented with the wild-type allele of whiB7 (n = 3). Statistical analyses were performed using Mann–Whitney U test. Error bars represent SD. P-value: *: P < 0.0332, **: P < 0.0021, ***: P < 0.0002, and ****: P < 0.0001.

Fig 8

WhiB7-dependent and -independent impacts of the oxidative stress inducer diamide on the tolerance of the WT M. abscessus to cefoxitin. Bactericidal activity of cefoxitin on stationary phase cultures diluted 1:100 in fresh medium without or with diamide added (a) both during the growth phase to the stationary phase and during cefoxitin treatment (after dilution) for the WT and WTΔwhiB7 strains (n = 4) and (b) during the growth phase only for the WT strain (n = 2). Bactericidal activity of cefoxitin on stationary phase cultures diluted 1:100 in fresh medium (n = 3) (c) or log-phase cultures (n = 3) (d) of the WT and WTΔwhiB7 strains without or with diamide only added during the cefoxitin treatment. Inset boxes depict the temporal addition of diamide in experiments. Error bars represent SD.

Fig 9

Proposed mechanism for cefoxitin tolerance in MABserB2. Metabolic disorders when MABserB2 reaches the stationary phase cause oxidative stress through the generation of RMB such as ROS and RNS. This would activate a stress response resulting in a shift of the redox balance toward a more reducing cellular environment, as reflected by a higher NADH/NAD⁺ ratio. This reduced environment, along with a decreased availability of ribosomes in the stationary phase, would trigger the activation and overexpression of whiB7, which would also contribute to the adaptive stress response via upregulation of WhiB7 regulon genes involved in redox homeostasis. The stress response induced in MABserB2 during the stationary phase would make the bacteria more apt to efficiently antagonize the production of RMB generated by the antibiotic, resulting in the decreased cell death rate and higher tolerance.