c-di-AMP signaling plays important role in determining antibiotic tolerance phenotypes of Mycobacterium smegmatis

Abstract

In this study, we probe the role of secondary messenger c-di-AMP in drug tolerance, which includes both persister and resistant mutant characterization of Mycobacterium smegmatis. Specifically, with the use of c-di-AMP null and overproducing mutants, we showed how c-di-AMP plays a significant role in resistance mutagenesis against antibiotics with different mechanisms of action. We elucidated the specific molecular mechanism linking the elevated intracellular c-di-AMP level and high mutant generation and highlighted the significance of non-homology-based DNA repair. Further investigation enabled us to identify the unique mutational landscape of target and non-target mutation categories linked to intracellular c-di-AMP levels. Overall fitness cost of unique target mutations was estimated in different strain backgrounds, and then we showed the critical role of c-di-AMP in driving epistatic interactions between resistance genes, resulting in the evolution of multi-drug tolerance. Finally, we identified the role of c-di-AMP in persister cells regrowth and mutant enrichment upon cessation of antibiotic treatment.

Figure 1

Modulating intracellular c-di-AMP concentration affects the number of spontaneous resistant mutants against ciprofloxacin and rifampicin. (A) ciprofloxacin mutation frequency and (B) rifampicin mutation frequency were calculated for M. smegmatis WT, M. smegmatis ΔdisA, M. smegmatis Δpde and their respective complementation strains [N = 3]. All the strains were grown till stationary phase. Similarly, (C) ciprofloxacin mutation rate and (D) rifampicin mutation rate were calculated for M. smegmatis WT, M. smegmatis ΔdisA, M. smegmatis Δpde along with respective complementation strains [N = 3]. All the graphs are plotted using GraphPad Prism8, unpaired t-test was used to calculate statistical significance: ***P < 0.001; **P < 0.01; *P < 0.05; ns = non- significant.

Figure 2

(A) A bigger fold change in rifampicin mutation rate was observed for M. smegmatis Δpde strain compared to M. smegmatis WT strain after UV induced mutagenesis [N = 3]. (B) Representative microscopic images were shown to compare cell damage, cell filamentation and chromosome disintegration in both M. smegmatis WT and M. smegmatis Δpde strain after a sublethal dose of UV exposure (0.125 mJ/cm²). A lack of cellular filamentation in Δpde strain was indicative of the absence of RecA-mediated SOS repair in Δpde strain; white arrows indicate severe DNA condensation and damage. Microscopy scale bars = 2 µm. A plasmid-based assay was performed to estimate the NHEJ-driven repair phenomenon. Though (C) NHEJ efficiency was comparable between M. smegmatis WT and Δpde strain [N = 3], a significant drop in (D) NHEJ repair fidelity (~ 19%) was observed due to error-prone lacZ reannealing [N = 3]. (E) Knockdown of the principal NHEJ component Ku [by antisense (AS) approach] reversed the hypermutation phenotype in M. smegmatis Δpde strain, estimated by resistance frequency calculation against ciprofloxacin [N = 3]. All the graphs are plotted using GraphPad Prism8 (N = 3), unpaired t-test was used to calculate statistical significance: ***P < 0.001; **P < 0.01; *P < 0.05; ns = non-significant.

Figure 3

(A) Increased c-di-AMP concentration determines the mutational landscape and results in the high probability of non-QRDR (Category-I) mutations in Δpde strain, which are only resistant at to 10X MIC, but do not survive at higher 60X MIC of ciprofloxacin. A two-way ANOVA test was performed to check statistical significance [N = 3]. (B) EtBr efflux assay demonstrated a high degree of efflux activity in category I cip^R (non-QRDR) mutant, but not in category II cip^R (with a QRDR SNP) mutant or parental strains [N = 3]. (C) High fitness cost associated with D94N mutation in the GyrA protein subunit was observed in the case of Δpde (cip^R category II mutant) when cells had growth deficiency in minimal media and the fitness cost was neutralized to the M. smegmatis Δpde parental strain level by acquiring a second compensatory mutation (rif^R) after prolonged growth in M9 minimal media for 5 days [N = 3]. Estimation of the specific role of high intracellular c-di-AMP concentration driving positive epistatic interactions: (D) increased rifampicin mutation frequency of a cip^R category II mutant to become double mutant (cip^R, rif^R) was only favored when intracellular c-di-AMP concentration was high (in Δpde background), but not in WT background [N = 3] and (E) rifampicin mutation frequency dropped in Δpde cip^R category II mutant when normal physiological level of c-di-AMP was restored with pMV361-pde complementation [N = 3]. All the graphs are plotted using GraphPad Prism8, unpaired t-test was used to calculate statistical significance: ***P < 0.001; **P < 0.01; *P < 0.05; ns = non-significant.

Figure 4

(A) Regrowth of persisters (after 10X ciprofloxacin treatment and subsequent washing of the drug) was significantly decelerated by high c-di-AMP concentration in Δpde strain, unlike WT and the complementation strain (Δpde + pMV361-pde) [N = 3]. (B) Slower resuscitation of Δpde surviving cells results from a rapid enrichment of genetic mutants, around 93% of mutant population enrichment happened in Δpde strain within 24 h of the killing, whereas in the case of WT strain ~ 52% of cells became ciprofloxacin mutants [N = 3]. (C) Significantly slower induction of the P_rpfA (as a GFP readout) in Δpde background confirms the molecular basis of slower persisters regrowth [N = 3], when the c-di- AMP level was normalized in the same strain by a disA gene frameshift mutation, P_rpfA induction was almost restored to the WT level. All the graphs are plotted using GraphPad Prism8, unpaired t-test was used to calculate statistical significance: ***P < 0.001; **P < 0.01; *P < 0.05; ns = non-significant.

Figure 5

Schematic representation of our proposed model showing how high c-di-AMP concentration in M. smegmatis drives error-prone DNA repair which results in a significant change in the mutational landscape and fitness of a cell and finally promotes multi-drug resistance. The figure was created using the paid license from BioRender (www.biorender.com).