The efflux pumps Rv1877 and Rv0191 play differential roles in the protection of Mycobacterium tuberculosis against chemical stress

Abstract

Background: It was previously shown that GlnA3_sc enabled Streptomyces coelicolor to survive in excess polyamines. However, subsequent studies revealed that Rv1878, the corresponding Mycobacterium tuberculosis (M.tb) ortholog, was not essential for the detoxification of spermine (Spm), in M.tb. On the other hand, the multi-drug efflux pump Rv1877 was previously shown to enable export of a wide range of compounds, while Rv0191 was shown to be more specific to chloramphenicol.

Rationale: Therefore, we first wanted to determine if detoxification of Spm by efflux can be achieved by any efflux pump, or if that was dependent upon the function of the pump. Next, since Rv1878 was found not to be essential for the detoxification of Spm, we sought to follow-up on the investigation of the physiological role of Rv1878 along with Rv1877 and Rv0191.

Approach: To evaluate the specificity of efflux pumps in the mycobacterial tolerance to Spm, we generated unmarked ∆rv1877 and ∆rv0191 M.tb mutants and evaluated their susceptibility to Spm. To follow up on the investigation of any other physiological roles they may have, we characterized them along with the ∆rv1878 M.tb mutant.

Results: The ∆rv1877 mutant was sensitive to Spm stress, while the ∆rv0191 mutant was not. On the other hand, the ∆rv1878 mutant grew better than the wild-type during iron starvation yet was sensitive to cell wall stress. The proteins Rv1877 and Rv1878 seemed to play physiological roles during hypoxia and acidic stress. Lastly, the ∆rv0191 mutant was the only mutant that was sensitive to oxidative stress.

Conclusion: The multidrug MFS-type efflux pump Rv1877 is required for Spm detoxification, as opposed to Rv0191 which seems to play a more specific role. Moreover, Rv1878 seems to play a role in the regulation of iron homeostasis and the reconstitution of the cell wall of M.tb. On the other hand, the sensitivity of the ∆rv0191 mutant to oxidative stress, suggests that Rv0191 may be responsible for the transport of low molecular weight thiols.

Keywords: MFS-type pump; Mycobacterium tuberculosis; Rv0191; Rv1877; Rv1878; cell wall stress; iron homeostasis; spermine stress.

Figure 1

Genotyping of mutants. (A) Southern blot design for the identification of the ∆rv1877 mutant. The restriction enzyme RSrII was used to digest M.tb genomic DNA, and the PCR fragment flanking the upstream region (US) was used as the probe. (B) Genotyping reveals a 5,980 bp band for the wild-type (WT) and a 16,002 bp band for the mutant. (C) Southern blot design for the identification of the ∆rv0191 mutant. The restriction enzymes AleI and SrfI were used to digest M.tb genomic DNA, and the PCR fragment flanking the downstream region (DS) was used as the probe. (D) Genotyping reveals a 4,939 bp band for the wild-type (WT) and a 9,794 bp band for the mutant.

Figure 2

Susceptibility of the mutants to Spm stress. (A) The ∆rv1877 mutant was exposed for 3 h to 2 mM Spm in Sauton’s media. Survival percentage was evaluated relative to the untreated DMSO control. A t-test between the wild-type and the mutant was performed using Prism 10 to determine statistical significance resulting to p = 0.015, and between the complement and the mutant to yield a p value of 0.0071. (B) The ∆rv0191 mutant was treated similarly. There was no difference observed. (C) The ∆rv1877 mutant was exposed for 7 days to 250 μM and 500 μM Spm in PBS. The survival percentage was derived relative to the CFUs obtained from day-1. A marginal sensitivity relative to the wild-type and an almost (p = 0.06, 2-way Anova) statistically significant sensitivity relative its complement was found. (D) The ∆rv0191 mutant was treated similarly, and it survived better than the wild-type under this condition. Alpha was set to 0.05, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 during the t-test.

Figure 3

Characterization of the mutants during oxidative stress (OS) and cell wall stress (0.5% SDS). (A) The ∆rv1877 mutant was exposed for 3 h to 2 mM CuOOH (OS). Survival percentage was evaluated relative to the untreated DMSO control since reagent was diluted in DMSO before each experiment. The mutant displayed no sensitivity. (B) The ∆rv1878 mutant was exposed similarly. It displayed only a marginal sensitivity. (C) The ∆rv0191 mutant was exposed similarly. It displayed a statistically significant sensitivity with p = 0.011. (D) The ∆rv1877 mutant was exposed for 2 days to 0.5%SDS. The survival percentage was derived relative to the CFUs obtained from day-1. It displayed a sensitivity that was not statistically significant. (E) The ∆rv1878 mutant was exposed similarly. It displayed a sensitivity that was statistically significant with a p value of 0.028. (F) The ∆rv0191 mutant was exposed similarly. It displayed no sensitivity to the stress. Alpha was set to 0.05, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 during the t-test.

Figure 4

Evaluation of the physiological of the mutants during hypoxia. (A) An oxygen meter was placed in the hypoxia system, and the percentage of oxygen displayed on the meter was recorded every10-20 min. (B) Using the integrated software in the lightcycler480, the expression level of specific genes (in the anaerobic condition) was determined relative to the house-keeping gene sigA, then the absolute value was determined relative to a standard curve, finally the fold change was derived relative to the expression levels in the aerobic agitated cultures. The gene rv1877, rv1878 and acr, were marginally upregulated. (C) The ∆rv1877 mutant seemed not to be sensitive. (D) The ∆rv1878 displayed a marginal sensitivity that was partially reversed in its complement. (E) The ∆rv0191 mutant also displayed no sensitivity. (F) When the survival percentage of the mutant was normalized to the wild-type’s, the ∆rv1877 mutant displayed a statistically significant sensitivity (p = 0.03), but this was not complemented. (G) The ∆rv1878 mutant still displayed a marginal sensitivity 4 h. The ∆rv0191 mutant remained not sensitive. Alpha was set to 0.05, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 during the t-test.

Figure 5

Evaluation of the physiological role of the mutants during iron starvation (IS). (A) The ∆rv1877 mutant was exposed for more than 2 days, to IS. Survival percentage was derived relative to CFUs obtained from day-1. It displayed a statically significant resistance at day-5 (p = 0.004); however, this phenotype was not reversed in its complement. (B) The ∆rv1878 mutant was treated similarly, and it displayed a statically resistance at day-2 (p = 0.04), which was reversed in its complemented strain. (C) The levels of ferric and ferrous irons were quantified in the wild-type and the ∆rv1878 mutant. The levels obtained in the mutants were normalized to the wild-type’s to obtain the fold Fe-levels. The mutant seemed to store more iron in ferric form, compared to the wild-type. (D) The ∆rv0191 exposed to IS, also displayed a statically resistance phenotype at day-5 (p = 0.01), which was partially reversed in its complement. Alpha was set to 0.05, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 during the t-test.

Figure 6

Evaluation of the survival of the mutants in acidified media. (A) When the ∆rv1877 mutant was exposed to acidified Sauton’s media for 24 h, it displayed a marginal sensitivity to acid stress. (B) For further analysis, the survival percentage of each strain was divided to the survival percentage of the wild-type to obtain the fold difference (FD), and in this case the mutant was statistically sensitive to acid stress (p = 0.0001). (C) However, it was not sensitive to acidified 7H9, 6d. Even when it was analyzed differently by using the FD. (E) The ∆rv1878 mutant was treated as described in 7a, and it was not sensitive to acidified Sauton’s media. (F) Neither was it, even when it was analyzed differently by using the FD. (G) However, it was significantly (p = 0.03) sensitive in acidified 7H9. (H) And also significantly sensitive (p = 0.0211) when it was analyzed differently by using the FD. (I) The ∆rv0191 was treated as described in 7a, and it was not sensitive in acidified Sauton’s media. (J) Neither was it, when the FD was derived. (K) Nor in acidified 7H9. (L) Also, not when data were further analyzed using the FD. Alpha was set to 0.05, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001during the t-test.