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. 2024 May 8;15(5):e0037424.
doi: 10.1128/mbio.00374-24. Epub 2024 Apr 2.

Differential impacts of DNA repair machinery on fluoroquinolone persisters with different chromosome abundances

Juechun Tang #  1 , Allison M Herzfeld #  2   3 , Gabrielle Leon  1 , Mark P Brynildsen  1   2
Affiliations

Differential impacts of DNA repair machinery on fluoroquinolone persisters with different chromosome abundances

Juechun Tang et al. mBio. .

Abstract

DNA repair machinery has been found to be indispensable for fluoroquinolone (FQ) persistence of Escherichia coli. Previously, we found that cells harboring two copies of the chromosome (2Chr) in stationary-phase cultures were more likely to yield FQ persisters than those with one copy of the chromosome (1Chr). Furthermore, we found that RecA and RecB were required to observe that difference, and that loss of either more significantly impacted 2Chr persisters than 1Chr persisters. To better understand the survival mechanisms of persisters with different chromosome abundances, we examined their dependencies on different DNA repair proteins. Here, we show that lexA3 and ∆recN negatively impact the abundances of 2Chr persisters to FQs, without significant impacts on 1Chr persisters. In comparison, ∆xseA, ∆xseB, and ∆uvrD preferentially depress 1Chr persistence to levels that were near the limit of detection. Collectively, these data show that the DNA repair mechanisms used by persisters vary based on chromosome number, and suggest that efforts to eradicate FQ persisters will likely have to take heterogeneity in single-cell chromosome abundance into consideration.

Importance: Persisters are rare phenotypic variants in isogenic populations that survive antibiotic treatments that kill the other cells present. Evidence has accumulated that supports a role for persisters in chronic and recurrent infections. Here, we explore how an under-appreciated phenotypic variable, chromosome copy number (#Chr), influences the DNA repair systems persisters use to survive fluoroquinolone treatments. We found that #Chr significantly biases the DNA repair systems used by persisters, which suggests that #Chr heterogeneity should be considered when devising strategies to eradicate these troublesome bacterial variants.

Keywords: ciprofloxacin; levofloxacin; lexA; persistence; recN; uvrD; xseA; xseB.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Exonuclease VII mutants show impaired persistence levels to FQ treatments. Wild type, ∆xseA, ∆xseB, or ∆xseA∆xseB were grown to stationary phase as described in Materials and Methods and treated with (A) 5 μg/mL LEVO or (B) 1 μg/mL CIP. Data denote means ± SEMs for three or more biological replicates. Statistical analyses were performed using one-way analysis of variance assessing the effects of gene deletion to log-transformed survival fraction after 5 h FQ treatment followed by Tukey honestly significant difference (HSD) post hoc tests for multiple comparisons [LEVO: F(3,18) = 94.2, P = 3.4e − 11; CIP: F(3,9) = 99.7, P = 5.7e − 6]. Asterisk (⁠∗) denotes statistical significance (adjusted P < 0.05) between indicated mutant strain and wild type.
Fig 2
Fig 2
Complementation and genomic replacement establish the importance of ExoVII to FQ persistence. All strains were grown to stationary phase and treated with 5 µg/mL LEVO for 5 h. (A) Complementation of xseB via a low copy plasmid restores persistence to wild-type levels. (B) Replacement of xseA via a genomic integration technique. The nonfunctional truncated xseA, xseA150, and mutations in the DNA-binding domain [xseA (F63A) kanR] or catalytic domain [xseA (D155A) kanR] of XseA showed persister levels similar to that of ∆xseA, whereas restoration of the wild-type allele (xseA+ kanR) did not have an impact on persistence. Data denote means ± SEM for three or more biological replicates. Statistical analyses were performed using one-way analysis of variance at 5 h FQ treatment time point followed by Tukey HSD post hoc tests for multiple comparisons [XseB: F(2,10) = 5.8, P = 0.021; XseA: F(5,20) = 80.8, P = 1.4e − 12]. Asterisk (⁠∗) denotes statistical significance (adjusted P < 0.05) in log-transformed survival between indicated mutants (+/− plasmid) and wild type (+/− plasmid).
Fig 3
Fig 3
Exonucleases with redundant function with ExoVII for other processes do not impact FQ persistence. ΔxonA, ΔexoX, ΔrecJ, ΔsbcC, and ΔsbcD were grown to stationary phase and treated with 5 µg/mL LEVO for 5 h. All tested mutants showed comparable survival as wild type. Data denote means ± SEMs for three or more biological replicates. Statistical analyses were performed using one-way analysis of variance assessing the effects of gene deletion to survival fraction at 5 h FQ treatment time point [F(5,15) = 1.6, P = 0.2].
Fig 4
Fig 4
DNA repair mutants differentially impact 1Chr and 2Chr persister levels. Survival of sorted subpopulations in wild-type and DNA repair mutants after 5 h of treatment with 5 µg/mL LEVO. Data denote mean ± SEM for three or more biological replicates. Statistical analyses were conducted on the log-transformed survival after 5 h LEVO treatment on (i) 1Chr cells, (ii) 2Chr cells, (iii) total population, and (iv) fold-change in survival between 2Chr and 1Chr cells, respectively, using one-way analysis of variance followed by Tukey HSD post hoc tests [1Chr: F(6,22) = 50.7, P = 9.0e − 12; 2Chr: F(6,22) = 27.4, P = 3.8e − 9; total: F(6,22) = 25.7, P = 7.0e − 9; 2Chr/1Chr: F(6,22) = 18.9, P = 1.3e − 7]. Values in red denote statistical significance (adjusted P < 0.05) between indicated mutant strain and wild type using the same measurement (column name). lexA3 and ∆recN showed significant declines in 2Chr survival when compared with wild-type 2Chr cells, whereas ∆xseA, ∆xseB, ∆xseA∆xseB, and ∆uvrD showed reductions in survival for both 1Chr and 2Chr cells, with a greater impact on 1Chr cells. IData from reference (41) based on sorting protocol with slight modifications from present study. Statistical analyses for ∆recA and ∆recB were performed following the same procedure as other mutant strains except that we compared them with the wild-type data obtained using the same sorting protocol [1Chr: F(2,10) = 26.0, P = 0.0001; 2Chr: F(2,10) = 65.9, P = 1.7e − 6; total: F(2,10) = 56.9, P = 3.5e − 6; 2Chr/1Chr: F(2,10) = 27.0, P = 9.2e − 5].
Fig 5
Fig 5
Complementation and genomic replacement restore FQ persistence to XseA and UvrD mutants. Survival of sorted subpopulations of (A) xseA150, (B) xseA+ kanR, (D) ∆uvrD + pUA66 (empty vector), and (E) uvrD-complemented strain following LEVO persister assay. Untreated controls denote samples treated with sterile water. (C) Survival of wild-type + pUA66, ∆uvrD + pUA66, and uvrD-complemented strain following the standard persistence assay protocol. Data denote mean ± SEM of two or more biological replicates. Statistical analyses were performed using one-way analysis of variance at 5 h FQ treatment time point followed by Tukey HSD post hoc test for multiple comparisons [F(2, 7) =146.5, P = 1.94e − 6]. Asterisk (⁠∗) denotes statistical significance (adjusted P < 0.05) in log-transformed survival between indicated mutant with plasmid and wild type with plasmid.
Fig 6
Fig 6
UvrD and ExoVII impact 1Chr survival more than that of 2Chr cells after CIP treatment. Survival of wild-type, ∆xseA, and ∆uvrD in 1Chr-sorted, 2Chr-sorted, and total-sorted subpopulations after 5 h of treatment with 1 µg/mL CIP. Data denote mean ± SEM of three or more biological replicates. Statistical analyses were conducted on log-transformed survival after 5 h CIP treatment of (i) 1Chr cells, (ii) 2Chr cells, (iii) total population, and (iv) fold-change in survival between 2Chr and 1Chr cells, respectively, using one-way analysis of variance followed by Tukey HSD post hoc tests. [1Chr: F(2,8) =48.1, P = 3.5e − 5; 2Chr: F(2,8) = 26.1, P = 0.0003; total: F(2,8) = 43.5, P = 5.0e − 5; 2Chr/1Chr: F(2,8) = 12.3, P = 0.003]. Values in red denote statistical significance (adjusted P < 0.05) between indicated mutant strain and wild type of the same #Chr. Both ∆xseA and ∆uvrD showed a greater impact on 1Chr survival than 2Chr survival.
Fig 7
Fig 7
ExoVII is necessary for FQ persisters only in the presence of UvrD. Wild type, ∆xseA, ∆uvrD, or ∆uvrD∆xseA were grown to stationary phase as described in Materials and Methods and treated with 5 μg/mL LEVO. Data denote means ± SEMs of three or more biological replicates. Statistical analyses were performed using one-way analysis of variance assessing the effects of gene deletion(s) to log-transformed survival fraction after 5 h FQ treatment followed by Tukey HSD post hoc tests for multiple comparisons [F(3,18) = 39.5, P = 4.0e − 8]. Asterisk (⁠∗) denotes statistical significance (adjusted P < 0.05) between indicated strain and double-mutant ∆uvrD∆xseA.
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