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Ranking of persister genes in the same Escherichia coli genetic background demonstrates varying importance of individual persister genes in tolerance to different antibiotics.

Wu N, He L, Cui P, Wang W, Yuan Y, Liu S, Xu T, Zhang S, Wu J, Zhang W, Zhang Y - Front Microbiol (2015)

Bottom Line: We found that oxyR, dnaK, sucB, relA, rpoS, clpB, mqsR, and recA were prominent persister genes involved in persistence to multiple antibiotics.These results indicate that varying hierarchy and importance of persister genes exist and that persister genes can be divided into those involved in shallow persistence and those involved in deep persistence.These findings have implications for improved understanding of persistence phenomenon and developing new drugs targeting persisters for more effective cure of persistent infections.

View Article: PubMed Central - PubMed

Affiliation: Key Lab of Molecular Virology, Institute of Medical Microbiology, Department of Infectious Diseases, Huashan Hospital, Fudan University Shanghai, China.

ABSTRACT
Despite the identification of many genes and pathways involved in the persistence phenomenon of bacteria, the relative importance of these genes in a single organism remains unclear. Here, using Escherichia coli as a model, we generated mutants of 21 known candidate persister genes and compared the relative importance of these mutants in persistence to various antibiotics (ampicillin, gentamicin, norfloxacin, and trimethoprim) at different times. We found that oxyR, dnaK, sucB, relA, rpoS, clpB, mqsR, and recA were prominent persister genes involved in persistence to multiple antibiotics. These genes map to the following pathways: antioxidative defense pathway (oxyR), global regulators (dnaK, clpB, and rpoS), energy production (sucB), stringent response (relA), toxin-antitoxin (TA) module (mqsR), and SOS response (recA). Among the TA modules, the ranking order was mqsR, lon, relE, tisAB, hipA, and dinJ. Intriguingly, rpoS deletion caused a defect in persistence to gentamicin but increased persistence to ampicillin and norfloxacin. Mutants demonstrated dramatic differences in persistence to different antibiotics at different time points: some mutants (oxyR, dnaK, phoU, lon, recA, mqsR, and tisAB) displayed defect in persistence from early time points, while other mutants (relE, smpB, glpD, umuD, and tnaA) showed defect only at later time points. These results indicate that varying hierarchy and importance of persister genes exist and that persister genes can be divided into those involved in shallow persistence and those involved in deep persistence. Our findings suggest that the persistence phenomenon is a dynamic process with different persister genes playing roles of variable significance at different times. These findings have implications for improved understanding of persistence phenomenon and developing new drugs targeting persisters for more effective cure of persistent infections.

No MeSH data available.


Related in: MedlinePlus

Deletion mutant ranking depends on cell survival over time under conditions of ampicillin exposure. Stationary phase cultures of W3110 (gray bars) and single-gene mutants (white bars) were diluted 100-fold and exposed to 100 μg/ml ampicillin for 4, 8, and 24 h. The data for each mutant was plotted and compared with that of W3110. Error bars indicate the standard deviation (n = 3). The asterisk indicates statistical significance as determined using Mann–Whitney U tests (∗P < 0.05).
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Figure 1: Deletion mutant ranking depends on cell survival over time under conditions of ampicillin exposure. Stationary phase cultures of W3110 (gray bars) and single-gene mutants (white bars) were diluted 100-fold and exposed to 100 μg/ml ampicillin for 4, 8, and 24 h. The data for each mutant was plotted and compared with that of W3110. Error bars indicate the standard deviation (n = 3). The asterisk indicates statistical significance as determined using Mann–Whitney U tests (∗P < 0.05).

Mentions: To determine the persister levels for the persister gene deletion mutants, the stationary phase cultures of the mutants and the wild-type strain W3110 were exposed to the following four antibiotics, respectively, including ampicillin (100 μg/ml), norfloxacin (4 μg/ml), gentamicin (20 μg/ml), and trimethoprim (64 μg/ml). The survival of the mutants at different time points under exposure of a single antibiotic was assessed. The results showed that about half the mutants were more susceptible to most antibiotics than the parent strain W3110. Upon treatment with ampicillin, the persister levels of most knockout strains decreased significantly at 2 h, while those of the wild-type strain decreased from 4 h antibiotic exposure (data not shown). It is worth noting that persister levels in oxyR, dnaK, recA, lon, relA, glpD, mqsR, phoU, and sucB mutants were below the limit of detection (10 CFU/ml) after ampicillin exposure after 1 day, whereas about three orders of magnitude (0.01%) of the parent control strain W3110 cells still remained (Figure 1). The persister gene mutants were ranked from 4 h, because almost all the mutants demonstrated the same magnitude of decrease before this time point. The results showed that deletion of recA, lon, oxyR, phoU, dnaK, and mqsR significantly decreased persister formation (>6.9-fold) compared with W3110. At 8 h, in addition to the six genes above, the relA, sucB, and glpD mutants also displayed a dramatic decrease in persister levels. Furthermore, the relE and clpB mutants showed a significant decrease at 24 h. Therefore, the 11 genes could be divided into three groups according to the time points when a significant defect in persistence was observed, suggesting that different persister genes play roles of variable importance at different time points during the persistence phenomenon.


Ranking of persister genes in the same Escherichia coli genetic background demonstrates varying importance of individual persister genes in tolerance to different antibiotics.

Wu N, He L, Cui P, Wang W, Yuan Y, Liu S, Xu T, Zhang S, Wu J, Zhang W, Zhang Y - Front Microbiol (2015)

Deletion mutant ranking depends on cell survival over time under conditions of ampicillin exposure. Stationary phase cultures of W3110 (gray bars) and single-gene mutants (white bars) were diluted 100-fold and exposed to 100 μg/ml ampicillin for 4, 8, and 24 h. The data for each mutant was plotted and compared with that of W3110. Error bars indicate the standard deviation (n = 3). The asterisk indicates statistical significance as determined using Mann–Whitney U tests (∗P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588708&req=5

Figure 1: Deletion mutant ranking depends on cell survival over time under conditions of ampicillin exposure. Stationary phase cultures of W3110 (gray bars) and single-gene mutants (white bars) were diluted 100-fold and exposed to 100 μg/ml ampicillin for 4, 8, and 24 h. The data for each mutant was plotted and compared with that of W3110. Error bars indicate the standard deviation (n = 3). The asterisk indicates statistical significance as determined using Mann–Whitney U tests (∗P < 0.05).
Mentions: To determine the persister levels for the persister gene deletion mutants, the stationary phase cultures of the mutants and the wild-type strain W3110 were exposed to the following four antibiotics, respectively, including ampicillin (100 μg/ml), norfloxacin (4 μg/ml), gentamicin (20 μg/ml), and trimethoprim (64 μg/ml). The survival of the mutants at different time points under exposure of a single antibiotic was assessed. The results showed that about half the mutants were more susceptible to most antibiotics than the parent strain W3110. Upon treatment with ampicillin, the persister levels of most knockout strains decreased significantly at 2 h, while those of the wild-type strain decreased from 4 h antibiotic exposure (data not shown). It is worth noting that persister levels in oxyR, dnaK, recA, lon, relA, glpD, mqsR, phoU, and sucB mutants were below the limit of detection (10 CFU/ml) after ampicillin exposure after 1 day, whereas about three orders of magnitude (0.01%) of the parent control strain W3110 cells still remained (Figure 1). The persister gene mutants were ranked from 4 h, because almost all the mutants demonstrated the same magnitude of decrease before this time point. The results showed that deletion of recA, lon, oxyR, phoU, dnaK, and mqsR significantly decreased persister formation (>6.9-fold) compared with W3110. At 8 h, in addition to the six genes above, the relA, sucB, and glpD mutants also displayed a dramatic decrease in persister levels. Furthermore, the relE and clpB mutants showed a significant decrease at 24 h. Therefore, the 11 genes could be divided into three groups according to the time points when a significant defect in persistence was observed, suggesting that different persister genes play roles of variable importance at different time points during the persistence phenomenon.

Bottom Line: We found that oxyR, dnaK, sucB, relA, rpoS, clpB, mqsR, and recA were prominent persister genes involved in persistence to multiple antibiotics.These results indicate that varying hierarchy and importance of persister genes exist and that persister genes can be divided into those involved in shallow persistence and those involved in deep persistence.These findings have implications for improved understanding of persistence phenomenon and developing new drugs targeting persisters for more effective cure of persistent infections.

View Article: PubMed Central - PubMed

Affiliation: Key Lab of Molecular Virology, Institute of Medical Microbiology, Department of Infectious Diseases, Huashan Hospital, Fudan University Shanghai, China.

ABSTRACT
Despite the identification of many genes and pathways involved in the persistence phenomenon of bacteria, the relative importance of these genes in a single organism remains unclear. Here, using Escherichia coli as a model, we generated mutants of 21 known candidate persister genes and compared the relative importance of these mutants in persistence to various antibiotics (ampicillin, gentamicin, norfloxacin, and trimethoprim) at different times. We found that oxyR, dnaK, sucB, relA, rpoS, clpB, mqsR, and recA were prominent persister genes involved in persistence to multiple antibiotics. These genes map to the following pathways: antioxidative defense pathway (oxyR), global regulators (dnaK, clpB, and rpoS), energy production (sucB), stringent response (relA), toxin-antitoxin (TA) module (mqsR), and SOS response (recA). Among the TA modules, the ranking order was mqsR, lon, relE, tisAB, hipA, and dinJ. Intriguingly, rpoS deletion caused a defect in persistence to gentamicin but increased persistence to ampicillin and norfloxacin. Mutants demonstrated dramatic differences in persistence to different antibiotics at different time points: some mutants (oxyR, dnaK, phoU, lon, recA, mqsR, and tisAB) displayed defect in persistence from early time points, while other mutants (relE, smpB, glpD, umuD, and tnaA) showed defect only at later time points. These results indicate that varying hierarchy and importance of persister genes exist and that persister genes can be divided into those involved in shallow persistence and those involved in deep persistence. Our findings suggest that the persistence phenomenon is a dynamic process with different persister genes playing roles of variable significance at different times. These findings have implications for improved understanding of persistence phenomenon and developing new drugs targeting persisters for more effective cure of persistent infections.

No MeSH data available.


Related in: MedlinePlus