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Endopeptidase-mediated beta lactam tolerance.

Dörr T, Davis BM, Waldor MK - PLoS Pathog. (2015)

Bottom Line: In response to a wide variety of cell wall--acting antibiotics, this pathogen loses its rod shape, indicative of cell wall degradation, and becomes spherical.Other autolysins proved dispensable for this process.Our findings suggest the enzymes that mediate cell wall degradation are critical for determining bacterial cell fate--sphere formation vs. lysis--after treatment with antibiotics that target cell wall synthesis.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Brigham and Women's Hospital and Howard Hughes Medical Institute, Boston, Massachusetts, United States of America; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
In many bacteria, inhibition of cell wall synthesis leads to cell death and lysis. The pathways and enzymes that mediate cell lysis after exposure to cell wall-acting antibiotics (e.g. beta lactams) are incompletely understood, but the activities of enzymes that degrade the cell wall ('autolysins') are thought to be critical. Here, we report that Vibrio cholerae, the cholera pathogen, is tolerant to antibiotics targeting cell wall synthesis. In response to a wide variety of cell wall--acting antibiotics, this pathogen loses its rod shape, indicative of cell wall degradation, and becomes spherical. Genetic analyses revealed that paradoxically, V. cholerae survival via sphere formation required the activity of D,D endopeptidases, enzymes that cleave the cell wall. Other autolysins proved dispensable for this process. Our findings suggest the enzymes that mediate cell wall degradation are critical for determining bacterial cell fate--sphere formation vs. lysis--after treatment with antibiotics that target cell wall synthesis.

No MeSH data available.


Related in: MedlinePlus

Inhibition of cell-wall synthesis in V. cholerae leads to sphere formation.(A) Kinetics of viable V. cholerae cell counts after cells were exposed to various antibiotics that inhibit cell wall synthesis. Cells were grown to ~ 2–3 x 108 cfu/ml and then exposed to 100 μg/ml of penicillin G (penG), phosphomycin (phos), D-cycloserine (cyc), ampicillin (amp) or no antibiotic (no AB) (T0). Data shown are averages of two independent experiments; error bars represent standard deviation. (B) Images of V. cholerae cells at different time points after exposure to penicillin G from the experiment shown in (A). (C) Morphology of wt exponential phase cells exposed to 100 μg/ml cyc, phos, amp or 1 μg/ml meropenem (mero) for 3 h. (D) Time lapse images of wt cells plated on an agarose pad containing 100 μg/ml pen G. Frames are 5 min apart, scale bar = 2 μm. Arrowheads point to selected blebs. (E) Localization of blebs after exposure to pen G. Three different time lapse series with ~ 40 cells each were obtained as described in (D) and bleb location as the ratio of distance from an arbitrarily chosen pole divided by cell length was measured using ImageJ software. (F) Time lapse images of wt cells stained with the fluorescent D amino acid analogue HADA (50 μM) for 30 min and washed 2 x to remove excess dye prior to imaging on an agarose pad containing pen G. Images were brightness/contrast-adjusted to compensate for photobleaching. Frames are 5 min apart, scale bar = 2 μm. HADA stain is false-colored in red.
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ppat.1004850.g001: Inhibition of cell-wall synthesis in V. cholerae leads to sphere formation.(A) Kinetics of viable V. cholerae cell counts after cells were exposed to various antibiotics that inhibit cell wall synthesis. Cells were grown to ~ 2–3 x 108 cfu/ml and then exposed to 100 μg/ml of penicillin G (penG), phosphomycin (phos), D-cycloserine (cyc), ampicillin (amp) or no antibiotic (no AB) (T0). Data shown are averages of two independent experiments; error bars represent standard deviation. (B) Images of V. cholerae cells at different time points after exposure to penicillin G from the experiment shown in (A). (C) Morphology of wt exponential phase cells exposed to 100 μg/ml cyc, phos, amp or 1 μg/ml meropenem (mero) for 3 h. (D) Time lapse images of wt cells plated on an agarose pad containing 100 μg/ml pen G. Frames are 5 min apart, scale bar = 2 μm. Arrowheads point to selected blebs. (E) Localization of blebs after exposure to pen G. Three different time lapse series with ~ 40 cells each were obtained as described in (D) and bleb location as the ratio of distance from an arbitrarily chosen pole divided by cell length was measured using ImageJ software. (F) Time lapse images of wt cells stained with the fluorescent D amino acid analogue HADA (50 μM) for 30 min and washed 2 x to remove excess dye prior to imaging on an agarose pad containing pen G. Images were brightness/contrast-adjusted to compensate for photobleaching. Frames are 5 min apart, scale bar = 2 μm. HADA stain is false-colored in red.

Mentions: We observed that mid- to late exponential phase cultures of V. cholerae treated with high doses of penicillin G or ampicillin (100 μg/ml, 20 x MIC) failed to divide, but did not show a decline in viable cells (i.e., cfu) (Fig 1A, S1A Fig). Similarly, inhibition of early steps in PG synthesis by D-cycloserine, an inhibitor of D-Ala-D-Ala ligase (100 μg/ml, 2 x MIC), or phosphomycin, an inhibitor of MurA (100 μg/ml, 2x MIC), did not appreciably affect the survival of V. cholerae. Thus, although antibiotics targeting cell wall synthesis are effective in preventing V. cholerae proliferation, they do not induce the cell death typically observed in dividing cells of other species. Due to V. cholerae’s “tolerance” of these chemotherapeutic agents, their effects are not irreversible.


Endopeptidase-mediated beta lactam tolerance.

Dörr T, Davis BM, Waldor MK - PLoS Pathog. (2015)

Inhibition of cell-wall synthesis in V. cholerae leads to sphere formation.(A) Kinetics of viable V. cholerae cell counts after cells were exposed to various antibiotics that inhibit cell wall synthesis. Cells were grown to ~ 2–3 x 108 cfu/ml and then exposed to 100 μg/ml of penicillin G (penG), phosphomycin (phos), D-cycloserine (cyc), ampicillin (amp) or no antibiotic (no AB) (T0). Data shown are averages of two independent experiments; error bars represent standard deviation. (B) Images of V. cholerae cells at different time points after exposure to penicillin G from the experiment shown in (A). (C) Morphology of wt exponential phase cells exposed to 100 μg/ml cyc, phos, amp or 1 μg/ml meropenem (mero) for 3 h. (D) Time lapse images of wt cells plated on an agarose pad containing 100 μg/ml pen G. Frames are 5 min apart, scale bar = 2 μm. Arrowheads point to selected blebs. (E) Localization of blebs after exposure to pen G. Three different time lapse series with ~ 40 cells each were obtained as described in (D) and bleb location as the ratio of distance from an arbitrarily chosen pole divided by cell length was measured using ImageJ software. (F) Time lapse images of wt cells stained with the fluorescent D amino acid analogue HADA (50 μM) for 30 min and washed 2 x to remove excess dye prior to imaging on an agarose pad containing pen G. Images were brightness/contrast-adjusted to compensate for photobleaching. Frames are 5 min apart, scale bar = 2 μm. HADA stain is false-colored in red.
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ppat.1004850.g001: Inhibition of cell-wall synthesis in V. cholerae leads to sphere formation.(A) Kinetics of viable V. cholerae cell counts after cells were exposed to various antibiotics that inhibit cell wall synthesis. Cells were grown to ~ 2–3 x 108 cfu/ml and then exposed to 100 μg/ml of penicillin G (penG), phosphomycin (phos), D-cycloserine (cyc), ampicillin (amp) or no antibiotic (no AB) (T0). Data shown are averages of two independent experiments; error bars represent standard deviation. (B) Images of V. cholerae cells at different time points after exposure to penicillin G from the experiment shown in (A). (C) Morphology of wt exponential phase cells exposed to 100 μg/ml cyc, phos, amp or 1 μg/ml meropenem (mero) for 3 h. (D) Time lapse images of wt cells plated on an agarose pad containing 100 μg/ml pen G. Frames are 5 min apart, scale bar = 2 μm. Arrowheads point to selected blebs. (E) Localization of blebs after exposure to pen G. Three different time lapse series with ~ 40 cells each were obtained as described in (D) and bleb location as the ratio of distance from an arbitrarily chosen pole divided by cell length was measured using ImageJ software. (F) Time lapse images of wt cells stained with the fluorescent D amino acid analogue HADA (50 μM) for 30 min and washed 2 x to remove excess dye prior to imaging on an agarose pad containing pen G. Images were brightness/contrast-adjusted to compensate for photobleaching. Frames are 5 min apart, scale bar = 2 μm. HADA stain is false-colored in red.
Mentions: We observed that mid- to late exponential phase cultures of V. cholerae treated with high doses of penicillin G or ampicillin (100 μg/ml, 20 x MIC) failed to divide, but did not show a decline in viable cells (i.e., cfu) (Fig 1A, S1A Fig). Similarly, inhibition of early steps in PG synthesis by D-cycloserine, an inhibitor of D-Ala-D-Ala ligase (100 μg/ml, 2 x MIC), or phosphomycin, an inhibitor of MurA (100 μg/ml, 2x MIC), did not appreciably affect the survival of V. cholerae. Thus, although antibiotics targeting cell wall synthesis are effective in preventing V. cholerae proliferation, they do not induce the cell death typically observed in dividing cells of other species. Due to V. cholerae’s “tolerance” of these chemotherapeutic agents, their effects are not irreversible.

Bottom Line: In response to a wide variety of cell wall--acting antibiotics, this pathogen loses its rod shape, indicative of cell wall degradation, and becomes spherical.Other autolysins proved dispensable for this process.Our findings suggest the enzymes that mediate cell wall degradation are critical for determining bacterial cell fate--sphere formation vs. lysis--after treatment with antibiotics that target cell wall synthesis.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Brigham and Women's Hospital and Howard Hughes Medical Institute, Boston, Massachusetts, United States of America; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
In many bacteria, inhibition of cell wall synthesis leads to cell death and lysis. The pathways and enzymes that mediate cell lysis after exposure to cell wall-acting antibiotics (e.g. beta lactams) are incompletely understood, but the activities of enzymes that degrade the cell wall ('autolysins') are thought to be critical. Here, we report that Vibrio cholerae, the cholera pathogen, is tolerant to antibiotics targeting cell wall synthesis. In response to a wide variety of cell wall--acting antibiotics, this pathogen loses its rod shape, indicative of cell wall degradation, and becomes spherical. Genetic analyses revealed that paradoxically, V. cholerae survival via sphere formation required the activity of D,D endopeptidases, enzymes that cleave the cell wall. Other autolysins proved dispensable for this process. Our findings suggest the enzymes that mediate cell wall degradation are critical for determining bacterial cell fate--sphere formation vs. lysis--after treatment with antibiotics that target cell wall synthesis.

No MeSH data available.


Related in: MedlinePlus