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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

V. cholerae endopeptidase are required to prevent lysis in response to inhibition of cell wall synthesis.(A) Wild type V. cholerae and a Δendo (ΔshyABC ΔnlpC ΔtagE1 ΔtagE2 Ptac:shyA) mutant were grown in the presence or absence of 200 μM IPTG for 1.5 h (= T0) and then exposed to pen G. Cfu/ml at the indicated time points were normalized to cfu/ml at T0 and shown as fold change in viability. Data are mean of two biological replicates; error bars represent standard deviation. (B) Representative images of V. cholerae Δendo cells at different time points after exposure to penicillin G from the experiment shown in (A). (C) Time lapse images of Δendo cells initially grown for 1.5 h in either the presence or absence of IPTG (25 μM) and subsequently imaged on agarose pads containing 100 μg/ml pen G. The pad for ShyA+ cells also contained IPTG. The control panel depicts cells applied to an agarose pad containing neither antibiotic nor IPTG. Frames in the lower (control) panel are 10 min apart; in the other panels, frames are 5 min apart. Scale bar = 2 μm.
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ppat.1004850.g004: V. cholerae endopeptidase are required to prevent lysis in response to inhibition of cell wall synthesis.(A) Wild type V. cholerae and a Δendo (ΔshyABC ΔnlpC ΔtagE1 ΔtagE2 Ptac:shyA) mutant were grown in the presence or absence of 200 μM IPTG for 1.5 h (= T0) and then exposed to pen G. Cfu/ml at the indicated time points were normalized to cfu/ml at T0 and shown as fold change in viability. Data are mean of two biological replicates; error bars represent standard deviation. (B) Representative images of V. cholerae Δendo cells at different time points after exposure to penicillin G from the experiment shown in (A). (C) Time lapse images of Δendo cells initially grown for 1.5 h in either the presence or absence of IPTG (25 μM) and subsequently imaged on agarose pads containing 100 μg/ml pen G. The pad for ShyA+ cells also contained IPTG. The control panel depicts cells applied to an agarose pad containing neither antibiotic nor IPTG. Frames in the lower (control) panel are 10 min apart; in the other panels, frames are 5 min apart. Scale bar = 2 μm.

Mentions: Since amidases and the 5 LTGs were not critical for sphere formation, we turned our focus towards endopeptidases, two of which (ShyA and ShyC) are synthetically lethal and essential for cell elongation in V. cholerae [25]. The V. cholerae genome encodes five periplasmic M23 endopeptidases and one P60 family endopeptidase (NlpC). We constructed a mutant that lacks all predicted non-PBP endopeptidases and expresses inducible shyA from a neutral chromosomal locus (ΔshyA ΔshyB ΔshyC ΔnlpC ΔtagE1 ΔtagE2 Ptac:shyA; Δendo). In this background, depletion of shyA slowed growth, similar to our previous observations with a ΔshyA ΔshyC Ptac:shyA strain, which is defective in cell elongation but proficient in cell division [25](Fig 4A). Importantly, untreated Δendo cells did not lyse even after extended ShyA depletion (Fig 4C and “control”). Unexpectedly, however, exposure of ShyA-depleted Δendo cells to penicillin G resulted in rapid loss of viability and concomitant lysis of the majority of the population (Fig 4A and 4B). Lysis could be prevented and sphere formation restored by expression of shyA (Fig 4C). A similar pattern was observed with D-cycloserine and phosphomycin (S7 Fig). Notably, analysis of the dynamics of cell lysis using time lapse microscopy revealed that cell disintegration did not proceed through a spherical intermediate (Fig 4C). These results suggest that, paradoxically, the presence of a D,D endopeptidase (ShyA), a putative ‘autolysin’, prevents lysis and enables formation of viable spheres after exposure of V. cholerae to a beta lactam antibiotic.


Endopeptidase-mediated beta lactam tolerance.

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

V. cholerae endopeptidase are required to prevent lysis in response to inhibition of cell wall synthesis.(A) Wild type V. cholerae and a Δendo (ΔshyABC ΔnlpC ΔtagE1 ΔtagE2 Ptac:shyA) mutant were grown in the presence or absence of 200 μM IPTG for 1.5 h (= T0) and then exposed to pen G. Cfu/ml at the indicated time points were normalized to cfu/ml at T0 and shown as fold change in viability. Data are mean of two biological replicates; error bars represent standard deviation. (B) Representative images of V. cholerae Δendo cells at different time points after exposure to penicillin G from the experiment shown in (A). (C) Time lapse images of Δendo cells initially grown for 1.5 h in either the presence or absence of IPTG (25 μM) and subsequently imaged on agarose pads containing 100 μg/ml pen G. The pad for ShyA+ cells also contained IPTG. The control panel depicts cells applied to an agarose pad containing neither antibiotic nor IPTG. Frames in the lower (control) panel are 10 min apart; in the other panels, frames are 5 min apart. Scale bar = 2 μm.
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Related In: Results  -  Collection

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ppat.1004850.g004: V. cholerae endopeptidase are required to prevent lysis in response to inhibition of cell wall synthesis.(A) Wild type V. cholerae and a Δendo (ΔshyABC ΔnlpC ΔtagE1 ΔtagE2 Ptac:shyA) mutant were grown in the presence or absence of 200 μM IPTG for 1.5 h (= T0) and then exposed to pen G. Cfu/ml at the indicated time points were normalized to cfu/ml at T0 and shown as fold change in viability. Data are mean of two biological replicates; error bars represent standard deviation. (B) Representative images of V. cholerae Δendo cells at different time points after exposure to penicillin G from the experiment shown in (A). (C) Time lapse images of Δendo cells initially grown for 1.5 h in either the presence or absence of IPTG (25 μM) and subsequently imaged on agarose pads containing 100 μg/ml pen G. The pad for ShyA+ cells also contained IPTG. The control panel depicts cells applied to an agarose pad containing neither antibiotic nor IPTG. Frames in the lower (control) panel are 10 min apart; in the other panels, frames are 5 min apart. Scale bar = 2 μm.
Mentions: Since amidases and the 5 LTGs were not critical for sphere formation, we turned our focus towards endopeptidases, two of which (ShyA and ShyC) are synthetically lethal and essential for cell elongation in V. cholerae [25]. The V. cholerae genome encodes five periplasmic M23 endopeptidases and one P60 family endopeptidase (NlpC). We constructed a mutant that lacks all predicted non-PBP endopeptidases and expresses inducible shyA from a neutral chromosomal locus (ΔshyA ΔshyB ΔshyC ΔnlpC ΔtagE1 ΔtagE2 Ptac:shyA; Δendo). In this background, depletion of shyA slowed growth, similar to our previous observations with a ΔshyA ΔshyC Ptac:shyA strain, which is defective in cell elongation but proficient in cell division [25](Fig 4A). Importantly, untreated Δendo cells did not lyse even after extended ShyA depletion (Fig 4C and “control”). Unexpectedly, however, exposure of ShyA-depleted Δendo cells to penicillin G resulted in rapid loss of viability and concomitant lysis of the majority of the population (Fig 4A and 4B). Lysis could be prevented and sphere formation restored by expression of shyA (Fig 4C). A similar pattern was observed with D-cycloserine and phosphomycin (S7 Fig). Notably, analysis of the dynamics of cell lysis using time lapse microscopy revealed that cell disintegration did not proceed through a spherical intermediate (Fig 4C). These results suggest that, paradoxically, the presence of a D,D endopeptidase (ShyA), a putative ‘autolysin’, prevents lysis and enables formation of viable spheres after exposure of V. cholerae to a beta lactam antibiotic.

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