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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 other bacteria does not always lead to cell lysis.(A) Influence of shyA or yebA overexpression on viability of E. coli EDL933 treated with meropenem. EDL933 carrying either pBAD33 (plasmid vector) or its derivatives encoding yebA or shyA was grown to exponential phase in the presence of arabinose, then meropenem (1 μg/ml) was added at (T0) and viable cell counts determined at the indicated times. Data are average of three independent experiments; error bars represent standard deviation. (B) Representative images from the 3 h time point of an experiment similar to the one depicted in (A), with or without the addition of 10 mM Mg2+ in the growth medium. (C) Time lapse images of P. aeruginosa, A. baumannii, V. cholerae and EDL933 cells exposed to meropenem. Cells were grown in LB until OD600 ~ 0.5 and then applied to an agarose pad (10% LB/PBS) containing meropenem (4 μg/ml) as well as MgSO4 (10 mM)/CaCl2 (1 mM) for P. aeruginosa and A. baumannii only. Frames are 9 min apart, scale bar = 5 μm. No AB = no antibiotic added.
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ppat.1004850.g005: Inhibition of cell wall synthesis in other bacteria does not always lead to cell lysis.(A) Influence of shyA or yebA overexpression on viability of E. coli EDL933 treated with meropenem. EDL933 carrying either pBAD33 (plasmid vector) or its derivatives encoding yebA or shyA was grown to exponential phase in the presence of arabinose, then meropenem (1 μg/ml) was added at (T0) and viable cell counts determined at the indicated times. Data are average of three independent experiments; error bars represent standard deviation. (B) Representative images from the 3 h time point of an experiment similar to the one depicted in (A), with or without the addition of 10 mM Mg2+ in the growth medium. (C) Time lapse images of P. aeruginosa, A. baumannii, V. cholerae and EDL933 cells exposed to meropenem. Cells were grown in LB until OD600 ~ 0.5 and then applied to an agarose pad (10% LB/PBS) containing meropenem (4 μg/ml) as well as MgSO4 (10 mM)/CaCl2 (1 mM) for P. aeruginosa and A. baumannii only. Frames are 9 min apart, scale bar = 5 μm. No AB = no antibiotic added.

Mentions: Since ShyA could prevent beta lactam-mediated lysis in V. cholerae, we tested whether it could protect a heterologous organism from lysis after inhibition of cell wall synthesis. We overproduced ShyA in the EHEC isolate EDL933 and measured its survival after exposure to meropenem. Overexpression of shyA alone did not influence EHEC growth. However, expression of this endopeptidase increased EHEC’s capacity to survive meropenem exposure by ~10-fold compared to an empty vector control, overexpression of yebA, E. coli’s ShyA homologue (Fig 5A) or overexpression of ShyA carrying an active site mutation (H375A). Meropenem-treated EDL933 expressing ShyA formed spheres, while the control cells carrying the empty vector rapidly lysed (Fig 5B). These data suggest that ShyA activity is linked to survival in the presence of beta lactam antibiotics, and that ShyA-mediated cleavage of the cell wall may differ from YebA-mediated cleavage events, although it is theoretically possible that these results only reflect differences in the expression levels for the two proteins.


Endopeptidase-mediated beta lactam tolerance.

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

Inhibition of cell wall synthesis in other bacteria does not always lead to cell lysis.(A) Influence of shyA or yebA overexpression on viability of E. coli EDL933 treated with meropenem. EDL933 carrying either pBAD33 (plasmid vector) or its derivatives encoding yebA or shyA was grown to exponential phase in the presence of arabinose, then meropenem (1 μg/ml) was added at (T0) and viable cell counts determined at the indicated times. Data are average of three independent experiments; error bars represent standard deviation. (B) Representative images from the 3 h time point of an experiment similar to the one depicted in (A), with or without the addition of 10 mM Mg2+ in the growth medium. (C) Time lapse images of P. aeruginosa, A. baumannii, V. cholerae and EDL933 cells exposed to meropenem. Cells were grown in LB until OD600 ~ 0.5 and then applied to an agarose pad (10% LB/PBS) containing meropenem (4 μg/ml) as well as MgSO4 (10 mM)/CaCl2 (1 mM) for P. aeruginosa and A. baumannii only. Frames are 9 min apart, scale bar = 5 μm. No AB = no antibiotic added.
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ppat.1004850.g005: Inhibition of cell wall synthesis in other bacteria does not always lead to cell lysis.(A) Influence of shyA or yebA overexpression on viability of E. coli EDL933 treated with meropenem. EDL933 carrying either pBAD33 (plasmid vector) or its derivatives encoding yebA or shyA was grown to exponential phase in the presence of arabinose, then meropenem (1 μg/ml) was added at (T0) and viable cell counts determined at the indicated times. Data are average of three independent experiments; error bars represent standard deviation. (B) Representative images from the 3 h time point of an experiment similar to the one depicted in (A), with or without the addition of 10 mM Mg2+ in the growth medium. (C) Time lapse images of P. aeruginosa, A. baumannii, V. cholerae and EDL933 cells exposed to meropenem. Cells were grown in LB until OD600 ~ 0.5 and then applied to an agarose pad (10% LB/PBS) containing meropenem (4 μg/ml) as well as MgSO4 (10 mM)/CaCl2 (1 mM) for P. aeruginosa and A. baumannii only. Frames are 9 min apart, scale bar = 5 μm. No AB = no antibiotic added.
Mentions: Since ShyA could prevent beta lactam-mediated lysis in V. cholerae, we tested whether it could protect a heterologous organism from lysis after inhibition of cell wall synthesis. We overproduced ShyA in the EHEC isolate EDL933 and measured its survival after exposure to meropenem. Overexpression of shyA alone did not influence EHEC growth. However, expression of this endopeptidase increased EHEC’s capacity to survive meropenem exposure by ~10-fold compared to an empty vector control, overexpression of yebA, E. coli’s ShyA homologue (Fig 5A) or overexpression of ShyA carrying an active site mutation (H375A). Meropenem-treated EDL933 expressing ShyA formed spheres, while the control cells carrying the empty vector rapidly lysed (Fig 5B). These data suggest that ShyA activity is linked to survival in the presence of beta lactam antibiotics, and that ShyA-mediated cleavage of the cell wall may differ from YebA-mediated cleavage events, although it is theoretically possible that these results only reflect differences in the expression levels for the two proteins.

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