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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

Deletion of multiple lytic transglycosylases alters the kinetics of V. cholerae sphere formation after inhibition of cell wall synthesis.(A and B) The Δ5LTG mutant (ΔmltABDFΔslt70) was treated as described in Fig 1A and Fig 1B. No AB = no antibiotic added. (C) Time lapse images of Δ5LTG cells plated on agarose pads containing 100 μg/ml pen G. Frames are 5 min apart, scale bar = 5 μm; red arrowheads point to polar blebs. (D) Quantification of bleb location for time lapses described in C. For details, see Methods. (E) HADA incorporation during exposure to cell wall synthesis inhibitors in broth culture. Cells were grown to exponential phase in the presence of HADA (50 μM), then exposed to 100 μg/ml pen G or phos with HADA remaining in the growth medium. At the indicated time points, samples were washed twice prior to imaging. Images were minimally processed (background subtraction) and are comparable to each other, but not to the one depicted in Fig 1F. Scale bar = 5 μm. Yellow arrowheads point to polar appendages containing PG.
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ppat.1004850.g003: Deletion of multiple lytic transglycosylases alters the kinetics of V. cholerae sphere formation after inhibition of cell wall synthesis.(A and B) The Δ5LTG mutant (ΔmltABDFΔslt70) was treated as described in Fig 1A and Fig 1B. No AB = no antibiotic added. (C) Time lapse images of Δ5LTG cells plated on agarose pads containing 100 μg/ml pen G. Frames are 5 min apart, scale bar = 5 μm; red arrowheads point to polar blebs. (D) Quantification of bleb location for time lapses described in C. For details, see Methods. (E) HADA incorporation during exposure to cell wall synthesis inhibitors in broth culture. Cells were grown to exponential phase in the presence of HADA (50 μM), then exposed to 100 μg/ml pen G or phos with HADA remaining in the growth medium. At the indicated time points, samples were washed twice prior to imaging. Images were minimally processed (background subtraction) and are comparable to each other, but not to the one depicted in Fig 1F. Scale bar = 5 μm. Yellow arrowheads point to polar appendages containing PG.

Mentions: We also assessed the role of lytic transglycosylases (LTGs) in V. cholerae’s response to antibiotics that inhibit PG synthesis. The V. cholerae genome encodes 6 predicted LTGs (mltA, mltB, mltC, mltD, mltF, and slt70). We found that a strain lacking 5 of these (ΔmltABDFΔslt70; Δ5LTG) was viable; however, we were unable to obtain a mutant lacking all six. Exposure of Δ5LTG to penicillin resulted in a ~1 log reduction in viability that was not accompanied by lysis, whereas phosphomycin or D-cycloserine did not reduce this strain’s viability (Fig 3A and Fig 3B). Similarly, deletion of certain lytic transglycosylases sensitizes other bacteria to beta lactam antibiotics [13,14], and this has recently been proposed to reflect the LTGs’ role in a quality control mechanism during cell wall synthesis [31].


Endopeptidase-mediated beta lactam tolerance.

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

Deletion of multiple lytic transglycosylases alters the kinetics of V. cholerae sphere formation after inhibition of cell wall synthesis.(A and B) The Δ5LTG mutant (ΔmltABDFΔslt70) was treated as described in Fig 1A and Fig 1B. No AB = no antibiotic added. (C) Time lapse images of Δ5LTG cells plated on agarose pads containing 100 μg/ml pen G. Frames are 5 min apart, scale bar = 5 μm; red arrowheads point to polar blebs. (D) Quantification of bleb location for time lapses described in C. For details, see Methods. (E) HADA incorporation during exposure to cell wall synthesis inhibitors in broth culture. Cells were grown to exponential phase in the presence of HADA (50 μM), then exposed to 100 μg/ml pen G or phos with HADA remaining in the growth medium. At the indicated time points, samples were washed twice prior to imaging. Images were minimally processed (background subtraction) and are comparable to each other, but not to the one depicted in Fig 1F. Scale bar = 5 μm. Yellow arrowheads point to polar appendages containing PG.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401780&req=5

ppat.1004850.g003: Deletion of multiple lytic transglycosylases alters the kinetics of V. cholerae sphere formation after inhibition of cell wall synthesis.(A and B) The Δ5LTG mutant (ΔmltABDFΔslt70) was treated as described in Fig 1A and Fig 1B. No AB = no antibiotic added. (C) Time lapse images of Δ5LTG cells plated on agarose pads containing 100 μg/ml pen G. Frames are 5 min apart, scale bar = 5 μm; red arrowheads point to polar blebs. (D) Quantification of bleb location for time lapses described in C. For details, see Methods. (E) HADA incorporation during exposure to cell wall synthesis inhibitors in broth culture. Cells were grown to exponential phase in the presence of HADA (50 μM), then exposed to 100 μg/ml pen G or phos with HADA remaining in the growth medium. At the indicated time points, samples were washed twice prior to imaging. Images were minimally processed (background subtraction) and are comparable to each other, but not to the one depicted in Fig 1F. Scale bar = 5 μm. Yellow arrowheads point to polar appendages containing PG.
Mentions: We also assessed the role of lytic transglycosylases (LTGs) in V. cholerae’s response to antibiotics that inhibit PG synthesis. The V. cholerae genome encodes 6 predicted LTGs (mltA, mltB, mltC, mltD, mltF, and slt70). We found that a strain lacking 5 of these (ΔmltABDFΔslt70; Δ5LTG) was viable; however, we were unable to obtain a mutant lacking all six. Exposure of Δ5LTG to penicillin resulted in a ~1 log reduction in viability that was not accompanied by lysis, whereas phosphomycin or D-cycloserine did not reduce this strain’s viability (Fig 3A and Fig 3B). Similarly, deletion of certain lytic transglycosylases sensitizes other bacteria to beta lactam antibiotics [13,14], and this has recently been proposed to reflect the LTGs’ role in a quality control mechanism during cell wall synthesis [31].

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