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Protein complexes and proteolytic activation of the cell wall hydrolase RipA regulate septal resolution in mycobacteria.

Chao MC, Kieser KJ, Minami S, Mavrici D, Aldridge BB, Fortune SM, Alber T, Rubin EJ - PLoS Pathog. (2013)

Bottom Line: Peptidoglycan hydrolases are a double-edged sword.They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria.Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America.

ABSTRACT
Peptidoglycan hydrolases are a double-edged sword. They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria. How bacteria ensure that peptidoglycan hydrolases function only in the correct spatial and temporal context remains largely unknown. Here, we demonstrate that dysregulation converts the essential mycobacterial peptidoglycan hydrolase RipA to an autolysin that compromises cellular structural integrity. We find that mycobacteria control RipA activity through two interconnected levels of regulation in vivo-protein interactions coordinate PG hydrolysis, while proteolysis is necessary for RipA enzymatic activity. Dysregulation of RipA protein complexes by treatment with a peptidoglycan synthase inhibitor leads to excessive RipA activity and impairment of correct morphology. Furthermore, expression of a RipA dominant negative mutant or of differentially processed RipA homologues reveals that RipA is produced as a zymogen, requiring proteolytic processing for activity. The amount of RipA processing differs between fast-growing and slow-growing mycobacteria and correlates with the requirement for peptidoglycan hydrolase activity in these species. Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.

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RipA depletion protects cells from meropenem-induced lysis.(A) A RipA conditional depletion strain of M. smegmatis was grown in the presence of inducer (RipA replete conditions) and 10 µg/mL of meropenem. Cells were imaged for morphological changes at various time points after meropenem addition. During meropenem treatment, septal and polar bulging were observed (arrows). Cell membranes were stained with FM4-64. Scale bar represents 2 µm. (B) Lysis of M. smegmatis cells from (A) was also characterized by OD600 at various times post meropenem treatment. (C) The RipA conditional depletion strain of M. smegmatis was grown with anhydrotetracylcine (aTc) inducer (RipA replete) or pre-depleted (RipA deplete) in the absence of aTc for 6 hours. These cells were then treated with 10 µg/mL of meropenem for 6 hours, washed, serially diluted and plated for CFU enumeration. (**, the difference between survival in RipA replete cells and RipA depleted cells in the presence of meropenem was significant with a p-value = 0.0006). (D) Cells post meropenem treatment in (C) were imaged by fluorescence microscopy to determine morphological changes. The plasma membrane was visualized with FM4-64. Scale bar represents 2 µm.
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ppat-1003197-g002: RipA depletion protects cells from meropenem-induced lysis.(A) A RipA conditional depletion strain of M. smegmatis was grown in the presence of inducer (RipA replete conditions) and 10 µg/mL of meropenem. Cells were imaged for morphological changes at various time points after meropenem addition. During meropenem treatment, septal and polar bulging were observed (arrows). Cell membranes were stained with FM4-64. Scale bar represents 2 µm. (B) Lysis of M. smegmatis cells from (A) was also characterized by OD600 at various times post meropenem treatment. (C) The RipA conditional depletion strain of M. smegmatis was grown with anhydrotetracylcine (aTc) inducer (RipA replete) or pre-depleted (RipA deplete) in the absence of aTc for 6 hours. These cells were then treated with 10 µg/mL of meropenem for 6 hours, washed, serially diluted and plated for CFU enumeration. (**, the difference between survival in RipA replete cells and RipA depleted cells in the presence of meropenem was significant with a p-value = 0.0006). (D) Cells post meropenem treatment in (C) were imaged by fluorescence microscopy to determine morphological changes. The plasma membrane was visualized with FM4-64. Scale bar represents 2 µm.

Mentions: We first treated M. smegmatis with 10 µg/mL meropenem and assessed morphological changes over time by microscopy. Treated cells filament and swell at the poles and septa, which are the sites of mycobacterial PG incorporation (Figure 2A, arrows). This morphological toxicity correlated with a decrease in optical density over time, which suggested lysis (Figure 2B). This was borne out by CFU analysis, which showed that 80% of treated cells were killed within 6 hours of meropenem treatment (Figure 2C, bar 2).


Protein complexes and proteolytic activation of the cell wall hydrolase RipA regulate septal resolution in mycobacteria.

Chao MC, Kieser KJ, Minami S, Mavrici D, Aldridge BB, Fortune SM, Alber T, Rubin EJ - PLoS Pathog. (2013)

RipA depletion protects cells from meropenem-induced lysis.(A) A RipA conditional depletion strain of M. smegmatis was grown in the presence of inducer (RipA replete conditions) and 10 µg/mL of meropenem. Cells were imaged for morphological changes at various time points after meropenem addition. During meropenem treatment, septal and polar bulging were observed (arrows). Cell membranes were stained with FM4-64. Scale bar represents 2 µm. (B) Lysis of M. smegmatis cells from (A) was also characterized by OD600 at various times post meropenem treatment. (C) The RipA conditional depletion strain of M. smegmatis was grown with anhydrotetracylcine (aTc) inducer (RipA replete) or pre-depleted (RipA deplete) in the absence of aTc for 6 hours. These cells were then treated with 10 µg/mL of meropenem for 6 hours, washed, serially diluted and plated for CFU enumeration. (**, the difference between survival in RipA replete cells and RipA depleted cells in the presence of meropenem was significant with a p-value = 0.0006). (D) Cells post meropenem treatment in (C) were imaged by fluorescence microscopy to determine morphological changes. The plasma membrane was visualized with FM4-64. Scale bar represents 2 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585148&req=5

ppat-1003197-g002: RipA depletion protects cells from meropenem-induced lysis.(A) A RipA conditional depletion strain of M. smegmatis was grown in the presence of inducer (RipA replete conditions) and 10 µg/mL of meropenem. Cells were imaged for morphological changes at various time points after meropenem addition. During meropenem treatment, septal and polar bulging were observed (arrows). Cell membranes were stained with FM4-64. Scale bar represents 2 µm. (B) Lysis of M. smegmatis cells from (A) was also characterized by OD600 at various times post meropenem treatment. (C) The RipA conditional depletion strain of M. smegmatis was grown with anhydrotetracylcine (aTc) inducer (RipA replete) or pre-depleted (RipA deplete) in the absence of aTc for 6 hours. These cells were then treated with 10 µg/mL of meropenem for 6 hours, washed, serially diluted and plated for CFU enumeration. (**, the difference between survival in RipA replete cells and RipA depleted cells in the presence of meropenem was significant with a p-value = 0.0006). (D) Cells post meropenem treatment in (C) were imaged by fluorescence microscopy to determine morphological changes. The plasma membrane was visualized with FM4-64. Scale bar represents 2 µm.
Mentions: We first treated M. smegmatis with 10 µg/mL meropenem and assessed morphological changes over time by microscopy. Treated cells filament and swell at the poles and septa, which are the sites of mycobacterial PG incorporation (Figure 2A, arrows). This morphological toxicity correlated with a decrease in optical density over time, which suggested lysis (Figure 2B). This was borne out by CFU analysis, which showed that 80% of treated cells were killed within 6 hours of meropenem treatment (Figure 2C, bar 2).

Bottom Line: Peptidoglycan hydrolases are a double-edged sword.They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria.Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America.

ABSTRACT
Peptidoglycan hydrolases are a double-edged sword. They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria. How bacteria ensure that peptidoglycan hydrolases function only in the correct spatial and temporal context remains largely unknown. Here, we demonstrate that dysregulation converts the essential mycobacterial peptidoglycan hydrolase RipA to an autolysin that compromises cellular structural integrity. We find that mycobacteria control RipA activity through two interconnected levels of regulation in vivo-protein interactions coordinate PG hydrolysis, while proteolysis is necessary for RipA enzymatic activity. Dysregulation of RipA protein complexes by treatment with a peptidoglycan synthase inhibitor leads to excessive RipA activity and impairment of correct morphology. Furthermore, expression of a RipA dominant negative mutant or of differentially processed RipA homologues reveals that RipA is produced as a zymogen, requiring proteolytic processing for activity. The amount of RipA processing differs between fast-growing and slow-growing mycobacteria and correlates with the requirement for peptidoglycan hydrolase activity in these species. Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.

Show MeSH
Related in: MedlinePlus