Limits...
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.

Show MeSH

Related in: MedlinePlus

Dysregulation of RipA in M. smegmatis causes growth attenuation.(A) Micrographs of RipA dysregulated strains after 24 hours of depletion or induction. RipA depletion was achieved by placing ripA under the control of an anhydrotetracycline (aTc) inducible promoter [18]. Depletion occurred over 24 hours by growing this strain in the absence of aTc. Alternately, wildtype RipA (RipASm) or catalytically inactive (RipASm C408A) M. smegmatis RipA was overexpressed in wildtype cells. In the C408A overexpression strain, both chaining (white arrows) and bulging (red arrows) cells were observed. Membranes were stained with TMA-DPH. Scale bar represents 2 µm. (B) M. smegmatis strains overexpressing wildtype (RipASm) and catalytically inactive (C408A) RipA were induced with aTc and growth assessed by OD600 over time. (C) M. smegmatis was induced with aTc to overexpress either wildtype RipASm or inactive RipASm C408A. Colony forming units (CFUs) were enumerated at the indicated time course by serial dilution and plating onto inducer-free agar. (D) Time-lapse microscopy was used to visualize M. smegmatis grown on an agar pad with aTc to overexpress wildtype RipASm (left panels) or catalytically inactive RipASm C408A (right panels). A GFP reporter was used to visualize RipA induction as well as determine cytokinesis and cell lysis. Presented here are frames from agar pads at four time points post-induction, showing individual cells before and after cell lysis, as detected by loss of GFP signal (arrows). Scale bar represents 2 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585148&req=5

ppat-1003197-g001: Dysregulation of RipA in M. smegmatis causes growth attenuation.(A) Micrographs of RipA dysregulated strains after 24 hours of depletion or induction. RipA depletion was achieved by placing ripA under the control of an anhydrotetracycline (aTc) inducible promoter [18]. Depletion occurred over 24 hours by growing this strain in the absence of aTc. Alternately, wildtype RipA (RipASm) or catalytically inactive (RipASm C408A) M. smegmatis RipA was overexpressed in wildtype cells. In the C408A overexpression strain, both chaining (white arrows) and bulging (red arrows) cells were observed. Membranes were stained with TMA-DPH. Scale bar represents 2 µm. (B) M. smegmatis strains overexpressing wildtype (RipASm) and catalytically inactive (C408A) RipA were induced with aTc and growth assessed by OD600 over time. (C) M. smegmatis was induced with aTc to overexpress either wildtype RipASm or inactive RipASm C408A. Colony forming units (CFUs) were enumerated at the indicated time course by serial dilution and plating onto inducer-free agar. (D) Time-lapse microscopy was used to visualize M. smegmatis grown on an agar pad with aTc to overexpress wildtype RipASm (left panels) or catalytically inactive RipASm C408A (right panels). A GFP reporter was used to visualize RipA induction as well as determine cytokinesis and cell lysis. Presented here are frames from agar pads at four time points post-induction, showing individual cells before and after cell lysis, as detected by loss of GFP signal (arrows). Scale bar represents 2 µm.

Mentions: When RipA is depleted, daughter cells are unable to separate and instead, grow as chains (Figure 1A). While cells require peptidoglycan hydrolysis to accomplish cell separation, excessive cell wall degradation can compromise structural integrity and lead to lysis. We hypothesized that RipA sits in this precarious situation, where the cell cannot tolerate either too little or too much RipA activity.


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)

Dysregulation of RipA in M. smegmatis causes growth attenuation.(A) Micrographs of RipA dysregulated strains after 24 hours of depletion or induction. RipA depletion was achieved by placing ripA under the control of an anhydrotetracycline (aTc) inducible promoter [18]. Depletion occurred over 24 hours by growing this strain in the absence of aTc. Alternately, wildtype RipA (RipASm) or catalytically inactive (RipASm C408A) M. smegmatis RipA was overexpressed in wildtype cells. In the C408A overexpression strain, both chaining (white arrows) and bulging (red arrows) cells were observed. Membranes were stained with TMA-DPH. Scale bar represents 2 µm. (B) M. smegmatis strains overexpressing wildtype (RipASm) and catalytically inactive (C408A) RipA were induced with aTc and growth assessed by OD600 over time. (C) M. smegmatis was induced with aTc to overexpress either wildtype RipASm or inactive RipASm C408A. Colony forming units (CFUs) were enumerated at the indicated time course by serial dilution and plating onto inducer-free agar. (D) Time-lapse microscopy was used to visualize M. smegmatis grown on an agar pad with aTc to overexpress wildtype RipASm (left panels) or catalytically inactive RipASm C408A (right panels). A GFP reporter was used to visualize RipA induction as well as determine cytokinesis and cell lysis. Presented here are frames from agar pads at four time points post-induction, showing individual cells before and after cell lysis, as detected by loss of GFP signal (arrows). Scale bar represents 2 µm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003197-g001: Dysregulation of RipA in M. smegmatis causes growth attenuation.(A) Micrographs of RipA dysregulated strains after 24 hours of depletion or induction. RipA depletion was achieved by placing ripA under the control of an anhydrotetracycline (aTc) inducible promoter [18]. Depletion occurred over 24 hours by growing this strain in the absence of aTc. Alternately, wildtype RipA (RipASm) or catalytically inactive (RipASm C408A) M. smegmatis RipA was overexpressed in wildtype cells. In the C408A overexpression strain, both chaining (white arrows) and bulging (red arrows) cells were observed. Membranes were stained with TMA-DPH. Scale bar represents 2 µm. (B) M. smegmatis strains overexpressing wildtype (RipASm) and catalytically inactive (C408A) RipA were induced with aTc and growth assessed by OD600 over time. (C) M. smegmatis was induced with aTc to overexpress either wildtype RipASm or inactive RipASm C408A. Colony forming units (CFUs) were enumerated at the indicated time course by serial dilution and plating onto inducer-free agar. (D) Time-lapse microscopy was used to visualize M. smegmatis grown on an agar pad with aTc to overexpress wildtype RipASm (left panels) or catalytically inactive RipASm C408A (right panels). A GFP reporter was used to visualize RipA induction as well as determine cytokinesis and cell lysis. Presented here are frames from agar pads at four time points post-induction, showing individual cells before and after cell lysis, as detected by loss of GFP signal (arrows). Scale bar represents 2 µm.
Mentions: When RipA is depleted, daughter cells are unable to separate and instead, grow as chains (Figure 1A). While cells require peptidoglycan hydrolysis to accomplish cell separation, excessive cell wall degradation can compromise structural integrity and lead to lysis. We hypothesized that RipA sits in this precarious situation, where the cell cannot tolerate either too little or too much RipA activity.

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