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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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Truncation causes RipATB to become active in M. smegmatis.(A) M. tuberculosis and M. smegmatis RipA were truncated to the active endopeptidase domain (AD) and fused to the RipA secretion signal to generate RipATB-AD and RipASm-AD. Micrographs of M. smegmatis overexpressing RipATB-AD or RipASm-AD were taken at 6 and 20 hours post induction with aTc. Membranes were visualized by FM4-64 staining. Scale bar represents 2 µm. (B) M. smegmatis strains were constructed to overexpress full length RipATB and RipASm and truncated RipATB-AD, RipASm-AD constructs under the control of aTc. Growth of these strains in the presence of inducer was assessed over time by OD600. These strains grown in the absence of inducer as controls are shown in Figure S8.
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ppat-1003197-g006: Truncation causes RipATB to become active in M. smegmatis.(A) M. tuberculosis and M. smegmatis RipA were truncated to the active endopeptidase domain (AD) and fused to the RipA secretion signal to generate RipATB-AD and RipASm-AD. Micrographs of M. smegmatis overexpressing RipATB-AD or RipASm-AD were taken at 6 and 20 hours post induction with aTc. Membranes were visualized by FM4-64 staining. Scale bar represents 2 µm. (B) M. smegmatis strains were constructed to overexpress full length RipATB and RipASm and truncated RipATB-AD, RipASm-AD constructs under the control of aTc. Growth of these strains in the presence of inducer was assessed over time by OD600. These strains grown in the absence of inducer as controls are shown in Figure S8.

Mentions: However, it could be formally possible that RipATB cannot recognize M. smegmatis peptidoglycan or is not intrinsically active enough in M. smegmatis to cause morphological defects. To test if RipATB can be enzymatically functional in M. smegmatis, we deleted the predicted N-terminal inhibitory segment by fusing the truncated active domain of RipATB to the RipA secretion signal peptide (RipATB-AD). As a control, we also produced a construct in which the M. smegmatis RipA active domain (RipASm-AD) can be secreted. None of the strains produced growth defects when uninduced (Figure S8). As expected, RipASm-AD like full length RipASm, was fully functional when induced and disrupted cell wall integrity, leading to bulging of the cells and a concomitant growth defect (Figure 6A,B). When RipATB-AD was secreted, we found that it was also functional and behaved in the same way as RipASm-AD (Figure 6A,B). Thus, the catalytic domain of RipATB can be active in M. smegmatis, but full length RipATB is not toxic because it does not undergo efficient processing in M. smegmatis.


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)

Truncation causes RipATB to become active in M. smegmatis.(A) M. tuberculosis and M. smegmatis RipA were truncated to the active endopeptidase domain (AD) and fused to the RipA secretion signal to generate RipATB-AD and RipASm-AD. Micrographs of M. smegmatis overexpressing RipATB-AD or RipASm-AD were taken at 6 and 20 hours post induction with aTc. Membranes were visualized by FM4-64 staining. Scale bar represents 2 µm. (B) M. smegmatis strains were constructed to overexpress full length RipATB and RipASm and truncated RipATB-AD, RipASm-AD constructs under the control of aTc. Growth of these strains in the presence of inducer was assessed over time by OD600. These strains grown in the absence of inducer as controls are shown in Figure S8.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003197-g006: Truncation causes RipATB to become active in M. smegmatis.(A) M. tuberculosis and M. smegmatis RipA were truncated to the active endopeptidase domain (AD) and fused to the RipA secretion signal to generate RipATB-AD and RipASm-AD. Micrographs of M. smegmatis overexpressing RipATB-AD or RipASm-AD were taken at 6 and 20 hours post induction with aTc. Membranes were visualized by FM4-64 staining. Scale bar represents 2 µm. (B) M. smegmatis strains were constructed to overexpress full length RipATB and RipASm and truncated RipATB-AD, RipASm-AD constructs under the control of aTc. Growth of these strains in the presence of inducer was assessed over time by OD600. These strains grown in the absence of inducer as controls are shown in Figure S8.
Mentions: However, it could be formally possible that RipATB cannot recognize M. smegmatis peptidoglycan or is not intrinsically active enough in M. smegmatis to cause morphological defects. To test if RipATB can be enzymatically functional in M. smegmatis, we deleted the predicted N-terminal inhibitory segment by fusing the truncated active domain of RipATB to the RipA secretion signal peptide (RipATB-AD). As a control, we also produced a construct in which the M. smegmatis RipA active domain (RipASm-AD) can be secreted. None of the strains produced growth defects when uninduced (Figure S8). As expected, RipASm-AD like full length RipASm, was fully functional when induced and disrupted cell wall integrity, leading to bulging of the cells and a concomitant growth defect (Figure 6A,B). When RipATB-AD was secreted, we found that it was also functional and behaved in the same way as RipASm-AD (Figure 6A,B). Thus, the catalytic domain of RipATB can be active in M. smegmatis, but full length RipATB is not toxic because it does not undergo efficient processing in M. smegmatis.

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