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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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Proteolyzied RipASm C408A overexpression correlates with morphological toxicity.(A) Micrographs of M. smegmatis grown in various concentrations of inducer to overexpress RipASm C408A-FLAG. Scale bar represents 2 µm. (B) Total cell lysates from M. smegmatis overexpressing RIpASm C408A-FLAG under various inducer concentrations were probed with anti-FLAG antibody. Both full length (arrow) and processed RipASm C408A species (brackets) were detected, including two major processed species: a band at 25 kDa and a doublet around 12 kDa. (C) Samples from (B) were probed with anti-RipA antibody (Figure S7) and densitometry analysis performed. The levels of full length (black), processed (red) and total (blue) recombinant RipASm C408A proteins were compared to endogenous RipA levels. The fold overexpression of RipASm C408A relative to endogenous RipA was graphed as a function of inducer concentration.
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ppat-1003197-g004: Proteolyzied RipASm C408A overexpression correlates with morphological toxicity.(A) Micrographs of M. smegmatis grown in various concentrations of inducer to overexpress RipASm C408A-FLAG. Scale bar represents 2 µm. (B) Total cell lysates from M. smegmatis overexpressing RIpASm C408A-FLAG under various inducer concentrations were probed with anti-FLAG antibody. Both full length (arrow) and processed RipASm C408A species (brackets) were detected, including two major processed species: a band at 25 kDa and a doublet around 12 kDa. (C) Samples from (B) were probed with anti-RipA antibody (Figure S7) and densitometry analysis performed. The levels of full length (black), processed (red) and total (blue) recombinant RipASm C408A proteins were compared to endogenous RipA levels. The fold overexpression of RipASm C408A relative to endogenous RipA was graphed as a function of inducer concentration.

Mentions: The observation of RipA cleavage suggested this process could be required for the protein's function in vivo. To test whether RipA processing is correlated with division, we titrated overexpression of RipASm C408A and quantified the amount of induction needed to mediate chaining. We found that low level overexpression with 30 ng/mL inducer was sufficient to cause chaining (Figure 4A) without saturating the processing machinery, since these cells did not accumulate full length RipA (Figure 4B). Instead, mildly overexpressed RipA was processed down to two sets of smaller species at around 23 kDa and 12 kDa (Figure 4B). We also saw a dose-dependent saturation of the endogenous processing capacity, with high induction leading to accumulation of full length RipA (Figure 4B), as well as loss of processed endogenous RipA (Figure S6A). When we quantified recombinant protein levels by comparing densitometry with endogenous RipA protein (Figure 4C), we found that even mild RipASm C408A overexpression at 30 ng/mL of inducer (processed recombinant protein is approximately 10% of endogenous wild type RipA levels) was sufficient to cause chaining and cellular toxicity (Figure 4A). Together with qPCR data showing that RipASm C408A induction does not affect endogenous RipA transcription (Figure S6B), these data suggest direct competition between the RipAsm C408A mutant and endogenous RipA for processing machinery, and that the processed, not full length, form of RipA is required for division.


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)

Proteolyzied RipASm C408A overexpression correlates with morphological toxicity.(A) Micrographs of M. smegmatis grown in various concentrations of inducer to overexpress RipASm C408A-FLAG. Scale bar represents 2 µm. (B) Total cell lysates from M. smegmatis overexpressing RIpASm C408A-FLAG under various inducer concentrations were probed with anti-FLAG antibody. Both full length (arrow) and processed RipASm C408A species (brackets) were detected, including two major processed species: a band at 25 kDa and a doublet around 12 kDa. (C) Samples from (B) were probed with anti-RipA antibody (Figure S7) and densitometry analysis performed. The levels of full length (black), processed (red) and total (blue) recombinant RipASm C408A proteins were compared to endogenous RipA levels. The fold overexpression of RipASm C408A relative to endogenous RipA was graphed as a function of inducer concentration.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585148&req=5

ppat-1003197-g004: Proteolyzied RipASm C408A overexpression correlates with morphological toxicity.(A) Micrographs of M. smegmatis grown in various concentrations of inducer to overexpress RipASm C408A-FLAG. Scale bar represents 2 µm. (B) Total cell lysates from M. smegmatis overexpressing RIpASm C408A-FLAG under various inducer concentrations were probed with anti-FLAG antibody. Both full length (arrow) and processed RipASm C408A species (brackets) were detected, including two major processed species: a band at 25 kDa and a doublet around 12 kDa. (C) Samples from (B) were probed with anti-RipA antibody (Figure S7) and densitometry analysis performed. The levels of full length (black), processed (red) and total (blue) recombinant RipASm C408A proteins were compared to endogenous RipA levels. The fold overexpression of RipASm C408A relative to endogenous RipA was graphed as a function of inducer concentration.
Mentions: The observation of RipA cleavage suggested this process could be required for the protein's function in vivo. To test whether RipA processing is correlated with division, we titrated overexpression of RipASm C408A and quantified the amount of induction needed to mediate chaining. We found that low level overexpression with 30 ng/mL inducer was sufficient to cause chaining (Figure 4A) without saturating the processing machinery, since these cells did not accumulate full length RipA (Figure 4B). Instead, mildly overexpressed RipA was processed down to two sets of smaller species at around 23 kDa and 12 kDa (Figure 4B). We also saw a dose-dependent saturation of the endogenous processing capacity, with high induction leading to accumulation of full length RipA (Figure 4B), as well as loss of processed endogenous RipA (Figure S6A). When we quantified recombinant protein levels by comparing densitometry with endogenous RipA protein (Figure 4C), we found that even mild RipASm C408A overexpression at 30 ng/mL of inducer (processed recombinant protein is approximately 10% of endogenous wild type RipA levels) was sufficient to cause chaining and cellular toxicity (Figure 4A). Together with qPCR data showing that RipASm C408A induction does not affect endogenous RipA transcription (Figure S6B), these data suggest direct competition between the RipAsm C408A mutant and endogenous RipA for processing machinery, and that the processed, not full length, form of RipA is required for division.

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