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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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Processed RipA is toxic to M. tuberculosis.(A) Full length RipATB and truncated RipATB-AD was overexpressed in M. tuberculosis for several days with addition of aTc. Cells were analyzed by microscopy for changes in morphology. Membranes were stained with FM4-64. Scale bar represents 2 µM. (B) Growth of M. tuberculosis induced with aTc and overexpressing full length RipATB was measured by OD600. (C) Growth of M. tuberculosis induced with aTc and overexpressing truncated RipATB-AD was measured by OD600. (D) M. tuberculosis RipA (RipATB) was overexpressed in M. tuberculosis by induction (lane 2) with aTc for 48 hours. RipATB was then detected by anti-RipA Western blot analysis. Uninduced lysates were used a control (lane 1). Full length RipA (arrow) and processed forms (brackets) were detected. (E) Total overexpressed protein was quantified from (D) by performing densitometry analysis on bands apparent in the induced strain that are absent from the uninduced control. Fold change in total RipATB overexpression relative to endogenous full length RipA signal was graphed.
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ppat-1003197-g007: Processed RipA is toxic to M. tuberculosis.(A) Full length RipATB and truncated RipATB-AD was overexpressed in M. tuberculosis for several days with addition of aTc. Cells were analyzed by microscopy for changes in morphology. Membranes were stained with FM4-64. Scale bar represents 2 µM. (B) Growth of M. tuberculosis induced with aTc and overexpressing full length RipATB was measured by OD600. (C) Growth of M. tuberculosis induced with aTc and overexpressing truncated RipATB-AD was measured by OD600. (D) M. tuberculosis RipA (RipATB) was overexpressed in M. tuberculosis by induction (lane 2) with aTc for 48 hours. RipATB was then detected by anti-RipA Western blot analysis. Uninduced lysates were used a control (lane 1). Full length RipA (arrow) and processed forms (brackets) were detected. (E) Total overexpressed protein was quantified from (D) by performing densitometry analysis on bands apparent in the induced strain that are absent from the uninduced control. Fold change in total RipATB overexpression relative to endogenous full length RipA signal was graphed.

Mentions: Given the potentially toxic nature of hyperactive RipA we hypothesized that RipA processing and activation may be less robust in slow-growing mycobacteria in order to match their much slower rate of growth and consequent lower requirement for peptidoglycan hydrolysis. To investigate this model, we first determined whether RipA is processed in M. tuberculosis by overexpressing RipATB. By Western blot analysis, we found multiple immunoreactive smaller species of RipATB, suggesting processing in M. tuberculosis (Figure 7D, brackets). However, the induction of RipATB in M. tuberculosis did not produce morphological changes or growth defects, even after five days of induction (Figure 7A,B). This overexpression produced about 3 fold more protein (most of which is in the processed form) than endogenous full length RipA (Figure 7E), which is similar to the amount of overexpression needed to observe cell chaining in M. smegmatis with the RipASm C408A allele (Figure 4C). The lack of morphological changes in M. tuberculosis is also in contrast with the marked lethality of RipASm overexpression in M. smegmatis (Figure 1).


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)

Processed RipA is toxic to M. tuberculosis.(A) Full length RipATB and truncated RipATB-AD was overexpressed in M. tuberculosis for several days with addition of aTc. Cells were analyzed by microscopy for changes in morphology. Membranes were stained with FM4-64. Scale bar represents 2 µM. (B) Growth of M. tuberculosis induced with aTc and overexpressing full length RipATB was measured by OD600. (C) Growth of M. tuberculosis induced with aTc and overexpressing truncated RipATB-AD was measured by OD600. (D) M. tuberculosis RipA (RipATB) was overexpressed in M. tuberculosis by induction (lane 2) with aTc for 48 hours. RipATB was then detected by anti-RipA Western blot analysis. Uninduced lysates were used a control (lane 1). Full length RipA (arrow) and processed forms (brackets) were detected. (E) Total overexpressed protein was quantified from (D) by performing densitometry analysis on bands apparent in the induced strain that are absent from the uninduced control. Fold change in total RipATB overexpression relative to endogenous full length RipA signal was graphed.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003197-g007: Processed RipA is toxic to M. tuberculosis.(A) Full length RipATB and truncated RipATB-AD was overexpressed in M. tuberculosis for several days with addition of aTc. Cells were analyzed by microscopy for changes in morphology. Membranes were stained with FM4-64. Scale bar represents 2 µM. (B) Growth of M. tuberculosis induced with aTc and overexpressing full length RipATB was measured by OD600. (C) Growth of M. tuberculosis induced with aTc and overexpressing truncated RipATB-AD was measured by OD600. (D) M. tuberculosis RipA (RipATB) was overexpressed in M. tuberculosis by induction (lane 2) with aTc for 48 hours. RipATB was then detected by anti-RipA Western blot analysis. Uninduced lysates were used a control (lane 1). Full length RipA (arrow) and processed forms (brackets) were detected. (E) Total overexpressed protein was quantified from (D) by performing densitometry analysis on bands apparent in the induced strain that are absent from the uninduced control. Fold change in total RipATB overexpression relative to endogenous full length RipA signal was graphed.
Mentions: Given the potentially toxic nature of hyperactive RipA we hypothesized that RipA processing and activation may be less robust in slow-growing mycobacteria in order to match their much slower rate of growth and consequent lower requirement for peptidoglycan hydrolysis. To investigate this model, we first determined whether RipA is processed in M. tuberculosis by overexpressing RipATB. By Western blot analysis, we found multiple immunoreactive smaller species of RipATB, suggesting processing in M. tuberculosis (Figure 7D, brackets). However, the induction of RipATB in M. tuberculosis did not produce morphological changes or growth defects, even after five days of induction (Figure 7A,B). This overexpression produced about 3 fold more protein (most of which is in the processed form) than endogenous full length RipA (Figure 7E), which is similar to the amount of overexpression needed to observe cell chaining in M. smegmatis with the RipASm C408A allele (Figure 4C). The lack of morphological changes in M. tuberculosis is also in contrast with the marked lethality of RipASm overexpression in M. smegmatis (Figure 1).

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