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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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RipASm but not RipATB is proteolytically processed in M. smegmatis.(A) Whole cell lysates were made from cells depleted for RipA (Depl), wildtype M. smegmatis (WT), and cells overexpressing either wildtype RipASm (RipASm), RipATB (RipATB), or catalytically inactive RipASm C408A (C408A). The lysates were probed with anti-RipA polyclonal antibody. Full length RipA (arrow), and proteolytically cleaved endogenous RipA (red arrow) and recombinant species (brackets) were detected. (B) Coomassie stain of RipA-expressing M. smegmatis cells showing total protein loaded. Lanes are identical to (A). As RipASm C408A induction is particularly strong, this sample was deliberately underloaded to prevent its signal in (A) from overwhelming detection of less abundant species. (C) Western blots with anti-RipA (left panel) and anti-FLAG antibodies (right panel). Culture filtrates were collected from wildtype M. smegmatis (WT) and cells in which chromosomal RipA was C-terminally fused to a FLAG epitope (FL). A truncated RipA species that is shifted by the FLAG tag was observed (asterisk).
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ppat-1003197-g003: RipASm but not RipATB is proteolytically processed in M. smegmatis.(A) Whole cell lysates were made from cells depleted for RipA (Depl), wildtype M. smegmatis (WT), and cells overexpressing either wildtype RipASm (RipASm), RipATB (RipATB), or catalytically inactive RipASm C408A (C408A). The lysates were probed with anti-RipA polyclonal antibody. Full length RipA (arrow), and proteolytically cleaved endogenous RipA (red arrow) and recombinant species (brackets) were detected. (B) Coomassie stain of RipA-expressing M. smegmatis cells showing total protein loaded. Lanes are identical to (A). As RipASm C408A induction is particularly strong, this sample was deliberately underloaded to prevent its signal in (A) from overwhelming detection of less abundant species. (C) Western blots with anti-RipA (left panel) and anti-FLAG antibodies (right panel). Culture filtrates were collected from wildtype M. smegmatis (WT) and cells in which chromosomal RipA was C-terminally fused to a FLAG epitope (FL). A truncated RipA species that is shifted by the FLAG tag was observed (asterisk).

Mentions: When we used a RipA polyclonal antibody that recognizes a C-terminal epitope, we observed truncated RipA species from mycobacteria by Western blotting. When we overexpressed RipASm, we found several bands smaller than the predicted full length protein (Figure 3A, lane 3). Likewise, we saw these truncated bands when we overexpressed RipASm C408A in M. smegmatis (Figure 3A, brackets). These products were not due to non-specific cytoplasmic degradation of overexpressed RipA, as we fractionated RipASm C408A overexpressing cells and found that RipA processed species were enriched in the cell wall fraction (Figure S4A). The efficiency of fractionating mycobacteria was confirmed by Western blotting against RpoB (cytosolic) and mycobacterial antigen 85 (cell wall) markers (Figures S4B, S4C). Thus, these results suggest that RipA undergoes physiological post-translational processing in the periplasmic or cell wall compartment.


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)

RipASm but not RipATB is proteolytically processed in M. smegmatis.(A) Whole cell lysates were made from cells depleted for RipA (Depl), wildtype M. smegmatis (WT), and cells overexpressing either wildtype RipASm (RipASm), RipATB (RipATB), or catalytically inactive RipASm C408A (C408A). The lysates were probed with anti-RipA polyclonal antibody. Full length RipA (arrow), and proteolytically cleaved endogenous RipA (red arrow) and recombinant species (brackets) were detected. (B) Coomassie stain of RipA-expressing M. smegmatis cells showing total protein loaded. Lanes are identical to (A). As RipASm C408A induction is particularly strong, this sample was deliberately underloaded to prevent its signal in (A) from overwhelming detection of less abundant species. (C) Western blots with anti-RipA (left panel) and anti-FLAG antibodies (right panel). Culture filtrates were collected from wildtype M. smegmatis (WT) and cells in which chromosomal RipA was C-terminally fused to a FLAG epitope (FL). A truncated RipA species that is shifted by the FLAG tag was observed (asterisk).
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Related In: Results  -  Collection

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ppat-1003197-g003: RipASm but not RipATB is proteolytically processed in M. smegmatis.(A) Whole cell lysates were made from cells depleted for RipA (Depl), wildtype M. smegmatis (WT), and cells overexpressing either wildtype RipASm (RipASm), RipATB (RipATB), or catalytically inactive RipASm C408A (C408A). The lysates were probed with anti-RipA polyclonal antibody. Full length RipA (arrow), and proteolytically cleaved endogenous RipA (red arrow) and recombinant species (brackets) were detected. (B) Coomassie stain of RipA-expressing M. smegmatis cells showing total protein loaded. Lanes are identical to (A). As RipASm C408A induction is particularly strong, this sample was deliberately underloaded to prevent its signal in (A) from overwhelming detection of less abundant species. (C) Western blots with anti-RipA (left panel) and anti-FLAG antibodies (right panel). Culture filtrates were collected from wildtype M. smegmatis (WT) and cells in which chromosomal RipA was C-terminally fused to a FLAG epitope (FL). A truncated RipA species that is shifted by the FLAG tag was observed (asterisk).
Mentions: When we used a RipA polyclonal antibody that recognizes a C-terminal epitope, we observed truncated RipA species from mycobacteria by Western blotting. When we overexpressed RipASm, we found several bands smaller than the predicted full length protein (Figure 3A, lane 3). Likewise, we saw these truncated bands when we overexpressed RipASm C408A in M. smegmatis (Figure 3A, brackets). These products were not due to non-specific cytoplasmic degradation of overexpressed RipA, as we fractionated RipASm C408A overexpressing cells and found that RipA processed species were enriched in the cell wall fraction (Figure S4A). The efficiency of fractionating mycobacteria was confirmed by Western blotting against RpoB (cytosolic) and mycobacterial antigen 85 (cell wall) markers (Figures S4B, S4C). Thus, these results suggest that RipA undergoes physiological post-translational processing in the periplasmic or cell wall compartment.

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