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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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RipA is differentially active in a species-specific manner.(A) Fluorescence micrographs of M. smegmatis strains induced with aTc to overexpress M. tuberculosis RipA (RipATB) or M. smegmatis RipA (RipASm). Membranes were stained with TMA-DPH. Scale bar represents 2 µm. (B) M. smegmatis strains in (A) were induced with aTc to overexpress RipATB and RipASm constructs. Growth of these strains was monitored over time by OD600.
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ppat-1003197-g005: RipA is differentially active in a species-specific manner.(A) Fluorescence micrographs of M. smegmatis strains induced with aTc to overexpress M. tuberculosis RipA (RipATB) or M. smegmatis RipA (RipASm). Membranes were stained with TMA-DPH. Scale bar represents 2 µm. (B) M. smegmatis strains in (A) were induced with aTc to overexpress RipATB and RipASm constructs. Growth of these strains was monitored over time by OD600.

Mentions: However, though correlated with function, the processed species we observed could be the product of an inactivating event. To test this, we took advantage of the observation that the M. tuberculosis homologue of RipA (RipATB) functions differently in M. smegmatis than its native counterpart, despite having the same general domain architecture (Figure S7). In contrast to RipASm, which is toxic when overexpressed, overexpression of RipATB in M. smegmatis, surprisingly caused no toxicity or cell morphological differences (Figure 5A,B), despite similar protein levels (Figure 3A). We examined whether RipASm toxicity was correlated with its processing by performing Western blot analysis on RipATB overexpressing M. smegmatis. In contrast to overexpression of RipASm, when wildtype RipATB is overexpressed we observed only a single full length band at 55 kDa (Figure 3A, right arrow). The absence of processing correlates with the lack of detectable RipATB toxicity in M. smegmatis. Thus, we hypothesize that proteolytic cleavage is required for activating RipA in vivo.


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)

RipA is differentially active in a species-specific manner.(A) Fluorescence micrographs of M. smegmatis strains induced with aTc to overexpress M. tuberculosis RipA (RipATB) or M. smegmatis RipA (RipASm). Membranes were stained with TMA-DPH. Scale bar represents 2 µm. (B) M. smegmatis strains in (A) were induced with aTc to overexpress RipATB and RipASm constructs. Growth of these strains was monitored over time by OD600.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003197-g005: RipA is differentially active in a species-specific manner.(A) Fluorescence micrographs of M. smegmatis strains induced with aTc to overexpress M. tuberculosis RipA (RipATB) or M. smegmatis RipA (RipASm). Membranes were stained with TMA-DPH. Scale bar represents 2 µm. (B) M. smegmatis strains in (A) were induced with aTc to overexpress RipATB and RipASm constructs. Growth of these strains was monitored over time by OD600.
Mentions: However, though correlated with function, the processed species we observed could be the product of an inactivating event. To test this, we took advantage of the observation that the M. tuberculosis homologue of RipA (RipATB) functions differently in M. smegmatis than its native counterpart, despite having the same general domain architecture (Figure S7). In contrast to RipASm, which is toxic when overexpressed, overexpression of RipATB in M. smegmatis, surprisingly caused no toxicity or cell morphological differences (Figure 5A,B), despite similar protein levels (Figure 3A). We examined whether RipASm toxicity was correlated with its processing by performing Western blot analysis on RipATB overexpressing M. smegmatis. In contrast to overexpression of RipASm, when wildtype RipATB is overexpressed we observed only a single full length band at 55 kDa (Figure 3A, right arrow). The absence of processing correlates with the lack of detectable RipATB toxicity in M. smegmatis. Thus, we hypothesize that proteolytic cleavage is required for activating RipA in vivo.

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