Research Article: Protein Complexes and Proteolytic Activation of the Cell Wall Hydrolase RipA Regulate Septal Resolution in Mycobacteria

Date Published: February 28, 2013

Publisher: Public Library of Science

Author(s): Michael C. Chao, Karen J. Kieser, Shoko Minami, Daniela Mavrici, Bree B. Aldridge, Sarah M. Fortune, Tom Alber, Eric J. Rubin, Adrie J. C. Steyn.


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.

Partial Text

Mycobacterium tuberculosis is the causative agent of tuberculosis and accounts for up to 10 million symptomatic infections a year [1]. The spread of multi-, extensively- and now totally- drug resistant strains [2] has created a pressing need to understand essential mycobacterial processes in an effort to define novel targets for chemotherapy. One highly essential bacterial process is peptidoglycan (PG) synthesis and remodeling, which is critical for providing structural integrity in nearly all bacteria. PG forms a continuous macromolecular mesh that is part of the bacterial cell wall and is required for correct cellular morphology and opposition to osmotic forces. Despite extensive biochemical and genetic characterization of the enzymes responsible for the synthesis and degradation of PG (reviewed in [3], [4]), the mechanism by which these enzymes coordinate their activities remains poorly defined. It is clear, however, that dysregulation of this homeostatic balance frequently has lethal effects on the bacterium—inactivation of peptidoglycan synthases, either through the use of penicillin derivatives or overexpression of dominant negative forms of PG synthetic enzymes, induces lysis of cells [5], [6]. In many cases, this lethality can be suppressed by inactivation of several peptidoglycan hydrolases [5], [7], suggesting that PG hydrolase autolysin activity is restrained by functional interactions with PG synthases. This idea is consistent with a ‘make-then-break’ approach to cell wall synthesis where new PG subunits are first incorporated before the existing sacculus is cleaved to allow expansion [8]. One example of this is the formation of the septal PG—cells ensure that the septal PG is formed before PG hydrolases cleave apart the daughter cells.

Bacteria rely on peptidoglycan (PG) for shape and structure. The prevailing view of PG remodeling requires the concerted action of synthetic enzymes ligating new subunits into the existing PG lattice followed by hydrolysis of the PG sacculus by autolysins to allow cellular expansion or division. This process is accomplished through the action of large holoenzyme complexes in the periplasm consisting of both PG synthetic and hydrolytic enzymes. Disruption of PG synthesis in these complexes can dysregulate cognate PG hydrolases, which can then become autolysins that lyse the cell [26]. Thus, the coordination and regulation of PG hydrolases is a critical process for the survival of the bacterium.




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