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

Chao, Michael C.; Kieser, Karen J.; Minami, Shoko; Mavrici, Daniela; Aldridge, Bree B.; Fortune, Sarah M.; Alber, Tom; Rubín, Eric J.

doi:10.1371/journal.ppat.1003197

Cited by 50 publications

(55 citation statements)

References 39 publications

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“…If the critical PG hydrolases are not activated, the cells form long chains that are covalently connected (5). Conversely, if PG hydrolases are activated in a manner that is not coordinated with cell division, the cells are prone to lysis (6). Many of these enzymes form multiprotein complexes in which the hydrolytic activity is controlled by divisome proteins and coordinated with PG synthases (7,8).…”

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confidence: 99%

Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC

Mavrici

Marakalala

Holton

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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137

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Bacterial growth and cell division are coordinated with hydrolysis of the peptidoglycan (PG) layer of the cell wall, but the mechanisms of regulation of extracellular PG hydrolases are not well understood. Here we report the biochemical, structural, and genetic analysis of the Mycobacterium tuberculosis homolog of the transmembrane PG-hydrolase regulator, FtsX. The purified FtsX extracellular domain binds the PG peptidase Rv2190c/RipC N-terminal segment, causing a conformational change that activates the enzyme. Deletion of ftsEX and ripC caused similar phenotypes in Mycobacterium smegmatis, as expected for genes in a single pathway. The crystal structure of the FtsX extracellular domain reveals an unprecedented fold containing two lobes connected by a flexible hinge. Mutations in the hydrophobic cleft between the lobes reduce RipC binding in vitro and inhibit FtsX function in M. smegmatis. These studies suggest how FtsX recognizes RipC and support a model in which a conformational change in FtsX links the cell division apparatus with PG hydrolysis.bacterial cell wall | extracellular signaling | divisome | long-range conformational change

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confidence: 99%

Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC

Mavrici

Marakalala

Holton

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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137

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“…The inability of the ripAB operon to complement the lysozyme defect may be the result of excessive endopeptidase activity in the presence of lysozyme. We have noted that wild-type cells bearing the ripAB operon on a multicopy plasmid are very sick (data not shown), suggesting that higher than normal levels of endopeptidases are detrimental, which has been noted before with RipA (31). The ripAB plasmid is not detrimental to the ⌬ripA mutant in any other assay but the lysozyme assay, suggesting that the damage inflicted by the lysozyme is important in this phenomenon.…”

Section: Discussionmentioning

confidence: 55%

“…It was previously shown that this is likely a species-specific cleavage event (31); thus, the failure of the M. tuberculosis ripAB operon to complement the ⌬ripA mutant is likely because the protease of M. smegmatis does not recognize the M. tuberculosis RipA protein. We noticed a similar loss of complementation in the lysozyme phenotype of the ⌬ripA mutant with the M. tuberculosis ripAB operon.…”

Section: Discussionmentioning

confidence: 99%

“…RipA has an N-terminal domain that occludes the active site of the enzyme, which may have to be removed in order for the enzyme to function on high-molecular-weight PG substrates (17,18,31). It was previously shown that this is likely a species-specific cleavage event (31); thus, the failure of the M. tuberculosis ripAB operon to complement the ⌬ripA mutant is likely because the protease of M. smegmatis does not recognize the M. tuberculosis RipA protein.…”

Section: Discussionmentioning

confidence: 99%

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The RipA and RipB Peptidoglycan Endopeptidases Are Individually Nonessential to Mycobacterium smegmatis

Martinelli

Pavelka

2016

J Bacteriol

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Mycobacteria possess a series of Rip peptidoglycan endopeptidases that have been characterized in various levels of detail. The RipA and RipB proteins have been extensively studied and are DL-endopeptidases, and RipA has been considered essential to Mycobacterium smegmatis and Mycobacterium tuberculosis. We show here that the ripA and ripB genes are individually dispensable in M. smegmatis and that at least one of the genes must be expressed for viability. We characterized strains carrying inframe deletion mutations of ripA and ripB and found that both mutant strains exhibited increased susceptibility to a limited number of antibiotics and to detergent but that only the ⌬ripA mutant displayed hypersusceptibility to lysozyme. We also constructed and characterized ⌬ripD and ⌬ripA ⌬ripD mutants and found that the single mutant had only an intermediate lysozyme hypersusceptibility phenotype compared to that of wild-type cells while loss of ripD in the ⌬ripA background partially rescued the antibiotic and lysozyme phenotypes of the ⌬ripA mutant. IMPORTANCEWe show that the RipA endopeptidase, which has been considered essential for cell division in certain mycobacteria, is not essential but that at least it or a similar protein, RipB, must be expressed by the bacteria for viability. This work is the first description of strains carrying single deletion mutations of RipA, RipB, and a novel endopeptidase-like protein, RipD. O ne of the most important characteristics of mycobacteria is a complex cell envelope, the biosynthesis of which is the target of several antimycobacterial drugs (1-3). Our research is focused upon the assembly and maintenance of the peptidoglycan (PG) layer, which surrounds the entirety of the cell and covalently anchors the other components of the cell envelope. The biosynthesis of the PG layer is inhibited by ␤-lactam antibiotics, which have recently been reevaluated as potential antitubercular drugs (4, 5).The mycobacterial PG is composed of glycan strands of Nacetylglucosamine and N-acetyl/N-glycolylmuramic acid with a high degree of interpeptide cross-links (6-9). Assembly is orchestrated by a variety of transglycosylases and transpeptidases, including the penicillin binding proteins (PBPs) PonA1, PonA2, PbpA, and PbpB, as well as several LD-transpeptidases (reviewed in reference 10). In order to incorporate new precursor material into the preexisting cell wall, the old cell wall material must first be metabolized by lytic transglycosylases, endopeptidases, and amidases (11). Cell wall turnover has been extensively studied in Gram-negative bacteria, but comparatively little is known about the balance of degradation/synthesis of the PG of mycobacteria. Most of the research has focused on the Rpf resuscitation-promoting factors, which are PG glycan hydrolases, and on several PG endopeptidases (11,12).The endopeptidases belong to the NlpC/P60 superfamily (13), with the first NlpC/P60 mycobacterial proteins identified in Mycobacterium marinum and given the names iipA and iipB (invasion and ...

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“…The C‐terminal domain of PonA1 crosslinks the penultimate d ‐alanine to the d ‐meso‐diaminopimelic acid between parallel peptidoglycan strands. PonA1 localizes to the poles and septa of M. smegmatis and is required for maintaining normal cell length in both M. tuberculosis and M. smegmatis 15, 20, 21. Changes to the level of PonA1 protein result in a structurally abnormal cell shape, suggesting that PonA1 is critical for maintaining physiological cell wall synthesis and cell shape during growth.…”

Section: Introductionmentioning

confidence: 99%

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance

et al. 2016

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Mycobacterium tuberculosis is a human respiratory pathogen that causes the deadly disease tuberculosis. The rapid global spread of antibiotic‐resistant M. tuberculosis makes tuberculosis infections difficult to treat. To overcome this problem new effective antimicrobial strategies are urgently needed. One promising target for new therapeutic approaches is PonA1, a class A penicillin‐binding protein, which is required for maintaining physiological cell wall synthesis and cell shape during growth in mycobacteria. Here, crystal structures of the transpeptidase domain, the enzymatic domain responsible for penicillin binding, of PonA1 from M. tuberculosis in the inhibitor‐free form and in complex with penicillin V are reported. We used site‐directed mutagenesis, antibiotic profiling experiments, and fluorescence thermal shift assays to measure PonA1's sensitivity to different classes of β‐lactams. Structural comparison of the PonA1 apo‐form and the antibiotic‐bound form shows that binding of penicillin V induces conformational changes in the position of the loop β4′‐α3 surrounding the penicillin‐binding site. We have also found that binding of different antibiotics including penicillin V positively impacts protein stability, while other tested β‐lactams such as clavulanate or meropenem resulted in destabilization of PonA1. Our antibiotic profiling experiments indicate that the transpeptidase activity of PonA1 in both M. tuberculosis and M. smegmatis mediates tolerance to specific cell wall‐targeting antibiotics, particularly to penicillin V and meropenem. Because M. tuberculosis is an important human pathogen, these structural data provide a template to design novel transpeptidase inhibitors to treat tuberculosis infections.DatabaseStructural data are available in the PDB database under the accession numbers 5CRF and 5CXW.

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

Cited by 50 publications

References 39 publications

Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC

Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC

The RipA and RipB Peptidoglycan Endopeptidases Are Individually Nonessential to Mycobacterium smegmatis

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance

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