Probing the Catalytic Activity of a Cell Division-Specific Transpeptidase In Vivo with β-Lactams

127

Bacterial cell division is a complex, multimolecular process that requires biosynthesis of new peptidoglycan by penicillin-binding proteins (PBPs) during cell wall elongation and septum formation steps. Streptococcus pneumoniae has three bifunctional (class A) PBPs that catalyze both polymerization of glycan chains (glycosyltransfer) and cross-linking of pentapeptidic bridges (transpeptidation) during the peptidoglycan biosynthetic process. In addition to playing important roles in cell division, PBPs are also the targets for ␤-lactam antibiotics and thus play key roles in drug-resistance mechanisms. The crystal structure of a soluble form of pneumococcal PBP1b (PBP1b*) has been solved to 1.9 Å, thus providing previously undescribed structural information regarding a class A PBP from any organism. PBP1b* is a three-domain molecule harboring a short peptide from the glycosyltransferase domain bound to an interdomain linker region, the transpeptidase domain, and a C-terminal region. The structure of PBP1b* complexed with ␤-lactam antibiotics reveals that ligand recognition requires a conformational modification involving conserved elements within the cleft. The open and closed structures of PBP1b* suggest how class A PBPs may become activated as novel peptidoglycan synthesis becomes necessary during the cell division process. In addition, this structure provides an initial framework for the understanding of the role of class A PBPs in the development of antibiotic resistance.antibiotic ͉ cell wall ͉ transpeptidase ͉ glycosyltransferase S treptococcus pneumoniae is a major human pathogen, being the causative agent of pneumonia and meningitis, which victimize Ͼ1 million individuals yearly, especially in developing countries. Treatment of such infections has, for Ͼ60 years, depended on ␤-lactam antibiotics, which perturb the cell wall biosynthetic machinery by targeting penicillin-binding proteins (PBPs). PBPs catalyze the last steps in the synthesis of the peptidoglycan, a 3D cross-linked mesh composed of repeating disaccharide units linked to peptidic bridges (1); stability of the peptidoglycan not only is necessary for bacterial shape and morphology, but its synthesis is an absolute requirement for normal cell division and growth processes. Thus, targeting of the peptidoglycan biosynthetic machinery by ␤-lactams often leads to cell lysis and death.

Section: Discussionmentioning

confidence: 48%

Active site restructuring regulates ligand recognition in class A penicillin-binding proteins

Machebœuf

Guilmi

Job

et al. 2005

127

“…Although FtsI inhibitors are often used to induce E. coli to filament (11), scattered reports indicate that they can also induce rapid cell lysis (12,13). We found that midcell lysis is reproducible and requires cells to be growing exponentially in rich media at low cell densities [Ͻ10 8 cells/ml, supporting information (SI) Fig.…”

Section: Cephalexin Induces Filamentation Followed By Lysis At Low Cellmentioning

confidence: 80%

Rapid β-lactam-induced lysis requires successful assembly of the cell division machinery

Chung

Yao

Goehring³

et al. 2009

113

116

␤-lactam antibiotics inhibit penicillin binding proteins (PBPs) involved in peptidoglycan synthesis. Although inhibition of peptidoglycan biosynthesis is generally thought to induce cell lysis, the pattern and mechanism of cell lysis can vary substantially. ␤-lactams that inhibit FtsI, the only division specific PBP, block cell division and result in growth as filaments. These filaments ultimately lyse through a poorly understood mechanism. Here we find that one such ␤-lactam, cephalexin, can, under certain conditions, lead instead to rapid lysis at nascent division sites through a process that requires the complete and ordered assembly of the divisome, the essential machinery involved in cell division. We propose that this assembly process (in which the localization of cell wall hydrolases depends on properly targeted FtsN, which in turn depends on the presence of FtsI) ensures that the biosynthetic machinery to form new septa is in place before the machinery to degrade septated daughter cells is enabled. ␤-lactams that target FtsI subvert this mechanism by inhibiting FtsI without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. One seemingly paradoxical implication of our results is that ␤-lactam therapy may be improved by promoting active cell division.amidase ͉ cell wall hydrolase ͉ ftsN ͉ penicillin-binding proteins ͉ peptidoglycan

“…To further investigate whether cell wall synthesis limits the progression of septum closure, we analyzed septum closure rates in a temperature-sensitive mutant strain, MCI23, in which the total cellular activity of the essential cell wall transpeptidase, FtsI (also known as PBP3), is reduced even at permissive temperatures, likely because of decreased thermostability of the protein (101). FtsI localizes to the septum at the onset of constriction (102,103) and exhibits stimulated transpeptidase activity in dividing cells compared with nondividing cells (104). If FtsI's PG synthesis activity, rather than Z-ring contraction, were a primary driver of septum closure, we would expect the lowered FtsI activity in the MCI23 strain to result in a reduced rate of septum closure.…”

Section: Decreased Activity Of Pg Synthesis Enzyme Ftsi Decreases Septummentioning

confidence: 99%

Defining the rate-limiting processes of bacterial cytokinesis

Coltharp

Buss

Plumer

et al. 2016

162

216

Bacterial cytokinesis is accomplished by the essential ‘divisome’ machinery. The most widely conserved divisome component, FtsZ, is a tubulin homolog that polymerizes into the ‘FtsZ-ring’ (‘Z-ring’). Previous in vitro studies suggest that Z-ring contraction serves as a major constrictive force generator to limit the progression of cytokinesis. Here, we applied quantitative superresolution imaging to examine whether and how Z-ring contraction limits the rate of septum closure during cytokinesis in Escherichia coli cells. Surprisingly, septum closure rate was robust to substantial changes in all Z-ring properties proposed to be coupled to force generation: FtsZ’s GTPase activity, Z-ring density, and the timing of Z-ring assembly and disassembly. Instead, the rate was limited by the activity of an essential cell wall synthesis enzyme and further modulated by a physical divisome–chromosome coupling. These results challenge a Z-ring–centric view of bacterial cytokinesis and identify cell wall synthesis and chromosome segregation as limiting processes of cytokinesis.