Inactivation Of Penicillin-binding Proteins Research Articles

In Vivo Probe of Lipid II‐Interacting Proteins

β-Lactams represent one of the most important classes of antibiotics discovered to date. These agents block Lipid II processing and cell wall biosynthesis through inactivation of penicillin-binding proteins (PBPs). PBPs enzymatically load cell wall building blocks from Lipid II carrier molecules onto the growing cell wall scaffold during growth and division. Lipid II, a bottleneck in cell wall biosynthesis, is the target of some of the most potent antibiotics in clinical use. Despite the immense therapeutic value of this biosynthetic pathway, the PBP-Lipid II association has not been established in live cells. To determine this key interaction, we designed an unnatural d-amino acid dipeptide that is metabolically incorporated into Lipid II molecules. By hijacking the peptidoglycan biosynthetic machinery, photoaffinity probes were installed in combination with click partners within Lipid II, thereby allowing, for the first time, demonstration of PBP interactions in vivo with Lipid II.

Penicillin binding protein 5 affects cell diameter, contour, and morphology of Escherichia coli.

Although general physiological functions have been ascribed to the high-molecular-weight penicillin binding proteins (PBPs) of Escherichia coli, the low-molecular-weight PBPs have no well-defined biological roles. When we examined the morphology of a set of E. coli mutants lacking multiple PBPs, we observed that strains expressing active PBP 5 produced cells of normal shape, while mutants lacking PBP 5 produced cells with altered diameters, contours, and topological features. These morphological effects were visible in untreated cells, but the defects were exacerbated in cells forced to filament by inactivation of PBP 3 or FtsZ. After filamentation, cellular diameter varied erratically along the length of individual filaments and many filaments exhibited extensive branching. Also, in general, the mean diameter of cells lacking PBP 5 was significantly increased compared to that of cells from isogenic strains expressing active PBP 5. Expression of cloned PBP 5 reversed the effects observed in DeltadacA mutants. Although deletion of PBP 5 was required for these phenotypes, the absence of additional PBPs magnified the effects. The greatest morphological alterations required that at least three PBPs in addition to PBP 5 be deleted from a single strain. In the extreme cases in which six or seven PBPs were deleted from a single mutant, cells and cell filaments expressing PBP 5 retained a normal morphology but cells and filaments lacking PBP 5 were aberrant. In no case did mutation of another PBP produce the same drastic morphological effects. We conclude that among the low-molecular-weight PBPs, PBP 5 plays a principle role in determining cell diameter, surface uniformity, and overall topology of the peptidoglycan sacculus.

Inactivation of Escherichia coli penicillin-binding proteins by human neutrophils

Neutrophils use a variety of microbicidal mechanisms in their role as one of the primary arms of the human host defense system. We have previously observed that a cell-free system containing myeloperoxidase (MPO), one of the major components of the neutrophil's oxidative antimicrobial systems, inactivated microbial penicillin-binding proteins (PBPs), which mediate the formation of the peptidoglycan layer of eubacterial cell walls. This is a potentially important mechanism of MPO-mediated bacterial toxicity. Since numerous other microbicidal systems, both oxidative and nonoxidative, are used by whole neutrophils, we investigated the effect of intact neutrophils on Escherichia coli PBPs. Penicillin binding activity was progressively reduced by neutrophil exposure for all PBPs. Loss of penicillin binding activity correlated well with loss of microbial viability for almost all PBPs. Azide-treated neutrophils, MPO-deficient neutrophils, and chronic granulomatous disease neutrophils inactivated E. coli PBPs in a manner similar to that of normal neutrophils, suggesting that MPO-independent, and even oxygen-independent, microbicidal systems are also involved in inactivation of PBPs. PBP inactivation, an antimicrobial strategy used by beta-lactam-producing molds (and now by physicians), may be an important microbicidal mechanism used by human neutrophils.

Bacteriostatic and bactericidal activities of beta-lactams against Streptococcus (Enterococcus) faecium are associated with saturation of different penicillin-binding proteins

The MICs and MBCs of benzylpenicillin, ampicillin, cefotaxime, and methicillin were evaluated against a Streptococcus (Enterococcus) faecium wild-type strain and against three mutants hyperproducing PBP 5 in cells incubated at both optimal and suboptimal temperatures. In the wild-type strain grown at optimal temperature, the MBCs of all beta-lactams were significantly greater than the MICs (bacteriostatic effect). As opposed to this, in the same cells grown at suboptimal temperature and in the mutants hyperproducing PBP 5 at all temperatures, the MICs of all antibiotics coincided with the MBCs (bactericidal effect). Under all conditions in which the MIC and MBC were the same, with all antibiotics, growth inhibition occurred only at the minimal concentration saturating all penicillin-binding proteins (PBPs) (or at higher concentrations). On the contrary, under conditions in which the MIC was lower than the MBC, only some of the PBPs were saturated (or bound) at both the MIC and the MBC, PBP 5 in no case being either saturated or bound. Under all conditions in which saturation of all PBPs was needed for growth inhibition, cells died at all antibiotic MBCs with kinetics which were much faster than those with which they died at the MBCs under conditions in which not all PBPs were saturated (or bound). In addition, under the former conditions, antibiotic concentrations above the MBCs did not significantly accelerate cell death kinetics, while under the latter conditions there was an acceleration in kinetics with increasing antibiotic concentrations up to full saturation of PBPs. It is suggested that the killing that occurs when all PBPs are saturated is a direct consequence of inactivation of PBP functions, while killing occurring when only some of them are saturated or bound is also (or mainly) an indirect consequence of inability of cells to grow and that, in S. faecium, the targets for growth inhibition and cell killing reside in different PBPs: for the latter effect, inactivation of one (or more) of the high-molecular-weight PBPs is sufficient, whereas in the former case inactivation of PBP 5 is necessary (after saturation of all other PBPs).

Spherical E. coli due to elevated levels of D-alanine carboxypeptidase.

The characteristic shape of bacterial cells is maintained by the strength and form of the murein layer of the cell wall1. Thus, rod-shaped bacteria need to synthesize a rod-shaped murein. The shape of the murein may be determined by the properties of the penicillin-binding proteins (PBPs) that catalyse the insertion of murein precursors into the cell wall2. In Escherichia coli inhibition of PBPs produces characteristic effects on bacterial morphology. For example, inactivation of PBP 2 results in an inability to synthesize cylindrical murein during cell elongation and the growth of E. coli as spherical cells2. We report here that osmotically stable spherical cells of E. coli can also be produced by an increase in the level of PBP 5, a D-alanine carboxypeptidase3, and show that these cells, and those produced by inactivation of PBP 2, have the same abnormality in the structure of the newly inserted murein.