Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin

Williamson, Russell; Tomasz, Alexander

doi:10.1111/j.1432-1033.1985.tb09126.x

Cited by 45 publications

(34 citation statements)

References 41 publications

(5 reference statements)

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“…Earlier studies have shown that bacterial cells can continue cell wall synthesis and growth with less than the total normal complement of wall synthetic enzymes, suggesting that only a subclass of all these proteins detectable by the penicillin-binding technique is actually essential (3,22). We introduced an experimental design that allowed us to further dissect this question.…”

Section: Discussionmentioning

confidence: 99%

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Newly made enzymes determine ongoing cell wall synthesis and the antibacterial effects of cell wall synthesis inhibitors

Tuomanen¹

1986

J Bacteriol

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Cell wall synthesis can continue with less than the total complement of cell wall synthetic enzymes present in normal growing cells. A method was developed to investigate whether there exists an excess of cell wall-synthesizing enzymes (penicillin-binding proteins rPBPs]) which all remain functional or whether a mixed population of functional and nonfunctional enzymes charicterize normal cells. Surprisingly, cells in which less than 10% of the PBPs were functional could grow at a normal rate, as evidenced by increases in viable counts, culture turbidity, and rates of peptidoglycan, protein, and RNA synthesis. This subset of functional enzymes was biosynthetically new. Penicillin-induced lysis occurred contingent on the acylation of this same small fraction of PBPs, the copy number and affinities of which were below the level of detection by current fluorographic assay techniques. We propose that PBPs have a short functional half-life and that cell wall synthesis and bacterial lysis reflect the activity of newly synthesized PBPs.Cell wall synthetic enzymes are integral membrane proteins. The fact that these proteins form covalent complexes with radioactive penicillin allows the quick detection and quantitation of these important membrane proteins without need of purification or other complex strategies that are used for thti detection and titration of other protein membrane coniponnts. The availability of such a sensitive and specific assay opens up the possibility of studying the biosynthesis, membrane incorporation, and regulation of this set of protein molecules of great biological importance.The catalytic functions of cell wall synthetic enzymes (penicillin-binding protein [PBPs]) involve a strong topographic element: these enzymes, while anchored in the plasma membrane, catalyze the building and rearrangement of covalent bonds in the macromolecular sheets of cell wall exterior to the plasma membrane. In the bacteria studied in this report, this incorporation reaction is restricted to a limited number of anatomical areas of the cell suirface, most commonly at the cell equator (1, 6). PBPs are minor protein constituents of the plasma membrane which have been estimated to represent about 3,000 molecules per cell in Escherichia coli (17) and about 20,000 molecules in pneumococci (21). However, only 1,000 molecules per cell in E. coli and 2/3 of the total number of PBPs in pneumococci appear to perform essential functions. Additional studies haveshown that apparently normal wall synthesis (and cell multiplication) can continue with less than a full complement of these essential PBPs (3, 22), indicating either that these proteins are present in excess or that only some fraction of all the proteins estimated by the PBP assay are functional. The purpose of the studies described in this paper was to test this point. Our experimental strategy exploited conditions of penicillin tolerance in which cell wall synthetic enzymes could be fully acylated with penicillin without killing or lysis of the cells. We carefully exam...

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Section: Discussionmentioning

confidence: 99%

“…shown that apparently normal wall synthesis (and cell multiplication) can continue with less than a full complement of these essential PBPs (3,22), indicating either that these proteins are present in excess or that only some fraction of all the proteins estimated by the PBP assay are functional.…”

mentioning

confidence: 99%

Newly made enzymes determine ongoing cell wall synthesis and the antibacterial effects of cell wall synthesis inhibitors

Tuomanen¹

1986

J Bacteriol

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“…The virtually complete inhibition of the maturation process by cefotaxime, a relatively poor inhibitor of PBP 2b, without a comparably severe inhibition of the formation of the nascept wall,'suggests that PBP 2b may perform an important role in this process (i.e., the initial incorporation of precursors into the wall). Such a role has already been suggested for PBP 2b on the basis of earlier studies (27).…”

Section: Discussionmentioning

confidence: 52%

“…The cell wall of this * Corresponding author. bacterium does not seem to undergo turnover (27), and in the case of pneumococci it is possible to design a method that allows a physical separation of newly made from mature cell wall segments which can then be compared for compositional differences by direct chemical analysis.The method is based on the high degree of specificity of the pneumococcal autolysin (N-acetylmuramoyl-L-alanine amidase) in dissecting out wall segments with choline-containing teichoic acid chains from amid wall segments that contain ethanolamine (EA) teichoic acids and are resistant to the hydrolytic action of the amidase (19). In the basic experimental design, we grew pneumococci in EA medium.…”

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confidence: 99%

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Changes in composition of peptidoglycan during maturation of the cell wall in pneumococci

Laitinen

Tomasz

1990

J Bacteriol

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An experimental system which allows the selective reisolation and structural analysis of a newly made (nascent) segment of pneumococcal peptidoglycan at various times after its incorporation into the preexisting old ceH wall was developed. Age-related changes were observed in each one of the major nine wall peptide components resolvable by a high-performance liquid chromatography method. The nascent wall segment (made in 1.7% of a generation time) contained 60% of its peptides as the alanyl-isoglutamyl-lysine tripeptide monomer, 12% as the directly cross-linked peptide dimer (tri-tetra peptide), and a total of 2% as the two major peptide trimers. In the mature wall segment reisolated 1 h later (1 generation time), the proportion of the tripeptide monomer dropped to 40%, while the major dimer and trimers increased to 23% and 8%, respectively. The age-related structural changes were completely inhibited by cefotaxime. The observations indicate that covalent bonds in the structure of pneumococcal peptidoglycan undergo substantial secondary rearrangements after incorporation into the preexisting wall. These changes are likely to be related to the movement of the conserved cell wall segments within the cell surface during cell division.Streptococcus pneumoniae is a gram-positive coccus with a single wall growth zone located at the cellular equator, where newly made wall units of peptidoglycan and teichoic acid are added to the preexisting wall (2). The wall material becomes part of a conserved segregational unit, which then moves in a symmetrical fashion to the "left" and "right" of the cellular equator during the enlargement of the bacterial cell (25). Virtually nothing is known about the possible structural changes that may accompany these cell divisionrelated movements of the cell wall units on the pneumococcal cell surface.Previous studies with Escherichia coli (4), Neisseria gonorrhoeae (6, 16, 17), Staphylococcus aureus (21), and Streptococcus faecalis (5) resulted in data indicating that newly made peptidoglycan in each of these bacteria had a relative abundance of un-cross-linked stem peptides, some of which then became cross-linked in a maturation process that followed the incorporation of wall precursors into the cell wall. All these experiments were done by using radioactively labeled compounds in pulse-chase experiments. The results have unambiguously shown that the peptidoglycans of both gram-positive and gram-negative bacteria undergo secondary structural changes after their covalent linkage to the preexisting wall. Nevertheless, the existence of pools of unknown size of the radioactive compounds used and the phenomenon of cell wall turnover (which exists in most of the systems used) introduce elements of uncertainty into the qualitative interpretation of the pulse-chase experiments. Recent studies of E. coli indicate that peptidoglycan material released during turnover can be selectively reutilized for wall synthesis (15) and that these recycled compounds can enter the biosynthetic pathway closer to th...

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Combating Multidrug‐Resistant Bacteria by Integrating a Novel Target Site Penetration and Receptor Binding Assay Platform Into Translational Modeling

Lang

Shah

Tao

et al. 2021

Clin Pharma and Therapeutics

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Multidrug-resistant bacteria are causing a serious global health crisis. A dramatic decline in antibiotic discovery and development investment by pharmaceutical industry over the last decades has slowed the adoption of new technologies. It is imperative that we create new mechanistic insights based on latest technologies, and use translational strategies to optimize patient therapy. Although drug development has relied on minimal inhibitory concentration testing and established in vitro and mouse infection models, the limited understanding of outer membrane permeability in Gramnegative bacteria presents major challenges. Our team has developed a platform using the latest technologies to characterize target site penetration and receptor binding in intact bacteria that inform translational modeling and guide new discovery. Enhanced assays can quantify the outer membrane permeability of β-lactam antibiotics and β-lactamase inhibitors using multiplex liquid chromatography tandem mass spectrometry. While β-lactam antibiotics are known to bind to multiple different penicillin-binding proteins (PBPs), their binding profiles are almost always studied in lysed bacteria. Novel assays for PBP binding in the periplasm of intact bacteria were developed and proteins identified via proteomics. To characterize bacterial morphology changes in response to PBP binding, high-throughput flow cytometry and time-lapse confocal microscopy with fluorescent probes provide unprecedented mechanistic insights. Moreover, novel assays to quantify cytosolic receptor binding and intracellular drug concentrations inform target site occupancy. These mechanistic data are integrated by quantitative and systems pharmacology modeling to maximize bacterial killing and minimize resistance in in vitro and mouse infection models. This translational approach holds promise to identify antibiotic combination dosing strategies for patients with serious infections.

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Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin

Cited by 45 publications

References 41 publications

Newly made enzymes determine ongoing cell wall synthesis and the antibacterial effects of cell wall synthesis inhibitors

Newly made enzymes determine ongoing cell wall synthesis and the antibacterial effects of cell wall synthesis inhibitors

Changes in composition of peptidoglycan during maturation of the cell wall in pneumococci

Combating Multidrug‐Resistant Bacteria by Integrating a Novel Target Site Penetration and Receptor Binding Assay Platform Into Translational Modeling

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