scholarly journals Mechanism of β-Lactam Action in Streptococcus pneumoniae: the Piperacillin Paradox

2014 ◽  
Vol 59 (1) ◽  
pp. 609-621 ◽  
Author(s):  
Jules Philippe ◽  
Benoit Gallet ◽  
Cécile Morlot ◽  
Dalia Denapaite ◽  
Regine Hakenbeck ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Streptococcus Pneumoniae ◽  
Mode Of Action ◽  
Time Course ◽  
Binding Proteins ◽  
Epifluorescence Microscopy ◽  
Human Pathogen ◽  
Content Type ◽  
Penicillin Binding Proteins

ABSTRACTThe human pathogenStreptococcus pneumoniaehas been treated for decades with β-lactam antibiotics. Its resistance is now widespread, mediated by the expression of mosaic variants of the target enzymes, the penicillin-binding proteins (PBPs). Understanding the mode of action of β-lactams, not only in molecular detail but also in their physiological consequences, will be crucial to improving these drugs and any counterresistances. In this work, we investigate the piperacillin paradox, by which this β-lactam selects primarily variants of PBP2b, whereas its most reactive target is PBP2x. These PBPs are both essential monofunctional transpeptidases involved in peptidoglycan assembly. PBP2x participates in septal synthesis, while PBP2b functions in peripheral elongation. The formation of the “lemon”-shaped cells induced by piperacillin treatment is consistent with the inhibition of PBP2x. Following the examination of treated and untreated cells by electron microscopy, the localization of the PBPs by epifluorescence microscopy, and the determination of the inhibition time course of the different PBPs, we propose a model of peptidoglycan assembly that accounts for the piperacillin paradox.

scholarly journals A CozE Homolog Contributes to Cell Size Homeostasis of Streptococcus pneumoniae

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Gro Anita Stamsås ◽  
Marine Restelli ◽  
Adrien Ducret ◽  
Céline Freton ◽  
Pierre Simon Garcia ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Cell Size ◽  
Binding Proteins ◽  
Integral Membrane Protein ◽  
Antibiotics Resistance ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
Double Deletion ◽  
Size And Morphology ◽  
Septal Localization

ABSTRACT Control of peptidoglycan assembly is critical to maintain bacterial cell size and morphology. Penicillin-binding proteins (PBPs) are crucial enzymes for the polymerization of the glycan strand and/or their cross-linking via peptide branches. Over the last few years, it has become clear that PBP activity and localization can be regulated by specific cognate regulators. The first regulator of PBP activity in Gram-positive bacteria was discovered in the human pathogen Streptococcus pneumoniae. This regulator, named CozE, controls the activity of the bifunctional PBP1a to promote cell elongation and achieve a proper cell morphology. In this work, we studied a previously undescribed CozE homolog in the pneumococcus, which we named CozEb. This protein displays the same membrane organization as CozE but is much more widely conserved among Streptococcaceae genomes. Interestingly, cozEb deletion results in cells that are smaller than their wild-type counterparts, which is the opposite effect of cozE deletion. Furthermore, double deletion of cozE and cozEb results in poor viability and exacerbated cell shape defects. Coimmunoprecipitation further showed that CozEb is part of the same complex as CozE and PBP1a. However, although we confirmed that CozE is required for septal localization of PBP1a, the absence of CozEb has no effect on PBP1a localization. Nevertheless, we found that the overexpression of CozEb can compensate for the absence of CozE in all our assays. Altogether, our results show that the interplay between PBP1a and the cell size regulators CozE and CozEb is required for the maintenance of pneumococcal cell size and shape. IMPORTANCE Penicillin-binding proteins (PBPs), the proteins catalyzing the last steps of peptidoglycan assembly, are critical for bacteria to maintain cell size, shape, and integrity. PBPs are consequently attractive targets for antibiotics. Resistance to antibiotics in Streptococcus pneumoniae (the pneumococcus) are often associated with mutations in the PBPs. In this work, we describe a new protein, CozEb, controlling the cell size of pneumococcus. CozEb is a highly conserved integral membrane protein that works together with other proteins to regulate PBPs and peptidoglycan synthesis. Deciphering the intricate mechanisms by which the pneumococcus controls peptidoglycan assembly might allow the design of innovative anti-infective strategies, for example, by resensitizing resistant strains to PBP-targeting antibiotics.

scholarly journals Genomic Analyses of DNA Transformation and Penicillin Resistance in Streptococcus pneumoniae Clinical Isolates

2013 ◽  
Vol 58 (3) ◽  
pp. 1397-1403 ◽  
Author(s):  
Fereshteh Fani ◽  
Philippe Leprohon ◽  
George G. Zhanel ◽  
Michel G. Bergeron ◽  
Marc Ouellette
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Proteins ◽  
Resistance Mechanisms ◽  
Clinical Isolates ◽  
Penicillin Resistance ◽  
Dna Transformation ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
Genomic Analyses ◽  
Genome Scale

ABSTRACTAlterations in penicillin-binding proteins, the target enzymes for β-lactam antibiotics, are recognized as primary penicillin resistance mechanisms inStreptococcus pneumoniae. Few studies have analyzed penicillin resistance at the genome scale, however, and we report the sequencing ofS. pneumoniaeR6 transformants generated while reconstructing the penicillin resistance phenotypes from three penicillin-resistant clinical isolates by serial genome transformation. The genome sequences of the three last-level transformants T2-18209, T5-1983, and T3-55938 revealed that 16.2 kb, 82.7kb, and 137.2 kb of their genomes had been replaced with 5, 20, and 37 recombinant sequence segments derived from their respective parental clinical isolates, documenting the extent of DNA transformation between strains. A role in penicillin resistance was confirmed for some of the mutations identified in the transformants. Several multiple recombination events were also found to have happened at single loci coding for penicillin-binding proteins (PBPs) that increase resistance. Sequencing of the transformants with MICs for penicillin similar to those of the parent clinical strains confirmed the importance of mosaic PBP2x, -2b, and -1a as a driving force in penicillin resistance. A role in resistance for mosaic PBP2a was also observed for two of the resistant clinical isolates.

scholarly journals Profiling of β-Lactam Selectivity for Penicillin-Binding Proteins in Streptococcus pneumoniae D39

2015 ◽  
Vol 59 (6) ◽  
pp. 3548-3555 ◽  
Author(s):  
Ozden Kocaoglu ◽  
Ho-Ching T. Tsui ◽  
Malcolm E. Winkler ◽  
Erin E. Carlson
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Proteins ◽  
Morphological Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sublethal Concentration ◽  
Live Cells ◽  
Bacterial Cell Division ◽  
Content Type ◽  
Penicillin Binding Proteins

ABSTRACTSelective fluorescent β-lactam chemical probes enable the visualization of the transpeptidase activity of penicillin-binding proteins (PBPs) at different stages of bacterial cell division. To facilitate the development of new fluorescent probes for PBP imaging, we evaluated 20 commercially available β-lactams for selective PBP inhibition in an unencapsulated derivative of the D39 strain ofStreptococcus pneumoniae. Live cells were treated with β-lactam antibiotics at different concentrations and subsequently incubated with Bocillin FL (Boc-FL; fluorescent penicillin) to saturate uninhibited PBPs. Fluorophore-labeled PBPs were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence scanning. Among 20 compounds tested, carbapenems (doripenem and meropenem) were coselective for PBP1a, PBP2x, and PBP3, while six of the nine penicillin compounds were coselective for PBP2x and PBP3. In contrast, the seven cephalosporin compounds tested display variability in their PBP-binding profiles. Three cephalosporin compounds (cefoxitin, cephalexin, and cefsulodin) and the monobactam aztreonam exhibited selectivity for PBP3, while only cefuroxime (a cephalosporin) was selective for PBP2x. Treatment ofS. pneumoniaecultures with a sublethal concentration of cefuroxime that inhibited 60% of PBP2x activity and less than 20% of the activity of other PBPs resulted in formation of elongated cells. In contrast, treatment ofS. pneumoniaecultures with concentrations of aztreonam and cefoxitin that inhibited up to 70% of PBP3 activity and less than 30% of other PBPs resulted in no discernible morphological changes. Additionally, correlation of the MIC and IC50s for each PBP, with the exception of faropenem, amdinocillin (mecillinam), and 6-APA, suggests that pneumococcal growth inhibition is primarily due to the inhibition of PBP2x.

scholarly journals Increasing Ceftriaxone Resistance and Multiple Alterations of Penicillin-Binding Proteins among Penicillin-Resistant Streptococcus pneumoniae Isolates in Taiwan

2007 ◽  
Vol 51 (9) ◽  
pp. 3404-3406 ◽  
Author(s):  
Cheng-Hsun Chiu ◽  
Lin-Hui Su ◽  
Yhu-Chering Huang ◽  
Jui-Chia Lai ◽  
Hsiu-Ling Chen ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Binding Proteins ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Penicillin Binding Proteins

ABSTRACT The rate of nonsusceptibility of penicillin-resistant Streptococcus pneumoniae strains to ceftriaxone increased significantly in Taiwan in 2005. Approximately 90% of the ceftriaxone-nonsusceptible isolates were found to be of four major serotypes (serotypes 6B, 14, 19F, and 23F). Seven amino acid alterations in the penicillin-binding protein 2B transpeptidase-encoding region specifically contributed to the resistance.

scholarly journals Susceptibility of Methicillin-Resistant Staphylococcus aureus to Five Quinazolinone Antibacterials

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Sara Ceballos ◽  
Choon Kim ◽  
Yuanyuan Qian ◽  
Shahriar Mobashery ◽  
Mayland Chang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Binding Proteins ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Methicillin Resistant ◽  
Content Type ◽  
Penicillin Binding Proteins

ABSTRACT The in vitro activities of five quinazolinone antibacterials, compounds Q1 to Q5, were tested against 210 strains of methicillin-resistant Staphylococcus aureus (MRSA). The MIC50/MIC90 values (in μg/ml) were as follows: Q1, 0.5/2; Q2, 1/4; Q3, 2/4; Q4, 0.06/0.25; and Q5, 0.125/0.5. Several strains with high MIC values (from 8 to >32 μg/ml) for some of these compounds exhibited amino acid changes in the penicillin-binding proteins, which are targeted by these antibacterials.

scholarly journals Refining the Pneumococcal Competence Regulon by RNA Sequencing

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Jelle Slager ◽  
Rieza Aprianto ◽  
Jan-Willem Veening
Keyword(s):  
Antibiotic Resistance ◽  
Streptococcus Pneumoniae ◽  
Rna Sequencing ◽  
Genome Annotation ◽  
Stress Factors ◽  
Microarray Technology ◽  
Human Pathogen ◽  
Exogenous Dna ◽  
Content Type ◽  
Opportunistic Human Pathogen

ABSTRACTCompetence for genetic transformation allows the opportunistic human pathogenStreptococcus pneumoniaeto take up exogenous DNA for incorporation into its own genome. This ability may account for the extraordinary genomic plasticity of this bacterium, leading to antigenic variation, vaccine escape, and the spread of antibiotic resistance. The competence system has been thoroughly studied, and its regulation is well understood. Additionally, over the last decade, several stress factors have been shown to trigger the competent state, leading to the activation of several stress response regulons. The arrival of next-generation sequencing techniques allowed us to update the competence regulon, the latest report on which still depended on DNA microarray technology. Enabled by the availability of an up-to-date genome annotation, including transcript boundaries, we assayed time-dependent expression of all annotated features in response to competence induction, were able to identify the affected promoters, and produced a more complete overview of the various regulons activated during the competence state. We show that 4% of all annotated genes are under direct control of competence regulators ComE and ComX, while the expression of a total of up to 17% of all genes is affected, either directly or indirectly. Among the affected genes are various small RNAs with an as-yet-unknown function. Besides the ComE and ComX regulons, we were also able to refine the CiaR, VraR (LiaR), and BlpR regulons, underlining the strength of combining transcriptome sequencing (RNA-seq) with a well-annotated genome.IMPORTANCEStreptococcus pneumoniaeis an opportunistic human pathogen responsible for over a million deaths every year. Although both vaccination programs and antibiotic therapies have been effective in prevention and treatment of pneumococcal infections, respectively, the sustainability of these solutions is uncertain. The pneumococcal genome is highly flexible, leading to vaccine escape and antibiotic resistance. This flexibility is predominantly facilitated by competence, a state allowing the cell to take up and integrate exogenous DNA. Thus, it is essential to obtain a detailed overview of gene expression during competence. This is stressed by the fact that administration of several classes of antibiotics can lead to competence. Previous studies on the competence regulon were performed with microarray technology and were limited to an incomplete set of known genes. Using RNA sequencing combined with an up-to-date genome annotation, we provide an updated overview of competence-regulated genes.

scholarly journals Cellular Response of the Amoeba Acanthamoeba castellanii to Chlorine, Chlorine Dioxide, and Monochloramine Treatments

2011 ◽  
Vol 77 (14) ◽  
pp. 4974-4980 ◽  
Author(s):  
Emerancienne Mogoa ◽  
Charles Bodet ◽  
Franck Morel ◽  
Marie-Hélène Rodier ◽  
Bernard Legube ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Flow Cytometry ◽  
Chlorine Dioxide ◽  
Mode Of Action ◽  
Cellular Response ◽  
Acanthamoeba Castellanii ◽  
Size Reduction ◽  
Resistant Bacteria ◽  
Free Living ◽  
Content Type

ABSTRACTAcanthamoeba castellaniiis a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested onA. castellaniitrophozoites. Doses of disinfectants leading to up to a 3-log reduction were compared by flow cytometry and electron microscopy. Chlorine treatment led to size reduction, permeabilization, and retraction of pseudopods. In addition, treatment with chlorine dioxide led to a vacuolization of the cytoplasm. Monochloramine had a dose-dependent effect. At the highest doses monochloramine treatment resulted in almost no changes in cell size and permeability, as shown by flow cytometry, but the cell surface became smooth and dense, as seen by electron microscopy. We show that these disinfectants globally induced size reduction, membrane permeabilization, and morphological modifications but that they have a different mode of action onA. castellanii.

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