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 Capsule Homology Does Not Increase the Frequency of Transformation of Linked Penicillin Binding Proteins PBP 1a and PBP 2x in Streptococcus pneumoniae

2005 ◽  
Vol 49 (4) ◽  
pp. 1591-1592 ◽  
Author(s):  
Krzysztof Trzciński ◽  
Adam MacNeil ◽  
Keith P. Klugman ◽  
Marc Lipsitch
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Proteins ◽  
Penicillin Resistance ◽  
Penicillin Binding Proteins ◽  
Transformation Rates

ABSTRACT Penicillin resistance is mainly confined to a limited number of Streptococcus pneumoniae serotypes. Given linkage between the capsular biosynthesis locus and two penicillin binding proteins, we tested whether capsule homology increases transformation rates of penicillin resistance. Transformation rates in homologous donor-recipient pairs were no higher than expected, falsifying this hypothesis.

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 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 Identical Penicillin-Binding Domains in Penicillin-Binding Proteins of Streptococcus pneumoniae Clinical Isolates with Different Levels of β-Lactam Resistance

2005 ◽  
Vol 49 (7) ◽  
pp. 2895-2902 ◽  
Author(s):  
Laurent Chesnel ◽  
Raphaël Carapito ◽  
Jacques Croizé ◽  
Otto Dideberg ◽  
Thierry Vernet ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Proteins ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Penicillin G ◽  
Resistance Level ◽  
Penicillin Binding Proteins ◽  
Binding Domains ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT We have sequenced the penicillin-binding domains of the complete repertoire of penicillin-binding proteins and MurM from 22 clinical isolates of Streptococcus pneumoniae that span a wide range of β-lactam resistance levels. Evidence of mosaicism was found in the genes encoding PBP 1a, PBP 2b, PBP 2x, MurM, and, possibly, PBP 2a. Five isolates were found to have identical PBP and MurM sequences, even though the MICs for penicillin G ranged from 0.25 to 2.0 mg/liter. When the sequences encoding PBP 1a, PBP 2b, and PBP 2x from one of these isolates were used to transform laboratory strain R6, the resulting strain had a resistance level higher than that of the less resistant isolates carrying that PBP set but lower than that of the most resistant isolates carrying that PBP set. This result demonstrates that if the R6 strain is arbitrarily defined as the standard genotype, some wild genetic backgrounds can either increase or decrease the PBP-based resistance phenotype.

scholarly journals Activities of Ceftobiprole and Other β-Lactams against Streptococcus pneumoniae Clinical Isolates from the United States with Defined Substitutions in Penicillin-Binding Proteins PBP 1a, PBP 2b, and PBP 2x

2006 ◽  
Vol 50 (7) ◽  
pp. 2530-2532 ◽  
Author(s):  
Todd A. Davies ◽  
Wenchi Shang ◽  
Karen Bush
Keyword(s):  
United States ◽  
Streptococcus Pneumoniae ◽  
Binding Proteins ◽  
Binding Protein ◽  
The United States ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Penicillin Binding Protein ◽  
Penicillin Binding Proteins

ABSTRACT The activities of ceftobiprole and other β-lactams were examined with 30 Streptococcus pneumoniae isolates containing multiple pbp1a, pbp2b, and pbp2x mutations. The highest ceftobiprole MIC was 1 μg/ml, while the comparator MICs were 16 to 64 μg/ml. Fifty percent inhibitory concentrations for penicillin-binding protein 2x were 0.5 μg/ml (ceftobiprole) and 4 μg/ml (ceftriaxone) in a penicillin- and ceftriaxone-resistant isolate.

scholarly journals Penicillin-binding proteins 2b and 2x of Streptococcus pneumoniae are primary resistance determinants for different classes of beta-lactam antibiotics.

1996 ◽  
Vol 40 (4) ◽  
pp. 829-834 ◽  
Author(s):  
T Grebe ◽  
R Hakenbeck
Keyword(s):  
Streptococcus Pneumoniae ◽  
Clinical Isolate ◽  
Binding Proteins ◽  
Point Mutations ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Beta Lactam ◽  
Penicillin Binding Proteins ◽  
Beta Lactam Antibiotics ◽  
High Level

High-level resistance to beta-lactam antibiotics in Streptococcus pneumoniae is mediated by successive alterations in essential penicillin-binding proteins (PBPs). In the present work, single amino acid changes in S. pneumoniae PBP 2x and PBP 2b that result in reduced affinity for the antibiotic and that confer first-level beta-lactam resistance are defined. Point mutations in the PBP genes were generated by PCR-derived mutagenesis. Those conferring maximal resistance to either cefotaxime (pbp2x) or piperacillin (pbp2b) were obtained after transformation of the susceptible laboratory strain R6 with the PCR-amplified PBP genes and selection on agar with various concentrations of the antibiotic. In the case of PBP 2x, transformants for which the cefotaxime MIC was 0.16 microgram/ml contained the substitution of a Thr for an Ala at position 550 (Thr550-->Ala), close to the PBP homology box Lys547SerGly, a mutation frequently observed in laboratory mutants and in a high-level cefotaxime-resistant clinical isolate as well. After further selection, transformants resisting 0.3 microgram of cefotaxime per ml were obtained; they contained the substitution Gly550 as the result of two mutations in the same codon. In PBP 2b, Thr446-->Ala, adjacent to another homology box Ser443SerAsn, was the mutation selected with piperacillin. This substitution has been described in all clinical isolates with a low-affinity PBP 2b but was distinct from point mutations found in laboratory mutants. Both pbp2b with the single mutation and a mosaic pbp2b of a clinical isolate conferred a twofold increase in piperacillin resistance. Attempts to select PBP 2b variants at higher piperacillin concentrations were unsuccessful. The mutated PBP 2b also markedly reduced the lytic response to piperacillin, suggesting that such a mutation is an important step in resistance development in clinical isolates.

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