Penicillin-binding Protein 2x of Streptococcus pneumoniae: Three New Mutational Pathways for Remodelling an Essential Enzyme into a Resistance Determinant

Maurer, Patrick; Koch, Barbara; Zerfaß, Ilka; Krauß, Jan; Linden, Mark van der; Frère, Jean‐Marie; Contreras‐Martel, Carlos; Hakenbeck, Regine

doi:10.1016/j.jmb.2007.12.058

Cited by 36 publications

(29 citation statements)

References 42 publications

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“…Bioinformatic analysis of S. pneumoniae strains whose genomes have been sequenced indicated that the luxS gene is located in a region that contains genes involved in division and cell wall biosynthesis. Specifically, luxS is situated upstream (ϳ6.4 kb) of the gene cps4A (encoding a capsule polysaccharide biosynthesis protein) and ϳ4 kb downstream of the pbp2X gene, whose encoded protein is involved in cell wall biosynthesis and is a target of ␤-lactam antibiotics (30,57), followed by mraY (encoding a phospho-N-acetylmuramoyl-pentapeptide-transferase [ϳ3 kb]) (29) and, ϳ294 bp downstream, the clpL gene, which encodes a putative ATPdependent Clp protease (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

The LuxS-Dependent Quorum-Sensing System Regulates Early Biofilm Formation by Streptococcus pneumoniae Strain D39

et al. 2011

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Streptococcus pneumoniae is the leading cause of death in children worldwide and forms highly organized biofilms in the nasopharynx, lungs, and middle ear mucosa. The luxS-controlled quorum-sensing (QS) system has recently been implicated in virulence and persistence in the nasopharynx, but its role in biofilms has not been studied. Here we show that this QS system plays a major role in the control of S. pneumoniae biofilm formation. Our results demonstrate that the luxS gene is contained by invasive isolates and normal-flora strains in a region that contains genes involved in division and cell wall biosynthesis. The luxS gene was maximally transcribed, as a monocistronic message, in the early mid-log phase of growth, and this coincides with the appearance of early biofilms. Demonstrating the role of the LuxS system in regulating S. pneumoniae biofilms, at 24 h postinoculation, two different D39⌬luxS mutants produced ϳ80% less biofilm biomass than wild-type (WT) strain D39 did. Complementation of these strains with luxS, either in a plasmid or integrated as a single copy in the genome, restored their biofilm level to that of the WT. Moreover, a soluble factor secreted by WT strain D39 or purified AI-2 restored the biofilm phenotype of D39⌬luxS. Our results also demonstrate that during the early mid-log phase of growth, LuxS regulates the transcript levels of lytA, which encodes an autolysin previously implicated in biofilms, and also the transcript levels of ply, which encodes the pneumococcal pneumolysin. In conclusion, the luxS-controlled QS system is a key regulator of early biofilm formation by S. pneumoniae strain D39.

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Section: Resultsmentioning

confidence: 99%

The LuxS-Dependent Quorum-Sensing System Regulates Early Biofilm Formation by Streptococcus pneumoniae Strain D39

et al. 2011

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“…Fine-tuning of the MICs revealed subtle differences mediated by a single point mutation, with T338G appearing the most effective one and which might have larger effects in the context of other mutations in PBP2x and other PBPs as well. The cefotaxime MIC increased only by approximately twofold, whereas the mutation G601V has been shown to result in a fourfold increase (40) and mutations at T550 result in MICs of 0.2 to 0.3 g/ml (11,19).…”

Section: Discussionmentioning

confidence: 99%

“…After separation on SDS-PAGE, proteins were transferred onto a polyvinylidene difluoride membrane. PBPs were visualized after incubation with affinity-purified PBP2x antibodies (1:10,000 in phosphate-buffered saline) (40) or PBP1a antibodies (1:3,000 in phosphate-buffered saline) (21) followed by incubation with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (Sigma-Aldrich, St. Louis, MO) and staining with 4-nitrobluetetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate.…”

Section: Methodsmentioning

confidence: 99%

An Important Site in PBP2x of Penicillin-Resistant Clinical Isolates of Streptococcus pneumoniae : Mutational Analysis of Thr338

Zerfaß

Hakenbeck

Denapaite

2009

Antimicrob Agents Chemother

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Penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae represents a primary resistance determinant for beta-lactams, and low-affinity PBP2x variants can easily be selected with cefotaxime. Penicillinresistant clinical isolates of S. pneumoniae frequently contain in their mosaic PBP2x the mutation T338A adjacent to the active site S337, and T338P as well as T338G substitutions are also known. Site-directed mutagenesis has now documented that a single point mutation at position T338 confers selectable levels of beta-lactam resistance preferentially to oxacillin. Despite the moderate impact on beta-lactam susceptibility,

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“…The peptidoglycan (PG) is an important component of the cell surface of all bacteria (reviewed in references 8, 61, 65-67, and 73) and is the target for several antibiotics, including ␤-lactam antibiotics and vancomycin, used to treat pneumococcal infections (55,68). Notably, resistance of S. pneumoniae to ␤-lactam antibiotics is increasing at an alarming rate due to multiple amino acid changes in the pneumococcal penicillin binding proteins (PBPs) (13,23,33,36,72).…”

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Characterization of Mutants Deficient in the l,d -Carboxypeptidase (DacB) and WalRK (VicRK) Regulon, Involved in Peptidoglycan Maturation of Streptococcus pneumoniae Serotype 2 Strain D39

2011

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Peptidoglycan (PG) hydrolases play critical roles in the remodeling of bacterial cell walls during division. PG hydrolases have been studied extensively in several bacillus species, such as Escherichia coli and Bacillus subtilis, but remain relatively uncharacterized in ovococcus species, such as Streptococcus pneumoniae (pneumococcus). In this work, we identified genes that encode proteins with putative PG hydrolytic domains in the genome of S. pneumoniae strain D39. Knockout mutations in these genes were constructed, and the resulting mutants were characterized in comparison with the parent strain for growth, cell morphology, PG peptide incorporation, and in some cases, PG peptide composition. In addition, we characterized deletion mutations in nonessential genes of unknown function in the WalRK Spn two-component system regulon, which also contains the essential pcsB cell division gene. Several mutants did not show overt phenotypes, which is perhaps indicative of redundancy. In contrast, two new mutants showed distinct defects in PG biosynthesis. One mutation was in a gene designated dacB (spd_0549), which we showed encodes an L,D-carboxypeptidase involved in PG maturation. Notably, dacB mutants, similar to dacA (D,D-carboxypeptidase) mutants, exhibited defects in cell shape and septation, consistent with the idea that the availability of PG peptide precursors is important for proper PG biosynthesis. Epistasis analysis indicated that DacA functions before DacB in D-Ala removal, and immunofluorescence microscopy showed that DacA and DacB are located over the entire surface of pneumococcal cells. The other mutation was in WalRK Spn regulon gene spd_0703, which encodes a putative membrane protein that may function as a type of conserved streptococcal shape, elongation, division, and sporulation (SEDS) protein.

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Penicillin-binding Protein 2x of Streptococcus pneumoniae: Three New Mutational Pathways for Remodelling an Essential Enzyme into a Resistance Determinant

Cited by 36 publications

References 42 publications

The LuxS-Dependent Quorum-Sensing System Regulates Early Biofilm Formation by Streptococcus pneumoniae Strain D39

The LuxS-Dependent Quorum-Sensing System Regulates Early Biofilm Formation by Streptococcus pneumoniae Strain D39

An Important Site in PBP2x of Penicillin-Resistant Clinical Isolates of Streptococcus pneumoniae : Mutational Analysis of Thr338

Characterization of Mutants Deficient in the l,d -Carboxypeptidase (DacB) and WalRK (VicRK) Regulon, Involved in Peptidoglycan Maturation of Streptococcus pneumoniae Serotype 2 Strain D39

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