Role of interspecies transfer of chromosomal genes in the evolution of penicillin resistance in pathogenic and commensal Neisseria species

Summary The two pathogenic species of Neisseria, N. meningitidis and N. gonorrhoeae, have evolved resistance to penicillin by alterations in chromosomal genes encoding the high molecular weight penicillin-binding proteins, or PBPs. The PBP 2 gene (penA) has been sequenced from over 20 Neisseria isolates, including susceptible and resistant strains of the two pathogenic species, and five human commensal species. The genes from penicillin-susceptible strains of N. meningitidis and N. gonorrhoeae are very uniform, whereas those from penicillin-resistant strains consist of a mosaic of regions resembling those in susceptible strains of the same species, interspersed with regions resembling those in one, or in some cases, two of the commensal species. The mosaic structure is interpreted as having arisen from the horizontal transfer, by genetic transformation, of blocks of DNA, usually of a few hundred base pairs. The commensal species identified as donors in these interspecies recombinational events (N. flavescens and N. cinerea) are intrinsically more resistant to penicillin than typical isolates of the pathogenic species. Transformation has apparently provided N. meningitidis and N. gonorrhoeae with a mechanism by which they can obtain increased resistance to penicillin by replacing their penA genes (or the relevant parts of them) with the penA genes of related species that fortuitously produce forms of PBP 2 that are less susceptible to inhibition by the antibiotic. The ends of the diverged blocks of DNA in the penA genes of different penicillin-resistant strains are located at the same position more often than would be the case if they represent independent crossovers at random points along the gene. Some of these common crossover points may represent common ancestry, but reasons are given for thinking that some may represent independent events occurring at recombinational hotspots. Offprint requests to: B.G. Spratt     

The structural modifications induced by the M339F substitution in PBP2x from Streptococcus pneumoniae further decreases the susceptibility to beta-lactams of resistant strains

PBP2x is a primary determinant of beta-lactams resistance in Streptococcus pneumoniae. Altered PBP2x with multiple mutations have a reduced "affinity" for the antibiotics. An important polymorphism is found in PBP2x sequences from clinical resistant strains. To understand the mechanism of resistance, it is necessary to identify and characterize the relevant substitutions. Many similar PBP2x sequences from resistant isolates have the previously studied T338A mutation, adjacent to the active site Ser337. We report here the structural and functional analysis of the M339F substitution that is found in a subset of these sequences, originating from highly resistant strains. The M339F mutation causes a 4-10-fold reduction of the reaction rate with beta-lactams, depending on the molecular context. In addition, release of the inactivated antibiotic from the active site is up to 3-fold faster as a result from the M339F mutation. These effects measured in vitro are correlated with the level of beta-lactam resistance in vivo conferred by several PBP2x variants. Thus, a single amino acid difference between similar PBP2x from clinical isolates can strongly modulate the degree of beta-lactam resistance. The crystal structure of the double mutant T338A/M339F solved to a resolution of 2.4 A shows a distortion of the active site and a reorientation of the hydroxyl group of the active site Ser337, which can explain the kinetic effects of the mutations.     

Penicillin-binding protein 2b of Streptococcus pneumoniae in piperacillin-resistant laboratory mutants.

In Streptococcus pneumoniae, alterations in penicillin-binding protein 2b (PBP 2b) that reduce the affinity for penicillin binding are observed during development of beta-lactam resistance. The development of resistance was now studied in three independently obtained piperacillin-resistant laboratory mutants isolated after several selection steps on increasing concentrations of the antibiotic. The mutants differed from the clinical isolates in major aspects: first-level resistance could not be correlated with alterations in the known PBP genes, and the first PBP altered was PBP 2b. The point mutations occurring in the PBP 2b genes were characterized. Each mutant contained one single point mutation in the PBP 2b gene. In one mutant, this resulted in a mutation of Gly-617 to Ala within one of the homology boxes common to all PBPs, and in the other two cases, the same Gly-to-Asp substitution at the end of the penicillin-binding domain had occurred. The sites affected were homologous to those determined previously in the S. pneumoniae PBP 2x of mutants resistant to cefotaxime, indicating that, in both PBPs, similar sites are important for interaction with the respective beta-lactams.     

Molecular characteristics of penicillin-binding protein 2b, 2x, and 1a sequences in penicillin-nonsusceptible Streptococcus pneumoniae isolates in Shenyang, China

The aim of this study was to investigate the nature of the amino acid motifs found in penicillin-binding proteins (PBP) 2b, 2x, and 1a of penicillin-nonsusceptible Streptococcus pneumoniae isolates from Shenyang, China, and to obtain information regarding the prevalence of alterations within the motifs or in positions flanking the motifs. For 18 clinical isolates comprising 4 penicillin-susceptible S. pneumoniae, 5 penicillin-intermediate S. pneumoniae, and 9 penicillin-resistant S. pneumoniae. the DNA sequences of PBP2b, PBP2x, and PBP1a transpeptidase domains were determined and then genotyped by multilocus sequence typing. Sequence analysis revealed that most penicillin-nonsusceptible S. pneumoniae isolates (penicillin MIC > or = 1.5 microg/mL and cefotaxime MIC > or = 2 microg/mL) shared identical PBP2b, PBP2x, and PBP1a amino acid profiles. Most penicillin-resistant S. pneumoniae isolates were ST320 (4-16-19-15-6-20-1), the double-locus variant of the Taiwan19F-14 clone. This study will serve as a basis for future monitoring of genetic changes associated with the emergence and spread of beta-lactam resistance in Shenyang, China.     

肺炎链球菌pbp2b和pbp1a基因突变与青霉素耐药的相关性研究

目的探讨耐青霉素肺炎链球菌pbp2b和pbp1 a基因的突变与青霉素耐药的关系,为明了肺炎链球菌的耐药性变异机制,防治其感染提供实验依据。方法从呼吸道感染患儿痰标本中分离肺炎链球菌163株,液体培养基连续稀释法测定其对青霉素的最小抑菌浓度(M IC),套式聚合酶链反应(nPCR)扩增pbp2b和pbp1 a基因,扩增产物直接DNA测序,所测序列与青霉素敏感株(SPN R6)的基因序列进行比较,并分析其氨基酸结构的改变。结果 163株肺炎链球菌中检出青霉素敏感菌75株,中度敏感17株,青霉素耐药菌71株(44%)。耐药菌中58株存在pbp2b突变(81.7%),其中,56株为点突变,2株为CCT插入突变;在27株有pbp2b基因突变的B型和C型耐药菌中,21株出现了不同程度的pbp1 a基因突变。PBP2B氨基酸结构改变以苏氨酸变为丙氨酸、精氨酸变为赖氨酸为主,PBP1A以丙氨酸变为苏氨酸、谷氨酸变为天门冬氨酸为主。结论肺炎链球菌的pbp2b和pbp1 a基因突变与对青霉素的耐药性密切相关,PBP2b突变导致低水平耐药;PBP2b和PBP1A突变导致高水平耐药。     

The CiaRH system of Streptococcus pneumoniae prevents lysis during stress induced by treatment with cell wall inhibitors and by mutations in pbp2x involved in beta-lactam resistance

The two-component signal-transducing system CiaRH of Streptococcus pneumoniae plays an important role during the development of beta-lactam resistance in laboratory mutants. We show here that a functional CiaRH system is required for survival under many different lysis-inducing conditions. Mutants with an activated CiaRH system were highly resistant to lysis induced by a wide variety of early and late cell wall inhibitors, such as cycloserine, bacitracin, and vancomycin, and were also less susceptible to these drugs. In contrast, loss-of-function CiaRH mutants were hypersusceptible to these drugs and were apparently unable to maintain a stationary growth phase in normal growth medium and under choline deprivation as well. Moreover, disruption of CiaR in penicillin-resistant mutants with an altered pbp2x gene encoding low-affinity PBP2x resulted in severe growth defects and rapid lysis. This phenotype was observed with pbp2x genes containing point mutations selected in the laboratory and with highly altered mosaic pbp2x genes from penicillin-resistant clinical isolates as well. This documents for the first time that PBP2x mutations required for development of beta-lactam resistance are functionally not neutral and are tolerated only in the presence of the CiaRH system. This might explain why cia mutations have not been observed in penicillin-resistant clinical isolates. The results document that the CiaRH system is required for maintenance of the stationary growth phase and for prevention of autolysis triggered under many different conditions, suggesting a major role for this system in ensuring cell wall integrity.     

Separation of abnormal cell wall composition from penicillin resistance through genetic transformation of Streptococcus pneumoniae.

Compared with most penicillin-susceptible isolates of Streptococcus pneumoniae, penicillin-resistant clinical isolate Hun 663 contains mosaic penicillin-binding protein (PBP) genes encoding PBPs with reduced penicillin affinities, anomalous molecular sizes, and also cell walls of unusual chemical composition. Chromosomal DNA prepared from Hun 663 was used to transform susceptible recipient cells to donor level penicillin resistance, and a resistant transformant was used next as the source of DNA in the construction of a second round of penicillin-resistant transformants. The greatly reduced penicillin affinity of the high-molecular-weight PBPs was retained in all transformants through both genetic crosses. On the other hand, PBP pattern and abnormal cell wall composition, both of which are stable, clone-specific properties of strain Hun 663, were changed: individual transformants showed a variety of new, abnormal PBP patterns. Furthermore, while the composition of cell walls resembled that of the DNA donor in the first-round transformants, it became virtually identical to that of susceptible pneumococci in the second-round transformants. The findings indicate that genetic elements encoding the low affinity of PBPs and the penicillin resistance of the bacteria are separable from determinants that are responsible for the abnormal cell wall composition that often accompanies penicillin resistance in clinical strains of pneumococci.     

Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae

Clinical isolates of Streptococcus pneumoniae that have greatly increased levels of resistance to penicillin (greater than 1000-fold) have been reported from South Africa during the last ten years. Penicillin resistance in these strains is entirely due to the development of penicillin-binding proteins (PBPs) with decreased affinity for penicillin. We have cloned and sequenced the coding region for the transpeptidase domain of penicillin-binding protein 2B from three penicillin-sensitive strains of S. pneumoniae and from a penicillin-resistant South African strain. The amino acid sequences of the transpeptidase domains of PBP2B of the three penicillin-sensitive strains were identical and there were only between one and four differences in the nucleotide sequences of their coding regions. The corresponding region of the PBP2B gene from the penicillin-resistant strain differed by 74 nucleotide substitutions which resulted in 17 alterations in the amino acid sequence of PBP2B. The most remarkable alteration that has occurred during the development of the 'penicillin-resistant' form of PBP2B is the substitution of seven consecutive residues in a region that is predicted to form a loop at the bottom of the penicillin-binding site.     

Correlation between substitutions in penicillin-binding protein 1 and amoxicillin resistance in Helicobacter pylori

The correlation between the substitutions of penicillin-binding protein 1 (PBP1) and amoxicillin resistance was studied for the determination of the substitutions in PBP1 which confer amoxicillin resistance in Helicobacter pylori. By the comparison of the amino acid sequences of PBP1 in the amoxicillinresistant (n=3), low-susceptible (n=3), and susceptible (n=13) H. pylori isolates, the substitution Asn562-->Tyr, which is adjacent to KTG motif (555-557), was common and specific to amoxicillin-resistant H. pylori. Additionally, all amoxicillin-resistant isolates had multiple substitutions such as Ser414-->Arg in the transpeptidase region of PBP1 of H. pylori. Furthermore all transformants obtained by the natural transformation using the pbp1 genes of amoxicillin-resistant H. pylori isolates had multiple substitutions including Asn562-->Tyr. These results suggest that multiple amino acid substitutions in the transpeptidase region of PBP1 are closely related to amoxicillin resistance in H. pylori.     

Genetics of resistance to third-generation cephalosporins in clinical isolates of Streptococcus pneumoniae

Resistance to third-generation cephalosporins in a clinical isolate of Streptococcus pneumoniae was shown to be due to the production of altered forms of penicillin-binding proteins (PBPs) 2X and 1A. The cloned PBP2X gene from the resistant strain was able to transform a susceptible strain to an intermediate level of resistance. The resulting transformant could be transformed to the full level of resistance of the clinical isolate using the cloned PBP1A gene from the latter strain. Chromosomal DNA from the resistant strain (and from other resistant strains) could readily transform a susceptible strain to the full level of resistance to third-generation cephalosporins (greater than 250-fold for cefotaxime; greater than 100-fold for ceftriaxone) in a single step (transformation frequency of about 10(-5)). The resistant transformants obtained with chromosomal DNA were shown by gene fingerprinting to have gained both the PBP1A and PBP2X genes from the DNA donor.     

Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae.

Penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae is one of the high-molecular-weight PBPs involved in the development of intrinsic beta-lactam resistance. Point mutations in the PBP 2x genes (pbpX) have now been characterized in five independent spontaneous laboratory mutants in order to identify protein regions which are important for interaction with beta-lactam antibiotics. All mutant genes contained two to four mutations resulting in amino acid substitutions within the penicillin-binding domain of PBP 2x, and none of the mutants carried an identical set of mutations. For one particular mutant, C606, carrying four mutations in pbpX, the mutations at positions 601 and 597 conferred first- and second-level resistance when introduced into the susceptible parent strain S. pneumoniae R6. However, the other two mutations, at amino acid positions 289 and 422, which were originally selected at the fifth and sixth isolation steps, did not contribute at all to resistance in similar experiments. This suggests that they are phenotypically expressed only in combination with mutations in other genes. Three PBP 2x regions were mutated in from two to all four mutants carrying a low-affinity PBP 2x. However, in a fifth mutant containing a PBP 2x with apparent zero affinity for beta-lactams, the three mutations in pbpX mapped at entirely different positions. This demonstrates that different mutational pathways exist for remodeling this PBP during resistance development.     

Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506

Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin-binding proteins (PBPs) which reduce their affinity for beta-lactam antibiotics. PBP2x is one of the high-Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime-resistant laboratory mutants. In this study, we have sequenced a 2564 base-pair chromosomal fragment from the penicillin-sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base-pair open reading frame was found which coded for a protein having an Mr of 82.35kD, a value which is in good agreement with the Mr of 80-85 kD measured by SDS-gel electrophoresis of the PBP2x protein itself. The N-terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime-resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the beta-lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from beta-lactam-resistant strains are known. The penicillin-binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N-terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin-resistant Staphylococcus aureus PBP2'. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low-Mr PBPs.     

A PBP2x from a clinical isolate of Streptococcus pneumoniae exhibits an alternative mechanism for reduction of susceptibility to beta-lactam antibiotics

The human pathogen Streptococcus pneumoniae is one of the main causative agents of respiratory tract infections. At present, clinical isolates of S. pneumoniae often exhibit decreased susceptibility toward beta-lactams, a phenomenon linked to multiple mutations within the penicillin-binding proteins (PBPs). PBP2x, one of the six PBPs of S. pneumoniae, is the first target to be modified under antibiotic pressure. By comparing 89 S. pneumoniae PBP2x sequences from clinical and public data bases, we have identified one major group of sequences from drug-sensitive strains as well as two distinct groups from drug-resistant strains. The first group includes proteins that display high similarity to PBP2x from the well characterized resistant strain Sp328. The second group includes sequences in which a signature mutation, Q552E, is found adjacent to the third catalytic motif. In this work, a PBP2x from a representative strain from the latter group (S. pneumoniae 5259) was biochemically and structurally characterized. Phenotypical analyses of transformed pneumococci show that the Q552E substitution is responsible for most of the reduction of strain susceptibility toward beta-lactams. The crystal structure of 5259-PBP2x reveals a change in polarity and charge distribution around the active site cavity, as well as rearrangement of strand beta3, emulating structural changes observed for other PBPs that confer drug resistance to Gram-positive pathogens. Interestingly, the active site of 5259-PBP2x is in closed conformation, whereas that of Sp328-PBP2x is open. Consequently, S. pneumoniae has evolved to employ the same protein in two distinct mechanisms of antibiotic resistance.     

浙江地区青霉素耐药肺炎链球菌PBPs基因及氨基酸序列研究

为阐明温州地区青霉素耐药肺炎链球菌（PRSP）的青霉素结合蛋白（PBPs）的基因和氨基酸序列的变异特点,对温州医学院自2000年11月～2004年1月收集的26份肺炎链球菌进行分离、鉴定及青霉素药敏实验,并对每株链球菌的PBP1A、PBP2B、PBP2X基因进行PCR扩增和直接测序,通过序列比对与生物信息学分析。结果表明,研究中的PBP1A的主要突变位点是保守基序KTG之后的4个连续氨基酸替换Thr574Ala、Ser575Thr、Gln576Gly、Phe577Tyr和保守序列STMK内的氨基酸替换Thr371Ser;PBP2B的主要突变位点是保守序列SSN之后的氨基酸替换Thr451Ala;PBP2X的主要突变位点是保守基序STMK 内的氨基酸替换Thr338Ala。以上突变类型以及菌株的青霉素耐药水平与文献报道相符。研究检测的PRSP的PBPs基因中暂未发现本地区特有的（新的）基因突变,也未检测出文献报道的某些与青霉素抗性相关的氨基酸替换。     

Amino acid variations of cytochrome P-450 lanosterol 14 alpha-demethylase (CYP51A1) from fluconazoleresistant clinical isolates of Candida albicans

We studied six clinical isolates of Candida albicans. All six isolates showed high level resistance to fluconazole (minimum inhibitory concentrations 64 microg/ml) with varying degrees of cross-resistance to other azoles but not to amphotericin B. Neither higher dosage nor upregulation of the gene encoding the cytochrome P- 450 lanosterol 14 alpha-demethylase (CYP51A1 or P-450LDM) was responsible for fluconazole resistance. The resistant and the susceptible isolates accumulated similar amounts of azoles. To examine whether resistance to fluconazole in these clinical isolates of C. albicans is mediated by an altered target of azole action, we cloned the structural gene encoding P-450LDM from the fluconazole resistant isolates. The amino acid sequences of the P-450LDMs from the isolates were deduced from the gene sequences and compared to the P-450LDM sequence of the fluconazole-susceptible C. albicans B311. The enzymes from the clinical isolates showed 2 to 7 amino acid variations scattered across the molecules encompassing 10 different loci. One-half of the amino acid changes obtained were conserved substitutions (E116D, K143R, E266D, D278E, R287K) compared to the susceptible strain. Non-conserved substitutions were T128K, R267H, S405F, G450E and G464S, three of which are in and around the hemebinding region of the molecule. R287K is the only amino acid change that was found in all six clinical isolates. One or more of these mutational alterations may lead to the expression of an azole-resistant enzyme.     

Horizontal transfer of multiple penicillin-binding protein genes, and capsular biosynthetic genes, in natural populations of Streptococcus pneumoniae

Multiply antibiotic-resistant serotype 23F isolates of Streptococcus pneumoniae are prevalent in Spain and have also been recovered recently in the United Kingdom and the United States. Analysis of populations of these isolates by multilocus enzyme electrophoresis, and restriction endonuclease cleavage electrophoretic profiling of penicillin-binding protein (PBP) genes, has demonstrated that these isolates are a single clone (Muñoz et al., 1991). Here we report studies of non-serotype 23F penicillin-resistant pneumococci isolated in Spain and the United Kingdom. One of the isolates expressed serotype 19 capsule but was otherwise indistinguishable from the serotype 23F clone on the basis of multilocus enzyme electrophoresis, antibiotic resistance profiling, and restriction endonuclease patterns of genes encoding PBP1A, PBP2B and PBP2X, a result which suggests that horizontal transfer of capsular biosynthesis genes had occurred. These same techniques revealed that six other resistant isolates, all expressing serotype 9 polysaccharide capsule, represent a clone. Interestingly, the chromosomal lineage of this clone is not closely related to the 23F clone; however, the serotype 9 and 23F clones harbour apparently identical PBP1 A, -2B and -2X genes. To explain these data, we favour the interpretation that horizontal gene transfer in natural populations has distributed genes encoding altered forms of PBP1A, -2B and -2X to distinct evolutionary lineages of S. pneumoniae.     

Mosaic pbpX genes of major clones of penicillin-resistant Streptococcus pneumoniae have evolved from pbpX genes of a penicillin-sensitive Streptococcus oralis

Penicillin-resistant clinical isolates of Streptococcus pneumoniae contain mosaic penicillin-binding protein (PBP) genes that encode PBPs with decreased affinity for β-lactam antibiotics. The mosaic blocks are believed to be the result of gene transfer of homologous PBP genes from related penicillin-resistant species. We have now identified a gene homologous to the pneumococcal PBP2x gene (pbpX) in a penicillin-sensitive Streptococcus oralis isolate M3 from South Africa that diverged by almost 20% from pbpX of penicillin-sensitive pneumococci, and a central sequence block of a mosaic pbpX gene of Streptococcus mitis strain NCTC 10712. In contrast, it differed by only 2-4% of the 1 to 1.5 kb mosaic block in pbpX genes of three genetically unrelated penicillin-resistant S. pneumoniae isolates, two of them representing clones of serotype 6B and 23F, which are prevalent in Spain and are also already found in other countries. With low concentrations of cefotaxime, transformants of the sensitive S. pneumoniae R6 strain could be selected containing pbpX genes from either S. mitis NCTC 10712 or S. oralis M3, demonstrating that genetic exchange can already occur between β-lactam-sensitive species. These data are in agreement with the assumption that PBPs as penicillin-resistance determinants have evolved by the accumulation of point mutations in genes of sensitive commensal species.     

Interspecies recombination between the penA genes of Neisseria meningitidis and commensal Neisseria species during the emergence of penicillin resistance in N. meningitidis: natural events and laboratory simulation.

The penicillin-binding protein 2 genes (penA) of penicillin-resistant Neisseria meningitidis have a mosaic structure that has arisen by the introduction of regions from the penA genes of Neisseria flavescens or Neisseria cinerea. Chromosomal DNA from both N. cinerea and N. flavescens could transform a penicillin-susceptible isolate of N. meningitidis to increased resistance to penicillin. With N. flavescens DNA, transformation to resistance was accompanied by the introduction of the N. flavescens penA gene, providing a laboratory demonstration of the interspecies recombinational events that we believe underlie the development of penicillin resistance in many meningococci in nature. Surprisingly, with N. cinerea DNA, the penicillin-resistant transformants did not obtain the N. cinerea penA gene. However, the region of the penA gene derived from N. cinerea in N. meningitidis K196 contained an extra codon (Asp-345A) which was not found in any of the four N. cinerea isolates that we examined and which is known to result in a decrease in the affinity of PBP 2 in gonococci.     

Kinetics of beta-lactam interactions with penicillin-susceptible and -resistant penicillin-binding protein 2x proteins from Streptococcus pneumoniae. Involvement of acylation and deacylation in beta-lactam resistance.

Kinetic interactions of beta-lactam antibiotics such as penicillin-G and cefotaxime with normal, penicillin-susceptible PBP2x from Streptococcus pneumoniae and a penicillin-resistant PBP2x (PBP2x(R)) from a resistant clinical isolate (CS109) of the bacterium have been extensively characterized using electrospray mass spectrometry coupled with a fast reaction (quench flow) technique. Kinetic evidence for a two-step acylation of PBP2x by penicillin-G has been demonstrated, and the dissociation constant, K(d) of 0.9 mm, and the acylation rate constant, k(2) of 180 s(-1), have been determined for the first time. The millimolar range K(d) implies that the beta-lactam fits to the active site pocket of the penicillin-sensitive PBP rather poorly, whereas the extremely fast k(2) value indicates that this step contributes most of the binding affinity of the beta-lactam. The values of K(d) (4 mm) and k(2) (0.56 s(-1)) were also determined for PBP2x(R). The combined value of k(2)/K(d), known as overall binding efficiency, for PBP2x(R) (137 m(-1) s(-1)) was over 1000-fold slower than that for PBP2x (200,000 m(-1) s(-1)), indicating that a major part is played by the acylation steps in penicillin resistance. Most of the decreased binding efficiency of PBP2x(R) comes from the decreased ( approximately 300-fold) k(2). Kinetic studies of cefotaxime acylation of the two PBP2x proteins confirmed all of the above findings. Deacylation rate constants (k(3)) for the third step of the interactions were determined to be 8 x 10(-6) s(-1) for penicilloyl-PBP2x and 5.7 x 10(-4) s(-1) for penicilloyl-PBP2x(R), corresponding to over 70-fold increase of the deacylation rate for the resistant PBP2x(R). Similarly, over 80-fold enhancement of the deacylation rate was found for cefotaxime-PBP2x(R) complex (k(3) = 3 x 10(-4) s(-1)) as compared with that of cefotaxime-PBP2x complex (3.5 x 10(-6) s(-1)). This is the first time that such a significant increase of k(3) values was found for a beta-lactam-resistant penicillin-binding protein. These data indicate that the deacylation step also plays a role, which is much more important than previously thought, in PBP2x(R) resistance to beta-lactams.     

Common alterations in PBP1a from resistant Streptococcus pneumoniae decrease its reactivity toward beta-lactams: structural insights

The development of high level beta-lactam resistance in the pneumococcus requires the expression of an altered form of PBP1a, in addition to modified forms of PBP2b and PBP2x, which are necessary for the appearance of low levels of resistance. Here, we present the crystal structure of a soluble form of PBP1a from the highly resistant Streptococcus pneumoniae strain 5204 (minimal inhibitory concentration of cefotaxime is 12 mg.liter(-1)). Mutations T371A, which is adjacent to the catalytic nucleophile Ser370, and TSQF(574-577)NTGY, which lie in a loop bordering the active site cleft, were investigated by site-directed mutagenesis. The consequences of these substitutions on reaction kinetics with beta-lactams were probed in vitro, and their effect on resistance was measured in vivo. The results are interpreted in the framework of the crystal structure, which displays a narrower, discontinuous active site cavity, compared with that of PBP1a from the beta-lactam susceptible strain R6, as well as a reorientation of the catalytic Ser370.