Ureidopenicillins Are Potent Inhibitors of Penicillin-Binding Protein 2 from Multidrug-Resistant Neisseria gonorrhoeae H041.

Effective treatment of gonorrhea is threatened by the increasing prevalence of Neisseria gonorrhoeae strains resistant to the extended-spectrum cephalosporins (ESCs). Recently, we demonstrated the promise of the third-generation cephalosporin cefoperazone as an antigonococcal agent due to its rapid second-order rate of acylation against penicillin-binding protein 2 (PBP2) from the ESC-resistant strain H041 and robust antimicrobial activity against H041. Noting the presence of a ureido moiety in cefoperazone, we evaluated a subset of structurally similar ureido ß-lactams, including piperacillin, azlocillin, and mezlocillin, for activity against PBP2 from H041 using biochemical and structural analyses. We found that the ureidopenicillin piperacillin has a second-order rate of acylation against PBP2 that is 12-fold higher than cefoperazone and 85-fold higher than ceftriaxone and a lower MIC against H041 than ceftriaxone. Surprisingly, the affinity of ureidopenicillins for PBP2 is minimal, indicating that their inhibitory potency is due to a higher rate of the acylation step of the reaction compared to cephalosporins. Enhanced acylation results from the combination of a penam scaffold with a 2,3-dioxopiperazine-containing R1 group. Crystal structures show that the ureido ß-lactams overcome the effects of resistance mutations present in PBP2 from H041 by eliciting conformational changes that are hindered when PBP2 interacts with the weaker inhibitor ceftriaxone. Overall, our results support the potential of piperacillin as a treatment for gonorrhea and provide a framework for the future design of ß-lactams with improved activity against ESC-resistant N. gonorrhoeae.

Development and validation of multiplex assays for mouse and human IgG and IgA to Neisseria gonorrhoeae antigens.

There is an urgent need for vaccines against Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea. Vaccination with an outer-membrane vesicle (OMV)-based Neisseria meningitidis (Nm) vaccine provides some protection from Ng; however, the mechanisms underlying this cross-protection are unknown. To address this need, we developed multiplexed bead-based assays for the relative quantification of human and mouse IgG and IgA against Ng antigens. The assays were evaluated for analyte independence, dilutional linearity, specificity, sensitivity, intra- and inter-assay variability, and robustness to sample storage conditions. The assay was then used to test samples from mice and humans immunized with an Nm-OMV vaccine.

Preclinical safety and efficacy characterization of an LpxC inhibitor against Gram-negative pathogens.

The UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase LpxC is an essential enzyme in the biosynthesis of lipid A, the outer membrane anchor of lipopolysaccharide and lipooligosaccharide in Gram-negative bacteria. The development of LpxC-targeting antibiotics toward clinical therapeutics has been hindered by the limited antibiotic profile of reported non-hydroxamate inhibitors and unexpected cardiovascular toxicity observed in certain hydroxamate and non-hydroxamate-based inhibitors. Here, we report the preclinical characterization of a slow, tight-binding LpxC inhibitor, LPC-233, with low picomolar affinity. The compound is a rapid bactericidal antibiotic, unaffected by established resistance mechanisms to commercial antibiotics, and displays outstanding activity against a wide range of Gram-negative clinical isolates in vitro. It is orally bioavailable and efficiently eliminates infections caused by susceptible and multidrug-resistant Gram-negative bacterial pathogens in murine soft tissue, sepsis, and urinary tract infection models. It displays exceptional in vitro and in vivo safety profiles, with no detectable adverse cardiovascular toxicity in dogs at 100 milligrams per kilogram. These results establish the feasibility of developing oral LpxC-targeting antibiotics for clinical applications.

Commensal Neisseria species share immune suppressive mechanisms with Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a highly adapted human sexually transmitted pathogen that can cause symptomatic infections associated with localized inflammation as well as asymptomatic and subclinical infections, particularly in females. Gonococcal infection in humans does not generate an effective immune response in most cases, which contributes to both transmission of the pathogen and reinfection after treatment. Neisseria gonorrhoeae is known to evade and suppress human immune responses through a variety of mechanisms. Commensal Neisseria species that are closely related to N. gonorrhoeae, such as N. cinerea, N. lactamica, N. elongata, and N. mucosa, rarely cause disease and instead asymptomatically colonize mucosal sites for prolonged periods of time without evoking clearing immunologic responses. We have shown previously that N. gonorrhoeae inhibits the capacity of antigen-pulsed dendritic cells to induce CD4+ T cell proliferation in vitro. Much of the suppressive effects of N. gonorrhoeae on dendritic cells can be recapitulated either by outer-membrane vesicles released from the bacteria or by purified PorB, the most abundant outer-membrane protein in Neisseria gonorrhoeae. We show here that three commensal Neisseria species, N. cinerea, N. lactamica and N. mucosa, show a comparable capacity to suppress dendritic cell-induced T cell proliferation in vitro through mechanisms similar to those demonstrated previously for N. gonorrhoeae, including inhibition by purified PorB. Our findings suggest that some immune-evasive properties of pathogenic N. gonorrhoeae are shared with commensal Neisseria species and may contribute to the ability of both pathogens and commensals to cause prolonged mucosal colonization in humans.

Loss of P2Y1 receptor desensitization does not impact hemostasis or thrombosis despite increased platelet reactivity in vitro.

BACKGROUND: The hemostatic plug formation at sites of vascular injury is strongly dependent on rapid platelet activation and integrin-mediated adhesion and aggregation. However, to prevent thrombotic complications, platelet aggregate formation must be a self-limiting process. The second-wave mediator adenosine diphosphate (ADP) activates platelets via Gq-coupled P2Y1 and Gi-coupled P2Y12 receptors. After ADP exposure, the P2Y1 receptor undergoes rapid phosphorylation-induced desensitization, a negative feedback mechanism believed to be critical for limiting thrombus growth. OBJECTIVE: The objective of this study was to examine the role of rapid P2Y1 receptor desensitization on platelet function and thrombus formation in vivo. METHODS: We analyzed a novel knock-in mouse strain expressing a P2Y1 receptor variant that cannot be phosphorylated beyond residue 340 (P2Y1340-0P), thereby preventing the desensitization of the receptor. RESULTS: P2Y1340-0P mice followed a Mendelian inheritance pattern, and peripheral platelet counts were comparable between P2Y1340-0P/340-0P and control mice. In vitro, P2Y1340-0P/340-0P platelets were hyperreactive to ADP, showed a robust activation response to the P2Y1 receptor-selective agonist, MRS2365, and did not desensitize in response to repeated ADP challenge. We observed increased calcium mobilization, protein kinase C substrate phosphorylation, alpha granule release, activation of the small GTPase Rap1, and integrin inside-out activation/aggregation. This hyperreactivity, however, did not lead to increased platelet adhesion or excessive plug formation under physiological shear conditions. CONCLUSION: Our studies demonstrate that receptor phosphorylation at the C-terminus is critical for P2Y1 receptor desensitization in platelets and that impaired desensitization leads to increased P2Y1 receptor signaling in vitro. Surprisingly, desensitization of the P2Y1 receptor is not required for limiting platelet adhesion/aggregation at sites of vascular injury, likely because ADP is degraded quickly or washed away in the bloodstream.

Developing evidence-based resources for evaluating postgraduate trainees in the biomedical sciences.

Postgraduate trainees elevate the academic strength of institutions by conducting research, promoting innovation, securing grant funding, training undergraduate students, and building alliances. Rigorous and systematic program evaluation can help ensure that postgraduate training programs are achieving the program's intended outcomes. The purpose of this project was to develop evidence-based evaluation tools that could be shared across federally funded biomedical training programs to enhance program evaluation capacity. This manuscript describes the evidence-based process used to determine program evaluation needs of these programs at a research-intensive university. Using a multi-phased sequential exploratory mixed methods approach, data were collected from trainees, employers, leaders, and program directors. Data analyses included document analysis of program plans, inductive coding of focus groups and interviews, and descriptive analysis of surveys. Two overarching categories-Trainee Skills and Program Characteristics-were identified including six themes each. Program directors prioritized communication, social and behavioral skills, and collaboration as the trainee skills that they needed the most help evaluating. Furthermore, program directors prioritized the following program characteristics as those that they needed the most help evaluating: training environment, trainee outcomes, and opportunities offered. Surveys, interview scripts, and related resources for the categories and themes were developed and curated on a publicly available website for program directors to use in their program evaluations.

Mutations in PBP2 from ceftriaxone-resistant Neisseria gonorrhoeae alter the dynamics of the ß3-ß4 loop to favor a low-affinity drug-binding state.

Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended ß3-ß4 loop conformation and a high-affinity state with an inward ß3-ß4 loop conformation. Histidine-514, which is located at the boundary of the ß4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward ß3-ß4 loop conformation or stabilizing the extended ß3-ß4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations.

Draft Genome Sequences of Three Penicillin-Resistant Neisseria gonorrhoeae Strains Isolated in Cincinnati, Ohio, in 1994.

Here, we report the draft genome sequences of three penicillin-resistant Neisseria gonorrhoeae isolates. We include associated data on MICs and genetic relationships to other N. gonorrhoeae strains collected from across the United States. Resistance mutations known to contribute to reduced penicillin susceptibility are annotated in each genome.

Molecular Features of Cephalosporins Important for Activity against Antimicrobial-Resistant Neisseria gonorrhoeae.

The increasing prevalence of Neisseria gonorrhoeae strains exhibiting decreased susceptibility to extended-spectrum cephalosporins (ESCs) presents a challenge for the successful treatment of gonorrhea infections. To address this challenge, we evaluated a panel of 23 cephalosporins against penicillin-binding protein 2 (PBP2) from the ESC-resistant (ESCR) N. gonorrhoeae strain H041 to determine which molecular features are important for antimicrobial activity. Structure-activity relationships (SARs) developed from acylation rate constants against PBP2 and antimicrobial susceptibilities against the H041 strain of N. gonorrhoeae, and interpreted against docking models, reveal that cephalosporins possessing large, lipophilic R1 side chains and electronegative R2 side chains with planar groups are associated with higher acylation rates against PBP2, but also that these same amphipathic features can lower antimicrobial activity. Based on these studies, we tested cefoperazone, one of the most effective ESCs for targeting PBP2, in the female mouse model infected with H041 and showed that it was equally or more effective than ceftriaxone or gentamicin for clearing infections. Taken together, our results reveal that two U.S. Food and Drug Administration (FDA)-approved agents (cefoperazone, ceftaroline) and one FDA-qualified infectious disease product (ceftobiprole) have potential as first-line treatments for gonorrhea and provide a framework for the future design of cephalosporins with improved activity against ESC-resistant N. gonorrhoeae.

Mutations in penicillin-binding protein 2 from cephalosporin-resistant Neisseria gonorrhoeae hinder ceftriaxone acylation by restricting protein dynamics.

The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs). ESC resistance is conferred by mosaic variants of penicillin-binding protein 2 (PBP2) that have diminished capacity to form acylated adducts with cephalosporins. To elucidate the molecular mechanisms of ESC resistance, we conducted a biochemical and high-resolution structural analysis of PBP2 variants derived from the decreased-susceptibility N. gonorrhoeae strain 35/02 and ESC-resistant strain H041. Our data reveal that mutations both lower affinity of PBP2 for ceftriaxone and restrict conformational changes that normally accompany acylation. Specifically, we observe that a G545S substitution hinders rotation of the ß3 strand necessary to form the oxyanion hole for acylation and also traps ceftriaxone in a noncanonical configuration. In addition, F504L and N512Y substitutions appear to prevent bending of the ß3-ß4 loop that is required to contact the R1 group of ceftriaxone in the active site. Other mutations also appear to act by reducing flexibility in the protein. Overall, our findings reveal that restriction of protein dynamics in PBP2 underpins the ESC resistance of N. gonorrhoeae.

Recognition of the ß-lactam carboxylate triggers acylation of Neisseria gonorrhoeae penicillin-binding protein 2.

Resistance of Neisseria gonorrhoeae to extended-spectrum cephalosporins (ESCs) has become a major threat to human health. The primary mechanism by which N. gonorrhoeae becomes resistant to ESCs is by acquiring a mosaic penA allele, encoding penicillin-binding protein 2 (PBP2) variants containing up to 62 mutations compared with WT, of which a subset contribute to resistance. To interpret molecular mechanisms underpinning cephalosporin resistance, it is necessary to know how PBP2 is acylated by ESCs. Here, we report the crystal structures of the transpeptidase domain of WT PBP2 in complex with cefixime and ceftriaxone, along with structures of PBP2 in the apo form and with a phosphate ion bound in the active site at resolutions of 1-7-1.9 Å. These structures reveal that acylation of PBP2 by ESCs is accompanied by rotation of the Thr-498 side chain in the KTG motif to contact the cephalosporin carboxylate, twisting of the ß3 strand to form the oxyanion hole, and rolling of the ß3-ß4 loop toward the active site. Recognition of the cephalosporin carboxylate appears to be the key trigger for formation of an acylation-competent state of PBP2. The structures also begin to explain the impact of mutations implicated in ESC resistance. In particular, a G545S mutation may hinder twisting of ß3 because its side chain hydroxyl forms a hydrogen bond with Thr-498. Overall, our data suggest that acylation is initiated by conformational changes elicited or trapped by binding of ESCs and that these movements are restricted by mutations associated with resistance against ESCs.

Antimicrobial Resistance in Neisseria gonorrhoeae: Proceedings of the STAR Sexually Transmitted Infection-Clinical Trial Group Programmatic Meeting.

The goal of the Sexually Transmitted Infection Clinical Trial Group's Antimicrobial Resistance (AMR) in Neisseria gonorrhoeae (NG) meeting was to assemble experts from academia, government, nonprofit and industry to discuss the current state of research, gaps and challenges in research and technology and priorities and new directions to address the continued emergence of multidrug-resistant NG infections. Topics discussed at the meeting, which will be the focus of this article, include AMR NG global surveillance initiatives, the use of whole genome sequencing and bioinformatics to understand mutations associated with AMR, mechanisms of AMR, and novel antibiotics, vaccines and other methods to treat AMR NG. Key points highlighted during the meeting include: (i) US and International surveillance programs to understand AMR in NG; (ii) the US National Strategy for combating antimicrobial-resistant bacteria; (iii) surveillance needs, challenges, and novel technologies; (iv) plasmid-mediated and chromosomally mediated mechanisms of AMR in NG; (v) novel therapeutic (eg, sialic acid analogs, factor H [FH]/Fc fusion molecule, monoclonal antibodies, topoisomerase inhibitors, fluoroketolides, LpxC inhibitors) and preventative (eg, peptide mimic) strategies to combat infection. The way forward will require renewed political will, new funding initiatives, and collaborations across academic and commercial research and public health programs.

PubMLST for Antigen Allele Mining to Inform Development of Gonorrhea Protein-Based Vaccines.

Neisseria gonorrhoeae (Ng) is a human-specific pathogen and the etiological agent of gonorrhea, a sexually transmitted infection with a significant global health burden. While often asymptomatic, untreated gonorrhea can lead to pelvic inflammatory disease, ectopic pregnancy, infertility, and increased transmission/acquisition of HIV. A protective gonorrhea vaccine may be the only way to control disease transmission in the future due to the inexorable development of antibiotic resistance. Subunit antigens are proven candidates for vaccine development due to their safety, cost-effectiveness, and rapid preparation. To inform protein-based gonorrhea vaccine design by including different antigen variants, herein we present bioinformatics mining of alleles and single nucleotide/amino acid polymorphisms using DNA/protein sequences of all Ng isolates deposited into the PubMLST database and MtrE and BamA as model antigens. We also present phylogenetic analyses that can be performed using sequence data to gain insights into the evolutionary relationships between the polymorphisms found among the population of isolates using a convenient tool: Molecular Evolutionary Genetics Analysis (MEGA) software. Finally, we perform antigen polymorphism mapping onto the MtrE and BamA structures. This methodology can be applied for rational vaccine design to increase vaccine coverage and cross-protection by heteroligand presentation achieved via inclusion of diverse antigen variants and is relevant to over 100 different species and genera deposited into the PubMLST family of databases.

Properly folded and functional PorB from Neisseria gonorrhoeae inhibits dendritic cell stimulation of CD4+ T cell proliferation.

Neisseria gonorrhoeae is an exclusive human pathogen that evades the host immune system through multiple mechanisms. We have shown that N. gonorrhoeae suppresses the capacity of antigen-presenting cells to induce CD4+ T cell proliferation. In this study, we sought to determine the gonococcal factors involved in this adaptive immune suppression. We show that suppression of the capacity of antigen-pulsed dendritic cells to induce T cell proliferation is recapitulated by administration of a high-molecular-weight fraction of conditioned medium from N. gonorrhoeae cultures, which includes outer membrane vesicles that are shed during growth of the bacteria. N. gonorrhoeae PorB is the most abundant protein in N. gonorrhoeae-derived vesicles, and treatment of dendritic cells with purified recombinant PorB inhibited the capacity of the cells to stimulate T cell proliferation. This immunosuppressive feature of purified PorB depended on proper folding of the protein. PorB from N. gonorrhoeae, as well as other Neisseria species and other Gram-negative bacterial species, are known to activate host Toll-like receptor 2 (TLR2) signaling. Published studies have demonstrated that purified Neisseria PorB forms proteinacious nanoparticles, termed proteosomes, when detergent micelles are removed. Unlike folded, detergent-solubilized PorB, PorB proteosomes stimulate immune responses. We now demonstrate that the formation of PorB proteosomes from structurally intact PorB eliminates the immunosuppressive property of the protein while enhancing TLR2 stimulation. These findings suggest that gonococcal PorB present in shed outer membrane vesicles plays a role in suppression of adaptive immune responses to this immune-evasive pathogen.

In Vivo-Selected Compensatory Mutations Restore the Fitness Cost of Mosaic penA Alleles That Confer Ceftriaxone Resistance in Neisseria gonorrhoeae.

Resistance to ceftriaxone in Neisseria gonorrhoeae is mainly conferred by mosaic penA alleles that encode penicillin-binding protein 2 (PBP2) variants with markedly lower rates of acylation by ceftriaxone. To assess the impact of these mosaic penA alleles on gonococcal fitness, we introduced the mosaic penA alleles from two ceftriaxone-resistant (Cror) clinical isolates (H041 and F89) into a Cros strain (FA19) by allelic exchange and showed that the resultant Cror mutants were significantly outcompeted by the Cros parent strain in vitro and in a murine infection model. Four Cror compensatory mutants of FA19 penA41 were isolated independently from mice that outcompeted the parent strain both in vitro and in vivo One of these compensatory mutants (LV41C) displayed a unique growth profile, with rapid log growth followed by a sharp plateau/gradual decline at stationary phase. Genome sequencing of LV41C revealed a mutation (G348D) in the acnB gene encoding the bifunctional aconitate hydratase 2/2 methylisocitrate dehydratase. Introduction of the acnBG348D allele into FA19 penA41 conferred both a growth profile that phenocopied that of LV41C and a fitness advantage, although not as strongly as that exhibited by the original compensatory mutant, suggesting the existence of additional compensatory mutations. The mutant aconitase appears to be a functional knockout with lower activity and expression than wild-type aconitase. Transcriptome sequencing (RNA-seq) analysis of FA19 penA41 acnBG348D revealed a large set of upregulated genes involved in carbon and energy metabolism. We conclude that compensatory mutations can be selected in Cror gonococcal strains that increase metabolism to ameliorate their fitness deficit.IMPORTANCE The emergence of ceftriaxone-resistant (Cror) Neisseria gonorrhoeae has led to the looming threat of untreatable gonorrhea. Whether Cro resistance is likely to spread can be predicted from studies that compare the relative fitnesses of susceptible and resistant strains that differ only in the penA gene that confers Cro resistance. We showed that mosaic penA alleles found in Cror clinical isolates are outcompeted by the Cros parent strain in vitro and in vivo but that compensatory mutations that allow ceftriaxone resistance to be maintained by increasing bacterial fitness are selected during mouse infection. One compensatory mutant that was studied in more detail had a mutation in acnB, which encodes the aconitase that functions in the tricarboxylic acid (TCA) cycle. This study illustrates that compensatory mutations can be selected during infection, which we hypothesize may allow the spread of Cro resistance in nature. This study also provides novel insights into gonococcal metabolism and physiology.

Control of gdhR Expression in Neisseria gonorrhoeae via Autoregulation and a Master Repressor (MtrR) of a Drug Efflux Pump Operon.

The MtrCDE efflux pump of Neisseria gonorrhoeae contributes to gonococcal resistance to a number of antibiotics used previously or currently in treatment of gonorrhea, as well as to host-derived antimicrobials that participate in innate defense. Overexpression of the MtrCDE efflux pump increases gonococcal survival and fitness during experimental lower genital tract infection of female mice. Transcription of mtrCDE can be repressed by the DNA-binding protein MtrR, which also acts as a global regulator of genes involved in important metabolic, physiologic, or regulatory processes. Here, we investigated whether a gene downstream of mtrCDE, previously annotated gdhR in Neisseria meningitidis, is a target for regulation by MtrR. In meningococci, GdhR serves as a regulator of genes involved in glucose catabolism, amino acid transport, and biosynthesis, including gdhA, which encodes an l-glutamate dehydrogenase and is located next to gdhR but is transcriptionally divergent. We report here that in N. gonorrhoeae, expression of gdhR is subject to autoregulation by GdhR and direct repression by MtrR. Importantly, loss of GdhR significantly increased gonococcal fitness compared to a complemented mutant strain during experimental murine infection. Interestingly, loss of GdhR did not influence expression of gdhA, as reported for meningococci. This variance is most likely due to differences in promoter localization and utilization between gonococci and meningococci. We propose that transcriptional control of gonococcal genes through the action of MtrR and GdhR contributes to fitness of N. gonorrhoeae during infection.IMPORTANCE The pathogenic Neisseria species are strict human pathogens that can cause a sexually transmitted infection (N. gonorrhoeae) or meningitis or fulminant septicemia (N. meningitidis). Although they share considerable genetic information, little attention has been directed to comparing transcriptional regulatory systems that modulate expression of their conserved genes. We hypothesized that transcriptional regulatory differences exist between these two pathogens, and we used the gdh locus as a model to test this idea. For this purpose, we studied two conserved genes (gdhR and gdhA) within the locus. Despite general conservation of the gdh locus in gonococci and meningococci, differences exist in noncoding sequences that correspond to promoter elements or potential sites for interacting with DNA-binding proteins, such as GdhR and MtrR. Our results indicate that implications drawn from studying regulation of conserved genes in one pathogen are not necessarily translatable to a genetically related pathogen.

Alanine 501 Mutations in Penicillin-Binding Protein 2 from Neisseria gonorrhoeae: Structure, Mechanism, and Effects on Cephalosporin Resistance and Biological Fitness.

Resistance of Neisseria gonorrhoeae to expanded-spectrum cephalosporins such as ceftriaxone and cefixime has increased markedly in the past decade. The primary cephalosporin resistance determinant is a mutated penA gene, which encodes the essential peptidoglycan transpeptidase, penicillin-binding protein 2 (PBP2). Decreased susceptibility and resistance can be conferred by mosaic penA alleles containing upward of 60 amino acid changes relative to wild-type PBP2, or by nonmosaic alleles with relatively few mutations, the most important of which occurs at Ala501 located near the active site of PBP2. Recently, fully cefixime- and ceftriaxone-resistant clinical isolates that harbored a mosaic penA allele with an A501P mutation were identified. To examine the potential of mutations at Ala501 to increase resistance to expanded-spectrum cephalosporins, we randomized codon 501 in a mosaic penA allele and transformed N. gonorrhoeae to increased cefixime resistance. Interestingly, only five substitutions of Ala501 (A501V, A501T, A501P, A501R, and A501S) that increased resistance and preserved essential transpeptidase function were isolated. To understand their structural implications, these mutations were introduced into the nonmosaic PBP2-6140CT, which contains four C-terminal mutations present in PBP2 from the penicillin-resistant strain FA6140. The crystal structure of PBP2-6140CT-A501T was determined and revealed ordering of a loop near the active site and a new hydrogen bond involving Thr501 that connects the loop and the SxxK conserved active site motif. The structure suggests that increased rigidity in the active site region is a mechanism for cephalosporin resistance mediated by Ala501 mutations in PBP2.

Synthesis and Kinetic Analysis of Two Conformationally Restricted Peptide Substrates of Escherichia coli Penicillin-Binding Protein 5.

Escherichia coli PBP5 (penicillin-binding protein 5) is a dd-carboxypeptidase involved in bacterial cell wall maturation. Beyond the C-terminal d-alanyl-d-alanine moiety, PBP5, like the essential high-molecular mass PBPs, has little specificity for other elements of peptidoglycan structure, at least as elicited in vitro by small peptidoglycan fragments. On the basis of the crystal structure of a stem pentapeptide derivative noncovalently bound to E. coli PBP6 (Protein Data Bank entry 3ITB ), closely similar in structure to PBP5, we have modeled a pentapeptide structure at the active site of PBP5. Because the two termini of the pentapeptide are directed into solution in the PBP6 crystal structure, we then modeled a 19-membered cyclic peptide analogue by cross-linking the terminal amines by succinylation. An analogous smaller, 17-membered cyclic peptide, in which the l-lysine of the original was replaced by l-diaminobutyric acid, could also be modeled into the active site. We anticipated that, just as the reactivity of stem peptide fragments of peptidoglycan with PBPs in vivo may be entropically enhanced by immobilization in the polymer, so too would that of our cyclic peptides with respect to their acyclic analogues in vitro. This paper describes the synthesis of the peptides described above that were required to examine this hypothesis and presents an analysis of their structures and reaction kinetics with PBP5.

Complete Genome Sequences of Three Neisseria gonorrhoeae Laboratory Reference Strains, Determined Using PacBio Single-Molecule Real-Time Technology.

Neisseria gonorrhoeae, the etiological agent that causes the sexually transmitted infection gonorrhea, is a significant public health concern due to the emergence of antimicrobial resistance. We report the complete genome sequences of three reference isolates with varied antimicrobial susceptibility that will aid in elucidating the genetic mechanisms that confer resistance.