RESUMEN
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.
RESUMEN
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.
Asunto(s)
Ceftriaxona/química , Farmacorresistencia Bacteriana , Neisseria gonorrhoeae/enzimología , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/química , Sustitución de Aminoácidos , Sitios de Unión , Mutación Missense , Neisseria gonorrhoeae/genética , Estructura Secundaria de Proteína , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/genética , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Resistencia a las Cefalosporinas , Neisseria gonorrhoeae/metabolismo , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/metabolismo , Acetilación/efectos de los fármacos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Ceftriaxona/farmacología , Mutación Missense , Neisseria gonorrhoeae/genética , Estructura Secundaria de Proteína , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/genéticaRESUMEN
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.
Asunto(s)
D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/genética , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/metabolismo , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/ultraestructura , Acilación , Alelos , Sitios de Unión/efectos de los fármacos , Dominio Catalítico , Cefixima/farmacología , Ceftriaxona/farmacología , Resistencia a las Cefalosporinas , Cefalosporinas/farmacología , Gonorrea/genética , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Proteínas de Unión a las Penicilinas/químicaRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Proliferación Celular , Células Dendríticas/inmunología , Gonorrea/inmunología , Neisseria gonorrhoeae/inmunología , Porinas/química , Pliegue de Proteína , Linfocitos T CD4-Positivos/microbiología , Células Cultivadas , Células Dendríticas/microbiología , Gonorrea/microbiología , Humanos , Activación de Linfocitos , Porinas/metabolismo , Transducción de Señal , Receptor Toll-Like 2/metabolismoRESUMEN
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.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple , Gonorrea/tratamiento farmacológico , Procesos de Grupo , Neisseria gonorrhoeae/efectos de los fármacos , Enfermedades de Transmisión Sexual/tratamiento farmacológico , Antibacterianos/efectos adversos , Antibacterianos/uso terapéutico , Programas de Optimización del Uso de los Antimicrobianos/métodos , Vacunas Bacterianas/uso terapéutico , Barbitúricos/uso terapéutico , Monitoreo Epidemiológico , Humanos , Isoxazoles , Macrólidos/uso terapéutico , Pruebas de Sensibilidad Microbiana , Morfolinas , Mutación , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/inmunología , Neisseria gonorrhoeae/aislamiento & purificación , Oxazolidinonas , Salud Pública/métodos , Compuestos de Espiro/uso terapéutico , Inhibidores de Topoisomerasa/uso terapéutico , Triazoles/uso terapéutico , Organización Mundial de la SaludRESUMEN
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.
Asunto(s)
Alanina , Resistencia a las Cefalosporinas/genética , Mutación , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genética , Proteínas de Unión a las Penicilinas/química , Proteínas de Unión a las Penicilinas/genética , Alelos , Dominio Catalítico , Enlace de Hidrógeno , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Neisseria gonorrhoeae/fisiología , Proteínas de Unión a las Penicilinas/metabolismo , Estabilidad Proteica , TemperaturaRESUMEN
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.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Proteínas de Unión a las Penicilinas/metabolismo , Dominio Catalítico , Cinética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , Conformación Proteica , Especificidad por SustratoRESUMEN
UNLABELLED: In strains of Neisseria gonorrhoeae harboring the mtr and penB determinants that decrease permeation of antibiotics into the periplasm, mutation or deletion of the PilQ secretin of type IV pili increases resistance to penicillin by â¼3-fold, indicating a role for PilQ in antibiotic permeation. In this study, we examined spontaneously arising mutants with decreased susceptibility to penicillin. One class of mutants had a phenotype indistinguishable from that of a previously characterized pilQ2 mutation that interfered with the formation of SDS-resistant PilQ multimers. A second class of mutants contained frameshift mutations in genes upstream of pilQ in the pilMNOPQ operon that increased resistance to levels similar to those of the pilQ2 mutation. In-frame deletions of these genes were constructed, but only the frameshift mutations increased antibiotic resistance, suggesting that the mutations had polar effects on PilQ. Consistent with this result, titration of wild-type PilQ levels revealed a direct correlation between resistance and expression levels of PilQ. To determine which form of PilQ, the monomer or the multimer, was responsible for antibiotic permeation, we manipulated and quantified these forms in different mutants. Deletion of PilW, which is responsible for the maturation of PilQ into SDS-resistant multimers, had no effect on resistance. Moreover, Western blot analysis revealed that while SDS-resistant multimer levels were decreased by 26% in frameshift mutants, the levels of PilQ monomers were decreased by 48%. These data suggest that immature, SDS-labile complexes, not mature, SDS-resistant PilQ complexes, serve as the route of entry of antibiotics into the periplasm. IMPORTANCE: The capacity of antibiotics to reach their target is crucial for their activity. In Neisseria gonorrhoeae, the PilQ secretin of type IV pili plays an important role in antibiotic influx when diffusion of antibiotics through porins is limited (e.g., in most resistant strains). On Western blots, PilQ exists both as a mature higher-order multimer and an immature, SDS-labile monomer. In this study, we examined spontaneously arising mutations in PilQ and in the genes upstream of PilQ in the pilMNOPQ operon that increase resistance to penicillin. We provide evidence that PilQ monomers associate by mass action to form immature multimers and that these complexes likely mediate the diffusion of antibiotics across the outer membrane.
Asunto(s)
Antibacterianos/farmacología , Proteínas Fimbrias/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/metabolismo , Dodecil Sulfato de Sodio/química , Antibacterianos/metabolismo , Proteínas Fimbrias/genética , Pruebas de Sensibilidad Microbiana , Mutación , Neisseria gonorrhoeae/genética , Resistencia a las PenicilinasRESUMEN
A hallmark of penicillin-binding protein 2 (PBP2) from penicillin-resistant strains of Neisseria gonorrhoeae is insertion of an aspartate after position 345. The insertion resides on a loop near the active site and is immediately adjacent to an existing aspartate (Asp346) that forms a functionally important hydrogen bond with Ser363 of the SxN conserved motif. Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function. Although the kinetic consequences of the Asp insertion are well-established, how it impacts the structure of PBP2 is unknown. Here, we report the 2.2 Å resolution crystal structure of a truncated construct of PBP2 containing all five mutations present in PBP2 from the penicillin-resistant strain 6140, including the Asp insertion. Commensurate with the strict specificity for the Asp insertion over similar amino acids, the insertion does not cause disordering of the structure, but rather induces localized flexibility in the ß2c-ß2d loop. The crystal structure resolves the ambiguity of whether the insertion is Asp345a or Asp346a (due to the adjacent Asp) because the hydrogen bond between Asp346 and Ser362 is preserved and the insertion is therefore Asp346a. The side chain of Asp346a projects directly toward the ß-lactam-binding site near Asn364 of the SxN motif. The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the ß-lactam ring during acylation because of the negative charge of its side chain.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/metabolismo , Acilación , Ácido Aspártico/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Mutagénesis Insercional , Neisseria gonorrhoeae/genética , Resistencia a las Penicilinas/genética , Resistencia a las Penicilinas/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina , Electricidad EstáticaRESUMEN
Biased agonism describes a multistate model of G protein-coupled receptor activation in which each ligand induces a unique structural conformation of the receptor, such that the receptor couples differentially to G proteins and other intracellular proteins. P2Y receptors are G protein-coupled receptors that are activated by endogenous nucleotides, such as adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP). A previous report suggested that UTP may be a biased agonist at the human P2Y11 receptor, as it increased cytosolic [Ca2+], but did not induce accumulation of inositol phosphates, whereas ATP did both. The mechanism of action of UTP was unclear, so the aim of this study was to characterise the interaction of UTP with the P2Y11 receptor in greater detail. Intracellular Ca2+ was monitored in 1321N1 cells stably expressing human P2Y11 receptors using the Ca2+-sensitive fluorescent indicator, fluo-4. ATP evoked a rapid, concentration-dependent rise in intracellular Ca2+, but surprisingly, even high concentrations of UTP were ineffective. In contrast, UTP was slightly, but significantly more potent than ATP in evoking a rise in intracellular Ca2+ in 1321N1 cells stably expressing the human P2Y2 receptor, with no difference in the maximum response. Thus, the lack of response to UTP at hP2Y11 receptors was not due to a problem with the UTP solution. Furthermore, coapplying a high concentration of UTP with ATP did not inhibit the response to ATP. Thus, contrary to a previous report, we find no evidence for an agonist action of UTP at the human P2Y11 receptor, nor does UTP act as an antagonist.
Asunto(s)
Receptores Purinérgicos P2/metabolismo , Transducción de Señal/fisiología , Uridina Trifosfato/metabolismo , Astrocitoma/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
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.
Asunto(s)
Ceftriaxona , Gonorrea , Humanos , Ceftriaxona/metabolismo , Ceftriaxona/farmacología , Neisseria gonorrhoeae/genética , Gonorrea/tratamiento farmacológico , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Cefoperazona/farmacología , Cefalosporinas/farmacología , Cefalosporinas/metabolismo , Piperacilina/metabolismo , Piperacilina/farmacología , beta-Lactamas/farmacologíaRESUMEN
The P2Y(4) receptor is selectively targeted to the apical membrane in polarized epithelial cell lines and has been shown to play a key role in intestinal chloride secretion. In this study, we delimit a 23 amino acid sequence within the P2Y(4) receptor C-tail that directs its apical targeting. Using a mutagenesis approach, we found that four hydrophobic residues near the COOH-terminal end of the signal are necessary for apical sorting, whereas two basic residues near the NH(2)-terminal end of the signal are involved to a lesser extent. Interestingly, mutation of the key hydrophobic residues results in a basolateral enrichment of the receptor construct, suggesting that the apical targeting sequence may prevent insertion or disrupt stability of the receptor at the basolateral membrane. The signal is not sequence specific, as an inversion of the 23 amino acid sequence does not disrupt apical targeting. We also show that the apical targeting sequence is an autonomous signal and is capable of redistributing the normally basolateral P2Y(12) receptor, suggesting that the apical signal is dominant over the basolateral signal in the main body of the P2Y(12) receptor. The targeting sequence is unique to the P2Y(4) receptor, and sequence alignments of the COOH-terminal tail of mammalian orthologs reveal that the hydrophobic residues in the targeting signal are highly conserved. These data define the novel apical sorting signal of the P2Y(4) receptor, which may represent a common mechanism for trafficking of epithelial transmembrane proteins.
Asunto(s)
Polaridad Celular/fisiología , Citoplasma/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Purinérgicos P2/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Membrana Celular/fisiología , Células Cultivadas , Células Epiteliales/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Transporte de Proteínas , Receptores Purinérgicos P2/genética , Transducción de SeñalRESUMEN
The recent identification of a high-level-ceftriaxone-resistant (MIC = 2 to 4 µg/ml) isolate of Neisseria gonorrhoeae from Japan (H041) portends the loss of ceftriaxone as an effective treatment for gonococcal infections. This is of grave concern because ceftriaxone is the last remaining option for first-line empirical antimicrobial monotherapy. The penA gene from H041 (penA41) is a mosaic penA allele similar to mosaic alleles conferring intermediate-level cephalosporin resistance (Ceph(i)) worldwide but has 13 additional mutations compared to the mosaic penA gene from the previously studied Ceph(i) strain 35/02 (penA35). When transformed into the wild-type strain FA19, the penA41 allele confers 300- and 570-fold increases in the MICs for ceftriaxone and cefixime, respectively. In order to understand the mechanisms involved in high-level ceftriaxone resistance and to improve surveillance and epidemiology during the potential emergence of ceftriaxone resistance, we sought to identify the minimum number of amino acid alterations above those in penA35 that confer high-level resistance to ceftriaxone. Using restriction fragment exchange and site-directed mutagenesis, we identified three mutations, A311V, T316P, and T483S, that, when incorporated into the mosaic penA35 allele, confer essentially all of the increased resistance of penA41. A311V and T316P are close to the active-site nucleophile Ser310 that forms the acyl-enzyme complex, while Thr483 is predicted to interact with the carboxylate of the ß-lactam antibiotic. These three mutations have thus far been described only for penA41, but dissemination of these mutations in other mosaic alleles would spell the end of ceftriaxone as an effective treatment for gonococcal infections.
Asunto(s)
Antibacterianos/farmacología , Ceftriaxona/farmacología , Resistencia a las Cefalosporinas/genética , Cefalosporinas/farmacología , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genética , Proteínas de Unión a las Penicilinas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Gonorrea , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutación , Neisseria gonorrhoeae/clasificación , Estructura Secundaria de Proteína , Alineación de SecuenciaRESUMEN
The orphan receptor GPR17 has been reported to be activated by UDP, UDP-sugars, and cysteinyl leukotrienes, and coupled to intracellular Ca(2+) mobilization and inhibition of cAMP accumulation, but other studies have reported either a different agonist profile or lack of agonist activity altogether. To determine if GPR17 is activated by uracil nucleotides and leukotrienes, the hemagglutinin-tagged receptor was expressed in five different cell lines and the signaling properties of the receptor were investigated. In C6, 1321N1, or Chinese hamster ovary (CHO) cells stably expressing GPR17, UDP, UDP-glucose, UDP-galactose, and cysteinyl leukotriene C4 (LTC4) all failed to promote inhibition of forskolin-stimulated cAMP accumulation, whereas both UDP and UDP-glucose promoted marked inhibition (>80%) of forskolin-stimulated cAMP accumulation in C6 and CHO cells expressing the P2Y14 receptor. Likewise, none of these compounds promoted accumulation of inositol phosphates in COS-7 or human embryonic kidney 293 cells transiently transfected with GPR17 alone or cotransfected with Gαq/i5, which links Gi-coupled receptors to the Gq-regulated phospholipase C (PLC) signaling pathway, or PLCε, which is activated by the Gα12/13 signaling pathway. Moreover, none of these compounds promoted internalization of GPR17 in 1321N1-GPR17 cells. Consistent with previous reports, coexpression experiments of GPR17 with cysteinyl leukotriene receptor 1 (CysLTR1) suggested that GPR17 acts as a negative regulator of CysLTR1. Taken together, these data suggest that UDP, UDP-glucose, UDP-galactose, and LTC4 are not the cognate ligands of GPR17.
Asunto(s)
Cisteína/metabolismo , Leucotrienos/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Nucleótidos de Uracilo/metabolismo , Animales , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Cisteína/farmacología , Células HEK293 , Humanos , Leucotrienos/farmacología , Nucleótidos de Uracilo/farmacología , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato Glucosa/farmacologíaRESUMEN
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.
Asunto(s)
Gonorrea , Neisseria , Humanos , Neisseria gonorrhoeae , Gonorrea/microbiología , Linfocitos T CD4-Positivos , Proteínas de la Membrana/metabolismoRESUMEN
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.
Asunto(s)
Trombosis , Lesiones del Sistema Vascular , Ratones , Animales , Agregación Plaquetaria , Plaquetas/metabolismo , Hemostasis , Trombosis/genética , Trombosis/prevención & control , Trombosis/metabolismo , Adenosina Difosfato/farmacología , Integrinas/metabolismo , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismoRESUMEN
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.
Asunto(s)
Bacterias Gramnegativas , Lípido A , Animales , Ratones , Perros , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antibacterianos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Inhibidores Enzimáticos/químicaRESUMEN
Insertion of an aspartate residue at position 345a in penicillin-binding protein 2 (PBP 2), which lowers the rate of penicillin acylation by ~6-fold, is commonly observed in penicillin-resistant strains of Neisseria gonorrhoeae. Here, we show that insertions of other amino acids also lower the penicillin acylation rate of PBP 2, but none supported growth of N. gonorrhoeae, indicating loss of essential transpeptidase activity. The Asp345a mutation likely acts by altering the interaction between its adjacent residue, Asp346, in the ß2a-ß2d hairpin loop and Ser363, the middle residue of the SXN active site motif. Because the adjacent aspartate creates ambiguity in the position of the insertion, we also examined if insertions at position 346a could confer decreased susceptibility to penicillin. However, only aspartate insertions were identified, indicating that only an Asp-Asp couple can confer resistance and retain transpeptidase function. The importance of the Asp346-Ser363 interaction was assessed by mutation of each residue to Ala. Although both mutants lowered the acylation rate of penicillin G by 5-fold, neither could support growth of N. gonorrhoeae, again indicating loss of transpeptidase function. Interaction between a residue in the equivalent of the ß2a-ß2d hairpin loop and the middle residue of the SXN motif is observed in crystal structures of other Class B PBPs, and its importance is also supported by multisequence alignments. Overall, these results suggest that this conserved interaction can be manipulated (e.g., by insertion) to lower the acylation rate by ß-lactam antibiotics and increase resistance, but only if essential transpeptidase activity is preserved.
Asunto(s)
Neisseria gonorrhoeae/metabolismo , Proteínas de Unión a las Penicilinas/genética , Dominio Catalítico , Secuencia Conservada , Modelos Moleculares , Mutación , Neisseria gonorrhoeae/genética , Proteínas de Unión a las Penicilinas/químicaRESUMEN
The P2Y(1) receptor is localized to the basolateral membrane of polarized Madin-Darby canine kidney (MDCK) cells. In the present study, we identified a 25-residue region within the C-terminal tail (C-tail) of the P2Y(1) receptor that directs basolateral sorting. Deletion of this sorting signal caused redirection of the receptor to the apical membrane, indicating that the region from the N-terminus to transmembrane domain 7 (TM7) contains an apical-sorting signal that is overridden by a dominant basolateral signal in the C-tail. Location of the signal relative to TM7 is crucial, because increasing its distance from the end of TM7 resulted in loss of basolateral sorting. The basolateral-sorting signal does not use any previously established basolateral-sorting motifs, i.e. tyrosine-containing or di-hydrophobic motifs, for function, and it is functional even when inverted or when its amino acids are scrambled, indicating that the signal is sequence independent. Mutagenesis of different classes of amino acids within the signal identified charged residues (five basic and four acidic amino acids in 25 residues) as crucial determinants for sorting function, with amidated amino acids having a lesser role. Mutational analyses revealed that whereas charge balance (+1 overall) of the signal is unimportant, the total number of charged residues (nine), either positive or negative, is crucial for basolateral targeting. These data define a new class of targeting signal that relies on total charge and might provide a common mechanism for polarized trafficking of epithelial proteins.