RESUMEN
The human pathogen Streptococcus pneumoniae has been treated for decades with ß-lactam antibiotics. Its resistance is now widespread, mediated by the expression of mosaic variants of the target enzymes, the penicillin-binding proteins (PBPs). Understanding the mode of action of ß-lactams, not only in molecular detail but also in their physiological consequences, will be crucial to improving these drugs and any counterresistances. In this work, we investigate the piperacillin paradox, by which this ß-lactam selects primarily variants of PBP2b, whereas its most reactive target is PBP2x. These PBPs are both essential monofunctional transpeptidases involved in peptidoglycan assembly. PBP2x participates in septal synthesis, while PBP2b functions in peripheral elongation. The formation of the "lemon"-shaped cells induced by piperacillin treatment is consistent with the inhibition of PBP2x. Following the examination of treated and untreated cells by electron microscopy, the localization of the PBPs by epifluorescence microscopy, and the determination of the inhibition time course of the different PBPs, we propose a model of peptidoglycan assembly that accounts for the piperacillin paradox.
Asunto(s)
Proteínas de Unión a las Penicilinas/antagonistas & inhibidores , Piperacilina/farmacología , Streptococcus pneumoniae/efectos de los fármacos , Resistencia betalactámica , Aminoaciltransferasas/antagonistas & inhibidores , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Pruebas de Sensibilidad Microbiana , Terapia Molecular Dirigida , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/fisiología , Resistencia betalactámica/efectos de los fármacosRESUMEN
Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex and the crystal structure of the isolated NTNH protein. Unexpectedly, the BoNT/X complex is stable and protease-resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo. Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents very weak ganglioside binding and exposed hydrophobic surfaces.
RESUMEN
Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions are largely unknown. We now show that the Escherichia coli peptidoglycan glycosyltrasferase-transpeptidase PBP1A interacts with the cell elongation-specific transpeptidase PBP2 in vitro and in the cell. Cells lacking PBP1A are thinner and initiate cell division later in the cell cycle. PBP1A localizes mainly to the cylindrical wall of the cell, supporting its role in cell elongation. Our in vitro peptidoglycan synthesis assays provide novel insights into the cooperativity of peptidoglycan synthases with different activities. PBP2 stimulates the glycosyltransferase activity of PBP1A, and PBP1A and PBP2 cooperate to attach newly synthesized peptidoglycan to sacculi. PBP2 has peptidoglycan transpeptidase activity in the presence of active PBP1A. Our data also provide a possible explanation for the depletion of lipid II precursors in penicillin-treated cells.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/crecimiento & desarrollo , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano Glicosiltransferasa/metabolismo , Peptidoglicano/biosíntesis , Pared Celular/metabolismo , Escherichia coli/enzimologíaRESUMEN
Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex at 3.1 Å resolution. Unexpectedly, the BoNT/X complex is stable and protease resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo . Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents weak ganglioside binding and exposed hydrophobic surfaces.
RESUMEN
During voluntary behaviors, animals need to disable any reflexes that could interfere with the intended movements. With the optomotor response, flies stabilize a straight flight path by correcting for unintended deviations sensed as the panoramic motion of the surround. HS cells of the fly are thought to mediate optomotor responses to horizontal motion. During spontaneous flight turns, an efference copy acts on HS cells with the right sign to counteract the visual input elicited by the fly's own behavior. Here, we investigated, whether looming-elicited turns in flying Drosophila have a similar effect on HS cells. We show that looming stimuli themselves can influence the processing of panoramic motion stimuli in HS cells and that an inhibitory efference copy suppresses excitatory motion responses during turns in both directions, but only in a subset of HS cells. Our findings support the notion that the processing of sensory information is finely tuned to behavioral context.
RESUMEN
Resistance to ß-lactam antibacterials, importantly via production of ß-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) ß-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C ßlactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied ß-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D ßlactamases, our data support the proposal that bicyclic boronates are broad-spectrum ßlactamase inhibitors that work by mimicking a high energy 'tetrahedral' intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful ß-lactamase inhibition.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Ácidos Borónicos/farmacología , Inhibidores de beta-Lactamasas/farmacología , Antibacterianos , Proteínas Bacterianas/genética , Ácidos Borónicos/química , Cristalografía por Rayos X , Ciclización , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Escherichia coli/enzimología , Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/clasificación , beta-Lactamasas/genéticaRESUMEN
The morphogenesis of ovococci has been reviewed extensively. Recent results have provided new insights concerning the mechanisms of elongation in ovoid bacteria. We present here the proteins involved in the elongation (firmly established and more or less hypothetical) and discuss the relationship between elongation and division of ovococci.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enterococcus faecalis/metabolismo , Enterococcus/metabolismo , Lactococcus lactis/metabolismo , Proteínas de la Membrana/metabolismo , Streptococcus pneumoniae/metabolismo , Proteínas Bacterianas/química , Membrana Celular/química , Membrana Celular/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Enterococcus/química , Enterococcus/ultraestructura , Enterococcus faecalis/química , Enterococcus faecalis/ultraestructura , Lactococcus lactis/química , Lactococcus lactis/ultraestructura , Proteínas de la Membrana/química , Peptidoglicano/metabolismo , Mapeo de Interacción de Proteínas , Streptococcus pneumoniae/química , Streptococcus pneumoniae/ultraestructuraRESUMEN
The synthesis of peptidoglycan, the major component of the bacterial cell wall, is essential to cell survival, yet its mechanism remains poorly understood. In the present work, we have isolated several membrane protein complexes consisting of the late division proteins of Streptococcus pneumoniae: DivIB, DivIC, FtsL, PBP2x and FtsW, or subsets thereof. We have co-expressed membrane proteins from S. pneumoniae in Escherichia coli. By combining two successive affinity chromatography steps, we obtained membrane protein complexes with a very good purity. These complexes are functional, as indicated by the retained activity of PBP2x to bind a fluorescent derivative of penicillin and to hydrolyze the substrate analogue S2d. Moreover, we have evidenced the stabilizing role of protein-protein interactions within each complex. This work paves the way for a complete reconstitution of peptidoglycan synthesis in vitro, which will be critical to the elucidation of its intricate regulation mechanisms.
Asunto(s)
Proteínas Bacterianas/metabolismo , División Celular , Pared Celular/metabolismo , Proteínas de la Membrana/metabolismo , Streptococcus pneumoniae/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Hidrólisis , Cinética , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteolisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Streptococcus pneumoniae/genéticaRESUMEN
Understanding the molecular basis of bacterial cell wall assembly is of paramount importance in addressing the threat of increasing antibiotic resistance worldwide. Streptococcus pneumoniae presents a particularly acute problem in this respect, as it is capable of rapid evolution by homologous recombination with related species. Resistant strains selected by treatment with ß-lactams express variants of the target enzymes that do not recognize the drugs but retain their activity in cell wall building, despite the antibiotics being mimics of the natural substrate. Until now, the crucial transpeptidase activity that is inhibited by ß-lactams was not amenable to in vitro investigation with enzymes from Gram-positive organisms, including streptococci, staphylococci, or enterococci pathogens. We report here for the first time the in vitro assembly of peptidoglycan using recombinant penicillin-binding proteins from pneumococcus and the precursor lipid II. The two required enzymatic activities, glycosyl transferase for elongating glycan chains and transpeptidase for cross-linking stem-peptides, were observed. Most importantly, the transpeptidase activity was dependent on the chemical nature of the stem-peptide. Amidation of the second residue glutamate into iso-glutamine by the recently discovered amido-transferase MurT/GatD is required for efficient cross-linking of the peptidoglycan.
Asunto(s)
Pared Celular/enzimología , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano Glicosiltransferasa/metabolismo , Peptidoglicano/biosíntesis , Peptidil Transferasas/metabolismo , Streptococcus pneumoniae/enzimología , Antibacterianos/farmacología , Pared Celular/química , Pared Celular/efectos de los fármacos , Pared Celular/genética , Ingeniería Genética , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Recombinación Homóloga , Proteínas de Unión a las Penicilinas/química , Proteínas de Unión a las Penicilinas/genética , Peptidoglicano/química , Peptidoglicano/genética , Peptidoglicano Glicosiltransferasa/química , Peptidoglicano Glicosiltransferasa/genética , Peptidil Transferasas/química , Peptidil Transferasas/genética , Streptococcus pneumoniae/química , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genética , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/química , Uridina Difosfato Ácido N-Acetilmurámico/metabolismo , Resistencia betalactámica/efectos de los fármacos , Resistencia betalactámica/genética , beta-Lactamas/farmacologíaRESUMEN
A total of 221 isolates of M. tuberculosis were sampled from hospitals and the general population in the northern plain of Vietnam, one of the most populated region of the country. Genotypic composition and diversity were characterized, and we investigated how they are affected by sampling (hospital vs. general population), correcting for potential confounding effects (location, age and gender of the patients). Spoligotyping and 12 MIRU-VNTR typing were used as first line. Then 15 MIRU-VNTR standard set was used, making 21 MIRU-VNTR typing for the clustered isolates. Result showed that 8 lineages and 13 sub-lineages were circulating in the region. The most predominant lineages were Beijing (38.5%) and EAI (38.5%). Others appeared with small proportions H (1.4%), LAM (1.8%), T (8.1%), X (0.9%), MANU (2.3%), and Zero (0.4%). Higher clustering rate was found in the hospital samples (17.9% in urban and 19.2% in rural areas) compared to the population ones (0%). The typical Vietnamese EAI4-VNM sub-lineage of EAI lineage accounted for 67% of EAI strains and was associated with older ages. Beijing genotypes were associated with younger, urban population and were characterized by high clustering rates. These characteristics strongly suggest that Beijing strains are invading the population, replacing the local EAI-VNM4, thus predicting a more serious tuberculosis situation in the future in the absence of more effective control strategies.