Evaluación del mecanismo de corte del RNA por la toxina bacteriana Kid y de su actividad inhibora de la traducción

Leída en Unversidad Complutense de Madrid. Facultad de Ciencias Biológicas en 11-27-2009; 198 págs.Kid , the toxin of the parD (kis, kid) maintenance system of plasmid R1 is an endoribonuclease that preferentially cuts RNA at the 5´ of A in the core sequence 5´-UA(A/C)-3´ and inhibits protein synthesis. A mechanism of the RNase activity of this toxin has been proposed based in the analysis of RNA cleavage products and in a model of the structure of an RNA substrate with the Kid toxin. The model tentatively identified residues of the catalytic site and others involved in general and specific binding of the Kid toxin to an RNA substrate. Inhibition of protein synthesis by Kid is supposed to be the consequence of a ribosome independent cleavage of mRNA. In this thesis we aimed to evaluate the model on the binding and cleavage of RNA by the Kid toxin analysing the effect of specific mutations affecting key residues proposed by the model. We also aimed to analyze the possible involvement of the releasing factor RF1 in the activity of the toxin as protein synthesis inhibitor with the help of novel mutants in the gene of this factor that were previously isolated in our laboratory. A evaluation of the model was required as the structure of the Kid toxin and the RNA substrate was a model relaying on the docking of an RNA substrate on the protein. This evaluation was performed by native mass spectrometry, a novel non-disruptive development of mass spectrometry that allowed the identification of protein-protein and protein-nucleic acids complexes in solution at physiological pH. Using this methodology, which is particularly useful for comparisons of the different mutational variants of the same protein, we analysed the effects on RNA binding and cleavage of specific mutations in some of the key residues identified by the model. A collection of Kid proteins containing single changes in residues of the proposed catalytic site, R73H, D75E, D75N and H17P were isolated and purified. In vivo analysis showed that the mutations abolished the toxicity of the Kid protein but conserved the corregulatory potential of the wild-type protein. Further RNA cleavage assays done using mass spectrometry analysis on two different RNA substrates, 5´-AUACA-3´ and 5´-UUACU-3´, indicated that the mutations abolished or reduced drastically the RNase activity of the toxin without altering substantially its capacity to bind to the mimetic substrate, 5´-AdUACA-3´. The model also proposed the existence of two possible and symmetric RNA binding sites on the dimeric structure of the active toxin and identified residues involved in general binding to the substrate, such as R85, or in specific interactions with the bases at the core sequence 5´-UAC-3´ such as A55, T46 and T69. With the exceptions of T46G mutant, these mutants showed a reduced but measurable RNase activity. R85W showed a drastic reduction in RNA binding and has a concomitant reduction in RNase activity. Kid mutants retaining RNase activity, also showed the potential to inhibit protein synthesis in vitro and the toxicity of the Kid wt protein in vivo. While supporting the available model the results obtained reveal additional complexities on the role of T46. The possible involvement of interactions of Kid toxin with components of the ribosome machinery in the mechanism of protein synthesis inhibition was opened by the accidental discovery of novel mutations in prfA. This gene encodes the polypeptide releasing factor RF1Programa de Formación del Gobierno Vasco (BFI 05.35)Peer reviewe

Evaluación del mecanismo de corte de RNA por la toxina bacteriana Kid y de su actividad inhibidora de la traducción

Kid, la toxina endoribonucleasa del sistema Toxina-Antitoxina parD del plásmido R1, corta ARN en la secuencia 5́-UA(A/C)-3́ inhibiendo síntesis de proteínas, efecto atribuido a la rotura del ARNm independientemente de ribosomas. En la evaluación del modelo de unión y corte del ARN por Kid se ha analizado, por espectrometría de masas, el efecto de mutaciones en residuos claves del modelo. Las mutaciones R73H, D75E, D75N y H17P, afectando residuos del centro catalítico propuesto, inactivan la toxicidad de Kid. Ensayos de corte de los sustratos 5́-AUACA-3́ y 5́-UUACU-3́, indican que las mutaciones anulan la actividad ARNasa de la toxina sin afectar su unión al sustrato 5́-AdUACA-3́. El análisis de mutaciones en residuos implicados en unión, indica que interaccionan con el sustrato 5́-AdUACA-3́ con menor eficiencia que la proteína silvestre y que, exceptuando a T46G, conservan actividad ARNasa. Los mutantes que retenían actividad, mostraron inhibición de síntesis de proteínas in vitro y de toxicidad in vivo. Los resultados apoyan el modelo propuesto indicando que T46 está implicado en unión a ARN participando más directamente en su actividad ARNasa. El descubrimiento de mutaciones en prfA abrió la posibilidad de analizar las interacciones de Kid con componentes ribosomales. prfA codifica el factor de terminación de la traducción RF1 de E. coli. Las mutaciones afectando residuos localizados cerca del sitio de reconocimiento de codones, mostraron reducción en la eficiencia de terminación en codones UAG, sin afectar su estabilidad in vivo. Los mutantes mostraron hipersensibilidad a antibióticos aminoglicósidos y a Kid cuya actividad ARNasa es, en principio, independiente del proceso de traducción. La hipersensibilidad a Kid, dependiente de su actividad ARNasa, se suprimió al sobreproducir RF1 silvestre o la antitoxina Kis. Estos datos aportan la primera evidencia de la participación de RF1 en la ruta de toxicidad de Kid, añadiendo complejidades a la actividad de Kid

Bacterial toxin-antitoxin systems targeting translation

10 p.-2 fig.Toxin-antitoxin systems (TAS) emerged more than 25 years ago and have since developed as an important field in molecular microbiology. TAS are autoregulated operons coding a stable toxin and an unstable antitoxin found in the plasmids and chromosomes of Bacteria and Archaea. The conditional activation of their toxins interferes with cell growth/viability and, depending on the context, can influence plasmid maintenance,stress management, bacterial persistence, cell differentiation and, it is likely also bacterial virulence. This review summarizes recent results on the parD system of plasmid R1 and on the chromosomal relBE systems found in Escherichia coli and in Streptococcus pneumoniae with a focus on the RNase activity of their toxins,their regulation and their biomedical applications and implications.The authors acknowledge the financial support of the MICINN, the Spanish Ministry of Science and Innovation, (CSD2008-00013; BFU2008- 00179-E/BMC)Peer reviewe

Effectiveness of COVID-19 Vaccination Mandates and Incentives in Europe

During 2021–2022 many countries in the European region of the World Health Organization (WHO) adopted mandatory and incentive-based vaccination measures to stimulate immunization against COVID-19. The measures ranged from positive incentive-based programs (i.e., cash incentives, meal discounts, and lotteries) to introducing COVID-19 certificates and enforcing the universal mandatory vaccination with fines. We assessed the effect of such interventions on COVID-19 vaccine uptake in the population of eight countries within the region. An interrupted time series (ITS) analysis was performed using an autoregressive integrated moving average (ARIMA) approach to account for autocorrelation and seasonality. The results showed the immediate positive impact of vaccination incentives on vaccine uptake in most cases, with the highest impact being cash incentives for the population (1197 per million population per day). Discount incentives did not show any significant impact. The introduction of COVID-19 certificates was associated with a significant immediate or gradual increase in daily administered vaccine doses in all the countries included in the study, up to 117,617 doses gained per million per month. The effect of mandatory vaccination for all or some groups of the population varied from a continuous decrease in daily administered doses (332 per million capita per day), no significant effect, or a delayed or temporary increase (1489 per million capita per day)

405. Serum Antibody Responses Against Carbapenem-Resistant Klebsiella pneumoniae in Infected Patients

Abstract Background Capsular polysaccharide (CPS) of Carbapenem-resistant K. pneumoniae ST258 (CR-Kp) is a potential vaccine target. CPS of these isolates generally falls within 2 homology groups named clade 1 and clade 2. We and others have made antibodies (Abs) that act against clade2 CR-Kp but failed to make therapeutic Abs against clade1 CR-Kp. Previous studies had shown that studying patient’s antibody responses could help in identifying suitable candidates for developing immunotherapies. Thus, we sought to identify potential vaccine candidates by investigating the humoral response CPS in CR-Kp-infected patients. Methods 24 CR-Kp isolates and corresponding serums were collected from inpatients at Stony Brook Hospital. CPS was isolated and purified by size-exclusion column chromatography from CR-Kp strains 34 (clade 2), 36 (clade 1), and 38 (clade-Other). Anti-CPS Abs in patient’s serum were detected by enzyme-linked immunosorbent assay (ELISA) and bulk Abs from positive serum were purified using an affinity column. These Abs were tested for activity against CR-Kp by serum bactericidal and agglutination assays. Results 50% of clade2 CR-Kp-infected patients had humoral responses against CPS34. 77% of clade 1-infected patients sera cross-reacted wtih CPS34, but none of them developed Abs against CPS36. Interestingly, 90% of clade1 and 60% of clade 2-infected patients, respectively, showed Abs binding to CPS38. Thus, we selectively purified Anti-CPS Abs from two clade-Other-infected patients and observed that they were cross-reactive with all three CPS. Further, these Anti-CPS Abs agglutinated all tested CR-Kp isolates (34, 36, and 38) when compared with control human IgG (P &lt; 0.005). Additionally, these Anti-CPS Abs promoted killing of clade2 bacteria and inhibited the growth of clade1 bacteria in Ab-mediated serum bactericidal assay. These data elucidate that humoral responses developed in clade-Other CR-Kp-infected patients have therapeutic potential. Conclusion With the unavailability of effective antimicrobials for CR-Kp, approaches like developing novel anti-CPS vaccine could serve as an alternate therapy. Our data suggest that developing immunotherapies targeting CPS38 could potentially provide protection across both clade1 and clade2 bacteria in clinical settings. Disclosures All authors: No reported disclosures. </jats:sec