RESUMEN
Recent efforts in understanding the course and severity of SARS-CoV-2 infections have highlighted both potentially beneficial and detrimental effects of cross-reactive antibodies derived from memory immunity. Specifically, due to a significant degree of sequence similarity between SARS-CoV-2 and other members of the coronavirus family, memory B-cells that emerged from previous infections with endemic human coronaviruses (HCoVs) could be reactivated upon encountering the newly emerged SARS-CoV-2, thus prompting the production of cross-reactive antibodies. Determining the affinity and concentration of these potentially cross-reactive antibodies to the new SARS-CoV-2 antigens is therefore particularly important when assessing both existing immunity against common HCoVs and adverse effects like antibody-dependent enhancement (ADE) in COVID-19. However, these two fundamental parameters cannot easily be disentangled by surface-based assays like enzyme-linked immunosorbent assays (ELISAs), which are routinely used to assess cross-reactivity. Here, we have used microfluidic antibody affinity profiling (MAAP) to quantitatively evaluate the humoral immune response in COVID-19 convalescent patients by determining both antibody affinity and concentration against spike antigens of SARS-CoV-2 directly in nine convalescent COVID-19 patient and three pre-pandemic sera that were seropositive for common HCoVs. All 12 sera contained low concentrations of high-affinity antibodies against spike antigens of HCoV-NL63 and HCoV-HKU1, indicative of past exposure to these pathogens, while the affinity against the SARS-CoV-2 spike protein was lower. These results suggest that cross-reactivity as a consequence of memory reactivation upon an acute SARS-CoV-2 infection may not be a significant factor in generating immunity against SARS-CoV-2.
Asunto(s)
COVID-19 , SARS-CoV-2 , Anticuerpos Antivirales , Afinidad de Anticuerpos , Humanos , Microfluídica , Glicoproteína de la Espiga del CoronavirusRESUMEN
The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, K d, of anti-receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect-based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations.
Asunto(s)
Anticuerpos Antivirales/sangre , COVID-19/inmunología , Microfluídica/métodos , SARS-CoV-2/inmunología , Adulto , Anciano , Enzima Convertidora de Angiotensina 2/sangre , Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos , Linfocitos B/inmunología , Linfocitos B/virología , COVID-19/sangre , COVID-19/etiología , Reacciones Cruzadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Glicoproteína de la Espiga del Coronavirus/sangre , Glicoproteína de la Espiga del Coronavirus/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
The humoral immune response plays a key role in suppressing the pathogenesis of SARS-CoV-2. The molecular determinants underlying the neutralization of the virus remain, however, incompletely understood. Here, we show that the ability of antibodies to disrupt the binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell, the key molecular event initiating SARS-CoV-2 entry into host cells, is controlled by the affinity of these antibodies to the viral antigen. By using microfluidic antibody-affinity profiling, we were able to quantify the serum-antibody mediated inhibition of ACE2-spike binding in two SARS-CoV-2 seropositive individuals. Measurements to determine the affinity, concentration, and neutralization potential of antibodies were performed directly in human serum. Using this approach, we demonstrate that the level of inhibition in both samples can be quantitatively described using the dissociation constants (KD) of the binary interactions between the ACE2 receptor and the spike protein as well as the spike protein and the neutralizing antibody. These experiments represent a new type of in-solution receptor binding competition assay, which has further potential applications, ranging from decisions on donor selection for convalescent plasma therapy, to identification of lead candidates in therapeutic antibody development, and vaccine development.
Asunto(s)
COVID-19 , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2 , Afinidad de Anticuerpos , COVID-19/terapia , Humanos , Inmunización Pasiva , Peptidil-Dipeptidasa A/genética , Glicoproteína de la Espiga del Coronavirus/genética , Sueroterapia para COVID-19RESUMEN
For proper chromosome segregation in mitosis, sister kinetochores must interact with microtubules from opposite spindle poles (chromosome bi-orientation) [1, 2]. To promote bi-orientation, Aurora B kinase disrupts aberrant kinetochore-microtubule interactions [3-6]. It has long been debated how Aurora B halts this action when bi-orientation is established and tension is applied across sister kinetochores. A popular explanation for it is that, upon bi-orientation, sister kinetochores are pulled in opposite directions, stretching the outer kinetochores [7, 8] and moving Aurora B substrates away from Aurora-B-localizing sites at centromeres (spatial separation model) [3, 5, 9]. This model predicts that Aurora B localization at centromeres is required for bi-orientation. However, this notion was challenged by the observation that Bir1 (yeast survivin), which recruits Ipl1-Sli15 (yeast Aurora B-INCENP) to centromeres, can become dispensable for bi-orientation [10]. This raised the possibility that Aurora B localization at centromeres is dispensable for bi-orientation. Alternatively, there might be a Bir1-independent mechanism for recruiting Ipl1-Sli15 to centromeres or inner kinetochores [5, 9]. Here, we show that the COMA inner kinetochore sub-complex physically interacts with Sli15, recruits Ipl1-Sli15 to the inner kinetochore, and promotes chromosome bi-orientation, independently of Bir1, in budding yeast. Moreover, using an engineered recruitment of Ipl1-Sli15 to the inner kinetochore when both Bir1 and COMA are defective, we show that localization of Ipl1-Sli15 at centromeres or inner kinetochores is required for bi-orientation. Our results give important insight into how Aurora B disrupts kinetochore-microtubule interaction in a tension-dependent manner to promote chromosome bi-orientation.
Asunto(s)
Aurora Quinasas/genética , Centrómero/metabolismo , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Aurora Quinasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Chromatin remodelling is involved in the transcriptional regulation of the RNA polymerase I transcribed variant surface glycoprotein (VSG) expression sites (ESs) of Trypanosoma brucei. We show that the T. bruceiâ FACT complex contains the Pob3 and Spt16 subunits, and plays a key role in ES silencing. We see an inverse correlation between transcription and condensed chromatin, whereby FACT knockdown results in ES derepression and more open chromatin around silent ES promoters. Derepressed ESs show increased sensitivity to micrococcal nuclease (MNase) digestion, and a decrease in histones at silent ES promoters but not telomeres. In contrast, FACT knockdown results in more histones at the active ES, correlated with transcription shut-down. ES promoters are derepressed in cells stalled at the G2/M cell cycle stage after knockdown of FACT, but not in G2/M cells stalled after knockdown of cyclin 6. This argues that the observed ES derepression is a direct consequence of histone chaperone activity by FACT at the G2/M cell cycle stage which could affect transcription elongation, rather than an indirect consequence of a cell cycle checkpoint. These experiments highlight the role of the FACT complex in cell cycle-specific chromatin remodelling within VSG ESs.
Asunto(s)
Regulación de la Expresión Génica , Histonas/metabolismo , Factores de Transcripción/metabolismo , Trypanosoma brucei brucei/fisiología , Glicoproteínas Variantes de Superficie de Trypanosoma/biosíntesis , Ciclo Celular , Cromatina/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMEN
The African trypanosome Trypanosoma brucei monoallelically expresses one of more than 1000 Variant Surface Glycoprotein (VSG) genes. The active VSG is transcribed from one of about 15 telomeric VSG expression sites (ESs). It is unclear how monoallelic expression of VSG is controlled, and how inactive VSG ESs are silenced. Here, we show that blocking synthesis of the T. brucei FACT subunit TbSpt16 triggers a G2/early M phase cell cycle arrest in both bloodstream and insect form T. brucei. Segregation of T. brucei minichromosomes in these stalled cells is impaired, implicating FACT in maintenance of centromeres. Strikingly, knock-down of TbSpt16 results in 20- to 23-fold derepression of silent VSG ES promoters in bloodstream form T. brucei, with derepression specific to the G2/M cell cycle stage. In insect form T. brucei TbSpt16 knock-down results in 16- to 25-fold VSG ES derepression. Using chromatin immunoprecipitation (ChIP), TbSpt16 was found to be particularly enriched at the promoter region of silent but not active VSG ESs in bloodstream form T. brucei. The chromatin remodeler FACT is therefore implicated in maintenance of repressed chromatin present at silent VSG ES promoters, but is also essential for chromosome segregation presumably through maintenance of functional centromeres.
Asunto(s)
Ciclo Celular , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismo , Secuencia de Aminoácidos , Inmunoprecipitación de Cromatina , Replicación del ADN , ADN Protozoario/biosíntesis , Técnicas de Silenciamiento del Gen , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superficie de Trypanosoma/genéticaRESUMEN
Phylogenetic analyses based on defined proteins or different RNA species have revealed that the order kinetoplastida belongs to the early-branching eukaryotes and may thus contain organisms in which complex cellular events are easier to analyze. This view was further supported by results from a bioinformatic survey that suggested that nearly half of the autophagy-related proteins existent in yeast are missing in trypanosomatids. On the other hand, these organisms have evolved a highly sophisticated machinery to escape from the different host immune-response strategies and have learned to cope with extremely variable environmental conditions by morphological and functional reorganization of the cell. For both the stress response and the differentiation processes, autophagy seems to be an indispensable prerequisite. So far autophagy has not been systematically investigated in trypanosomatids. Here we present technical information on how to handle the different parasites belonging to this order and give an overview of the current status of autophagy research in these organisms.
Asunto(s)
Autofagia/fisiología , Bioensayo/métodos , Kinetoplastida/fisiología , Modelos Biológicos , Secuencia de Aminoácidos , Animales , Técnicas de Cultivo de Célula , Biología Computacional , Homeostasis , Humanos , Kinetoplastida/genética , Kinetoplastida/patogenicidad , Kinetoplastida/ultraestructura , Datos de Secuencia Molecular , Orgánulos/metabolismo , Orgánulos/ultraestructura , Interferencia de ARN , Alineación de SecuenciaRESUMEN
Dihydroxyacetone (DHA) was examined to explore its trypanocidal activity. The compound is easily taken up by trypanosomes via its aquaglyceroporins but is not converted to a glycolytic intermediate due to the lack of a respective kinase. Investigating the DHA-induced cell death it became evident that parasites die by autophagy rather than by necrosis or apoptosis. Since autophagy is not well studied in African trypanosomes our work offers a way to investigate the importance of autophagy for trypanosomes not only for stress coping but also for organelle reconstruction during differentiation.
Asunto(s)
Autofagia/fisiología , Dihidroxiacetona/farmacología , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , África , Animales , Autofagia/efectos de los fármacos , Muerte Celular , Relación Dosis-Respuesta a Droga , Concentración 50 Inhibidora , Cinética , Trypanosoma brucei brucei/ultraestructuraRESUMEN
We evaluated the effects of dihydroxyacetone (DHA) on Trypanosoma brucei bloodstream forms. DHA is considered an energy source for many different cell types. T. brucei takes up DHA readily due to the presence of aquaglyceroporins. However, the parasite is unable to use it as a carbon source because of the absence of DHA kinase (DHAK). We could not find a homolog of the relevant gene in the genomic database of T. brucei and have been unable to detect DHAK activity in cell lysates of the parasite, and the parasite died quickly if DHA was the sole energy source in the medium. In addition, during trypanosome cultivation, DHA induced growth inhibition with a 50% inhibitory concentration of about 1 mM, a concentration that is completely innocuous to mammals. DHA caused cell cycle arrest in the G(2)/M phase of up to 70% at a concentration of 2 mM. Also, DHA-treated parasites showed profound ultrastructural alterations, including an increase of vesicular structures within the cytosol and the presence of multivesicular bodies, myelin-like structures, and autophagy-like vacuoles, as well as a marked disorder of the characteristic mitochondrion structure. Based on the toxicity of DHA for trypanosomes compared with mammals, we consider DHA a starting point for a rational design of new trypanocidal drugs.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Dihidroxiacetona/farmacología , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/ultraestructuraRESUMEN
Trypanosoma brucei, a protozoan parasite causing sleeping sickness, is transmitted by the tsetse fly and undergoes a complex lifecycle including several defined stages within the insect vector and its mammalian host. In the latter, differentiation from the long slender to the short stumpy form is induced by a yet unknown factor of trypanosomal origin. Here we describe that some thiazolidinediones are also able to induce differentiation. In higher eukaryotes, thiazolidinediones are involved in metabolism and differentiation processes mainly by binding to the intracellular receptor peroxisome proliferator activated receptor gamma. Our studies focus on the effects of troglitazone on bloodstream form trypanosomes. Differentiation was monitored using mitochondrial markers (membrane potential, succinate dehydrogenase activity, inhibition of oxygen uptake by KCN, amount of cytochrome transcripts), morphological changes (Transmission EM and light microscopy), and transformation experiments (loss of the Variant Surface Glycoprotein coat and increase of dihydroliponamide dehydrogenase activity). To further investigate the mechanisms responsible for these changes, microarray analyses were performed, showing an upregulation of expression site associated gene 8 (ESAG8), a potential differentiation regulator.