RESUMO
Maternal immunity impacts the infant, but how is unclear. To understand the implications of the immune exposures of vaccination and infection in pregnancy for neonatal immunity, we evaluated antibody functions in paired peripheral maternal and cord blood. We compared those who in pregnancy received mRNA coronavirus disease 2019 (COVID-19) vaccine, were infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the combination. We found that vaccination enriched a subset of neutralizing activities and Fc effector functions that was driven by IgG1 and was minimally impacted by antibody glycosylation in maternal blood. In paired cord blood, maternal vaccination also enhanced IgG1. However, Fc effector functions compared to neutralizing activities were preferentially transferred. Moreover, changes in IgG posttranslational glycosylation contributed more to cord than peripheral maternal blood antibody functional potency. These differences were enhanced with the combination of vaccination and infection as compared to either alone. Thus, Fc effector functions and antibody glycosylation highlight underexplored maternal opportunities to safeguard newborns.
Assuntos
COVID-19 , Recém-Nascido , Lactente , Feminino , Gravidez , Humanos , COVID-19/prevenção & controle , SARS-CoV-2 , Imunoglobulina G , Vacinas contra COVID-19 , Vacinação , Anticorpos AntiviraisRESUMO
Because novel SARS-CoV-2 variants continue to emerge, immunogenicity of XBB.1.5 monovalent vaccines against live clinical isolates needs to be evaluated. We report boosting of IgG (2.1×), IgA (1.5×), and total IgG/A/M (1.7×) targeting the spike receptor-binding domain and neutralizing titers against WA1 (2.2×), XBB.1.5 (7.4×), EG.5.1 (10.5×), and JN.1 (4.7×) variants.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , Imunidade Humoral , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , COVID-19/imunologia , SARS-CoV-2/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Glicoproteína da Espícula de Coronavírus/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Feminino , Imunogenicidade da Vacina , AdultoRESUMO
The unprecedented severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has called for substantial investigations into the capacity of the human immune system to protect against reinfection and keep pace with the evolution of SARS-CoV-2. We evaluated the magnitude and durability of the SARS-CoV-2-specific antibody responses against parental WA-1 SARS-CoV-2 receptor-binding domain (RBD) and a representative variant of concern (VoC) RBD using antibodies from 2 antibody compartments: long-lived plasma cell-derived plasma antibodies and antibodies encoded by SARS-CoV-2-specific memory B cells (MBCs). Thirty-five participants naturally infected with SARS-CoV-2 were evaluated; although only 25 of 35 participants had VoC RBD-reactive plasma antibodies, 34 of 35 (97%) participants had VoC RBD-reactive MBC-derived antibodies. Our finding that 97% of previously infected individuals have MBCs specific for variant RBDs provides reason for optimism regarding the capacity of vaccination, prior infection, and/or both, to elicit immunity with the capacity to limit disease severity and transmission of VoCs as they arise and circulate.
Assuntos
COVID-19 , Células B de Memória , SARS-CoV-2/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Humanos , Índice de Gravidade de Doença , Glicoproteína da Espícula de CoronavírusRESUMO
The lipid composition of HIV-1 virions is enriched in sphingomyelin (SM), but the roles that SM or other sphingolipids (SLs) might play in the HIV-1 replication pathway have not been elucidated. In human cells, SL levels are regulated by ceramide synthase (CerS) enzymes that produce ceramides, which can be converted to SMs, hexosylceramides, and other SLs. In many cell types, CerS2, which catalyzes the synthesis of very long chain ceramides, is the major CerS. We have examined how CerS2 deficiency affects the assembly and infectivity of HIV-1. As expected, we observed that very long chain ceramide, hexosylceramide, and SM were reduced in CerS2 knockout cells. CerS2 deficiency did not affect HIV-1 assembly or the incorporation of the HIV-1 envelope (Env) protein into virus particles, but it reduced the infectivites of viruses produced in the CerS2-deficient cells. The reduced viral infection levels were dependent on HIV-1 Env, since HIV-1 particles that were pseudotyped with the vesicular stomatitis virus glycoprotein did not exhibit reductions in infectivity. Moreover, cell-cell fusion assays demonstrated that the functional defect of HIV-1 Env in CerS2-deficient cells was independent of other viral proteins. Overall, our results indicate that the altered lipid composition of CerS2-deficient cells specifically inhibit the HIV-1 Env receptor binding and/or fusion processes.
Assuntos
Deleção de Genes , Infecções por HIV/genética , HIV-1/fisiologia , Proteínas de Membrana/genética , Esfingosina N-Aciltransferase/genética , Proteínas Supressoras de Tumor/genética , Ceramidas/genética , Ceramidas/metabolismo , Células HEK293 , Infecções por HIV/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Internalização do VírusRESUMO
In this perspective article, we describe the current status of lipid tools for studying host lipid-virus interactions at the cellular level. We discuss the potential lipidomic changes that viral infections impose on host cells and then outline the tools available and the resulting options to investigate the host cell lipid interactome. The future outcome will reveal new targets for treating virus infections.
Assuntos
Viroses , Interações Hospedeiro-Patógeno , Humanos , Lipídeos , Viroses/tratamento farmacológicoRESUMO
BACKGROUND: COVID-19 pandemic has a devastating impact on the economies and health care system of sub-Saharan Africa. Healthcare workers (HWs), the main actors of the health system, are at higher risk because of their occupation. Serology-based estimates of SARS-CoV-2 infection among HWs represent a measure of HWs' exposure to the virus and could be used as a guide to the prevalence of SARS-CoV-2 in the community and valuable in combating COVID-19. This information is currently lacking in Ethiopia and other African countries. This study aimed to develop an in-house antibody testing assay, assess the prevalence of SARS-CoV-2 antibodies among Ethiopian high-risk frontline HWs. METHODS: We developed and validated an in-house Enzyme-Linked Immunosorbent Assay (ELISA) for specific detection of anti-SARS-CoV-2 receptor binding domain immunoglobin G (IgG) antibodies. We then used this assay to assess the seroprevalence among HWs in five public hospitals located in different geographic regions of Ethiopia. From consenting HWs, blood samples were collected between December 2020 and February 2021, the period between the two peaks of COVID-19 in Ethiopia. Socio-demographic and clinical data were collected using questionnaire-based interviews. Descriptive statistics and bivariate and multivariate logistic regression were used to determine the overall and post-stratified seroprevalence and the association between seropositivity and potential risk factors. RESULTS: Our successfully developed in-house assay sensitivity was 100% in serum samples collected 2- weeks after the first onset of symptoms whereas its specificity in pre-COVID-19 pandemic sera was 97.7%. Using this assay, we analyzed a total of 1997 sera collected from HWs. Of 1997 HWs who provided a blood sample, and demographic and clinical data, 51.7% were females, 74.0% had no symptoms compatible with COVID-19, and 29.0% had a history of contact with suspected or confirmed patients with SARS-CoV-2 infection. The overall seroprevalence was 39.6%. The lowest (24.5%) and the highest (48.0%) seroprevalence rates were found in Hiwot Fana Specialized Hospital in Harar and ALERT Hospital in Addis Ababa, respectively. Of the 821 seropositive HWs, 224(27.3%) of them had a history of symptoms consistent with COVID-19 while 436 (> 53%) of them had no contact with COVID-19 cases as well as no history of COVID-19 like symptoms. A history of close contact with suspected/confirmed COVID-19 cases is associated with seropositivity (Adjusted Odds Ratio (AOR) = 1.4, 95% CI 1.1-1.8; p = 0.015). CONCLUSION: High SARS-CoV-2 seroprevalence levels were observed in the five Ethiopian hospitals. These findings highlight the significant burden of asymptomatic infection in Ethiopia and may reflect the scale of transmission in the general population.
Assuntos
COVID-19 , Pandemias , COVID-19/diagnóstico , COVID-19/epidemiologia , Etiópia/epidemiologia , Feminino , Pessoal de Saúde , Humanos , SARS-CoV-2 , Estudos SoroepidemiológicosRESUMO
As a complement to vaccines, small-molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, which cause COVID-19. Affinity selection-mass spectrometry was used for the discovery of botanical ligands to the SARS-CoV-2 spike protein. Cannabinoid acids from hemp (Cannabis sativa) were found to be allosteric as well as orthosteric ligands with micromolar affinity for the spike protein. In follow-up virus neutralization assays, cannabigerolic acid and cannabidiolic acid prevented infection of human epithelial cells by a pseudovirus expressing the SARS-CoV-2 spike protein and prevented entry of live SARS-CoV-2 into cells. Importantly, cannabigerolic acid and cannabidiolic acid were equally effective against the SARS-CoV-2 alpha variant B.1.1.7 and the beta variant B.1.351. Orally bioavailable and with a long history of safe human use, these cannabinoids, isolated or in hemp extracts, have the potential to prevent as well as treat infection by SARS-CoV-2.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Canabinoides/farmacologia , SARS-CoV-2/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Antivirais/química , Antivirais/metabolismo , Benzoatos/farmacologia , COVID-19/prevenção & controle , Canabinoides/química , Canabinoides/metabolismo , Chlorocebus aethiops , Humanos , Ligantes , Espectrometria de Massas , Modelos Moleculares , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo , Células VeroRESUMO
Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12(-/-) Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12(-/-) cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades.
Assuntos
Membrana Celular/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Ubiquitina Tiolesterase/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Núcleo Celular/metabolismo , Separação Celular , Citosol/metabolismo , Endopeptidases/metabolismo , Ácidos Graxos Insaturados/farmacologia , Humanos , Células Jurkat , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Transporte Proteico , Reprodutibilidade dos Testes , Especificidade por Substrato/efeitos dos fármacos , Linfócitos T/metabolismo , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/deficiência , Proteases Específicas de Ubiquitina/metabolismoRESUMO
SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS)1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog, ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with Glu permitting SMS-catalyzed CPE production and Asp confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.
Assuntos
Domínio Catalítico , Mutagênese Sítio-Dirigida , Esfingolipídeos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Domínios Proteicos , Especificidade por Substrato , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.
Assuntos
Candida albicans , Candidíase/imunologia , Interações Hospedeiro-Patógeno/imunologia , Fagocitose/fisiologia , Esfingolipídeos/biossíntese , Animais , Candida albicans/imunologia , Linhagem Celular , Cromatografia em Camada Fina , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Modelos Animais de Doenças , Citometria de Fluxo , Técnicas de Inativação de Genes , Humanos , Espectrometria de Massas , CamundongosAssuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , COVID-19/diagnóstico , Teste Sorológico para COVID-19 , Humanos , Isotipos de Imunoglobulinas/sangue , Testes de Neutralização , Reação em Cadeia da PolimeraseRESUMO
SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS) 1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, sphingomyelin synthase-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmatic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with glutamic acid permitting SMS-catalyzed CPE production and aspartic acid confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.
Assuntos
Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Engenharia de Proteínas , Esfingomielinas/biossíntese , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Sistema Livre de Células , Química Click , Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Células HeLa , Humanos , Proteínas de Membrana/química , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Esfingomielinas/genética , Transferases (Outros Grupos de Fosfato Substituídos)/químicaRESUMO
Cells synthesize ceramides in the endoplasmic reticulum (ER) as precursors for sphingolipids to form an impermeable plasma membrane. As ceramides are engaged in apoptotic pathways, cells would need to monitor their levels closely to avoid killing themselves during sphingolipid biosynthesis. How this is accomplished remains to be established. Here we identify SMSr (SAMD8), an ER-resident ceramide phosphoethanolamine (CPE) synthase, as a suppressor of ceramide-mediated cell death. Disruption of SMSr catalytic activity causes a rise in ER ceramides and their mislocalization to mitochondria, triggering a mitochondrial pathway of apoptosis. Blocking de novo ceramide synthesis, stimulating ceramide export from the ER or targeting a bacterial ceramidase to mitochondria rescues SMSr-deficient cells from apoptosis. We also show that SMSr-catalyzed CPE production, although essential, is not sufficient to suppress ceramide-induced cell death and that SMSr-mediated ceramide homeostasis requires the N-terminal sterile α-motif, or SAM domain, of the enzyme. These results define ER ceramides as bona fide transducers of mitochondrial apoptosis and indicate a primary role of SMSr in monitoring ER ceramide levels to prevent inappropriate cell death during sphingolipid biosynthesis.
Assuntos
Apoptose , Ceramidas/metabolismo , Mitocôndrias/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Biocatálise , Ceramidases/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Marcação de Genes , Células HeLa , Humanos , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Esfingomielinas/metabolismoRESUMO
Cells genetically deficient in sphingomyelin synthase-1 (SGMS1) or blocked in their synthesis pharmacologically through exposure to a serine palmitoyltransferase inhibitor (myriocin) show strongly reduced surface display of influenza virus glycoproteins hemagglutinin (HA) and neuraminidase (NA). The transport of HA to the cell surface was assessed by accessibility of HA on intact cells to exogenously added trypsin and to HA-specific antibodies. Rates of de novo synthesis of viral proteins in wild-type and SGMS1-deficient cells were equivalent, and HA negotiated the intracellular trafficking pathway through the Golgi normally. We engineered a strain of influenza virus to allow site-specific labeling of HA and NA using sortase. Accessibility of both HA and NA to sortase was blocked in SGMS1-deficient cells and in cells exposed to myriocin, with a corresponding inhibition of the release of virus particles from infected cells. Generation of influenza virus particles thus critically relies on a functional sphingomyelin biosynthetic pathway, required to drive influenza viral glycoproteins into lipid domains of a composition compatible with virus budding and release.
Assuntos
Vias Biossintéticas/fisiologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Esfingomielinas/biossíntese , Animais , Transporte Biológico/fisiologia , Cães , Ácidos Graxos Monoinsaturados/farmacologia , Imunofluorescência , Glicoproteínas de Hemaglutininação de Vírus da Influenza/fisiologia , Interações Hospedeiro-Patógeno , Células Madin Darby de Rim Canino , Polietilenoglicóis , Serina C-Palmitoiltransferase/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , TripsinaRESUMO
A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport.
Assuntos
ADP Ribose Transferases/toxicidade , Toxinas Bacterianas/toxicidade , Exotoxinas/toxicidade , Furina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Virulência/toxicidade , Rede trans-Golgi/metabolismo , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Sequência de Bases , Primers do DNA , Endocitose , Exotoxinas/genética , Mutação , Reação em Cadeia da Polimerase , Transporte Proteico , Proteólise , Receptores Acoplados a Proteínas G/fisiologia , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosaAssuntos
Flavivirus/fisiologia , Flavivirus/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Metabolismo dos Lipídeos , Animais , Colesterol/biossíntese , Retículo Endoplasmático/virologia , Ácidos Graxos/biossíntese , Flaviviridae/genética , Flaviviridae/patogenicidade , Flaviviridae/fisiologia , Flavivirus/genética , Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , Interações Hospedeiro-Patógeno/genética , Humanos , Modelos Biológicos , Tropismo Viral , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Proteínas de Homeodomínio/imunologia , Lectinas Tipo C/imunologia , Macrófagos Peritoneais/imunologia , Neuropeptídeos/imunologia , Fagocitose/imunologia , Proteínas Tirosina Quinases/imunologia , Actinas/genética , Actinas/imunologia , Actinas/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Candida albicans/metabolismo , Candidíase/genética , Candidíase/metabolismo , Candidíase/patologia , Linhagem Celular , Diglicerídeos/genética , Diglicerídeos/imunologia , Diglicerídeos/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fagocitose/genética , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/imunologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismoRESUMO
Functions and cell biology of the sphingolipids sphingosine and sphinganine in cells are not well understood. While some signaling roles for sphingosine have been elucidated, the closely related sphinganine has been described only insofar as it does not elicit many of the same signaling responses. Here, we prepared multifunctionalized derivatives of the two lipid species that differ only in a single double bond of the carbon backbone. Using these novel probes, we were able to define their spatiotemporal distributions within cells. Furthermore, we used these tools to systematically map the protein interactomes of both lipids. The lipid-protein conjugates, prepared through photo-crosslinking in live cells and extraction via click chemistry to azide beads, revealed significant differences in the captured proteins, highlighting their distinct roles in various cellular processes. This work elucidates mechanistic differences between these critical lipids and sets the foundation for further studies of the cellular functions of sphingosine and sphinganine.
Assuntos
Esfingolipídeos , Esfingosina , Esfingosina/análogos & derivados , Esfingolipídeos/metabolismo , Esfingosina/metabolismoRESUMO
As novel SARS-CoV-2 variants continue to emerge, the updated XBB.1.5 monovalent vaccines remain to be evaluated in terms of immunogenicity against live clinical isolates. We report boosting of IgG(2.1X), IgA(1.5X), and total IgG/A/M(1.7X) antibodies targeting the spike receptor-binding domain and neutralizing titers against WA1(2.2X), XBB.1.5(7.4X), EG.5.1(10.5X), and JN.1(4.7X) variants.