RESUMEN
Cytotoxic T lymphocytes (CTLs) mediate host defense against viral and intracellular bacterial infections and tumors. However, the magnitude of CTL response and their function needed to confer heterosubtypic immunity against influenza virus infection are unknown. We addressed the role of CD8+ T cells in the absence of any cross-reactive antibody responses to influenza viral proteins using an adenoviral vector expressing a 9mer amino acid sequence recognized by CD8+ T cells. Our results indicate that both CD8+ T cell frequency and function are crucial for heterosubtypic immunity. Low morbidity, lower viral lung titers, low to minimal lung pathology, and better survival upon heterosubtypic virus challenge correlated with the increased frequency of NP-specific CTLs. NP-CD8+ T cells induced by differential infection doses displayed distinct RNA transcriptome profiles and functional properties. CD8+ T cells induced by a high dose of influenza virus secreted significantly higher levels of IFN-γ and exhibited higher levels of cytotoxic function. The mice that received NP-CD8+ T cells from the high-dose virus recipients through adoptive transfer had lower viral titers following viral challenge than those induced by the low dose of virus, suggesting differential cellular programming by antigen dose. Enhanced NP-CD8+ T-cell functions induced by a higher dose of influenza virus strongly correlated with the increased expression of cellular and metabolic genes, indicating a shift to a more glycolytic metabolic phenotype. These findings have implications for developing effective T cell vaccines against infectious diseases and cancer. IMPORTANCE: Cytotoxic T lymphocytes (CTLs) are an important component of the adaptive immune system that clears virus-infected cells or tumor cells. Hence, developing next-generation vaccines that induce or recall CTL responses against cancer and infectious diseases is crucial. However, it is not clear if the frequency, function, or both are essential in conferring protection, as in the case of influenza. In this study, we demonstrate that both CTL frequency and function are crucial for providing heterosubtypic immunity to influenza by utilizing an Ad-viral vector expressing a CD8 epitope only to rule out the role of antibodies, single-cell RNA-seq analysis, as well as adoptive transfer experiments. Our findings have implications for developing T cell vaccines against infectious diseases and cancer.
RESUMEN
Preventing and controlling influenza virus infection remains a global public health challenge, as it causes seasonal epidemics to unexpected pandemics. These infections are responsible for high morbidity, mortality, and substantial economic impact. Vaccines are the prophylaxis mainstay in the fight against influenza. However, vaccination fails to confer complete protection due to inadequate vaccination coverages, vaccine shortages, and mismatches with circulating strains. Antivirals represent an important prophylactic and therapeutic measure to reduce influenza-associated morbidity and mortality, particularly in high-risk populations. Here, we review current FDA-approved influenza antivirals with their mechanisms of action, and different viral- and host-directed influenza antiviral approaches, including immunomodulatory interventions in clinical development. Furthermore, we also illustrate the potential utility of machine learning in developing next-generation antivirals against influenza.
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Vacunas contra la Influenza , Gripe Humana , Infecciones por Orthomyxoviridae , Orthomyxoviridae , Humanos , Gripe Humana/tratamiento farmacológico , Gripe Humana/prevención & control , Antivirales/farmacología , Antivirales/uso terapéutico , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Vacunas contra la Influenza/uso terapéuticoRESUMEN
MOV10 is an RNA helicase required for organismal development and is highly expressed in postnatal brain. MOV10 is an AGO2-associated protein that is also necessary for AGO2-mediated silencing. AGO2 is the primary effector of the miRNA pathway. MOV10 has been shown to be ubiquitinated, leading to its degradation and release from bound mRNAs, but no other posttranslational modifications with functional implications have been described. Using mass spectrometry, we show that MOV10 is phosphorylated in cells at the C-terminus, specifically at serine 970 (S970). Substitution of S970 to phospho-mimic aspartic acid (S970D) blocked unfolding of an RNA G-quadruplex, similar to when the helicase domain was mutated (K531A). In contrast, the alanine substitution (S970A) of MOV10 unfolded the model RNA G-quadruplex. To examine its role in cells, our RNA-seq analysis showed that the expression of S970D causes decreased expression of MOV10 enhanced Cross-Linking Immunoprecipitation targets compared to WT. Introduction of S970A had an intermediate effect, suggesting that S970 was protective of mRNAs. In whole-cell extracts, MOV10 and its substitutions bound AGO2 comparably; however, knockdown of AGO2 abrogated the S970D-induced mRNA degradation. Thus, MOV10 activity protects mRNA from AGO2; phosphorylation of S970 restricts this activity resulting in AGO2-mediated mRNA degradation. S970 is positioned C-terminal to the defined MOV10-AGO2 interaction site and is proximal to a disordered region that likely modulates AGO2 interaction with target mRNAs upon phosphorylation. In summary, we provide evidence whereby MOV10 phosphorylation facilitates AGO2 association with the 3'UTR of translating mRNAs that leads to their degradation.
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MicroARNs , ARN Helicasas , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , MicroARNs/genética , Encéfalo/metabolismo , ADN Helicasas/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismoRESUMEN
Babesia duncani is the causative agent of babesiosis in the western United States. The indirect fluorescent antibody (IFA) assay is the diagnostic test of choice for detection of B. duncani-specific antibodies. However, this test requires parasitized red blood cells harvested from infected hamsters, and test results are often difficult to interpret. To simplify serological testing for B. duncani, a proteomics approach was employed to identify candidate immunodiagnostic antigens. Several proteins were identified by electrospray ionization mass spectrometric analysis, and four recombinant protein constructs were expressed and used in a multiplex bead assay (MBA) to detect B. duncani-specific antibodies. Two antigens, AAY83295.1 and AAY83296.1, performed well with high sensitivities and specificities. AAY83295.1 had a higher sensitivity (100%) but lower specificity (89%) than AAY83296.1, which had a sensitivity of 90% and a specificity of 96%. Combining these two antigens did not improve the performance of the assay. This MBA could be useful for diagnosis, serosurveillance, and blood donor screening for B. duncani infection.
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Babesia , Babesiosis , Animales , Anticuerpos Antiprotozoarios , Babesia/genética , Babesiosis/diagnóstico , Cricetinae , Eritrocitos , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunoglobulina G , Estados UnidosRESUMEN
Human genital Chlamydia infection is a major public health concern due to the serious reproductive system complications. Chlamydia binds several receptor tyrosine kinases (RTKs) on host cells, including the epidermal growth factor receptor (EGFR), and activates cellular signaling cascades for host invasion, cytoskeletal remodeling, optimal inclusion development, and induction of pathogenic epithelial-mesenchyme transition (EMT). Chlamydia also upregulates transforming growth factor beta (TGF-ß) expression, whose signaling pathway synergizes with the EGFR cascade, but its role in infectivity, inclusions, and EMT induction is unknown. We hypothesized that the EGFR and TGF-ß signaling pathways cooperate during chlamydial infection for optimal inclusion development and stable EMT induction. The results revealed that Chlamydia upregulated TGF-ß expression as early as 6 h postinfection of epithelial cells and stimulated both the EGFR and TGF-ß signaling pathways. Inhibition of either the EGFR or TGF-ßR1 signaling substantially reduced inclusion development; however, the combined inhibition of both EGFR and TGF-ßR1 signaling reduced inclusions by over 90% and prevented EMT induction. Importantly, EGFR inhibition suppressed TGF-ß expression, and an inhibitory thrombospondin-1 (Tsp1)-based peptide inhibited chlamydia-induced EMT, revealing a major source of active TGF-ß during infection. Finally, TGF-ßR signaling inhibition suppressed the expression of transforming acidic coiled-coil protein-3 (TACC3), which stabilizes EGFR signaling, suggesting reciprocal regulation between TGF-ß and EGFR signaling during chlamydial infection. Thus, RTK-mediated host invasion by chlamydia upregulated TGF-ß expression and signaling, which cooperated with other cellular signaling cascades and cytoskeletal remodeling to support optimal inclusion development and EMT induction. This finding may provide new targets for chlamydial disease biomarkers and prevention.
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Infecciones por Chlamydia/fisiopatología , Chlamydia/crecimiento & desarrollo , Células Epiteliales/microbiología , Receptores ErbB/metabolismo , Interacciones Huésped-Patógeno , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular , Endocitosis , Transición Epitelial-Mesenquimal , Cuerpos de Inclusión/microbiología , Ratones , Modelos BiológicosRESUMEN
LL-37, the only human cathelicidin that is released during inflammation, is a potent regulator of immune responses by facilitating delivery of oligonucleotides to intracellular TLR-9, thereby enhancing the response of human plasmacytoid dendritic cells (pDCs) to extracellular DNA. Although important for pathogen recognition, this mechanism may facilitate development of autoimmune diseases. In this article, we show that citrullination of LL-37 by peptidyl-arginine deiminases (PADs) hindered peptide-dependent DNA uptake and sensing by pDCs. In contrast, carbamylation of the peptide (homocitrullination of Lys residues) had no effect. The efficiency of LL-37 binding to oligonucleotides and activation of pDCs was found to be inversely proportional to the number of citrullinated residues in the peptide. Similarly, preincubation of carbamylated LL-37 with PAD2 abrogated the peptide's ability to bind DNA. Conversely, LL-37 with Arg residues substituted by homoarginine, which cannot be deiminated, elicited full activity of native LL-37 regardless of PAD2 treatment. Taken together, the data showed that citrullination abolished LL-37 ability to bind DNA and altered the immunomodulatory function of the peptide. Both activities were dependent on the proper distribution of guanidinium side chains in the native peptide sequence. Moreover, our data suggest that cathelicidin/LL-37 is citrullinated by PADs during NET formation, thus affecting the inflammatory potential of NETs. Together this may represent a novel mechanism for preventing the breakdown of immunotolerance, which is dependent on the response of APCs to self-molecules (including cell-free DNA); overactivation may facilitate development of autoimmunity.
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Péptidos Catiónicos Antimicrobianos/metabolismo , Ácidos Nucleicos Libres de Células/inmunología , Citrulinación/fisiología , ADN/inmunología , Células Dendríticas/inmunología , Tolerancia Inmunológica/inmunología , Desiminasas de la Arginina Proteica/metabolismo , Animales , Autoinmunidad/inmunología , Transporte Biológico , Línea Celular , Citrulina/metabolismo , ADN/metabolismo , Humanos , Ratones , Células RAW 264.7 , CatelicidinasRESUMEN
LL-37, the only member of the mammalian cathelicidin in humans, plays an essential role in innate immunity by killing pathogens and regulating the inflammatory response. However, at an inflammatory focus, arginine residues in LL-37 can be converted to citrulline via a reaction catalyzed by peptidyl-arginine deiminases (PAD2 and PAD4), which are expressed in neutrophils and are highly active during the formation of neutrophil extracellular traps (NETs). Citrullination impairs the bactericidal activity of LL-37 and abrogates its immunomodulatory functions. Therefore, we hypothesized that citrullination-resistant LL-37 variants would retain the functionality of the native peptide in the presence of PADs. To test this hypothesis, we synthetized LL-37 in which arginine residues were substituted by homoarginine (hArg-LL-37). Bactericidal activity of hArg-LL-37 was comparable with that of native LL-37, but neither treatment with PAD4 nor exposure to NETs affected the antibacterial and immunomodulatory activities of hArg-LL-37. Importantly, the susceptibilities of LL-37 and hArg-LL-37 to degradation by proteases did not significantly differ. Collectively, we demonstrated that citrullination-resistant hArg-LL-37 is an attractive lead compound for the generation of new agents to treat bacterial infections and other inflammatory diseases associated with enhanced PAD activity. Moreover, our results provide a proof-of-concept for synthesis of therapeutic peptides using homoarginine.
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Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Hidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/química , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Citrulinación/efectos de los fármacos , Citocinas/metabolismo , Activación Enzimática , Humanos , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Pruebas de Sensibilidad Microbiana , Arginina Deiminasa Proteína-Tipo 4/genética , Arginina Deiminasa Proteína-Tipo 4/aislamiento & purificación , Proteolisis , Células RAW 264.7 , CatelicidinasRESUMEN
The unfolded protein response (UPR) contributes to chlamydial pathogenesis, as a source of lipids and ATP during replication, and for establishing the initial anti-apoptotic state of host cell that ensures successful inclusion development. The molecular mechanism(s) of UPR induction by Chlamydia is unknown. Chlamydia use type III secretion system (T3SS) effector proteins (e.g, the Translocated Actin-Recruiting Phosphoprotein (Tarp) to stimulate host cell's cytoskeletal reorganization that facilitates invasion and inclusion development. We investigated the hypothesis that T3SS effector-mediated assembly of myosin-II complex produces activated non-muscle myosin heavy chain II (NMMHC-II), which then binds the UPR master regulator (BiP) and/or transducers to induce UPR. Our results revealed the interaction of the chlamydial effector proteins (CT228 and Tarp) with components of the myosin II complex and UPR regulator and transducer during infection. These interactions caused the activation and binding of NMMHC-II to BiP and IRE1α leading to UPR induction. In addition, specific inhibitors of myosin light chain kinase, Tarp oligomerization and myosin ATPase significantly reduced UPR activation and Chlamydia replication. Thus, Chlamydia induce UPR through T3SS effector-mediated activation of NMMHC-II components of the myosin complex to facilitate infectivity. The finding provides greater insights into chlamydial pathogenesis with the potential to identify therapeutic targets and formulations.
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Chlamydia muridarum/patogenicidad , Chlamydia trachomatis/patogenicidad , Interacciones Microbiota-Huesped/fisiología , Respuesta de Proteína Desplegada/fisiología , Animales , Infecciones por Chlamydia/etiología , Infecciones por Chlamydia/metabolismo , Infecciones por Chlamydia/microbiología , Chlamydia muridarum/metabolismo , Chlamydia trachomatis/metabolismo , Células HeLa , Humanos , Cuerpos de Inclusión/metabolismo , Ratones , Miosina Tipo II/metabolismo , Sistemas de Secreción Tipo III/metabolismoRESUMEN
Host defense peptides, also known as antimicrobial peptides, are key elements of innate host defense. One host defense peptide with well-characterized antimicrobial activity is the human cathelicidin, LL-37. LL-37 has been shown to be upregulated at sites of infection and inflammation and is regarded as one of the primary innate defense molecules against bacterial and viral infection. Human exposure to combustion-derived or engineered nanoparticles is of increasing concern, and the implications of nanomaterial exposure on the human immune response is poorly understood. However, it is widely acknowledged that nanoparticles can interact strongly with several immune proteins of biological significance, with these interactions resulting in structural and functional changes of the proteins involved. This study investigated whether the potent antibacterial and antiviral functions of LL-37 were inhibited by exposure to, and interaction with, carbon nanoparticles, together with characterizing the nature of the interaction. LL-37 was exposed to carbon black nanoparticles in vitro, and the antibacterial and antiviral functions of the peptide were subsequently assessed. We demonstrate a substantial loss of antimicrobial function when the peptide was exposed to low concentrations of nanomaterials, and we further show that the nanomaterial-peptide interaction resulted in a significant change in the structure of the peptide. The human health implications of these findings are significant, as, to our knowledge, this is the first evidence that nanoparticles can alter host defense peptide structure and function, indicating a new role for nanoparticle exposure in increased disease susceptibility.
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Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Carbono , Nanopartículas/química , Nanopartículas/toxicidad , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Antivirales/farmacología , Bacterias/efectos de los fármacos , Humanos , Inflamación , Rhinovirus/efectos de los fármacos , CatelicidinasRESUMEN
Bacterial nucleoid-associated proteins (NAPs) are critical to genome integrity and chromosome maintenance. Post-translational modifications of bacterial NAPs appear to function similarly to their better studied mammalian counterparts. The histone-like NAP HupB from Mycobacterium tuberculosis (Mtb) was previously observed to be acetylated by the acetyltransferase Eis, leading to genome reorganization. We report biochemical and structural aspects of acetylation of HupB by Eis. We also found that the SirT-family NAD+-dependent deacetylase Rv1151c from Mtb deacetylated HupB in vitro and characterized the deacetylation kinetics. We propose that activities of Eis and Rv1151c could regulate the acetylation status of HupB to remodel the mycobacterial chromosome in response to environmental changes.
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Acetiltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Mycobacterium tuberculosis/metabolismo , Acetilación , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Clonación Molecular , Cristalografía por Rayos X , Farmacorresistencia Bacteriana Múltiple/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/fisiología , Histona Desacetilasas/genética , Histonas/genética , Cinética , Lisina/química , Modelos Moleculares , Mycobacterium tuberculosis/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espectrometría de Masas en TándemRESUMEN
The reproductive system complications of genital chlamydial infection include fallopian tube fibrosis and tubal factor infertility. However, the molecular pathogenesis of these complications remains poorly understood. The induction of pathogenic epithelial-mesenchymal transition (EMT) through microRNA (miRNA) dysregulation was recently proposed as the pathogenic basis of chlamydial complications. Focusing on fibrogenesis, we investigated the hypothesis that chlamydia-induced fibrosis is caused by EMT-driven generation of myofibroblasts, the effector cells of fibrosis that produce excessive extracellular matrix (ECM) proteins. The results revealed that the targets of a major category of altered miRNAs during chlamydial infection are key components of the pathophysiological process of fibrogenesis; these target molecules include collagen types I, III, and IV, transforming growth factor ß (TGF-ß), TGF-ß receptor 1 (TGF-ßR1), connective tissue growth factor (CTGF), E-cadherin, SRY-box 7 (SOX7), and NFAT (nuclear factor of activated T cells) kinase dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1a (Dyrk1a). Chlamydial induction of EMT resulted in the generation of α-smooth muscle actin (α-SMA)-positive myofibroblasts that produced ECM proteins, including collagen types I and III and fibronectin. Furthermore, the inhibition of EMT prevented the generation of myofibroblasts and production of ECM proteins during chlamydial infection. These findings may provide useful avenues for targeting EMT or specific components of the EMT pathways as a therapeutic intervention strategy to prevent chlamydia-related complications.
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Infecciones por Chlamydia/complicaciones , Infecciones por Chlamydia/patología , Chlamydia/patogenicidad , Transición Epitelial-Mesenquimal/fisiología , Fibrosis/etiología , Fibrosis/patología , Actinas/metabolismo , Animales , Cadherinas/metabolismo , Línea Celular , Infecciones por Chlamydia/microbiología , Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Fibrosis/microbiología , Ratones , MicroARNs/metabolismo , Miofibroblastos/microbiología , Miofibroblastos/patología , Factores de Transcripción NFATC/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factores de Transcripción SOXF/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Chlamydia is an obligate intracellular bacterium that relies on host cells for essential nutrients and adenosine triphosphate (ATP) for a productive infection. Although the unfolded protein response (UPR) plays a major role in certain microbial infectivity, its role in chlamydial pathogenesis is unknown. We hypothesized that Chlamydia induces UPR and exploits it to upregulate host cell uptake and metabolism of glucose, production of ATP, phospholipids, and other molecules required for its replicative development and host survival. Using a combination of biochemical and pathway inhibition assays, we showed that the 3 UPR pathway transducers-protein kinase RNA-activated (PKR)-like ER kinase (PERK), inositol-requiring enzyme-1α (IRE1α), and activating transcription factor-6α (ATF6α)-were activated during Chlamydia infection. The kinase activity of PERK and ribonuclease (RNase) of IRE1α mediated the upregulation of hexokinase II and production of ATP via substrate-level phosphorylation. In addition, the activation of PERK and IRE1α promoted autophagy formation and apoptosis resistance for host survival. Moreover, the activation of IRE1α resulted in the generation of spliced X-box binding protein 1 (sXBP1) and upregulation of lipid production. The vital role of UPR pathways in Chlamydia development and pathogenesis could lead to the identification of potential molecular targets for therapeutics against Chlamydia.
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Infecciones por Chlamydia/microbiología , Chlamydia/patogenicidad , Respuesta de Proteína Desplegada , Factor de Transcripción Activador 6/metabolismo , Animales , Apoptosis , Supervivencia Celular , Infecciones por Chlamydia/metabolismo , Endorribonucleasas/metabolismo , Activación Enzimática , Femenino , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , eIF-2 Quinasa/metabolismoRESUMEN
Cathelicidin LL-37 plays an essential role in innate immunity by killing invading microorganisms and regulating the inflammatory response. These activities depend on the cationic character of the peptide, which is conferred by arginine and lysine residues. At inflammatory foci in vivo, LL-37 is exposed to peptidyl arginine deiminase (PAD), an enzyme released by inflammatory cells. Therefore, we hypothesized that PAD-mediated citrullination of the arginine residues within LL-37 will abrogate its immunomodulatory functions. We found that, when citrullinated, LL-37 was at least 40 times less efficient at neutralizing the proinflammatory activity of LPS due to a marked decrease in its affinity for endotoxin. Also, the ability of citrullinated LL-37 to quench macrophage responses to lipoteichoic acid and poly(I:C) signaling via TLR2 and TLR3, respectively, was significantly reduced. Furthermore, in stark contrast to native LL-37, the modified peptide completely lost the ability to prevent morbidity and mortality in a mouse model of d-galactosamine-sensitized endotoxin shock. In fact, administration of citrullinated LL-37 plus endotoxin actually exacerbated sepsis due to the inability of LL-37 to neutralize LPS and the subsequent enhancement of systemic inflammation due to increased serum levels of IL-6. Importantly, serum from septic mice showed increased PAD activity, which strongly correlated with the level of citrullination, indicating that PAD-driven protein modification occurs in vivo. Because LL-37 is a potential treatment for sepsis, its administration should be preceded by a careful analysis to ensure that the citrullinated peptide is not generated in treated patients.
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Péptidos Catiónicos Antimicrobianos/inmunología , Citrulina/inmunología , Inmunidad Innata , Macrófagos/inmunología , Sepsis/inmunología , Sepsis/prevención & control , Animales , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Línea Celular , Citrulina/genética , Femenino , Humanos , Hidrolasas/genética , Hidrolasas/inmunología , Inductores de Interferón/farmacología , Interleucina-6/genética , Interleucina-6/inmunología , Lipopolisacáridos/inmunología , Macrófagos/patología , Masculino , Ratones , Poli I-C/farmacología , Sepsis/genética , Sepsis/patología , Ácidos Teicoicos/inmunología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , CatelicidinasRESUMEN
Transglutaminases are best known for their ability to catalyze protein cross-linking reactions that impart chemical and physical resilience to cellular structures. Here, we report the crystal structure and characterization of Tgl, a transglutaminase from the bacterium Bacillus subtilis. Tgl is produced during sporulation and cross-links the surface of the highly resilient spore. Tgl-like proteins are found only in spore-forming bacteria of the Bacillus and Clostridia classes, indicating an ancient origin. Tgl is a single-domain protein, produced in active form, and the smallest transglutaminase characterized to date. We show that Tgl is structurally similar to bacterial cell wall endopeptidases and has an NlpC/P60 catalytic core, thought to represent the ancestral unit of the cysteine protease fold. We show that Tgl functions through a unique partially redundant catalytic dyad formed by Cys116 and Glu187 or Glu115. Strikingly, the catalytic Cys is insulated within a hydrophobic tunnel that traverses the molecule from side to side. The lack of similarity of Tgl to other transglutaminases together with its small size suggests that an NlpC/P60 catalytic core and insulation of the active site during catalysis may be essential requirements for protein cross-linking.
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Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Transglutaminasas/química , Proteínas Bacterianas/genética , Dominio Catalítico , Reactivos de Enlaces Cruzados/química , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Mutación Puntual , Conformación Proteica , Pliegue de Proteína , Transglutaminasas/genéticaRESUMEN
Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) share virologic and epidemiologic features and cause clinically similar respiratory illness predominantly in young children. In a previous study of acute febrile respiratory illness in Bangladesh, we tested paired serum specimens from 852 children presenting fever and cough for diagnostic increases in titers of antibody to hRSV and hMPV by enzyme immunoassay (EIA). Unexpectedly, of 93 serum pairs that showed a ≥ 4-fold increase in titers of antibody to hRSV, 24 (25.8%) showed a concurrent increase in titers of antibody to hMPV; of 91 pairs showing an increase to hMPV, 13 (14.3%) showed a concurrent increase to hRSV. We speculated that common antigens shared by these viruses explain this finding. Since the nucleocapsid (N) proteins of these viruses show the greatest sequence homology, we tested hyperimmune antisera prepared for each virus against baculovirus-expressed recombinant N (recN) proteins for potential cross-reactivity. The antisera were reciprocally reactive with both proteins. To localize common antigenic regions, we first expressed the carboxy domain of the hMPV N protein that was the most highly conserved region within the hRSV N protein. Although reciprocally reactive with antisera by Western blotting, this truncated protein did not react with hMPV IgG-positive human sera by EIA. Using 5 synthetic peptides that spanned the amino-terminal portion of the hMPV N protein, we identified a single peptide that was cross-reactive with human sera positive for either virus. Antiserum prepared for this peptide was reactive with recN proteins of both viruses, indicating that a common immunoreactive site exists in this region.
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Anticuerpos Antivirales/sangre , Reacciones Cruzadas , Metapneumovirus/inmunología , Proteínas de la Nucleocápside/inmunología , Virus Sincitiales Respiratorios/inmunología , Bangladesh , Western Blotting , Preescolar , Femenino , Humanos , Técnicas para Inmunoenzimas , Lactante , Masculino , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/virologíaRESUMEN
Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.
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Adhesinas Bacterianas/inmunología , Antígenos Bacterianos/inmunología , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Femenino , Glicosilación , Humanos , Masculino , Manosa/genética , Manosa/inmunología , Ratones , Ratones Endogámicos BALB C , Mycobacterium bovis/genética , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Linfocitos T/metabolismo , Tuberculosis/genética , Tuberculosis/inmunología , Tuberculosis/metabolismo , Vacunas contra la Tuberculosis/genéticaRESUMEN
Chikungunya virus (CHIKV) and the closely related onyong-nyong virus (ONNV) are arthritogenic arboviruses that have caused significant, often debilitating, disease in millions of people. However, despite their kinship, they are vectored by different mosquito subfamilies that diverged 180 million years ago (anopheline versus culicine subfamilies). Previous work indicated that the nonstructural protein 3 (nsP3) of these alphaviruses was partially responsible for this vector specificity. To better understand the cellular components controlling alphavirus vector specificity, a cell culture model system of the anopheline restriction of CHIKV was developed along with a protein expression strategy. Mosquito proteins that differentially interacted with CHIKV nsP3 or ONNV nsP3 were identified. Six proteins were identified that specifically bound ONNV nsP3, ten that bound CHIKV nsP3 and eight that interacted with both. In addition to identifying novel factors that may play a role in virus/vector processing, these lists included host proteins that have been previously implicated as contributing to alphavirus replication.
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Alphavirus , Fiebre Chikungunya , Virus Chikungunya , Culicidae , Humanos , Animales , Culicidae/metabolismo , Mosquitos Vectores , Virus Chikungunya/metabolismo , Alphavirus/genética , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
Monkeypox virus (MPXV), a member of the Orthopoxvirus (OPXV) genus, is a zoonotic virus, endemic to central and western Africa that can cause smallpox-like symptoms in humans with fatal outcomes in up to 15% of patients. The incidence of MPXV infections in the Democratic Republic of the Congo, where the majority of cases have occurred historically, has been estimated to have increased as much as 20-fold since the end of smallpox vaccination in 1980. Considering the risk global travel carries for future disease outbreaks, accurate epidemiological surveillance of MPXV is warranted as demonstrated by the recent Mpox outbreak, where the majority of cases were occurring in non-endemic areas. Serological differentiation between childhood vaccination and recent infection with MPXV or other OPXVs is difficult due to the high level of conservation within OPXV proteins. Here, a peptide-based serological assay was developed to specifically detect exposure to MPXV. A comparative analysis of immunogenic proteins across human OPXVs identified a large subset of proteins that could potentially be specifically recognized in response to a MPXV infection. Peptides were chosen based upon MPXV sequence specificity and predicted immunogenicity. Peptides individually and combined were screened in an ELISA against serum from well-characterized Mpox outbreaks, vaccinee sera, and smallpox sera collected prior to eradication. One peptide combination was successful with ~86% sensitivity and ~90% specificity. The performance of the assay was assessed against the OPXV IgG ELISA in the context of a serosurvey by retrospectively screening a set of serum specimens from the region in Ghana believed to have harbored the MPXV-infected rodents involved in the 2003 United States outbreak.
RESUMEN
A pneumococcal serotyping/genotyping system (PSGS) was developed based upon targeted PCR, followed by electrospray ionization mass spectrometry and amplicon base composition analysis. Eight multiplex PCRs, 32 targeting serotype-determining capsular biosynthetic loci, and 8 targeting multilocus sequence typing (MLST) loci were employed for each of 229 highly diverse Streptococcus pneumoniae isolates. The most powerful aspect of the PSGS system was the identification of capsular serotypes accounting for the majority of invasive and carried pneumococcal strains. Altogether, 45 different serotypes or serogroups were correctly predicted among the 196 resolvable isolates, with only 2 unexpected negative results. All 33 isolates that represented 23 serotypes not included in the PSGS yielded negative serotyping results. A genotyping database was constructed using the base compositions of 65- to 100-bp sections of MLST alleles compiled within http://www.mlst.net. From this database, one or more MLST sequence types (STs) that comprised a PSGS genotype were identified. The end result of more PSGS genotypes (163) than conventional STs actually tested (155) was primarily due to amplification failures of 1 to 3 targets. In many instances, the PSGS genotype could provide resolution of single- and double-locus variants. This molecular serotyping/genotyping scheme is well suited to rapid characterization of large sets of pneumococcal isolates.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Reacción en Cadena de la Polimerasa/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/genética , ADN Bacteriano/genética , Genotipo , HumanosRESUMEN
Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins-18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue "demidefensin" precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclin genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection.