RESUMEN
Human coronaviruses (HCoV) are respiratory pathogens with neuroinvasive, neurotropic, and neurovirulent properties, highlighting the importance of studying the potential implication of these viruses in neurological diseases. The OC43 strain (HCoV-OC43) was reported to induce neuronal cell death, which may participate in neuropathogenesis. Here, we show that HCoV-OC43 harboring two point mutations in the spike glycoprotein (rOC/Us183-241) was more neurovirulent than the wild-type HCoV-OC43 (rOC/ATCC) in mice and induced more cell death in murine and human neuronal cells. To evaluate the role of regulated cell death (RCD) in HCoV-OC43-mediated neural pathogenesis, we determined if knockdown of Bax, a key regulator of apoptosis, or RIP1, a key regulator of necroptosis, altered the percentage of neuronal cell death following HCoV-OC43 infection. We found that Bax-dependent apoptosis did not play a significant role in RCD following infection, as inhibition of Bax expression mediated by RNA interference did not confer cellular protection against the cell death process. On the other hand, we demonstrated that RIP1 and MLKL were involved in neuronal cell death, as RIP1 knockdown and chemical inhibition of MLKL significantly increased cell survival after infection. Taken together, these results indicate that RIP1 and MLKL contribute to necroptotic cell death after HCoV-OC43 infection to limit viral replication. However, this RCD could lead to neuronal loss in the mouse CNS and accentuate the neuroinflammation process, reflecting the severity of neuropathogenesis. IMPORTANCE: Because they are naturally neuroinvasive and neurotropic, human coronaviruses are suspected to participate in the development of neurological diseases. Given that the strain OC43 is neurovirulent in mice and induces neuronal cell death, we explored the neuronal response to infection by characterizing the activation of RCD. Our results revealed that classical apoptosis associated with the Bax protein does not play a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins usually associated with necroptosis (an RCD back-up system when apoptosis is not adequately induced), both play a pivotal role in the process. As necroptosis disrupts cellular membranes and allows the release of damage-associated molecular patterns (DAMP) and possibly induces the production of proinflammatory cytokines, it may represent a proinflammatory cell death mechanism that contributes to excessive neuroinflammation and neurodegeneration and eventually to neurological disorders after a coronavirus infection.
Asunto(s)
Infecciones por Coronavirus/genética , Coronavirus Humano OC43/patogenicidad , Proteínas Activadoras de GTPasa/genética , Interacciones Huésped-Patógeno , Proteínas Quinasas/genética , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Muerte Celular , Línea Celular , Línea Celular Tumoral , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/mortalidad , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Embrión de Mamíferos , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Mutación , Neuronas/patología , Neuronas/virología , Cultivo Primario de Células , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Glicoproteína de la Espiga del Coronavirus/metabolismo , Análisis de Supervivencia , Virulencia , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Human coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S) glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43). In an attempt to study the role of this protein in virus spread within the central nervous system (CNS) and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G758 to RRSR↓R758), which introduces a putative furin-like cleavage (↓) site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies.
Asunto(s)
Enfermedades del Sistema Nervioso Central/virología , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/patogenicidad , Proproteína Convertasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Coronavirus Humano OC43/aislamiento & purificación , Coronavirus Humano OC43/fisiología , Glicoproteínas/metabolismo , Humanos , Ratones , VirulenciaRESUMEN
In humans, viral infections of the respiratory tract are a leading cause of morbidity and mortality worldwide. Several recognized respiratory viral agents have a neuroinvasive capacity since they can spread from the respiratory tract to the central nervous system (CNS). Once there, infection of CNS cells (neurotropism) could lead to human health problems, such as encephalitis and long-term neurological diseases. Among the various respiratory viruses, coronaviruses are important pathogens of humans and animals. Human Coronaviruses (HCoV) usually infect the upper respiratory tract, where they are mainly associated with common colds. However, in more vulnerable populations, such as newborns, infants, the elderly, and immune-compromised individuals, they can also affect the lower respiratory tract, leading to pneumonia, exacerbations of asthma, respiratory distress syndrome, or even severe acute respiratory syndrome (SARS). The respiratory involvement of HCoV has been clearly established since the 1960s. In addition, for almost three decades now, the scientific literature has also demonstrated that HCoV are neuroinvasive and neurotropic and could induce an overactivation of the immune system, in part by participating in the activation of autoreactive immune cells that could be associated with autoimmunity in susceptible individuals. Furthermore, it was shown that in the murine CNS, neurons are the main target of infection, which causes these essential cells to undergo degeneration and eventually die by some form of programmed cell death after virus infection. Moreover, it appears that the viral surface glycoprotein (S) represents an important factor in the neurodegenerative process. Given all these properties, it has been suggested that these recognized human respiratory pathogens could be associated with the triggering or the exacerbation of neurological diseases for which the etiology remains unknown or poorly understood.
Asunto(s)
Enfermedades Virales del Sistema Nervioso Central/virología , Coronavirus/patogenicidad , Infecciones del Sistema Respiratorio/virología , Animales , Apoptosis , Sistema Nervioso Central/virología , HumanosRESUMEN
In humans, viral infections of the respiratory tract are a leading cause of morbidity and mortality worldwide. Among the various respiratory viruses, coronaviruses are important ubiquitous pathogens of humans and animals. Since the late 1960's, human coronaviruses (HCoV) are recognized pathogens of the upper respiratory tract, being mainly associated with mild pathologies such as the common cold. However, in vulnerable populations, (newborns, infants, the elderly and immune-compromised individuals), they can affect the lower respiratory tract, leading to pneumonia, exacerbations of asthma, respiratory distress syndrome or even severe acute respiratory syndrome (SARS). For almost three decades now, the scientific literature has also demonstrated that HCoV are neuroinvasive and neurotropic: neurons are often the target cell in the central nervous system (CNS), inducing neurodegeneration and eventually death. Moreover, HCoV can contribute to an overactivation of the immune system that could lead to autoimmunity in the CNS of susceptible individuals. Given all these properties, it has been suggested that HCoV could be associated with the triggering or the exacerbation of human neurological diseases for which the etiology remains unknown or poorly understood.
RESUMEN
Human coronaviruses (HCoV) are recognized respiratory pathogens. Some HCoV strains, including HCoV-OC43, can invade the central nervous system, where they infect neurons, with unclear consequences. We have previously reported that HCoV-OC43 infection of human neurons activates the unfolded-protein response and caspase-3 and induces cell death and that the viral spike (S) glycoprotein is involved in the process. We now report on underlying mechanisms associated with the induction of programmed cell death (PCD) after infection by the reference HCoV-OC43 virus (rOC/ATCC) and a more neurovirulent and cytotoxic HCoV-OC43 variant harboring two point mutations in the S glycoprotein (rOC/U(S183-241)). Even though caspase-3 and caspase-9 were both activated after infection, the use of caspase inhibitors neither reduced nor delayed virus-induced PCD, suggesting that these proteases are not essential in the process. On the other hand, the proapoptotic proteins BAX, cytochrome c (CytC), and apoptosis-inducing factor (AIF) were relocalized toward the mitochondria, cytosol, and nucleus, respectively, after infection by both virus variants. Moreover, LA-N-5 neuronal cells treated with cyclosporine (CsA), an inhibitor of the mitochondrial permeabilization transition pore (mPTP), or knocked down for cyclophilin D (CypD) were completely protected from rOC/ATCC-induced neuronal PCD, underlining the involvement of CypD in the process. On the other hand, CsA and CypD knockdown had moderate effects on rOC/U(S183-241)-induced PCD. In conclusion, our results are consistent with mitochondrial AIF and cyclophilin D being central in HCoV-OC43-induced PCD, while caspases appear not to be essential.
Asunto(s)
Apoptosis , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Infecciones por Coronavirus/enzimología , Coronavirus Humano OC43/fisiología , Ciclofilinas/metabolismo , Neuronas/citología , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismo , Caspasa 3/genética , Caspasa 9/genética , Línea Celular , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/fisiopatología , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/genética , Peptidil-Prolil Isomerasa F , Ciclofilinas/genética , Humanos , Neuronas/enzimología , Neuronas/virologíaRESUMEN
Prophages contribute to the evolution and virulence of most bacterial pathogens, but their role in Clostridium difficile is unclear. Here we describe the isolation of four Myoviridae phages, ΦMMP01, ΦMMP02, ΦMMP03, and ΦMMP04, that were recovered as free viral particles in the filter-sterilized stool supernatants of patients suffering from C. difficile infection (CDI). Furthermore, identical prophages were found in the chromosomes of C. difficile isolated from the corresponding fecal samples. We therefore provide, for the first time, evidence of in vivo prophage induction during CDI. We completely sequenced the genomes of ΦMMP02 and ΦMMP04, and bioinformatics analyses did not reveal the presence of virulence factors but underlined the unique character of ΦMMP04. We also studied the mobility of ΦMMP02 and ΦMMP04 prophages in vitro. Both prophages were spontaneously induced, with 4 to 5 log PFU/ml detected in the culture supernatants of the corresponding lysogens. When lysogens were grown in the presence of subinhibitory concentrations of ciprofloxacin, moxifloxacin, levofloxacin, or mitomycin C, the phage titers further increased, reaching 8 to 9 log PFU/ml in the case of ΦMMP04. In summary, our study highlights the extensive genetic diversity and mobility of C. difficile prophages. Moreover, antibiotics known to represent risk factors for CDI, such as quinolones, can stimulate prophage mobility in vitro and probably in vivo as well, which underscores their potential impact on phage-mediated horizontal gene transfer events and the evolution of C. difficile.
Asunto(s)
Clostridioides difficile , Infecciones por Clostridium/virología , Heces/virología , Myoviridae/aislamiento & purificación , Myoviridae/fisiología , Activación Viral , Antibacterianos/farmacología , Compuestos Aza/farmacología , Secuencia de Bases , Ciprofloxacina/farmacología , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Clostridioides difficile/virología , Infecciones por Clostridium/genética , Infecciones por Clostridium/microbiología , ADN Viral/genética , Heces/microbiología , Fluoroquinolonas , Variación Genética , Genoma Viral , Humanos , Levofloxacino , Lisogenia , Pruebas de Sensibilidad Microbiana , Mitomicina/farmacología , Datos de Secuencia Molecular , Moxifloxacino , Myoviridae/genética , Ofloxacino/farmacología , Profagos/genética , Profagos/aislamiento & purificación , Profagos/fisiología , Quinolinas/farmacología , Análisis de Secuencia de ADN , Activación Viral/genéticaRESUMEN
TcdA and TcdB exotoxins are the main virulence factors of Clostridium difficile, one of the most deadly nosocomial pathogens. Recent data suggest that prophages can influence the regulation of toxin expression. Here we present the characterization of ÏCD38-2, a pac-type temperate Siphoviridae phage that stimulates toxin expression when introduced as a prophage into C. difficile. Host range analysis showed that ÏCD38-2 was able to infect 99/207 isolates of C. difficile representing 11 different PCR ribotypes. Of 89 isolates corresponding to the NAP1/027 hypervirulent strain, which recently caused several outbreaks in North America and Europe, 79 (89%) were sensitive to ÏCD38-2. The complete double-stranded DNA (dsDNA) genome was determined, and a putative function could be assigned to 24 of the 55 open reading frames. No toxins or virulence factors could be identified based on bioinformatics analyses. Our data also suggest that ÏCD38-2 replicates as a circular plasmid in C. difficile lysogens. Upon introduction of ÏCD38-2 into a NAP1/027 representative isolate, up to 1.6- and 2.1-fold more TcdA and TcdB, respectively, were detected by immunodot blotting in culture supernatants of the lysogen than in the wild-type strain. In addition, real-time quantitative reverse transcriptase PCR (qRT-PCR) analyses showed that the mRNA levels of all five pathogenicity locus (PaLoc) genes were higher in the CD274 lysogen. Our study provides the first genomic sequence of a new pac-type Siphoviridae phage family member infecting C. difficile and brings further evidence supporting the role of prophages in toxin production in this important nosocomial pathogen.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Toxinas Bacterianas/biosíntesis , Clostridioides difficile/patogenicidad , Clostridioides difficile/virología , Enterotoxinas/biosíntesis , Regulación Bacteriana de la Expresión Génica , Lisogenia , Profagos/genética , Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/microbiología , ADN Viral/química , ADN Viral/genética , Europa (Continente) , Perfilación de la Expresión Génica , Genoma Viral , Especificidad del Huésped , Immunoblotting , Datos de Secuencia Molecular , América del Norte , Sistemas de Lectura Abierta , Profagos/crecimiento & desarrollo , Profagos/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Siphoviridae/genética , Siphoviridae/crecimiento & desarrollo , Siphoviridae/aislamiento & purificaciónRESUMEN
Among the various respiratory viruses infecting human beings, coronaviruses are important pathogens, which usually infect the upper respiratory tract, where they are mainly associated with common colds. However, in more vulnerable populations, such as newborns, infants, the elderly and immune-compromised individuals, these opportunistic pathogens can also affect the lower respiratory tract, leading to pneumonia, exacerbations of asthma, and various types of respiratory distress syndrome. The respiratory involvement of human coronaviruses has been clearly established since the 1960s. Nevertheless, for almost three decades now, data reported in the scientific literature has also demonstrated that, like it was described for other human viruses, coronaviruses have neuroinvasive capacities since they can spread from the respiratory tract to the central nervous system (CNS). Once there, infection of CNS cells (neurotropism) could lead to human health problems, such as encephalitis and long-term neurological diseases. Neuroinvasive coronaviruses could damage the CNS as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuroimmunopathology) and/or viral replication, which directly induces damage to CNS cells (virus-induced neuropathology). Given all these properties, it has been suggested that these opportunistic human respiratory pathogens could be associated with the triggering or the exacerbation of neurologic diseases for which the etiology remains poorly understood. Herein, we present host and viral factors that participate in the regulation of the possible pathogenic processes associated with CNS infection by human coronaviruses and we try to decipher the intricate interplay between virus and host target cells in order to characterize their role in the virus life cycle as well as in the capacity of the cell to respond to viral invasion.