RESUMO
Enterotoxigenic Escherichia coli (ETEC) infections are highly prevalent in developing countries, where clinical presentations range from asymptomatic colonization to severe cholera-like illness. The molecular basis for these varied presentations, which may involve strain-specific virulence features as well as host factors, has not been elucidated. We demonstrate that, when challenged with ETEC strain H10407, originally isolated from a case of cholera-like illness, blood group A human volunteers developed severe diarrhea more frequently than individuals from other blood groups. Interestingly, a diverse population of ETEC strains, including H10407, secrete the EtpA adhesin molecule. As many bacterial adhesins also agglutinate red blood cells, we combined the use of glycan arrays, biolayer inferometry, and noncanonical amino acid labeling with hemagglutination studies to demonstrate that EtpA is a dominant ETEC blood group A-specific lectin/hemagglutinin. Importantly, we have also shown that EtpA interacts specifically with glycans expressed on intestinal epithelial cells from blood group A individuals and that EtpA-mediated bacterial-host interactions accelerate bacterial adhesion and effective delivery of both the heat-labile and heat-stable toxins of ETEC. Collectively, these data provide additional insight into the complex molecular basis of severe ETEC diarrheal illness that may inform rational design of vaccines to protect those at highest risk.
Assuntos
Sistema ABO de Grupos Sanguíneos/metabolismo , Diarreia , Escherichia coli Enterotoxigênica , Células Epiteliais/metabolismo , Infecções por Escherichia coli/metabolismo , Mucosa Intestinal/metabolismo , Adesinas de Escherichia coli/metabolismo , Diarreia/metabolismo , Diarreia/microbiologia , Diarreia/patologia , Escherichia coli Enterotoxigênica/metabolismo , Escherichia coli Enterotoxigênica/patogenicidade , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Infecções por Escherichia coli/patologia , Feminino , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Índice de Gravidade de DoençaRESUMO
Heartland virus is a suspected tickborne pathogen in the United States. We describe a case of hemophagocytic lymphohistiocytosis, then death, in an immunosuppressed elderly man in Missouri, USA, who was infected with Heartland virus.
Assuntos
Infecções por Bunyaviridae/complicações , Infecções por Bunyaviridae/virologia , Linfo-Histiocitose Hemofagocítica/complicações , Linfo-Histiocitose Hemofagocítica/epidemiologia , Phlebovirus/isolamento & purificação , Idoso , Anticorpos Antivirais/sangue , Infecções por Bunyaviridae/sangue , Infecções por Bunyaviridae/epidemiologia , Evolução Fatal , Humanos , Hospedeiro Imunocomprometido , Masculino , Missouri/epidemiologia , Phlebovirus/genéticaRESUMO
The presence of the endogenous Leishmania RNA virus 1 (LRV1) replicating stably within some parasite species has been associated with the development of more severe forms of leishmaniasis and relapses after drug treatment in humans. Here, we show that the disease-exacerbatory role of LRV1 relies on type I IFN (type I IFNs) production by macrophages and signaling in vivo. Moreover, infecting mice with the LRV1-cured Leishmania guyanensis (LgyLRV1- ) strain of parasites followed by type I IFN treatment increased lesion size and parasite burden, quantitatively reproducing the LRV1-bearing (LgyLRV1+ ) infection phenotype. This finding suggested the possibility that exogenous viral infections could likewise increase pathogenicity, which was tested by coinfecting mice with L. guyanensis and lymphocytic choriomeningitis virus (LCMV), or the sand fly-transmitted arbovirus Toscana virus (TOSV). The type I IFN antiviral response increased the pathology of L. guyanensis infection, accompanied by down-regulation of the IFN-γ receptor normally required for antileishmanial control. Further, LCMV coinfection of IFN-γ-deficient mice promoted parasite dissemination to secondary sites, reproducing the LgyLRV1+ metastatic phenotype. Remarkably, LCMV coinfection of mice that had healed from L. guyanensis infection induced reactivation of disease pathology, overriding the protective adaptive immune response. Our findings establish that type I IFN-dependent responses, arising from endogenous viral elements (dsRNA/LRV1), or exogenous coinfection with IFN-inducing viruses, are able to synergize with New World Leishmania parasites in both primary and relapse infections. Thus, viral infections likely represent a significant risk factor along with parasite and host factors, thereby contributing to the pathological spectrum of human leishmaniasis.
Assuntos
Interferon Tipo I/imunologia , Leishmania guyanensis , Leishmaniose Mucocutânea/imunologia , Leishmaniavirus/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Febre por Flebótomos/imunologia , Vírus da Febre do Flebótomo Napolitano/imunologia , Animais , Coinfecção , Interferon Tipo I/genética , Leishmania guyanensis/imunologia , Leishmania guyanensis/virologia , Leishmaniose Mucocutânea/genética , Leishmaniose Mucocutânea/patologia , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/patologia , Camundongos , Camundongos Knockout , Febre por Flebótomos/genética , Febre por Flebótomos/patologiaRESUMO
The endogenous double-stranded RNA (dsRNA) virus Leishmaniavirus (LRV1) has been implicated as a pathogenicity factor for leishmaniasis in rodent models and human disease, and associated with drug-treatment failures in Leishmania braziliensis and Leishmania guyanensis infections. Thus, methods targeting LRV1 could have therapeutic benefit. Here we screened a panel of antivirals for parasite and LRV1 inhibition, focusing on nucleoside analogs to capitalize on the highly active salvage pathways of Leishmania, which are purine auxotrophs. Applying a capsid flow cytometry assay, we identified two 2'-C-methyladenosine analogs showing selective inhibition of LRV1. Treatment resulted in loss of LRV1 with first-order kinetics, as expected for random virus segregation, and elimination within six cell doublings, consistent with a measured LRV1 copy number of about 15. Viral loss was specific to antiviral nucleoside treatment and not induced by growth inhibitors, in contrast to fungal dsRNA viruses. Comparisons of drug-treated LRV1+ and LRV1- lines recapitulated LRV1-dependent pathology and parasite replication in mouse infections, and cytokine secretion in macrophage infections. Agents targeting Totiviridae have not been described previously, nor are there many examples of inhibitors acting against dsRNA viruses more generally. The compounds identified here provide a key proof-of-principle in support of further studies identifying efficacious antivirals for use in in vivo studies of LRV1-mediated virulence.
Assuntos
Antivirais/farmacologia , Leishmania braziliensis/virologia , Leishmania guyanensis/virologia , Leishmaniavirus/efeitos dos fármacos , Nucleosídeos/farmacologia , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Leishmaniose/parasitologia , Leishmaniavirus/genética , Leishmaniavirus/metabolismo , Camundongos Endogâmicos C57BL , Nucleotídeos/farmacologiaRESUMO
Recent studies have shown that a cytoplasmic virus called Leishmaniavirus (LRV) is present in some Leishmania species and acts as a potent innate immunogen, aggravating lesional inflammation and development in mice. In humans, the presence of LRV in Leishmania guyanensis and in L. braziliensis was significantly correlated with poor treatment response and symptomatic relapse. So far, no clinical effort has used LRV for prophylactic purposes. In this context, we designed an original vaccine strategy that targeted LRV nested in Leishmania parasites to prevent virus-related complications. To this end, C57BL/6 mice were immunized with a recombinant LRV1 Leishmania guyanensis viral capsid polypeptide formulated with a T helper 1-polarizing adjuvant. LRV1-vaccinated mice had significant reduction in lesion size and parasite load when subsequently challenged with LRV1+ Leishmania guyanensis parasites. The protection conferred by this immunization could be reproduced in naïve mice via T-cell transfer from vaccinated mice but not by serum transfer. The induction of LRV1 specific T cells secreting IFN-γ was confirmed in vaccinated mice and provided strong evidence that LRV1-specific protection arose via a cell mediated immune response against the LRV1 capsid. Our studies suggest that immunization with LRV1 capsid could be of a preventive benefit in mitigating the elevated pathology associated with LRV1 bearing Leishmania infections and possibly avoiding symptomatic relapses after an initial treatment. This novel anti-endosymbiotic vaccine strategy could be exploited to control other infectious diseases, as similar viral infections are largely prevalent across pathogenic pathogens and could consequently open new vaccine opportunities.
Assuntos
Proteínas do Capsídeo/imunologia , Leishmania guyanensis/virologia , Leishmaniose/prevenção & controle , Leishmaniavirus/imunologia , Animais , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/genética , Feminino , Humanos , Imunidade Celular , Leishmania guyanensis/genética , Leishmania guyanensis/imunologia , Leishmania guyanensis/fisiologia , Leishmaniose/imunologia , Leishmaniose/parasitologia , Leishmaniavirus/genética , Leishmaniavirus/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Simbiose , Linfócitos T/imunologia , VacinaçãoRESUMO
JC virus (JCV) is a human polyomavirus that infects the central nervous system (CNS) of immunocompromised patients. JCV granule cell neuronopathy (JCV-GCN) is caused by infection of cerebellar granule cells, causing ataxia. A 77-year-old man with iatrogenic lymphopenia presented with severe ataxia and was diagnosed with JCV-GCN. His ataxia and cerebrospinal fluid (CSF) improved with intravenous immunoglobulin, high-dose intravenous methylprednisolone, mirtazapine, and mefloquine. Interleukin-7 (IL-7) therapy reconstituted his lymphocytes and reduced his CSF JCV load. One month after IL-7 therapy, he developed worsening ataxia and CSF inflammation, which raised suspicion for immune reconstitution inflammatory syndrome. Steroids were restarted and his ataxia stabilized.
Assuntos
Ataxia/tratamento farmacológico , Síndrome do Hamartoma Múltiplo/tratamento farmacológico , Hospedeiro Imunocomprometido , Interleucina-7/uso terapêutico , Leucoencefalopatia Multifocal Progressiva/tratamento farmacológico , Linfopenia/tratamento farmacológico , Malformações do Desenvolvimento Cortical do Grupo I/tratamento farmacológico , Idoso , Ataxia/diagnóstico , Ataxia/imunologia , Ataxia/virologia , Doença Crônica , Síndrome do Hamartoma Múltiplo/diagnóstico , Síndrome do Hamartoma Múltiplo/imunologia , Síndrome do Hamartoma Múltiplo/virologia , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Vírus JC/imunologia , Vírus JC/patogenicidade , Vírus JC/fisiologia , Leucoencefalopatia Multifocal Progressiva/diagnóstico , Leucoencefalopatia Multifocal Progressiva/imunologia , Leucoencefalopatia Multifocal Progressiva/virologia , Linfopenia/diagnóstico , Linfopenia/imunologia , Linfopenia/virologia , Masculino , Malformações do Desenvolvimento Cortical do Grupo I/diagnóstico , Malformações do Desenvolvimento Cortical do Grupo I/imunologia , Malformações do Desenvolvimento Cortical do Grupo I/virologia , Mefloquina/uso terapêutico , Metilprednisolona/uso terapêutico , Mianserina/análogos & derivados , Mianserina/uso terapêutico , Mirtazapina , Proteínas Recombinantes/uso terapêuticoRESUMO
Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.
Assuntos
Antiprotozoários/farmacologia , Leishmaniose Mucocutânea/tratamento farmacológico , Leishmaniavirus/efeitos dos fármacos , Oligorribonucleotídeos Antissenso/farmacologia , RNA de Cadeia Dupla/antagonistas & inibidores , RNA Viral/antagonistas & inibidores , Animais , Antiprotozoários/química , Antiprotozoários/metabolismo , Expressão Gênica , Sequências Repetidas Invertidas , Leishmania braziliensis/patogenicidade , Leishmania braziliensis/virologia , Leishmania guyanensis/patogenicidade , Leishmania guyanensis/virologia , Leishmaniose Mucocutânea/parasitologia , Leishmaniose Mucocutânea/virologia , Leishmaniavirus/genética , Leishmaniavirus/metabolismo , Macrófagos/parasitologia , Macrófagos/virologia , Camundongos , Oligorribonucleotídeos Antissenso/genética , Oligorribonucleotídeos Antissenso/metabolismo , Interferência de RNA/efeitos dos fármacos , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Simbiose/genética , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Because O blood group has been associated with more severe cholera infections, it has been hypothesized that cholera toxin (CT) may bind non-O blood group antigens of the intestinal mucosae, thereby preventing efficient interaction with target GM1 gangliosides required for uptake of the toxin and activation of cyclic adenosine monophosphate (cAMP) signaling in target epithelia. Herein, we show that after exposure to CT, human enteroids expressing O blood group exhibited marked increase in cAMP relative to cells derived from blood group A individuals. Likewise, using CRISPR/Cas9 engineering, a functional group O line (HT-29-A(-/-)) was generated from a parent group A HT-29 line. CT stimulated robust cAMP responses in HT-29-A(-/-) cells relative to HT-29 cells. These findings provide a direct molecular link between blood group O expression and differential cellular responses to CT, recapitulating clinical and epidemiologic observations.
Assuntos
Sistema ABO de Grupos Sanguíneos/metabolismo , Toxina da Cólera/farmacologia , AMP Cíclico/biossíntese , Gangliosídeo G(M1)/metabolismo , Vibrio cholerae/química , Sistema ABO de Grupos Sanguíneos/química , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Sistemas CRISPR-Cas , Engenharia Celular , Cólera , Toxina da Cólera/isolamento & purificação , Colforsina/farmacologia , Gangliosídeo G(M1)/química , Galactose/química , Galactose/metabolismo , Células HT29 , Humanos , Ligação Proteica , Índice de Gravidade de Doença , Vibrio cholerae/metabolismoRESUMO
Propionibacterium acnes is a known cause of postneurosurgical meningitis; however, it is rarely implicated in de novo meningitis. Herein we report a case of a 49-year-old male with de novo meningitis caused by P. acnes with metastatic melanoma as the only identified risk factor for his infection.
Assuntos
Infecções por Bactérias Gram-Positivas/diagnóstico , Melanoma/complicações , Melanoma/secundário , Neoplasias Meníngeas/complicações , Neoplasias Meníngeas/secundário , Meningites Bacterianas/diagnóstico , Propionibacterium acnes/isolamento & purificação , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/patologia , Humanos , Masculino , Melanoma/patologia , Neoplasias Meníngeas/patologia , Meningite , Meningites Bacterianas/microbiologia , Meningites Bacterianas/patologia , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence. METHODOLOGY/PRINCIPAL FINDINGS: This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice. CONCLUSIONS/SIGNIFICANCE: We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.
Assuntos
Leishmania/virologia , Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/isolamento & purificação , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Humanos , Immunoblotting/métodos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA de Cadeia Dupla/imunologia , RNA Viral/genética , Análise de Sequência de DNA , Virologia/métodosRESUMO
BACKGROUND: Target repurposing utilizes knowledge of "druggable" targets obtained in one organism and exploits this information to pursue new potential drug targets in other organisms. Here we describe such studies to evaluate whether inhibitors targeting the kinase domain of the mammalian Target of Rapamycin (mTOR) and human phosphoinositide-3-kinases (PI3Ks) show promise against the kinetoplastid parasites Trypanosoma brucei, T. cruzi, Leishmania major, and L. donovani. The genomes of trypanosomatids encode at least 12 proteins belonging to the PI3K protein superfamily, some of which are unique to parasites. Moreover, the shared PI3Ks differ greatly in sequence from those of the human host, thereby providing opportunities for selective inhibition. METHODOLOGY/PRINCIPAL FINDINGS: We focused on 8 inhibitors targeting mTOR and/or PI3Ks selected from various stages of pre-clinical and clinical development, and tested them against in vitro parasite cultures and in vivo models of infection. Several inhibitors showed micromolar or better efficacy against these organisms in culture. One compound, NVP-BEZ235, displayed sub-nanomolar potency, efficacy against cultured parasites, and an ability to clear parasitemia in an animal model of T. brucei rhodesiense infection. CONCLUSIONS/SIGNIFICANCE: These studies strongly suggest that mammalian PI3/TOR kinase inhibitors are a productive starting point for anti-trypanosomal drug discovery. Our data suggest that NVP-BEZ235, an advanced clinical candidate against solid tumors, merits further investigation as an agent for treating African sleeping sickness.