RESUMO
Sequencing of Ebola virus (EBOV) genomes during the 2014-2016 epidemic identified several naturally occurring, dominant mutations potentially impacting virulence or tropism. In this study, we characterized EBOV variants carrying one of the following substitutions: A82V in the glycoprotein (GP), R111C in the nucleoprotein (NP), or D759G in the RNA-dependent RNA polymerase (L). Compared with the wild-type (WT) EBOV C07 isolate, NP and L mutants conferred a replication advantage in monkey Vero E6, human A549, and insectivorous bat Tb1.Lu cells, while L mutants displayed a disadvantage in human Huh7 cells. The replication of the GP mutant was significantly delayed in Tb1.Lu cells and similar to that of the WT in other cells. The L mutant was less virulent, as evidenced by increased survival for mice and a significantly delayed time to death for ferrets, but increased lengths of the period of EBOV shedding may have contributed to the prolonged epidemic. Our results show that single substitutions can have observable impacts on EBOV pathogenicity and provide a framework for the study of other mutations.IMPORTANCE During the Ebola virus (EBOV) disease outbreak in West Africa in 2014-2016, it was discovered that several mutations in the virus emerged and became prevalent in the human population. This suggests that these mutations may play a role impacting viral fitness. We investigated three of these previously identified mutations (in the glycoprotein [GP], nucleoprotein [NP], or RNA-dependent RNA polymerase [L]) in cell culture, as well as in mice and ferrets, by generating recombinant viruses (based on an early West African EBOV strain) each carrying one of these mutations. The NP and L mutations appear to decrease virulence, whereas the GP mutation slightly increases virulence but mainly impacts viral tropism. Our results show that these single mutations can impact EBOV virulence in animals and have implications for the rational design of efficacious antiviral therapies against these infections.
Assuntos
Substituição de Aminoácidos , Ebolavirus/fisiologia , Ebolavirus/patogenicidade , Proteínas Virais/genética , Células A549 , Animais , Linhagem Celular , Quirópteros , Chlorocebus aethiops , Ebolavirus/genética , Humanos , Células Vero , Virulência , Replicação Viral , Eliminação de Partículas ViraisRESUMO
Ebola virus (EBOV) infections result in aggressive hemorrhagic fever in humans, with fatality rates reaching 90% and with no licensed specific therapeutics to treat ill patients. Advances over the past 5 years have firmly established monoclonal antibody (MAb)-based products as the most promising therapeutics for treating EBOV infections, but production is costly and quantities are limited; therefore, MAbs are not the best candidates for mass use in the case of an epidemic. To address this need, we generated EBOV-specific polyclonal F(ab')2 fragments from horses hyperimmunized with an EBOV vaccine. The F(ab')2 was found to potently neutralize West African and Central African EBOV in vitro Treatment of nonhuman primates (NHPs) with seven doses of 100 mg/kg F(ab')2 beginning 3 or 5 days postinfection (dpi) resulted in a 100% survival rate. Notably, NHPs for which treatment was initiated at 5 dpi were already highly viremic, with observable signs of EBOV disease, which demonstrated that F(ab')2 was still effective as a therapeutic agent even in symptomatic subjects. These results show that F(ab')2 should be advanced for clinical testing in preparation for future EBOV outbreaks and epidemics.IMPORTANCE EBOV is one of the deadliest viruses to humans. It has been over 40 years since EBOV was first reported, but no cure is available. Research breakthroughs over the past 5 years have shown that MAbs constitute an effective therapy for EBOV infections. However, MAbs are expensive and difficult to produce in large amounts and therefore may only play a limited role during an epidemic. A cheaper alternative is required, especially since EBOV is endemic in several third world countries with limited medical resources. Here, we used a standard protocol to produce large amounts of antiserum F(ab')2 fragments from horses vaccinated with an EBOV vaccine, and we tested the protectiveness in monkeys. We showed that F(ab')2 was effective in 100% of monkeys even after the animals were visibly ill with EBOV disease. Thus, F(ab')2 could be a very good option for large-scale treatments of patients and should be advanced to clinical testing.
Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Fragmentos Fab das Imunoglobulinas/imunologia , Macaca mulatta/virologia , Animais , Anticorpos Antivirais/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/veterinária , Cavalos/imunologia , Imunização , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Imunoterapia/métodosRESUMO
BACKGROUND: Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrPd). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay. No harmonized method for producing the protein has been agreed upon, in part due to the variety of substrates that have been applied in RT-QuIC. METHODS: This study examines the effects of two different rPrP refolding protocols on the production, QuIC performance, and structure characteristics of two constructs of rPrP commonly used in QuIC: full length hamster and a sheep-hamster chimeric rPrP. RESULTS: Under the described conditions, the best performing substrate was the chimeric sheep-hamster rPrP produced by shorter guanidine-HCl exposure and faster gradient elution. CONCLUSIONS: The observation that different rPrP production protocols influence QuIC performance indicates that caution should be exercised when comparing inter-laboratory QuIC results.
Assuntos
Bioensaio/métodos , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cricetinae , Proteínas Priônicas/genética , Proteínas Priônicas/isolamento & purificação , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , OvinosRESUMO
Real-time quaking-induced conversion (RT-QuIC) has been proposed as a sensitive diagnostic test for sporadic Creutzfeldt-Jakob disease; however, before this assay can be introduced into clinical practice, its reliability and reproducibility need to be demonstrated. Two international ring trials were undertaken in which a set of 25 cerebrospinal fluid samples were analyzed by a total of 11 different centers using a range of recombinant prion protein substrates and instrumentation. The results show almost complete concordance between the centers and demonstrate that RT-QuIC is a suitably reliable and robust technique for clinical practice. Ann Neurol 2016;80:160-165.
Assuntos
Síndrome de Creutzfeldt-Jakob/líquido cefalorraquidiano , Síndrome de Creutzfeldt-Jakob/diagnóstico , Agregação Patológica de Proteínas/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Internacionalidade , Masculino , Pessoa de Meia-Idade , Príons/química , Proteínas Recombinantes/química , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The Prion Laboratory Section of the Public Health Agency of Canada supports heath care professionals dealing with patients suspected to have Creutzfeldt-Jakob disease (CJD) by testing cerebrospinal fluid (CSF) for protein markers of CJD. To better serve Canadian diagnostic requirements, a quaking-induced conversion (QuIC)-based assay has been added to the test panel. The QuIC tests exploit the ability of disease-associated prion protein, found in the CSF of a majority of CJD patients, to convert a recombinant prion protein (rPrP) into detectable amounts of a misfolded, aggregated form of rPrP. The rPrP aggregates interact with a specific dye, causing a measurable change in the dye's fluorescence emission spectrum. Optimal test and analysis parameters were empirically determined. Taking both practical and performance considerations into account, an endpoint QuIC (EP-QuIC) configuration was chosen. EP-QuIC uses a thermo-mixer to perform the shaking necessary to produce the quaking-induced conversions. Fluorescence readings are obtained from a microwell fluorescence reader only at the beginning and the end of EP-QuIC reactions. Samples for which the relative fluorescence unit ratio between the initial and final readings represent a ≥4 increase in signal intensity in at least two of the three replicates are classified as positive. A retrospective analysis of 91 CSF samples that included 45 confirmed cases of CJD and 46 non-CJD cases was used to estimate the performance characteristics of the EP-QuIC assay. The diagnostic sensitivity and specificity of the EP-QuIC test of this set of samples were 98 and 91%, respectively.
Assuntos
Síndrome de Creutzfeldt-Jakob/diagnóstico , Diagnóstico , Testes Diagnósticos de Rotina/métodos , Proteínas Priônicas/líquido cefalorraquidiano , Canadá , Líquido Cefalorraquidiano/química , Humanos , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing.
Assuntos
Antígenos de Bactérias/análise , Toxinas Bacterianas/genética , Técnicas de Tipagem Bacteriana/métodos , Escherichia coli/classificação , Técnicas de Genotipagem/métodos , Espectrometria de Massas/métodos , Antígenos O/genética , Antígenos de Bactérias/genética , Escherichia coli/química , Escherichia coli/genética , Infecções por Escherichia coli/diagnóstico , HumanosRESUMO
BACKGROUND: Escherichia coli H antigen typing with antisera, a useful method for flagella clinical identification and classification, is a time-consuming process because of the need to induce flagella growth and the occurrence of undetermined strains. We developed an alternative rapid and analytically sensitive mass spectrometry (MS) method, termed MS-based H antigen typing (MS-H), and applied it at the protein sequence level for H antigen typing. We also performed a comparison with traditional serotyping on reference strains and clinical isolates. METHODS: On the basis of international guidelines, the analytical selectivity and sensitivity, imprecision, correlation, repeatability, and reproducibility of the MS-H platform was evaluated using reference strains. Comparison of MS-H typing and serotyping was performed using 302 clinical isolates from 5 Canadian provinces, and discrepant results between the 2 platforms were resolved through whole genome sequencing. RESULTS: Repeated tests on reference strain EDL933 demonstrated a lower limit of the measuring interval at the subsingle colony (16.97 µg or 1.465 × 10(7) cells) level and close correlation (r(2) > 0.99) between cell culture biomass and sequence coverage. The CV was <10.0% among multiple repeats with 4 reference strains. Intra- and interlaboratory tests demonstrated that the MS-H method was robust and reproducible under various sample preparation and instrumentation conditions. Using discrepancy analysis via whole genome sequencing, performed on isolates with discrepant results, MS-H accurately identified 12.3% more isolates than conventional serotyping. CONCLUSIONS: MS-H typing of E. coli is useful for fast and accurate flagella typing and could be very useful during E. coli outbreaks.
Assuntos
Antígenos de Bactérias/análise , Antígenos de Bactérias/química , Escherichia coli/química , Flagelos/química , Espectrometria de Massas/métodos , Sorotipagem/métodos , Sorotipagem/normas , Antígenos de Bactérias/imunologia , Canadá , Escherichia coli/imunologia , Escherichia coli/isolamento & purificação , Flagelos/imunologia , Sensibilidade e EspecificidadeRESUMO
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has gained popularity in recent years for rapid bacterial identification, mostly at the genus or species level. In this study, a rapid method to identify the Escherichia coli flagellar antigen (H antigen) at the subspecies level was developed using a MALDI-TOF MS platform with high specificity and sensitivity. Flagella were trapped on a filter membrane, and on-filter trypsin digestion was performed. The tryptic digests of each flagellin then were collected and analyzed by MALDI-TOF MS through peptide mass fingerprinting. Sixty-one reference strains containing all 53 H types and 85 clinical strains were tested and compared to serotyping designations. Whole-genome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix for MALDI-TOF MS, with higher confidence during protein identification. After method optimization, reference strains representing all 53 E. coli H types were identified correctly by MALDI-TOF MS. A custom E. coli flagellar/H antigen database was crucial for clearly identifying the E. coli H antigens. Of 85 clinical isolates tested by MALDI-TOF MS-H, 75 identified MS-H types (88.2%) matched results obtained from traditional serotyping. Among 10 isolates where the results of MALDI-TOF MS-H and serotyping did not agree, 60% of H types characterized by whole-genome sequencing agreed with those identified by MALDI-TOF MS-H, compared to only 20% by serotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coli H antigen identification, especially during E. coli outbreaks.
Assuntos
Antígenos de Bactérias/análise , Técnicas de Tipagem Bacteriana/métodos , Escherichia coli/química , Escherichia coli/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Humanos , Sensibilidade e Especificidade , Sorotipagem/métodos , Fatores de TempoRESUMO
Forty-three reference strains involving the 24 most common serovars of Salmonella enterica were examined by using a mass spectrometry-based H antigen typing platform (MS-H). The results indicate that MS-H can be used as a sensitive, rapid, and straightforward approach for the typing of Salmonella flagella at the molecular level without antiserum and phase inversion.
Assuntos
Antígenos de Bactérias/química , Cromatografia Líquida/métodos , Flagelos/química , Salmonella enterica/química , Salmonella enterica/classificação , Espectrometria de Massas em Tandem/métodos , Técnicas Bacteriológicas/métodos , HumanosRESUMO
BACKGROUND: The presence of Campylobacter jejuni temperate bacteriophages has increasingly been associated with specific biological effects. It has recently been demonstrated that the presence of the prophage CJIE1 is associated with increased adherence and invasion of C. jejuni isolates in cell culture assays. RESULTS: Quantitative comparative proteomics experiments were undertaken using three closely related isolates with CJIE1 and one isolate without CJIE1 to determine whether there was a corresponding difference in protein expression levels. Initial experiments indicated that about 2% of the total proteins characterized were expressed at different levels in isolates with or without the prophage. Some of these proteins regulated by the presence of CJIE1 were associated with virulence or regulatory functions. Additional experiments were conducted using C. jejuni isolates with and without CJIE1 grown on four different media: Mueller Hinton (MH) media containing blood; MH media containing 0.1% sodium deoxycholate, which is thought to result in increased expression of virulence proteins; MH media containing 2.5% Oxgall; and MHwithout additives. These experiments provided further evidence that CJIE1 affected protein expression, including virulence-associated proteins. They also demonstrated a general bile response involving a majority of the proteome and clearly showed the induction of almost all proteins known to be involved with iron acquisition. The data have been deposited to the ProteomeXchange with identifiers PXD000798, PXD000799, PXD000800, and PXD000801. CONCLUSION: The presence of the CJIE1 prophage was associated with differences in protein expression levels under different conditions. Further work is required to determine what genes are involved in causing this phenomenon.
Assuntos
Proteínas de Bactérias/biossíntese , Ácidos e Sais Biliares/metabolismo , Campylobacter jejuni/metabolismo , Campylobacter jejuni/virologia , Regulação Bacteriana da Expressão Gênica , Prófagos/genética , Proteínas de Bactérias/genética , Meios de Cultura/química , DNA Bacteriano/química , DNA Bacteriano/genética , Dados de Sequência Molecular , Proteoma/análise , Análise de Sequência de DNARESUMO
Inactivation of intact influenza viruses using formaldehyde or ß-propiolactone (BPL) is essential for vaccine production and safety. The extent of chemical modifications of such reagents on viral proteins needs to be extensively investigated to better control the reactions and quality of vaccines. We have evaluated the effect of BPL inactivation on two candidate re-assortant vaccines (NIBRG-121xp and NYMC-X181A) derived from A/California/07/2009 pandemic influenza viruses using high-resolution FT-ICR MS-based proteomic approaches. We report here an ultra performance LC MS/MS method for determining full-length protein sequences of hemagglutinin and neuraminidase through protein delipidation, various enzymatic digestions, and subsequent mass spectrometric analyses of the proteolytic peptides. We also demonstrate the ability to reliably identify hundreds of unique sites modified by propiolactone on the surface of glycoprotein antigens. The location of these modifications correlated with changes to protein folding, conformation, and stability, but demonstrated no effect on protein disulfide linkages. In some cases, these modifications resulted in suppression of protein function, an effect that correlated with the degree of change of the modified amino acids' side chain length and polarity.
Assuntos
Vacinas contra Influenza/química , Neuraminidase/química , Propiolactona/química , Proteínas de Ligação a RNA/química , Proteínas do Core Viral/química , Proteínas Virais/química , Inativação de Vírus , Sequência de Aminoácidos , Antígenos Virais/química , Cisteína/química , Hemaglutininas/química , Proteínas do Nucleocapsídeo , Polissacarídeos/química , Espectrometria de Massas em TandemRESUMO
Ebola virus (EBOV) is responsible for numerous devastating outbreaks throughout Africa, including the 2013-2016 West African outbreak as well as the two recent outbreaks in the Democratic Republic of the Congo (DRC), one of which is ongoing. Although EBOV disease (EVD) has typically been considered a highly lethal acute infection, increasing evidence suggests that the virus can persist in certain immune-privileged sites and occasionally lead to EVD recrudescence. Little is understood about the processes that contribute to EBOV persistence and recrudescence, in part because of the rarity of these phenomena but also because of the absence of an animal model that recapitulates them. Here, we describe a case of EBOV persistence associated with atypical EVD in a nonhuman primate (NHP) following inoculation with EBOV and treatment with an experimental monoclonal antibody cocktail. Although this animal exhibited only mild signs of acute EVD, it developed severe disease 2 weeks later and succumbed shortly thereafter. Viremia was undetectable at the time of death, despite abundant levels of viral RNA in most tissues, each of which appeared to harbor a distinct viral quasispecies. Remarkably, sequence analysis identified a single mutation in glycoprotein (GP) that not only resisted antibody-mediated neutralization but also increased viral growth kinetics and virulence. Overall, this report represents the most thoroughly characterized case of atypical EVD in an NHP described thus far, and it provides valuable insight into factors that may contribute to EBOV persistence and recrudescent disease.IMPORTANCE Ebola virus remains a global threat to public health and biosecurity, yet we still know relatively little about its pathogenesis and the complications that arise following recovery. With nearly 20,000 survivors from the 2013-2016 West African outbreak, as well as over 1,000 survivors of the recent outbreak in the DRC, we must consider the consequences of virus persistence and recrudescent disease, even if they are rare. In this study, we describe a case of atypical Ebola virus disease in a nonhuman primate after treatment with a monoclonal antibody. Not only does this study underscore the potential for atypical disease presentations, but it also emphasizes the importance of considering how medical countermeasures might relate to these phenomena, especially as antibodies are incorporated into the standard of care. The results presented herein provide a foundation from which we can continue to investigate these facets of Ebola virus disease.
Assuntos
Anticorpos Monoclonais/imunologia , Ebolavirus/genética , Glicoproteínas/genética , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Mutação , África , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes , Anticorpos Antivirais/imunologia , Citocinas , Surtos de Doenças , Feminino , Furões , Doença pelo Vírus Ebola/tratamento farmacológico , Masculino , Primatas , RNA Viral/isolamento & purificaçãoRESUMO
Ebola virus (EBOV) is a highly pathogenic filovirus that causes outbreaks of a severe hemorrhagic fever known as EBOV disease (EVD). Ebola virus disease is characterized in part by a dysregulated immune response and massive production of both pro- and anti-inflammatory cytokines. To better understand the immune response elicited by EVD in the context of treatment with experimental anti-EBOV antibody cocktails, we analyzed 29 cytokines in 42 EBOV-infected rhesus macaques. In comparison to the surviving treated animals, which exhibited minimal aberrations in only a few cytokine levels, nonsurviving animals exhibited a dramatically upregulated inflammatory response that was delayed by antibody treatment.
RESUMO
Mass spectrometry (MS) has been widely used in recent years for bacterial identification and typing. Single bacterial colonies are regarded as pure cultures of bacteria grown from single cells. In this chapter, we describe a method for identifying bacteria at the species level with 100% accuracy using the proteomes of bacterial cultures from single colonies. In this chapter, six reference strains of gram-negative and gram-positive bacteria are analyzed, producing results of high reproducibility, as examples of bacterial identification through the application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and a custom database. Details on sample preparation and identification of Streptococcus pneumoniae are also described.
Assuntos
Proteínas de Bactérias/análise , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Streptococcus pneumoniae/metabolismoRESUMO
The development of an effective vaccine against HIV infection remains a global priority. Dendritic cell (DC)-based HIV immunotherapeutic vaccine is a promising approach which aims at optimizing the HIV-specific immune response using primed DCs to promote and enhance both the cellular and humoral arms of immunity. Since the Ebola virus envelope glycoprotein (EboGP) has strong DC-targeting ability, we investigated whether EboGP is able to direct HIV particles towards DCs efficiently and promote potent HIV-specific immune responses. Our results indicate that the incorporation of EboGP into non-replicating virus-like particles (VLPs) enhances their ability to target human monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs). Also, a mucin-like domain deleted EboGP (EboGPΔM) can further enhanced the MDDCs and MDMs-targeting ability. Furthermore, we investigated the effect of EboGP on HIV immunogenicity in mice, and the results revealed a significantly stronger HIV-specific humoral immune response when immunized with EboGP-pseudotyped HIV VLPs compared with those immunized with HIV VLPs. Splenocytes harvested from mice immunized with EboGP-pseudotyped HIV VLPs secreted increased levels of macrophage inflammatory proteins-1α (MIP-1α) and IL-4 upon stimulation with HIV Env and/or Gag peptides compared with those harvested from mice immunized with HIV VLPs. Collectively, this study provides evidence for the first time that the incorporation of EboGP in HIV VLPs can facilitate DC and macrophage targeting and induce more potent immune responses against HIV.
Assuntos
Vacinas contra a AIDS/imunologia , Células Dendríticas/efeitos dos fármacos , Anticorpos Anti-HIV/biossíntese , Infecções por HIV/prevenção & controle , Macrófagos/efeitos dos fármacos , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas do Envelope Viral/genética , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Animais , Quimiocina CCL3/genética , Quimiocina CCL3/imunologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Ebolavirus/química , Feminino , Expressão Gênica , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Imunização , Imunogenicidade da Vacina , Interleucina-4/genética , Interleucina-4/imunologia , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Cultura Primária de Células , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas do Envelope Viral/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
After we published our preliminary study on the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and curated E. coli toxin databases on the identification of E. coli Shiga toxins (Stxs) in the Journal of Proteomics in year 2018, we were encouraged to further refine the method and test clinical isolates. In this study, different concentrations of mitomycin C (MMC) and ciprofloxacin (CF), two common antibiotic/chemotherapy agents capable of stimulating Stx production, were first tested and compared on three reference strains and eight clinical isolates to observe the toxin induction and subsequent identification. Notably, no differences were observed between the two agents other than the concentrations applied. Seventeen more clinical isolates were then tested using fixed MMC and CF concentrations and sample amount. This study confirms that the majority of stx2-positive E. coli strains can be stimulated to produce sufficient toxin for confident identification. This does not occur with stx1-positive E. coli isolates, however, despite the fact that both Stxs can be identified for several isolates without MMC or CF stimulation. BIOLOGICAL SIGNIFICANCE: Stxs, especially Stx2, are very important causes of severe food-borne disease, even death. This study confirms that receptor analogue-based affinity enrichment of Stxs, after MMC or CF treatment of E. coli, is useful for fast and accurate Stx2 identification through LC-MS/MS.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteômica , Toxina Shiga I , Toxina Shiga II , Escherichia coli Shiga Toxigênica/metabolismo , Cromatografia Líquida , Humanos , Toxina Shiga I/análise , Toxina Shiga I/metabolismo , Toxina Shiga II/análise , Toxina Shiga II/metabolismo , Espectrometria de Massas em TandemRESUMO
Toxin expression is a key factor in Shiga toxin (Stx)-producing E. coli, a common pathogen involved in foodborne disease outbreaks. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) based approach has been used in this study to identify commonly reported E. coli toxins, with a focus on Shiga toxins (Stxs). Different sample preparation methods using variable culture conditions and concentrations of mitomycin C (MMC), a common antibiotic/chemotherapy agent capable of stimulating Stx production, were first tested on reference strains EDL933 and 90-2380 by LC-MS/MS detection of tryptic digests of receptor-analogue affinity binding enriched Stx preparations from culture supernatants and lysates. A curated E. coli protein toxin database was also used for faster and more straightforward toxin identification. With eight more genetically confirmed E. coli strains examined to verify the method, this preliminary study indicates that receptor-analogue based affinity enrichment on cell lysate or supernatant is a sensitive and accurate method for Stx identification. BIOLOGICAL SIGNIFICANCE: The existence of Stx is very important for identifying Stx-producing E. coli and implementing a clinical treatment regime. This study demonstrates for the first time that using a curated E. coli toxin database, together with receptor-analogue-based affinity enrichment of Stxs after MMC treatment of E. coli, is an easy and appropriate approach for fast and accurate Stx identification through LC-MS/MS.
Assuntos
Bases de Dados de Proteínas , Proteínas de Escherichia coli/metabolismo , Toxina Shiga/metabolismo , Escherichia coli Shiga Toxigênica/metabolismo , Cromatografia Líquida , Espectrometria de Massas em TandemRESUMO
Biochemical methods such as metabolite testing and serotyping are traditionally used in clinical microbiology laboratories to identify and categorize microorganisms. Due to the large variety of bacteria, identifying representative metabolites is tedious, while raising high-quality antisera or antibodies unique to specific biomarkers used in serotyping is very challenging, sometimes even impossible. Although serotyping is a certified approach for differentiating bacteria such as E. coli and Salmonella at the subspecies level, the method is tedious, laborious, and not practical during an infectious disease outbreak. Mass spectrometry (MS) platforms, especially matrix assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS), have recently become popular in the field of bacterial identification due to their fast speed and low cost. In the past few years, we have used liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches to solve various problems hindering serotyping and have overcome some insufficiencies of the MALDI-TOF-MS platform. The current article aims to review the characteristics, advantages, and disadvantages of MS-based platforms over traditional approaches in bacterial identification and categorization.