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The tRNA (m1G37) methyltransferase TrmD catalyzes m1G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa (PaTrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of PaTrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates. Analysis of steady-state kinetics with S-adenosyl-l-methionine (SAM) and tRNALeu(GAG) showed that PaTrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNALeu(GAG) bind to PaTrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM (Ki = 0.41 ± 0.07 µM) and uncompetitive for tRNA (Ki = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric PaTrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into PaTrmD as a potential target for the development of antibiotics.
Assuntos
Pseudomonas aeruginosa/enzimologia , tRNA Metiltransferases/metabolismo , Catálise , Cristalografia por Raios X , Cinética , Ligação Proteica , Conformação Proteica , RNA de Transferência/metabolismo , S-Adenosilmetionina/metabolismo , Especificidade por Substrato , tRNA Metiltransferases/química , tRNA Metiltransferases/isolamento & purificaçãoRESUMO
The misincorporation of 2'-deoxyribonucleotides (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fidelity of coding RNAs and the mutagenic evolution of viral RNA genomes. However, quantitative appreciation for the degree to which dN misincorporation occurs is limited by the lack of analytical tools. Here, we report a method to hydrolyze RNA to release 2'-deoxyribonucleotide-ribonucleotide pairs (dNrN) that are then quantified by chromatography-coupled mass spectrometry (LC-MS). Using this platform, we found misincorporated dNs occurring at 1 per 103 to 105 ribonucleotide (nt) in mRNA, rRNAs and tRNA in human cells, Escherichia coli, Saccharomyces cerevisiae and, most abundantly, in the RNA genome of dengue virus. The frequency of dNs varied widely among organisms and sequence contexts, and partly reflected the in vitro discrimination efficiencies of different RNA polymerases against 2'-deoxyribonucleoside 5'-triphosphates (dNTPs). Further, we demonstrate a strong link between dN frequencies in RNA and the balance of dNTPs and ribonucleoside 5'-triphosphates (rNTPs) in the cellular pool, with significant stress-induced variation of dN incorporation. Potential implications of dNs in RNA are discussed, including the possibilities of dN incorporation in RNA as a contributing factor in viral evolution and human disease, and as a host immune defense mechanism against viral infections.
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Composição de Bases , Desoxirribonucleotídeos/química , RNA/química , RNA/genética , Ribonucleotídeos , Estresse Fisiológico/genética , Animais , Linhagem Celular , Cromatografia Líquida , Células Eucarióticas/metabolismo , Humanos , Hidrólise , Mamíferos , Mutagênese , Células Procarióticas/metabolismo , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Viral/química , RNA Viral/genética , Espectrometria de Massas em TandemRESUMO
Bacteria respond to environmental stresses using a variety of signaling and gene expression pathways, with translational mechanisms being the least well understood. Here, we identified a tRNA methyltransferase in Pseudomonas aeruginosa PA14, trmJ, which confers resistance to oxidative stress. Analysis of tRNA from a trmJ mutant revealed that TrmJ catalyzes formation of Cm, Um, and, unexpectedly, Am. Defined in vitro analyses revealed that tRNAMet(CAU) and tRNATrp(CCA) are substrates for Cm formation, tRNAGln(UUG), tRNAPro(UGG), tRNAPro(CGG) and tRNAHis(GUG) for Um, and tRNAPro(GGG) for Am. tRNASer(UGA), previously observed as a TrmJ substrate in Escherichia coli, was not modified by PA14 TrmJ. Position 32 was confirmed as the TrmJ target for Am in tRNAPro(GGG) and Um in tRNAGln(UUG) by mass spectrometric analysis. Crystal structures of the free catalytic N-terminal domain of TrmJ show a 2-fold symmetrical dimer with an active site located at the interface between the monomers and a flexible basic loop positioned to bind tRNA, with conformational changes upon binding of the SAM-analog sinefungin. The loss of TrmJ rendered PA14 sensitive to H2O2 exposure, with reduced expression of oxyR-recG, katB-ankB, and katE These results reveal that TrmJ is a tRNA:Cm32/Um32/Am32 methyltransferase involved in translational fidelity and the oxidative stress response.
Assuntos
Proteínas de Bactérias/química , Estresse Oxidativo , Pseudomonas aeruginosa/enzimologia , RNA de Transferência/metabolismo , tRNA Metiltransferases/química , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Sequência de Bases , Domínio Catalítico , Cristalografia por Raios X , Peróxido de Hidrogênio/farmacologia , Metilação , Modelos Moleculares , Pseudomonas aeruginosa/efeitos dos fármacos , RNA Bacteriano/química , tRNA Metiltransferases/fisiologiaRESUMO
A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria.
Assuntos
Mycobacterium bovis/genética , RNA Bacteriano/genética , RNA Ribossômico 5S/genética , RNA não Traduzido/genética , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão/métodos , Processamento Pós-Transcricional do RNA , RNA Bacteriano/isolamento & purificação , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/isolamento & purificação , RNA Ribossômico 5S/isolamento & purificação , RNA de Transferência/genética , RNA de Transferência/isolamento & purificação , RNA não Traduzido/isolamento & purificação , Reprodutibilidade dos Testes , Ribonucleosídeos/genéticaRESUMO
A renewed interest in non-coding RNA (ncRNA) has led to the discovery of novel RNA species and post-transcriptional ribonucleoside modifications, and an emerging appreciation for the role of ncRNA in RNA epigenetics. Although much can be learned by amplification-based analysis of ncRNA sequence and quantity, there is a significant need for direct analysis of RNA, which has led to numerous methods for purification of specific ncRNA molecules. However, no single method allows purification of the full range of cellular ncRNA species. To this end, we developed a multidimensional chromatographic platform to resolve, isolate and quantify all canonical ncRNAs in a single sample of cells or tissue, as well as novel ncRNA species. The applicability of the platform is demonstrated in analyses of ncRNA from bacteria, human cells and plasmodium-infected reticulocytes, as well as a viral RNA genome. Among the many potential applications of this platform are a system-level analysis of the dozens of modified ribonucleosides in ncRNA, characterization of novel long ncRNA species, enhanced detection of rare transcript variants and analysis of viral genomes.
Assuntos
RNA não Traduzido/isolamento & purificação , Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Fluorometria , Humanos , MicroRNAs/isolamento & purificação , Mycobacterium bovis/genética , Plasmodium berghei/genética , RNA Bacteriano/isolamento & purificação , RNA de Protozoário/isolamento & purificação , RNA Ribossômico/isolamento & purificação , RNA de Transferência/isolamento & purificação , RNA Viral/isolamento & purificaçãoRESUMO
Mycobacteria adapt to infection stresses by entering a reversible non-replicating persistence (NRP) with slow or no cell growth and broad antimicrobial tolerance. Hypoxia and nutrient deprivation are two well-studied stresses commonly used to model the NRP, yet little is known about the molecular differences in mycobacterial adaptation to these distinct stresses that lead to a comparable NRP phenotype. Here we performed a multisystem interrogation of the Mycobacterium bovis BCG (BCG) starvation response, which revealed a coordinated metabolic shift away from the glycolysis of nutrient-replete growth to depletion of lipid stores, lipolysis, and fatty acid ß-oxidation in NRP. This contrasts with BCG's NRP hypoxia response involving a shift to cholesterol metabolism and triglyceride storage. Our analysis reveals cryptic metabolic vulnerabilities of the starvation-induced NRP state, such as their newfound hypersensitivity to H2O2. These observations pave the way for developing precision therapeutics against these otherwise drug refractory pathogens.
Assuntos
Adaptação Fisiológica , Mycobacterium bovis , Mycobacterium bovis/metabolismo , Glicólise , Reprogramação MetabólicaRESUMO
Rapid and sensitive detection of pathogens in various samples is crucial for disease diagnosis, environmental surveillance, as well as food and water safety monitoring. However, the low abundance of pathogens (<10 CFU) in large volume (1 mL-1 L) samples containing vast backgrounds critically limits the sensitivity of even the most advanced techniques, such as digital PCR. Therefore, there is a critical need for sample preparation that can enrich low-abundance pathogens from complex and large-volume samples. This study develops an efficient electrostatic microfiltration (EM)-based sample preparation technique capable of processing ultra-large-volume (≥500 mL) samples at high throughput (≥10 mL min-1). This approach achieves a significant enrichment (>8000×) of extremely-low-abundance pathogens (down to level of 0.02 CFU mL-1, i.e., 10 CFU in 500 mL). Furthermore, EM-enabled sample preparation facilitates digital amplification techniques sensitively detecting broad pathogens, including bacteria, fungi, and viruses from various samples, in a rapid (≤3 h) sample-to-result workflow. Notably, the operational ease, portability, and compatibility/integrability with various downstream detection platforms highlight its great potential for widespread applications across diverse settings.
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The abundance and low production cost of biomaterial cellulose paper have attracted attention for many applications. Point-of-care (PoC) diagnostic tests have been successfully developed using patterned cellulose paper. Although PoC diagnostic tests are rapid and simple to perform, their sample processing throughput is limited, allowing for only one sample to be evaluated at a time, which restricts potential applications. Thus, it was appealing to expand cellulose-based PoC tests to high-throughput versions to increase their applicability. Here, we present the development of a high-throughput cellulose-based 96-well plate vertical flow pull-down assay that can process 96 tests, is easy to prepare, and can be customized for different detection targets. The device has two key features: (i) patterned cellulose paper for 96 tests that do not require pre-immobilization of capturing reagents, and (ii) reusable sturdy housing. We believe that a variety of applications, including laboratory testing, population surveillance tests, and sizable clinical trials for diagnostic tests, can benefit from the adoption of this cellulose-based 96-well plate assay.
Assuntos
Celulose , Testes ImediatosRESUMO
Helicobacter cinaedi, a common human intestinal bacterium, has been implicated in various enteric and systemic diseases in normal and immunocompromised patients. Protection against oxidative stress is a crucial component of bacterium-host interactions. Alkyl hydroperoxide reductase C (AhpC) is an enzyme responsible for detoxification of peroxides and is important in protection from peroxide-induced stress. H. cinaedi possesses a single ahpC, which was investigated with respect to its role in bacterial survival during oxidative stress. The H. cinaedi ahpC mutant had diminished resistance to organic hydroperoxide toxicity but increased hydrogen peroxide resistance compared with the wild-type (WT) strain. The mutant also exhibited an oxygen-sensitive phenotype and was more susceptible to killing by macrophages than the WT strain. In vivo experiments in BALB/c and BALB/c interleukin-10 (IL-10)(-/-) mice revealed that the cecal colonizing ability of the ahpC mutant was significantly reduced. The mutant also had diminished ability to induce bacterium-specific immune responses in vivo, as shown by immunoglobulin (IgG2a and IgG1) serum levels. Collectively, these data suggest that H. cinaedi ahpC not only contributes to protecting the organism against oxidative stress but also alters its pathogenic properties in vivo.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter/patogenicidade , Interleucina-10/imunologia , Viabilidade Microbiana , Estresse Oxidativo , Peroxidases/metabolismo , Estresse Fisiológico , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Ceco/microbiologia , Feminino , Deleção de Genes , Helicobacter/efeitos dos fármacos , Helicobacter/enzimologia , Interações Hospedeiro-Patógeno , Peróxido de Hidrogênio/toxicidade , Interleucina-10/deficiência , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Oxigênio/toxicidade , Peroxidases/genética , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
As the COVID-19 pandemic continues, countries around the world are switching toward vaccinations and boosters to combat the pandemic. However, waning immunity against SARS-CoV-2 wild-type (WT) and variants have been widely reported. Booster vaccinations have shown to be able to increase immunological protection against new variants; however, the protection observed appears to decrease quickly over time suggesting a second booster shot may be appropriate. Moreover, heterogeneity and waning of the immune response at the individual level was observed suggesting a more personalized vaccination approach should be considered. To evaluate such a personalized strategy, it is important to have the ability to rapidly evaluate the level of neutralizing antibody (nAbs) response against variants at the individual level and ideally at a point of care setting. Here, we applied the recently developed cellulose pulled-down virus neutralization test (cpVNT) to rapidly assess individual nAb levels to WT and variants of concerns in response to booster vaccination. Our findings confirmed significant heterogeneity of nAb responses against a panel of SARS-CoV-2 variants, and indicated a strong increase in nAb response against variants of concern (VOCs) upon booster vaccination. For instance, the nAb response against current predominant omicron variant was observed with medians of 88.1% (n = 6, 95% CI = 73.2% to 96.2%) within 1-month postbooster and 70.7% (n = 22, 95% CI = 66.4% to 81.8%) 3 months postbooster. Our data show a point of care (POC) test focusing on nAb response levels against VOCs can guide decisions on the potential need for booster vaccinations at individual level. Importantly, it also suggests the current booster vaccines only give a transient protective response against some VOC and new more targeted formulations of a booster vaccine against specific VOC may need to be developed in the future. IMPORTANCE Vaccination against SARS-CoV-2 induces protection through production of neutralization antibodies (nAb). The level of nAb is a major indicator of immunity against SARS-CoV-2 infection. We developed a rapid point-of-care test that can monitor the nAb level from a drop of finger stick blood. Here, we have implemented the test to monitor individual nAb level against wild-type and variants of SARS-CoV-2 at various time points of vaccination, including post-second-dose vaccination and postbooster vaccination. Huge diversity of nAb levels were observed among individuals as well as increment in nAb levels especially against Omicron variant after booster vaccination. This study evaluated the performance of this point-of-care test for personalized nAb response tracking. It verifies the potential of using a rapid nAb test to guide future vaccination regimens at both the individual and population level.
Assuntos
COVID-19 , Vacinas , Humanos , SARS-CoV-2/genética , Anticorpos Antivirais , Pandemias , COVID-19/prevenção & controle , Anticorpos Neutralizantes , VacinaçãoRESUMO
Surveillance of SARS-CoV-2 infection is critical for controlling the current pandemic. Antigen rapid tests (ARTs) provide a means for surveillance. Available lateral flow assay format ARTs rely heavily on nitrocellulose paper, raising challenges in supply shortage. Vertical flow assay (VFA) with cellulose paper as test material attracts much attention as a complementary test approach. However, current reported VFAs are facing challenges in reading the test signal from the bottom face of the test cassette, complicating the test workflow and hindering translation into rapid test application. Here, we address this gap with an enhanced VFA against SARS-CoV-2 N protein that adapts a cellulose pull-down test format allowing (1) one-step sample application at the top of the test cassette and (2) readout of the test signal from the top. We also demonstrate the feasibility of translating the enhanced VFA into a point-of-care application that can help in SARS-CoV-2 surveillance.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Humanos , Pandemias , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e EspecificidadeRESUMO
There is clinical need for a quantifiable point-of-care (PoC) SARS-CoV-2 neutralizing antibody (nAb) test that is adaptable with the pandemic's changing landscape. Here, we present a rapid and semi-quantitative nAb test that uses finger stick or venous blood to assess the nAb response of vaccinated population against wild-type (WT), alpha, beta, gamma, and delta variant RBDs. It captures a clinically relevant range of nAb levels, and effectively differentiates prevaccination, post first dose, and post second dose vaccination samples within 10 min. The data observed against alpha, beta, gamma, and delta variants agrees with published results evaluated in established serology tests. Finally, our test revealed a substantial reduction in nAb level for beta, gamma, and delta variants between early BNT162b2 vaccination group (within 3 months) and later vaccination group (post 3 months). This test is highly suited for PoC settings and provides an insightful nAb response in a postvaccinated population.
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Background: Neutralizing antibodies (NAbs) prevent pathogens from infecting host cells. Detection of SARS-CoV-2 NAbs is critical to evaluate herd immunity and monitor vaccine efficacy against SARS-CoV-2, the virus that causes COVID-19. All currently available NAb tests are lab-based and time-intensive. Method: We develop a 10 min cellulose pull-down test to detect NAbs against SARS-CoV-2 from human plasma. The test evaluates the ability of antibodies to disrupt ACE2 receptor-RBD complex formation. The simple, portable, and rapid testing process relies on two key technologies: (i) the vertical-flow paper-based assay format and (ii) the rapid interaction of cellulose binding domain to cellulose paper. Results: Here we show the construction of a cellulose-based vertical-flow test. The developed test gives above 80% sensitivity and specificity and up to 93% accuracy as compared to two current lab-based methods using COVID-19 convalescent plasma. Conclusions: A rapid 10 min cellulose based test has been developed for detection of NAb against SARS-CoV-2. The test demonstrates comparable performance to the lab-based tests and can be used at Point-of-Care. Importantly, the approach used for this test can be easily extended to test RBD variants or to evaluate NAbs against other pathogens.
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BACKGROUND & AIMS: The commensal microbiota is believed to have an important role in regulating immune responsiveness and preventing intestinal inflammation. Intestinal microbes produce signals that regulate inflammation via Toll-like receptor (TLR) signaling, but the mechanisms of this process are poorly understood. We investigated the role of the anti-inflammatory cytokine interleukin (IL)-10 in this signaling pathway using a mouse model of colitis. METHODS: Clinical, histopathologic, and functional parameters of intestinal inflammation were evaluated in TLR4(-/-), IL-10(-/-), and TLR4(-/-) x IL-10(-/-) mice that were free of specific pathogens and in TLR4(-/-) x IL-10(-/-) mice following eradication and reintroduction of Helicobacter hepaticus. Regulatory T-cell (Treg) function was evaluated by crossing each of the lines with transgenic mice that express green fluorescent protein under control of the endogenous regulatory elements of Foxp3. Apoptotic cells in the colonic lamina propria were detected by a TUNEL assay. RESULTS: TLR4-mediated signals have 2 interrelated roles in promoting inflammation in TLR4(-/-) x IL-10(-/-) mice. In the absence of TLR4-mediated signals, secretion of proinflammatory and immunoregulatory cytokines is dysregulated. Tregs (Foxp3(+)) that secrete interferon-gamma and IL-17 accumulate in the colonic lamina propria of TLR4(-/-) x IL-10(-/-) mice and do not prevent inflammation. Aberrant control of epithelial cell turnover results in the persistence of antigen-presenting cells that contain apoptotic epithelial fragments in the colonic lamina propria of Helicobacter-infected TLR4(-/-) mice. CONCLUSIONS: In mice that lack both IL-10- and TLR4-mediated signals, aberrant regulatory T-cell function and dysregulated control of epithelial homeostasis combine to exacerbate intestinal inflammation.
Assuntos
Colite/imunologia , Células Epiteliais/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter hepaticus/imunologia , Mediadores da Inflamação/metabolismo , Interleucina-10/deficiência , Linfócitos T Reguladores/imunologia , Receptor 4 Toll-Like/metabolismo , Animais , Apoptose , Colite/microbiologia , Colite/patologia , Colite/prevenção & controle , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Fatores de Transcrição Forkhead/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/patologia , Interferon gama/metabolismo , Interleucina-10/genética , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prolapso Retal/imunologia , Prolapso Retal/microbiologia , Baço/imunologia , Baço/microbiologia , Linfócitos T Reguladores/microbiologia , Células Th1/imunologia , Células Th1/microbiologia , Fatores de Tempo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genéticaRESUMO
Among the >120 modified ribonucleosides in the prokaryotic epitranscriptome, many tRNA modifications are critical to bacterial survival, which makes their synthetic enzymes ideal targets for antibiotic development. Here we performed a structure-based design of inhibitors of tRNA-(N1G37) methyltransferase, TrmD, which is an essential enzyme in many bacterial pathogens. On the basis of crystal structures of TrmDs from Pseudomonas aeruginosa and Mycobacterium tuberculosis, we synthesized a series of thienopyrimidinone derivatives with nanomolar potency against TrmD in vitro and discovered a novel active site conformational change triggered by inhibitor binding. This tyrosine-flipping mechanism is uniquely found in P. aeruginosa TrmD and renders the enzyme inaccessible to the cofactor S-adenosyl-l-methionine (SAM) and probably to the substrate tRNA. Biophysical and biochemical structure-activity relationship studies provided insights into the mechanisms underlying the potency of thienopyrimidinones as TrmD inhibitors, with several derivatives found to be active against Gram-positive and mycobacterial pathogens. These results lay a foundation for further development of TrmD inhibitors as antimicrobial agents.
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Inibidores Enzimáticos/farmacologia , Pirimidinas/farmacologia , Tirosina/farmacologia , tRNA Metiltransferases/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Pseudomonas aeruginosa/enzimologia , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade , Tirosina/química , tRNA Metiltransferases/metabolismoRESUMO
Acute diarrheal illness is a global health problem that may be exacerbated by concurrent infection. Using Citrobacter rodentium, a murine model of attaching and effacing diarrheagenic Escherichia coli, we demonstrate that persistent Helicobacter hepaticus infection modulates host responses to diarrheal disease, resulting in delayed recovery from weight loss and from tissue damage. Chronic colitis in concurrently infected mice is characterized by macrophage and Foxp3(+) regulatory T-cell accumulation. Prolonged disease is also associated with increased interleukin-17 expression, which may be due to suppression of gamma interferon during the acute phase of diarrheal infection. This new model of polymicrobial infection provides insight into the mechanism by which subclinical infection can exacerbate morbidity due to an unrelated self-limiting infection.
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Disenteria/microbiologia , Infecções por Enterobacteriaceae/complicações , Infecções por Helicobacter/complicações , Animais , Citrobacter rodentium , Colite/imunologia , Colite/microbiologia , Citocinas/biossíntese , Citocinas/imunologia , Modelos Animais de Doenças , Disenteria/imunologia , Disenteria/patologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/patologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/patologia , Helicobacter hepaticus , Imuno-Histoquímica , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologiaRESUMO
PURPOSE OF REVIEW: There have been considerable advances in our understanding of the molecular pathogenesis of enteropathogenic Escherichia coli and enterohemorrhagic E. coli infection. Given the difficulty of infecting laboratory mice with these diarrhea-causing pathogens, a growing number of studies have found the murine bacterial pathogen Citrobacter rodentium to provide a robust, relevant in-vivo model system. RECENT FINDINGS: All inbred strains and outbred stocks of laboratory mice studied to date have been found to be susceptible to C. rodentium infection. The natural course of disease ranges from subclinical epithelial hyperplasia in the colon, to clinical diarrhea and colitis, to fatal infection, depending on the age, genetic background, and health status of the host. Infection is self-limiting, leading to disease resolution and protective immunity. Here we review recent discoveries related to bacterial virulence determinants, epithelial hyperplasia, innate and adaptive immune responses, and mechanisms of diarrhea. SUMMARY: Infection of laboratory mice with C. rodentium provides a useful in-vivo model for studying the pathogenesis of infectious gastroenteritis and acute diarrheal illness, and for preclinical evaluation of candidate preventive and therapeutic agents.
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Citrobacter rodentium/patogenicidade , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Animais , Diarreia/microbiologia , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Camundongos , Camundongos Endogâmicos , Probióticos , VirulênciaRESUMO
The role of reactive oxygen species (ROS) in microbial metabolism and stress response has emerged as a major theme in microbiology and infectious disease. Reactive fluorescent dyes have the potential to advance the study of ROS in the complex intracellular environment, especially for high-content and high-throughput analyses. However, current dye-based approaches to measuring intracellular ROS have the potential for significant artifacts. Here, we describe a robust platform for flow cytometric quantification of ROS in bacteria using fluorescent dyes, with ROS measurements in 10s-of-1000s of individual cells under a variety of conditions. False positives and variability among sample types (e.g., bacterial species, stress conditions) are reduced with a flexible four-step gating scheme that accounts for side- and forward-scattered light (morphological changes), background fluorescence, DNA content, and dye uptake to identify cells producing ROS. Using CellROX Green dye with Escherichia coli, Mycobacterium smegmatis, and Mycobacterium bovis BCG as diverse model bacteria, we show that (1) the generation of a quantifiable CellROX Green signal for superoxide, but not hydrogen peroxide-induced hydroxyl radicals, validates this dye as a superoxide detector; (2) the level of dye-detectable superoxide does not correlate with cytotoxicity or antibiotic sensitivity; (3) the non-replicating, antibiotic tolerant state of nutrient-deprived mycobacteria is associated with high levels of superoxide; and (4) antibiotic-induced production of superoxide is idiosyncratic with regard to both the species and the physiological state of the bacteria. We also show that the gating method is applicable to other fluorescent indicator dyes, such as the 5-carboxyfluorescein diacetate acetoxymethyl ester and 5-cyano-2,3-ditolyl tetrazolium chloride for cellular esterase and reductive respiratory activities, respectively. These results demonstrate that properly controlled flow cytometry coupled with fluorescent probes provides precise and accurate quantitative analysis of ROS generation and metabolic changes in stressed bacteria.
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Here we describe an analytical platform for systems-level quantitative analysis of modified ribonucleosides in any RNA species, with a focus on stress-induced reprogramming of tRNA as part of a system of translational control of cell stress response. This chapter emphasizes strategies and caveats for each of the seven steps of the platform workflow: (1) RNA isolation, (2) RNA purification, (3) RNA hydrolysis to individual ribonucleosides, (4) chromatographic resolution of ribonucleosides, (5) identification of the full set of modified ribonucleosides, (6) mass spectrometric quantification of ribonucleosides, (6) interrogation of ribonucleoside datasets, and (7) mapping the location of stress-sensitive modifications in individual tRNA molecules. We have focused on the critical determinants of analytical sensitivity, specificity, precision, and accuracy in an effort to ensure the most biologically meaningful data on mechanisms of translational control of cell stress response. The methods described here should find wide use in virtually any analysis involving RNA modifications.
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Espectrometria de Massas/métodos , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/química , Ribonucleosídeos/química , Biossíntese de Proteínas/genética , RNA de Transferência/genética , Ribonucleosídeos/genéticaRESUMO
Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress-response mechanism involving selective translation of codon-biased mRNA for crucial proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography-mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA. The protocol includes tRNA purification by HPLC, enzymatic hydrolysis, reversed-phase HPLC resolution of the ribonucleosides, and identification and quantification of individual ribonucleosides by LC-MS via dynamic multiple reaction monitoring (DMRM). In this approach, the relative proportions of modified ribonucleosides are quantified in several micrograms of tRNA in a 15-min LC-MS run. This protocol can be modified to analyze other types of RNA by modifying the steps for RNA purification as appropriate. By comparison, traditional methods for detecting modified ribonucleosides are labor- and time-intensive, they require larger RNA quantities, they are modification-specific or require radioactive labeling.