RESUMO
Measles virus (MeV) is a member of the family Paramixoviridae that causes a highly contagious respiratory disease but has emerged as a promising oncolytic platform. Previous studies of MeV entry focused on the identification of cellular receptors. However, the endocytic and trafficking pathways utilized during MeV entry remain poorly described. The contribution of each endocytic pathway has been examined in cells that express the MeV receptors SLAM (signaling lymphocyte-activating molecule) and PVRL4 (poliovirus receptor-like 4) (nectin-4). Recombinant MeVs expressing either firefly luciferase or green fluorescent protein together with a variety of inhibitors were used. The results showed that MeV uptake was dynamin independent in the Vero.hPVRL4, Vero.hSLAM, and PVRL4-positive MCF7 breast cancer cell lines. However, MeV infection was blocked by 5-(N-ethyl-N-propyl)amiloride (EIPA), the hallmark inhibitor of macropinocytosis, as well as inhibitors of actin polymerization. By using phalloidin staining, MeV entry was shown to induce actin rearrangements and the formation of membrane ruffles accompanied by transient elevated fluid uptake. Small interfering RNA (siRNA) knockdown of p21-activated kinase 1 (PAK1) demonstrated that MeV enters both Vero.hPVRL4 and Vero.hSLAM cells in a PAK1-independent manner using a macropinocytosis-like pathway. In contrast, MeV entry into MCF7 human breast cancer cells relied upon Rac1 and its effector PAK1 through a PVRL4-mediated macropinocytosis pathway. MeV entry into DLD-1 colon and HTB-20 breast cancer cells also appeared to use the same pathway. Overall, these findings provide new insight into the life cycle of MeV, which could lead to therapies that block virus entry or methods that improve the uptake of MeV by cancer cells during oncolytic therapy.IMPORTANCE In the past decades, measles virus (MeV) has emerged as a promising oncolytic platform. Previous studies concerning MeV entry focused mainly on the identification of putative receptors for MeV. Nectin-4 (PVRL4) was recently identified as the epithelial cell receptor for MeV. However, the specific endocytic and trafficking pathways utilized during MeV infections are poorly documented. In this study, we demonstrated that MeV enters host cells via a dynamin-independent and actin-dependent endocytic pathway. Moreover, we show that MeV gains entry into MCF7, DLD-1, and HTB-20 cancer cells through a PVRL4-mediated macropinocytosis pathway and identified the typical cellular GTPase and kinase involved. Our findings provide new insight into the life cycle of MeV, which may lead to the development of therapies that block the entry of the virus into the host cell or alternatively promote the uptake of oncolytic MeV into cancer cells.
Assuntos
Moléculas de Adesão Celular/metabolismo , Vírus do Sarampo/fisiologia , Pinocitose , Internalização do Vírus , Actinas/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Neoplasias da Mama , Linhagem Celular , Chlorocebus aethiops , Neoplasias do Colo , Células Epiteliais/virologia , Feminino , Humanos , Células MCF-7 , Vírus do Sarampo/efeitos dos fármacos , Vírus do Sarampo/genética , Vírus Oncolíticos/fisiologia , Pinocitose/efeitos dos fármacos , RNA Interferente Pequeno/genética , Células Vero , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismoRESUMO
The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein.IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z-d-Phe-l-Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion.
Assuntos
Vírus do Sarampo/efeitos dos fármacos , Vírus do Sarampo/genética , Mutação , Oligopeptídeos/farmacologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Animais , Antivirais/farmacologia , Benzamidas/farmacologia , Chlorocebus aethiops , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Fusão de Membrana/efeitos dos fármacos , Modelos Moleculares , Ligação Proteica , Células Vero , Proteínas Virais de Fusão/química , Internalização do Vírus/efeitos dos fármacosRESUMO
Vaccine and laboratory adapted strains of measles virus can use CD46 as a receptor to infect many human cell lines. However, wild type isolates of measles virus cannot use CD46, and they infect activated lymphocytes, dendritic cells, and macrophages via the receptor CD150/SLAM. Wild type virus can also infect epithelial cells of the respiratory tract through an unidentified receptor. We demonstrate that wild type measles virus infects primary airway epithelial cells grown in fetal calf serum and many adenocarcinoma cell lines of the lung, breast, and colon. Transfection of non-infectable adenocarcinoma cell lines with an expression vector encoding CD150/SLAM rendered them susceptible to measles virus, indicating that they were virus replication competent, but lacked a receptor for virus attachment and entry. Microarray analysis of susceptible versus non-susceptible cell lines was performed, and comparison of membrane protein gene transcripts produced a list of 11 candidate receptors. Of these, only the human tumor cell marker PVRL4 (Nectin 4) rendered cells amenable to measles virus infections. Flow cytometry confirmed that PVRL4 is highly expressed on the surfaces of susceptible lung, breast, and colon adenocarcinoma cell lines. Measles virus preferentially infected adenocarcinoma cell lines from the apical surface, although basolateral infection was observed with reduced kinetics. Confocal immune fluorescence microscopy and surface biotinylation experiments revealed that PVRL4 was expressed on both the apical and basolateral surfaces of these cell lines. Antibodies and siRNA directed against PVRL4 were able to block measles virus infections in MCF7 and NCI-H358 cancer cells. A virus binding assay indicated that PVRL4 was a bona fide receptor that supported virus attachment to the host cell. Several strains of measles virus were also shown to use PVRL4 as a receptor. Measles virus infection reduced PVRL4 surface expression in MCF7 cells, a property that is characteristic of receptor-associated viral infections.
Assuntos
Biomarcadores Tumorais/imunologia , Moléculas de Adesão Celular/metabolismo , Células Epiteliais/metabolismo , Vírus do Sarampo/patogenicidade , Receptores Virais/metabolismo , Animais , Moléculas de Adesão Celular/genética , Linhagem Celular , Células Dendríticas/imunologia , Células Dendríticas/virologia , Regulação para Baixo , Células Epiteliais/citologia , Regulação da Expressão Gênica , Humanos , Linfócitos/imunologia , Linfócitos/virologia , Vírus do Sarampo/imunologia , Camundongos , Análise em Microsséries , Microscopia Confocal/métodos , RNA Interferente Pequeno/metabolismo , Ligação Viral , Replicação ViralRESUMO
CD8 T cells play a central role in antiviral immunity. Type I interferons are among the earliest responders after virus exposure and can cause extensive reprogramming and antigen-independent bystander activation of CD8 T cells. Although bystander activation of pre-existing memory CD8 T cells is known to play an important role in host defense and immunopathology, its impact on naïve CD8 T cells remains underappreciated. Here we report that exposure to reovirus, both in vitro or in vivo, promotes bystander activation of naïve CD8 T cells within 24 hours and that this distinct subtype of CD8 T cell displays an innate, antiviral, type I interferon sensitized signature. The induction of bystander naïve CD8 T cells is STAT1 dependent and regulated through nicotinamide phosphoribosyl transferase (NAMPT)-mediated enzymatic actions within NAD+ salvage metabolic biosynthesis. These findings identify a novel aspect of CD8 T cell activation following virus infection with implications for human health and physiology.
Assuntos
NAD , Viroses , Humanos , Linfócitos T CD8-Positivos , Antígenos , AntiviraisRESUMO
Hepatitis C virus (HCV) infection causes significant morbidity, and efficient mouse models would greatly facilitate virus studies and the development of effective vaccines and new therapeutic agents. Entry factors, innate immunity, and host factors needed for viral replication represent the initial barriers that restrict HCV infection of mouse cells. Experiments in this paper consider early postentry steps of viral infection and investigate the roles of interferon regulatory factors (IRF-3 and IRF-9) and microRNA (miR-122) in promoting HCV replication in mouse embryo fibroblasts (MEFs) that contain viral subgenomic replicons. While wild-type murine fibroblasts are restricted for HCV RNA replication, deletion of IRF-3 alone can facilitate replicon activity in these cells. This effect is thought to be related to the inactivation of the type I interferon synthesis mediated by IRF-3. Additional deletion of IRF-9 to yield IRF-3(-/-) IRF-9(-/-) MEFs, which have blocked type I interferon signaling, did not increase HCV replication. Expression of liver-specific miR-122 in MEFs further stimulated the synthesis of HCV replicons in the rodent fibroblasts. The combined effects of miR-122 expression and deletion of IRF-3 produced a cooperative stimulation of HCV subgenome replication. miR-122 and IRF-3 are independent host factors that are capable of influencing HCV replication, and our findings could help to establish mouse models and other cell systems that support HCV growth and particle formation.
Assuntos
Fibroblastos/virologia , Hepacivirus/imunologia , Hepacivirus/fisiologia , Fator Regulador 3 de Interferon/deficiência , MicroRNAs/biossíntese , Replicação Viral , Animais , Fator Regulador 3 de Interferon/imunologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/deficiência , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/imunologia , Camundongos , Camundongos Knockout , Deleção de SequênciaRESUMO
Understanding the efficacy and durability of heterologous immunization schedules against SARS-CoV-2 is critical, as supply demands and vaccine choices become significant issues in the global vaccination strategy. Here we characterize the neutralizing antibodies produced in two subjects who received combination immunizations against SARS-CoV-2, first with Covishield (Oxford-AstraZeneca) vaccine, followed 33 days later with a second dose (booster) shot of the Pfizer-BioNTech vaccine. Serum samples were collected 25 days following the primary vaccination and 13 days after the secondary Pfizer vaccination. Both subjects exhibited increased levels of isotype IgG and IgM antibodies directed against the entire spike protein following immunizations. These antibodies also exhibited increased reactivity with the receptor binding domain (RBD) in the spike protein and neutralized the infectivity of replicating vesicular stomatitis virus (VSV) that contains the COVID-19 coronavirus S protein gene in place of its normal G glycoprotein. This VSV pseudovirus also contains the reporter gene for enhanced green fluorescent protein (eGFP). Antibody titers against the spike protein and serum neutralization titers against the reporter virus are reported for the 2 heterologous vaccinated individuals and compared to a positive control derived from a convalescent patient and a negative control from an unexposed individual. The Pfizer-BioNTech vaccine increased antibody binding to the spike protein and RBD, and approached levels found in the convalescent positive control. Neutralizing antibodies against the VSV-S pseudovirus in the 2 subjects also approached levels in the convalescent sera. These results firmly validate the value of the Pfizer-BioNTech vaccine in boosting immunity following initial Covishield inoculation.
Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Imunidade Humoral/efeitos dos fármacos , Anticorpos Neutralizantes/imunologia , COVID-19/prevenção & controle , Estudos de Casos e Controles , Feminino , Humanos , Masculino , SARS-CoV-2RESUMO
Streptococcus mutans in dental biofilms is regularly exposed to cycles of acidic pH during the ingestion of fermentable dietary carbohydrates. The ability of S. mutans to tolerate low pH is crucial for its virulence and pathogenesis in dental caries. To better understand its acid tolerance mechanisms, we performed genome-wide transcriptional analysis of S. mutans in response to an acidic pH signal. The preliminary results showed that adaptation of S. mutans to pH 5.5 induced differential expression of nearly 14 % of the genes in the genome, including 169 upregulated genes and 108 downregulated genes, largely categorized into nine functional groups. One of the most interesting findings was that the genes encoding multiple two-component systems (TCSs), including CiaHR, LevSR, LiaSR, ScnKR, Hk/Rr1037/1038 and ComDE, were upregulated during acid adaptation. Real-time qRT-PCR confirmed the same trend in the expression profiles of these genes at pH 5.5. To determine the roles of these transduction systems in acid adaptation, mutants with a deletion of the histidine-kinase-encoding genes were constructed and assayed for the acid tolerance response (ATR). The results revealed that inactivation of each of these systems resulted in a mutant that was impaired in ATR, since pre-exposure of these mutants to pH 5.5 did not induce the same level of protection against lethal pH levels as the parent did. A competitive fitness assay showed that all the mutants were unable to compete with the parent strain for persistence in dual-strain mixed cultures at acidic pH, although, with the exception of the mutant in liaS, little effect was observed at neutral pH. The evidence from this study suggests that the multiple TCSs are required for S. mutans to orchestrate its signal transduction networks for optimal adaptation to acidic pH.
Assuntos
Ácidos/toxicidade , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Transdução de Sinais , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/fisiologia , Estresse Fisiológico , Deleção de Genes , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The genome of Streptococcus mutans harbours 13 two-component signal transduction systems (TCSTSs). Of these, a peptide-mediated quorum-sensing system, ComCDE, and the HK/RR11 two-component system are well known to regulate several virulence-associated traits in in vitro experiments, including genetic competence, bacteriocin production, biofilm formation and stress responses. In this study, we investigated the hypothesis that inactivation of ComCDE, HK/RR11 or both systems would attenuate the virulence and cariogenicity of S. mutans. The results showed that simultaneous inactivation of both signal transduction systems additively attenuated S. mutans virulence and cariogenicity, since inactivation of either of these systems alone did not result in the same degree of effect. The double deletion mutant SMcde-hk11 was defective in genetic competence, had a reduced acid production, was unable to grow at pH 5.0 and formed an abnormal biofilm with reduced biomass. Animal studies showed that this mutant had reduced capabilities for oral colonization, succession and initiation of dental caries. A competitive index (CI) analysis using a mixed-infection animal model revealed that all the mutants, particularly SMcde-hk11, had reduced fitness in their ecological niches and were unable to compete with the wild-type strain for persistence in dental biofilms. The evidence from this study suggests that the ComCDE and HK/RR11 signal transduction systems can be considered to be novel targets for the development of strategies in the prevention and treatment of S. mutans infections.
Assuntos
Proteínas de Bactérias/genética , Cárie Dentária/microbiologia , Percepção de Quorum , Infecções Estreptocócicas/microbiologia , Streptococcus mutans/metabolismo , Streptococcus mutans/patogenicidade , Animais , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Feminino , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Humanos , Boca/microbiologia , Mutação , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Streptococcus mutans/genética , VirulênciaRESUMO
Pyruvate-dependent reduction of NADP has been demonstrated in cell extracts of the human gastric pathogen Helicobacter pylori. However, NADP is not a substrate of purified pyruvate:ferredoxin oxidoreductase (PFOR), suggesting that other redox active enzymes mediate this reaction. Here we show that fqrB (HP1164), which is essential and highly conserved among the epsilonproteobacteria, exhibits NADPH oxidoreductase activity. FqrB was purified by nickel interaction chromatography following overexpression in Escherichia coli. The protein contained flavin adenine dinucleotide and exhibited NADPH quinone reductase activity with menadione or benzoquinone and weak activity with cytochrome c, molecular oxygen, and 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB). FqrB exhibited a ping-pong catalytic mechanism, a k(cat) of 122 s(-1), and an apparent K(m) of 14 muM for menadione and 26 muM for NADPH. FqrB also reduced flavodoxin (FldA), the electron carrier of PFOR. In coupled enzyme assays with purified PFOR and FldA, FqrB reduced NADP in a pyruvate- and reduced coenzyme A (CoA)-dependent manner. Moreover, in the presence of NADPH, CO(2), and acetyl-CoA, the PFOR:FldA:FqrB complex generated pyruvate via CO(2) fixation. PFOR was the rate-limiting enzyme in the complex, and nitazoxanide, a specific inhibitor of PFOR of H. pylori and Campylobacter jejuni, also inhibited NADP reduction in cell-free lysates. These capnophilic (CO(2)-requiring) organisms contain gaps in pathways of central metabolism that would benefit substantially from pyruvate formation via CO(2) fixation. Thus, FqrB provides a novel function in pyruvate metabolism and, together with production of superoxide anions via quinone reduction under high oxygen tensions, contributes to the unique microaerobic lifestyle that defines the epsilonproteobacterial group.
Assuntos
Campylobacter jejuni/metabolismo , Helicobacter pylori/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , NADP/metabolismo , Piruvato Sintase/metabolismo , Ácido Pirúvico/metabolismo , Sequência de Aminoácidos , Campylobacter jejuni/enzimologia , Campylobacter jejuni/genética , Biologia Computacional , Flavodoxina/metabolismo , Helicobacter pylori/enzimologia , Helicobacter pylori/genética , Cinética , Dados de Sequência Molecular , NAD(P)H Desidrogenase (Quinona)/genética , Oxirredução , Piruvato Sintase/genética , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Nitazoxanide (NTZ) exhibits broad-spectrum activity against anaerobic bacteria and parasites and the ulcer-causing pathogen Helicobacter pylori. Here we show that NTZ is a noncompetitive inhibitor (K(i), 2 to 10 microM) of the pyruvate:ferredoxin/flavodoxin oxidoreductases (PFORs) of Trichomonas vaginalis, Entamoeba histolytica, Giardia intestinalis, Clostridium difficile, Clostridium perfringens, H. pylori, and Campylobacter jejuni and is weakly active against the pyruvate dehydrogenase of Escherichia coli. To further mechanistic studies, the PFOR operon of H. pylori was cloned and overexpressed in E. coli, and the multisubunit complex was purified by ion-exchange chromatography. Pyruvate-dependent PFOR activity with NTZ, as measured by a decrease in absorbance at 418 nm (spectral shift from 418 to 351 nm), unlike the reduction of viologen dyes, did not result in the accumulation of products (acetyl coenzyme A and CO(2)) and pyruvate was not consumed in the reaction. NTZ did not displace the thiamine pyrophosphate (TPP) cofactor of PFOR, and the 351-nm absorbing form of NTZ was inactive. Optical scans and (1)H nuclear magnetic resonance analyses determined that the spectral shift (A(418) to A(351)) of NTZ was due to protonation of the anion (NTZ(-)) of the 2-amino group of the thiazole ring which could be generated with the pure compound under acidic solutions (pK(a) = 6.18). We propose that NTZ(-) intercepts PFOR at an early step in the formation of the lactyl-TPP transition intermediate, resulting in the reversal of pyruvate binding prior to decarboxylation and in coordination with proton transfer to NTZ. Thus, NTZ might be the first example of an antimicrobial that targets the "activated cofactor" of an enzymatic reaction rather than its substrate or catalytic sites, a novel mechanism that may escape mutation-based drug resistance.
Assuntos
Antiprotozoários/farmacologia , Bactérias Anaeróbias/enzimologia , Campylobacter jejuni/enzimologia , Inibidores Enzimáticos , Helicobacter pylori/enzimologia , Parasitos/enzimologia , Complexo Piruvato Desidrogenase/antagonistas & inibidores , Tiazóis/farmacologia , Acetilcoenzima A/antagonistas & inibidores , Acetilcoenzima A/biossíntese , Animais , Bactérias Anaeróbias/efeitos dos fármacos , Campylobacter jejuni/efeitos dos fármacos , Dióxido de Carbono/metabolismo , Clonagem Molecular , Clostridium/efeitos dos fármacos , Clostridium/enzimologia , Meios de Cultura , Helicobacter pylori/efeitos dos fármacos , Cinética , L-Lactato Desidrogenase/metabolismo , Espectroscopia de Ressonância Magnética , Nitrocompostos , Parasitos/efeitos dos fármacos , Ácido Pirúvico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiamina Pirofosfato/metabolismoRESUMO
The location of Helicobacter pylori in the gastric mucosa of mammals is defined by natural pH gradients within the gastric mucus, which are more alkaline proximal to the mucosal epithelial cells and more acidic toward the lumen. We have used a microscope slide-based pH gradient assay and video data collection system to document pH-tactic behavior. In response to hydrochloric acid (HCl), H. pylori changes its swimming pattern from straight-line random swimming to arcing or circular patterns that move the motile population away from the strong acid. Bacteria in more-alkaline regions did not swim toward the acid, suggesting the pH taxis is a form of negative chemotaxis. To identify the chemoreceptor(s) responsible for the transduction of pH-tactic signals, a vector-free allelic replacement strategy was used to construct mutations in each of the four annotated chemoreceptor genes (tlpA, tlpB, tlpC, and tlpD) in H. pylori strain SS1 and a motile variant of strain KE26695. All deletion mutants were motile and displayed normal chemotaxis in brucella soft agar, but only tlpB mutants were defective for pH taxis. tlpD mutants exhibited more tumbling and arcing swimming, while tlpC mutants were hypermotile and responsive to acid. While tlpA, tlpC, and tlpD mutants colonized mice to near wild-type levels, tlpB mutants were defective for colonization of highly permissive C57BL/6 interleukin-12 (IL-12) (p40-/-)-deficient mice. Complementation of the tlpB mutant (tlpB expressed from the rdxA locus) restored pH taxis and infectivity for mice. pH taxis, like motility and urease activity, is essential for colonization and persistence in the gastric mucosa, and thus TlpB function might represent a novel target in the development of therapeutics that blind tactic behavior.
Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia/fisiologia , Mucosa Gástrica/microbiologia , Helicobacter pylori/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Quimiotaxia/genética , Deleção de Genes , Infecções por Helicobacter/microbiologia , Concentração de Íons de Hidrogênio , Interleucina-12/genética , Camundongos , Camundongos Knockout , MutaçãoRESUMO
Tellurite resistance (Te(r)) is widespread in nature and it is shown here that the natural resistance of Proteus mirabilis to tellurite is due to a chromosomally located orthologue of plasmid-borne ter genes found in enteric bacteria. The P. mirabilis ter locus (terZABCDE) was identified in a screen of Tn5lacZ-generated mutants of which one contained an insertion in terC. The P. mirabilis terC mutant displayed increased susceptibility to tellurite (Te(s)) and complementation with terC carried on a multicopy plasmid restored high-level Te(r). Primer extension analysis revealed a single transcriptional start site upstream of terZ, but only with RNA harvested from bacteria grown in the presence of tellurite. Northern blotting and reverse transcriptase-PCR (RT-PCR) analyses confirmed that the ter operon was inducible by tellurite and to a lesser extent by oxidative stress inducers such as hydrogen peroxide and methyl viologen (paraquat). Direct and inverted repeat sequences were identified in the ter promoter region as well as motifs upstream of the -35 hexamer that resembled OxyR-binding sequences. Finally, the 390 bp intergenic promoter region located between orf3 and terZ showed no DNA sequence identity with any other published ter sequences, whereas terZABCDE genes exhibited 73-85 % DNA sequence identity. The ter operon was present in all clinical isolates of P. mirabilis and Proteus vulgaris tested and is inferred for Morganella and Providencia spp. based on screening for high level Te(r) and preliminary PCR analysis. Thus, a chromosomally located inducible tellurite resistance operon appears to be a common feature of the genus Proteus.
Assuntos
Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Proteus mirabilis/efeitos dos fármacos , Telúrio/farmacologia , Proteínas de Bactérias/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Dados de Sequência Molecular , Paraquat/farmacologia , Proteus mirabilis/genética , Proteus mirabilis/crescimento & desenvolvimento , Análise de Sequência de DNARESUMO
Nitazoxanide (NTZ) is a redox-active nitrothiazolyl-salicylamide prodrug that kills Helicobacter pylori and also many anaerobic bacterial, protozoan, and helminthic species. Here we describe development and use of a spectrophotometric assay, based on nitroreduction of NTZ at 412 nm, to identify H. pylori enzymes responsible for its activation and mode of action. Three enzymes that reduce NTZ were identified: two related NADPH nitroreductases, which also mediate susceptibility to metronidazole (MTZ) (RdxA and FrxA), and pyruvate oxidoreductase (POR). Recombinant His-tagged RdxA, FrxA, and POR, overexpressed in nitroreductase-deficient Escherichia coli, each rapidly reduced NTZ, whereas only FrxA and to a lesser extent POR reduced nitrofuran substrates (furazolidone, nitrofurantoin, and nitrofurazone). POR exhibited no MTZ reductase activity either in extracts of H. pylori or following overexpression in E. coli; RdxA exhibited no nitrofuran reductase activity, and FrxA exhibited no MTZ reductase activity. Analysis of mutation to rifampin resistance (Rif(r)) indicated that NTZ was not mutagenic and that nitrofurans were only weakly mutagenic. Alkaline gel DNA electrophoresis indicated that none of these prodrugs caused DNA breakage. In contrast, MTZ caused DNA damage and was strongly mutagenic. We conclude that POR, an essential enzyme, is responsible for most or all of the bactericidal effects of NTZ against H. pylori. While loss-of-function mutations in rdxA and frxA produce a Mtz(r) phenotype, they do not contribute much to the innate susceptibility of H. pylori to NTZ or nitrofurans.
Assuntos
Helicobacter pylori/enzimologia , Cetona Oxirredutases/metabolismo , Metronidazol/metabolismo , Nitrofuranos/metabolismo , Nitrorredutases/metabolismo , Tiazóis/metabolismo , Biotransformação , Clonagem Molecular , Dano ao DNA , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Helicobacter pylori/efeitos dos fármacos , Cetona Oxirredutases/genética , Metronidazol/farmacologia , Mutação , Nitrocompostos , Nitrofuranos/farmacologia , Nitrorredutases/genética , Oxirredução , Piruvato Sintase , Especificidade por Substrato , Tiazóis/farmacologiaRESUMO
Legionella pneumophila displays a biphasic developmental cycle in which replicating forms (RFs) differentiate postexponentially into highly infectious, cyst-like mature intracellular forms (MIFs). Using comparative protein profile analyses (MIFs versus RFs), we identified a 20-kDa protein, previously annotated as "Mip-like" protein, that was enriched in MIFs. However, this 20-kDa protein shared no similarity with Mip, a well-characterized peptidyl-prolyl isomerase of L. pneumophila, and for clarity we renamed it MagA (for "MIF-associated gene"). We monitored MagA levels across the growth cycle (in vitro and in vivo) by immunoblotting and established that MagA levels increased postexponentially in vitro (approximately 3-fold) and nearly 10-fold during MIF morphogenesis in HeLa cells. DNA sequence analysis of the magA locus revealed an upstream divergently transcribed gene, msrA, encoding a peptide methionine sulfoxide reductase and a shared promoter region containing direct and indirect repeat sequences as well as -10 hexamers often associated with stationary-phase regulation. While MagA has no known function, it contains a conserved CXXC motif commonly found in members of the thioredoxin reductase family and in AhpD reductases that are associated with alkylhydroperoxide reductase (AhpC), suggesting a possible role in protection from oxidative stress. MIFs from L. pneumophila strain Lp02 containing a magA deletion exhibited differences in Giménez staining, as well as an apparent increase in cytopathology to HeLa cells, but otherwise were unaltered in virulence traits. As demonstrated by this study, MagA appears to be a MIF-specific protein expressed late in intracellular growth that may serve as a useful marker of development.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Legionella pneumophila/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Sequência de Bases , Biomarcadores , Mapeamento Cromossômico , Células HeLa , Humanos , Legionella pneumophila/crescimento & desenvolvimento , Dados de Sequência Molecular , Regiões Promotoras GenéticasRESUMO
The identification of genes associated with colonization and persistence of Helicobacter pylori in the gastric mucosa has been limited by the lack of robust animal models that support infection by strains whose genomes have been completely sequenced. Here we report that an interleukin-12 (IL-12)-deficient mouse (IL-12(-/-) p40 subunit knockout in C57BL/6 mouse) is permissive for infection by a motile variant (KE88-3887) of The Institute For Genomic Research-sequenced strain (KE26695) of H. pylori. The IL-12-deficient mouse was also more permissive for colonization by the mouse-colonizing Sydney 1 strain of H. pylori than were wild-type C57BL/6 mice. Differences in colonization efficiency were demonstrated by mouse challenge with SS1 strains containing loss-of-function mutations in two genes (hspR and hrcA), whose products negatively regulate several heat shock genes. At 5 weeks postinfection, double-knockout mutants (SS1 hspR hrcA) efficiently colonized IL-12-deficient mice (5 of 5 animals compared to 4 of 10 for C57BL6 mice) and bacterial counts were higher in stomachs of IL-12-deficient mice (10(6) versus 10(5) CFU/g of stomach, respectively). IL-12-deficient mice were efficiently colonized by KE88-3887 (29 of 30), but not by nonmotile KE26695, and bacterial numbers (10(4) to 10(5) CFU/g of stomach) were unchanged over an 8-week period postinfection. In contrast, C57BL/6 mice were inefficiently colonized by KE88-3887 (8 of 20 animals with bacterial loads at the limit of detection, approximately 10(3) CFU/g), and infection did not persist much beyond 5 weeks. Cytokine responses (tumor necrosis factor alpha and gamma interferon), pathology, and antral-predominant infection were indistinguishable between IL-12-deficient and C57BL/6 mice. The increased permissiveness of the IL-12-deficient mouse for infection with H. pylori should facilitate whole-genome-based strategies to study genes associated with virulence and immune modulation.
Assuntos
Proteínas de Bactérias , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/etiologia , Helicobacter pylori/fisiologia , Interleucina-12/fisiologia , Animais , Proteínas de Ligação a DNA , Feminino , Proteínas de Choque Térmico/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Proteínas Repressoras/fisiologiaRESUMO
Nucleotide sequence analysis of an approximately 80-kb genomic region revealed an approximately 65-kb locus that bears hallmarks of a pathogenicity island. This locus includes homologues of a type IV secretion system, mobile genetic elements, and known virulence factors. Comparative studies with other Legionella pneumophila strains and serogroups indicated that this approximately 65-kb locus is unique to L. pneumophila serogroup 1 Philadelphia-1 strains.