RESUMO
Vibrio parahaemolyticus is the leading bacterial cause of gastroenteritis associated with seafood consumption worldwide. Not all members of the species are thought to be pathogenic, thus identification of virulent organisms is essential to protect public health and the seafood industry. Correlations of human disease and known genetic markers (e.g. thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH)) appear complex. Some isolates recovered from patients lack these factors, while their presence has become increasingly noted in isolates recovered from the environment. Here, we used whole-genome sequencing in combination with mammalian and insect models of infection to assess the pathogenic potential of V. parahaemolyticus isolated from European Atlantic shellfish production areas. We found environmental V. parahaemolyticus isolates harboured multiple virulence-associated genes, including TDH and/or TRH. However, carriage of these factors did not necessarily reflect virulence in the mammalian intestine, as an isolate containing TDH and the genes coding for a type 3 secretion system (T3SS) 2α virulence determinant, appeared avirulent. Moreover, environmental V. parahaemolyticus lacking TDH or TRH could be assigned to groups causing low and high levels of mortality in insect larvae, with experiments using defined bacterial mutants showing that a functional T3SS1 contributed to larval death. When taken together, our findings highlight the genetic diversity of V. parahaemolyticus isolates found in the environment, their potential to cause disease and the need for a more systematic evaluation of virulence in diverse V. parahaemolyticus to allow better genetic markers.
Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Proteínas Hemolisinas , Vibrioses , Vibrio parahaemolyticus , Fatores de Virulência , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/patogenicidade , Vibrio parahaemolyticus/classificação , Vibrio parahaemolyticus/isolamento & purificação , Animais , Virulência/genética , Europa (Continente) , Proteínas Hemolisinas/genética , Fatores de Virulência/genética , Vibrioses/microbiologia , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Humanos , Sequenciamento Completo do Genoma , Fenótipo , Frutos do Mar/microbiologia , Larva/microbiologia , Sistemas de Secreção Tipo III/genética , Genoma Bacteriano , Alimentos Marinhos/microbiologiaRESUMO
Electrophilic small molecules with novel reactivity are powerful tools that enable activity-based protein profiling and covalent inhibitor discovery. Here, we report a reactive heterocyclic scaffold, 4-chloro-pyrazolopyridine (CPzP) for selective modification of proteins via a nucleophilic aromatic substitution (SNAr) mechanism. Chemoproteomic profiling reveals that CPzPs engage cysteines within functionally diverse protein sites including ribosomal protein S5 (RPS5), inosine monophosphate dehydrogenase 2 (IMPDH2), and heat shock protein 60 (HSP60). Through the optimization of appended recognition elements, we demonstrate the utility of CPzP for covalent inhibition of prolyl endopeptidase (PREP) by targeting a noncatalytic active-site cysteine. This study suggests that the proteome reactivity of CPzPs can be modulated by both electronic and steric features of the ring system, providing a new tunable electrophile for applications in chemoproteomics and covalent inhibitor design.
Assuntos
Cisteína , Pirazóis , Piridinas , Piridinas/química , Piridinas/farmacologia , Cisteína/química , Pirazóis/química , Pirazóis/farmacologia , Humanos , Ligantes , Descoberta de DrogasRESUMO
Strategies to target specific protein cysteines are critical to covalent probe and drug discovery. 3-Bromo-4,5-dihydroisoxazole (BDHI) is a natural product-inspired, synthetically accessible electrophilic moiety that has previously been shown to react with nucleophilic cysteines in the active site of purified enzymes. Here, we define the global cysteine reactivity and selectivity of a set of BDHI-functionalized chemical fragments using competitive chemoproteomic profiling methods. Our study demonstrates that BDHIs capably engage reactive cysteine residues in the human proteome and the selectivity landscape of cysteines liganded by BDHI is distinct from that of haloacetamide electrophiles. Given its tempered reactivity, BDHIs showed restricted, selective engagement with proteins driven by interactions between a tunable binding element and the complementary protein sites. We validate that BDHI forms covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), emerging anticancer targets. BDHI electrophile was further exploited in Bruton's tyrosine kinase (BTK) inhibitor design using a single-step late-stage installation of the warhead onto acrylamide-containing compounds. Together, this study expands the spectrum of optimizable chemical tools for covalent ligand discovery and highlights the utility of 3-bromo-4,5-dihydroisoxazole as a cysteine-reactive electrophile.
Assuntos
Produtos Biológicos , Cisteína , Humanos , Cisteína/química , Descoberta de Drogas , Acrilamida , Domínio Catalítico , Peptidilprolil Isomerase de Interação com NIMARESUMO
Tetrodotoxin (TTX), a potent neurotoxin mostly associated with pufferfish poisoning, is also found in bivalve shellfish. Recent studies into this emerging food safety threat reported TTX in a few, mainly estuarine, shellfish production areas in some European countries, including the United Kingdom. A pattern in occurrences has started to emerge, however the role of temperature on TTX has not been investigated in detail. Therefore, we conducted a large systematic TTX screening study, encompassing over 3500 bivalve samples collected throughout 2016 from 155 shellfish monitoring sites along the coast of Great Britain. Overall, we found that only 1.1 % of tested samples contained TTX above the reporting limit of 2 µg/kg whole shellfish flesh and these samples all originated from ten shellfish production sites in southern England. Subsequent continuous monitoring of selected areas over a five-year period showed a potential seasonal TTX accumulation in bivalves, starting in June when water temperatures reached around 15 °C. For the first time, satellite-derived data were also applied to investigate temperature differences between sites with and without confirmed presence of TTX in 2016. Although average annual temperatures were similar in both groups, daily mean values were higher in summer and lower in winter at sites where TTX was found. Here, temperature also increased significantly faster during late spring and early summer, the critical period for TTX. Our study supports the hypothesis that temperature is one of the key triggers of events leading to TTX accumulation in European bivalves. However, other factors are also likely to play an important role, including the presence or absence of a de novo biological source, which remains elusive.
Assuntos
Bivalves , Frutos do Mar , Animais , Tetrodotoxina , Temperatura , Alimentos MarinhosRESUMO
Animal models represent part of the arsenal available to researchers studying the pathophysiology of potentially deadly human pathogens such as Shiga toxin-producing Escherichia coli (STEC). The optimal model may differ depending on what aspects of pathogen biology, disease progression, or host response are under study. Here, we provide detailed protocols for the infant rabbit model of STEC, which largely reproduces the intestinal disease seen following natural oral infection, and share insights from studies examining O157 and non-O157 serotypes.
Assuntos
Infecções por Escherichia coli , Escherichia coli O157 , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/patologia , Escherichia coli O157/metabolismo , Escherichia coli O157/patogenicidade , Humanos , CoelhosRESUMO
A potent and heat-stable tetrodotoxin (TTX) has been found to accumulate in various marine bivalve species, including Pacific oysters (Crassostrea gigas), raising a food safety concern. While several studies on geographical occurrence of TTX have been conducted, there is a lack of knowledge about the distribution of the toxin within and between bivalves. We, therefore, measured TTX in the whole flesh, mantle, gills, labial palps, digestive gland, adductor muscle and intravalvular fluid of C. gigas using liquid chromatography-tandem mass spectrometry. Weekly monitoring during summer months revealed the highest TTX concentrations in the digestive gland (up to 242 µg/kg), significantly higher than in other oyster tissues. Intra-population variability of TTX, measured in the whole flesh of each of twenty animals, reached 46% and 32% in the two separate batches, respectively. In addition, an inter-population study was conducted to compare TTX levels at four locations within the oyster production area. TTX concentrations in the whole flesh varied significantly between some of these locations, which was unexplained by the differences in weight of flesh. This is the first study examining TTX distribution in C. gigas and the first confirmation of the preferential accumulation of TTX in oyster digestive gland.
Assuntos
Crassostrea/química , Venenos/análise , Tetrodotoxina/análise , Poluentes Químicos da Água/análise , Animais , Trato Gastrointestinal/química , Brânquias/química , Espectrometria de Massas em Tandem/métodos , Distribuição Tecidual/fisiologiaRESUMO
Vibrio cholerae O1 El Tor is an aquatic Gram-negative bacterium responsible for the current seventh pandemic of the diarrheal disease, cholera. A previous whole-genome analysis on V. cholerae O1 El Tor strains from the 2010 epidemic in Pakistan showed that all strains contained the V. cholerae pathogenicity island-1 and the accessory colonisation gene acfC (VC_0841). Here we show that acfC possess an open reading frame of 770 bp encoding a protein with a predicted size of 28 kDa, which shares high amino acid similarity with two adhesion proteins found in other enteropathogens, including Paa in serotype O45 porcine enteropathogenic Escherichia coli and PEB3 in Campylobacter jejuni. Using a defined acfC deletion mutant, we studied the specific role of AcfC in V. cholerae O1 El Tor environmental survival, colonisation and virulence in two infection model systems (Galleria mellonella and infant rabbits). Our results indicate that AcfC might be a periplasmic sulfate-binding protein that affects chemotaxis towards mucin and bacterial infectivity in the infant rabbit model of cholera. Overall, our findings suggest that AcfC contributes to the chemotactic response of WT V. cholerae and plays an important role in defining the overall distribution of the organism within the intestine.
Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia , Vibrio cholerae O1/metabolismo , Vibrio cholerae O1/patogenicidade , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Adesão Celular , Células HT29 , Humanos , Intestino Delgado/microbiologia , Mutação , Periplasma/metabolismo , Transporte Proteico , Coelhos , Sulfatos/metabolismo , Vibrio cholerae O1/citologia , Vibrio cholerae O1/genética , VirulênciaRESUMO
The QseEF histidine kinase/response regulator system modulates expression of enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica serovar Typhimurium virulence genes in response to the host neurotransmitters epinephrine and norepinephrine. qseG, which encodes an outer membrane lipoprotein, is cotranscribed with qseEF in these enteric pathogens, but there is little knowledge of its role in virulence. Here, we found that in EHEC QseG interacts with the type III secretion system (T3SS) gate protein SepL and modulates the kinetics of attaching and effacing (AE) lesion formation on tissue-cultured cells. Moreover, an EHEC ΔqseG mutant had reduced intestinal colonization in an infant rabbit model. Additionally, in Citrobacter rodentium, an AE lesion-forming pathogen like EHEC, QseG is required for full virulence in a mouse model. In S Typhimurium, we found that QseG regulates the phase switch between the two flagellin types, FliC and FljB. In an S Typhimurium ΔqseG mutant, the phase-variable promoter for fljB is preferentially switched into the "on" position, leading to overproduction of this phase two flagellin. In infection of tissue-cultured cells, the S Typhimurium ΔqseG mutant provokes increased inflammatory cytokine production versus the wild type; in vivo, in a murine infection model, the ΔqseG strain caused a more severe inflammatory response and was attenuated versus the wild-type strain. Collectively, our findings demonstrate that QseG is important for full virulence in several enteric pathogens and controls flagellar phase variation in S Typhimurium, and they highlight both the complexity and conservation of the regulatory networks that control the virulence of enteric pathogens.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Citrobacter rodentium/fisiologia , Escherichia coli Êntero-Hemorrágica/fisiologia , Proteínas de Escherichia coli/metabolismo , Flagelos/fisiologia , Salmonella typhimurium/fisiologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Flagelina/biossíntese , Regulação Bacteriana da Expressão Gênica , Camundongos , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Coelhos , Deleção de Sequência , Transcrição Gênica , VirulênciaRESUMO
UNLABELLED: Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, which is largely used as a surrogate EHEC model for murine infections, are exposed to several host neurotransmitters in the gut. An important chemical exchange within the gut involves the neurotransmitters epinephrine and/or norepinephrine, extensively reported to increase virulence gene expression in EHEC, acting through two bacterial adrenergic sensors: QseC and QseE. However, EHEC is unable to establish itself and cause its hallmark lesions, attaching and effacing (AE) lesions, on murine enterocytes. To address the role of these neurotransmitters during enteric infection, we employed C. rodentium Both EHEC and C. rodentium harbor the locus of enterocyte effacement (LEE) that is necessary for AE lesion formation. Here we show that expression of the LEE, as well as that of other virulence genes in C. rodentium, is also activated by epinephrine and/or norepinephrine. Both QseC and QseE are required for LEE gene activation in C. rodentium, and the qseC and qseE mutants are attenuated for murine infection. C. rodentium has a decreased ability to colonize dopamine ß-hydroxylase knockout (Dbh(-/-)) mice, which do not produce epinephrine and norepinephrine. Both adrenergic sensors are required for C. rodentium to sense these neurotransmitters and activate the LEE genes during infection. These data indicate that epinephrine and norepinephrine are sensed by bacterial adrenergic receptors during enteric infection to promote activation of their virulence repertoire. This is the first report of the role of these neurotransmitters during mammalian gastrointestinal (GI) infection by a noninvasive pathogen. IMPORTANCE: The epinephrine and norepinephrine neurotransmitters play important roles in gut physiology and motility. Of note, epinephrine and norepinephrine play a central role in stress responses in mammals, and stress has profound effects on GI function. Bacterial enteric pathogens exploit these neurotransmitters as signals to coordinate the regulation of their virulence genes. The bacterial QseC and QseE adrenergic sensors are at the center of this regulatory cascade. C. rodentium is a noninvasive murine pathogen with a colonization mechanism similar to that of EHEC, enabling the investigation of host signals in mice. The presence of these neurotransmitters in the gut is necessary for C. rodentium to fully activate its virulence program, in a QseC/QseE-dependent manner, to successfully colonize its murine host. Our study data provide the first example of epinephrine and norepinephrine signaling within the gut to stimulate infection by a bacterial pathogen in a natural animal infection.
Assuntos
Citrobacter rodentium/patogenicidade , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli Êntero-Hemorrágica/patogenicidade , Trato Gastrointestinal/microbiologia , Regulação Bacteriana da Expressão Gênica , Fosfoproteínas/genética , Receptores Adrenérgicos/genética , Animais , Citrobacter rodentium/genética , Dopamina beta-Hidroxilase/genética , Enterócitos/microbiologia , Escherichia coli Êntero-Hemorrágica/genética , Epinefrina/genética , Epinefrina/metabolismo , Infecções por Escherichia coli , Proteínas de Escherichia coli/genética , Genes Bacterianos , Interações Hospedeiro-Patógeno , Camundongos , Camundongos Knockout , Norepinefrina/genética , Norepinefrina/metabolismo , Vasoconstritores , Virulência/genéticaRESUMO
Global change has caused a worldwide increase in reports of Vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals. In Europe, higher prevalence of human infections followed regional climatic trends with outbreaks occurring during episodes of unusually warm weather. Similar patterns were also observed in Vibrio-associated diseases affecting marine organisms such as fish, bivalves and corals. Basic knowledge is still lacking on the ecology and evolutionary biology of these bacteria as well as on their virulence mechanisms. Current limitations in experimental systems to study infection and the lack of diagnostic tools still prevent a better understanding of Vibrio emergence. A major challenge is to foster cooperation between fundamental and applied research in order to investigate the consequences of pathogen emergence in natural Vibrio populations and answer federative questions that meet societal needs. Here we report the proceedings of the first European workshop dedicated to these specific goals of the Vibrio research community by connecting current knowledge to societal issues related to ocean health and food security.
RESUMO
Microbial pathogens that colonize multiple tissues commonly produce adhesive surface proteins that mediate attachment to cells and/or extracellular matrix in target organs. Many of these 'adhesins' bind to multiple ligands, complicating efforts to understand the role of each ligand-binding activity. Borrelia burgdorferi, the causative agent of Lyme disease, produces BBK32, first identified as a fibronectin-binding adhesin that promotes skin and joint colonization. BBK32 also binds to glycosaminoglycan (GAG), which, like fibronectin is ubiquitously present on cell surfaces. To determine which binding activity is relevant for BBK32-promoted infectivity, we generated a panel of BBK32 truncation and internal deletion mutants, and identified variants specifically defective for binding to either fibronectin or GAG. These variants promoted bacterial attachment to different mammalian cell types in vitro, suggesting that fibronectin and GAG binding may play distinct roles during infection. Intravenous inoculation of mice with a high-passage non-infectious B. burgdorferi strain that produced wild-type BBK32 or BBK32 mutants defective for GAG or fibronectin binding, revealed that only GAG-binding activity was required for significant localization to joints at 60 min post-infection. An otherwise infectious B. burgdorferi strain producing BBK32 specifically deficient in fibronectin binding was fully capable of both skin and joint colonization in the murine model, whereas a strain producing BBK32 selectively attenuated for GAG binding colonized the inoculation site but not knee or tibiotarsus joints. Thus, the BBK32 fibronectin- and GAG-binding activities are separable in vivo, and BBK32-mediated GAG binding, but not fibronectin binding, contributes to joint colonization.
Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/metabolismo , Glicosaminoglicanos/metabolismo , Adesinas Bacterianas/genética , Animais , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Fibronectinas/metabolismo , Articulações/microbiologia , Doença de Lyme , Camundongos , Ligação Proteica , Deleção de SequênciaRESUMO
Classical studies have focused on the role that individual regulators play in controlling virulence gene expression. An emerging theme, however, is that bacterial metabolism also plays a key role in this process. Our previous work identified a series of proteins that were implicated in the regulation of virulence. One of these proteins was AdhE, a bi-functional acetaldehyde-CoA dehydrogenase and alcohol dehydrogenase. Deletion of its gene (adhE) resulted in elevated levels of extracellular acetate and a stark pleiotropic phenotype: strong suppression of the Type Three Secretion System (T3SS) and overexpression of non-functional flagella. Correspondingly, the adhE mutant bound poorly to host cells and was unable to swim. Furthermore, the mutant was significantly less virulent than its parent when tested in vivo, which supports the hypothesis that attachment and motility are central to the colonization process. The molecular basis by which AdhE affects virulence gene regulation was found to be multifactorial, involving acetate-stimulated transcription of flagella expression and post-transcriptional regulation of the T3SS through Hfq. Our study reveals fascinating insights into the links between bacterial physiology, the expression of virulence genes, and the underlying molecular mechanism mechanisms by which these processes are regulated.
Assuntos
Acetatos/metabolismo , Álcool Desidrogenase/metabolismo , Aldeído Oxirredutases/metabolismo , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Álcool Desidrogenase/genética , Aldeído Oxirredutases/genética , Animais , Modelos Animais de Doenças , Infecções por Escherichia coli/patologia , Escherichia coli O157/enzimologia , Escherichia coli O157/fisiologia , Proteínas de Escherichia coli/genética , Flagelos/fisiologia , Regulação Bacteriana da Expressão Gênica , Coelhos , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Many pathogens produce the ß-(1-6)-linked poly-N-acetylglucosamine (PNAG) surface polysaccharide that is being developed as a broadly protective antimicrobial vaccine. However, it is unknown whether systemically injected PNAG vaccines or antibodies would provide protective immunity against pathogens confined to the gastrointestinal tract such as Shiga toxin (Stx)-producing Escherichia coli (STEC), an important group of gastrointestinal (GI) pathogens for which effective immunotherapeutics are lacking. To ascertain whether systemic IgG antibody to PNAG impacts this infectious situation, a vaccine consisting of a synthetic nonamer of nonacetylated PNAG, 9GlcNH2, conjugated to the Shiga toxin 1b subunit (9GlcNH2-Stx1b) was produced. Rabbit antibodies raised to the conjugate vaccine were tested for bacterial killing and toxin neutralization in vitro and protection against infection in infant mice. Cell surface PNAG was detected on all 9 STEC isolates tested, representing 6 STEC serogroups, including E. coli O157:H7. Antibody to the 9GlcNH2-Stx1b conjugate neutralized Stx1 potently and Stx2 modestly. For O157:H7 and O104:H4 STEC strains, antibodies elicited by the 9GlcNH2-Stx1b conjugate possessed opsonic killing and bactericidal activity. Following intraperitoneal injection, antibodies to both PNAG and Stx were needed for infant mouse protection against O157 STEC. These antibodies also mediated protection against the Stx2-producing O104:H4 strain that was the cause of a recent outbreak in Germany, although sufficient doses of antibody to PNAG alone were protective against this strain in infant mice. Our observations suggest that vaccination against both PNAG and Stx, using a construct such as the 9GlcNH2-Stx1b conjugate vaccine, would be protective against a broad range of STEC serogroups. IMPORTANCE The presence of poly-N-acetylglucosamine (PNAG) on many pathogens presents an opportunity to target this one structure with a multispecies vaccine. Whether antibodies to PNAG can protect against pathogens confined to the gastrointestinal tract is not known. As Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are serious causes of infection whose virulence is dependent on elaboration of Stx, we prepared a vaccine containing a synthetic nonamer of PNAG (9GlcNH2) conjugated to Shiga toxin 1b subunit (9GlcNH2-Stx1b) to evaluate bacterial killing, toxin neutralization, and protective efficacy in infant mice. All nine (100%) clinical strains of STEC from different serogroups expressed PNAG. Vaccine-induced antibody mediated in vitro killing of STEC and neutralization of both Stx1 and Stx2. Passive administration of antibody to the conjugate showed protection requiring immunity to both PNAG and Stx for O157 strains, although for an O104 strain, antibody to PNAG alone was protective. Immunity to PNAG may contribute to protection against STEC infections.
Assuntos
Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/imunologia , Toxina Shiga/imunologia , Escherichia coli Shiga Toxigênica/imunologia , beta-Glucanas/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Antitoxinas/sangue , Modelos Animais de Doenças , Infecções por Escherichia coli/imunologia , Vacinas contra Escherichia coli/administração & dosagem , Camundongos , Viabilidade Microbiana/imunologia , Proteínas Opsonizantes/sangue , Coelhos , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologiaRESUMO
The outbreak of diarrhoea and haemolytic uraemic syndrome that occurred in Germany in 2011 was caused by a Shiga toxin-producing enteroaggregative Escherichia coli (EAEC) strain. The strain was classified as EAEC owing to the presence of a plasmid (pAA) that mediates a characteristic pattern of aggregative adherence on cultured cells, the defining feature of EAEC that has classically been associated with virulence. Here we describe an infant rabbit-based model of intestinal colonization and diarrhoea caused by the outbreak strain, which we use to decipher the factors that mediate the pathogen's virulence. Shiga toxin is the key factor required for diarrhoea. Unexpectedly, we observe that pAA is dispensable for intestinal colonization and development of intestinal pathology. Instead, chromosome-encoded autotransporters are critical for robust colonization and diarrhoeal disease in this model. Our findings suggest that conventional wisdom linking aggregative adherence to EAEC intestinal colonization is false for at least a subset of strains.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Escherichia coli/fisiologia , Plasmídeos/fisiologia , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/patogenicidade , Animais , Diarreia/metabolismo , Diarreia/microbiologia , Diarreia/fisiopatologia , Modelos Animais de Doenças , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/fisiopatologia , Feminino , Intestinos/microbiologia , Intestinos/patologia , Masculino , Filogenia , CoelhosRESUMO
The first major outbreaks caused by enterohemorrhagic Escherichia coli (EHEC) raised public and medical awareness of the risks associated with acquiring this potentially deadly infection. The widespread presence of these organisms in the environment, the severity of the clinical sequelae, and the lack of treatment options and effective preventive measures demand that we obtain a better understanding of how this group of organisms cause disease. Animal models allow study of the processes and factors that contribute to disease and, as such, form a valuable tool in the repertoire of infectious disease researchers. Yet despite more than 30 years of research, it seems that no single model host reproduces the full spectrum of clinical disease induced by EHEC in humans. In the first part of this review, a synopsis of what is known about EHEC infections is garnered from human outbreaks and biopsy specimens. The main features and limitations of EHEC infection models that are based on the three most commonly used species (pigs, rabbits, and mice) are described within a historical context. Recent advances are highlighted, and a brief overview of models based on other species is given. Finally, the impact of the host on moderating EHEC infection is considered in light of growing evidence for the need to consider the biology and virulence strategies of EHEC in the context of its niche within the intestine.
Assuntos
Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Animais , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Coelhos , SuínosRESUMO
BACKGROUND/OBJECTIVES: The present study was aimed at the issue of whether peripheral nerve injury-induced chronic pain is maintained by supraspinal structures governing descending facilitation to the spinal dorsal horn, or whether altered peripheral nociceptive mechanisms sustain central hyperexcitability and, in turn, neuropathic pain. We examined this question by determining the contribution of peripheral/spinal mechanisms, isolated from supraspinal influence(s), in cutaneous hypersensitivity in an animal model of peripheral neuropathy. METHODS: Adult rats were spinalized at T8-T9; 8 days later, peripheral neuropathy was induced by implanting a 2-mm polyethylene cuff around the left sciatic nerve. Hind paw withdrawal responses to mechanical or thermal plantar stimulation were evaluated using von Frey filaments or a heat lamp, respectively. RESULTS: Spinalized rats without cuff implantation exhibited a moderate decrease in mechanical withdrawal threshold on ~day 10 (P < 0.05) and in thermal withdrawal threshold on ~day 18 (P < 0.05). However, cuff-implanted spinalized rats developed a more rapid and significant decrease in mechanical (~day 4; P < 0.001) and thermal (~day 10; P < 0.05) withdrawal thresholds that remained significantly decreased through the duration of the study. CONCLUSIONS: Our findings demonstrate an aberrant peripheral/spinal mechanism that induces and maintains thermal and to a greater degree tactile cutaneous hypersensitivity in the cuff model of neuropathic pain, and raise the prospect that altered peripheral/spinal nociceptive mechanisms in humans with peripheral neuropathy may have a pathologically relevant role in both inducing and sustaining neuropathic pain.
Assuntos
Estado de Descerebração/fisiopatologia , Neuralgia/fisiopatologia , Doenças do Sistema Nervoso Periférico/fisiopatologia , Animais , Membro Posterior/fisiologia , Temperatura Alta , Masculino , Medição da Dor , Limiar da Dor , Estimulação Física , Ratos , Ratos Sprague-Dawley , Neuropatia Ciática/etiologia , Neuropatia Ciática/fisiopatologiaRESUMO
In the last decade, the U.S. Food and Drug Administration (FDA) has issued several warnings and recalls for food products that exceed FDA standards for lead. Products containing chili peppers and salt were often suspected as sources of lead contamination, and included items such as candy that are routinely investigated. However, products such as hot sauces that contain similar ingredients have not been the focus of evaluations. This study quantified lead concentrations in imported hot sauces, evaluated product compliance to existing United States standards, and calculated potential dietary lead exposure for children using the Integrated Exposure Uptake Biokinetic Model. Finally, recommendations for reducing the risk of lead exposure from hot sauces are provided. Twenty-five (25) bottles of imported hot sauces manufactured in Mexico and South America were purchased in Clark County, Nevada. All hot sauces were analyzed for lead concentrations, pH, and leaded packaging. Hot sauces were analyzed by inductively coupled plasma mass spectrometry and packaging was analyzed using x-ray fluorescence technology. Four brands of hot sauces (16%) exceeded 0.1 ppm lead, the current FDA action level for lead in candy. Hot sauces with lead concentrations >0.1 ppm lead contained salt and were manufactured in Mexico. Subsequent analysis of additional lots of hot sauces exceeding 0.1 ppm lead revealed inconsistent lead concentrations between and within manufacturer lots. The lead concentrations of the plastic hot sauce lids ranged from below the limit of detection to 2,028 ppm lead. There was no association between lead concentrations in hot sauces and pepper type. These results indicate the need for more rigorous screening protocols for products imported from Mexico, the establishment of an applicable standard for hot sauce, and resources to allow for the enforcement of existing food safety policies. The data reported herein represent the first known investigation of lead concentrations in hot sauces.
Assuntos
Capsicum/química , Contaminação de Alimentos/análise , Chumbo/análise , Capsicum/economia , Qualidade de Produtos para o Consumidor , Contaminação de Alimentos/economia , México , Nevada , América do SulRESUMO
Vibrio parahaemolyticus type III secretion system 2 (T3SS2) is essential for the organism's virulence, but the effectors required for intestinal colonization and induction of diarrhea by this pathogen have not been identified. Here, we identify a type III secretion system (T3SS2)-secreted effector, VopZ, that is essential for V. parahaemolyticus pathogenicity. VopZ plays distinct, genetically separable roles in enabling intestinal colonization and diarrheagenesis. Truncation of VopZ prevents V. parahaemolyticus colonization, whereas deletion of VopZ amino acids 38-62 abrogates V. parahaemolyticus-induced diarrhea and intestinal pathology but does not impair colonization. VopZ inhibits activation of the kinase TAK1 and thereby prevents the activation of MAPK and NF-κB signaling pathways, which lie downstream. In contrast, the VopZ internal deletion mutant cannot counter the activation of pathways regulated by TAK1. Collectively, our findings suggest that VopZ's inhibition of TAK1 is critical for V. parahaemolyticus to induce diarrhea and intestinal pathology.
Assuntos
Proteínas de Bactérias/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Vibrio parahaemolyticus/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Interleucina-8/antagonistas & inibidores , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fosforilação , Coelhos , Transdução de Sinais , Transfecção , Vibrioses/metabolismo , Vibrioses/microbiologia , Vibrioses/patologia , Vibrio parahaemolyticus/patogenicidade , Fatores de Virulência/genéticaRESUMO
The mammalian gastrointestinal tract provides a complex and competitive environment for the microbiota. Successful colonization by pathogens requires scavenging nutrients, sensing chemical signals, competing with the resident bacteria and precisely regulating the expression of virulence genes. The gastrointestinal pathogen enterohaemorrhagic Escherichia coli (EHEC) relies on inter-kingdom chemical sensing systems to regulate virulence gene expression. Here we show that these systems control the expression of a novel two-component signal transduction system, named FusKR, where FusK is the histidine sensor kinase and FusR the response regulator. FusK senses fucose and controls expression of virulence and metabolic genes. This fucose-sensing system is required for robust EHEC colonization of the mammalian intestine. Fucose is highly abundant in the intestine. Bacteroides thetaiotaomicron produces multiple fucosidases that cleave fucose from host glycans, resulting in high fucose availability in the gut lumen. During growth in mucin, B. thetaiotaomicron contributes to EHEC virulence by cleaving fucose from mucin, thereby activating the FusKR signalling cascade, modulating the virulence gene expression of EHEC. Our findings suggest that EHEC uses fucose, a host-derived signal made available by the microbiota, to modulate EHEC pathogenicity and metabolism.