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1.
Antimicrob Agents Chemother ; 67(7): e0009023, 2023 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-37278639

RESUMO

Mycobacterium abscessus infections are difficult to treat and are often considered untreatable without tissue resection. Due to the intrinsic drug-resistant nature of the bacteria, combination therapy of three or more antibiotics is recommended. A major challenge in treating M. abscessus infections is the absence of a universal combination therapy with satisfying clinical success rates, leaving clinicians to treat infections using antibiotics lacking efficacy data. We systematically measured drug combinations in M. abscessus to establish a resource of drug interaction data and identify patterns of synergy to help design optimized combination therapies. We measured 191 pairwise drug combination effects among 22 antibacterials and identified 71 synergistic pairs, 54 antagonistic pairs, and 66 potentiator-antibiotic pairs. We found that commonly used drug combinations in the clinic, such as azithromycin and amikacin, are antagonistic in the lab reference strain ATCC 19977, whereas novel combinations, such as azithromycin and rifampicin, are synergistic. Another challenge in developing universally effective multidrug therapies for M. abscessus is the significant variation in drug response between isolates. We measured drug interactions in a focused set of 36 drug pairs across a small panel of clinical isolates with rough and smooth morphotypes. We observed strain-dependent drug interactions that cannot be predicted from single-drug susceptibility profiles or known drug mechanisms of action. Our study demonstrates the immense potential to identify synergistic drug combinations in the vast drug combination space and emphasizes the importance of strain-specific combination measurements for designing improved therapeutic interventions.


Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Humanos , Azitromicina/farmacologia , Azitromicina/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Amicacina/farmacologia , Amicacina/uso terapêutico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Interações Medicamentosas , Testes de Sensibilidade Microbiana
2.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G227-G237, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32597706

RESUMO

Antibiotic treatment is a standard therapy for Clostridioides difficile infection, but dysbiosis of the gut microbiota due to antibiotic exposure is also a major risk factor for the disease. Following an initial episode of C. difficile infection, a relentless cycle of recurrence can occur, where persistent treatment-related dysbiosis predisposes the patient to subsequent relapse. This study uses a longitudinal study design to compare the effects of a narrow-spectrum (ridinilazole) or broad-spectrum antibiotic (vancomycin) on intestinal bile acid profiles and their associations with gut bacteria over the course of C. difficile infection treatment. At the end of treatment (day 10), subjects receiving vancomycin showed a nearly 100-fold increase in the ratio of conjugated to secondary bile acids in their stool compared with baseline, whereas subjects receiving ridinilazole maintained this ratio near baseline levels. Correlation analysis detected significant positive associations between secondary bile acids and several Bacteroidales and Clostridiales families. These families were depleted in the vancomycin group but preserved at near-baseline abundance in the ridinilazole group. Enterobacteriaceae, which expanded to a greater extent in the vancomycin group, correlated negatively and positively with secondary and conjugated primary bile acids, respectively. Bile acid ratios at the end of treatment were significantly different between those who recurred and those who did not. These results indicate that a narrow-spectrum antibiotic maintains an intestinal bile acid profile associated with a lowered risk of recurrence.NEW & NOTEWORTHY This is the first study to demonstrate in humans the relationships between Clostridioides difficile antibiotic treatment choice and bile acid metabolism both during therapy and after treatment cessation. The results show a microbiota- and metabolome-preserving property of a novel narrow-spectrum agent that correlates with the agent's favorable sustained clinical response rates compared with broad-spectrum antibiotic treatment.


Assuntos
Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Ácidos e Sais Biliares/química , Clostridiales/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Piridinas/farmacologia , Ácidos e Sais Biliares/metabolismo , Fezes/química , Microbioma Gastrointestinal/fisiologia , Humanos
3.
J Antimicrob Chemother ; 73(8): 2078-2084, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29718329

RESUMO

Objectives: We evaluated the antimicrobial susceptibility and ribotypes of Clostridium difficile isolates from participants in a Phase 2 study of ridinilazole, a novel targeted-spectrum agent for treatment of C. difficile infection. Methods: Participants received ridinilazole (200 mg twice daily) or vancomycin (125 mg four times daily) for 10 days (ClinicalTrials.gov: NCT02092935). The MICs of ridinilazole and comparators for C. difficile isolates from stool samples were determined by agar dilution. Toxin gene profiling was performed by multiplex PCR and ribotype identification by capillary electrophoresis. Results: Eighty-nine isolates were recovered from 88/100 participants (one participant had two strains at baseline). The median colony count (cfu/g stool) was 1.9 × 104 (range: 2.5 × 102-7.0 × 106). Twelve participants (three received ridinilazole and nine received vancomycin) experienced recurrence, confirmed by immunoassays for free toxin in stool samples. The ribotype of eight out of nine isolates obtained at recurrence matched those of the initial isolates. All isolates, including those obtained at recurrence, were susceptible to ridinilazole within the expected range [median (range) MIC: 0.12 (0.06-0.5) mg/L]. The median (range) vancomycin MIC was 1 (0.5-4.0) mg/L. At baseline, 13.6% and 13.3% of samples in the ridinilazole and vancomycin groups were positive for VRE, increasing to 23.7% and 29.7% by day 40, respectively. Common ribotypes included 014-20 (14 isolates), 027 (13), 106 (7), 002 (7), 078-126 (4), 001 (4), 087 (3) and 198 (3). Toxin gene profiling of nearly all baseline isolates (98.9%) revealed a binary toxin gene (cdtA/cdtB) prevalence of 35%. Conclusions: Ridinilazole potently inhibited recovered C. difficile isolates. Recurrence was not associated with altered susceptibility.


Assuntos
Benzimidazóis/farmacologia , Clostridioides difficile/classificação , Clostridioides difficile/efeitos dos fármacos , Infecções por Clostridium/tratamento farmacológico , Piridinas/farmacologia , Vancomicina/farmacologia , Antibacterianos/farmacologia , Método Duplo-Cego , Genes Bacterianos , Humanos , Testes de Sensibilidade Microbiana , Ribotipagem
4.
Anaerobe ; 51: 68-72, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29709869

RESUMO

Extraintestinal infection with Clostridium difficile has been reported but remains uncommon. Treatment of this unusual complication is complex given the limitations of current therapeutic options. Here we report a novel case of recurrent extraintestinal C. difficile infection that occurred following fecal microbiota transplantation. Using whole genome sequencing, we confirmed recrudescence rather than reinfection was responsible. The patient ultimately responded to prolonged, targeted antimicrobial therapy informed by susceptibility testing.


Assuntos
Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/epidemiologia , Transplante de Microbiota Fecal/efeitos adversos , Antibacterianos/uso terapêutico , Clostridioides difficile/classificação , Clostridioides difficile/genética , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Resultado do Tratamento , Sequenciamento Completo do Genoma
5.
Infect Immun ; 84(1): 138-48, 2016 01.
Artigo em Inglês | MEDLINE | ID: mdl-26483408

RESUMO

Infection with enterohemorrhagic Escherichia coli (EHEC) can result in severe disease, including hemorrhagic colitis and the hemolytic uremic syndrome. Shiga toxins (Stx) are the key EHEC virulence determinant contributing to severe disease. Despite inhibiting protein synthesis, Shiga toxins paradoxically induce the expression of proinflammatory cytokines from various cell types in vitro, including intestinal epithelial cells (IECs). This effect is mediated in large part by the ribotoxic stress response (RSR). The Shiga toxin-induced RSR is known to involve the activation of the stress-activated protein kinases (SAPKs) p38 and JNK. In some cell types, Stx also can induce the classical mitogen-activated protein kinases (MAPKs) or ERK1/2, but the mechanism(s) by which this activation occurs is unknown. In this study, we investigated the mechanism by which Stx activates ERK1/2s in IECs and the contribution of ERK1/2 activation to interleukin-8 (IL-8) expression. We demonstrate that Stx1 activates ERK1/2 in a biphasic manner: the first phase occurs in response to StxB1 subunit, while the second phase requires StxA1 subunit activity. We show that the A subunit-dependent ERK1/2 activation is mediated through ZAK-dependent signaling, and inhibition of ERK1/2 activation via the MEK1/2 inhibitors U0126 and PD98059 results in decreased Stx1-mediated IL-8 mRNA. Finally, we demonstrate that ERK1/2 are activated in vivo in the colon of Stx2-intoxicated infant rabbits, a model in which Stx2 induces a primarily neutrophilic inflammatory response. Together, our data support a role for ERK1/2 activation in the development of Stx-mediated intestinal inflammation.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Butadienos/farmacologia , Linhagem Celular , Escherichia coli Êntero-Hemorrágica/patogenicidade , Ativação Enzimática , Células Epiteliais/imunologia , Infecções por Escherichia coli/microbiologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides/farmacologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Células HEK293 , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Inflamação/imunologia , Interleucina-8/biossíntese , Interleucina-8/genética , Mucosa Intestinal/imunologia , MAP Quinase Quinase Quinases , Nitrilas/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Coelhos
6.
Infect Immun ; 83(1): 28-38, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25312954

RESUMO

Shiga toxins (Stx) are a family of cytotoxic proteins that can cause hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy, following infections by Shiga toxin-producing Escherichia coli (STEC). Renal failure is a key feature of HUS and a major cause of childhood renal failure worldwide. There are currently no specific therapies for STEC-associated HUS, and the mechanism of Stx-induced renal injury is not well understood primarily due to a lack of fully representative animal models and an inability to monitor disease progression on a molecular or cellular level in humans at early stages. Three-dimensional (3D) tissue models have been shown to be more in vivo-like in their phenotype and physiology than 2D cultures for numerous disease models, including cancer and polycystic kidney disease. It is unknown whether exposure of a 3D renal tissue model to Stx will yield a more in vivo-like response than 2D cell culture. In this study, we characterized Stx2-mediated cytotoxicity in a bioengineered 3D human renal tissue model previously shown to be a predictor of drug-induced nephrotoxicity and compared its response to Stx2 exposure in 2D cell culture. Our results demonstrate that although many mechanistic aspects of cytotoxicity were similar between 3D and 2D, treatment of the 3D tissues with Stx resulted in an elevated secretion of the kidney injury marker 1 (Kim-1) and the cytokine interleukin-8 compared to the 2D cell cultures. This study represents the first application of 3D tissues for the study of Stx-mediated kidney injury.


Assuntos
Rim/efeitos dos fármacos , Organoides/efeitos dos fármacos , Toxina Shiga II/toxicidade , Receptor Celular 1 do Vírus da Hepatite A , Humanos , Glicoproteínas de Membrana/análise , Modelos Biológicos , Técnicas de Cultura de Órgãos , Receptores Virais/análise
8.
Curr Top Microbiol Immunol ; 357: 41-65, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22057792

RESUMO

Shiga toxins and ricin are potent inhibitors of protein synthesis. In addition to causing inhibition of protein synthesis, these toxins activate proinflammatory signaling cascades that may contribute to the severe diseases associated with toxin exposure. Treatment of cells with Shiga toxins and ricin have been shown to activate a number of signaling pathways including those associated with the ribotoxic stress response, Nuclear factor kappa B activation, inflammasome activation, the unfolded protein response, mTOR signaling, hemostasis, and retrograde trafficking. In this chapter, we review our current understanding of these signaling pathways as they pertain to intoxication by Shiga toxins and ricin.


Assuntos
Ricina/farmacologia , Toxinas Shiga/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Proteínas de Transporte/metabolismo , Estresse do Retículo Endoplasmático , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , NF-kappa B/metabolismo , Iniciação Traducional da Cadeia Peptídica , Transporte Proteico , Toxinas Shiga/metabolismo , Estresse Fisiológico
9.
Infect Immun ; 80(8): 2858-67, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22689816

RESUMO

Shiga-toxigenic Escherichia coli (STEC) O113:H21 strains that lack the locus of enterocyte effacement (LEE) efficiently invade eukaryotic cells in vitro, unlike LEE-positive O157:H7 strains. We used a fliC deletion mutant of the O113:H21 STEC strain 98NK2 (98NK2ΔfliC) to show that invasion of colonic epithelial (HCT-8) cells is heavily dependent on production of flagellin, even though adherence to the cells was actually enhanced in the mutant. Flagellin binds and signals through Toll-like receptor 5 (TLR5), but there was no evidence that either TLR5, the adaptor protein myeloid differentiation primary response gene 88 (MyD88), or the serine kinase interleukin-1 receptor-associated kinase (IRAK) were required for invasion of HCT-8 cells by strain 98NK2, as judged by transfection, RNA knockdown, or inhibitor studies. However, pretreatment of cells with anti-asialo-GM1 significantly decreased 98NK2 invasion (by 40.8%), while neuraminidase treatment (which cleaves terminal sialic acid residues, thus converting GM1 into asialo-GM1) significantly increased invasion (by 70.7%). Pretreatment of HCT-8 cells with either the cholesterol-depleting agent methyl-ß-cyclodextrin (MßCD) or the tyrosine kinase inhibitor genistein significantly decreased invasion by 98NK2, indicating a potential role for lipid rafts in the invasion mechanism. Confocal microscopy also showed invading 98NK2 colocalized with lipid raft markers caveolin-1 and GM1. Interestingly, anti-asialo-GM1, neuraminidase, MßCD, and genistein have similar effects on the vestigial level of STEC invasion seen for STEC strain 98NK2ΔfliC, indicating that lipid rafts mediate a common step in flagellin-dependent and flagellin-independent cellular invasion.


Assuntos
Colo/citologia , Células Epiteliais/microbiologia , Flagelina/metabolismo , Microdomínios da Membrana/fisiologia , Escherichia coli Shiga Toxigênica/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/citologia , Flagelina/genética , Gangliosídeo G(M1)/genética , Gangliosídeo G(M1)/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/genética , Receptor 5 Toll-Like/metabolismo
10.
Nature ; 443(7111): 548-52, 2006 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-17024087

RESUMO

AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.


Assuntos
Toxinas Bacterianas/metabolismo , Citotoxinas/metabolismo , Retículo Endoplasmático/metabolismo , Escherichia coli/enzimologia , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/metabolismo , Subtilisina/metabolismo , Animais , Sítios de Ligação , Bovinos , Chlorocebus aethiops , Chaperona BiP do Retículo Endoplasmático , Escherichia coli/patogenicidade , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Camundongos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutagênese , Especificidade por Substrato , Células Vero
11.
Curr Opin Microbiol ; 65: 183-190, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34929548

RESUMO

The formation of attaching and effacing (A/E) lesions on intestinal epithelium, combined with Shiga toxin production, are hallmarks of enterohemorrhagic Escherichia coli (EHEC) infection that can lead to lethal hemolytic uremic syndrome. Although an animal infection model that fully recapitulates human disease remains elusive, mice orally infected with Citrobacter rodentium(ϕStx2dact), a natural murine pathogen lysogenized with an EHEC-derived Shiga toxin 2-producing bacteriophage, develop intestinal A/E lesions and toxin-dependent systemic disease. This model has facilitated investigation of how: (A) phage gene expression and prophage induction contribute to disease and are potentially triggered by antibiotic treatment; (B) virulence gene expression is altered by microbiota and the colonic metabolomic milieu; and (C) innate immune signaling is affected by Stx. Thus, the model provides a unique tool for accessing diverse aspects of EHEC pathogenesis.


Assuntos
Bacteriófagos , Escherichia coli Êntero-Hemorrágica , Infecções por Escherichia coli , Síndrome Hemolítico-Urêmica , Animais , Bacteriófagos/metabolismo , Citrobacter rodentium/genética , Citrobacter rodentium/metabolismo , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/metabolismo , Feminino , Síndrome Hemolítico-Urêmica/genética , Síndrome Hemolítico-Urêmica/metabolismo , Síndrome Hemolítico-Urêmica/patologia , Humanos , Mucosa Intestinal/metabolismo , Masculino , Camundongos
12.
J Infect Dis ; 202(9): 1415-23, 2010 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-20874089

RESUMO

Subtilase cytotoxin (SubAB), produced by certain virulent Shiga toxigenic Escherichia coli strains, causes hemolytic uremic syndrome-like pathology in mice, including extensive microvascular thrombosis. SubAB acts by specifically cleaving the essential endoplasmic reticulum chaperone binding immunoglobulin protein (BiP). BiP has been reported to inhibit the activation of tissue factor (TF), the major initiator of extrinsic coagulation. We hypothesized that the apparent prothrombotic effect of SubAB in vivo may involve the stimulation of TF­dependent procoagulant activity. TF­dependent procoagulant activity, TF messenger RNA (mRNA) levels, and BiP cleavage were therefore examined in human macrophage cells and primary human umbilical vein endothelial cells exposed to SubAB. In both types of cells, SubAB significantly increased TF­dependent procoagulant activity, induced TF mRNA expression, and mediated BiP cleavage. No effects were seen when cells were treated with a nonproteolytic mutant toxin, SubAA272B. Our results suggest that the procoagulant effect of SubAB may be dependent on both the up­regulation of TF expression and the activation of TF by means of BiP cleavage.


Assuntos
Proteínas de Escherichia coli/toxicidade , Proteínas de Choque Térmico/metabolismo , Escherichia coli Shiga Toxigênica/patogenicidade , Subtilisinas/toxicidade , Tromboplastina/biossíntese , Animais , Coagulação Sanguínea/efeitos dos fármacos , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Células Endoteliais/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Humanos , Macrófagos/efeitos dos fármacos , Camundongos , Subtilisinas/metabolismo , Trombose/induzido quimicamente
13.
Infect Immun ; 78(7): 2984-94, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20439475

RESUMO

Shiga toxins expressed in the intestinal lumen during infection with Shiga-toxigenic Escherichia coli must translocate across the epithelium and enter the systemic circulation to cause systemic (pathological) effects, including hemolytic uremic syndrome. The transepithelial migration of polymorphonuclear leukocytes in response to chemokine expression by intestinal epithelial cells is thought to promote uptake of Stx from the intestinal lumen by compromising the epithelial barrier. In the present study, we investigated the hypothesis that flagellin acts in conjunction with Shiga toxin to augment this chemokine expression. We investigated the relative contributions of nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) signaling to transcription and translation of interleukin-8. Using reporter gene constructs, we showed that flagellin-mediated interleukin-8 gene transcription is heavily dependent on both NF-kappaB and extracellular signal-regulated kinase 1 and 2 (ERK-1/2) activation. In contrast, inhibition of p38 has no detectable effect on interleukin-8 gene transcription, even though flagellin-mediated activation of host p38 is critical for maximal interleukin-8 protein expression. Inhibition of MAPK-interacting kinase 1 suggests that p38 signaling affects the posttranscriptional regulation of interleukin-8 protein expression induced by flagellin. Cotreatment with Stx2 and flagellin results in a synergistic upregulation of c-Jun N-terminal protein kinases (JNKs), p38 activation, and a superinduction of interleukin-8 mRNA. This synergism was also evident at the protein level, with increased interleukin-8 protein detectable following cotreatment with flagellin and Stx2. We propose that flagellin, in conjunction with Shiga toxin, synergistically upregulates stress-activated protein kinases, resulting in superinduction of interleukin-8 and, ultimately, absorption of Stx into the systemic circulation.


Assuntos
Infecções por Escherichia coli/microbiologia , Flagelina/metabolismo , Interleucina-8/fisiologia , Toxina Shiga II/metabolismo , Escherichia coli Shiga Toxigênica/patogenicidade , Linhagem Celular , Clonagem Molecular , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Interleucina-8/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , NF-kappa B/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
14.
Cell Microbiol ; 10(7): 1468-77, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18331592

RESUMO

Shiga toxins (Stxs) and ricin initiate damage to host cells by cleaving a single adenine residue on the alpha-sarcin loop of the 28S ribosomal RNA. This molecular insult results in a cascade of intracellular events termed the ribotoxic stress response (RSR). Although Stxs and ricin have been shown to cause the RSR, the mitogen-activated protein kinase kinase kinase (MAP3K) that transduces the signal from intoxicated ribosomes to activate SAPKinases has remained elusive. We show in vitro that DHP-2 (7-[3-fluoro-4-aminophenyl-(4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl))]-quinoline), a zipper sterile-alpha-motif kinase (ZAK)-specific inhibitor, blocks Stx2/ricin-induced SAPKinase activation. Treatment of cells with DHP-2 also blocks Stx2/ricin-mediated upregulation of the proinflammatory cytokine interleukin-8 and results in a modest but statistically significant improvement in cell viability following Stx2/ricin treatment. Finally we show that siRNA directed against the N-terminus of ZAK diminishes Stx2/Ricin-induced SAPKinase activation. Together, these data demonstrate that a ZAK isoform(s) is the MAP3Kinase that transduces the RSR. Therefore, ZAKalpha and/or beta isoforms may act as potential therapeutic target(s) for treating Stx/ricin-associated illnesses. Furthermore, a small molecule inhibitor like DHP-2 may prove valuable in preventing the Stx/ricin-induced proinflammatory and/or apoptotic effects that are thought to contribute to pathogenesis by Stx-producing Escherichia coli and ricin.


Assuntos
Citocinas/metabolismo , Regulação da Expressão Gênica , Proteínas Quinases/metabolismo , Ricina/metabolismo , Toxina Shiga II/metabolismo , Idoso , Animais , Linhagem Celular , Criança , Chlorocebus aethiops , Citocinas/genética , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Interleucina-8/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinases , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases/genética , Pirazóis/metabolismo , Quinolinas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Escherichia coli Shiga Toxigênica/metabolismo , Escherichia coli Shiga Toxigênica/patogenicidade , Células Vero , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
Cell Microbiol ; 10(3): 795-806, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18042253

RESUMO

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. Its cytotoxic activity is due to its capacity to enter cells and specifically cleave the endoplasmic reticulum (ER) chaperone BiP. However, its trafficking within target cells has not been investigated previously. In Vero cells, fluorescence colocalization with subcellular markers established that SubAB is trafficked from the cell surface to the ER via a retrograde pathway similar, but not identical, to those of Shiga toxin (Stx) and cholera toxin (Ctx), with their pathways converging at the Golgi. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalization is exclusively clathrin-dependent.


Assuntos
Clatrina/metabolismo , Retículo Endoplasmático/enzimologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/antagonistas & inibidores , Chaperonas Moleculares/antagonistas & inibidores , Escherichia coli Shiga Toxigênica/fisiologia , Subtilisinas/metabolismo , Animais , Arsenicais/farmacologia , Chlorocebus aethiops , Clatrina/antagonistas & inibidores , Retículo Endoplasmático/química , Chaperona BiP do Retículo Endoplasmático , Inibidores Enzimáticos/farmacologia , Complexo de Golgi/química , Células HeLa , Humanos , Microscopia de Fluorescência , Células Vero
16.
Cell Microbiol ; 10(9): 1775-86, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18433465

RESUMO

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB(5) cytotoxins produced by Shiga toxigenic Escherichia coli. Its cytotoxic activity is due to its capacity to enter cells and specifically cleave the essential endoplasmic reticulum (ER) chaperone BiP (GRP78). In the present study, we have examined its capacity to trigger the three ER stress-signalling pathways in Vero cells. Activation of PKR-like ER kinase was demonstrated by phosphorylation of eIF2alpha, which occurred within 30 min of toxin treatment, and correlated with inhibition of global protein synthesis. Activation of inositol-requiring enzyme 1 was demonstrated by splicing of X-box-binding protein 1 mRNA, while activating transcription factor 6 activation was demonstrated by depletion of the 90 kDa uncleaved form, and appearance of the 50 kDa cleaved form. The rapidity with which ER stress-signalling responses are triggered by exposure of cells to SubAB is consistent with the hypothesis that cleavage by the toxin causes BiP to dissociate from the signalling molecules.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Escherichia coli Shiga Toxigênica/metabolismo , Subtilisinas/metabolismo , eIF-2 Quinase/metabolismo , Animais , Chlorocebus aethiops , Transdução de Sinais , Células Vero
17.
PLoS One ; 13(8): e0199810, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30071046

RESUMO

Ridinilazole, a novel targeted antibacterial being developed for the treatment of C. difficile infection (CDI) and prevention of recurrence, was shown in a recent Phase 2 study to be superior to vancomycin with regard to the primary efficacy measure, sustained clinical response (SCR), with the superiority being driven primarily by marked reductions in the rates of CDI recurrence within 30 days. Tolerability of ridinilazole was comparable to that of vancomycin. The current nested cohort study compared the effects of ridinilazole and vancomycin on fecal microbiota during and after treatment among participants in the Phase 2 study. Changes in the microbiota were assessed using qPCR and high-throughput sequencing on participants' stools collected at multiple time-points (baseline [Day 1], Day 5, end-of-treatment [EOT; Day 10], Day 25, end-of-study [EOS; Day 40], and at CDI recurrence). qPCR analyses showed profound losses of Bacteroides, C. coccoides, C. leptum, and Prevotella groups at EOT with vancomycin treatment, while ridinilazole-treated participants had a modest decrease in C. leptum group levels at EOT, with levels recovering by Day 25. Vancomycin-treated participants had a significant increase in the Enterobacteriaceae group, with this increase persisting beyond EOT. At EOT, alpha diversity decreased with both antibiotics, though to a significantly lesser extent with ridinilazole (p <0.0001). Beta diversity analysis showed a significantly larger weighted Unifrac distance from baseline-to-EOT with vancomycin. Taxonomically, ridinilazole had a markedly narrower impact, with modest reductions in relative abundance in Firmicutes taxa. Microbiota composition returned to baseline sooner with ridinilazole than with vancomycin. Vancomycin treatment resulted in microbiome-wide changes, with significant reductions in relative abundances of Firmicutes, Bacteroidetes, Actinobacteria, and a profound increase in abundance of Proteobacteria. These findings demonstrate that ridinilazole is significantly less disruptive to microbiota than vancomycin, which may contribute to the reduced CDI recurrence observed in the Phase 2 study.


Assuntos
Antibacterianos/uso terapêutico , Benzimidazóis/uso terapêutico , Piridinas/uso terapêutico , Vancomicina/uso terapêutico , Actinobacteria/efeitos dos fármacos , Actinobacteria/isolamento & purificação , Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/microbiologia , Estudos de Coortes , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Análise Discriminante , Fezes/microbiologia , Firmicutes/efeitos dos fármacos , Firmicutes/isolamento & purificação , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Análise de Componente Principal , Proteobactérias/efeitos dos fármacos , Proteobactérias/isolamento & purificação , Piridinas/farmacologia , Vancomicina/farmacologia
18.
Clin Infect Dis ; 43(12): 1587-95, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17109294

RESUMO

In 1982, hemorrhagic colitis and hemolytic-uremic syndrome were linked to infection with Escherichia coli O157:H7, a serotype now classified as Shiga toxin-producing E. coli (STEC). Thereafter, hemorrhagic colitis and hemolytic-uremic syndrome associated with non-O157 STEC serogroups were reported, with the frequency of non-O157 STEC illness rivaling that of O157:H7 in certain geographic regions. In the United States, non-O157 E. coli may account for up to 20%-50% of all STEC infections. A high index of suspicion, paired with options to test for non-O157 STEC infection, are necessary for early recognition and appropriate treatment of these infections. Supportive care without the use of antibiotics is currently considered to be optimal treatment for all STEC infections. This commentary provides a perspective on the non-O157 STEC as human pathogens, how and when the clinician should approach the diagnosis of these organisms, and the challenges ahead.


Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli O157/patogenicidade , Virulência , Técnicas de Tipagem Bacteriana , Colite/etiologia , Colite/microbiologia , Infecções por Escherichia coli/complicações , Escherichia coli O157/classificação , Síndrome Hemolítico-Urêmica/etiologia , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Sorotipagem , Toxinas Shiga
19.
Toxins (Basel) ; 8(9)2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27598200

RESUMO

Ricin activates the proinflammatory ribotoxic stress response through the mitogen activated protein 3 kinase (MAP3K) ZAK, resulting in activation of mitogen activated protein kinases (MAPKs) p38 and JNK1/2. We had a novel zak-/- mouse generated to study the role of ZAK signaling in vivo during ricin intoxication. To characterize this murine strain, we intoxicated zak-/- and zak+/+ bone marrow-derived murine macrophages with ricin, measured p38 and JNK1/2 activation by Western blot, and measured zak, c-jun, and cxcl-1 expression by qRT-PCR. To determine whether zak-/- mice differed from wild-type mice in their in vivo response to ricin, we performed oral ricin intoxication experiments with zak+/+ and zak-/- mice, using blinded histopathology scoring of duodenal tissue sections to determine differences in tissue damage. Unlike macrophages derived from zak+/+ mice, those derived from the novel zak-/- strain fail to activate p38 and JNK1/2 and have decreased c-jun and cxcl-1 expression following ricin intoxication. Furthermore, compared with zak+/+ mice, zak-/- mice have decreased duodenal damage following in vivo ricin challenge. zak-/- mice demonstrate a distinct ribotoxic stress-associated phenotype in response to ricin and therefore provide a new animal model for in vivo studies of ZAK signaling.


Assuntos
Duodeno/efeitos dos fármacos , MAP Quinase Quinase Quinases/deficiência , Macrófagos/efeitos dos fármacos , Ricina/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Animais , Células Cultivadas , Quimiocina CXCL1/metabolismo , Duodeno/enzimologia , Duodeno/patologia , Ativação Enzimática , Genótipo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinases/genética , Macrófagos/enzimologia , Macrófagos/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
Clin Infect Dis ; 38(9): 1298-303, 2004 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15127344

RESUMO

Large-scale outbreaks of Shiga toxin-producing Escherichia coli (STEC) infection have revealed the great disease-causing potential of this organism, especially among children and elderly persons. Approximately 5%-10% of people with STEC infection will develop hemolytic-uremic syndrome (HUS), approximately 10% of those who develop HUS will die or have permanent renal failure, and up to 50% of those who develop HUS will develop some degree of renal impairment. Important concepts in understanding the pathogenesis and prevention of STEC-associated HUS are emerging, although no specific therapy yet exists. Optimal management of STEC infection includes intravenous hydration, avoidance of antimotility agents and antimicrobials, and monitoring for sequelae. Antimicrobials may have a potentially harmful role, possibly by inducing intestinal production of Shiga toxin during the diarrheal phase of illness. A recent clinical trial evaluating an intraluminal Shiga toxin-binding agent to ameliorate HUS showed no improvement in outcome. Interventions to prevent HUS from developing in STEC-infected children are under investigation. Prevention of exposure to STEC remains important, and animal vaccines to prevent stool shedding of STEC among food animals are in development.


Assuntos
Infecções por Escherichia coli/fisiopatologia , Escherichia coli/química , Síndrome Hemolítico-Urêmica/etiologia , Nefropatias/etiologia , Toxina Shiga/toxicidade , Idoso , Animais , Criança , Pré-Escolar , Surtos de Doenças , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/mortalidade , Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/administração & dosagem , Síndrome Hemolítico-Urêmica/epidemiologia , Síndrome Hemolítico-Urêmica/mortalidade , Síndrome Hemolítico-Urêmica/prevenção & controle , Humanos , Nefropatias/microbiologia , Pessoa de Meia-Idade
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