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1.
Eur J Med Chem ; 94: 8-21, 2015 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-25747496

RESUMO

A range of hexadentate 3-hydroxypyridin-4-ones have been synthesized. These compounds were found to possess a high affinity for iron(III), with logK1 values of about 34 and pFe values over 30. Antimicrobial assays indicated that they can inhibit the growth of three clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) and three clinical isolates of Pseudomonas, suggesting that hexadentate 3-hydroxypyridin-4-ones have potential application in the treatment of wound infections.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Piridinas/química , Antibacterianos/síntese química , Técnicas de Química Sintética , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Ferro/química , Quelantes de Ferro/química , Quelantes de Ferro/farmacologia , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade
2.
Exp Parasitol ; 132(4): 513-8, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23000484

RESUMO

Staphylococcus aureus is a leading cause of nosocomial infections. Haematogenous spread is a pre-requisite but it is not clear how S. aureus survive the onslaught of macrophages. Acanthamoeba is a protozoan pathogen that is remarkably similar to macrophages, particularly in their cellular structure (morphological and ultra-structural features), molecular motility, biochemical physiology, ability to capture prey by phagocytosis and interactions with microbial pathogens. Thus, we hypothesize that S. aureus exhibit similarities in their interactions with Acanthamoeba and ThP1 macrophage-like cells. Here, we studied interactions of methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA) and Staphylococcus epidermidis (SE) with Acanthamoeba castellanii belonging to the T4 genotype and macrophage-like cells (ThP1). The findings revealed that both MRSA and MSSA exhibited similarities in their binding/association and invasion of A. castellanii and ThP1 cells. Long-term incubation showed that MRSA and MSSA can survive intracellularly of both Acanthamoeba and ThP1 cells. Overall, these findings suggest that Acanthamoeba exhibit similar characteristics with ThP1 macrophage-like cells in their interaction with MRSA and MSSA. Additionally it was shown that bacteria survive inside Acanthamoeba during the encystment process as evidenced by bacterial recovery from mature cysts. Given that Acanthamoeba cysts are airborne, these findings suggest that cysts may act as "Trojan horse" to help spread MRSA to susceptible hosts.


Assuntos
Acanthamoeba castellanii/microbiologia , Macrófagos/microbiologia , Staphylococcus aureus/fisiologia , Análise de Variância , Humanos , Leucemia Mielomonocítica Aguda , Staphylococcus aureus Resistente à Meticilina/fisiologia , Sepse/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/fisiologia , Células Tumorais Cultivadas
3.
Appl Microbiol Biotechnol ; 75(1): 133-40, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17225099

RESUMO

Acanthamoeba is an opportunistic protozoan pathogen that can cause blinding keratitis as well as fatal granulomatous encephalitis. One of the distressing aspects in combating Acanthamoeba infections is the prolonged and problematic treatment. For example, current treatment against Acanthamoeba keratitis requires early diagnosis followed by hourly topical application of a mixture of drugs that can last up to a year. The aggressive and prolonged management is due to the ability of Acanthamoeba to rapidly adapt to harsh conditions and switch phenotypes into a resistant cyst form. One possibility of improving the treatment of Acanthamoeba infections is to inhibit the ability of these parasites to switch into the cyst form. The cyst wall is partially made of cellulose. Here, we tested whether a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), can enhance the effects of the antiamoebic drug pentamidine isethionate (PMD). Our findings revealed that DCB can block Acanthamoeba encystment and may improve the antiamoebic effects of PMD. Using in vitro assays, the findings revealed that DCB enhanced the inhibitory effects of PMD on Acanthamoeba binding to and cytotoxicity of the host cells, suggesting the cellulose biosynthesis pathway as a novel target for the improved treatment of Acanthamoeba infections.


Assuntos
Ceratite por Acanthamoeba/tratamento farmacológico , Acanthamoeba castellanii/efeitos dos fármacos , Amebicidas/farmacologia , Celulose/biossíntese , Nitrilas/farmacologia , Pentamidina/farmacologia , Ceratite por Acanthamoeba/parasitologia , Acanthamoeba castellanii/crescimento & desenvolvimento , Acanthamoeba castellanii/isolamento & purificação , Acanthamoeba castellanii/metabolismo , Amebicidas/toxicidade , Animais , Encéfalo/irrigação sanguínea , Células Cultivadas , Celulose/antagonistas & inibidores , Sinergismo Farmacológico , Endotélio Vascular/citologia , Humanos , Microcirculação , Nitrilas/toxicidade , Pentamidina/toxicidade
4.
BMC Microbiol ; 6: 42, 2006 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-16672059

RESUMO

BACKGROUND: Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined. RESULTS: Using an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF) suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10-phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC). Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like) suggesting a role in blood-brain barrier perturbations. CONCLUSION: Overall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s) in invasion of the brain tissue by amoebae.


Assuntos
Acanthamoeba/enzimologia , Acanthamoeba/patogenicidade , Amebíase/parasitologia , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Encefalite/parasitologia , Metaloproteases/metabolismo , Serina Endopeptidases/metabolismo , Acanthamoeba/classificação , Acanthamoeba/genética , Ceratite por Acanthamoeba/parasitologia , Ceratite por Acanthamoeba/fisiopatologia , Amebíase/fisiopatologia , Animais , Barreira Hematoencefálica , Encéfalo/irrigação sanguínea , Encéfalo/parasitologia , Células Cultivadas , Infecções Protozoárias do Sistema Nervoso Central/fisiopatologia , Encefalite/fisiopatologia , Endotélio Vascular/citologia , Matriz Extracelular/metabolismo , Humanos , Metaloproteases/química , Microcirculação , Serina Endopeptidases/química
5.
J Infect ; 51(2): 150-6, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16038767

RESUMO

OBJECTIVES: Granulomatous amoebic encephalitis (GAE) is a serious human infection with fatal consequences, however, the pathogenic mechanisms associated with this disease remain unclear. Several lines of evidence suggest that haematogenous spread is a prerequisite for Acanthamoeba encephalitis but it is not clear how circulating amoebae cross the blood-brain barrier to gain entry into the central nervous system. Objectives of this study were to determine the effects of Acanthamoeba on the permeability of an in vitro blood-brain barrier model and factors contributing to these changes. METHODS: Using human brain microvascular endothelial cells, an in vitro blood-brain barrier model was constructed in 24-well Transwell plates. Acanthamoeba (GAE isolate belonging to T1 genotype) or its conditioned media were used to determine permeability changes. Zymography assays were performed to characterise Acanthamoeba proteases. In addition, the ability of Acanthamoeba to bind brain microvascular endothelial cells was determined using adhesion assays. RESULTS: We observed that Acanthamoeba produced an increase of more than 45% in the blood-brain barrier permeability. Acanthamoeba-conditioned media exhibited similar effects indicating Acanthamoeba-mediated blood-brain barrier permeability is contact-independent. Prior treatment of conditioned media with phenylmethyl sulfonyl fluoride (PMSF, serine protease inhibitor), abolished permeability changes indicating the role of serine proteases. Of interest, methyl alpha-d-mannopyranoside inhibited Acanthamoeba binding to human brain microvascular endothelial cells but had no effect on Acanthamoeba-mediated blood-brain barrier permeability. Zymography assays revealed that Acanthamoeba produced two major proteases, one of which was inhibited by PMSF (serine protease inhibitor) and the second with 1,10-phenanthroline (metalloprotease inhibitor). CONCLUSIONS: We have for the first time shown that Acanthamoeba produces human brain microvascular endothelial cells permeability, which can be blocked by PMSF. A metalloprotease of approx. molecular weight of 150 kDa is produced by A. castellanii (GAE isolate belonging to T1 genotype) and its role in the disease is suggested.


Assuntos
Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/patogenicidade , Barreira Hematoencefálica/metabolismo , Metaloproteases/fisiologia , Serina Endopeptidases/fisiologia , Acanthamoeba castellanii/genética , Amebíase/parasitologia , Amebíase/fisiopatologia , Animais , Barreira Hematoencefálica/parasitologia , Adesão Celular/fisiologia , Linhagem Celular , Meios de Cultivo Condicionados , Encefalite/parasitologia , Encefalite/fisiopatologia , Células Endoteliais/parasitologia , Células Endoteliais/fisiologia , Genótipo , Humanos , Lectina de Ligação a Manose/fisiologia , Metaloproteases/metabolismo , Peptídeo Hidrolases/metabolismo , Permeabilidade , Serina Endopeptidases/metabolismo , Fatores de Virulência/classificação
6.
Acta Trop ; 95(2): 100-8, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15963936

RESUMO

Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment and can cause human infections. The life cycle of Acanthamoeba consists of an infective trophozoite form. However under harsh environmental conditions trophozoites differentiate into a double-walled, metabolically inactive and resistant cyst form. Research in Acanthamoeba has mostly focussed on the infective trophozoite form and its pathogenic mechanisms. In this study, we used Acanthamoeba isolates belonging to T1, T2, T3, T4, T7 genotypes and studied their cysts properties. We determined that food deprivation stimulates encystment in Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes in a sodium dodecyl sulfate (SDS)-resistant manner. In addition, increase in osmolarity triggered encystment in T1, T2, T3, T4 isolates (SDS-resistant) but T7 failed to encyst (SDS-labile). Adhesion assays revealed that Acanthamoeba cysts belonging to T1, T2, T3, T4, and T7 genotypes exhibited no and/or minimal binding (<5%) to the host cells. Fluorescein-labelled lectins showed that all Acanthamoeba isolates tested exhibited binding to concanavalin A, indicating the expression of mannosyl- and/or glucosyl-residues. Role of cysts in the transmission of infection is discussed further.


Assuntos
Acanthamoeba/classificação , Epitélio Corneano/citologia , Acanthamoeba/genética , Acanthamoeba/crescimento & desenvolvimento , Animais , Adesão Celular , Células Cultivadas , Epitélio Corneano/parasitologia , Genótipo , Humanos , Estágios do Ciclo de Vida , Concentração Osmolar
7.
Infect Immun ; 73(5): 2704-8, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15845472

RESUMO

Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and transmigrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate belonging to the T1 genotype on human brain microvascular endothelial cells, which constitute the blood-brain barrier. Using an apoptosis-specific enzyme-linked immunosorbent assay, we showed that Acanthamoeba induces programmed cell death in brain microvascular endothelial cells. Next, we observed that Acanthamoeba specifically activates phosphatidylinositol 3-kinase. Acanthamoeba-mediated brain endothelial cell death was abolished using LY294002, a phosphatidylinositol 3-kinase inhibitor. These results were further confirmed using brain microvascular endothelial cells expressing dominant negative forms of phosphatidylinositol 3-kinase. This is the first demonstration that Acanthamoeba-mediated brain microvascular endothelial cell death is dependent on phosphatidylinositol 3-kinase.


Assuntos
Acanthamoeba castellanii/patogenicidade , Apoptose , Encéfalo/irrigação sanguínea , Células Endoteliais/parasitologia , Microcirculação/parasitologia , Fosfatidilinositol 3-Quinases/metabolismo , Amebíase/parasitologia , Amebíase/fisiopatologia , Animais , Barreira Hematoencefálica , Células Cultivadas , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Infecções Protozoárias do Sistema Nervoso Central/fisiopatologia , Encefalite/parasitologia , Encefalite/fisiopatologia , Células Endoteliais/enzimologia , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/parasitologia , Humanos , Microcirculação/enzimologia , Microcirculação/fisiologia
8.
Microb Pathog ; 37(5): 231-9, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15519044

RESUMO

Acanthamoeba are opportunistic protozoan parasites that can cause fatal granulomatous amoebic encephalitis and eye keratitis, however the pathogenic mechanisms of Acanthamoeba remain unclear. In this study, we described the ability of live Acanthamoeba to hydrolyse extracellular ATP. Both clinical and non-clinical isolates belonging to genotypes, T1, T2, T3, T4 and T7 exhibited ecto-ATPase activities in vitro. Using non-denaturing polyacrylamide gel electrophoresis, ecto-ATPases were further characterized. All Acanthamoeba isolates tested, exhibited a single ecto-ATPase band (approximate molecular weight of 272 kDa). However, clinical isolates exhibited additional bands suggesting that ecto-ATPases may play a role in the pathogenesis of Acanthamoeba. This was supported using suramin (ecto-ATPase inhibitor), which inhibited Acanthamoeba-induced host cell cytotoxicity. Previously, we and others have shown that Acanthamoeba binds to host cells using their mannose-binding protein and binding can be blocked using exogenous alpha-mannose. In this study, we observed that alpha-mannose significantly increased ecto-ATPase activities of pathogenic Acanthamoeba belonging to T1, T2, T3 and T4 genotypes but had no effect on non-pathogenic Acanthamoeba (belonging to T7 genotype). Overall, we have shown, for the first time, that Acanthamoeba exhibit ecto-ATPase activities, which may play a role in the pathogenesis of Acanthamoeba as well as their potential role in the differentiation of pathogenic Acanthamoeba.


Assuntos
Acanthamoeba/enzimologia , Adenosina Trifosfatases/metabolismo , Acanthamoeba/genética , Adenosina Trifosfatases/genética , Animais , Adesão Celular , Células Epiteliais/citologia , Células Epiteliais/parasitologia , Epitélio Corneano/citologia , Epitélio Corneano/parasitologia , Regulação da Expressão Gênica , Humanos , Hidrólise , Manose/metabolismo , Lectina de Ligação a Manose/metabolismo , Suramina/farmacologia
9.
J Med Microbiol ; 53(Pt 10): 1007-1012, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15358823

RESUMO

The first isolation in the UK of Balamuthia mandrillaris amoebae from a fatal case of granulomatous amoebic meningoencephalitis is reported. Using primary cultures of human brain microvascular endothelial cells (HBMECs), amoebae were isolated from the brain and cerebrospinal fluid (CSF). The cultures showed a cytopathic effect at 20-28 days, but morphologically identifiable B. mandrillaris amoebae were seen in cleared plaques in subcultures at 45 days. The identification of the organism was later confirmed using PCR on Chelex-treated extracts. Serum taken while the patient was still alive reacted strongly with slide antigen prepared from cultures of the post-mortem isolate, and also with those from a baboon B. mandrillaris strain at 1:10,000 in indirect immunofluorescence, but with Acanthamoeba castellanii (Neff) at 1:160, supporting B. mandrillaris to be the causative agent. If the presence of amoebae in the post-mortem CSF reflects the condition in life, PCR studies on CSF and on biopsies of cutaneous lesions may also be a valuable tool. The role of HBMECs in understanding the interactions of B. mandrillaris with the blood-brain barrier is discussed.


Assuntos
Amebíase/parasitologia , Amoeba/isolamento & purificação , Encéfalo/parasitologia , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Líquido Cefalorraquidiano/parasitologia , Células Endoteliais/parasitologia , Granuloma/parasitologia , Animais , Barreira Hematoencefálica , Encéfalo/irrigação sanguínea , Humanos , Papio , Reação em Cadeia da Polimerase
10.
J Med Microbiol ; 53(Pt 8): 711-717, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15272056

RESUMO

Acanthamoeba can cause fatal granulomatous amoebic encephalitis (GAE) and eye keratitis. However, the pathogenesis and pathophysiology of these emerging diseases remain unclear. In this study, the effects of Acanthamoeba on the host cell cycle using human brain microvascular endothelial cells (HBMEC) and human corneal epithelial cells (HCEC) were determined. Two isolates of Acanthamoeba belonging to the T1 genotype (GAE isolate) and T4 genotype (keratitis isolate) were used, which showed severe cytotoxicity on HBMEC and HCEC, respectively. No tissue specificity was observed in their ability to exhibit binding to the host cells. To determine the effects of Acanthamoeba on the host cell cycle, a cell-cycle-specific gene array was used. This screened for 96 genes specific for host cell-cycle regulation. It was observed that Acanthamoeba inhibited expression of genes encoding cyclins F and G1 and cyclin-dependent kinase 6, which are proteins important for cell-cycle progression. Moreover, upregulation was observed of the expression of genes such as GADD45A and p130 Rb, associated with cell-cycle arrest, indicating cell-cycle inhibition. Next, the effect of Acanthamoeba on retinoblastoma protein (pRb) phosphorylation was determined. pRb is a potent inhibitor of G1-to-S cell-cycle progression; however, its function is inhibited upon phosphorylation, allowing progression into S phase. Western blotting revealed that Acanthamoeba abolished pRb phosphorylation leading to cell-cycle arrest at the G1-to-S transition. Taken together, these studies demonstrated for the first time that Acanthamoeba inhibits the host cell cycle at the transcriptional level, as well as by modulating pRb phosphorylation using host cell-signalling mechanisms. A complete understanding of Acanthamoeba-host cell interactions may help in developing novel strategies to treat Acanthamoeba infections.


Assuntos
Acanthamoeba/patogenicidade , Ciclo Celular , Células Endoteliais/parasitologia , Epitélio Corneano/parasitologia , Acanthamoeba/isolamento & purificação , Animais , Adesão Celular/fisiologia , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Células Cultivadas , Ciclina G , Ciclina G1 , Quinase 6 Dependente de Ciclina , Quinases Ciclina-Dependentes/biossíntese , Quinases Ciclina-Dependentes/genética , Ciclinas/biossíntese , Ciclinas/genética , Células Endoteliais/citologia , Células Epiteliais/citologia , Células Epiteliais/parasitologia , Epitélio Corneano/citologia , Fase G1 , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Fosforilação , Biossíntese de Proteínas , Proteínas/genética , Proteína do Retinoblastoma/metabolismo , Proteína p130 Retinoblastoma-Like , Fase S , Transcrição Gênica/genética
11.
Microb Pathog ; 35(6): 235-41, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14580387

RESUMO

Acanthamoeba are opportunistic protozoan parasites that can cause fatal granulomatous amoebic encephalitis, however, the pathogenic mechanisms associated with this disease remain unclear. One of the primary factors in Acanthamoeba encephalitis is the haematogenous spread, followed by invasion of the blood-brain barrier resulting in the transmigration of Acanthamoeba into the central nervous system. In this study, we have used human brain microvascular endothelial cells, which constitute the blood-brain barrier and studied their interactions with Acanthamoeba. Using in vitro cultures, we showed that Acanthamoeba isolates belonging to genotypes T3, T4 and T11, exhibited increased cytotoxicity on human brain microvascular endothelial cells as well as exhibited higher binding and were considered potential pathogens. In contrast, Acanthamoeba isolates belonging to genotypes T2 and T7 exhibited minimal cytotoxicity and significantly less binding to human brain microvascular endothelial cells (P< 0.01). Furthermore, exogenous alpha-mannose inhibited binding but increased cytotoxicity of human brain microvascular endothelial cells. This is the first demonstration of Acanthamoeba interactions with primary human brain microvascular endothelial cells.


Assuntos
Acanthamoeba/patogenicidade , Barreira Hematoencefálica/parasitologia , Encéfalo/irrigação sanguínea , Endotélio Vascular/parasitologia , Acanthamoeba/genética , Acanthamoeba/metabolismo , Animais , Encéfalo/parasitologia , Adesão Celular , Morte Celular , Linhagem Celular , Endotélio Vascular/enzimologia , Endotélio Vascular/patologia , Genótipo , Humanos , L-Lactato Desidrogenase/metabolismo , Manose/metabolismo , Lectina de Ligação a Manose/metabolismo
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