Staphylococcus aureus exhibit similarities in their interactions with Acanthamoeba and ThP1 macrophage-like cells.

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.

Role of human tear fluid in Acanthamoeba interactions with the human corneal epithelial cells.

Acanthamoeba keratitis is a painful and progressive sight-threatening infection. However, the precise mechanisms associated with the pathogenesis and pathophysiology of Acanthamoeba keratitis remain incompletely understood. Using tears from healthy individuals and an Acanthamoeba keratitis patient, we demonstrated that both subjects exhibited similar levels of Acanthamoeba-specific IgA as demonstrated by Western blotting and enzyme-linked immunosorbent assays. However, normal tears were slightly more potent in reducing Acanthamoeba binding to human corneal epithelial cells, compared with tears from Acanthamoeba keratitis patient (P>0.05 using paired T-test, one-tail distribution). Neither normal tears nor Acanthamoeba keratitis tears had any protective effects on Acanthamoeba-mediated corneal epithelial cell cytotoxicity. Both lysozyme and lactoferrin which are major constituents of the tear film and possess antibacterial properties exhibited no significant effects on Acanthamoeba binding to and cytotoxicity of human corneal epithelial cells. The role of contact lens wear in Acanthamoeba keratitis is discussed further.

The role of proteases in the differentiation of Acanthamoeba castellanii.

Proteases are significant determinants of protozoan pathogenicity and cytolysis of host cells. However, there is now growing evidence of their involvement in cellular differentiation. Acanthamoeba castellanii of the T4 genotype elaborates a number of proteases, which are inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. Using this and other selective protease inhibitors, in tandem with siRNA primers, specific to the catalytic site of Acanthamoeba serine proteases, we demonstrate that serine protease activity is crucial for the differentiation of A. castellanii. Furthermore, both encystment and excystment of A. castellanii was found to be dependent on serine protease function.

Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis.

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.

Escherichia coli interactions with Acanthamoeba: a symbiosis with environmental and clinical implications.

The ability of Acanthamoeba to feed on Gram-negative bacteria, as well as to harbour potential pathogens, such as Legionella pneumophila, Coxiella burnetii, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes and Mycobacterium avium, suggest that both amoebae and bacteria are involved in complex interactions, which may play important roles in the environment and in human health. In this study, Acanthamoeba castellanii (a keratitis isolate belonging to the T4 genotype) was used and its interactions with Escherichia coli (strain K1, a cerebrospinal fluid isolate from a meningitis patient, O18 : K1 : H7, and a K-12 laboratory strain, HB101) were studied. The invasive K1 isolate exhibited a significantly higher association with A. castellanii than the non-invasive K-12 isolate. Similarly, K1 showed significantly increased invasion and/or uptake by A. castellanii in gentamicin protection assays than the non-invasive K-12. Using several mutants derived from K1, it was observed that outer-membrane protein A (OmpA) and LPS were crucial bacterial determinants responsible for E. coli K1 interactions with A. castellanii. Once inside the cell, E. coli K1 remained viable and multiplied within A. castellanii, while E. coli K-12 was killed. Again, OmpA and LPS were crucial for E. coli K1 intracellular survival in A. castellanii. In conclusion, these findings suggest that E. coli K1 interactions with A. castellanii are carefully regulated by the virulence of E. coli.

Acanthamoeba isolates belonging to T1, T2, T3, T4 but not T7 encyst in response to increased osmolarity and cysts do not bind to human corneal epithelial cells.

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.

Hexadentate 3-hydroxypyridin-4-ones with high iron(III) affinity: design, synthesis and inhibition on methicillin resistant Staphylococcus aureus and Pseudomonas strains.

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.

Acanthamoeba induces cell-cycle arrest in host cells.

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.

Post-mortem culture of Balamuthia mandrillaris from the brain and cerebrospinal fluid of a case of granulomatous amoebic meningoencephalitis, using human brain microvascular endothelial cells.

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.

Cellulose biosynthesis pathway is a potential target in the improved treatment of Acanthamoeba keratitis.

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.

Use of in vitro assays to determine effects of human serum on biological characteristics of Acanthamoeba castellanii.

Normal human serum inhibits Acanthamoeba (encephalitis isolate) binding to and cytotoxicity of human brain microvascular endothelial cells, which constitute the blood-brain barrier. Zymographic assays revealed that serum inhibits extracellular protease activities of acanthamoebae. But it is most likely that inhibition of specific properties of acanthamoebae is a consequence of the initial amoebicidal-amoebistatic effects induced by serum. For example, serum exhibited amoebicidal effects; i.e., up to 50% of the exposed trophozoites were killed. The residual subpopulation, although viable, remained static over longer incubations. Interestingly, serum enhanced the phagocytic ability of acanthamoebae, as measured by bacterial uptake. Overall, our results demonstrate that human serum has inhibitory effects on Acanthamoeba growth and viability, protease secretions, and binding to and subsequent cytotoxicity for brain microvascular endothelial cells. Conversely, Acanthamoeba phagocytosis was stimulated by serum.

Extracellular proteases of Acanthamoeba castellanii (encephalitis isolate belonging to T1 genotype) contribute to increased permeability in an in vitro model of the human blood-brain barrier.

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.

Mechanisms associated with Acanthamoeba castellanii (T4) phagocytosis.

Using fluorescein isothiocyanate (FITC)-labelled Escherichia coli, phagocytosis in Acanthamoeba is studied. This assay is based on the quenching effect of trypan blue on FITC-labelled E. coli. Only intracellular E. coli retain their fluorescence, which are easily discriminated from non-fluorescent adherent bacteria. Acanthamoeba uptake of E. coli is significantly reduced in the presence of genistein, a protein tyrosine kinase inhibitor. In contrast, sodium orthovanadate (protein tyrosine phosphatase inhibitor) increases bacterial uptake by Acanthamoeba. Treatment of Acanthamoeba with cytochalasin D (actin polymerization inhibitor) abolished the ability of Acanthamoeba to phagocytose E. coli suggesting that tyrosine kinase-mediated signaling may play a role in Acanthamoeba phagocytosis. In addition, we showed that phosphatidylinositol 3-kinase (PI3K) plays an important role in Acanthamoeba uptake of E. coli. Role of mannose-binding protein in Acanthamoeba phagocytosis is discussed further.

Acanthamoeba castellanii induces host cell death via a phosphatidylinositol 3-kinase-dependent mechanism.

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.

Ecto-ATPases of clinical and non-clinical isolates of Acanthamoeba.

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.

Acanthamoeba interactions with human brain microvascular endothelial cells.

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.