In vitro acanthamoebicidal activity of fusaric acid and dehydrofusaric acid from an endophytic fungus Fusarium sp. Tlau3.

Acanthamoeba is a genus of free-living protozoa that can cause sight- and life-threatening diseases in man. Its control is still problematic due to the lack of effective and nontoxic acanthamoebicidal agents. Herein, we report the first finding of an in vitro killing effect of fusaric acid and dehydrofusaric acid, isolated from metabolites of the Fusarium fujikuroi species complex Tlau3, on Acanthamoeba trophozoites isolated from two clinical (AS, AR) and two soil (S3, S5) samples. AS, AR, and S3 were classified as members of the T4 genotype, whereas S5 belongs to T5. The fungal extract was found to exhibit acanthamoebicidal activity, and activity-guided fractionation led to the isolation and identification of active principles, fusaric acid and dehydrofusaric acid. Their effects were in concentration- and time-dependent manners. Fusaric acid and dehydrofusaric acid showed IC50 values against AS trophozoites of 0.31 and 0.34 µM, respectively. Commercial fusaric acid displayed the same acanthamoebicidal activity as that of the isolated fusaric acid, and therefore, commercial fusaric acid was used throughout this study. IC50 values of commercial fusaric acid against AR, S3, and S5 trophozoites were 0.33, 0.33, and 0.66 µM, respectively. Fusaric acid calcium salt has a history of usage as a hypotensive agent in humans with no observed toxicity. The present study suggests that fusaric acid may serve as a starting point for the development towards therapeutic and environmental acanthamoebicides with low toxicity to humans.

Thai Acanthamoeba isolate (T4) induced apoptotic death in neuroblastoma cells via the Bax-mediated pathway.

A Thai Acanthamoeba isolate named AS recovered from a corneal scraping of a keratitis patient was genotypically determined as T4. AS trophozoites were used for studying Acanthamoeba-induced apoptosis in mouse neuroblastoma NA cells during in vitro co-cultivation. The Acanthamoeba-exposed NA cells showed signs of apoptosis including cell shrinkage, nuclear condensation and DNA fragmentation. The effect was confirmed by DNA laddering electrophoresis. Involvement of caspase enzymes and mitochondrial pro- and anti-apoptotic proteins (Bax and Bcl-2) in AS-induced apoptosis was determined. The use of Z-VAD-FMK, a pan-caspase inhibitor, significantly reduced the apoptotic effect, while Bax/Bcl-2 ratio analysis showed a significant increase in the expression of apoptotic proteins in AS-exposed NA cells. These results strongly indicated that apoptosis induced by AS trophozoites is caspase-dependent and is mediated by over-expression of pro-apoptotic proteins in the mitochondrial pathway. This is the first report on the role of Bax in mediating apoptosis induced by Acanthamoeba.

Acanthamoebicidal activity of Fusarium sp. Tlau3, an endophytic fungus from Thunbergia laurifolia Lindl.

A fungal endophyte identified as Fusarium sp. Tlau3 was isolated from fresh twig of Thunbergia laurifolia Lindl., a Thai medicinal plant collected from the forest of Chiang Mai Province, Northern Thailand. The fungus was grown on a medium containing yeast extracts and sucrose. The fungal metabolites were isolated from the culture broth by dichloromethane extraction, isooctane/methanol then n-butanol/water partitions, and fractionation with Sephadex LH 20 column chromatography. Acanthamoebicidal fractions were found to induce the formation of large contractile vacuole (LCV) in trophozoites of an Acanthamoeba clinical isolate, leading to cell lysis under isotonic and hypotonic conditions within 1 h. In hypertonic condition, LCV formation was also induced but without cell lysis. Acridine orange staining of the treated cells revealed increased intracellular acidity, implying an increased proton pumping or a vacuolar proton-ATPase (V-ATPase) stimulation. Scanning electron microscopy showed cell membrane damage with intact cytoplasmic organelles. Our finding has indicated that contractile vacuoles of Acanthamoeba trophozoites are the primary target of the amoebicidal substance(s) from this endophytic fungus.

Orbital apex syndrome from gnathostomiasis.

A 16-year-old Thai girl presented with acute unilateral visual loss, proptosis, and ophthalmoparesis. CT demonstrated thickening and enhancement of orbital tissues including the orbital apex. A history of consumption of raw fish, together with the findings of cutaneous migratory swelling and eosinophilia, made the diagnosis of gnathostomiasis likely. Her serum was positive for Gnathostoma spinigerum using an immunoblotting technique. Parasites removed from the skin lesions revealed the typical head bulbs with 4 circumferential rows of hooklets and fine cuticular spines on their surface. Treatment with an antihelminthic and systemic corticosteroids led to resolution of orbital inflammation but left a persistent optic neuropathy marked by nerve fiber bundle visual field loss with normal visual acuity. Gnathostomiasis should be suspected in patients with an orbital apex syndrome who live or travel in an endemic area, have eaten raw fish, and develop a migratory skin rash.