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Background We previously reported that the tomato glycoalkaloid tomatine inhibited the growth of Trichomonas vaginalis strain G3, Tritrichomonas foetus strain D1, and Tritrichomonas foetus-like strain C1 that cause disease in humans and farm and domesticated animals. The increasing prevalence of antibiotic resistance requires development of new tools to enhance or replace medicinal antibiotics. Methods Wild tomato plants were harvested and divided into leaves, stems, and fruit of different colors: green, yellow, and red. Samples were freeze dried and ground with a handheld mill. The resulting powders were evaluated for their potential anti-microbial effects on protozoan parasites, bacteria, and fungi. A concentration of 0.02% (w/v) was used for the inhibition of protozoan parasites. A high concentration of 10% (w/v) solution was tested for bacteria and fungi as an initial screen to evaluate potential anti-microbial activity and results using this high concentration limits its clinical relevance. Results Natural powders derived from various parts of tomato plants were all effective in inhibiting the growth of the three trichomonads to varying degrees. Test samples from leaves, stems, and immature ‘green’ tomato peels and fruit, all containing tomatine, were more effective as an inhibitor of the D1 strain than those prepared from yellow and red tomato peels which lack tomatine. Chlorogenic acid and quercetin glycosides were present in all parts of the plant and fruit, while caffeic acid was only found in the fruit peels. Any correlation between plant components and inhibition of the G3 and C1 strains was not apparent, although all the powders were variably effective. Tomato leaf was the most effective powder in all strains, and was also the highest in tomatine. S. enterica showed a minor susceptibility while B. cereus and C. albicans fungi both showed a significant growth inhibition with some of the test powders. The powders inhibited growth of the pathogens without affecting beneficial lactobacilli found in the normal flora of the vagina. Conclusions The results suggest that powders prepared from tomato leaves, stems, and green tomato peels and to a lesser extent from peels from yellow and red tomatoes offer potential multiple health benefits against infections caused by pathogenic protozoa, bacteria, and fungi, without affecting beneficial lactobacilli that also reside in the normal flora of the vagina.

Human trichomoniasis, caused by the pathogenic parasitic protozoan Trichomonas vaginalis, is the most common non-viral sexually transmitted disease that contributes to reproductive morbidity in affected women and possibly to prostate cancer in men. Tritrichomonas foetus strains cause the disease trichomoniasis in farm animals (cattle, bulls, pigs) and diarrhea in domestic animals (cats and dogs). Because some T. vaginalis strains have become resistant to the widely used drug metronidazole, there is a need to develop alternative treatments, based on safe natural products that have the potential to replace and/or enhance the activity of lower doses of metronidazole. To help meet this need, this overview collates and interprets worldwide reported studies on the efficacy of structurally different classes of food, marine, and medicinal plant extracts and some of their bioactive pure compounds against T. vaginalis and T. foetus in vitro and in infected mice and women. Active food extracts include potato peels and their glycoalkaloids α-chaconine and α-solanine, caffeic and chlorogenic acids, and quercetin; the tomato glycoalkaloid α-tomatine; theaflavin-rich black tea extracts and bioactive theaflavins; plant essential oils and their compounds (+)-α-bisabolol and eugenol; the grape skin compound resveratrol; the kidney bean lectin, marine extracts from algae, seaweeds, and fungi and compounds that are derived from fungi; medicinal extracts and about 30 isolated pure compounds. Also covered are the inactivation of drug-resistant T. vaginalis and T. foetus strains by sensitized light; anti-trichomonad effects in mice and women; beneficial effects of probiotics in women; and mechanisms that govern cell death. The summarized findings will hopefully stimulate additional research, including molecular-mechanism-guided inactivations and human clinical studies, that will help ameliorate adverse effects of pathogenic protozoa.

Copper-promoted azide-alkyne cycloadditions were attempted to synthesize a series of variedly functionalized 1H-1,2,3-triazole-linked isatin-ferrocene, ferrocenylmethoxy-isatin and isatin-ferrocenyl-chalcone conjugates. The synthesized scaffolds were assayed for their inhibitory activity against T. vaginalis as well as several common normal human flora bacteria. The observed inhibitory activities against T. vaginalis and undetectable inhibition of microflora bacteria suggest that these compounds may be specific against trichomonad protozoa and could serve as a new scaffold for synthesis of novel compounds against this important human pathogen.

Clinical cases of metronidazole resistant Trichomonas vaginalis infections have spurred interest in drug discovery against this protozoal parasite. We have carried out structure-activity studies using mononuclear palladium(II) complexes containing salicylaldiminato thiosemicarbazones on a patient isolate of Trichomonas vaginalis highly resistant to the FDA-approved drug metronidazole. A small library of sixteen compounds were analysed on this resistant isolate. Interestingly, compared with our previous analysis of a metronidazole sensitive strain, susceptibility of this resistant isolate to four of the six most potent compounds was observed. Two compounds had similar IC50 values between the resistant strain and a previously analysed sensitive line. Palladium(II) salicylaldiminato thiosemicarbazone complexes may represent, with further development, a new drug discovery direction for treating clinical cases of metronidazole-resistant T. vaginalis. The most potent compound had an IC50 value of 15 μM on parasite growth and showed no effects on common normal flora bacteria and no morphological effects when tested on cultured mammalian cells.

Trichomoniasis in humans, caused by the protozoal parasite Trichomonas vaginalis, is the most common non-viral sexually transmitted disease, while Tritrichomonas foetus causes trichomonosis, an infection of the gastrointestinal tract and diarrhea in farm animals and domesticated cats. As part of an effort to determine the inhibitory effects of plant-based extracts and pure compounds, seven commercially available cherry tomato varieties were hand-peeled, freeze-dried, and pounded into powders. The anti-trichomonad inhibitory activities of these peel powders at 0.02% concentration determined using an in vitro cell assay varied widely from 0.0% to 66.7% against T. vaginalis G3 (human), from 0.9% to 66.8% for T. foetus C1 (feline), and from 0.0% to 81.3% for T. foetus D1 (bovine). The organic Solanum lycopersicum var. cerasiforme (D) peels were the most active against all three trichomonads, inhibiting 52.2% (G3), 66.8% (C1), and 81.3% (D1). Additional assays showed that none of the powders inhibited the growth of foodborne pathogenic bacteria, pathogenic fungi, or non-pathogenic lactobacilli. Tomato peel and pomace powders with high content of described biologically active compounds could serve as functional food and feed additives that might help overcome adverse effects of wide-ranging diseases and complement the treatment of parasites with the anti-trichomonad drug metronidazole.

Reaction of dihydroartemisinin (DHA) with 4-methyl-4′-carboxy-2,2′-bipyridine yielded the new ester derivative L1. Six novel organometallic half-sandwich chlorido Rh(III) and Ir(III) complexes (1–6) containing pentamethylcyclopentadienyl, (Cp*), tetramethylphenylcyclopentadienyl (Cpxph), or tetramethylbiphenylcyclopentadienyl (Cpxbiph), and N,N-chelated bipyridyl group of L1, have been synthesized and characterized. The complexes were screened for inhibitory activity against the Plasmodium falciparum 3D7 (sensitive), Dd2 (multi-drug resistant) and NF54 late stage gametocytes (LSGNF54), the parasite strain Trichomonas vaginalis G3, as well as A2780 (human ovarian carcinoma), A549 (human alveolar adenocarcinoma), HCT116 (human colorectal carcinoma), MCF7 (human breast cancer) and PC3 (human prostate cancer) cancer cell lines. They show nanomolar antiplasmodial activity, outperforming chloroquine and artemisinin. Their activities were also comparable to dihydroartemisinin. As anticancer agents, several of the complexes showed high inhibitory effects, with Ir(III) complex 3, containing the tetramethylbiphenylcyclopentadienyl ligand, having similar IC50 values (concentration for 50% of maximum inhibition of cell growth) as the clinical drug cisplatin (1.06–9.23 μM versus 0.24–7.2 μM, respectively). Overall, the iridium complexes (1–3) are more potent compared to the rhodium derivatives (4–6), and complex 3 emerges as the most promising candidate for future studies.

Plant-derived anthraquinones were evaluated in cell assays for their inhibitory activities against the parasitic protozoa Trichomonas vaginalis human strain G3 that causes the sexually transmitted disease trichomoniasis in women, Tritrichomonas foetus bovine strain D1 that causes sexually transmitted diseases in farm animals (bulls, cows, and pigs), Tritrichomonas foetus-like strain C1 that causes diarrhea in domestic animals (cats and dogs), and bacteria and fungi. The anthraquinones assessed for their inhibitory activity were anthraquinone, aloe-emodin (1,8-dihydroxy-3-hydroxymethylanthraquinone), anthrarufin (1, 5-dihydroxyanthraquinone), chrysazin (1,8-dihydroxyanthraquinone), emodin (1,3,8-trihydroxy-6-methylanthraquinone), purpurin (1,2,4-trihydroxyanthraquinone), and rhein (1,8-dihydroxy-3-carboxyanthraquinone). Their activities were determined in terms of IC50 values, defined as the concentration that inhibits 50% of the cells under the test conditions and calculated from linear dose response plots for the parasitic protozoa, and zone of inhibition for bacteria and fungi, respectively. The results show that the different substituents on the anthraquinone ring seem to influence the relative potency. Analysis of the structure–activity relationships in protozoa indicates that the aloe-emodin and chrysazin with the highest biological activities merit further study for their potential to help treat the diseases in women and domestic and farm animals. Emodin also exhibited antifungal activity against Candida albicans. The suggested mechanism of action and the additional reported beneficial biological properties of anthraquinones suggest that they have the potential to ameliorate a broad spectrum of human diseases.

Fourteen novel arene RuII , and cyclopentadienyl (Cpx ) RhIII and IrIII complexes containing an N,N'-chelated pyridylimino- or quinolylimino ligand functionalized with the antimalarial drug sulfadoxine have been synthesized and characterized, including three by X-ray crystallography. The rhodium and iridium complexes exhibited potent antiplasmodial activity with IC50 values of 0.10-2.0 μm in either all, or one of the three Plasmodium falciparum assays (3D7 chloroquine sensitive, Dd2 chloroquine resistant and NF54 sexual late stage gametocytes) but were only moderately active towards Trichomonas vaginalis. They were active in both the asexual blood stage and the sexual late stage gametocyte assays, whereas the clinical parent drug, sulfadoxine, was inactive. Five complexes were moderately active against Mycobacterium tuberculosis (IC50

The fungal antioxidant system is one of the targets of the redox-active polyene antifungal drugs, including amphotericin B (AMB), nystatin (NYS), and natamycin (NAT). Besides medical applications, NAT has been used in industry for preserving foods and crops. In this study, we investigated two parameters (pH and food ingredients) affecting NAT efficacy. In the human pathogen, Aspergillus fumigatus, NAT (2 to 16 μg mL−1) exerted higher activity at pH 5.6 than at pH 3.5 on a defined medium. In contrast, NAT exhibited higher activity at pH 3.5 than at pH 5.6 against foodborne fungal contaminants, Aspergillus flavus, Aspergillus parasiticus, and Penicillium expansum, with P. expansum being the most sensitive. In commercial food matrices (10 organic fruit juices), food ingredients differentially affected NAT antifungal efficacy. Noteworthily, NAT overcame tolerance of the A. fumigatus signaling mutants to the fungicide fludioxonil and exerted antifungal synergism with the secondary metabolite, kojic acid (KA). Altogether, NAT exhibited better antifungal activity at acidic pH against foodborne fungi, however, the ingredients from commercial food matrices presented greater impact on NAT efficacy compared to pH values. Comprehensive determination of parameters affecting NAT efficacy and improved food formulation will promote sustainable food/crop production, food safety, and public health.

Trichomonas vaginalis, the most common nonviral sexually transmitted parasite, causes ∼283 million trichomoniasis infections annually and is associated with pregnancy complications and increased risk of HIV-1 acquisition. The antimicrobial drug metronidazole is used for treatment, but in a fraction of clinical cases, the parasites can become resistant to this drug. We undertook sequencing of multiple clinical isolates and lab derived lines to identify genetic markers and mechanisms of metronidazole resistance. Reduced representation genome sequencing of ∼100 T. vaginalis clinical isolates identified 3,923 SNP markers and presence of a bipartite population structure. Linkage disequilibrium was found to decay rapidly, suggesting genome-wide recombination and the feasibility of genetic association studies in the parasite. We identified 72 SNPs associated with metronidazole resistance, and a comparison of SNPs within several lab-derived resistant lines revealed an overlap with the clinically resistant isolates. We identified SNPs in genes for which no function has yet been assigned, as well as in functionally-characterized genes relevant to drug resistance (e.g., pyruvate:ferredoxin oxidoreductase). Transcription profiles of resistant strains showed common changes in genes involved in drug activation (e.g., flavin reductase), accumulation (e.g., multidrug resistance pump), and detoxification (e.g., nitroreductase). Finally, we identified convergent genetic changes in lab-derived resistant lines of Tritrichomonas foetus, a distantly related species that causes venereal disease in cattle. Shared genetic changes within and between T. vaginalis and Tr. foetus parasites suggest conservation of the pathways through which adaptation has occurred. These findings extend our knowledge of drug resistance in the parasite, providing a panel of markers that can be used as a diagnostic tool.

A series of N-alkyl tethered C-5 functionalized bis-isatins were synthesized and evaluated for antimicrobial activity against pathogenic microorganisms. The preliminary evaluation studies revealed the compound 4t, with an optimal combination of bromo-substituent at the C-5 position of isatin ring along with propyl chain linker being most active among the synthesized series exhibiting an IC50 value of 3.72 μM against Trichomonas vaginalis while 4j exhibited an IC50 value of 14.8 μM against Naegleria fowleri, more effective than the standard drug Miltefosine. The compound 3f with an octyl spacer length was the most potent among the series against Giardia lamblia with an IC50 of 18.4 μM while 3d exhibited an IC50 of 23 μM against Entamoeba histolytica. This library was also screened against the fungal pathogen Aspergillus parasiticus. A number of the compounds demonstrated potency against this fungus, illustrating a possible broad-spectrum activity. Furthermore, an evaluation of these synthesized compounds against a panel of normal flora bacteria revealed them to be non-cytotoxic, demonstrating the selectivity of these compounds. This observation, in combination with previous studies that isatin is non-toxic to humans, presents a new possible scaffold for drug discovery against these important protozoal pathogens of humans and animals.

Trichomoniasis, caused by the protozoan parasite Trichomonas vaginalis , is the most common, non-viral, sexually transmitted infection in the world, but only two closely related nitro drugs are approved for its treatment. New antimicrobials against trichomoniasis remain an urgent need. Several organic gold compounds were tested for activity against T. vaginalis thioredoxin reductase (TrxR) in cell-free systems as well as for activity against different trichomonads in vitro and in a murine infection model. The organic gold(I) compounds auranofin and chloro(diethylphenylphosphine)gold(I) inhibited TrxR in a concentration-dependent manner in assays with recombinant purified reductase and in cytoplasmic extracts of T. vaginalis transfected with a haemagglutinin epitope-tagged form of the reductase. Auranofin potently suppressed the growth of three independent clinical T. vaginalis isolates as well as several strains of another trichomonad ( Tritrichomonas foetus ) in a 24 h-assay, with 50% inhibitory concentrations of 0.7–2.5 µM and minimum lethal concentrations of 2–6 µM. The drug also compromised the ability of the parasite to overcome oxidant stress, supporting the notion that auranofin acts, in part, by inactivating TrxR-dependent antioxidant defences. Chloro(diethylphenylphosphine)gold(I) was 10-fold less effective against T. vaginalis in vitro than auranofin. Oral administration of auranofin for 4 days cleared the parasites in a murine model of vaginal T. foetus infection without displaying any apparent adverse effects. The approved human drug auranofin may be a promising agent as an alternative treatment of trichomoniasis in cases when standard nitro drug therapies have failed.

Parasitic infections like amebiasis, trichomoniasis, and giardiasis are major health threats with their widespread harmful consequences in tropical and subtropical regions of the world. FDA approved various types of nitro-imidazoles such as ornidazole, benznidazole and secnidazole are widely used medications against anaerobic infections but have lower efficacies than metronidazole (MTZ). MTZ is the current drug of choice for amebiasis, giardiasis and trichomoniasis but it has several adverse effects and potential resistance is a concern. In order to develop alternative antimicrobials, a library of 1H-1,2,3-triazole-tethered metronidazole-isatin conjugates has been synthesized using Huisgen’s azide-alkyne cycloaddition reaction and evaluated for their amebicidal, anti-trichomonal and anti-giardial potential. Most of the synthesized conjugates exhibited comparable activities with that of the standard drug MTZ against Trichomonas vaginalis and Tritrichomonas foetus while better activities were observed against Entamoeba histolytica and Giardia lamblia. Conjugates 9d and 10a were found to be 2-3 folds more potent than MTZ against E. histolytica and 8-16 folds more potent than MTZ against G. lamblia. Further analysis of these compounds on fungi and bacteria did not show inhibitory activity, demonstrating their specific anti-protozoal properties.

Growing resistance to antimicrobial drugs is an urgent life‐threatening problem, forcing medicinal chemists to redesign current drug scaffolds. Derivatization of known drugs to circumvent microbial resistance mechanisms is now an established strategy. The international UK, South African, Australian and USA collaboration of P. Chellan, P. J. Sadler et al. reported in the Full Paper on page 10078 ff., shows that organometallic conjugation of the antibacterial drug sulfadoxine has a remarkable effect on activity. Sulfadoxine has little activity towards various microbial strains, including Plasmodium falciparum and Mycobacterium tuberculosis, but introduction of half‐sandwich rhodium and iridium fragments dramatically “switched on” activity, so providing new drug leads to fight resistance.

Bioisosteric ferrocenyl-containing quinolines and ferrocenylamines containing organosilanes and their carbon analogues, were prepared and fully characterised. The molecular structures of two ferrocenyl-containing quinolines, determined using single-crystal X-ray diffraction, revealed that the compounds crystallise in a folded conformation. The compounds were screened for their antiplasmodial activity against the chloroquine-sensitive (NF54) and CQ-resistant (Dd2) strains of P. falciparum, as well as for their cytotoxicity against Chinese Hamster Ovarian (CHO) cells. The ferrocenyl-containing quinolines displayed activities in the low nanomolar range (6–36 nM), and showed selectivity towards parasites. β-Haematin inhibition assays suggest that the compounds may in part act via the inhibition of haemozoin formation, while microsomal metabolic stability studies reveal that the ferrocenyl-containing quinolines are rapidly metabolised in liver microsomes. Further, antitrichomonal screening against the metronidazole-sensitive (G3) strain of the mucosal pathogen T. vaginalis revealed that the quinoline-based compounds displayed superior parasite growth inhibition when compared to the ferrocenylamines. The library was also tested E. coli and on Lactobacilli spp. found as part of the normal flora of the human microbiome and no effect on growth in vitro was observed, supporting the observation that these compounds are specific for eukaryotic pathogens.

A series of mono- and multimeric polyamine-containing ferrocenyl complexes containing a quinoline motif were prepared. The complexes were characterised by standard techniques. The molecular structure of the monomeric salicylaldimine derivative was elucidated using single crystal X-ray diffraction and was consistent with the proposed structure. The antiplasmodial activity of the compounds were evaluated in vitro against both the NF54 (chloroquine-sensitive) and K1 (chloroquine-resistant) strains of Plasmodium falciparum. The polyamine derivatives exhibit good resistance index values suggesting that these systems are beneficial in overcoming the resistance experienced by chloroquine. Mechanistic studies suggest that haemozoin formation may be the target of these quinoline complexes in the parasite. Some of the complexes exhibit moderate to high cytotoxicity against WHCO1 oesophageal cancer cells in vitro. The monomeric ferrocenyl-amine complexes exhibit potent activity against this particular cell line. The complexes were also screened against the G3 strain of Trichomonas vaginalis and the salicylaldimine complexes demonstrated promising activity at the tested concentration. All of these compounds show no inhibitory effect on several common normal flora bacteria, indicative of their selectivity for eukaryotic pathogens and cancer.

The resistance of Plasmodium falciparum, the causative agent of malaria, against quinine and chloroquine along with the lack of malaria vaccines has encouraged the development of various synthetic strategies towards biologically active scaffolds. An emerging strategy in medicinal chemistry, termed molecular hybridization, involves the covalent fusion of two or more drugs, active compounds, and/or pharmacophoric units into a hybrid compound, with fascinating activities and multiple but not essentially simultaneous pharmacological targets. 4-Aminoquinolines are considered as promising antimalarials and 4-aminoquinoline hybridization is considered as an attractive and feasible approach for the development of new molecular frameworks for averting and delaying the emergence of drug resistance along with improved efficacy. The present review article describes the recent developments on the 4-aminoquinoline-hybridization towards the development of new antimalarials.

Background Plants produce secondary metabolites that often possess widespread bioactivity, and are then known as phytochemicals. We previously determined that several phytochemical-rich food-derived preparations were active against pathogenic foodborne bacteria. Trichomonads produce disease (trichomoniasis) in humans and in certain animals. Trichomonads are increasingly becoming resistant to conventional modes of treatment. It is of interest to test bioactive, natural compounds for efficacy against these pathogens. Methods Using a cell assay, black tea, green tea, grape, pomegranate, and jujube extracts, as well as whole dried jujube were tested against three trichomonads: Trichomonas vaginalis strain G3 (found in humans), Tritrichomonas foetus strain D1 (found in cattle), and Tritrichomonas foetus-like organism strain C1 (found in cats). The most effective of the test substances was subsequently tested against two metronidazole-resistant Trichomonas vaginalis strains, and on normal mucosal flora. Results Black tea extract inhibited all the tested trichomonads, but was most effective against the T. vaginalis organisms. Inhibition by black tea was correlated with the total and individual theaflavin content of the two tea extracts determined by HPLC. Metronidazole-resistant Trichomonas vaginalis strains were also inhibited by the black tea extract. The response of the organisms to the remaining preparations was variable and unique. We observed no effect of the black tea extract on common normal flora bacteria. Conclusions The results suggest that the black tea, and to a lesser degree green tea, grape seed, and pomegranate extracts might present possible natural alternative therapeutic agents to treat Trichomonas vaginalis infections in humans and the related trichomonad infections in animals, without negatively affecting the normal flora.

A series of C-3 thiourea functionalized β -lactams, β -lactam-7-chloroquinoline conjugates and 7-chloroquinoline-thiohydantoin derivatives were prepared with the aim of probing antimalarial structure–activity relationships. 7-Chlorquinoline-thiohydantoin derivatives were found to be potent inhibitors of cultured Plasmodium falciparum , with the most potent and non-cytotoxic compound exhibiting an IC 50 of 39.8 nM. Studies of β -hematin formation suggested that inhibition of haemozoin formation could be primary mechanism of action, with IC 50 values comparable to those of chloroquine. Evaluation of cytotoxicity against HeLa cells demonstrated high selective indices.

Trichomonas vaginalis, the causative agent of trichomonosis, is the most common non-viral sexually transmitted disease. Infection with this protozoan may have serious consequences, especially for women. Currently, 5-nitroimidazole drugs are the treatment of choice for trichomonosis, but the emergence of drug resistant isolates has limited the effectiveness of this therapy. In this context, a library of isatin-mannich adducts and 4-aminoquinoline-isatin mannich conjugates was synthesized following the Cu-promoted Mannich reaction and evaluated for their preliminary in vitro analysis against T. vaginalis at 50 μM. The preliminary evaluation data revealed that the introduction of 4-aminoquinoline ring enhanced the activity profiles with compounds viz. 6a and 6c exhibiting percentage growth inhibition of 92.13 and 100 %, respectively. Compounds with high levels of potency were further analyzed to determine their IC50 values as well as cytotoxicity profiles against mammalian cells. The most active compound in the synthesized conjugates displayed an IC50 value of 23 μM against cultured G3 strain in TYM Diamond’s media of T. vaginalis and was non-toxic to cultured mammalian HeLa cells at the same concentration. Synthesis of N-alkylated isatin analogs, 4-aminoquinoline isatin hybrids, in vitro evaluation against trichomoniasis and cytotoxicities against HeLa cells.

Graphical abstract, Highlights • Trichomonad protozoa infecting free-ranging band-tailed pigeons were characterized. • Trichomonas gallinae and novel species T. stableri were isolated in sick birds. • T. stableri is significantly smaller in length and width than T. gallinae. • T. stableri is genetically more similar to T. vaginalis than to T. gallinae. • T. stableri is a secondary agent of trichomonosis in band-tailed pigeons., Trichomonas gallinae is a ubiquitous flagellated protozoan parasite, and the most common etiologic agent of epidemic trichomonosis in columbid and passerine species. In this study, free-ranging Pacific Coast band-tailed pigeons (Patagioenas fasciata monilis) in California (USA) were found to be infected with trichomonad protozoa that were genetically and morphologically distinct from T. gallinae. In microscopic analysis, protozoa were significantly smaller in length and width than T. gallinae and were bimodal in morphology. Phylogenetic analysis of the ITS1/5.8S/ITS2, rpb1, and hydrogenosomal Fe-hydrogenase regions revealed that the protozoan shares an ancestor with Trichomonas vaginalis, the sexually-transmitted agent of trichomoniasis in humans. Clinical and pathologic features of infected birds were similar to infections with T. gallinae. Evidence presented here strongly support taxonomical distinction of this parasite, which we hereby name Trichomonas stableri n. sp. This work contributes to a growing body of evidence that T. gallinae is not the sole etiologic agent of avian trichomonosis, and that the incorporation of molecular tools is critical in the investigation of infectious causes of mortality in birds.

Cu(I)Cl promoted synthesis of N-propargylated-isatin Mannich mono- and bis-adducts with an extension towards the synthesis of N-propargylated-isatin-7-chloroquinoline conjugates was described. The synthesized scaffolds were evaluated for their in vitro activity against the veterinary protozoal pathogen Tritrichomonas foetus and cytotoxicity against human prostate (PC-3) cancer cell line. The preliminary evaluation data revealed the enhancement in the activity profiles with the introduction of 7-chloroquinoline ring with the most active conjugates 7a, 7c and 7d exhibiting an IC50 of 22.2, 11.3 and 24.5 μM respectively against T. foetus and minimal toxicity against human prostate (PC-3) cell lines., Graphical abstract Synthesis of N-propargylated isatin-Mannich mono-/bis-adducts; isatin-7-chloroquinoline conjugates, their in vitro evaluation against Tritrichomonas foetus and cytotoxic studies., Highlights • Cu(I)Cl promoted synthesis of N-propargylated-isatin Mannich mono- and bis-adducts. • Introduction of 7-chloro-quinoline moiety remarkably enhanced the activity against Tritrichomonas foetus. • The most active and non-cytotoxic compound exhibited an IC50 value 11.3 μM.

The present study investigated the inhibitory effects of the commercial tetrasaccharide tomato glycoalkaloid tomatine and the aglycone tomatidine on three mucosal pathogenic protozoa that are reported to infect humans, cattle, and cats, respectively: Trichomonas vaginalis strain G3, Tritrichomonas foetus strain D1, and Tritrichomonas foetus strain C1. A preliminary screen showed that tomatine at 100 μM concentration completely inhibited the growth of all three trichomonads. In contrast, the inhibition of all three pathogens by tomatidine was much lower, suggesting the involvement of the lycotetraose carbohydrate side chain in the mechanism of inhibition. Midpoints of concentration–response sigmoid plots of tomatine on the three strains correspond to IC50 values, the concentration that inhibits 50% of growth of the pathogenic protozoa. The concentration data were used to calculate the IC50 values for G3, D1, and C1 of 7.9, 1.9, and 2.2 μM, respectively. The results show an approximately 4-fold variation fr...

BackgroundTrichomonas vaginalis, the most common non-viral sexually transmitted parasite, causes ~283 million trichomoniasis infections annually and is associated with complications during pregnancy and increased risk of HIV-1 acquisition. The antimicrobial drug metronidazole is used for treatment, but has lead to widespread drug resistance. We undertook sequencing of multiple clinical isolates and lab derived lines to identify genetic markers and mechanisms of metronidazole resistance.ResultsReduced representation genome sequencings of more than 100 T. vaginalis clinical isolates identified 3,923 SNP markers and presence of a bipartite population structure. Linkage disequilibrium decays rapidly, suggesting genome-wide recombination and the feasibility of genetic association studies in the parasite. We identified 72 SNPs associated with metronidazole resistance, and a comparison of SNPs within several lab-derived resistant lines revealed an overlap with the clinically resistant isolates. We identified SNPs in sets of genes for which no function has yet been assigned, as well as in functionally-characterized genes highly relevant to drug resistance (e.g., pyruvate:ferredoxin oxidoreductase (PFO)). Transcription profiling of these and other genes served as proxy for testing the functional consequences of multiple identified SNPs. Transcription profiles of lab-derived drug resistance strain as well as clinically resistant strain depict common regulation changes in carbohydrate metabolism and oxygen detoxification pathways correlated with Mz resistance. Finally, we identified convergent genetic changes in lab-derived resistant lines of Tritrichomonas foetus, a distantly-related species that causes venereal disease in cattle.ConclusionsOur observation of shared genetic changes within and between T. vaginalis and Tr. foetus parasites suggests conservation of the pathways through which adaptation has occurred. These findings extend our knowledge of drug resistance in the parasite, providing a panel of markers that can be used as a diagnostic tool.

Apicomplexan parasites cause some of the most devastating human diseases, including malaria, toxoplasmosis, and cryptosporidiosis. New drug discovery is imperative in light of increased resistance. In this digest article, we briefly explore some of the recent and promising developments in new drug discovery against two apicomplexan parasites, Cryptosporidium and Toxoplasma.

Tridentate cycloplatinated thiosemicarbazone complexes have been prepared from a biologically significant ligand, 3,4-dichloroacetophenone thiosemicarbazone (1). The tetranuclear complex 2 was prepared by reaction of the ligand with K-2[PtCl4]. Two mononuclear (3 and 4) and two dinuclear (5 and 6) complexes were isolated upon cleavage of the Pt-S-bridging bonds of the tetranuclear complex 2 with the appropriate phosphane ligand. Each complex was characterized using various analytical and spectroscopic techniques, and the molecular structures of 2-4 were also elucidated. The in vitro antiparasitic activities of these complexes against Plasmodium falciparum strains (D10 (chloroquine sensitive) and Dd2 (chloroquine resistant)) and Trichomonas vaginalis have been determined. Preliminary studies into their potential plasmodial target in the form of beta-hematin formation inhibition assays were also completed. Preliminary results suggest that ligand 1 and complex 3 do not hinder formation of beta-hematin. The antiproliferative activity of the complexes against the cisplatin-senstive A2780 and cisplatin-resistant A2780cisR human ovarian cancer cell lines has been evaluated. The complexes were found to exhibit moderate to weak inhibitory activities.

Due to the increased interest in their application in the treatment of infectious diseases, boron-containing compounds have received a significant coverage in the literature. Herein, a small set of novel cinnamoly-oxaborole amides were synthesized and screened against nagana Trypanosoma brucei brucei for antitrypanosomal activity. Compound 5g emerged as a new hit with an in vitro IC50 value of 0.086 &mu, M against T. b. brucei without obvious inhibitory activity against HeLa cell lines. The same series was also screened against other human pathogens, including Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), for which moderate to weak activity (10 to &gt, 125 &mu, M) was observed. Similarly, these compounds exhibited moderate activity against the human protozoal pathogen Trichomonas vaginalis with no observed effect on common microbiome bacterial species. The cross-species inhibitory activity presents the possibility of these compounds serving as broad-spectrum antibiotics for these prevalent three human pathogens.

The resistance of Plasmodium falciparum, the causative agent of malaria, against quinine and chloroquine along with the lack of malaria vaccines has encouraged the development of various synthetic strategies towards biologically active scaffolds. An emerging strategy in medicinal chemistry, termed molecular hybridization, involves the covalent fusion of two or more drugs, active compounds, and/or pharmacophoric units into a hybrid compound, with fascinating activities and multiple but not essentially simultaneous pharmacological targets. 4-Aminoquinolines are considered as promising antimalarials and 4-aminoquinoline hybridization is considered as an attractive and feasible approach for the development of new molecular frameworks for averting and delaying the emergence of drug resistance along with improved efficacy. The present review article describes the recent developments on the 4-aminoquinoline-hybridization towards the development of new antimalarials.

A series of mono- and bis-salicylaldimine ligands and their corresponding Rh(i) complexes were prepared. The compounds were characterised using standard spectroscopic techniques including NMR, IR spectroscopy and mass spectrometry. The salicylaldimine ligands and complexes were screened for antiparasitic activity against two strains of Plasmodium falciparum i.e. the NF54 CQ-sensitive and K1 CQ-resistant strain as well as against the G3 isolate of Trichomonas vaginalis. The monomeric salicylaldimine quinolines exhibited good activity against the NF54 strain and the dimeric salicylaldimine quinolines exhibited no cross resistance across the two strains. The binuclear 5-chloro Rh(i) complex displayed the best activity against the Trichomonas vaginalis parasite, possibly a consequence of its enhanced lipophilicity. The compounds were also screened for cytotoxicity in vitro against WHCO1 oesophageal cancer cells. The monomeric salicylaldimine quinolines exhibited high selectivity towards malaria parasites compared to cancer cells, while the dimeric compounds were less selective.

Highlights • Trichomonads represent emerging species of medical and veterinary importance. • Clinical and molecular evidence suggest a zoonotic potential for trichomonads. • Close relationship between avian and human trichomonads revealed in outbreaks., Trichomonads are common parasites of many vertebrate and invertebrate species, with four species classically recognized as human parasites: Dientamoeba fragilis, Pentatrichomonas hominis, Trichomonas vaginalis, and Trichomonas tenax. The latter two species are considered human-specific; by contrast, D. fragilis and P. hominis have been isolated from domestic and farm mammals, demonstrating a wide host range and potential zoonotic origin. Several new studies have highlighted the zoonotic dimension of trichomonads. First, species typically known to infect birds and domestic mammals have been identified in human clinical samples. Second, several phylogenetic analyses have identified animal-derived trichomonads as close sister taxa of the two human-specific species. It is our opinion, therefore, that these observations prompt further investigation into the importance of zoonotic trichomonads for human health.

Trypanosoma brucei and Trichomonas vaginalis are both parasitic protozoans that are known to share many similar biochemical pathways. Aristeromycin, as well as 5′-iodovinyl and 5′-oxime analogues of adenosine, are potent inhibitors of AdoHcy hydrolase in T. brucei, an enzyme that catalyses the hydrolysis of AdoHcy to adenosine and l -homocysteine. To help determine the role of this enzyme in T. vaginalis, we have tested a library of 5′-modified adenosine derivatives, including 5′-deoxy-5′-(iodomethylene)-adenosine and related 6-N-cyclopropyl analogues. Our results indicate that these inhibitors are effective at inhibiting the growth of T. vaginalis, by as much as 95%.

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum , can stunt infant growth and can be lethal in immunocompromised individuals. The most widely used drugs for treating cryptosporidiosis are nitazoxanide and paromomycin, although both exhibit limited efficacy. To investigate an alternative approach to therapy, we demonstrate that the clan CA cysteine protease inhibitor N -methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibits C. parvum growth in mammalian cell lines in a concentration-dependent manner. Further, using the C57BL/6 gamma interferon receptor knockout (IFN-γR-KO) mouse model, which is highly susceptible to C. parvum , oral or intraperitoneal treatment with K11777 for 10 days rescued mice from otherwise lethal infections. Histologic examination of untreated mice showed intestinal inflammation, villous blunting, and abundant intracellular parasite stages. In contrast, K11777-treated mice (210 mg/kg of body weight/day) showed only minimal inflammation and no epithelial changes. Three putative protease targets (termed cryptopains 1 to 3, or CpaCATL-1, -2, and -3) were identified in the C. parvum genome, but only two are transcribed in infected mammals. A homology model predicted that K11777 would bind to cryptopain 1. Recombinant enzymatically active cryptopain 1 was successfully targeted by K11777 in a competition assay with a labeled active-site-directed probe. K11777 exhibited no toxicity in vitro and in vivo , and surviving animals remained free of parasites 3 weeks after treatment. The discovery that a cysteine protease inhibitor provides potent anticryptosporidial activity in an animal model of infection encourages the investigation and development of this biocide class as a new, and urgently needed, chemotherapy for cryptosporidiosis.

In this study, we describe the synthesis of mono- and bis-1H-1,2,3-triazole-tethered β-lactam–isatin conjugates using copper-catalysed azide-alkyne cycloaddition reaction between mono- and di-propargylated azetidin-2-ones and N-alkylazido isatins. The synthesized conjugates were evaluated for their preliminary in vitro analysis against Trichomonas vaginalis at 50 μM. The efficacy of synthesized hybrids was observed to depend on the substituent at N-1 position of β-lactam ring, as well as the presence of single/double 1H-1,2,3-triazole linker. Among the synthesized conjugates, the presence of a p-tolyl substituent at N-1 of β-lactam ring was preferred for good activity profiles while the increase in spacer length did not influence the efficacy of the compounds. Compounds with high levels of potency were further analysed to determine their IC50 values, as well as cytotoxicity profiles against mammalian cells. The most active compound in the synthesized conjugates displayed an IC50 value of 10.49 μM against cultured G3 strain of T. vaginalis and was non-toxic to cultured mammalian HeLa cells at the same concentration. Electronic supplementary material The online version of this article (doi:10.1007/s00044-014-0956-6) contains supplementary material, which is available to authorized users.

Trichomonas vaginalis is a member of the Parabasalia, a group of single-celled eukaryotes within the clade Excavata, which also includes parasites of genera such as Giardia and Trypanosoma . The ability to culture the parasite under both aerobic and anaerobic conditions has allowed simple manipulation in the laboratory. Studies of the basic biology of this organism not only provide important information about its unique cellular and metabolic features and contribute to our understanding of the evolution of anaerobic parasites and diversity among eukaryotes, but also contribute to the design of new drugs to cure infections. The publication of the genome sequence in 20071 identified several unique characteristics of the organism, such as the large genome size (∼160 megabases), extensive gene duplication and presence of families of transposable elements. It also facilitated data mining and the generation of ‘omic’ data sets, especially from transcriptomic and proteomic studies.2 For example, purification of hydrogenosomes and the subsequent sequencing of proteins via …

A series of mono- and multimeric 4-amino-7-chloroquinoline and ferrocenyl thioureas have been prepared by the reaction of a 7-chloroquinoline methyl ester and a ferrocenylimine methyl ester with various amines. These compounds were characterized using standard spectroscopic and analytical techniques. The compounds were evaluated against the NF54 (CQ-sensitive) and Dd2 (CQ-resistant) strains of Plasmodium falciparum. The quinoline compounds show enhanced activity compared to the ferrocene compounds against this parasite. Compound 5 displays the most promising activity against the NF54 strain. Compounds 5 and 6 are effective at inhibiting β-hematin formation perhaps due to an increased number of quinoline moieties. The trimeric (12) and tetrameric (13) ferrocenyl compounds also inhibit β-hematin formation, albeit to a lesser degree compared to the quinoline thioureas. The compounds were also screened against the G3 strain of Trichomonas vaginalis and here the ferrocene-containing compounds show a slightly higher parasite growth inhibition compared to the quinoline thioureas. The quinoline compounds were also found to be more cytotoxic compared to the ferrocenyl compounds. Compound 6 displays good cytotoxicity against WHCO1 oesophageal cancer cells.

Anopheles gambiae is the mosquito vector responsible for transmitting Plasmodium falciparum, a malaria parasite of humans. With the emergence of genome projects for a variety of prokaryotic and eukaryotic microorganisms, there has been a long-standing interest in sequencing the genomes of the malaria parasite and its insect vector. This tour de force effort has now been completed and reported. The alignment of putative orthologs in An. gambiae with those of Drosophila melanogaster highlights several similarities and differences. These findings could have implications in: (1) identifying new targets for insecticide development; (2) strengthening our understanding of the developmental biology of mosquitoes; and (3) possibly controlling pathogen transmission. A brief overview of these interesting findings and the implications for further studies will be discussed here.

Trichomonas vaginalis, a human-infectious protozoan, can display resistance to treatment by metronidazole. A library of 3,4-dichloroaniline amides based on propanil, an herbicide, has been synthesized and screened to test susceptibility to these analogs. From this preliminary study, the most effective compound 15, inhibits growth of the organism by 66% and 69% on the two strains tested, T1 and G3, respectively.

Parasitic diseases are of enormous public health significance in developing countries—a situation compounded by the toxicity of and resistance to many current chemotherapeutics. We investigated a focused library of 18 structurally diverse bis-acridine compounds for in vitro bioactivity against seven protozoan and one helminth parasite species and compared the bioactivities and the cytotoxicities of these compounds toward various mammalian cell lines. Structure-activity relationships demonstrated the influence of both the bis-acridine linker structure and the terminal acridine heterocycle on potency and cytotoxicity. The bioactivity of polyamine-linked acridines required a minimum linker length of approximately 10 Å. Increasing linker length resulted in bioactivity against most parasites but also cytotoxicity toward mammalian cells. N alkylation, but less so N acylation, of the polyamine linker ameliorated cytotoxicity while retaining bioactivity with 50% effective concentration (EC 50 ) values similar to or better than those measured for standard drugs. Substitution of the polyamine for either an alkyl or a polyether linker maintained bioactivity and further alleviated cytotoxicity. Polyamine-linked compounds in which the terminal acridine heterocycle had been replaced with an aza-acridine also maintained acceptable therapeutic indices. The most potent compounds recorded low- to mid-nanomolar EC 50 values against Plasmodium falciparum and Trypanosoma brucei ; otherwise, low-micromolar potencies were measured. Importantly, the bioactivity of the library was independent of P. falciparum resistance to chloroquine. Compound bioactivity was a function of neither the potential to bis-intercalate DNA nor the inhibition of trypanothione reductase, an important drug target in trypanosomatid parasites. Our approach illustrates the usefulness of screening focused compound libraries against multiple parasite targets. Some of the bis-acridines identified here may represent useful starting points for further lead optimization.

The molecular basis of metronidazole resistance has been examined in anaerobic bacteria, such as Bacteroides, Clostridium, and Helicobacter, and anaerobic parasitic protists such as Giardia, Entamoeba, and trichomonads. A variety of enzymatic and cellular alterations have been shown to correlate with metronidazole susceptibility in these pathogens; however, a common theme has been revealed. Resistant cells are typically deficient in drug activation. The frequent correlation between metronidazole resistance and ineffective drug activation suggests that drug resistance is the result of modification of proteins involved in drug activation. Copyright 1999 Harcourt Publishers Ltd.

African sleeping sickness in humans and Nagana in cattle are caused by the protozoan parasite Trypanosoma brucei. The chemotherapy available for these infections is limited and causes serious side effects to their mammalian hosts. Of the four available drugs used in treatment, three of them were developed >50 years ago. With the emergence of drug-resistant strains of T. brucei, it has become more imperative that new drug targets are identified. To this end, a recent study by R. Zoraghi and T. Seebeck identify a potential metabolic window for intervention in trypanosomiasis [1xThe cAMP-specific phosphodiesterase TbPDE2C is an essential enzyme in bloodstream form of Trypanosoma brucei. Zoraghi, R. and Seebeck, T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4343–4348Crossref | PubMed | Scopus (54)See all References][1]. Previous studies have identified cyclic nucleotide-specific phosphodiesterases as candidate drug targets. To explore this possibility, a cAMP-specific phosphodiesterase from T. brucei was characterized called TbPDE2C. Chemical inhibitors targeted against this enzyme are capable of inhibiting enzymatic activity and blocking the proliferation of the bloodstream form (human-infective stage) of the parasite in vitro. Ablation of TbPDE2C mRNA expression by RNA interference demonstrated that TbPDE2C is essential for maintaining intracellular cAMP levels, and loss of TbPDE2C led to increased intracellular levels of cAMP. The bloodstream form of the parasite is sensitive to the elevated levels of cAMP. This increased concentration of cAMP leads to disruption of the nuclear membrane and cell division, and ultimately gives rise to death. These in vitro studies identifying TbPDE2C as a potential target against trypanosomiasis should be followed with animal studies of inhibitors against this enzyme. Simultaneously, crystallization of the protein and subsequent analysis of its three dimensional structure should stimulate further drug development.

Cryptococcus neoformans is an encapsulated fungus that develops predominantly within immunocompromised hosts. Recent studies have shed light on the molecular and cellular basis of its pathogenesis. For example, the capsular layer of C. neoformans is required for virulence; it also elicits an antiphagocytic effect in vitro and shedding of the capsule promotes immunosuppression in vivo. C. neoformans liberates parts of its capsule after ingestion, which leads to the development of a large intracellular phagosome compartment. Dissecting this unique mechanism of pathogenesis in vivo has been difficult. As a result, in vitro models have been the primary means of examining C. neoformans invasion of host cells.A recent study by Tucker and Casadevall [1xReplication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm. Tucker, S.C. and Casadevall, A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3165–3170Crossref | PubMed | Scopus (160)See all References][1] provides strong evidence that C. neoformans replicates in the cytoplasm of macrophages using a mechanism involved in increasing phagosome permeability, with concomitant formation of intracellular vesicles that contain capsular polysaccharide. Intracellular shedding of capsular substances was measured using an immunofluorescence detection approach coupled with phase imaging. Small vesicles in the cytoplasm that stained with anti-capsule antiserum could be seen and were distinct from the larger, more brightly stained intracellular fungal cells. The authors examined loss of phagosomal membrane integrity using transmission electron microscopy, which revealed discontinuity between the phagosomal membrane and fungal cells in compartments containing engulfed C. neoformans. To determine whether membrane dissolution in phagosomes could be a mechanism by which C. neoformans could sequester nutrients, they measured the permeability of preloaded intracellular vesicles containing fluorescent markers in both infected and uninfected macrophages. Phase images of cells infected with C. neoformans captured the movement and diffusion of the fluorescent markers after fusion with the phagosome, indicating that there was increased permeability of the phagosomal membrane. To substantiate this observation, pH measurements using lysosensor staining of acidic compartments revealed that phagocytic vesicles containing fungal cells were less acidic than uninfected control macrophages, suggesting a loss of phagosome membrane integrity.Collectively, these data demonstrate that phagocytic uptake of C. neoformans by macrophages subsequently leads to dissolution of the phagosomal compartment, enabling the fungus to replicate intracellularly. In addition, there is simultaneous accumulation of polysaccharide in cytoplasmic vesicles. The infection process ultimately culminates in the lysis and destruction of the host cell. These data present a microbial strategy of subverting intracellular destruction that is different to the mechanisms used by other well-studied intracellular pathogens. For example, Legionella pneumophila prevents lysosome–phagosome fusion, and Listeria monocytogenes uses vacuole acidification for enzyme activation and subsequent lysis of the phagosomal compartment. In C. neoformans pathogenesis, the role of these intracellular polysaccharide vesicles and permeabilizing phagosomal membranes might provide more insight into this interesting mechanism.

Cryptococcus neoformans is a facultative intracellular fungal pathogen that can cause life-threatening meningitis in immunocompromised patients. Its ecological niche is soil and the normal route of infection is inhalation of infectious particles. C. neoformans cells are encapsulated by a cell wall composed of polysaccharide, which protects the fungus from intracellular destruction by phagocytic cells. Legionella pneumophila was the first bacterial pathogen shown to multiply and persist in macrophages and amoebae. Amoebae and macrophages phagocytose particles into vacuoles and secrete lysosomal enzymes that digest the particles; the intracellular events after ingestion of L. pneumophila by amoebae and macrophages are similar.Amoebae of the genus Acanthamoeba feed on bacteria and fungi. Acanthamoeba is a freshwater and soil protozoan that was originally isolated from cultures of C. neoformans, and has been used to study bacteria–amoeba interactions. A previous study using fungal cells demonstrated that Acanthamoeba cells can phagocytose and kill 78%–99% of cells using several C. neoformans isolates. Streenbergen et al. [1xCryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages. Steenbergen, J.N. et al. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15245–15250CrossRef | PubMed | Scopus (230)See all References][1] have now tested the hypothesis that C. neoformans is capable of intracellular growth in amoebae and that the mechanism of subversion of macrophage function is selected to facilitate survival of interactions with environmental phagocytic cells.To examine the interaction between amoebae and fungal cells, several wild-type, capsular strains were compared with several mutant, acapsular strains (including a pseudohyphal mutant) in a multitude of cellular assays. The addition of fungal cells to amoebae resulted in phagocytosis of the fungal cells. The efficiency of phagocytosis was determined to be between 17%–23% for capsular strains and 34%–65% for mutants defective in capsule or pseudohyphal production. This strongly suggested that fungal cells lacking capsules were phagocytosed at a significantly higher rate than capsular cells, suggesting a role for this cellular structure in avoiding ingestion. The number of ‘buds’ formed on the surface of the amoebae after infection, an indicator of intracellular replication, was also determined. In wild-type, capsular strains, the number of buds increased over time. Mutant strains of C. neoformans, however, showed no significant levels of budding, suggesting that they were probably being destroyed intracellularly. Trypan-blue exclusion of amoebae revealed that mutant C. neoformans cells were not able to kill the amoebae, whereas wild-type strains readily lysed them. Scanning electron micrographs of infected amoebae indicated signs of intracellular development of wild-type strains, and lack of development of mutant strains. An amoeba-killing assay was also used to quantify the levels of killing of amoebae by fungal cells. Again, those cells with wild-type capsules had a greater number of colony-forming units than those lacking the polysaccharide coat. A striking contrast between this study and that of L. pneumophila–amoebae interactions should be noted here: fungal cells destroy amoebae, probably to prevent intracellular destruction, rather than using them as hosts for replication, as is the case with the bacterial pathogen.This elegant study details for the first time the cellular interaction between C. neoformans and amoebae, cells which fungal particles often encounter in nature. Amoebae are also similar to macrophages found in the human host, and therefore provide insight into pathogenic mechanisms in the human host. This study also develops a useful tool to test the role of potential fungal virulence factors. One can also extrapolate from this biological example and begin to address questions regarding the evolutionary adaptation of pathogens to their vertebrate hosts.