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Eleftherios Mylonakis

Publications and source records attributed to Eleftherios Mylonakis.

At least 19 recordsLinked to original sources
PubMed2006

New agents for the treatment of fungal infections: clinical efficacy and gaps in coverage.

The incidence of fungal infections has increased globally, and the introduction of the newer triazoles and echinocandin antifungals is a more-than-welcome and long overdue development. In this report, we review the clinical trials evaluating the therapeutic efficacy of these new antifungal agents and examine possible gaps in coverage. Voriconazole has become the primary treatment for most forms of invasive aspergillosis in a number of centers, posaconazole offers a broad antifungal spectrum, and echinocandins are fungicidal against most Candida species. Moreover, the new agents are active against some fungi that are resistant to amphotericin B, may have a role in the management of fever and neutropenia, and provide exciting options for combination antifungal therapy. However, significant questions remain, including the management of breakthrough infections and treatment failures and the efficacy of the new antifungal agents against less common fungi.

Antifungal Agents↗
PubMed2006

Using non-mammalian hosts to study fungal virulence and host defense.

Non-mammalian hosts have been used to study host-fungal interactions. Hosts such as Drosophila melanogaster, Caenorhabditis elegans, Acathamoeba castellanii, Dictyostelium discoideum, and Galleria mellonella have provided means to examine the physical barriers, cellular mechanisms and molecular elements of the host response. The Drosophila host-response to fungi is mediated through the Toll pathway, whereas in C. elegans the host-response is TIR-1-dependent. Virulence traits that are involved in mammalian infection are important for the interaction of fungi with these hosts. Screening of fungal virulence traits using mutagenized fungi to determine changes in fungal infectivity of non-mammalian hosts has been used to identify novel virulence proteins used to infect C. elegans such as Kin1 (a serine/threonine protein kinase) and Rom2 (a Rho1 guanyl-nucleotide exchange factor) from Cryptococcus neoformans. These heterologous non-mammalian hosts highlight the similarities and differences between different hosts in fungal pathogenesis and they complement studies in mammalian systems and those using other genetic approaches.

Amoeba↗
PubMed2006

The pursuit of cryptococcal pathogenesis: heterologous hosts and the study of cryptococcal host-pathogen interactions.

Analysis of the molecular mechanisms by which a pathogen interacts with the human host is most commonly performed using a mammalian model of infection. However, several virulence-related genes previously shown to be involved in mammalian infection with Cryptococcus neoformans have also been shown to play a role in the interaction of these pathogens with invertebrates, such as Acanthamoeba castellanii, Caenorhabditis elegans, Dictyostelium discoideum, Drosophila melanogaster and Galleria mellonella. The study of host-pathogen interactions using these model hosts has allowed rapid screening of mutant libraries and can be used for the study of evolutionarily preserved aspects of microbial virulence and host response.

Animals↗
PubMed2006

Characterization and regulation of the trehalose synthesis pathway and its importance in the pathogenicity of Cryptococcus neoformans.

The disaccharide trehalose has been found to play diverse roles, from energy source to stress protectant, and this sugar is found in organisms as diverse as bacteria, fungi, plants, and invertebrates but not in mammals. Recent studies in the pathobiology of Cryptococcus neoformans identified the presence of a functioning trehalose pathway during infection and suggested its importance for C. neoformans survival in the host. Therefore, in C. neoformans we created null mutants of the trehalose-6-phosphate (T6P) synthase (TPS1), trehalose-6-phophate phosphatase (TPS2), and neutral trehalase (NTH1) genes. We found that both TPS1 and TPS2 are required for high-temperature (37 degrees C) growth and glycolysis but that the block at TPS2 results in the apparent toxic accumulation of T6P, which makes this enzyme a fungicidal target. Sorbitol suppresses the growth defect in the tps1 and tps2 mutants at 37 degrees C, which supports the hypothesis that these sugars (trehalose and sorbitol) act primarily as stress protectants for proteins and membranes during exposure to high temperatures in C. neoformans. The essential nature of this pathway for disease was confirmed when a tps1 mutant strain was found to be avirulent in both rabbits and mice. Furthermore, in the system of the invertebrate C. elegans, in which high in vivo temperature is no longer an environmental factor, attenuation in virulence was still noted with the tps1 mutant, and this supports the hypothesis that the trehalose pathway in C. neoformans is involved in more host survival mechanisms than simply high-temperature stresses and glycolysis. These studies in C. neoformans and previous studies in other pathogenic fungi support the view of the trehalose pathway as a selective fungicidal target for use in antifungal development.

Animals↗
PubMed2006

Developments in the treatment of candidiasis: more choices and new challenges.

The incidence of oesophageal candidiasis, candidaemia and disseminated candidiasis has increased dramatically. In addition to the amphotericin B formulations and fluconazole, the echinocandins anidulafungin, caspofungin and micafungin and the newer triazoles posaconazole and voriconazole are in the last stages of development and are becoming available for the management of candidiasis. This review presents these new agents and addresses their role in the treatment of candidiasis. All new antifungal agents exhibit potent activity against Candida spp. and echinocandins are fungicidal against most Candida spp. but appear to be less potent against certain species, such as Candida parapsilosis and C. guilliermondii. Systemic antifungal therapy can now be individualised based on the severity of the infection, comorbid conditions and the Candida spp. causing the infection. Studies are needed to investigate the possible development of resistance and the efficacy of these antifungal agents against the more resistant Candida spp.

Animals↗
PubMed2006

Newer triazole antifungal agents: pharmacology, spectrum, clinical efficacy and limitations.

New triazole antifungals (voriconazole, posaconazole, ravuconazole and albaconazole) have been developed to meet the increasing need for new antifungals, and address the rising incidence of invasive fungal infections and the emergence of fungal resistance. This report describes the spectrum of activity of the newer-generation triazoles based on data from in vitro, animal and clinical studies. The authors discuss the use of these agents in combination with other antifungals, the extent of cross-resistance, their toxicity profile and pharmacokinetic properties. A total of two agents are currently available: voriconazole (which is becoming a primary treatment for the management of invasive aspergillosis) and posaconazole (which demonstrates a broad antifungal spectrum). A further two agents, albaconazole and ravuconazole, are undergoing early clinical evaluation and their future is uncertain. For all newer triazoles, concerns about emerging drug-resistant fungi and the incidence and management of breakthrough infections will dictate their role in antifungal prophylaxis and treatment.

Animals↗
PubMed2005

Cryptococcus neoformans gene involved in mammalian pathogenesis identified by a Caenorhabditis elegans progeny-based approach.

Caenorhabditis elegans can serve as a substitute host for the study of microbial pathogenesis. We found that mutations in genes of the fungal pathogen Cryptococcus neoformans involved in mammalian virulence allow C. elegans to produce greater numbers of progeny than when exposed to wild-type fungus. We used this property to screen a library of C. neoformans mutants for strains that permit larger C. elegans brood sizes. In this screen, we identified a gene homologous to Saccharomyces cerevisiae ROM2. C. neoformans rom2 mutation resulted in a defect in mating and growth defects at elevated temperature or in the presence of cell wall or hyperosmolar stresses. An effect of the C. neoformans rom2 mutation in virulence was confirmed in a murine inhalation infection model. We propose that a screen for progeny-permissive mutants of microorganisms can serve as a high-throughput method for identifying novel loci related to mammalian pathogenesis.

Animals↗
PubMed2005

Galleria mellonella as a model system to study Cryptococcus neoformans pathogenesis.

Evaluation of Cryptococcus neoformans virulence in a number of nonmammalian hosts suggests that C. neoformans is a nonspecific pathogen. We used the killing of Galleria mellonella (the greater wax moth) caterpillar by C. neoformans to develop an invertebrate host model system that can be used to study cryptococcal virulence, host immune responses to infection, and the effects of antifungal compounds. All varieties of C. neoformans killed G. mellonella. After injection into the insect hemocoel, C. neoformans proliferated and, despite successful phagocytosis by host hemocytes, killed caterpillars both at 37 degrees C and 30 degrees C. The rate and extent of killing depended on the cryptococcal strain and the number of fungal cells injected. The sequenced C. neoformans clinical strain H99 was the most virulent of the strains tested and killed caterpillars with inocula as low as 20 CFU/caterpillar. Several C. neoformans genes previously shown to be involved in mammalian virulence (CAP59, GPA1, RAS1, and PKA1) also played a role in G. mellonella killing. Combination antifungal therapy (amphotericin B plus flucytosine) administered before or after inoculation was more effective than monotherapy in prolonging survival and in decreasing the tissue burden of cryptococci in the hemocoel. The G. mellonella-C. neoformans pathogenicity model may be a substitute for mammalian models of infection with C. neoformans and may facilitate the in vivo study of fungal virulence and efficacy of antifungal therapies.

Animals↗
PubMed2005

Cryptococcus neoformans {alpha} strains preferentially disseminate to the central nervous system during coinfection.

Cryptococcus neoformans is a fungal pathogen that has evolved over the past 40 million years into three distinct varieties or sibling species (gattii, grubii, and neoformans). Each variety manifests differences in epidemiology and disease, and var. grubii strains are responsible for the vast majority of human disease. In previous studies, alpha strains were more virulent than congenic a strains in var. neoformans, whereas var. grubii congenic a and alpha strains exhibited equivalent levels of virulence. Here the role of mating type in the virulence of var. grubii was further characterized in a panel of model systems. Congenic var. grubii a and alpha strains had equivalent survival rates when cultured with amoebae, nematodes, and macrophages. No difference in virulence was observed between a and alpha congenic strains in multiple inbred-mouse genetic backgrounds, and there was no difference in accumulations in the central nervous system (CNS) late in infection. In contrast, during coinfections, a and alpha strains are equivalent in peripheral tissues but alpha cells have an enhanced predilection to penetrate the CNS. These studies reveal the first virulence difference between congenic a and alpha strains in the most common pathogenic variety and suggest an explanation for the prevalence of alpha strains in clinical isolates.

Animals↗
PubMed2004

Cryptococcus neoformans Kin1 protein kinase homologue, identified through a Caenorhabditis elegans screen, promotes virulence in mammals.

Cryptococcal infections are a global cause of significant morbidity and mortality. Recent studies support the hypothesis that virulence of Cryptococcus neoformans may have evolved via survival selection in environmental hosts, such as amoebae and free-living nematodes. We used killing of the nematode Caenorhabditis elegans by C. neoformans as an assay to screen a library of random C. neoformans insertion mutants. Of 350 mutants tested, seven were identified with attenuated virulence that persisted after crossing the mutation back into a wild-type strain. Genetic analysis of one strain revealed an insertion in a gene homologous to Saccharomyces cerevisiae KIN1, which encodes a serine/threonine protein kinase. C. neoformans kin1 mutants exhibited significant defects in virulence in murine inhalation and haematogenous infection models and displayed increased binding to alveolar and peritoneal macrophages. The kin1 mutant phenotypes were complemented by the wild-type KIN1 gene. These findings show that the C. neoformans Kin1 kinase homologue is required for full virulence in disparate hosts and that C. elegans can be used as a substitute host to identify novel factors involved in fungal pathogenesis in mammals.

Animals↗
PubMed2004

Challenge of Drosophila melanogaster with Cryptococcus neoformans and role of the innate immune response.

We found that the ingestion of Cryptococcus neoformans by Drosophila melanogaster resulted in the death of the fly but that the ingestion of Saccharomyces cerevisiae or the nonpathogenic Cryptococcus kuetzingii or Cryptococcus laurentii did not. The C. neoformans protein kinase A and RAS signal transduction pathways, previously shown to be involved in virulence in mammals, also played a role in killing Drosophila. Mutation of the Toll immune response pathway, the predominant antifungal pathway of the fly, did not play a role in Drosophila defense following ingestion of the yeast. However, the Toll pathway was necessary for the clearance of C. neoformans introduced directly into the hemolymph of D. melanogaster and for the survival of systemically infected flies.

Animals↗
PubMed2004

Contribution of gelatinase, serine protease, and fsr to the pathogenesis of Enterococcus faecalis endophthalmitis.

Gelatinase and serine protease were found to contribute in concert to pathogenesis in a rabbit model of endophthalmitis. However, a mutant defective in the fsr regulator was observed to be more attenuated than a mutant rendered defective in the expression of gelatinase and serine protease as the result of a polar transposon insertion into the former. This increased attenuation suggests that there are possible additional pleiotropic effects of the defect in fsr on expression of traits contributing to the pathogenesis of enterococcal infection.

Animals↗
PubMed2004

Cytotoxicity of hydrogen peroxide produced by Enterococcus faecium.

Although the opportunistic bacterial pathogen Enterococcus faecium is a leading source of nosocomial infections, it appears to lack many of the overt virulence factors produced by other bacterial pathogens, and the underlying mechanism of pathogenesis is not clear. Using E. faecium-mediated killing of the nematode worm Caenorhabditis elegans as an indicator of toxicity, we determined that E. faecium produces hydrogen peroxide at levels that cause cellular damage. We identified E. faecium transposon insertion mutants with altered C. elegans killing activity, and these mutants were altered in hydrogen peroxide production. Mutation of an NADH oxidase-encoding gene eliminated nearly all NADH oxidase activity and reduced hydrogen peroxide production. Mutation of an NADH peroxidase-encoding gene resulted in the enhanced accumulation of hydrogen peroxide. E. faecium is able to produce hydrogen peroxide by using glycerol-3-phosphate oxidase, and addition of glycerol to the culture medium enhanced the killing of C. elegans. Conversely, addition of glucose, which leads to the down-regulation of glycerol metabolism, prevented both C. elegans killing and hydrogen peroxide production. Lastly, detoxification of hydrogen peroxide either by exogenously added catalase or by a C. elegans transgenic strain overproducing catalase prevented E. faecium-mediated killing. These results suggest that hydrogen peroxide produced by E. faecium has cytotoxic effects and highlight the utility of C. elegans pathogenicity models for identifying bacterial virulence factors.

Animals↗
PubMed2004

Disruption of the Aspergillus fumigatus gene encoding nucleolar protein CgrA impairs thermotolerant growth and reduces virulence.

Aspergillus fumigatus CgrA is the ortholog of a yeast nucleolar protein that functions in ribosome synthesis. To determine how CgrA contributes to the virulence of A. fumigatus, a Delta cgrA mutant was constructed by targeted gene disruption, and the mutant was reconstituted to wild type by homologous introduction of a functional cgrA gene. The Delta cgrA mutant had the same growth rate as the wild type at room temperature. However, when the cultures were incubated at 37 degrees C, a condition that increased the growth rate of the wild-type and reconstituted strains approximately threefold, the Delta cgrA mutant was unable to increase its growth rate. The absence of cgrA function caused a delay in both the onset and rate of germination at 37 degrees C but had little effect on germination at room temperature. The Delta cgrA mutant was significantly less virulent than the wild-type or reconstituted strain in immunosuppressed mice and was associated with smaller fungal colonies in lung tissue. However, this difference was less pronounced in a Drosophila infection model at 25 degrees C, which correlated with the comparable growth rates of the two strains at this temperature. To determine the intracellular localization of CgrA, the protein was tagged at the C terminus with green fluorescent protein, and costaining with propidium iodide revealed a predominantly nucleolar localization of the fusion protein in living hyphae. Together, these findings establish the intracellular localization of CgrA in A. fumigatus and demonstrate that cgrA is required for thermotolerant growth and wild-type virulence of the organism.

Animals↗
PubMed2003

The art of serendipity: killing of Caenorhabditis elegans by human pathogens as a model of bacterial and fungal pathogenesis.

The nematode worm, Caenorhabditis elegans, has been used to develop a facile model system of host-pathogen interactions to identify basic evolutionarily conserved pathways associated with microbial pathogenesis. The model involves the killing of Caenorhabditis elegans by a variety of human pathogens. Several virulence-related genes in a variety of pathogens previously shown to be involved in mammalian infection have also been shown to play a role in Caenorhabditis elegans killing. Screening of large numbers of microbial mutants for attenuation in a mammalian model would require thousands of mice, rats or rabbits. In contrast, the Caenorhabditis elegans model allows rapid identification of mutants in microbial genes associated with pathogenesis and then these phenotypes can be confirmed in a relevant mammalian model.

Animals↗
PubMed2002

Killing of Caenorhabditis elegans by Cryptococcus neoformans as a model of yeast pathogenesis.

We found that the well-studied nematode Caenorhabditis elegans can use various yeasts, including Cryptococcus laurentii and Cryptococcus kuetzingii, as a sole source of food, producing similar brood sizes compared with growth on its usual laboratory food source Escherichia coli OP50. C. elegans grown on these yeasts had a life span similar to (C. laurentii) or longer than (C. kuetzingii) those fed on E. coli. However, the human pathogenic yeast Cryptococcus neoformans killed C. elegans, and the C. neoformans polysaccharide capsule as well as several C. neoformans genes previously shown to be involved in mammalian virulence were also shown to play a role in C. elegans killing. These included genes associated with signal transduction pathways (GPA1, PKA1, PKR1, and RAS1), laccase production (LAC1), and the alpha mating type. C. neoformans adenine auxotrophs, which are less virulent in mammals, were also less virulent in C. elegans. These results support the model that mammalian pathogenesis of C. neoformans may be a consequence of adaptations that have evolved during the interaction of C. neoformans with environmental predators such as free-living nematodes and amoebae and suggest that C. elegans can be used as a simple model host in which C. neoformans pathogenesis can be readily studied.

Animals↗
PubMed2002

Combination antiviral therapy for ganciclovir-resistant cytomegalovirus infection in solid-organ transplant recipients.

The resistance of cytomegalovirus (CMV) to ganciclovir is a factor in therapeutic failure and disease progression. The clinical significance of such resistance in solid-organ transplantation has not been completely established. Six patients who developed persistent infection due to ganciclovir-resistant CMV were treated with a combination of ganciclovir (50% of the therapeutic dose) and a daily dose of intravenous foscarnet that gradually increased to a maximum of 125 mg/kg. All patients responded clinically within 72-96 hours. Magnesium depletion occurred in all patients. No clinical or laboratory relapses have been observed in 6-30 months of follow-up. Gradually increasing doses of foscarnet combined with half-dose regimens of ganciclovir are safe and can be beneficial in organ transplant recipients with ganciclovir-resistant CMV infection. Larger studies are needed to identify the patients who are most likely to benefit from this regimen.

Adult↗