The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence.

Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize the A. fumigatus protein phosphatase 2A SitA, the Saccharomyces cerevisiae Sit4p homologue. The sitA gene is not an essential gene, and we were able to construct an A. fumigatus null mutant. The ΔsitA strain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ΔsitA strain is more sensitive to several metals and ions, such as MnCl2, CaCl2, and LiCl, but it is more resistant to ZnSO4. The ΔsitA strain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-α) response in mouse macrophages. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway.

The development of animal infection models and antifungal efficacy assays against clinical isolates of Trichosporon asahii, T. asteroides and T. inkin.

The present study developed Galleria mellonella and murine infection models for the study of Trichosporon infections. The utility of the developed animal models was demonstrated through the assessment of virulence and antifungal efficacy for 7 clinical isolates of Trichosporon asahii, T. asteroides and T. inkin. The susceptibility of the Trichosporon isolates to several common antifungal drugs was tested in vitro using the broth microdilution and the E-test methods. The E-test method depicted a lower minimal inhibitory concentration (MIC) for amphotericin and a slightly higher MIC for caspofungin, while MICs observed for the azoles were different but comparable between both methods. All three Trichosporon species established infection in both the G. mellonella and immunosuppressed murine models. Species and strain dependent differences were observed in both the G. mellonella and murine models. T. asahii was demonstrated to be more virulent than the other 2 species in both animal hosts. Significant differences in virulence were observed between strains for T. asteroides in the murine model. In both animal models, fluconazole and voriconazole were able to improve the survival of the animals compared to the untreated control groups infected with any of the 3 Trichosporon species. In G. mellonella, amphotericin was not able to reduce mortality in any of the 3 species. In contrast, amphotericin was able to reduce murine mortality in the T. asahii or T. inkin models, respectively. Hence, the developed animal infection models can be directly applicable to the future deeper investigation of the molecular determinants of Trichosporon virulence and antifungal resistance.

Comparative virulence of three species of Exophiala in mice.

The virulence of Exophiala dermatitidis, E. oligosperma and E. xenobiotica, three of the most common members of the genus that cause human infections, was evaluated using experimental models of disseminated infection in immunocompromised mice. Exophiala dermatitidis, and to a lesser extent E. oligosperma, were the two species causing the highest mortality, while mice infected with E. xenobiotica had the lowest mortality. Tissue burden and histopathology studies demonstrated the neurotropism of E. dermatitidis, while E. oligosperma and E. xenobiotica had a limited capacity for invading brain tissue. These models could be useful for testing new therapies against Exophiala infections.

Development of murine models of disseminated infection by Neoscytalidium dimidiatum.

We have developed two murine models of disseminated infections by Neoscytalidium dimidiatum, an emerging dematiaceous fungus. Immunosuppressed mice were challenged through the lateral tail vein with 1 x 10(5) or 1 x 10(6) CFU/ml and immunocompetent animals with 1 x 10(6) or 1 x 10(7) CFU/ml. N. dimidiatum var. dimidiatum was more virulent than the nonpigmented variety, N. dimidiatum var. hyalinum. All mice infected with N. dimidiatum var. dimidiatum died within 8 days while those infected with N. dimidiatum var. hyalinum survived to the end of the experiment. Fungal load in tissue was also higher in animals inoculated with N. dimidiatum var. dimidiatum. In general, of the five organs tested, spleens and kidneys were most affected.

Efficacy of posaconazole in a murine disseminated infection by Candida tropicalis.

We evaluated the efficacy of posaconazole against Candida tropicalis in a systemic infection model with immunosuppressed mice. Posaconazole at 50 mg/kg of body weight/day prolonged the survival of mice and reduced the fungal tissue burden of mice infected with any of the five strains tested, with the exception of one strain that had a high MIC against this drug. Our results demonstrate the efficacy of posaconazole in the treatment of invasive murine infection caused by C. tropicalis.

Paradoxical growth of Candida dubliniensis does not preclude in vivo response to echinocandin therapy.

Candida dubliniensis commonly shows paradoxical or trailing growth effects in vitro in the presence of echinocandins. We tested the in vitro activities of anidulafungin, caspofungin, and micafungin against clinical isolates of C. dubliniensis and evaluated the efficacy of these drugs in two murine models of systemic infection. The three echinocandins were similarly effective in the treatment of experimental disseminated infections with C. dubliniensis strains showing or not showing abnormal growth in vitro.

Efficacy of a new formulation of amphotericin B in murine disseminated infections by Candida glabrata or Candida tropicalis.

Amphotericin B microspheres (M-AMB) are a new, inexpensive formulation of amphotericin B. In this study, we tested the efficacy of this new formulation for treating murine disseminated infections by Candida glabrata or Candida tropicalis. M-AMB showed a similar efficacy to that of amphotericin B deoxycholate and liposomal amphotericin B in the treatment of both disseminated murine infections. Its low toxicity and inexpensive production costs make this formulation potentially attractive for the treatment of fungal infections.

Efficacy of triazoles in a murine disseminated infection by Candida krusei.

We evaluated the efficacies of posaconazole and voriconazole in comparison with that of amphotericin B in a systemic murine infection by Candida krusei. Posaconazole at 50 mg/kg/day and voriconazole at 40 and 60 mg/kg/day prolonged survival and reduced the fungal tissue burden in the kidneys of mice similarly to amphotericin B at 1.5 mg/kg/day and liposomal amphotericin B at 10 mg/kg/day. None of the treatments tested completely resolved the infection.

Efficacy of liposomal amphotericin B combined with gamma interferon or granulocyte-macrophage colony-stimulating factor for treatment of systemic zygomycosis in mice.

Granulocyte-macrophage colony-stimulating factor enhanced the efficacy of liposomal amphotericin B (LAMB) in a murine model of disseminated infection by Rhizopus oryzae, significantly prolonging survival and reducing tissue burden. The use of gamma interferon (IFN-gamma) alone was ineffective, and IFN-gamma combined with LAMB did not improve the results obtained with LAMB alone.

Effects of double and triple combinations of antifungal drugs in a murine model of disseminated infection by Scedosporium prolificans.

We have evaluated the efficacies of micafungin, amphotericin B, and voriconazole, alone and in double and triple combinations, in a murine model of systemic infection by Scedosporium prolificans. Micafungin combined with voriconazole or amphotericin B was the most effective, these being the only treatments able to prolong survival and to reduce the fungal load in the kidneys and brain. Triple combinations of these drugs did not improve the results obtained with double combinations.

Interactions between triazoles and amphotericin B in treatment of disseminated murine infection by Fusarium oxysporum.

We have evaluated and compared the efficacies of high doses of amphotericin B (AMB; 3 mg/kg of body weight/day), voriconazole (60 mg/kg), and posaconazole (PSC; 100 mg/kg) alone and combined in a murine model of disseminated infection by Fusarium oxysporum. The combination of AMB with PSC showed the best results, prolonging the survival of mice and reducing their organ fungal loads. This combination might constitute a therapeutic option for those infections where monotherapies fail.

Combined antifungal therapy in a murine model of disseminated infection by Cladophialophora bantiana.

We tested ten day courses of amphotericin B (AMB), micafungin (MFG), voriconazole (VRC), flucytosine (5FC) and posaconazole (PSC) alone and in double or triple combinations in the treatment of disseminated infections caused by Cladophialophora bantiana in a murine model. Animals were monitored for survival for 40 days. We found that PSC at 100 mg/kg or 5FC at 180 mg/kg prolonged survival over controls. The combinations PSC+MFG and PSC+5FC improved survival compared to MFG and 5FC alone, but were not superior to PSC alone. The triple combination of PSC+MFG+5FC improved the survival with respect to both the control group and the component monotherapies, but all the animals died during the experiment. When treatment with this triple therapy was extended up to 30 days, half of the animals survived for at least 10 months. Combination therapy with the three drugs (PSC, MFG and 5FC) appears to be a promising option for the treatment of C. bantiana infections.

In vitro interactions of itraconazole and micafungin against clinically important filamentous fungi.

The in vitro interactions between itraconazole and micafungin against 133 strains of filamentous fungi of clinical interest were evaluated using a checkerboard method. Overall, synergistic interactions were observed against 30% of the strains tested. In the cases of Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Fonsecaea spp. and Sporothrix schenckii, synergistic interactions were observed against > or = 50% of the strains tested. For the rest of the fungi the interaction was indifferent; antagonism was not observed in any case.

Posaconazole combined with amphotericin B, an effective therapy for a murine disseminated infection caused by Rhizopus oryzae.

In a murine model of disseminated zygomycosis, low doses of amphotericin B (0.3 mg/kg body weight/day) combined with posaconazole (40 mg/kg/day) prolonged survival and reduced tissue burden with respect to that of controls and that of both drugs administered alone. Results were similar to those obtained with amphotericin B given alone at 0.8 mg/kg/day.

Role of the white collar 1 photoreceptor in carotenogenesis, UV resistance, hydrophobicity, and virulence of Fusarium oxysporum.

Knockout mutants of Fusarium oxysporum lacking the putative photoreceptor Wc1 were impaired in aerial hyphae, surface hydrophobicity, light-induced carotenogenesis, photoreactivation after UV treatment, and upregulation of photolyase gene transcription. Infection experiments with tomato plants and immunodepressed mice revealed that Wc1 is dispensable for pathogenicity on plants but required for full virulence on mammals.

Combined therapy in treatment of murine infection by Fusarium solani.

OBJECTIVES: We evaluated the efficacy of voriconazole, amphotericin B and micafungin alone and combined in a murine model of disseminated infection by Fusarium solani. METHODS: Immunosuppressed mice were treated with voriconazole at 60 mg/kg/day, amphotericin B at 3 mg/kg/day, micafungin at 10 mg/kg/day or combinations of these antifungal drugs. For survival studies, treatment began 1 day after infection and continued for 10 days. For tissue burden studies, animals were sacrificed on day 6 of the treatment and the fungal load in the kidneys and spleens was measured. The experiments were carried out using two different clinical strains of F. solani. RESULTS: Only the combination of voriconazole plus amphotericin B prolonged the survival of the mice versus the controls for the two strains tested. However, this combination only reduced tissue burden in the kidney and spleen of mice infected by one strain. The other treatments were clearly less effective. CONCLUSIONS: Voriconazole plus amphotericin B may have a role in the treatment of fusariosis.

Rho1 has distinct functions in morphogenesis, cell wall biosynthesis and virulence of Fusarium oxysporum.

Rho-type GTPases regulate polarized growth in yeast by reorganization of the actin cytoskeleton and through signalling pathways that control the expression of cell wall biosynthetic genes. We report the cloning and functional analysis of rho1 from Fusarium oxysporum, a soilborne fungal pathogen causing vascular wilt on plants and opportunistic infections in humans. F. oxysporum strains carrying either a Deltarho1 loss-of-function mutation or a rho1(G14V) gain-of-function allele were viable, but displayed a severely restricted colony phenotype which was partially relieved by the osmotic stabilizer sorbitol, indicating structural alterations in the cell wall. Consistent with this hypothesis, Deltarho1 strains showed increased resistance to cell wall-degrading enzymes and staining with Calcofluor white, as well as changes in chitin and glucan synthase gene expression and enzymatic activity. Re-introduction of a functional rho1 allele into the Deltarho1 mutant fully restored the wild-type phenotype. The Deltarho1 strain had dramatically reduced virulence on tomato plants, but was as virulent as the wild type on immunodepressed mice. Thus, Rho1 plays a key role during fungal infection of plants, but not of mammalian hosts.

Efficacy of a new formulation of amphotericin B in a murine model of disseminated infection by Candida glabrata.

OBJECTIVES: Amphotericin B poly-aggregates are a new formulation of amphotericin B, which can be obtained cheaply. In this study, we tested the efficacy of this new formulation for treating a disseminated infection by Candida glabrata in a murine model. METHODS: Mice were rendered neutropenic by intraperitoneal cyclophosphamide and intravenous 5-fluorouracil administration and infected intravenously with 2 x 10(8) cfu of C. glabrata. The efficacy of the new formulation of amphotericin B was evaluated by survival and tissue burden studies. The experiments were repeated using three different clinical strains of C. glabrata. RESULTS AND CONCLUSIONS: Amphotericin B poly-aggregates showed an efficacy similar to that of amphotericin B deoxycholate and liposomal amphotericin B in the treatment of a disseminated murine infection by C. glabrata.

Micafungin combined with fluconazole, an effective therapy for murine blastoschizomycosis.

OBJECTIVES: To study the efficacy of micafungin combined with fluconazole in a murine model of disseminated blastoschizomycosis. METHODS: Mice were treated with fluconazole at 40 or 80 mg/kg/day given orally, with micafungin at 10 mg/kg/day given subcutaneously or with a combination of micafungin with fluconazole at the doses described above. Treatment began 1 day after infection and continued for 6 days post-infection. Tissue burden studies were performed 1 day after the treatment finished. The experiments were performed with three different strains. RESULTS: The two combinations tested significantly improved the survival of mice with respect to the control group and reduced the tissue burden significantly with respect to the control and their respective monotherapies in most organs tested. All animals that received the combination of micafungin with fluconazole at 80 mg/kg/day survived up to the end of the experiment. CONCLUSIONS: The combination of micafungin with fluconazole is promising for the treatment of disseminated blastoschizomycosis.