Jianjun Qiao

The Sho1 Sensor Regulates Growth, Morphology, and Oxidant Adaptation in Aspergillus fumigatus but Is Not Essential for Development of Invasive Pulmonary Aspergillosis

ABSTRACT Aspergillus fumigatus is an important opportunistic fungal pathogen. This organism must be able to adapt to stress changes in the microenvironment during host invasion and systemic spread. The high-osmolarity-glycerol (HOG) mitogen-activated protein kinase (HOG-MAPK) signaling pathway plays an important role in regulating morphology, growth, and adaptation to stress and virulence in a number of fungal pathogens. The Sho1 adaptor protein is one important element of the two upstream branches of the HOG-MAPK pathway in Saccharomyces cerevisiae, a signal transduction cascade involved in adaptation to stress. We constructed a sho1 mutant of A. fumigatus, MA21. Both the growth and germination rates of the mutant were reduced, and the MA21 strain had an irregular hyphal morphology characterized by reduced production of phialides and conidia. This gene deletion mutant was sensitive to 2.5 mM hydrogen peroxide and 15 μM menadione, but it appeared to be minimally sensitive to diamide compared to the wild-type strain. In an immunosuppressed mouse model, the mutant was as virulent as the wild-type or complemented strains. These data support the idea that the loss of sho1, a highly conserved gene among fungi, regulates radial hyphal growth and delays germination of A. fumigatus conidia. In addition, the sho1 gene has a visible effect in the adaptation to oxidative stress in A. fumigatus similar to that in S. cerevisiae.

Antifungal activity of statins against Aspergillus species.

The cholesterol-lowering agents known as statins have in vitro activities against human pathogenic fungi, such as Candida species, Cryptococcus neoformans, and Zygomycetes. Synergy between statins and azoles against these fungi has also been reported. We evaluated the in vitro activities of two statins, lovastatin and simvastatin, alone and in combination with azoles and amphotericin B, against clinical isolates of Aspergillus spp. A disk diffusion assay showed that both statins were active against Aspergillus spp. The minimal inhibitory concentration (MIC) ranges for lovastatin and simvastatin against Aspergillus spp. were 16 to >256 microg/ml and 4 to >256 microg/ml, respectively. Although both statins were fungicidal for A. fumigatus, the MICs were vastly higher than clinically achievable concentrations. The results of a combined agar dilution-Epsilometer test as well as a disk diffusion assay showed that neither statin had any effect on the in vitro activities of itraconazole, voriconazole, or amphotericin B against Aspergillus spp.

Afyap1, encoding a bZip transcriptional factor of Aspergillus fumigatus, contributes to oxidative stress response but is not essential to the virulence of this pathogen in mice immunosuppressed by cyclophosphamide and triamcinolone

Aspergillus fumigatus, an important human fungal pathogen, encounters high levels of reactive oxygen species following its ingestion by phagocytes. Reactive oxygen species are important mediators of the fungicidal activities of phagocytes. In yeasts, YAP1 encodes for transcriptional factors that contribute to their oxidative stress response and given the importance of the stress response, we hypothesized that the YAP1 homologue in A. fumigatus plays a similar role in this fungus. In this study, we found that Afyap1, the Yap1 homologue of A. fumigatus, confers protection against oxidative stress. Replacement of Afyap1 with the marker gene pyrG (DeltaAfyap1) resulted in hypersensitivity of A. fumigatus to oxidants such as H(2)O(2) and menadione. In contrast, an A. fumigatus strain harboring multiple-copy Afyap1 was resistant to these two oxidants as well as the oxidant diamide. However, DeltaAfyap1 and strain harboring multiple-copy Afyap1 were comparable in their virulence to a wild-type A. fumigatus strain in a murine model of invasive pulmonary aspergillosis. Taken together, these results demonstrate that Afyap1 is involved in oxidative stress response but is not an essential virulence factor for A. fumigatus.

The effects of temperature, pH, and salinity on the growth and dimorphism of Penicillium marneffei

Penicillium marneffei is an important thermal dimorphic fungus that is endemic to Southeast Asia and China and causes penicilliosis, an AIDS-defining disease. Dimorphic switching is considered an important growth characteristic associated with its pathogenicity. In recent years, the molecular mechanisms underlying both dimorphic switching and monomorphic growth have been studied. However, little is known about the physical and chemical factors that impact either dimorphic switching or monomorphic growth of this organism. Further, the natural history of the disease is unclear. Our experiments focus upon the effects of temperature, pH and salinity on both growth phases of P. marneffei. We compared 11 isolates of P. marneffei and found that all could grow at a wide temperature range (8.0-39.8 degrees C), but growth was dramatically inhibited at 40 degrees C. The morphological switch from hyphae to yeast growth was initiated at 32 degrees C. However, the sensitivity to elevated temperatures during this transition varied among isolates. Both hyphae and yeast growth forms grew much better at acidic (pH 5, 6) and neutral pH than at alkaline conditions. While similar sensitivities were observed at high concentrations of NaCl and CaCl(2), in general, yeast cells displayed a greater sensitivity to both compounds. Our data demonstrate that isolate differences occur in growth patterns. Importantly, the growth requirements defined in our study may shed light on the environmental conditions that favor its survival, a subject that is not completely resolved in the current literature.

Truncated Afyap1 attenuates antifungal susceptibility of Aspergillus fumigatus to voriconazole and confers adaptation of the fungus to oxidative stress.

In yeasts truncated YAP1 homologues confer antifungal resistance. Our previous work has identified Afyap1, a YAP1 homologue, in Aspergillus fumigatus and found that it is responsible for oxidative stress in vitro. In order to decipher whether truncated Afyap1 involves in antifungal resistance mechanism and in oxidative stress adaptation in A. fumigatus, we introduce a putatively hyperactive truncated Afyap1 into wild-type A. fumigatus. We found that the resulted A. fumigatus containing truncated Afyap1 attenuated susceptibility to voriconazole and resistance to various oxidants. However, the Afyap1 deletion mutant and the strain harboring multiple copies of full-length Afyap1 had voriconazole susceptibility comparable with that of a wild-type A. fumigatus strain. Our study demonstrates that the truncated Afyap1 may involve in antifungal resistance to voriconazole in A. fumigatus and that the truncated Afyap1 sufficiently confers tolerance to oxidative stress in A. fumigatus.

In vitro interactions of micafungin with amphotericin B, itraconazole or fluconazole against the pathogenic phase of Penicillium marneffei

OBJECTIVESnPenicillium marneffei infection is an important disease among human immunodeficiency virus patients in south-east Asia, including southern China. However, therapeutic strategies are limited. Combination regimens with synergistic drugs could provide additional options for treating penicilliosis. We evaluated the in vitro efficacy of combining micafungin with amphotericin B, itraconazole or fluconazole against the pathogenic yeast form of P. marneffei.nnnMETHODSnTwenty isolates of P. marneffei were assayed. Drug interactions were assessed with the chequerboard technique using the CLSI (formerly the NCCLS) microdilution method (M27-A2) with minor modifications. The fractional inhibitory concentration index (FICI) was used to classify drug interactions. Results were interpreted as follows: synergy, FICI < or =0.5; no interaction, FICI >0.5 and < or =4.0; or antagonism, FICI >4.0.nnnRESULTSnThe in vitro interactions of micafungin combined with itraconazole showed the highest percentage of synergic interaction (65%); for the micafungin/amphotericin B combination, 50% of the isolates had synergic interaction. Micafungin significantly enhanced the antifungal activity of amphotericin B and itraconazole against P. marneffei. Micafungin, however, did not enhance the activity of fluconazole and no synergism was observed with this combination. Antagonism was not detected for any of the antifungal combinations assayed.nnnCONCLUSIONSnThe results of this study suggest that micafungin enhances the efficacy of itraconazole or amphotericin B in vitro and indicate that an echinocandin, such as micafungin, might have a potential role in combination therapy among patients infected with P. marneffei.