Molecular Evaluation of the Plasma Membrane Proton Pump from Aspergillus fumigatus

105

Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections, including invasive aspergillosis. It has been our aim to understand the molecular targets of AMB in Aspergillus fumigatus by genomic and proteomic approaches. In transcriptomic analysis, a total of 295 genes were found to be differentially expressed (165 upregulated and 130 downregulated), including many involving the ergosterol pathway, cell stress proteins, cell wall proteins, transport proteins, and hypothetical proteins. Proteomic profiles of A. fumigatus alone or A. fumigatus treated with AMB showed differential expression levels for 85 proteins (76 upregulated and 9 downregulated). Forty-eight of them were identified with high confidence and belonged to the above-mentioned categories. Differential expression levels for Rho-GDP dissociation inhibitor (Rho-GDI), secretory-pathway GDI, clathrin, Sec 31 (a subunit of the exocyst complex), and RAB GTPase Ypt51 in response to an antifungal drug are reported here for the first time and may represent a specific response of A. fumigatus to AMB. The expression of some of these genes was validated by real-time reverse transcription-PCR. The AMB responsive genes/proteins observed to be differentially expressed in A. fumigatus may be further explored for novel drug development.Invasive aspergillosis, caused primarily by Aspergillus fumigatus, has emerged as the leading cause of mortality among immunocompromised patients with underlying hematological diseases or bone marrow transplantation (7, 38). Amphotericin B (AMB), a broad-spectrum fungicidal agent, has been widely used to treat patients with invasive aspergillosis. AMB is reported to be fungicidal (MFC/MIC Յ 4) against all A. fumigatus and Aspergillus flavus isolates but not on Aspergillus terreus isolates (25). Its therapeutic use is limited by its toxicity (nephrotoxicity, cytotoxicity, and hepatotoxicity, etc.) in the host (13) and development of resistance in fungal isolates (42). However, use of lipid formulations of AMB, administration by the inhalation route, and development of less toxic analogues have facilitated better therapeutic outcomes (35). In general, it is known that AMB intercalates with ergosterol of the fungal cell membrane and forms pores resulting in leakage of fungal cell components, which leads to death via osmotic collapse (15, 22).It also promotes oxidative damage to cell membranes through generation of reactive oxygen species (ROS) (43) and damage to DNA resulting in loss of cell viability, a characteristic of apoptosis (31).Earlier efforts reported genome-wide expression analysis to understand the mechanism of action and specific effects of AMB and other antifungal drugs on nonfilamentous fungal species, such as Saccharomyces cerevisiae and Candida albicans (1,24,46). However, in an earlier study of interaction of A. fumigatus with voriconazole (11), decreased mRNA expression of ergosterol biosynthesis genes was observed, which was in contrast with previous reports of S....

Section: Discussionmentioning

confidence: 75%

Proteomic and Transcriptomic Analysis of Aspergillus fumigatus on Exposure to Amphotericin B

Gautam

Shankar

Madan

et al. 2008

105

“…The H ϩ -ATPase from Saccharomyces cerevisiae has been shown to be essential by gene-disruption experiments (50), and it displays a number of biochemical and genetic properties that make it attractive as a drug discovery target (41). The pumps mentioned above are present also in A. fumigatus (6). These pumps can be blocked by different compounds, including the novel compound conjugated styryl ketone, which was shown to be active when it was tested against A. fumigatus in vitro and in vivo (34,35); but no drug combination was tested against Aspergillus in those studies.…”

Section: Discussionmentioning

confidence: 99%

Potent Synergistic In Vitro Interaction between Nonantimicrobial Membrane-Active Compounds and Itraconazole against Clinical Isolates of Aspergillus fumigatus Resistant to Itraconazole

Afeltra

Vitale

Mouton

et al. 2004

To develop new approaches for the treatment of invasive infections caused byInvasive aspergillosis causes approximately 30% of invasive fungal infections in patients treated for cancer (11). Until 1990 only one drug was available for the treatment of invasive Aspergillus disease, amphotericin B, which must be given intravenously and which has a number of serious toxicities. In 1990 itraconazole (ITZ) capsules became available and Aspergillus species were included in the spectrum of activity of the drug, although the drug was mainly used in the prophylactic setting due to poor bioavailability (11). Ten years later an intravenous formulation of ITZ became available and allowed the drug to be used for the empirical or preemptive treatment of high-risk patients. With the registration of voriconazole and caspofungin, the arsenal of available drugs has increased further. However, despite antifungal therapy, the rate of mortality in patients with invasive aspergillosis remains very high, and clearly, new therapeutic approaches are needed. Combination therapy is one approach that can be used to improve the efficacy of antimicrobial therapy for difficult-to-treat infections, such as human immunodeficiency virus and mycobacterial infections. By analogy, the combination of ITZ with other compounds could represent a possible approach for the treatment of patients with invasive aspergillosis or patients infected with strains with reduced susceptibilities to antifungal agents. Resistance to antifungal azoles has been studied in yeasts and molds, especially Aspergillus. Resistance mechanisms include changes in the cellular azole content (an altered uptake or efflux mechanism), mutations in sterol desaturation during ergosterol biosynthesis, and mutations in or elevated levels of 14␣-demethylase (12, 42). The recent discovery of drug effluxmediated resistance mechanisms in yeasts and Aspergillus opens new therapeutic concepts. It has been recognized that Candida albicans and Aspergillus nidulans express multidrug efflux transporter (MET) genes belonging to different classes, i.e., the ATP-binding cassette (ABC) transporters and the major facilitators (13,48). The expression of these genes and their targeted deletion determine the level of azole resistance.In this study we investigated the in vitro interactions between ITZ and different nonantimicrobial membrane-active compounds against clinical ITZ-resistant (ITZ-R) and ITZsusceptible (ITZ-S) strains using four different drug interaction models. MATERIALS AND METHODSStrains. Fourteen clinical isolates of Aspergillus fumigatus were tested. These included seven ITZ-S isolates (isolates V09-22, V09-23, AZN5161, AZN7820, AZN8248, AZN9339, and AZN9362) and seven ITZ-R isolates (isolates V09-18, V09-19, AZN5241, AZN5242, AZN7720, AZN7722, and AZG7). The strains numbered AZN and V09 were obtained from the private collection of the Department of Medical Microbiology, University Medical Center Nijmegen, and strain AZG7 was obtained from the University Hospital Groningen, Groningen, The ...

“…Relative expression was evaluated using the Pfaffl method (33). The A. fumigatus PMA1 gene and beta-tubulin gene (BTU) were used to normalize the results (4,25). Two housekeeping genes were used because differences in expression were observed at different time points in A. fumigatus (unpublished data).…”

Section: Methodsmentioning

confidence: 99%

Establishing In Vitro-In Vivo Correlations for Aspergillus fumigatus : the Challenge of Azoles versus Echinocandins

Arendrup

Perkhofer

Howard

et al. 2008

101

Two clinical isolates of Aspergillus fumigatus, designated AT and DK, were recently obtained from patients failing caspofungin and itraconazole therapy, respectively. The isolates were tested by microdilution for susceptibility to itraconazole, voriconazole, posaconazole, ravuconazole, and caspofungin and by Etest for susceptibility to amphotericin B and caspofungin. Susceptibility testing documented that the DK isolate was azole resistant (itraconazole and posaconazole MICs, >4 g/ml; voriconazole MIC, 2 g/ml; ravuconazole MIC, 4 g/ml), and the resistance was confirmed in a hematogenous mouse model, with mortality and the galactomannan index as the primary and secondary end points. Sequencing of the cyp51A gene revealed the M220K mutation, conferring multiazole resistance. The Etest, but not microdilution, suggested that the AT isolate was resistant to caspofungin (MIC, >32 g/ml). In the animal model, this isolate showed reduced susceptibility to caspofungin. Sequencing of the FKS1 gene revealed no mutations; the enzyme retained full sensitivity in vitro; and investigation of the polysaccharide composition showed that the ␤-(1,3)-glucan proportion was unchanged. However, gene expression profiling by Northern blotting and real-time PCR demonstrated that the FKS gene was expressed at a higher level in the AT isolate than in the susceptible control isolate. To our knowledge, this is the first report to document the presence of multiazoleresistant clinical isolates in Denmark and to demonstrate reduced susceptibility to caspofungin in a clinical A. fumigatus isolate with increased expression of the FKS gene. Further research to determine the prevalence of resistance in A. fumigatus worldwide, and to develop easier and reliable tools for the identification of such isolates in routine laboratories, is warranted.