Keywords: amphotericin B resistant and susceptible A. terreus strain cluster, Galleria mellonella, invertebrate in vivo modelThe aim of this study was to investigate if the alternative in vivo model Galleria mellonella can be used (i) to determine differences in pathogenicity of amphotericin B (AMB) resistant and susceptible A. terreus isolates, (ii) to evaluate AMB efficacy in vivo (iii) and to correlate outcome to in vitro susceptibility data. Larvae were infected with 2 A. terreus AMB resistant (ATR) and 3 AMB susceptible (ATS) isolates and survival rates were correlated to physiological attributes and killing ability of larval haemocytes. Additionally, infected larvae were treated with different concentrations of L-AMB. Haemocyte density were ascertained to evaluate the influence of L-AMB on the larval immune cells. Larvae were sensitive to A. terreus infection in an inoculum-size and temperature dependent manner. In vitro susceptibility to L-AMB correlated with in vivo outcome of antifungal treatment, defining an AMB susceptible strain cluster of A. terreus. Susceptibility to L-AMB increased virulence potential in the larval model, but this increase was also in accordance with faster growth and less damage caused by larval haemocytes. L-AMB treatment primed the larval immune response by increasing haemocyte density. G. mellonella provides a convenient model for the in vivo screening of A. terreus virulence and treatment options, contributing to the generation of a hypothesis that can be further tested in refined experiments in mammalian models.

Amphotericin B (AMB) is the predominant antifungal drug, but the mechanism of resistance is not well understood. We compared the in vivo virulence of an AMB-resistant Aspergillus terreus (ATR) isolate with that of an AMB-susceptible A. terreus isolate (ATS) using a murine model for disseminated aspergillosis. Furthermore, we analyzed the molecular basis of intrinsic AMB resistance in vitro by comparing the ergosterol content, cell-associated AMB levels, AMB-induced intracellular efflux, and prooxidant effects between ATR and ATS. Infection of immunosuppressed mice with ATS or ATR showed that the ATS strain was more lethal than the ATR strain. However, AMB treatment improved the outcome in ATS-infected mice while having no positive effect on the animals infected with ATR. The in vitro data demonstrated that ergosterol content is not the molecular basis for AMB resistance. ATR absorbed less AMB, discharged more intracellular compounds, and had better protection against oxidative damage than the susceptible strain. Our experiments showed that ergosterol content plays a minor role in intrinsic AMB resistance and is not directly associated with intracellular cell-associated AMB content. AMB might exert its antifungal activity by oxidative injury rather than by an increase in membrane permeation. Invasive mold infections (IMI) are a significant determinant of morbidity and mortality in patients undergoing cancer chemotherapy, hematopoietic stem cell transplantation, or solid organ transplantation (1-3). These infections remain difficult to manage with therapeutic treatments because of a usually late diagnosis and complication of the treatment procedure by toxicity or interactions of drugs (4). The majority of IMI are caused by Aspergillus spp., and the most pathogenic species are Aspergillus fumigatus, Aspergillus terreus, and Aspergillus flavus (5). In particular, A. terreus, a widespread soil saprophyte and producer of several secondary metabolites, is a common cause of infection at the University Hospital of Innsbruck (UHI) in Austria (6-9). In vivo and in vitro data indicate that almost all A. terreus isolates are intrinsically resistant to amphotericin B (AMB), a fungicidal heptaene macrolide antimycotic, and a high mortality rate in patients is associated with this particular mold (10-12).Previous work has shown that AMB binds to ergosterol, the principal sterol in the fungal cell membrane, and forms aqueous pores in the lipid bilayers. Subsequently, proteins and amino acids leak out, which in turn leads to disrupted membrane proton gradients (13-16). AMB resistance is rare, and it has been suggested that for A. flavus and Candida albicans, the ergosterol content (17), the composition of the fungal cell wall (18), and the ability to produce catalase might play a role in AMB resistance (19). SokolAnderson et al. speculated that AMB causes cell death in C. albicans by oxidative damage (19). Despite intensive research for over 50 years, the exact mechanism of action of AMB is still incompletely understood, and the ...

The polyene antifungal amphotericin B (AmB) is widely used to treat life-threatening fungal infections. Even though AmB resistance is exceptionally rare in fungi, most Aspergillus terreus isolates exhibit an intrinsic resistance against the drug in vivo and in vitro. Heat shock proteins perform a fundamental protective role against a multitude of stress responses, thereby maintaining protein homeostasis in the organism. In this study, we elucidated the role of heat shock protein 70 (Hsp70) family members and compared resistant and susceptible A. terreus clinical isolates. The upregulation of cytoplasmic Hsp70 members at the transcriptional as well as translational levels was significantly higher with AmB treatment than without AmB treatment, particularly in resistant A. terreus isolates, thereby indicating a role of Hsp70 proteins in the AmB response. We found that Hsp70 inhibitors considerably increased the susceptibility of resistant A. terreus isolates to AmB but exerted little impact on susceptible isolates. Also, in in vivo experiments, using the Galleria mellonella infection model, cotreatment of resistant A. terreus strains with AmB and the Hsp70 inhibitor pifithrin-resulted in significantly improved survival compared with that achieved with AmB alone. Our results point to an important mechanism of regulation of AmB resistance by Hsp70 family members in A. terreus and suggest novel drug targets for the treatment of infections caused by resistant fungal isolates.