| Abstract |
INTRODUCTION: Aspergilli are saprophytic hyphomycetes that pose a lethal threat
to the immunocompromised patients, such as patients with hematological or solid
malignancies or organ recipients receiving immunosupression. Echinocandins are a
new category of potent antifungal agents against Aspergilli in vivo; however they have
mediocre in vitro activity against these fungi, such as in in vitro susceptibility studies.
This dissertation is trying to dissect the mechanisms that cause this lag in the in vitro
and the in vivo activity of these antifungal agents against Aspergilli.
MATERIALS AND METHODS: Several Aspergilli strains were used, along with
some Candida strains as controls, and susceptibility tests against echinocandins and
voriconazole were done to 6 different Aspergillus strains in standard susceptibility
conditions for hyphomycetes but also in cell culture medium in order to simulate the
in vivo conditions in the tissues of patients infected with Aspergillus. The same tests
were repeated with one Aspergillus fumigatus strain with caspofungin in several
media (the media were different as to buffer content (Hepes or MOPS) and
concentration, pH, CO2 and glucose concentration), and then in media with and
without bovine serum (Fetal Calf Serum – FCS), with and without Bovine Serum
Albumin (BSA) or BSA conjugated to FITC (Fluorescein isothiocyanate) – a
fluorescent molecule. In every experiment the Minimum Effective (concentration of
echinocandin that leads to production of short branched stubby hyphae) or Inhibitory
Concentration (MEC or MIC respectively) was noted, and the metabolic activity of
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the fungi was based on its ability to reduce XTT ((2,3)-bis (2-Methoxy 4-Nitro 5-
Sulphenyl) (2H) Tetrazolium Carboxanilide) to a chromogenic product or after
addition of CFDA (6-carboxyfluorescein diacetate) and estimation of the fluorescence
by the living biomass. Similar experiments were performed with anidulafungin and
micafungin in a smaller extent, in order to check if the phenotype takes place with
these echinocandins as well. The data were analyzed statistically and the different
conditions were compared based on the MECs, the concentration needed to reduce the
metabolic activity by 50% (Effective Concentration 50 – EC50), and the slope of the
curves generated with a non-linear regression (Hill’s equation). Confocal microscopy
experiments were performed with and without FITC-BSA in Aspergillus fumigatus a)
in different stages of its germination, b) with and without previous exposure to
caspofungin c) with and without previous exposure to high concentrations of BSA.
Additional confocal microscopy experiments were performed in Aspergillus fumigatus
with and without BSA, with caspofungin covalently bound to a fluorescent molecule
as well as scanning electron microscopy experiments with the same fungus with and
without BSA, with and without caspofungin. Finally, immunofluorescence confocal
microscopy experiments were performed with Aspergillus fumigatus hyphae grown
with different concentrations of caspofungin, with and without FCS, stained with a
primary murine anti-b-glucan antibody and a secondary fluorescent anti-murine
antibody.
RESULTS: The susceptibility tests of 6 Aspergillus strains to antifungals showed that
caspofungin demonstrates an increased activity in the cell culture medium that
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resembles the conditions found in the organisms of patients infected with Aspergillus;
this is not the case with voriconazole. This increased activity is documented by the
smaller MECs and EC50s in those conditions. I a series of experiments performed in
order to find the factor that mediates this lag in the caspofungin activity in the two
different media, FCS and more specifically BSA was found to mediate this effect.
This was confirmed with scanning electron microscopy experiments that extensively
described the damage at the hyphae and the hyphal tips. Next, confocal microscopy
experiments were performed, and showed that BSA is bound on Aspergilli, mostly at
the germinating forms, and that caspofungin binds more at the surface of the fungi in
the presence of BSA, while the increased caspofungin mediated b-glucan exposure is
even higher when BSA is present in the medium. Surprisingly, the abovementioned
phenotype was not confirmed with anidulafungin or micafungin.
DISCUSSION: The experiments described in this dissertation show that caspofungin
activity is increased in a cell culture medium that resembles more the physiologic
conditions in the tissues of patients infected with Aspergilli. Surprisingly, this is not
the case with anidulafungin or micafungin. This increased caspofungin activity, as
shown in the experiments in this dissertation, is associated with the increased BSA
binding on the fungal cell wall of Aspergillus, especially in the germinating forms of
the fungus. In the presence of caspofungin, BSA binding on the fungal cell wall is
even more increased and in the presence of BSA, caspofungin binding on the fungal
cell wall is increased, implying the possibility of BSA acting as a carrier molecule for
caspofungin, leading in a positive feedback loop, since the increased binding and
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activity of caspofungin would lead to even higher binding of BSA on the fungal cell
wall. Furthermore, since one of the current explanations of the lag in the in vivo and
the in vitro activity of echinocandins in the literature is the caspofungin induced
increased b-glucan exposure on the fungal cell wall surface, we performed confocal
microscopy experiments to Aspergillus fumigatus exposed to increasing caspofungin
concentrations and found that when the fungus had been exposed to BSA, b-glucan
exposure was higher. These two mechanisms could explain the lag in the in vivo and
the in vitro activity of echinocandins.
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