Non-invasive and invasive diagnoses of aspergillosis in a rat model by mass spectrometry

Dominika Luptáková, Tomáš Pluháček, Miloš Petřík, Jiří Novák, Andrea Palyzová, Lucie Sokolová, Anton Škríba, Blanka Šedivá, Karel Lemr, Vladimír Havlíček, Dominika Luptáková, Tomáš Pluháček, Miloš Petřík, Jiří Novák, Andrea Palyzová, Lucie Sokolová, Anton Škríba, Blanka Šedivá, Karel Lemr, Vladimír Havlíček

Abstract

Invasive pulmonary aspergillosis results in 450,000 deaths per year and complicates cancer chemotherapy, transplantations and the treatment of other immunosuppressed patients. Using a rat model of experimental aspergillosis, the fungal siderophores ferricrocin and triacetylfusarinine C were identified as markers of aspergillosis and quantified in urine, serum and lung tissues. Biomarkers were analyzed by matrix-assisted laser desorption ionization (MALDI) and electrospray ionization mass spectrometry using a 12T SolariX Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The limits of detection of the ferri-forms of triacetylfusarinine C and ferricrocin in the rat serum were 0.28 and 0.36 ng/mL, respectively. In the rat urine the respective limits of detection achieved 0.02 and 0.03 ng/mL. In the sera of infected animals, triacetylfusarinine C was not detected but ferricrocin concentration fluctuated in the 3-32 ng/mL range. Notably, the mean concentrations of triacetylfusarinine C and ferricrocin in the rat urine were 0.37 and 0.63 μg/mL, respectively. The MALDI FTICR mass spectrometry imaging illustrated the actual microbial ferricrocin distribution in the lung tissues and resolved the false-positive results obtained by the light microscopy and histological staining. Ferricrocin and triacetylfusarinine C detection in urine represents an innovative non-invasive indication of Aspergillus infection in a host.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Histological examination of rat lung tissue infected with Aspergillus fumigatus. GMS and eosin staining was used for the area definition, which was examined in parallel by MALDI-MSI (dashed line in black). Fungal hyphae are marked by red arrows in all panels (a–f). Descriptors: A (arteriole), Al (alveoli), B (primary bronchus or bronchiole), E (esophagus), H (hemorrhage), T (trachea), V (vein), N (nervus vagus).
Figure 2
Figure 2
Structures of Fe-TAFC (left) and Fe-FC (right).
Figure 3
Figure 3
Profiling of Fe-TAFC and Fe-FC in urine from infected (left six) and control (right three) animals. Data were generated by liquid chromatography and CASI-FTICR mass spectrometry. Bound iron concentration data were obtained using solution ICP-MS.
Figure 4
Figure 4
MALDI-CASI-FTICR mass spectrum of infected lung tissue with [FC + Na]+ ion signals (Panel A). Isotopic A + 1 ion is also labeled (m/z 741.3219). MALDI mass spectrum of control tissues does not reveal any fungal biomarkers (Panel B).
Figure 5
Figure 5
MALDI-CASI-FTICR-MS image (Bruker FlexImaging 4.1) of combined [FC + Na]+ and [FC + K]+ (±0.001 Da) distribution illuminated in green (A). Graphic output of our dereplication tool, i.e., CycloBranch, indicating the annotation of the siderophores (B).

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