Evidence supporting a role for mammalian chitinases in efficacy of caspofungin against experimental aspergillosis in immunocompromised rats

Abstract

Objectives: Caspofungin, currently used as salvage therapy for invasive pulmonary aspergillosis (IPA), strangely only causes morphological changes in fungal growth in vitro but does not inhibit the growth. In vivo it has good efficacy. Therefore the question arises how this in vivo activity is reached. Caspofungin is known to increase the amount of chitin in the fungal cell wall. Mammals produce two chitinases, chitotriosidase and AMCase, which can hydrolyse chitin. We hypothesized that the mammalian chitinases play a role in the in vivo efficacy of caspofungin.

Methods: In order to determine the role of chitotriosidase and AMCase in IPA, both chitinases were measured in rats which did or did not receive caspofungin treatment. In order to understand the role of each chitinase in the breakdown of the caspofungin-exposed cells, we also exposed caspofungin treated fungi to recombinant enzymes in vitro.

Results: IPA in immunocompromised rats caused a dramatic increase in chitinase activity. This increase in chitinase activity was still noted when rats were treated with caspofungin. In vitro, it was demonstrated that the action of both chitinases were needed to lyse the fungal cell wall upon caspofungin exposure.

Conclusion: Caspofungin seemed to alter the cell wall in such a way that the two chitinases, when combined, could lyse the fungal cell wall and assisted in clearing the fungal pathogen. We also found that both chitinases combined had a direct effect on the fungus in vitro.

Figure 1. Chitinase activity and fungal load in immunocompromised rats inoculated with A. fumigatus conidia.

Open squares: uninfected untreated rats; filled circles: infected untreated rats; grey triangles: infected rats, treated with caspofungin at 24 h post infection. Data are means of duplicates. Bars represent medians. For each group n≥4. A. Galactomannan (GM)-index, measured by Platelia assay. According to the manufacturer's manual, GM-index of <0,5 is considered negative; B, chitotriosidase activity, expressed in arbitrary units (a.u.); C, AMCase activity, expressed in arbitrary units (a.u.). * p<0.05; significant difference between the indicated groups.

Figure 2. Grocott staining (A, D, G) and presence of AMCase (B, E, H) and chitotriosidase (C, F, I) in several rats.

Panels A, B, and C show the lung of an uninfected rat. Panels D, E and F show the fungal focus in an infected, untreated rat. Panels G, H and I show the fungal focus in an infected, caspofungin treated rat. Original magnification ×400. All panels represent lungs on day 6 after inoculation. Slides were stained according to the described protocols. In Grocott staining (A, D, G), fungal hyphae are coloured black. Chitotriosidase- or AMCase-presenting cells are coloured red (B, C, E, F, H, I). In uninfected rats, normal morphology can be found in the lungs (A, B, C). In infected rats, normal morphology of alveoli is lost (D, E, F). Grocott staining shows many hyphae (D). An inflammatory response is found around the fungal focus, where chitotriosidase and AMCase are increasingly present (red zones) compared to an uninfected rat (E, F). After treatment with caspofungin, Grocott staining shows fungal material in all infected rats (G). AMCase bound fungal hyphae after treatment with caspofungin (H) and thus hyphae became visible. After treatment with caspofungin, chitotriosidase seemed to also bind the fungal cell wall and locate inside hyphal cells (I).

Figure 3. In vitro binding of recombinant chitinases to A. fumigatus hyphae.

Binding of recombinant chitotriosidase (A, B). Binding of recombinant chitotriosidase when incubated in combination with recombinant AMCase (C, D) and binding of recombinant AMCase (E, F). Panels A, C and E show unexposed hyphae. Panels B, D and F show caspofungin-exposed hyphae. A, B: Contrast and brightening were slightly modified in Photoshop due to the lack of colour. C, D, E, F: Photos were not modified in Photoshop. A–B Original magnification ×100. C–F Original magnification ×400. Slides were stained according to the described protocols. Binding of either recombinant enzyme is characterized by a red colour. Recombinant chitotriosidase did not bind to unexposed hyphae (A) or to caspofungin-exposed hyphae (B). When incubated with a combination of recombinant chitotriosidase and recombinant AMCase, recombinant chitotriosidase did bind to unexposed hyphae (arrow) and to conidial heads (C) and seemed to be taken up by the fungal cells after caspofungin exposure (D). Also the cell wall seemed to be lysed at several locations (arrows). Recombinant AMCase did bind to unexposed (E) and to caspofungin-exposed hyphae (F).

Figure 4. Calcofluor White staining of in vitro cultured A. fumigatus hyphae after incubation with recombinant chitotriosidase and recombinant AMCase.

When hyphae were cultured on Sabauroud's agar (A), the cell wall remained regular and intact after incubation with the two recombinant chitinases. When hyphae were cultured on Sabauroud's agar with 1 mg/L caspofungin (B), the cell wall was irregular and disrupted after incubation with the two recombinant chitinases.