AFM evaluation of a humanized recombinant antibody affecting C. auris cell wall and stability

Abstract

Fungal infections are increasingly impacting on the health of the population and particularly on subjects with a compromised immune system. The resistance phenomenon and the rise of new species carrying sometimes intrinsic and multi-drug resistance to the most commonly used antifungal drugs are greatly concerning healthcare organizations. As a result of this situation, there is growing interest in the development of therapeutic agents against pathogenic fungi. In particular, the Candida genus is responsible for severe life-threatening infections and among its species, C. auris is considered an urgent threat by the Center for Disease Control and Prevention, and is one of the three leading causes of morbidity and mortality worldwide. H5K1 is a humanized monoclonal antibody (hmAb) that selectively binds to β-1,3-glucans, vital components of the fungal cell wall. It has been previously demonstrated that it is active against Candida species, especially against C. auris, reaching its greatest potential when combined with commercially available antifungal drugs. Here we used atomic force microscopy (AFM) to assess the effects of H5K1, alone and in combination with fluconazole, caspofungin and amphotericin B, on C. auris cells. Through an extensive exploration we found that H5K1 has a significant role in the perturbation and remodeling of the fungal cell wall that is reflected in the loss of whole cell integrity. Moreover, it contributes substantially to the alterations in terms of chemical composition, stiffness and roughness induced specifically by caspofungin and amphotericin B. In addition to this, we demonstrated that AFM is a valuable technique to evaluate drug-microorganism interaction.

Fig. 1. Topographic images by NCM AFM of C. auris cell surface. 1 μm × 1 μm topographic analysis conducted in air to better detect morphological changes on the surface. (A) Control, (B) treatment with hmAb H5K1, (C) treatment with fluconazole, (D) treatment with the combination of hmAb H5K1 and fluconazole, (E) treatment with amphotericin B, (F) treatment with the combination hmAb H5K1 and amphotericin B, (G) treatment with caspofungin, (H) treatment with the combination of hmAb H5K1 and caspofungin. Matching data are illustrated in Fig. 2.

Fig. 2. Topographic spacing parameters as descriptor of C. auris cell surface irregularities. The spacing, the wavelength and the slope were evaluated. The spacing (Sm) describes the average spacing between topographic heigh variations in several random theoretical 1 μm line. The average wavelength (λa) indirectly expresses the mean distance between vertical variations becoming roughness descriptor. The average absolute slope (Δa) is considered ad hybrid parameter to obtain information about the depth and the height from topographic signal vertical variation. (A) spacing mean of the irregularities of the profile; (B) average wavelength; (C) average absolute slope of the wave function from topographic analyses. The statistical significances are referred to the comparison with the control sample.

Fig. 3. Sub-nanometric structures of C. auris cell surface. 0.5 μm × 0.5 μm analysis of the surface sub-domains. Images were obtained from FFT filtered amplitude signal analysis of images performed in NCM. This signal processing allows to highlight and contrast the topography-dependent subdomains. (A) Control, (B) treatment with hmAb H5K1, (C) treatment with fluconazole, (D) treatment with the combination of hmAb H5K1 and fluconazole, (E) treatment with amphotericin B, (F) treatment with the combination hmAb H5K1 and amphotericin B, (G) treatment with caspofungin, (H) treatment with the combination of hmAb H5K1 and caspofungin. Matching data are illustrated in Fig. 4.

Fig. 4. Average wavelength of the NCM amplitude signal profile. Amplitude λa (above mentioned) of amplitude profile is indirectly a descriptor of the ultrastructural domains organization of the cell surface. The statistical significances are referred to the comparison with the control. All treatments caused a spacing change in the surface molecules.

Fig. 5. Biochemical composition and distribution on C. auris surface. 1 μm × 1 μm analysis of the molecule arrangement on C. auris surface. Phase signals images were obtained in NCM and degree ranges normalized. (A) Control, (B) treatment with hmAb H5K1, (C) treatment with fluconazole, (D) treatment with the combination of hmAb H5K1 and fluconazole, (E) treatment with Amphotericin B, (F) treatment with the combination hmAb H5K1 and amphotericin B, (G) treatment with caspofungin, (H) treatment with the combination of hmAb H5K1 and caspofungin. Phase signals depend on chemical and nanomechanical properties of analyzed surfaces.

Fig. 6. RPV roughness (deg) of the NCM Phase signals. Phase signal distribution was measured as peak-to-valley mean-height roughness (RPV) and provides information about C. auris chemical surface homogeneity. The statistical significances are referred to the comparison with the control. All treatments caused distribution changes (incremented RPV) of chemical composition uniformity of surfaces.

Fig. 7. Force Modulation Mode (FMM) images obtained by amplitude (letters) and phase signals of the same wave function (marked letters). Images provide information about the local nanomechanics proprieties of C. auris surface. FMM amplitude describes the stiffness of the area: in bright the stiffest zones and in dark the softest zones. FMM Phase describes the elasticity of the analyzed area: in bright the most elastic zones while in dark the least elastic. (A/A′) Control, (B/B′) treatment with hmAb H5K1, (C/C′) treatment with fluconazole, (D/D′) treatment with the combination of hmAb H5K1 and fluconazole, (E/E′) treatment with amphotericin B, (F/F′) treatment with the combination hmAb H5K1 and amphotericin B, (G/G′) treatment with caspofungin, (H/H′) treatment with the combination of hmAb H5K1 and caspofungin. Signal analyses are reported in Fig. 8.

Fig. 8. FMM data representation of stiffness and elasticity mapping. Local stiffness and elasticity of C. auris cell surface. In (A) and (C) the FMM amplitude and phase average data are reported. In (B) and in (D) the roughness of FMM amplitude and phase signals are reported. The RPV increment suggests a nanomechanical alteration of cell surfaces after the treatments.

Fig. 9. Cell Stiffness expressed an nN obtained by F/D curves analysis. Almost all treatments were able to reduce the cell strength.