Inhibition of hydrophobic protein-mediated Candida albicans attachment to endothelial cells during physiologic shear flow

Abstract

Adhesion interactions during hematogenous dissemination of Candida albicans likely involve a complex array of host and fungal factors. Possible C. albicans factors include changes in cell surface hydrophobicity and exposed antigens that have been shown in static adhesion assays to influence attachment events. We used a novel in vitro shear analysis system to investigate host-pathogen interactions and the role of fungal cell surface hydrophobicity in adhesion events with human endothelial cells under simulated physiologic shear. Endothelial monolayers were grown in capillary tubes and tested with and without interleukin-1 beta activation in buffered medium containing human serum. Hydrophobic and hydrophilic stationary-phase C. albicans yeast cells were infused into the system under shear flow and found to adhere with widely varying efficiencies. The average number of adherent foci was determined from multiple fields, sampled via video microscopy, between 8 and 12 min after infusion. Hydrophobic C. albicans cells demonstrated significantly more heterotypic binding events (Candida-endothelial cell) and greater homotypic binding events (Candida-Candida) than hydrophilic yeast cells. Cytokine activation of the endothelium significantly increased binding by hydrophobic C. albicans compared to unactivated host cells. Preincubation of hydrophobic yeast cells with a monoclonal antibody against hydrophobic cell wall proteins significantly blocked adhesion interactions with the endothelial monolayers. Because the antibody also blocks C. albicans binding to laminin and fibronectin, results suggest that vascular adhesion events with endothelial cells and exposed extracellular matrix may be blocked during C. albicans dissemination. Future studies will address the protective efficacy of blocking or redirecting blood-borne fungal cells to favor host defense mechanisms.

FIG. 1

In vitro shear analysis of hydrophobic and hydrophilic C. albicans adhesion to activated endothelial cell monolayers. HUVECs were activated with IL-1β as described in Materials and Methods. Hydrophobic and hydrophilic yeast cells of C. albicans A9wt, LGH1095, and ATCC 90029 were cultured in YNB2G. For each isolate, hydrophobic cells demonstrated significantly higher average foci per field than hydrophilic cells (∗, P < 0.001 for each comparison, Mann-Whitney rank sum test). Error bars show the SD for the average foci per field (total bar height), while the upper white portion of each bar corresponds to the amount of homotypic binding, as described in the text.

FIG. 2

Effects of MAb 6C5 and 5D8 on hydrophobic C. albicans attachment to activated HUVECs. Hydrophobic C. albicans LGH1095 yeast cells (YNB2G, 23°C stationary phase) were suspended in assay medium with or without 60 μg of MAb 6C5, MAb 5D8, or control IgG2a (UPC-10; Sigma product no. M9144) per ml, incubated in an ice water slurry for 10 min, and infused into the shear assay system harboring IL-1β-activated HUVECs. Pretreatment with MAb 6C5 significantly decreased binding compared to the yeast-only control and compared to the yeast plus control IgG2a (P < 0.001 for each in pairwise multiple comparison, Tukey test).