Rhizopus oryzae adheres to, is phagocytosed by, and damages endothelial cells in vitro

Abstract

Rhizopus oryzae is the most common cause of zygomycosis, a life-threatening infection that usually occurs in immunocompromised patients. A characteristic hallmark of zygomycosis is angioinvasion by the fungus, resulting in thrombosis and subsequent tissue necrosis. Interactions between R. oryzae and vascular endothelial cells are therefore likely of central importance to the organism's pathogenetic strategy. We studied the ability of R. oryzae to adhere to and damage human umbilical vein endothelial cells (HUVECs) in vitro. We report that R. oryzae spores and germ tubes adhere to HUVECs, whereas only spores adhere to subendothelial matrix proteins. Additionally, R. oryzae damages endothelial cells. This endothelial cell damage requires direct contact and subsequent phagocytosis of the fungus. Surprisingly, R. oryzae viability was not required for damage, but phagocytosis was required for dead R. oryzae to cause damage. These results elucidate the nature of R. oryzae-endothelial cell interactions, which are likely central to the angioinvasion and tissue necrosis seen during zygomycotic infections. The fact that dead R. oryzae damage human endothelial cells may, in part, explain the lack of efficacy of fungicidal agents during clinical disease.

FIG. 1.

R. oryzae spores (A) and germ tubes (B) adhere to endothelial cells, whereas only spores adhere to the subendothelial matrix. *, P < 0.05 for endothelial cells versus all other substrates.

FIG. 2.

Interactions of R. oryzae spores and germ tubes with endothelial cells. Photomicrographs of endothelial cell monolayers infected with R. oryzae spores (A to C) and germ tubes (D to F) after 1 h (A and D), 4 h (B and E), and 8 h (C and F). Original magnification for A, B, D, and E is ×20 and for C and F is ×40. Arrows indicate organisms.

FIG. 3.

Damage to HUVECs is dose and time dependent. (A) Different inocula of R. oryzae germ tubes were incubated on endothelial cells for 5 h. *, P < 0.05 versus 10⁵ inoculum; **, P < 0.05 versus 10⁵ and 5 × 10⁵ inocula. (B) An inoculum of 10⁶ R. oryzae germ tubes was incubated with endothelial cells for various time intervals. *, P < 0.05 versus 2-h incubation.

FIG. 4.

Multiple R. oryzae strains damage endothelial cells more than Mucor. Germ tubes (5 × 10⁵) were cultured with endothelial cells for 5 h. *, P < 0.05 versus Mucor results.

FIG. 5.

R. oryzae-induced injury to HUVECs requires direct attachment and subsequent internalization by endothelial cells. Germ tubes (5 × 10⁵) were cultured with endothelial cells for 5 h. (A) Damage in the presence or absence of membrane inserts. †, P < 0.05 versus results with no membrane inserts. (B) Damage in the presence or absence of cytochalasin D. *, P < 0.05 versus results with no cytochalasin D; **, P < 0.05 versus results with all other groups.

FIG. 6.

Damage to HUVECs from R. oryzae is independent of organism viability. R. oryzae was killed with heat shock for 1 h at 60°C or incubation in 2.5% glutaraldehyde or 70% ethanol for 30 min. Germ tubes were washed extensively with PBS and then cultured (5 × 10⁵) with endothelial cells for 5 h.

FIG. 7.

Damage mediated by dead R. oryzae also requires phagocytosis by HUVECs. Germ tubes (5 × 10⁵) were cultured with endothelial cells for 5 h in the presence of 200 nM cytochalasin D. *, P < 0.05 versus results with no cytochalasin D.