Uptake ofAspergillus fumigatusConidia by Phagocytic and Nonphagocytic Cells In Vitro: Quantitation Using Strains Expressing Green Fluorescent Protein

Abstract

Several pathogenic fungal organisms enter eukaryotic cells and manipulate the host cell environment to favor their own growth and survival. Aspergillus fumigatus is a saprophytic fungus that causes invasive lung disease in the immunocompromised host. To determine whether A. fumigatus could enter eukaryotic cells, we studied the uptake of two different GFP-expressing A. fumigatus strains into A549 lung epithelial cells, human umbilical vein endothelial (HUVE) cells, and J774 murine macrophages in vitro. A549 cells internalized 30% of the bound conidia whereas HUVE and J774 cells internalized 50 and 90%, respectively. Conidia within A549 cells remained viable for 6 h; however, 60 to 80% of conidia within J774 cells were killed after only 4 h. Live and heat-killed conidia were internalized to the same extent by A549 cells. After 6 h, almost none of the conidia inside A549 cells had germinated, whereas extracellular conidia had developed germ tubes. Internalization of conidia by A549 cells was a temperature-dependent process and required rearrangement of the underlying host cell cytoskeleton; uptake was inhibited by 75% with 0.5 M cytochalasin D and by 65% with 5 M colchicine. Fluorescent labeling of infected A549 cells with rhodamine phalloidin provided visible evidence of cytoskeletal alteration as many of the intracellular conidia were contained in actin-coated phagosomes. These data provide evidence that significant numbers of A. fumigatus conidia can be internalized by nonprofessional phagocytes in vitro and these cells may serve as reservoirs for immune cell evasion and dissemination throughout the host.

Aspergillus fumigatus germ tube growth and not conidia ingestion induces expression of inflammatory mediator genes in the human lung epithelial cell line A549

“…Overexpression of chemokines and of chemokine receptor genes (IL-8 gene, CCL4, CXCL20 and CXCL2) has been described as early as 2 h in human monocyte models using cDNA microarray analysis (Cortez et al, 2006). In our study, we were unable to detect any significant increase before 8 h. If conidia ingestion were the triggering event, an inflammatory response would have occurred for both fungal species because the rate of internalized conidia was similar for both species: around 30-40 %, as reported previously (Wasylnka & Moore, 2002).…”

Host Cell Invasion by Medically Important Fungi

“…In addition, adherent extracellular conidia also germinate to produce hyphae, which in turn can invade neighboring epithelial cells (Fig. 1B), possibly via active penetration (Wasylnka and Moore 2002). Thus, both conidia and hyphae of A. fumigatus can adhere to and invade epithelial cells during the course of infection.…”

“…Both conidia and hyphae of A. fumigatus interact with a number of pulmonary cell types during different stages of invasive aspergillosis. Following inhalation, conidia that escape macrophage phagocytosis and killing can rapidly and avidly adhere to pulmonary alveolar epithelial cells and basement membrane components (Bromley and Donaldson 1996;DeHart et al 1997;Paris et al 1997;Wasylnka and Moore 2002). These adherent organisms invade alveolar epithelial cells by the process of induced endocytosis.…”

“…These adherent organisms invade alveolar epithelial cells by the process of induced endocytosis. Alveolar epithelial cells endocytose approximately one-third of adherent conidia through the formation of pseudopods followed by membrane invagination and internalization (Paris et al 1997;Wasylnka and Moore 2002). The endocytosed conidia are initially contained within acidic phagosomes where they are killed (Paris et al 1997;Wasylnka and Moore 2003).…”

Development of Worm-like Polymeric Drug Carriers with Multiple Ligands for Targeting Heterogeneous Breast Cancer Cells

“…Moreover, the conjugation of hydrophilic multiple ligands, such as cobalamin, folate, and cyclic RGD, seemed to provide flexibility at the interfacial curvature of the rod-like nanostructures, resulting in yielding flexible worm-like micelles (average 2.4 µm) (Figure 1). These results indicate that physical or chemical stimuli for non-spherical drug carriers may deform their morphologies, which will ultimately affect their pharmacokinetics or therapeutic efficacies 19,33,34 Figure 4 shows the distribution of ligands existing on wormlike surfaces. PLLA-b-PEG bock copolymers with fluorescent ligands (i.e., PLLA-b-PEG-folate-Cy5.5, PLLA-b-PEG-cobalamin-FITC, and PLLA-b-PEG-cyclic RGD-TMRHD) were self-assembled to visualize the surface of the worm-like micelles using a confocal microscope.…”