Role of the Inducible Adhesin CpAls7 in Binding of Candida parapsilosis to the Extracellular Matrix under Fluid Shear.

The yeast Candida parapsilosis is an increasingly common cause of systemic fungal infections among immunocompromised individuals, including premature infants. Adhesion to host surfaces is an important step in pathogenesis, but this process has not been extensively studied in this organism. A microfluidics assay was developed to test the ability of C. parapsilosis to adhere to immobilized host extracellular matrix proteins under physiological fluid shear conditions. Growth in mammalian tissue culture medium at 37°C for 3 to 6 h led to the induction of an adhesive phenotype at shear forces of 1 to 5 dynes/cm2 in some isolates of C. parapsilosis Glutamic acid, proline, and calcium appeared to be the minimally necessary requirements for increased adhesion in these assays. To determine whether genes homologous to the ALS gene family of C. albicans were important for the adhesive phenotype, the expression levels of 5 homologous C. parapsilosis genes were quantified by using quantitative PCR (qPCR) under conditions leading to increased adhesion. CPAR2_404800 (CpALS7) and CPAR2_404780 showed increased expression levels compared to those in control yeast. The extent of adhesion was variable among different isolates, and linear regression identified the expression of CpALS7 but not CPAR2_404780 as having a strong positive correlation with adhesion. A homozygous CpALS7 deletion strain was deficient in adhesion, whereas the expression of CpALS7 in Saccharomyces cerevisiae resulted in increased adhesion. Together, these data provide strong evidence that CpAls7 aids in the adherence of C. parapsilosis to the extracellular matrix under shear forces and support its previously reported role in virulence.

Protection of Candida parapsilosis from neutrophil killing through internalization by human endothelial cells.

Candida parapsilosis is a fungal pathogen that is associated with hematogenously disseminated disease in premature neonates, acutely ill or immunocompromised patients. In cell culture, C. parapsilosis cells are actively and avidly endocytosed by endothelial cells via actin polymerization mediated by N-WASP. Here we present evidence that C. parapsilosis that were internalized by endothelial cells remained alive, and avoided being acidified or otherwise damaged via the host cell. Internalized fungal cells reproduced intracellularly and eventually burst out of the host endothelial cell. When neutrophils were added to endothelium and C. parapsilosis, they patrolled the endothelial surface and efficiently killed most adherent fungal cells prior to endocytosis. But after endocytosis by endothelial cells, internalized fungal cells evaded neutrophil killing. Silencing endothelial N-WASP blocked endocytosis of C. parapsilosis and left fungal cells stranded on the cell surface, where they were susceptible to neutrophil killing. These observations suggest that for C. parapsilosis to escape from the bloodstream, fungi may adhere to and be internalized by endothelial cells before being confronted and phagocytosed by a patrolling leukocyte. Once internalized by endothelial cells, C. parapsilosis may safely replicate to cause further rounds of infection. Immunosurveillance of the intravascular lumen by leukocytes crawling on the endothelial surface and rapid killing of adherent yeast may play a major role in controlling C. parapsilosis dissemination and infected endothelial cells may be a significant reservoir for fungal persistence.

Human endothelial cells internalize Candida parapsilosis via N-WASP-mediated endocytosis.

Candida parapsilosis is a frequent cause of disseminated candidiasis and is associated with significant morbidity and mortality. Although important in pathogenesis, interactions of this organism with endothelial cells have received less attention than those of Candida albicans. Internalization of C. parapsilosis by monolayers of human endothelial cells was examined in an in vitro assay and compared to that of C. albicans. Both live and heat-killed yeast were efficiently internalized, with heat-killed yeast subsequently being detected in an acidic subcompartment. Internalization was marked by a process of engulfment by thin membrane extensions from the endothelium. Efficiency of internalization differed among different clinical isolates and species of yeast. Opsonization of C. parapsilosis by serum factors was not sufficient to cause endocytosis; instead, serum appeared to directly stimulate endothelial uptake. Colocalization of endothelial actin and N-WASP at sites of C. parapsilosis internalization was observed. A Förster-resonance energy transfer (FRET) probe for N-WASP activity showed active N-WASP at sites of internalization for both live and heat-killed C. parapsilosis and C. albicans. An actin nucleation inhibitor (cytochalasin D) and an N-WASP inhibitor (wiskostatin) both inhibited uptake of heat-killed C. parapsilosis, as did short interfering RNA-mediated ablation of N-WASP. Thus, endocytosis by endothelial cells may represent a means of traversal of the blood vessel wall by yeast during disseminated candidiasis, and N-WASP may play a key role in the process.

PT19c, Another Nonhypercalcemic Vitamin D2 Derivative, Demonstrates Antitumor Efficacy in Epithelial Ovarian and Endometrial Cancer Models.

Hypercalcemia remains a major impediment to the clinical use of vitamin D in cancer treatment. Approaches to remove hypercalcemia and development of nonhypercalcemic agents can lead to the development of vitamin D-based therapies for treatment of various cancers. In this report, in vitro and in vivo anticancer efficacy, safety, and details of vitamin D receptor (VDR) interactions of PT19c, a novel nonhypercalcemic vitamin D derived anticancer agent, are described. PT19c was synthesized by bromoacetylation of PTAD-ergocalciferol adduct. Broader growth inhibitory potential of PT19c was evaluated in a panel of chemoresistant breast, renal, ovarian, lung, colon, leukemia, prostate, melanoma, and central nervous system cancers cell line types of NCI60 cell line panel. Interactions of PT19c with VDR were determined by a VDR transactivation assay in a VDR overexpressing VDR-UAS-bla-HEK293 cells, in vitro VDR-coregulator binding, and molecular docking with VDR-ligand binding domain (VDR-LBD) in comparison with calcitriol. Acute toxicity of PT19c was determined in nontumored mice. In vivo antitumor efficacy of PT19c was determined via ovarian and endometrial cancer xenograft experiments. Effect of PT19c on actin filament organization and focal adhesion formation was examined by microscopy. PT19c treatment inhibited growth of chemoresistant NCI60 cell lines (log10GI50 ~ -4.05 to -6.73). PT19c (10 mg/kg, 35 days) reduced growth of ovarian and endometrial xenograft tumor without hypercalcemia. PT19c exerted no acute toxicity up to 400 mg/kg (QDx1) in animals. PT19c showed weak VDR antagonism, lack of VDR binding, and inverted spatial accommodation in VDR-LBD. PT19c caused actin filament dysfunction and inhibited focal adhesion in SKOV-3 cells. PT19c is a VDR independent nonhypercalcemic vitamin D-derived agent that showed noteworthy safety and efficacy in ovarian and endometrial cancer animal models and inhibited actin organization and focal adhesion in ovarian cancer cells.