Catherine M. Bendel

Molecular mimicry in Candida albicans. Role of an integrin analogue in adhesion of the yeast to human endothelium.

Hematogenous infection with the yeast Candida albicans now occurs with increasing frequency in the neonate, the immunocompromised patient, and the hyperglycemic or hyperalimented host. Yeast-phase C. albicans expresses a protein that is antigenically and structurally related to CD11b/CD18, a member of the beta 2 integrins and a well-characterized adhesin for mammalian neutrophils. Both the neutrophil protein and its analogue in C. albicans have an identical affinity for the C3 ligand iC3b, and both proteins are significantly increased in expression at 37 degrees C. Given these several similarities, we therefore studied the role of the integrin analogue on C. albicans in the adhesion of the yeast to human umbilical vein endothelium (HUVE). After growth of C. albicans in 20 mM D-glucose, as opposed to 20 mM L-glutamate, flow cytometric analysis with monoclonal antibodies recognizing the alpha-subunit of CD11b/CD18 demonstrated a 25.0% increase in mean channel fluorescence (range 18.4-31.8%), as well as an increased percentage of yeasts fluorescing (P less than 0.02). This increased intensity of fluorescence, which corresponds to increased expression of the integrin analogue, also correlated with a significant increase of 30-80% in adhesion of glucose-grown C. albicans to HUVE (P less than 0.02). Blockade of the integrin analogue on C. albicans by monoclonal antibodies recognizing adhesive epitopes on neutrophil CD11b/CD18 inhibited glucose-enhanced adhesion of C. albicans to HUVE. Incubation of glucose-grown C. albicans with saturating concentrations of purified human iC3b, the ligand for CD11b/CD18, reduced adhesion of the yeast to HUVE by 49.7%, whereas BSA in equimolar concentration had no effect (P less than 0.001). These results identify a glucose-responsive integrin analogue on C. albicans as one of possibly several cellular structures that mediate adhesion of the yeast to human endothelium.

Distinct mechanisms of epithelial adhesion for Candida albicans and Candida tropicalis. Identification of the participating ligands and development of inhibitory peptides.

The yeast Candida albicans is the leading cause of disseminated fungal infection in neonates, immunocompromised hosts, diabetics, and postoperative patients; Candida tropicalis is the second most frequent isolate. Because the integrin analogue in C. albicans shares antigenic, structural, and functional homologies with the beta 2-integrin subunits alpha M and alpha X, we investigated the role of integrin analogues in epithelial adhesion of C. albicans and C. tropicalis. On flow cytometry with the monoclonal antibody (mAb) OKM1, surface fluorescence was highest for C. albicans and significantly reduced for C. tropicalis (P < 0.001). However, adhesion to the human epithelial cell line HeLa S3 did not differ for these two candidal species: specific adhesion was highest for C. albicans at 44.0 +/- 1.8%, and only slightly lower for C. tropicalis at 38.8 +/- 3.6% (P = NS). The disparity between expression of the integrin analogue and epithelial adhesion suggested distinct mechanisms for this process in C. albicans versus C. tropicalis. Preincubation of C. albicans with anti-alpha M mAbs, with purified iC3b (the RGD ligand for the integrin analogue), or with 9-15-mer RGD peptides from iC3b all inhibited epithelial adhesion significantly (P < 0.001-0.04). Purified fibronectin or fibronectin-RGD peptides failed to block C. albicans adhesion. In contrast, epithelial adhesion of C. tropicalis was significantly inhibited by purified fibronectin and its RGD peptides (P < or = 0.021), but not by iC3b nor the iC3b-RGD peptides. Both iC3b and fibronectin were identified on the surface of epithelial cells after growth in serum-free medium. A polyclonal antibody to C3 inhibited C. albicans adhesion while a control antibody to fibronectin was ineffective; the converse was true for C. tropicalis. These results indicate that the pathogenic yeasts C. albicans and C. tropicalis recognize distinct RGD ligands present at the surface of the epithelial cell and that these interactions can be differentially inhibited by defined RGD peptides containing appropriate flanking sequences.

Colonization and epithelial adhesion in the pathogenesis of neonatal candidiasis

Candida species are important nosocomial pathogens in the newborn population, particularly among the premature very-low-birth-weight infants in neonatal intensive care units. Candida colonization of the neonatal skin and gastrointestinal tract is an important first step in the pathogenesis of invasive disease. C albicans is the most commonly isolated species in colonized or infected infants. Over the past decade the incidence of both colonization and infection with other Candida species, particularly C parapsilosis, has risen dramatically. Colonization of the infant occurs early in life and is affected by a variety of common practices in neonatal intensive care. Microbial factors also augment colonization, including the ability of Candida to adhere to human epithelium. A better understanding of the complex interactions between host risk factors and virulence traits of colonizing yeast may allow the risk of systemic spread to be reduced in the population of premature infants.

Effect of Fluconazole Prophylaxis on Candidiasis and Mortality in Premature Infants: A Randomized Clinical Trial

IMPORTANCE Invasive candidiasis in premature infants causes death and neurodevelopmental impairment. Fluconazole prophylaxis reduces candidiasis, but its effect on mortality and the safety of fluconazole are unknown. OBJECTIVE To evaluate the efficacy and safety of fluconazole in preventing death or invasive candidiasis in extremely low-birth-weight infants. DESIGN, SETTING, AND PATIENTS This study was a randomized, blinded, placebo-controlled trial of fluconazole in premature infants. Infants weighing less than 750 g at birth (N = 361) from 32 neonatal intensive care units (NICUs) in the United States were randomly assigned to receive either fluconazole or placebo twice weekly for 42 days. Surviving infants were evaluated at 18 to 22 months corrected age for neurodevelopmental outcomes. The study was conducted between November 2008 and February 2013. INTERVENTIONS Fluconazole (6 mg/kg of body weight) or placebo. MAIN OUTCOMES AND MEASURES The primary end point was a composite of death or definite or probable invasive candidiasis prior to study day 49 (1 week after completion of study drug). Secondary and safety outcomes included invasive candidiasis, liver function, bacterial infection, length of stay, intracranial hemorrhage, periventricular leukomalacia, chronic lung disease, patent ductus arteriosus requiring surgery, retinopathy of prematurity requiring surgery, necrotizing enterocolitis, spontaneous intestinal perforation, and neurodevelopmental outcomes-defined as a Bayley-III cognition composite score of less than 70, blindness, deafness, or cerebral palsy at 18 to 22 months corrected age. RESULTS Among infants receiving fluconazole, the composite primary end point of death or invasive candidiasis was 16% (95% CI, 11%-22%) vs 21% in the placebo group (95% CI, 15%-28%; odds ratio, 0.73 [95% CI, 0.43-1.23]; P = .24; treatment difference, -5% [95% CI, -13% to 3%]). Invasive candidiasis occurred less frequently in the fluconazole group (3% [95% CI, 1%-6%]) vs the placebo group (9% [95% CI, 5%-14%]; P = .02; treatment difference, -6% [95% CI, -11% to -1%]). The cumulative incidences of other secondary outcomes were not statistically different between groups. Neurodevelopmental impairment did not differ between the groups (fluconazole, 31% [95% CI, 21%-41%] vs placebo, 27% [95% CI, 18%-37%]; P = .60; treatment difference, 4% [95% CI, -10% to 17%]). CONCLUSIONS AND RELEVANCE Among infants with a birth weight of less than 750 g, 42 days of fluconazole prophylaxis compared with placebo did not result in a lower incidence of the composite of death or invasive candidiasis. These findings do not support the universal use of prophylactic fluconazole in extremely low-birth-weight infants. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00734539.

An Internal Polarity Landmark Is Important for Externally Induced Hyphal Behaviors in Candida albicans

ABSTRACT Directional growth is a function of polarized cells such as neurites, pollen tubes, and fungal hyphae. Correct orientation of the extending cell tip depends on signaling pathways and effectors that mediate asymmetric responses to specific environmental cues. In the hyphal form of the eukaryotic fungal pathogen Candida albicans, these responses include thigmotropism and galvanotropism (hyphal turning in response to changes in substrate topography and imposed electrical fields, respectively) and penetration into semisolid substrates. During vegetative growth in C. albicans, as in the model yeast Saccharomyces cerevisiae, the Ras-like GTPase Rsr1 mediates internal cellular cues to position new buds in a prespecified pattern on the mother cell cortex. Here, we demonstrate that Rsr1 is also important for hyphal tip orientation in response to the external environmental cues that induce thigmotropic and galvanotropic growth. In addition, Rsr1 is involved in hyphal interactions with epithelial cells in vitro and its deletion diminishes the hyphal invasion of kidney tissue during systemic infection. Thus, Rsr1, an internal polarity landmark in yeast, is also involved in polarized growth responses to asymmetric environmental signals, a paradigm that is different from that described for the homologous protein in S. cerevisiae. Rsr1 may thereby contribute to the pathogenesis of C. albicans infections by influencing hyphal tip responses triggered by interaction with host tissues.

Comparative virulence of Candida albicans yeast and filamentous forms in orally and intravenously inoculated mice.

ObjectiveCandida albicans, a dimorphic fungus that switches from yeast to filamentous forms, is a major cause of complicating systemic infection in intensive care patients. The aim of this study was to compare the pathogenic potential of C. albicans yeast and filamentous forms. DesignSeparate groups of mice were inoculated either intravenously or orally with C. albicans CAF2 (wild type), HLC54 (yeast forms defective in filament formation), or BCa2-10 (constitutively filamentous). Mice were killed 1, 7, 14, and 21 days after intravenous C. albicans and kidneys and liver were quantitatively cultured; cohort groups were observed for mortality. Mice were pretreated with antibiotics for 3 days before oral inoculation with C. albicans, and killed 3 days later with dexamethasone administered for the latter 3 days; at sacrifice, the mesenteric lymph nodes and kidneys were cultured to monitor extraintestinal dissemination of C. albicans. SettingUniversity teaching hospital research laboratory. SubjectsFemale, Swiss Webster, adult mice. Measurements and Main ResultsIn intravenously inoculated mice, mortality was highest with wild-type C. albicans CAF2 (92%), intermediate with HLC54 (56%), and not detected with constitutively filamentous BCa2-10 (0%); BCa2-10 was cleared from the kidney and liver, but CAF2 and HLC54 were recovered at approximately 105–7/g kidney and 104–5/g liver. There was only occasional mortality in orally inoculated mice and the numbers of cecal C. albicans CAF2 and HLC54 were similarly high (approximately 10/g), whereas numbers of cecal BCa2-10 were at least 100-fold lower. Extraintestinal dissemination was greatest with HLC54, intermediate with CAF2, and undetectable with BCa2-10. ConclusionsOf the three C. albicans strains studied, wild-type CAF2 was most virulent in intravenously inoculated mice and HLC54 (defective in filament formation) was most virulent in orally inoculated mice. The constitutively filamentous BCa2-10 was avirulent in both models, suggesting that filamentous forms by themselves might not be critically important for C. albicans virulence.

Abnormalities of the Central Visual Pathways in Prader–Willi Syndrome Associated with Hypopigmentation

Patients with oculocutaneous or ocular albinism have misrouting of optic fibers, with fibers from 20 degrees or more of the temporal retina crossing at the chiasm instead of projecting to the ipsilateral hemisphere. Misrouting can result in strabismus and nystagmus. Because patients with the Prader-Willi syndrome may also have hypopigmentation and strabismus, we wondered whether they too might have misrouting of optic fibers. We therefore studied six patients with Prader-Willi syndrome selected for a history of strabismus, using pattern-onset visually evoked potentials with binocular and monocular stimulation to look for evidence of misrouted retinal-ganglion fibers. Four had hypopigmentation, and three of these four had abnormal evoked potentials indistinguishable from those recorded in human albinos. The two with normal pigmentation had normal responses. These findings indicate that patients with Prader-Willi syndrome who have hypopigmentation have a brain abnormality characterized by misrouting of retinal-ganglion fibers at the optic chiasm--a finding previously reported only in forms of albinism.

Cecal Colonization and Systemic Spread of Candida albicans in Mice Treated with Antibiotics and Dexamethasone

Infections with Candida albicans have become a significant problem among very low birth weight infants in the neonatal intensive care unit. Risk factors are multiple and include administration of antibiotics and glucocorticoids, such as dexamethasone. Experiments were designed to study the combined effect of oral broad-spectrum antibiotics and parenteral dexamethasone on cecal colonization and extraintestinal dissemination of C. albicans in separate groups of mice that were orally inoculated with one of four C. albicans strains that were either wild-type INT1/INT1 or had one or more disruptions of the INT1 gene. Intestinal colonization was monitored by quantitative culture of the mouse cecum, and extraintestinal invasion was monitored by quantitative culture of the draining mesenteric lymph nodes and kidneys. At sacrifice, the average numbers of cecal C. albicans differed from 7.7 log10/g to 6.7 log10/g (p < 0.01) in mice orally inoculated with C. albicans containing two functional copies of INT1 and no functional copies of INT1, respectively. The incidence of extraintestinal dissemination to mesenteric lymph nodes and kidneys correspondingly varied from 57 to 13% (p < 0.01) and 83 to 4% (p < 0.01) in mice inoculated with these two C. albicans strains. Mice orally inoculated with C. albicans containing one functional copy of INT1 had intermediate levels of cecal colonization and extraintestinal dissemination. Thus, cecal colonization and extraintestinal dissemination of C. albicans was facilitated in antibiotic-treated mice given dexamethasone. In addition, the presence of two functional copies of the INT1 gene was associated with the greatest levels of cecal colonization and extraintestinal dissemination of C. albicans.

Gastrointestinal colonization by Candida albicans mutant strains in antibiotic-treated mice.

ABSTRACT Antibiotic-treated mice orally inoculated with one of threeCandida albicans strains (including two mutant strains) or indigenous Candida pelliculosa showed levels of candidal gastrointestinal colonization that were strain specific. However, regardless of strain, the numbers of viable candida were intermediate to high in the stomach, were consistently lowest in the upper small intestine, and increased progressively down the intestinal tract.

Adherence of yeast and filamentous forms of Candida albicans to cultured enterocytes

OBJECTIVE Systemic candidiasis is a major cause of complicating infections in intensive care units. Morbidity and mortality are high, even in those who receive appropriate antifungal therapy. Because the intestinal tract is considered a major portal of entry for systemic candidiasis, experiments were designed to clarify the ability of yeast and filamentous forms, as well as the INT1 gene product, to influence adherence of Candida albicans to the intestinal epithelium. DESIGN Controlled. SETTING University teaching hospital research laboratory. SUBJECTS Mature Caco-2 and HT-29 cultured enterocytes. INTERVENTIONS C. albicans INT1 mutant strains, defective in filament production, were used to observe the ultrastructural surface interactions of C. albicans with cultured intestinal epithelial cells, namely Caco-2 and HT-29 cells. These mutant strains also were used to quantify the effect of the INT1 gene product on C. albicans adherence (yeast and filamentous forms) to cultured enterocytes. Ultrastructural surface interactions of C. albicans with cultured enterocytes were observed with high resolution scanning electron microscopy. C. albicans adherence to cultured enterocytes was quantified by using a colorimetric enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS Both yeast and filamentous forms of C. albicans appeared tightly adherent to the apical surface of cultured enterocytes, and INT1 appeared to have little, if any, effect on these ultrastructural surface interactions. The distal ends of C. albicans filaments appeared to mediate adherence to enterocyte apical microvilli, and thigmotropism (contact guidance) appeared to play a role in C. albicans adherence. The absence of functional INT1 was associated with decreased adherence of C. albicans yeast forms to cultured enterocytes. CONCLUSIONS Although functional INT1 appeared to facilitate adherence of C. albicans yeast forms to cultured enterocytes, the role of INT1 in adherence of filamentous forms was unclear, and both yeast and filamentous forms could adhere to, and perhaps invade, the apical surface of cultured enterocytes.