Cas3p belongs to a seven-member family of capsule structure designer proteins

The polysaccharide capsule is the main virulence factor of the basidiomycetous yeast Cryptococcus neoformans. Four genes (CAP10, CAP59, CAP60, and CAP64) essential for capsule formation have been previously identified, although their roles in the biosynthetic pathway remain unclear. A genetic and bioinformatics approach allowed the identification of six CAP64-homologous genes, named CAS3, CAS31, CAS32, CAS33, CAS34, and CAS35, in the C. neoformans genome. This gene family is apparently specific in a subclass of the basidiomycete fungi. Single as well as double deletions of these genes in all possible combinations demonstrated that none of the CAP64-homologous genes were essential for capsule formation, although the cas35Delta strains displayed a hypocapsular phenotype. The chemical structure of the glucuronomannan (GXM) produced by the CAS family deletants revealed that these genes determined the position and the linkage of the xylose and/or O-acetyl residues on the mannose backbone. Hence, these genes are all involved in assembly of the GXM structure in C. neoformans.     

Haploid fruiting in Cryptococcus neoformans is not mating type alpha-specific

Under appropriate conditions, haploid Cryptococcus neoformans cells can undergo a morphological switch from a budding yeast form to develop hyphae and viable basidiospores, which resemble those produced by mating. This process, known as haploid fruiting, was previously thought to occur only in MATalpha strains. We identified two new strains of C. neoformans var. neoformans serotype D that are MATa type and are able to haploid fruit. Further, a MATa reference strain, B-3502, also produced hyphae and fruited after prolonged incubation on filament agar. Over-expression of STE12a dramatically enhanced the ability of all MATa strains tested to filament. Segregation analysis of haploid fruiting ability confirmed that haploid fruiting is not MATalpha-specific. Our results indicate that MATa cells are intrinsically able to haploid fruit and previous observations that they do not were probably biased by the examination of a small number of genetically related isolates that have been maintained in the laboratory for many years.     

Cryptococcus neoformans Overcomes Stress of Azole Drugs by Formation of Disomy in Specific Multiple Chromosomes

Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC, an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C. neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy.     

A Chronological History of the International Conference on Cryptococcus and Cryptococcosis (ICCC), an Invaluable Forum for Growth of the Cryptococcal Research Field and Clinical Practice

Cryptococcologists meet every 3 years to present their new research data and discuss the current state of cryptococcosis therapy at the International Conference on Cryptococcus and Cryptococcosis (ICCC). The ICCC has served as a unique forum where mycologists could interact and share their research data in a setting exclusively devoted to the study of Cryptococcus and cryptococcosis. This article presents an historical perspective on the ICCC meetings, beginning with the first ICCC that was held in Jerusalem, Israel in 1989. Subsequent ICCC meetings have grown, in terms of attendance and submitted abstracts. The history of the ICCC serves as a testimony to the remarkable progress that has been made in our basic understanding of the molecular biology, biochemistry, ecology, and taxonomy of Cryptococcus as well as the epidemiology, immunology, clinical manifestations, and treatment for cryptococcosis.     

Hyaluronic acid receptor CD44 deficiency is associated with decreased Cryptococcus neoformans brain infection

Cryptococcus neoformans is a pathogenic yeast that can invade the brain and cause meningoencephalitis. Our previous in vitro studies suggested that the interaction between C. neoformans hyaluronic acid and human brain endothelial CD44 could be the initial step of brain invasion. In this report, we used a CD44 knock-out (KO or CD44(-/-)) mouse model to explore the importance of CD44 in C. neoformans brain invasion. Our results showed that C. neoformans-infected CD44 KO mice survived longer than the infected wild-type mice. Consistent with our in vitro results, the brain and cerebrospinal fluid fungal burden was reduced in CD44-deficient mice. Histopathological studies showed smaller and fewer cystic lesions in the brains of CD44 KO mice. Interestingly, the cystic lesions contained C. neoformans cells embedded within their polysaccharide capsule and were surrounded by host glial cells. We also found that a secondary hyaluronic acid receptor, RHAMM (receptor of hyaluronan-mediated motility), was present in the CD44 KO mice. Importantly, our studies demonstrated an in vivo blocking effect of simvastatin. These results suggest that the CD44 and RHAMM receptors function on membrane lipid rafts during invasion and that simvastatin may have a potential therapeutic role in C. neoformans infections of the brain.     

Cryptococcus neoformans-Derived Microvesicles Enhance the Pathogenesis of Fungal Brain Infection

Cryptococcal meningoencephalitis is the most common fungal disease in the central nervous system. The mechanisms by which Cryptococcus neoformans invades the brain are largely unknown. In this study, we found that C. neoformans-derived microvesicles (CnMVs) can enhance the traversal of the blood-brain barrier (BBB) by C. neoformans in vitro. The immunofluorescence imaging demonstrates that CnMVs can fuse with human brain microvascular endothelial cells (HBMECs), the constituents of the BBB. This activity is presumably due to the ability of the CnMVs to activate HBMEC membrane rafts and induce cell fusogenic activity. CnMVs also enhanced C. neoformans infection of the brain, found in both infected brains and cerebrospinal fluid. In infected mouse brains, CnMVs are distributed inside and around C. neoformans-induced cystic lesions. GFAP (glial fibrillary acidic protein)-positive astrocytes were found surrounding the cystic lesions, overlapping with the 14-3-3-GFP (14-3-3-green fluorescence protein fusion) signals. Substantial changes could be observed in areas that have a high density of CnMV staining. This is the first demonstration that C. neoformans-derived microvesicles can facilitate cryptococcal traversal across the BBB and accumulate at lesion sites of C. neoformans-infected brains. Results of this study suggested that CnMVs play an important role in the pathogenesis of cryptococcal meningoencephalitis.     

Spatial and Temporal Analysis of Contact Rates in Female White-Tailed Deer

Abstract: White-tailed deer (Odocoileus virginianus) are important game mammals and potential reservoirs of diseases of domestic livestock; thus, diseases of deer are of great concern to wildlife managers. Contact, either direct or indirect, is necessary for disease transmission, but we know little about the ecological contexts that promote intrasexual contact among deer. Using pair-wise direct contacts estimated from Global Positioning System collar locations and joint utilization distributions (JUDs), we assessed habitats in which contacts occur to test whether direct contact rates among female white-tailed deer in different social groups differs among land-cover types. We also tested whether contact rates differed among seasons, lunar phases, and times of day. We obtained locations from 27 female deer for periods of 0.5–17 months during 2002–2006. We designated any simultaneous pair of locations for 2 deer <25 m apart as a direct contact. For each season, we used compositional analysis to compare land-cover types where 2 deer had contact to available land-cover weighted by their JUD. We used mixed-model logistic regression to test for effects of season, lunar phase, and time of day on contact rates. Contact rates during the gestation season were greater than expected from random use in forest and grassland cover, whereas contact rates during the fawning period were greater in agricultural fields than in other land-cover types. Contact rates were greatest during the rut and lowest in summer. Diel patterns of contact rates varied with season, and contact rates were elevated during full moon compared to other lunar periods. Both spatial and temporal analyses suggest that contact between female deer in different social groups occurs mainly during feeding, which highlights the potential impact of food distribution and habitat on contact rates among deer. By using methods to associate contacts and land-cover, we have created beneficial tools for more elaborate and detailed studies of disease transmission. Our methods can offer information necessary to develop spatially realistic models of disease transmission in deer.