Microsatellite Typing of Clinical and Environmental Cryptococcus neoformans var. grubii Isolates from Cuba Shows Multiple Genetic Lineages

Background

Human cryptococcal infections have been associated with bird droppings as a likely source of infection. Studies toward the local and global epidemiology of Cryptococcus spp. have been hampered by the lack of rapid, discriminatory, and exchangeable molecular typing methods.

Methodology/Principal Findings

We selected nine microsatellite markers for high-resolution fingerprinting from the genome of C. neoformans var. grubii. This panel of markers was applied to a collection of clinical (n = 122) and environmental (n = 68; from pigeon guano) C. neoformans var. grubii isolates from Cuba. All markers proved to be polymorphic. The average number of alleles per marker was 9 (range 5–51). A total of 104 genotypes could be distinguished. The discriminatory power of this panel of markers was 0.993. Multiple clusters of related genotypes could be discriminated that differed in only one or two microsatellite markers. These clusters were assigned as microsatellite complexes. The majority of environmental isolates (>70%) fell into 1 microsatellite complex containing only few clinical isolates (49 environmental versus 2 clinical). Clinical isolates were segregated over multiple microsatellite complexes.

Conclusions/Significance

A large genotypic variation exists in C. neoformans var. grubii. The genotypic segregation between clinical and environmental isolates from pigeon guano suggests additional source(s) of human cryptococcal infections. The selected panel of microsatellite markers is an excellent tool to study the epidemiology of C. neoformans var. grubii.     

Ancient Dispersal of the Human Fungal Pathogen Cryptococcus gattii from the Amazon Rainforest

Over the past two decades, several fungal outbreaks have occurred, including the high-profile ‘Vancouver Island’ and ‘Pacific Northwest’ outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.     

Chromoblastomycosis due to Fonsecaea pedrosoi and F. monophora in Cuba

We report two cases of chromoblastomycosis due to Fonsecaea pedrosoi and F. monophora in otherwise healthy Cuban males. Direct microscopic examination of biopsies revealed muriform cells, the hallmark of chromoblastomycosis. The suspected agents were recovered in culture, identified on the basis of morphological criteria and confirmed by sequencing of the internal transcribed spacer regions of rDNA. Final treatment consisted of surgical excision. The patients were successfully cured since there was no relapse after a follow-up of more than a year. In vitro antifungal susceptibility testing of both isolates showed that itraconazole and posaconazole had potent activity. High MICs of amphotericin B (2 μg/ml), fluconazole (>64 μg/ml), anidulafungin (8 μg/ml) and caspofungin (8 μg/ml) were found.     

Cladophialophora psammophila, a novel species of Chaetothyriales with a potential use in the bioremediation of volatile aromatic hydrocarbons

Cladophialophora is a genus of asexual black yeast-like fungi with one-celled, hydrophobic conidia which is predicted to have teleomorphs in the ascomycete genus Capronia, a member of the order Chaetothyriales. Cladophialophora species are relatively frequently involved in human disease ranging from mild cutaneous lesions to cerebral abscesses. Although the natural niche outside humans is unknown for most opportunistic Cladophialophora species, the fungi concerned are rarely isolated from environmental samples such as dead plant material, rotten wood, or soil. The objective of the present paper is to describe a novel species of Cladophialophora which was isolated from soil polluted with benzene, toluene, ethylbenzene, and xylene (BTEX). It proved to be able to grow with toluene and other related alkylbenzenes as its sole carbon and energy source. This strain is of interest for the biodegradation of toluene and other related xenobiotics under growth limiting conditions, particularly in air biofilters, dry and/or acidic soil. A preliminary genetic analysis using multilocus sequencing typing (MLST) and amplified fragments length polymorphism (AFLP) showed that this fungus was closely related to the pathogenic species Cladophialophora bantiana, sharing a C. bantiana-specific intron in SSU rDNA. However, it was unable to grow at 40°C and proved to be non-virulent in mice. The clear phylogenetic and ecophysiological delimitation of the species is fundamental to prevent biohazard in engineered bioremediation applications.     

Fibronectin modulates thymocyte-thymic epithelial cell interactions following Trypanosoma cruzi infection

Developing thymocytes interact with thymic epithelial cells (TECs) through cell-cell interactions, TEC-derived secretory moieties and extracellular matrix (ECM)-mediated interactions. These physiological interactions are crucial for normal thymocyte differentiation, but can be disrupted in pathological situations. Indeed, there is severe thymic atrophy in animals acutely infected with Trypanosoma cruzi due to CD4+CD8+ thymocyte depletion secondary to caspase-mediated apoptosis, together with changes in ECM deposition and thymocyte migration. We studied an in vitro model of TEC infection by T. cruzi and found that infected TEC cultures show a reduced number of cells, which was likely associated with decreased proliferative capacity, but not with increased cell death, as demonstrated by bromodeoxyuridine and annexin-V labelling. The infected TEC cultures exhibited increased expression of fibronectin (FN), laminin (LM) and type IV collagen. Importantly, treatment with FN increased the relative number of infected cells, whereas treatment with anti-FN or anti-LM antibodies resulted in lower infection rates. Consistent with these data, we observed increased thymocyte adhesion to infected TEC cultures. Overall, these results suggest that ECM molecules, particularly FN, facilitate infection of the thymic epithelium and that the consequent enhancement of ECM expression might be associated with changes in TEC-thymocyte interactions.     

Intrinsic regulation of substrate fluxes and energy conservation in Ca2+-ATPase

The Ca2+-ATPase of sarcoplasmic reticulum was utilized to demonstrate an intrinsic regulation of enzyme catalysis, whereby the ratio of forward and reverse flow is altered by the binding of Ca2+ and Pi to the enzyme. This is related to displacement of internal equilibria among intermediate enzyme ligand complexes, independent of the overall equilibrium of the ATP in equilibrium ADP + Pi transformation. A very high energy conservation with a velocity of reverse flow approaching that of forward flow, was obtained by increasing the enzyme affinity for Pi in the presence of Me2SO.     

Role of water in the energy of hydrolysis of phosphoanhydride and phosphoester bonds

The observed equilibrium constants for hydrolysis (Kobs) of a phosphoester and a phosphoanhydride bond were measured under a variety of conditions likely to alter the interactions of reactants and products with water. These included increasing the pH of the medium from 5.0 to 10.0, increasing the MgCl2 concentration form 0 to 200 mM, and decreasing the water activity of the medium by adding either dimethyl sulfoxide (50%, v/v) or polyethylene glycol 6,000-8,000 (50%, w/v). The Kobs for phosphoesters such as phosphoserine, glucose phosphate, glycerol phosphate, and ethylene glycol phosphate varied little over this wide range of conditions, the extreme values of Kobs being 12 and 200 M. In contrast, the Kobs for the phosphoanhydride bond of pyrophosphate varied from a value greater than 20,000 to 0.1 M. In totally aqueous media at a pH between 7.0 and 8.0 and in the presence of 0.5-1.0 mM MgCl2, the energy of hydrolysis of pyrophosphate was 1.2-4.0 kcal/mol greater than that of phosphoserine. However, when the water activity was decreased by adding polyethylene glycol to the medium within the same pH and MgCl2 concentration range, the energy of hydrolysis of phosphoserine became 2.0-2.5 kcal/mol greater than that of pyrophosphate. The results suggest that for phosphoesters, the solvation energies of reactants and products, unlike the case of phosphoanhydride bonds, are not the major factors in determining the energy of hydrolysis.     

Conformational fluctuations of the Ca2+-ATPase in the native membrane environment. Effects of pH, temperature, catalytic substrates, and thapsigargin

Digestion with proteinase K or trypsin yields complementary information on conformational transitions of the Ca(2+)-ATPase (SERCA) in the native membrane environment. Distinct digestion patterns are obtained with proteinase K, revealing interconversion of E1 and E2 or E1 approximately P and E2-P states. The pH dependence of digestion patterns shows that, in the presence of Mg(2+), conversion of E2 to E1 pattern occurs (even when Ca(2+) is absent) as H(+) dissociates from acidic residues. Mutational analysis demonstrates that the Glu(309) and Glu(771) acidic residues (empty Ca(2+)-binding sites I and II) are required for stabilization of E2. Glu(309) ionization is most important to yield E1. However, a further transition produced by Ca(2+) binding to E1 (i.e. E1.2Ca(2+)) is still needed for catalytic activation. Following ATP utilization, H(+)/Ca(2+) exchange is involved in the transition from the E1 approximately P.2Ca(2+) to the E2-P pattern, whereby alkaline pH will limit this conformational transition. Complementary experiments on digestion with trypsin exhibit high temperature dependence, indicating that, in the E1 and E2 ground states, the ATPase conformation undergoes strong fluctuations related to internal protein dynamics. The fluctuations are tightly constrained by ATP binding and phosphoenzyme formation, and this constraint must be overcome by thermal activation and substrate-free energy to allow enzyme turnover. In fact, a substantial portion of ATP free energy is utilized for conformational work related to the E1 approximately P.2Ca(2+) to E2-P transition, thereby disrupting high affinity binding and allowing luminal diffusion of Ca(2+). The E2 state and luminal path closure follow removal of conformational constraint by phosphate.