Agrobacterium tumefaciens-Mediated Transformation of Aspergillus fumigatus: an Efficient Tool for Insertional Mutagenesis and Targeted Gene Disruption

Agrobacterium tumefaciens was used to transform Aspergillus fumigatus by either random or site-directed integration of transforming DNA (T-DNA). Random mutagenesis via Agrobacterium tumefaciens-mediated transformation (ATMT) was accomplished with T-DNA containing a hygromycin resistance cassette. Cocultivation of A. fumigatus conidia and Agrobacterium (1:10 ratio) for 48 h at 24°C resulted in high frequencies of transformation (>100 transformants/10⁷ conidia). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. We chose alb1, a polyketide synthase gene, as the target gene for homologous integration because of the clear phenotype difference between the white colonies of Δalb1 mutant strains and the bluish-green colonies of wild-type strains. ATMT with a T-DNA-containing alb1 disruption construct resulted in 66% albino transformants. Southern analysis revealed that 19 of the 20 randomly chosen albino transformants (95%) were disrupted by homologous recombination. These results suggest that ATMT is an efficient tool for transformation, random insertional mutagenesis, and gene disruption in A. fumigatus.     

Restriction fragment polymorphism in mitochondrial DNA of Cryptococcus neoformans

The restriction patterns of mitochondrial DNA from 20 isolates of the two varieties of Cryptococcus neoformans were compared. The patterns exhibited extensive heterogeneity among the isolates regardless of their serotype or varietal status. Hybridizations with cloned fragments of the conserved cytochrome oxidase gene from Saccharomyces cerevisiae exhibited at least seven patterns among the 20 isolates. There were, however, similarities in the restriction patterns among isolates within the same serotype that were not shared by isolates of other serotypes. Intra-varietal similarities were observed in the restriction patterns among the isolates of C. neoformans var. neoformans which were not present in the restriction patterns among the isolates of C. neoformans var. gattii. Hybridization of some cloned mitochondrial DNA fragments to total DNA digests of various isolates revealed polymorphic as well as variety-specific patterns of homology. These findings agree with the antigenic heterogeneity among the isolates and support the current taxonomic classification of C. neoformans into two varieties.     

Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells is mediated through the lipid rafts-endocytic pathway via the dual specificity tyrosine phosphorylation-regulated kinase 3 (DYRK3)

Cryptococcus neoformans is a neurotropic fungal pathogen, which provokes the onset of devastating meningoencephalitis. We used human brain microvascular endothelial cells (HBMEC) as the in vitro model to investigate how C. neoformans traverses across the blood-brain barrier. In this study, we present several lines of evidence indicating that C. neoformans invasion is mediated through the endocytic pathway via lipid rafts. Human CD44 molecules from lipid rafts can directly interact with hyaluronic acid, the C. neoformans ligand. Bikunin, which perturbs CD44 function in the lipid raft, can block C. neoformans adhesion and invasion of HBMEC. The lipid raft marker, ganglioside GM1, co-localizes with CD44 on the plasma membrane, and C. neoformans cells can adhere to the host cell in areas where GM1 is enriched. These findings suggest that C. neoformans entry takes place on the lipid rafts. Upon C. neoformans engagement, GM1 is internalized through vesicular structures to the nuclear membrane. This endocytic redistribution process is abolished by cytochalasin D, nocodazole, or anti-DYRK3 (dual specificity tyrosine-phosphorylation-regulated kinase 3) siRNA. Concomitantly, the knockdown of DYRK3 significantly reduces C. neoformans invasion across the HBMEC monolayer in vitro. Our data demonstrate that the lipid raft-dependent endocytosis process mediates C. neoformans internalization into HBMEC and that the CD44 protein of the hosts, cytoskeleton, and intracellular kinase-DYRK3 are involved in this process.     

Parasites of fishes in the recently inundated ephemeral Lake Liambezi,Namibia

Lake Liambezi forms the periodic connection between the upper Zambezi, Kwando and Okavango rivers. A full parasitological assessment was conducted on 86 fish, representing 14 species in six families sampled in August 2011. Parasite diversity was low and dominated by species with complex life cycles involving intermediate hosts. Most prevalent were larval nematodes (Contracaecum sp.) infecting 12 and Trypanasoma sp. infecting nine of the 14 host species. The intra-erythrocytic parasite Babesiosoma mariae was found in the blood of Coptodon rendalli and Oreochromis andersonii with prevalence of 50% and 60%, respectively. The host-specific monogenean Annulotrema hepseti was recorded only from H. cuvieri with a prevalence of 100%. Notable absences were the copepod and branchiuran parasites that have direct lifecycles and usually occur in high prevalence and abundance in the region. Because parasites with direct life cycles can only be transported into the lake on the host fish, their absence suggests limited immigration of infected fishes into the lake. This suggests that internal recruitment dominates over immigration in the fish population dynamics in Lake Liambezi.     

Cryptotrichosporon anacardii gen. nov., sp. nov., a new trichosporonoid capsulate basidiomycetous yeast from Nigeria that is able to form melanin on niger seed agar

Five yeast isolates obtained from cashew tree flowers in Nigeria resembled Cryptococcus neoformans phenotypically by producing brown pigmented colonies on niger seed agar, expressing a capsule, and being able to grow at 37 degrees C. However, rRNA gene sequences, including the 18S rRNA gene, the D1/D2 domains of the 26S rRNA gene and the ITS1+2 regions, suggested that these yeasts form a basal lineage within the Trichosporonales (Tremellomycetidae, Hymenomycetes, Basidiomycota, Fungi). Since the isolates could not be identified with any known genus and species within the Trichosporonales, we describe them as Cryptotrichosporon anacardii gen. et sp. nov. with CBS 9551(T) (=NRRL Y-27671) as the type strain. The taxonomic conflict between phenetic and molecular classification schemes within this group of fungi is discussed, and is resolved in favor of the latter.     

Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion

Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus.     

Enigmatic relationship of two Microsporum species

Unusual cultures of a Microsporum species were isolated from eleven patients who had either tinea capitis or tinea corporis or both. Although the macroconidia produced in the primary cultures resembled those of M. canis, hyphal morphology and colony characteristics were different from any of the more commonly occurring Microsporum species. Careful examination of subcultures showed that the isolates were composed of two species; M. canis and M. distortum, whose relationship remains enigmatic.     

Ultrastructure of septal complex in Filobasidiella neoformans (Cryptococcus neoformans).

Electron microscopy of Filobasidiella neoformans, the perfect state of Cryptococcus neoformans, revealed basidiomycete doliporesepta between hyphal cells and also between clamp connections and adjacent cells. The pore-occluding material was a heterogeneous flattened plate with dark margins and a lighter center, as seen in the species of Filobasidium. Representative basidiomycete parenthesomes were lacking, and endoplasmic reticulum was seen in the dolipore region.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action in vivo and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Sublethal damage during cryopreservation of rainbow trout sperm

Although cellular damage during cryopreservation of freshwater fish spermatozoa has been reported in several studies, there is a lack of correlation between this damage and the fertility rates of eggs using postthawed milt. The apparent lack of such correlation may be due to other undetected sublethal cryodamage, which could affect the cell functionality and viability. This may be extremely important for freshwater fish spermatozoa whose ability to fertilize the egg requires dilution in water or hypoosmotic solutions, an hazardous environment for the cells. This study tested the change in cell permeability during cryopreservation, using Hoechst 33258 to assess cell permeability. The permeability of spermatozoa at different times after dilution in several hypoosmotic media were investigated. In the first trial, fresh semen, sperm diluted in freezing media (CPT), and freeze/thawed semen were studied. Three CPT were tested (Me2SO, DMA, and methanol). In the second trial, the addition of egg yolk as a membrane stabilizer was investigated. Samples were frozen at -20 degreesC/min in a programmable cooler and thawed in a 25 degreesC water bath. Dilution in the CPTs slightly increased the susceptibility of cells to damage but freezing/thawing caused a dramatic increase in the fragility of cells, which were killed in a few seconds after their contact with the hypoosmotic solutions. Egg yolk provided a significant protection to the membrane, allowing the cells a greater and more prolonged survival in the fertilization media. Samples frozen with Me2SO displayed the best results. These results are consistent with the achieved fertility rates that demonstrated sublethal cryodamage in the function of the sperm membrane that was not detected by standard procedures. Copyright 1998 Academic Press.