<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>, <Emphasis Type="Italic">Cryptococcus gattii</Emphasis>: Serotypes in Venezuela

Mycoses due to yeasts belonging to other genera than Candida have become common in the last years especially in immuno-compromised patients. Species of the anamorphic basidiomycetous yeast genus Trichosporon are such opportunistic human pathogenic yeasts which cause several diseases. In this study, Trichosporon faecale is reported in Germany for the first time. The isolate was taken from a human foot, where it was associated with a tinea pedis. The fungal isolate was identified by investigating the morphology, physiology by a commercial API 32 C-set and molecular data of SSU and LSU rDNA as well as the ITS region.     

<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Epidemiological Cutoff Values

Purpose of Review

The purpose of this review is to provide a current view of the importance of the determination and use of epidemiological cutoff values (ECVs) for Cryptococcus neoformans, since there are no clinical breakpoints (CBPs).

Recent Findings

ECVs have been proposed for some antifungal agents and C. neoformans, using standardized methodologies by the Clinical and Laboratory Standards Institute (CLSI) and by the European Committee for Antimicrobial Susceptibility Testing (EUCAST), based on the distribution of minimum inhibitory concentrations (MICs). There is no sufficient evidence for the determination of ECVs for C. neoformans using commercial methods; however, as these methods are routinely used in the microbiology laboratory, it is recommended for the establishment of local ECVs using these methods and following the criteria for their determination.

Summary

Due to the geographic and genetic variations inherent to C. neoformans, it is important to calculate ECVs, since they are useful in clinical practice to guide therapy in the absence of CBPs.

     

Phenotypic Switching of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> and <Emphasis Type="Italic">Cryptococcus</Emphasis><Emphasis Type="Italic">gattii</Emphasis>

Microorganisms that live in fluctuating environments must constantly adapt their behavior to survive. The host constitutes an important microenvironment in opportunistic and primary fungal pathogens like Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii). In clonal populations, adaptation may be achieved through the generation of diversity. For fungi phenotype switching constitutes a mechanism that allows them to change rapidly. Both C. neoformans and C. gattii undergo phenotypic switching, which allows them to be successful pathogens and cause persistent disease. Similar to other encapsulated microbes that exhibit phenotypic variation, phenotypic switching in Cryptococcus changes the polysaccharide capsule. Most importantly, in animal models phenotypic switching affects virulence and can change the outcome of infection. Virulence changes because C. neoformans and C. gattii switch variants elicit different inflammatory responses in the host. This altered host response can also affect the response to antifungal therapy and in some cases may even promote the selection of switch variants. This review highlights the similarity and differences between phenotypic switching in C. neoformans and C. gattii, the two dominant species that cause cryptococcosis in humans.     

Innate host defenses against <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, can cause life-threatening infections of the central nervous system in immunocompromised and immunocompetent individuals. Cryptococcal meningoencephalitis is the most common disseminated fungal infection in AIDS patients, and remains the third most common invasive fungal infection among organ transplant recipients. The administration of highly active antiretroviral therapy (HAART) has resulted in a decrease in the number of cases of AIDS-related cryptococcosis in developed countries, but in developing countries where HAART is not readily available, Cryptococcus is still a major concern. Therefore, there is an urgent need for the development of novel therapies and/or vaccines to combat cryptococcosis. Understanding the protective immune responses against Cryptococcus is critical for development of vaccines and immunotherapies to combat cryptococcosis. Consequently, this review focuses on our current knowledge of protective immune responses to C. neoformans, with an emphasis on innate immune responses.     

Requirement of the isocitrate lyase gene <Emphasis Type="Italic">ICL1</Emphasis> for <Emphasis Type="Italic">VPS41</Emphasis>-mediated starvation response in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cryptococcus neoformans is a major cause of fungal meningitis in individuals with impaired immunity. Our previous studies have shown that the VPS41 gene plays a critical role in the survival of Cryptococcus neoformans under nitrogen starvation; however, the molecular mechanisms underlying VPS41-mediated starvation response remain to be elucidated. In the present study, we show that, under nitrogen starvation, VPS41 strongly enhanced ICL1 expression in C. neoformans and that overexpression of ICL1 in the vps41 mutant dramatically suppressed its defects in starvation response due to the loss of VPS41 function. Moreover, targeted deletion of ICL1 resulted in a dramatic decline in viability of C. neoformans cells under nitrogen deprivation. Taken together, our data suggest a model in which VPS41 up-regulates ICL1 expression, directly or indirectly, to promote survival of C. neoformans under nitrogen starvation.     

Time-Kill Curves Studies with Amphotericin B Against <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>/<Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">gattii</Emphasis> Species Complex Clinical Isolates

Purpose of Review

We reviewed data on amphotericin B (AmB) tolerance among Cryptococcus neoformans/C. gattii species complex clinical isolates and present our results of large recent study on this issue.

Recent Findings

The standard method to detect antifungal susceptibility is based on MIC (minimal inhibitory concentration) determination; however, there is no interpretative clinical breakpoints defined for antifungal agents against Cryptococcus species, and to date, there is no correlation of MIC and clinical response. The time-kill curves (TKC) methodology seems to provide some correlation with outcome and it could identify distinct profiles of AmB-fungicidal activity.

Summary

Our group analyzed 83 human isolates from cryptococcosis cases. The isolates were tested by TKC and showed up 8.3% of tolerance to AmB. Importantly, the AmB-MIC was low for all isolates, including tolerant ones. Our findings are similar to other authors, due the ability of TKC to identify distinct AmB-fungicidal activity and detecting low susceptible isolates.

     

Disseminated <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis> and <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Co-Infection in Patients with AIDS

Histoplasmosis and cryptococcosis are the most prevalent systemic mycoses in HIV-infected patients. The authors report a 20-year-old Brazilian HIV-positive woman with concomitant disseminated histoplasmosis and cryptococcosis. In addition, we review the reported cases described in the medical literature.     

Experimental modulation of capsule size in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Experimental modulation of capsule size is an important technique for the study of the virulence of the encapsulated pathogen Cryptococcus neoformans. In this paper, we summarize the techniques available for experimental modulation of capsule size in this yeast and describe improved methods to induce capsule size changes. The response of the yeast to the various stimuli is highly dependent on the cryptococcal strain. A high CO₂ atmosphere and a low iron concentration have been used classically to increase capsule size. Unfortunately, these stimuli are not reliable for inducing capsular enlargement in all strains. Recently we have identified new and simpler conditions for inducing capsule enlargement that consistently elicited this effect. Specifically, we noted that mammalian serum or diluted Sabouraud broth in MOPS buffer pH 7.3 efficiently induced capsule growth. Media that slowed the growth rate of the yeast correlated with an increase in capsule size. Finally, we summarize the most commonly used media that induce capsule growth in C. neoformans. Published: March 3, 2004     

Growth and Mating of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> var. <Emphasis Type="Italic">grubii</Emphasis> on Woody Debris

A total of 36 Cryptococcus neoformans strains originating from South Africa were screened for wood degrading enzymes. All strains tested positive for cellulase activity while none where capable of xylan degradation. Three C. neoformans var. grubii strains, originating from clinical and environmental samples, representing the same genotype (VNI/AFLP1—C. neoformans var. grubii) and MATα, were evaluated for growth on debris of two common tree species in South Africa: Acacia mearnsii and Eucalyptus camaldulensis. The mating capability of all the C. neoformans strains was evaluated on similar debris. Strains grown on A. mearnsii yielded substantially greater yeast populations. A total of 26%, 6%, 46%, and 80% of the 36 C. neoformans strains tested were either able to mate or develop filaments when crossed on A. mearnsii and E. camaldulensis debris, V8 juice, and yeast carbon base (YCB) agar, respectively. Filamentation and monokaryotic fruiting was observed in 3% of strains when C. neoformans was cultured on either A. mearnsii, E. camaldulensis debris, or YCB. The results indicate that this fungus is capable of completing its life cycle and can produce basidiospores on woody debris. In the future, these findings should be considered when studying the epidemiology, microbial ecology, and proposed infection process of this global pathogen.     

Computational modeling and <Emphasis Type="Italic">in silico</Emphasis> analysis of differential regulation of <Emphasis Type="Italic">myo</Emphasis>-inositol catabolic enzymes in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Background  

Inositol is a key cellular metabolite for many organisms. Cryptococcus neoformans is an opportunistic pathogen which primarily infects the central nervous system, a region of high inositol concentration, of immunocompromised individuals. Through the use of myo-inositol oxygenase C. neoformans can catabolize inositol as a sole carbon source to support growth and viability.     

Characterization of<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> var.<Emphasis Type="Italic">neoformans</Emphasis> serotype A and A/D in samples from Egypt

The cryptococcal polysaccharide antigen was detected in 10 cerebrospinal fluid (CSF) and 23 serum samples from cryptococcal meningitis and intestinal cryptococcosis by the cryptococcal antigen latex agglutination system (CALAS). CALAS titers in CSF and serum samples of cryptococcal meningitis ranged over 8-2048 and 32-2048, respectively, while in cases of intestinal cryptococcosis, serum titers ranged over 8-2048. The isolates of yeast Cryptococcus neoformans were determined to be of serotype A or of the A/D pair. The total leukocyte count and biochemical parameters in CSF were significantly increased as indicators of microbial infection. Furthermore, the in vitro change of the teleomorph (sexual state) to the anamorph (asexual state) was also detected and the teleomorph state changed in vivo to the encapsulated anamoph state which is more virulent during infection in vivo than the yeast-like noncapsulated form. Two primers for internal transcribed spacer (ITS) regions of ribosomal DNA were used for molecular detection of C. neoformans. After PCR amplification, a DNA band of 415 bp, visualized on agarose gel, indicated the presence of C. neoformans cells in the tested CSF and serum samples. The primer sensitivity was also characterized using purified yeast chromosomal DNA as template; it was about 20 pg or more chromosomal DNA which represents about 10 cells of C. neoformans. The primers were also specific for ITS regions of C. neoformans and gave negative results with Candida albicans and E. coli chromosomal DNA templates.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Candida albicans</Emphasis>, a major human fungal pathogen

Candida albicans is the most common human fungal pathogen (Beck-Sague and Jarvis, 1993). It is normally a harmless commensal organism. However, it is a opportunistic pathogen for some immunologically weak and immunocompromised people. It is responsible for painful mucosal infections such as the vaginitis in women and oral-pharangeal thrush in AIDS patients. In certain groups of vulnerable patients it causes severe, life-threatening bloodstream infections and it causes severe, life-threatening bloodstream infections and subsequent infections in the internal organs. There are various fascinating features of the C. albicans life cycle and biology that have made the pathogen the subject of extensive research, including its ability to grow in unicellular yeast, psudohyphal, and hyphal forms (Fig. 1A); its ability to switch between different but stable phenotypic states, and the way that it retains the ability to mate but apparently loses the ability to go through meiosis to complete the sexual cycle. This research has been greatly facilitated by the derivation of the complete C. albicans genome sequence (Braun et al., 2005), the development of a variety of molecular tools for gene manipulation, and a store of underpinning knowledge of cell biology borrowed from the distantly related model yeast Saccharomyces cerevisiae (Berman and Sudbery, 2002; Noble and Johnson, 2007). This review will provide a brief overview of the importance of C. albicans as a public health issue, the experimental tools developed to study its fascinating biology, and some examples of how these have been applied.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Heteroresistance to Fluconazol in Clinical and Environmental Brazilian Strains of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>/<Emphasis Type="Italic">C. gattii</Emphasis> Species Complex

Purpose of Study

To review the literature on heteroresistance to fluconazole (FLC) and investigate the level of heteroresistance to FLC (LHF), we analyzed 100 clinical and environmental Brazilian Cryptococcus strains.

Recent Findings

Heteroresistance is a phenomenon described as the emergence of resistant subpopulation cells within a single susceptible strain that can tolerate higher concentrations of fluconazole above the minimal inhibitory concentration (MIC) level.

Summary

We found lower FLC-MICs (0.12–64 mg/L) than LHF (8–128 mg/L). Highly heteroresistant adapted subpopulations (256 mg/L) was found in minority (9%) strains, but importantly, 33% showed low FLC-MIC (8 mg/L). We concluded for similar LHF in both species, but higher LHF in clinical strains in comparison to environmental ones. Our findings stressed that the LHF is not correlated to species and pretty is strain-dependent and alert about high heteroresistant subpopulations that hardly reverts to the original LHF even upon the removal of drug pressure.

     

The chitobiose transporter, <Emphasis Type="Italic">chbC</Emphasis>, is required for chitin utilization in <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

Background  

The bacterium Borrelia burgdorferi, the causative agent of Lyme disease, is a limited-genome organism that must obtain many of its biochemical building blocks, including N-acetylglucosamine (GlcNAc), from its tick or vertebrate host. GlcNAc can be imported into the cell as a monomer or dimer (chitobiose), and the annotation for several B. burgdorferi genes suggests that this organism may be able to degrade and utilize chitin, a polymer of GlcNAc. We investigated the ability of B. burgdorferi to utilize chitin in the absence of free GlcNAc, and we attempted to identify genes involved in the process. We also examined the role of RpoS, one of two alternative sigma factors present in B. burgdorferi, in the regulation of chitin utilization.