Role of sphingomyelin synthase in controlling the antimicrobial activity of neutrophils against Cryptococcus neoformans

The key host cellular pathway(s) necessary to control the infection caused by inhalation of the environmental fungal pathogen Cryptococcus neoformans are still largely unknown. Here we have identified that the sphingolipid pathway in neutrophils is required for them to exert their killing activity on the fungus. In particular, using both pharmacological and genetic approaches, we show that inhibition of sphingomyelin synthase (SMS) activity profoundly impairs the killing ability of neutrophils by preventing the extracellular release of an antifungal factor(s). We next found that inhibition of protein kinase D (PKD), which controls vesicular sorting and secretion and is regulated by diacylglycerol (DAG) produced by SMS, totally blocks the extracellular killing activity of neutrophils against C. neoformans. The expression of SMS genes, SMS activity and the levels of the lipids regulated by SMS (namely sphingomyelin (SM) and DAG) are up-regulated during neutrophil differentiation. Finally, tissue imaging of lungs infected with C. neoformans using matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS), revealed that specific SM species are associated with neutrophil infiltration at the site of the infection. This study establishes a key role for SMS in the regulation of the killing activity of neutrophils against C. neoformans through a DAG-PKD dependent mechanism, and provides, for the first time, new insights into the protective role of host sphingolipids against a fungal infection.     

Cryptococcus neoformans, a fungus under stress

Cryptococcus neoformans is a human fungal pathogen that survives exposure to stresses during growth in the human host, including oxidative and nitrosative stress, high temperature, hypoxia, and nutrient deprivation. There have been many genes implicated in resistance to individual stresses. Notably, the catalases do not have the expected role in resistance to external oxidative stress, but specific peroxidases appear to be critical for resistance to both oxidative and nitrosative stresses. Signal transduction through the HOG1 and calcineurin/calmodulin pathways has been implicated in the stress response. Microarray and proteomic analyses have indicated that the common responses to stress are induction of metabolic and oxidative stress genes, and repression of genes encoding translational machinery.     

Human defenses against Cryptococcus neoformans: An update

Cryptococcus neoformans var. neoformans is an opportunistic fungal pathogen, especially in AIDS patients, and is found world-wide. On the other hand, Cryptococcus neoformans var. gatti (CN-g) is restricted to an association with two species of Eucalyptus trees. Alveolar macrophages (AM) constitute the first line of defense to Cryptococcus neoformans and offers some resistance. The inflammatory response to Cryptococcus neoformans with an influx of neutrophils and monocytes affords a second line of defense. Secretion of proinflammatory monokines by human AM is now being defined. The inflammatory phagocytes are efficient in killing Cryptococcus neoformans and offer strong resistance. T and B cell responses to infection, a third line of defense, results in production of lymphokines (IFNg, etc.) and specific antibodies. Enhancement of lymphocyte responses by IL-12 and IL-18 to Cryptococcus neoformans infection appears to be critical. Susceptibility of AIDS patients to Cryptococcus neoformans is associated with low CD4+ T cell counts and likely reduced efficacy of the second line of defense. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis of new antifungal peptides selective against Cryptococcus neoformans

Many drugs are available for the treatment of systemic or superficial mycoses, but only a limited number of them are effective antifungal drugs, devoid of toxic and undesirable side effects. Furthermore, resistance development and fungistatic rather than fungicidal activities represent limitations of current antifungal therapy. Therefore there remains an urgent need for a new generation of antifungal agents. According to a polypharmacological approach, the present work concerns the synthesis and antifungal activity of a set of peptides designed to simultaneously target the fungal cell surface and lanosterol demethylase, a key enzyme involved in ergosterol synthesis. Our peptides include amino acid sequences characteristic of membrane-active antimicrobial peptides (AMP), and due to the presence of His residues, they carry the imidazole ring characteristic of azole compounds. The peptides synthesized by us, were tested against different yeast species, and displayed general antifungal activity, with a therapeutically promising antifungal specificity against Cryptococcus neoformans.     

Natural cellular resistance of beige mice against Cryptococcus neoformans

Previous reports have demonstrated that natural killer (NK) cells are capable of inhibiting the growth of Cryptococcus neoformans in vitro, and recent studies indicate that adoptively transferred NK cell-enriched spleen cell populations enhance clearance of cryptococci from the tissues of cyclophosphamide-pretreated recipients. The primary objective of these studies was to confirm that NK cells participate in early clearance of C. neoformans in vivo. Secondarily, the anti-cryptococcal activities of polymorphonuclear leukocytes and macrophages were examined. Seven-week-old C57BL/6 bg/+ mice, which have normal levels of NK cell activity, were compared with their bg/bg littermates, which have impaired NK cell function. One and 3 days after injecting both groups of mice i.v. with 2 X 10(4) cryptococci, we assessed the NK cell activities in spleens, lungs, and livers and clearance of the organism from corresponding tissues as determined by the mean log10 numbers of cryptococcal colony-forming units (CFU) per organ. Three days postinfection, the mean numbers of cryptococcal CFU in lungs and spleens of bg/+ mice were significantly lower than in the corresponding organs of bg/bg mice. NK cell activities in spleens and lungs of bg/+ mice were significantly higher than were the NK cell activities in similar cell populations from bg/bg mice. In contrast, the mean numbers of cryptococcal CFU in livers of the two groups of animals were nearly equivalent, a situation not unexpected, since liver NK cell activities were extremely low and similar in both groups of animals. Although these data indicated a correlation between early clearance of cryptococci from tissues and levels of NK cell activities in the corresponding tissues, it was also possible that differences in phagocytic cell function between the bg/+ and bg/bg animals could account for the observed differences in clearance of cryptococci from the tissues. Therefore, phagocytic cells from the two groups of animals were compared with respect to their abilities to phagocytize and inhibit the growth of cryptococci and to their abilities to respond to chemotactic stimuli in vivo. Peritoneal PMNL from bg/+ and bg/bg mice were similar in their abilities to phagocytize and inhibit the growth of cryptococci, as well as in their chemotactic responses to viable cryptococci or sodium caseinate. In addition, there were no differences in splenic macrophage functions between the two groups of mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene.

The opportunistic fungal pathogen Cryptococcus neoformans has two mating types, MATa and MAT alpha. The MAT alpha strains are more virulent. Mating of opposite mating type haploid yeast cells results in the production of a filamentous hyphal phase. The MAT alpha locus has been isolated in this study in order to identify the genetic differences between mating types and their contribution to virulence. A 138-bp fragment of MAT alpha-specific DNA which cosegregates with alpha-mating type was isolated by using a difference cloning method. Overlapping phage and cosmid clones spanning the entire MAT alpha locus were isolated by using this MAT alpha-specific fragment as a probe. Mapping of these clones physically defined the MAT alpha locus to a 35- to 45-kb region which is present only in MAT alpha strains. Transformation studies with fragments of the MAT alpha locus identified a 2.1-kb XbaI-HindIII fragment that directs starvation-induced filament formation in MATa cells but not in MAT alpha cells. This 2.1-kb fragment contains a gene, MF alpha, with a small open reading frame encoding a pheromone precursor similar to the lipoprotein mating factors found in Saccharomyces cerevisiae, Ustilago maydis, and Schizosaccharomyces pombe. The ability of the MATa cells to express, process, and secrete the MAT alpha pheromone in response to starvation suggests similar mechanisms for these processes in both cell types. These results also suggest that the production of pheromone is under a type of nutritional control shared by the two cell types.     

Profiling a killer, the development of Cryptococcus neoformans

The ability of fungi to transition between unicellular and multicellular growth has a profound impact on our health and the economy. Many important fungal pathogens of humans, animals, and plants are dimorphic, and the ability to switch between morphological states has been associated with their virulence. Cryptococcus neoformans is a human fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised and, in some cases, immunocompetent hosts. Cryptococcus neoformans grows vegetatively as a budding yeast and switches to hyphal growth during the sexual cycle, which is important in the study of cryptococcal pathogenicity because spores resulting from sexual development are infectious propagules and can colonize the lungs of a host. In addition, sexual reproduction contributes to the genotypic variability of Cryptococcus species, which may lead to increased fitness and virulence. Despite significant advances in our understanding of the mechanisms behind the development of C. neoformans, our knowledge is still incomplete. Recent studies have led to the emergence of many intriguing questions and hypotheses. In this review, we describe and discuss the most interesting aspects of C. neoformans development and address their impact on pathogenicity.     

Micromorphology of Cryptococcus neoformans

Fine details of the internal and external morphology of Cryptococcus neoformans as seen in ultrathin sections are described and illustrated with electron micrographs. The capsule characteristic of this species contained microfibrils (30 to 40 A in diameter) that appeared to radiate from the cell wall and to coil and intertwine in various directions. These thin, uniformly structured, electron-dense filaments are believed to represent complex polysaccharide molecules. The internal morphology of C. neoformans was in many ways similar to that of yeasts studied by other authors. The cell was uninucleate with a single nucleolus. The nuclear envelope, a pair of unit membranes interrupted by pores, was typical of that found in eucaryotic organisms. Smooth endoplasmic reticulum, mitochondria, vacuoles, storage granules, and ribosomes were consistent features of the cytoplasm. In addition, C. neoformans presented membranous organelles derived from the plasma membrane and comparable to bacterial mesosomes and mitochondria of an annulate type.     

Epidemiology of Cryptococcus neoformans

The concept of the epidemiology of Cryptococcus neoformans as the causative agent of cryptococcosis and as a basidiomycetous yeast is based on the fact that bird manure has been until now its only known habitat but not plant material which likewise harbours various non-pathogenic Cryptococcus species.It could be shown that the possible influence of nutritional factors on the morphology and morphogenesis earns attention not only in view of the epidemiology of C. neoformans but of its perfect states, too.     

Synthetically modified l-histidine-rich peptidomimetics exhibit potent activity against Cryptococcus neoformans

We describe the synthesis and antimicrobial evaluation of structurally new peptidomimetics, rich in synthetically modified l-histidine. Two series of tripeptidomimetics were synthesized by varying lipophilicity at the C-2 position of l-histidine and at the N- and C-terminus. The data indicates that peptides (5f, 6f, 9f and 10f) possessing highly lipophilic adamantan-1-yl group displayed strong inhibition of Cryptococcus neoformans. Peptide 6f is the most potent of all with IC₅₀ and MFC values of 0.60 and 0.63 μg/mL, respectively, compared to the commercial drug amphotericin B (IC₅₀ = 0.69 and MFC = 1.25 μg/mL). The selectivity of these peptides to microbial pathogen was examined by a tryptophan fluorescence quenching study and transmission electron microscopy. These studies indicate that the peptides plausibly interact with the mimic membrane of pathogen by direct insertion, and results in disruption of membrane of pathogen.     

Characterization of a Phenol Oxidase from Cryptococcus neoformans var. neoformans

In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25 C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion-exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80,000 and a dimeric species (Mw = 160,000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH₂-terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.     

Superoxide dismutase in Cryptococcus neoformans varieties gattii, grubi, and neoformans

Some clear dissimilarities occur among the varieties of Cryptococcus neoformans but there are few studies about the differences among individual yeast antioxidant enzymes. The total superoxide dismutase (SOD) activities and the copper, zinc-depend SOD (Cu,ZnSOD) and manganese-dependent SOD (MnSOD) isoenzymes of five reference C. neoformans strains belonged to A, B, C, AD and D serotypes (Table I) and other nine C. neoformans isolates (Table II) were determined. There were significant differences (p < 0.01 and p < 0.05) in total SOD activity among the varietie gattii (serotype C) and the other varieties. Cu,ZnSOD showed difference (p < 0.05) between A and D serotypes. These results point out a variety and serotype-independent SOD activity in C. neoformans reference strains and the other isolates that were evaluated.     

Production of an antimicrobial substance against Cryptococcus neoformans by Paenibacillus brasilensis Sa3 isolated from the rhizosphere of Kalanchoe brasiliensis

An antifungal substance produced by Paenibacillus brasilensis strain Sa3 was preliminary characterized and showed to be stable after treatment with different enzymes and organic solvents and at a wide range of pH, and presented a molecular weight between 3 and 10 kDa. In vitro antagonism of this strain towards Cryptococcus neoformans was investigated by optical and electronic microscopic analyses and a fungicidal effect on C. neoformans was observed. Ultrastructural analysis showed intense changes on the fungus when it was paired cultured with strain Sa3, mainly the detachment of the capsule from the cell wall and the presence of altered organelles in the cytoplasm. This novel antifungal substance produced by P. brasilensis Sa3 may represent a new insight in antifungal therapy mainly against emergent fungi. Also, prospective studies on rhizobacteria of plants as Kalanchoe brasiliensis may offer a potential source for the discovery of bioactive compounds with medical value.     

In vitro activity of a liposomal nystatin formulation (Nyotran) against Cryptococcus neoformans

The in vitro antifungal activity of a new liposomal nystatin formulation (NISTL, Nyotran, Aronex Ltd., EE.UU.) was evaluated by a microdilution method with RPMI based on the M27A document of the National Committee for Clinical Laboratory Standards (NCCLS) against 22 isolates of Cryptococcus neoformans. This antifungal activity was compared with those of other seven antifungal agents, such as nystatin (NIST), amphotericin B deoxycholate, liposomal amphotericin B, amphotericin B lipid complex, amphotericin B colloidal dispersion, fluconazole, and itraconazole. NISTL was more active in vitrothan NIST, showing MIC values 2-3 fold smaller in 90% of the isolates. The results obtained suggest that this new formulation would be very helpful for the treatment of cryptococcosis.     

Biochemical variation of Cryptococcus neoformans

Ninety-seven strains of Cryptococcus neoformans and C. bacillisporus were examined for 44 biochemical characters and the results were analyzed numerically. One phenon emerged at the 86% level of similarity when strains were clustered according to their M-similarity values. All strains grew in ten carbon sources (D-glucose, D-galactose, arbutin, maltose, sucrose, D-melezitose, D-xylose, D-mannitol, D-glucitol, and meso-inositol), and also grew at 37 ° C and produced urease and phenoloxidase. None of them grew in melibiose, lactose, nor valine, and none reduced nitrate to nitrite. Comparison of selected biochemical characters, creatinine utilization, and serotypes of 49 aberrant strains is presented. Forty-eight of the 97 strains produced the Filobasidiella state either alone or when paired with a strain of compatible mating-type. Filobasidiella neoformans serotypes A and D were interfertile with compatible mating-types of F. bacillispora serotypes B and C. The 44 biochemical characters and 4 serotypes did not predict barriers to mating competence. The present study further substantiates that Filobasidiella neoformans and F. bacillispora are one species.     

Immunogenic fractions of Cryptococcus neoformans

Cryptococcus neoformans cell and culture supernatant extracts were fractionated by ion exchange and gel filtration column chromatography. Various fractions were used to immunize mice, and to assess the release of migration inhibition factor, delayed type hypersensitivity, and protective immunity after challenge with C. neoformans. Results suggest that the C. neoformans fractions, which protect mice, contain a high molecular weight, predominantly carbohydrate antigen that can be distinguished from the capsular polysaccharide.     

Auxotrophic Mutants of Cryptococcus neoformans

Auxotrophic mutants of Cryptococcus neoformans have been obtained by using the methods of mutagenesis and replica-plating.