The isolation of monoclonal antibodies to Cryptococcus neoformans antigens

In this work conditions for the reproduction of hybridoma technology, specially adapted to C. neoformans, for obtaining monoclonal hybridomas (McAb) to diagnostically significant antigens of C. neoformans, the infective agent of cryptococcosis, are presented. The advantages of using the short-time cycle of stimulation of mouse B lymphocytes with low doses of C. neoformans capsular polysaccharide and the effectiveness of the hybridization of mouse spleen cells with myeloma cells, line Sp2/0, are shown. Four lines of stable hybridomas, producing McAb to different epitopes of C. neoformans surface antigens, have been obtained. The specific activity of McAb has been studied in the indirect immunofluorescence assay, the cytochemical and solid-phase enzyme immunoassays (EIA). McAb 3E2, Cr2 and 2G9 have been shown to be suitable for use in diagnostic EIA systems.     

Regulatory Diversity of TUP1 in Cryptococcus neoformans

Cryptococcus neoformans serotype A strains, the major cause of cryptococcosis, are distributed worldwide, while serotype D strains are more concentrated in Central Europe. We have previously shown that deletion of the global regulator TUP1 in serotype D isolates results in a novel peptide-mediated, density-dependent growth phenotype that mimics quorum sensing and is not known to exist in other fungi. Unlike for tup1Δ strains of serotype D, the density-dependent growth phenotype was found to be absent in tup1Δ strains of serotype A which had been derived from several different genetic clusters. The serotype A H99 tup1Δ strain showed less retardation in the growth rate than tup1Δ strains of serotype D, but the mating efficiency was found to be similar in both serotypes. Deletion of TUP1 in the H99 strain resulted in significantly enhanced capsule production and defective melanin formation and also revealed a unique regulatory role of the TUP1 gene in maintaining iron/copper homeostasis. Differential expression of various genes involved in capsule formation and iron/copper homeostasis was observed between the wild-type and tup1Δ H99 strains. Furthermore, the H99 tup1Δ strain displayed pleiotropic effects which included sensitivity to sodium dodecyl sulfate, susceptibility to fluconazole, and attenuated virulence. These results demonstrate that the global regulator TUP1 has pathobiological significance and plays both conserved and distinct roles in serotype A and D strains of C. neoformans.The fungal Tup1 proteins function as global repressors which regulate a large number of genes associated with growth, morphological differentiation, and sexual and asexual reproduction. As a consequence, tup1 mutants are known to display numerous phenotypes (9, 19, 42). The deletion of TUP1 in Candida albicans results in constitutive filamentous growth with no budding yeast cells and is accompanied by loss of virulence (2, 32). In Penicillium marneffei, the only dimorphic species known in the genus Penicillium, deletion of the TUP1 homolog, tupA, confers reduced filamentation and abnormality in yeast morphogenesis (38). In the filamentous fungi Aspergillus nidulans and Neurospora crassa, deletion of the TUP1 homologs, rcoA and rco-1, respectively, severely affects growth and sexual and asexual reproduction (12, 46).Cryptococcus neoformans is a bipolar heterothallic basidiomycetous yeast with two serotypes, A and D, and the function of Tup1 has been studied only for serotype D strains (26, 27). While disruption of TUP1 in strains of serotype D did not affect yeast or hyphal cell morphology, it resulted in mating-type-dependent differences, including temperature-dependent growth, sensitivity to 0.8 M KCl, and expression of genes in several other biological pathways (26). Most importantly, tup1Δ strains displayed a peptide-mediated quorum-sensing-like phenomenon in both mating types of serotype D strains which has not been reported for any other fungal species (27).According to genome sequence data, the serotype A reference strain H99 shares 95% sequence identity with the serotype D reference strain JEC21 (29). However, serotype-specific differences between the two strains have been demonstrated in two major signaling pathways, the pheromone-responsive Cpk1 mitogen-activated protein kinase and cyclic AMP (cAMP) (5, 13, 41, 47). In addition, the high-osmolarity glycerol (HOG) pathway also showed regulatory disparity between the two serotypes (1, 8). Since the regulation of peptide-mediated quorum sensing by TUP1 is reported only for serotype D strains, we sought to determine whether the deletion of TUP1 in serotype A strains would have similar consequences. Surprisingly, we found striking differences in the phenotypes manifested by tup1Δ strains of the two serotypes. We report here the serotype-specific differences in TUP1 regulation between A and D strains and the novel regulatory role of TUP1 in maintaining iron/copper homeostasis in C. neoformans.     

Melanogenesis in Cryptococcus neoformans

Melanogenesis in Cryptococcus neoformans begins with the oxidation of dihydroxyphenylalanine by the enzyme phenol oxidase. The succeeding steps are very rapid. Two intermediates, dopachrome and 5,6-dihydroxyindole, have been isolated and characterized by high performance liquid chromatography. A pathway of melanin formation in C. neoformans is proposed, based on the presence of these intermediates.     

Persistence of infection in mice inoculated intranasally with Cryptococcus neoformans

Cryptococcus neoformans was instilled intranasally into mice which were periodically sacrificed to determine the course of infection. Cryptococci persisted within the nasal passages throughout the 90 day study. Extranasal dissemination began 14–28 days after instillation and was still demonstrable 90 days post-exposure. Ten percent mortality was observed in mice receiving 10⁶ cryptococci, while no mortality was observed in mice exposed to 10³ or 10⁴ cryptococci. Our research suggests that nasal colonization with C. neoformans can precede pulmonary and systemic cryptococcosis by weeks or months.     

The role of laccase in prostaglandin production by Cryptococcus neoformans

Recently, it has been demonstrated that the opportunistic fungal pathogen Cryptococcus neoformans can synthesize authentic immunomodulatory prostaglandins. The mechanism by which this takes place is unclear as there is no cyclooxygenase homologue in the cryptococcal genome. In this study, we show that cryptococcal production of both PGE₂ and PGF_2α can be chemically inhibited by caffeic acid, resveratrol and nordihydroguaiaretic acid. These polyphenolic molecules are frequently used as inhibitors of lipoxygenase enzymes; however, blast searches of the cryptococcal genome were unable to identify any homologues of mammalian, plant or fungal lipoxygenases. Next we investigated cryptococcal laccase, an enzyme known to bind polyphenols, and found that either antibody depletion or genetic deletion of the primary cryptococcal laccase ( lac1 Δ) resulted in a loss of cryptococcal prostaglandin production. To determine how laccase is involved, we tested recombinant laccase activity on the prostaglandin precursors, arachidonic acid (AA), PGG₂ and PGH₂. Using mass spectroscopy we determined that recombinant Lac1 does not modify AA or PGH₂, but does have a marked activity toward PGG₂ converting it to PGE₂ and 15-keto-PGE₂. These data demonstrate a critical role for laccase in cryptococcal prostaglandin production, and provides insight into a new and unique fungal prostaglandin pathway.     

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)     

Cryptococcus neoformans is resistant to surfactant protein A mediated host defense mechanisms

Initiation of a protective immune response to infection by the pathogenic fungus Cryptococcus neoformans is mediated in part by host factors that promote interactions between immune cells and C. neoformans yeast. Surfactant protein A (SP-A) contributes positively to pulmonary host defenses against a variety of bacteria, viruses, and fungi in part by promoting the recognition and phagocytosis of these pathogens by alveolar macrophages. In the present study we investigated the role of SP-A as a mediator of host defense against the pulmonary pathogen, C. neoformans. Previous studies have shown that SP-A binds to acapsular and minimally encapsulated strains of C. neoformans. Using in vitro binding assays we confirmed that SP-A does not directly bind to a fully encapsulated strain of C. neoformans (H99). However, we observed that when C. neoformans was incubated in bronchoalveolar fluid, SP-A binding was detected, suggesting that another alveolar host factor may enable SP-A binding. Indeed, we discovered that SP-A binds encapsulated C. neoformans via a previously unknown IgG dependent mechanism. The consequence of this interaction was the inhibition of IgG-mediated phagocytosis of C. neoformans by alveolar macrophages. Therefore, to assess the contribution of SP-A to the pulmonary host defenses we compared in vivo infections using SP-A null mice (SP-A-/-) and wild-type mice in an intranasal infection model. We found that the immune response assessed by cellular counts, TNFalpha cytokine production, and fungal burden in lungs and bronchoalveolar lavage fluids during early stages of infection were equivalent. Furthermore, the survival outcome of C. neoformans infection was equivalent in SP-A-/- and wild-type mice. Our results suggest that unlike a variety of bacteria, viruses, and other fungi, progression of disease with an inhalational challenge of C. neoformans does not appear to be negatively or positively affected by SP-A mediated mechanisms of pulmonary host defense.     

Reduced host resistance and Th1 response to Cryptococcus neoformans in interleukin-18 deficient mice

Using interleukin (IL)-18 deficient (IL-18(-/-)) mice, we examined the role of IL-18 in the host resistance and Th1 response against infection with Cryptococcus neoformans. Fungal clearance in the lung was reduced in IL-18(-/-) mice, although there was no significant change in the level of dissemination to the brain. The DTH response, as determined by footpad swelling, was also diminished in IL-18(-/-) mice compared to control wild-type (WT) mice. The levels of IL-12 and interferon (IFN)-gamma in the sera were significantly lower in IL-18(-/-) mice than in WT mice. Spleen cells from infected WT mice produced a high level of IFN-gamma upon stimulation with the microbe, while only a low level of IFN-gamma production was detected in spleen cells from infected IL-18(-/-) mice. Administration of IL-18 almost completely restored the reduced response in IL-18(-/-) mice, while IL-12 showed a marginal effect. These results demonstrated the important role of IL-18 in the resistance and Th1 response of mice to C. neoformans by potentiating the production of IFN-gamma.     

Pulmonary cell response in mice following intranasal instillation with Cryptococcus neoformans

Cryptococcus neoformans was instilled intranasally into mice which were sacrificed to determine the fate of instilled cryptococci and the pulmonary cellular response. During the first 12 h, instilled cryptococci decreased significantly in numbers and did not disseminate to extrapulmonary sites. There was a transient increase in neutrophils which may have been due to an influx of bacteria which accompanied the process of intranasal instillation.     

Urease activity in Cryptococcus neoformans and Cryptococcus gattii

Urease is an enzyme considered one of the main virulence factors in Cryptococcus neoformans. Quantitative differences in urease production between C. neoformans and the new species Cryptococcus gattii have not been so far documented. Using a standardized method, 25 isolates of C. neoformans and 19 of C. gattii were seeded in Christensen urea broth medium for urease activity detection. Approximately, the 50% of activity of one unit of commercial jack beans urease (A550=0.215) was considered as a reference to classified the Cryptococcus in two cathegories, low (A550<0.215) or high (A550=or>0.215) urease producers. After 72 hours of incubation, 76% of C. neoformans and 15.8% of C. gattii strains were high urease producers (p=0.016). Based on these results, the species C. neoformans appeared as the highest urease producer. Other virulence factors should also be investigated to explain C. gattii pathogenicity.     

Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice

A double mutant of Cryptococcus neoformans which lacked the ability to produce melanin (Mel-) on media containing diphenols and failed to grow at 37 degrees C (temperature sensitive, Tem-) was obtained by UV irradiation and subsequent cloning. The mutant showed two lesions in melanogenesis in that it lacked the active transport system for diphenolic compounds and also lacked phenoloxidase. Ultrastructures of the mutant and wild-type cells grown on a medium with or without L-dopa showed that only the wild-type cells grown on L-dopa medium formed a dark cell wall layer, presumably containing melanin. The mutant was crossed with a wild type, and the phenotypes of the progeny were analyzed. The analysis showed no linkage between the mating type and either Mel or Tem loci, but loose linkage was seen between Mel and Tem loci. The progeny, Mel+ Tem+, Mel+ Tem-, Mel- Tem+, and Mel- Tem-, were studied for their virulence in mice. Only Mel+ Tem+ types killed mice with an inoculum of 5 X 10(5) cells within 50 days.     

Phospholipase activity in Cryptococcus neoformans

Phospholipases have only been detected in a few fungi and yeasts, in particular in Candida albicans. Secreted phospholipases are considered by some researchers to be a potential factor of virulence and pathogenicity in C. albicans. Twenty-three Cryptococcus neoformans strains were tested in order to observe phospholipase production. Twenty-two of the 23 strains tested were able to produce phospholipases, and the ratio diameter of the colony to total diameter of the colony plus zone of precipitation (Pz) ranged between 0.271 and 0.949. C. neoformans, just like C. albicans, can be divided on the basis of the Pz into different strains according to their virulence and pathogenicity. There also appeared to be a correlation between the phospholipase production and the size of the capsule in the strains isolated from AIDS patients. For this reason, further studies on C. neoformans phospholipase activity would be useful in evaluating the virulence of different strains.     

Lipid metabolism in Cryptococcus neoformans

In recent years, lipids have been shown to act as signalling molecules not only in mammalian cells but also in many other eukaryotes. Whereas in mammalian cells lipids regulate cellular functions that play crucial roles in the regulation of pathobiological processes, such as cancer, cardiovascular and neurodegenerative disorders, and inflammation, in the fungus Cryptococcus neoformans lipids play key roles in the regulation of pathogenic traits required for the development of cryptococcosis, an infectious disease particularly frequent in immunocompromised individuals. In this minireview we discuss recent advances in the understanding of lipid metabolism in this important human pathogen, highlighting the potential of fungal lipid enzymatic pathways as promising new drug targets.     

Phenotypic switching in Cryptococcus neoformans

Cryptococcus neoformans strains exhibit considerable phenotype variability with regards to the capsular polysaccharide, sterol composition of the cell wall, and cell and colony morphology. Phenotypic changes can occur spontaneously during in vitro passage of strains or during chronic infection in vivo and may be associated with differences in virulence. Studies from our laboratory have demonstrated that phenotype variability can be the result of phenotypic switching. Phenotypic switching is defined as a reversible change of an observable colony phenotype that occurs at a frequency above the expected frequency for somatic mutations. This implies that phenotypic switching represents controlled and programmed changes in this pathogenic yeast rather than random mutations. We have shown that a phenotypic switch from a smooth colony phenotype to a mucoid colony phenotype occurs in vitro and in vivo during chronic infection of mice. More importantly we have now demonstrated that the switch is associated with an increase in virulence and a change in the host immune response. Implications of these findings for the pathogenesis of cryptococcosis are discussed.     

Melanin Biosynthesis in Cryptococcus neoformans

Pigment production by Cryptococcus neoformans is virulence associated. Dopamine- and 3,4-dihydroxyphenylalanine–melanin products were identified after acidic permanganate oxidation, alkaline hydrogen peroxide oxidation, or hydrolysis with hydriodic acid. These data provide direct chemical evidence for the formation of eumelanin polymers by catecholamine oxidation by laccase alone followed by oxidative coupling of dihydroxyindole.     

Creatinine metabolism in Cryptococcus neoformans and Cryptococcus bacillisporus.

The pathogenic species of Cryptococcus, C. neoformans and C. bacillisporus, utilized creatinine as a source of nitrogen but not of carbon. Chromatographic and autoradiographic studies suggest that creatinine metabolism in both species involves a single step resulting in the production of methylhydantoin and ammonia. The enzyme responsible for this step, creatinine deiminase, was produced by the cells only in the presence of creatinine in both species. The synthesis of creatinine deiminase was repressed by ammonia in C. neoformans, but not in C. bacillisporus. A possible explanation for this variation, based on the ecological differences between the two species, is discussed. A novel method for measuring creatinine deiminase activity is also described.