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Protective murine monoclonal antibodies to Cryptococcus neoformans.

Several murine monoclonal antibodies (MAbs) specific for the capsular glucuronoxylomannan of Cryptococcus neoformans were studied for their capacity to confer protection when passively administered to lethally infected mice. The MAb group studied recognized at least three distinct epitopes and included immunoglobulin M (IgM), IgG3, IgG1, and IgA isotypes. The protection model used A/J and BALB/c mice infected intraperitoneally with 10(8) cryptococci. The MAbs were administered either immediately preceding or, in one experiment, 24 to 48 h prior to infection. Protective efficacy was assessed by the ability of passively administered MAbs to prolong the survival of lethally infected mice. Three IgM MAbs, each of which recognized a distinct epitope, were able to prolong survival of lethally infected mice to different extents. A set of IgM, IgG3, IgG1 and IgA MAbs which utilize the same immunoglobulin gene elements and were derived from the same B-cell clone exhibited significant class differences in protective efficacy with IgA, IgG1 > IgM > IgG3. The results confirm that protective MAbs against C. neoformans capsular polysaccharide exist and strongly suggest that both epitope specificity and isotype are important determinants of protective efficacy.

Animals↗

Structure and biological activities of acapsular Cryptococcus neoformans 602 complemented with the CAP64 gene.

The extracellular polysaccharide capsule of Cryptococcus neoformans is a well-recognized virulence factor. Strain 602 is an acapsular clinical isolate of unknown serotype which has been widely used in studies of virulence and host-parasite interactions. In previous studies, strain 602 was compared with genetically unrelated strains of various serotypes because the wild-type equivalent of strain 602 was not available. We created an encapsulated strain, TYCC38-602, by transforming strain 602 with the CAP64 gene which was isolated from a serotype D strain. Serological tests and chemical analysis of the major polysaccharide capsule of TYCC38-602 indicated that strain 602 was originally derived from a serotype A strain. Restoration of the ability to produce a capsule enabled strain 602 to cause fatal infection in mice, whereas the acapsular strain 602 remained avirulent. Capsule-restored yeast cells of strain 602 activated the human complement system and bound C3 fragments in a manner that is characteristic of encapsulated cryptococci. In addition, the capsule in TYCC38-602 masked the ability of the organism to induce tumor necrosis factor alpha and subsequent nitric oxide synthase production in primed macrophage-like cells. These results indicate that the lack of capsule in strain 602 is the reason for its inability to cause fatal infection. Moreover, the acapsular phenotype accounts for differences in various biological activities of strain 602 compared to encapsulated strains. The results also indicate that the gene product of CAP64 does not contribute to serotype specificity of capsules in C. neoformans.

Animals↗

Serotype B/C Cryptococcus neoformans isolated from patients in nonendemic areas.

Of 90 clinical isolates of Cryptococcus neoformans studied, 3 were determined to be serotype B/C. The patients from whom these B/C isolates were obtained were identified as never having lived in or visited the areas associated with B/C serotypes. This finding suggests a broader geographic distribution of this serotype group than previously believed. The glycine-cycloheximide-phenol red medium described by Salkin and Hurd (J. Clin. Microbiol. 15:169-171, 1982) was shown to be more accurate in differentiating A/D and B/D serotype pairs of C. neoformans than the creatinine-dextrose-bromthymol blue medium described by Kwon-Chung et al. (Int. J. Syst. Bacteriol. 28:616-620, 1978).

Cryptococcosis↗

Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA.

A transformation scheme for Cryptococcus neoformans to yield high-frequency, integrative events was developed. Adenine auxotrophs from a clinical isolate of C. neoformans serotype A were complemented by the cryptococcal phosphoribosylaminoimidazole carboxylase gene (ade2) with a biolistic DNA delivery system. Comparison of two DNA delivery systems (electroporation versus a biolistic system) showed notable differences. The biolistic system did not require linear vectors and transformed each auxotrophic strain at similar frequencies. Examination of randomly selected transformants by biolistics showed that 15 to 40% were stable, depending on the recipient auxotroph, with integrative events identified in all stable transformants by DNA analysis. Although the ade2 cDNA copy transformed at a low frequency, DNA analysis found homologous recombination in each of these transformants. DNA analysis of stable transformants receiving genomic ade2 revealed ectopic integration in a majority of cases, but approximately a quarter of the transformants showed homologous recombination with vector integration or gene replacement. This system has the potential for targeted gene disruption, and its efficiency will also allow for screening of DNA libraries within C. neoformans. Further molecular strategies to study the pathobiology of this pathogenic yeast are now possible with this transformation system.

Blotting, Southern↗

Identification of extracellular phospholipase B, lysophospholipase, and acyltransferase produced by Cryptococcus neoformans.

We recently identified phospholipase activity as a potential virulence factor of Cryptococcus neoformans. We have now defined the nature of the phospholipase activity produced by a clinical isolate of C. neoformans var. neoformans, under native conditions, by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy and thin-layer chromatography (TLC) of radiolabelled substrates. Glycerophosphocholine was identified by NMR spectroscopy as the sole phospholipid degradation product of the reaction between substrate phosphatidylcholine (PC) and cryptococcal culture supernatants indicating the presence of phospholipase B (PLB). No lysophosphatidylcholine (lyso-PC) or products indicative of phospholipase C, phospholipase D, or other lipase activity were identified. Use of PC and lyso-PC containing radiolabelled acyl chains and separation of products by TLC confirmed the PLB and lysophospholipase (LPL) activities. Lysophospholipase transacylase (LPTA) activity was identified by the formation of radioactive PC from lyso-PC. Extracellular enzyme production was maximal after 6 to 10 h in fresh medium. Assay conditions were optimized for pH, linearity with time, enzyme concentration, and saturation by substrates to allow comparison with phospholipases from other organisms. LPL activity was 10- to 20-fold greater than PLB activity, with mean (+/- standard deviation) specific activities of 34.9 +/- 7.9 and 3.18 +/- 0.2 micromol of substrate hydrolyzed per min per mg of protein, respectively. The response of PLB to increasing substrate concentrations was bimodal, whereas inhibition of LPL and LPTA activities occurred at concentrations of substrate lyso-PC greater than 200 microM. Enzyme activities were stable at acid pH (3.8), with pH optima of 3.5 to 4.5. Activities were unchanged in the presence of exogenous serine protease inhibitors, divalent cations, and EDTA. We conclude that C. neoformans produces highly active extracellular PLB, LPL, and LPTA under native conditions.

Acyltransferases↗

Enhancement of HIV-1 infection by the capsular polysaccharide of Cryptococcus neoformans.

Patients with AIDS who become infected with Cryptococcus neoformans have a poor prognosis. We speculated that the presence of cryptococcal capsular polysaccharide may enhance HIV-1 infection. In an in-vitro study, the presence of cryptococcal polysaccharide significantly increased (p less than 0.05) production of p24 antigen after infection of H9 cells with HIV-1-infected H9 cells. We also found similar results when lymphocytes from an HIV-1-infected patient were co-cultured with mononuclear cells from an uninfected individual. Our findings suggest a new pathogenic role for the capsular polysaccharide--namely, the capacity to enhance HIV-1 infectivity.

Acquired Immunodeficiency Syndrome↗

Cch1 mediates calcium entry in Cryptococcus neoformans and is essential in low-calcium environments.

The ability of Cryptococcus neoformans to grow at the mammalian body temperature (37 degrees C to 39 degrees C) is a well-established virulence factor. Growth of C. neoformans at this physiological temperature requires calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase. When cytosolic calcium concentrations are low ( approximately 50 to 100 nM), calcineurin is inactive and becomes active only when cytosolic calcium concentrations rise ( approximately 1 to 10 microM) through the activation of calcium channels. In this study we analyzed the function of Cch1 in C. neoformans and found that Cch1 is a Ca(2+)-permeable channel that mediates calcium entry in C. neoformans. Analysis of the Cch1 protein sequence revealed differences in the voltage sensor (S4 regions), suggesting that Cch1 may have diminished voltage sensitivity or possibly an alternative gating mechanism. The inability of the cch1 mutant to grow under conditions of limited extracellular calcium concentrations ([Ca(2+)](extracellular), approximately 100 nM) suggested that Cch1 was required for calcium uptake in low-calcium environments. These results are consistent with the role of ScCch1 in mediating high-affinity calcium uptake in Saccharomyces cerevisiae. Although the growth defect of the cch1 mutant under conditions of limited [Ca(2+)](extracellular) ( approximately 100 nM) became more severe with increasing temperature (25 degrees C to 38.5 degrees ), this temperature sensitivity was not observed when the cch1 mutant was grown on rich medium ([Ca(2+)](extracellular), approximately 0.140 mM). Accordingly, the cch1 mutant strain displayed only attenuated virulence when tested in the mouse inhalation model of cryptococcosis, further suggesting that C. neoformans may have a limited requirement for Cch1 and that this requirement appears to include ion stress tolerance.

Amino Acid Sequence↗

Acquired resistance to Cryptococcus neoformans in adult mice vaccinated as newborns.

Although Cryptococcus neoformans causes serious infections in AIDS patients, cryptococcosis in immunologically immature infants, as in immunocompetent adults, is rare. To investigate the resistance of neonates to C. neoformans and to determine whether they could be efficiently vaccinated as neonates against challenge with the yeast as adults, the course of infection was monitored in the lungs of mice infected intranasally with yeast cells. Neonates were less able than adults to reduce yeast burdens less than 24 h postinoculation and less able to control the progressive growth of yeast over several weeks. However, in both neonates and adults, yeasts were substantially eradicated by 6 to 8 weeks after infection. Numbers of all classes of leukocytes recovered from lung lavages of infected neonates and adults were similar. Significant differences appeared only on day 14, when neonates had more neutrophils and adults had significantly more CD4+ CD45RB cells with low fluorescence intensity. When vaccinated neonates were rechallenged after reaching adulthood, they expressed resistance to C. neoformans as effectively as did mice immunized as adults and survived an intravenous challenge that was lethal for unimmunized controls. Thus, exposure of neonatal mice to viable C. neoformans yeast, which persists in the lungs for many weeks, does not result in immunological tolerance to a yeast challenge in adult mice, as predicted by immunological dogma, but instead immunizes them. Therefore, even in immunologically immature individuals, the immune system serves to protect against pathogens rather than simply to distinguish self from nonself.

Administration, Intranasal↗

Cryptococcus neoformans pulmonary infection in HIV-1-infected patients.

Cryptococcus neoformans (Cn) is a frequent pathogen in patients infected with the human immunodeficiency virus (HIV-1). We review the initial presentation and clinical course of 18 HIV-1-infected (HIV+) patients with a Cn pulmonary infection. Simultaneous positive cerebrospinal fluid (CSF) cultures were found in 10 (63%) of 16 examined. The most frequent presenting symptoms were fever (87%) and pulmonary complaints (60%). Although the most common chest radiographic finding was bilateral diffuse interstitial infiltrates, nodules and cavitary lesions were also seen. Nine (50%) of the 18 patients died within 6 weeks of diagnosis. Of six patients with an isolated Cn pulmonary infection, five have subsequently died. Three of these five patients did not receive maintenance therapy and had confirmed or probable relapse. Patients initially presenting with an isolated Cn pulmonary infection may later show disseminated disease, suggesting that such patients should receive both acute and maintenance therapy.

Adult↗

PAK kinases Ste20 and Pak1 govern cell polarity at different stages of mating in Cryptococcus neoformans.

Sexual identity and mating are linked to virulence of the fungal pathogen Cryptococcus neoformans. Cells of the alpha mating type are more prevalent and can be more virulent than a cells, and basidiospores are thought to be the infectious propagule. Mating in C. neoformans involves cell-cell fusion and the generation of dikaryotic hyphae, processes that involve substantial changes in cell polarity. Two p21-activated kinase (PAK) kinases, Pak1 and Ste20, are required for both mating and virulence in C. neoformans. We show here that Ste20 and Pak1 play crucial roles in polarized morphogenesis at different steps during mating: Pak1 functions during cell fusion, whereas Ste20 fulfills a distinct morphogenic role and is required to maintain polarity in the heterokaryotic mating filament. In conclusion, our studies demonstrate that PAK kinases are necessary for polar growth during mating and that polarity establishment is necessary for mating and may contribute to virulence of C. neoformans.

Cell Fusion↗

Immunohistochemical diagnosis of Cryptococcus neoformans var. gattii infection in chronic meningoencephalitis: the first case in Japan.

Cryptococcus neoformans (C. neoformans) var. gattii infection usually occurs in tropical and subtropical areas, and rarely in the northern hemisphere. We report the first Japanese with cryptococcal meningoencephalitis caused by C. neoformans var. gattii infection that occurred during a trip to Australia. This agent was identified in a cerebellar biopsy specimen by immunohistochemical technique with serotype-specific anti-sera. Because the meningitis caused by it did not respond well to conventional therapy, we used an aggressive therapeutic regimen to successfully treat the patient. Even in areas where C. neoformans var. gattii does not exist, this infection should be considered possible as a travel-related infection.

Aged↗

Variability of karyotypes and RAPD types in genetically related strains of Cryptococcus neoformans.

Variation in karyotypes and RAPD patterns of genetically related strains of Cryptococcus neoformans were analyzed. Capsular and filamentous mutants usually differ in their karyotypes from wild-types, but the RAPD patterns were found to be similar. Karyotype differences were observed in most heterothallic matings, but RAPD patterns remained identical. After self-sporulation of a diploid strain, minor chromosomal length polymorphism and minor changes in the RAPD types occurred. Three mechanisms, either alone or in combination, may in varying degrees contribute to the karyotype variation of C. neoformans: (1) mitotically induced changes; (2) karyotype changes as a result of meiotic recombination, and (3) mutagen-induced changes. The present data do not support the meiotic maintenance hypothesis, which claims that the amount of CLP generated is inversely proportional to the frequency of meiosis.

Cryptococcus neoformans↗

Dual infections with pigmented and albino strains of Cryptococcus neoformans in patients with or without human immunodeficiency virus infection in India.

Cryptococcus neoformans is an encapsulated yeast-like fungus of worldwide distribution. Melanin production is an important virulence factor of C. neoformans. We report the identification of distinct cryptococcal isolates with either pigmented or white colony phenotypes on L-dihydroxyphenylalanine agar plates in three patients who presented with meningitis to the All India Institute of Medical Sciences in India. Two of the patients were also infected with human immunodeficiency virus. Biochemical studies, India ink analysis, immunofluorescence with antibodies specific to capsular antigen, and serotyping confirmed that the melanotic and albino strains were C. neoformans serotypes A and D, respectively. Genotyping with M13 and [GACA]4 primers revealed that all the C. neoformans isolates were genetically different. The CNLAC1 gene associated with melanin production was identified in all the strains by PCR. Standard MIC testing revealed that the strains had similar susceptibilities to amphotericin B, but time-kill assays with the antifungal showed reduced susceptibility in melanin-producing strains. Infection studies with A/Jcr mice showed that the melanin-lacking yeast were less virulent than melanin-producing isolates. These findings indicate that these patients had dual infections with pigmented and albino strains of C. neoformans that were phenotypically and biologically different. Continued surveillance of primary isolates from patients with cryptococcosis by analyzing phenotypic differences and by molecular methods may reveal that mixed infections occur more commonly than is currently realized.

AIDS-Related Opportunistic Infections↗

First report of Cryptococcus neoformans var. gattii serotype B from Greece.

A plethora of cases of Cryptococcus neoformans infections have been recorded worldwide in immunocompromised individuals. The number of such cases showed a steady rise before 1981 and increased dramatically thereafter due to the AIDS epidemic. A similar pattern has been seen in Greece but, so far, infections appear to have been caused by C. neoformans var. neoformans. This paper describes for the first time two culture-proven C. n. var. gattii serotype B infections in Greece, one in an AIDS patient and one in a patient with systemic lupus erythematosus.

Acquired Immunodeficiency Syndrome↗

Distinct STRIPAK subunits drive conserved and subunit-specific signaling programs in Cryptococcus neoformans.

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a conserved protein phosphatase 2A (PP2A)-associated signaling hub that integrates kinase-phosphatase networks, yet its roles in human fungal pathogens remain poorly defined. Here, we dissected STRIPAK functions in the opportunistic pathogen Cryptococcus neoformans by combining genetic, genomic, virulence, and phosphoproteomic analyses across mutants lacking individual STRIPAK subunits. Loss of the core STRIPAK components via PPH22, FAR8, FAR9, or FAR11 mutations caused severe defects in growth, stress adaptation, cell cycle progression, and morphogenesis, accompanied by widespread aneuploidy and genome instability. In murine infection models, far11Δ strains were avirulent, whereas far9Δ mutants caused delayed but ultimately fatal disease and underwent host-associated genome remodeling, with recovered isolates exhibiting chromosome 11 amplification despite no consistent in vitro fitness advantage. In contrast, deletion of MOB3 produced a hypervirulent phenotype. mob3Δ cells exhibited enhanced transmigration across an in vitro blood-brain barrier model, increased survival in macrophages, and generated small-cell morphotypes, features associated with increased dissemination. Phosphoproteomic profiling revealed extensive and overlapping phosphorylation changes among core STRIPAK mutants, affecting pathways involved in signaling, cytoskeletal, cell cycle control, chromatin regulation, RNA metabolism, and stress responses. Conversely, mob3Δ mutants displayed a smaller, largely distinct phosphoproteomic signature. Network and functional enrichment analyses highlighted STRIPAK-dependent regulation of TORC2-associated signaling, MAPK/GTPase signaling, autophagy, nuclear transport, RNA processing, DNA replication, and ribosome biogenesis. Together, these findings establish STRIPAK as a coordinator of genome stability, morphological plasticity, stress adaptation, and virulence in C. neoformans, and demonstrate that individual STRIPAK subunits drive shared yet divergent signaling outputs that shape host-pathogen interactions.IMPORTANCEFungal pathogens must rapidly adapt their growth, morphology, and stress responses to survive within the host, requiring precise coordination of cellular signaling pathways. The conserved striatin-interacting phosphatase and kinase (STRIPAK) complex controls key developmental programs in eukaryotes, but its roles in fungal pathogenesis are not fully defined. We previously showed that STRIPAK is important for genome stability, development, and virulence in the opportunistic human fungal pathogen Cryptococcus neoformans. Here, we define how individual STRIPAK subunits differentially regulate fungal morphogenesis, genome plasticity, host adaptation, and virulence, revealing both shared and subunit-specific functions within this conserved signaling complex. Core STRIPAK mutants exhibit severe growth and stress-response defects and attenuation of virulence, whereas loss of the Mob3 subunit promotes hypervirulence by enhancing dissemination and persistence within the host. Phosphoproteomic profiling reveals that individual STRIPAK components exert shared yet distinct control over phosphorylation networks that shape host-pathogen interactions, establishing STRIPAK as a central signaling hub and a potential target for antifungal intervention.

Cryptococcus neoformans↗

In vitro susceptibility characteristics of Cryptococcus neoformans varieties from AIDS patients in Goiânia, Brazil.

Sixty clinical isolates of Cryptococcus neoformans from AIDS from Goiânia, state of Goiás, Brazil, were characterized according to varieties, serotypes and tested for antifungal susceptibility. To differentiate the two varieties was used L-canavanine-glycine-bromothymol blue medium and to separate the serotypes was used slide agglutination test with Crypto Check Iatron. The Minimal Inhibitory Concentration (MIC) of fluconazole, itraconazole, and amphotericin B were determined by the National Committee for Clinical Laboratory Standards macrodilution method. Our results identified 56 isolates as C. neoformans var. neoformans serotype A and 4 isolates as C. neoformans var. gattii serotype B. MIC values for C. neoformans var. gattii were higher than C. neoformans var. neoformans. We verified that none isolate was resistant to itraconazole and to amphotericin B, but one C. neoformans var. neoformans and three C. neoformans var. gattii isolates were resistant to fluconazole. The presence of C. neoformans var. gattii fluconazole resistant indicates the importance of determining not only the variety of C. neoformans infecting the patients but also measuring the MIC of the isolate in order to properly orient treatment.

AIDS-Related Opportunistic Infections↗

Variable efficacy of passive antibody administration against diverse Cryptococcus neoformans strains.

The efficacy of monoclonal antibody (MAb 2H1) against diverse strains of Cryptococcus neoformans was studied in a murine model of intravenous infection. For six of eight strains, administration of MAb prior to infection prolonged survival of mice. For two strains, 371 and SB4A, administration of MAb prior to infection did not prolong survival in multiple experiments with inocula ranging from 10(2) to 10(6) yeast cells per mouse. Mice infected with strains 371 and SB4A had fewer CFU than non-MAb-treated controls, but the CFU reduction was not sufficient to affect survival. Serum glucuronoxylomannan (GXM) levels varied for the different C. neoformans strains. For mice that did not receive MAb 2H1, there was a positive correlation between lung fungal burden and serum GXM levels. MAb 2H1-treated mice had significantly reduced serum GXM levels. The results indicate that the efficacy of MAb 2H1 administration in prolonging survival and/or reducing organ CFU varies with the C. neoformans strain.

Animals↗