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PubMed2005

Melanization of Cryptococcus neoformans affects lung inflammatory responses during cryptococcal infection.

The production of melanin pigments is associated with virulence for many microbes. Melanin is believed to contribute to microbial virulence by protecting microbial cells from oxidative attack during infection. However, there is also evidence from various systems that melanins have immunomodulatory properties, which conceivably could contribute to virulence by altering immune responses. To investigate the effect of melanin on the immune response, we compared the murine pulmonary responses to infection with melanized and nonmelanized Cryptococcus neoformans cells. Infection with melanized cells resulted in a greater fungal burden during the early stages of infection and was associated with higher levels of interleukin-4 and MCP-1 and greater numbers of infiltrating leukocytes. Infection with laccase-positive (melanotic) C. neoformans cells also elicited higher MCP-1 levels and more infiltrating leukocytes than did infection with laccase-negative cells. Melanization interfered with phagocytosis in vivo for encapsulated C. neoformans but not for nonencapsulated cells. The results provide strong evidence that cryptococcal melanization can influence the immune response to infection and suggest that immunomodulation is an additional mechanism by which the pigment contributes to virulence.

Animals↗
PubMed1996

Microdilution antifungal susceptibility testing of Candida albicans and Cryptococcus neoformans with and without agitation: an eight-center collaborative study.

The growth patterns observed in the trailing wells when fluconazole is being tested may give rise to readings that suggest resistance or increased MICs for known susceptible strains. We conducted a multicenter study to evaluate the intralaboratory and interlaboratory reproducibilities of a method that uses agitation to disperse these types of growth. Ten strains of Candida albicans and five strains of Cryptococcus neoformans were tested against fluconazole, flucytosine, and amphotericin B by using a microdilution adaptation of the proposed reference method of the National Committee for Clinical Laboratory Standards for yeasts (M27-T). The endpoint criterion used before agitation was consistent with the M27-T recommendation, while a criterion of 50% or more reduction of growth compared with the control was used after agitation. The results of this study showed that use of agitation and the modified endpoint criterion both improved intralaboratory and inter-laboratory agreement and increased the frequency of interpretable MICs. The MICs obtained by this method were comparable to those obtained by the broth macrodilution M27-T method. Like M27-T, this method was not able to definitely distinguish amphotericin B-susceptible from -resistant strains, although the MICs for the resistant strains were consistently higher than those for the susceptible ones. The findings imply that agitation should be seriously considered when antifungal agents, particularly fluconazole, are tested in a microdilution format.

Amphotericin B↗
PubMed1988

Enhanced binding of capsular polysaccharides of Cryptococcus neoformans to polystyrene microtitration plates for enzyme-linked immunosorbent assay.

A sensitive enzyme-linked immunosorbent assay (ELISA) to measure antibodies against capsular polysaccharide was developed, based on the enhanced binding of polysaccharide to polystyrene microtitration plates. The wells of the microtitration plate were primed with an adipic acid dihydrazide derivative of bovine serum albumin (AH-BSA) (100 micrograms/mL, 0.01 M NaPO4-0.14 M NaCl, pH 7.2 (PBS]. Capsular polysaccharide, the glucuronoxylomannan of Cryptococcus neoformans serotype A, was oxidized with NaIO4 for 5 min; the reaction was then quenched with ethylene glycol. The partially oxidized polysaccharide was dialyzed vs. PBS, and its concentration was adjusted to 50 micrograms/mL with PBS. This solution (100 microL/well) was covalently bound to the AH-BSA primed microtitration plates through formation of a Schiff base between the hydrazide group on the AH-BSA and the aldehyde groups on the polysaccharide. Antimouse IgG-alkaline phosphatase conjugate was used in an indirect ELISA to measure captured murine monoclonal antibodies directed against glucuronoxylomannan. Mean absorbances, after 15 min, were 0.13 in negative control wells, and greater than 0.7 in test wells. No intermediate steps were required to block nonspecific binding of antibody.

Antibodies↗
PubMed1995

In vitro determination of optimal antifungal combinations against Cryptococcus neoformans and Candida albicans.

There is currently no rapid, reliable, and reproducible in vitro technique to describe the growth-inhibitory interactions of antifungal drug combinations over a wide range of drug concentrations. We have developed a microdilution plate assay that was used to determine optimal drug combinations and concentrations of one-, two-, and three-drug regimens of amphotericin B (AmphB), fluconazole (FLU), and 5-fluorocytosine (5FC) for growth inhibition of three isolates each of Cryptococcus neoformans and Candida albicans. These growth inhibition data were then used in a multifactorial design technique to (i) generate contour and surface response plots to aid visual interpretation and (ii) develop mathematical equations describing the growth responses of the fungi to a wide range of antifungal concentrations and ratios. Our data indicated that (i) antifungal drug-drug interactions affecting yeast growth are complex functions of the drugs used in combination, their absolute concentrations, and also their relative (proportional) concentrations; (ii) AmphB-FLU combinations had additive effects against C. albicans over wide concentration ranges for each agent but were indifferent (i.e., were less than additive) in their inhibitory effect on C. neoformans; (iii) other two-drug combinations (FLU-5FC or AmphB-5FC) had indifferent effects on the growth of both fungi; and (iv) three-drug combinations (AmphB-FLU-5FC) showed an additive inhibitory effect on the growth of both C. albicans and C. neoformans. The finding that no antagonism was observed in combinations employing AmphB and FLU in this in vitro model is of critical importance since it argues against the current theoretical concept, based on the individual drug's mode of action, of antagonism between these two drugs. These microdilution techniques provide a method to determine rational regimens of antifungal agents in multidrug combinations for future testing to correlate in vitro activity with in vivo response. The use of this approach has made the evaluation of complex antifungal drug-drug interactions possible and provided important new information to the evolving field of antifungal drug combination.

Amphotericin B↗
PubMed1998

Cryptococcus neoformans differently regulates B7-1 (CD80) and B7-2 (CD86) expression on human monocytes.

To induce a specific response in primary resting T cells, two signals must be provided by antigen-presenting cells (APC). The first antigen-specific signal is mediated by formation of the T cell receptor major histocompatibility complex molecule ternary complexes. The second signal is delivered by interaction of either B7-1 or B7-2 expressed by APC with CD28 or CTLA-4 on T cells. In this study, we examined the modulation of B7-1 and B7-2 molecules on human monocytes exposed to encapsulated or acapsular Cryptococcus neoformans or Candida albicans. In our experimental system, C. albicans or acapsular C. neoformans are able to induce B7-1 expression while the encapsulated yeast is a poor stimulator. A modest increase of B7-2 expression was also observed after monocyte treatment with acapsular C. neoformans or C. albicans, while the encapsulated yeast was ineffective in inducing B7-2 molecules. Kinetic analysis showed the maximum expression of B7-1 after 24 to 48 h. Addition of the opsonic IgG1 mAb 2H1 to monocytes and C. neoformans significantly increased B7-1, but not B7-2, expression. The contribution of B7-1 and B7-2 co-stimulatory (CS) molecules to cryptococcal-specific T cell activation was analyzed and a substantial inhibition of T cell proliferation was observed. In this study we provide the first demonstration of fungal interference in the regulation of CS molecules. Our results suggest a potential mechanism for poor inflammatory responses observed in C. neoformans infections.

Antigen Presentation↗
PubMed1993

Modulation of I-A and I-E expression in macrophages by T-suppressor cells induced in Cryptococcus neoformans infected rats.

When the I-A and I-E expressions were assessed in peritoneal macrophages from Cryptococcus neoformans infected animals, a significant decrease in the former was observed when compared with normal macrophages (p < 0.001) whereas a significant increase in the I-E expression was observed when compared with controls (p < 0.005). On the other hand, when studying the in vitro action of Ts cells on the macrophages, it was observed that the I-A expression was significantly reduced in macrophages upon contact with Ts cells. Similar results were obtained when Ts cells were replaced by a soluble factor. In contrast, the I-E expression was significantly increased by in vitro action of the Ts cell or its soluble factor. Indomethacin partially restored I-A and I-E expression in the macrophages to control levels.

Animals↗
PubMed1996

Contribution of interferon-gamma in protecting mice during pulmonary and disseminated infection with Cryptococcus neoformans.

In the present study, the role of interferon-gamma (IFN-gamma) in the host resistance against Cryptococcus neoformans was examined using a murine model of pulmonary and disseminated infection. In this model, mice were infected intratracheally with live yeast cells, and the histological changes in the lungs and the number of microorganisms in the lung and brain were compared in mice treated and untreated with anti-IFN-gamma monoclonal antibody (mAb) to define the contribution of endogenously synthesized IFN-gamma in the natural course of infection. Administration of this mAb reduced the accumulation of inflammatory cells in the alveolar septa, peribronchial and perivascular areas, and promoted the expansive growth of microorganisms in the alveoli and destruction of alveolar structure. The neutralization of endogenous IFN-gamma by mAb increased the number of microorganisms in the lung and brain, and significantly shortened the survival time of infected mice. On the other hand, administration of IFN-gamma decreased the number of microorganisms in these organs, and significantly extended their survival time. Considered together, our results suggest that endogenous IFN-gamma protects mice from infection with C. neoformans by inducing a cellular inflammatory response, potentiating the clearance of microorganism from the lungs and preventing its dissemination into the central nervous system.

Animals↗
PubMed1983

Experimental hematogenous endophthalmitis due to Cryptococcus neoformans.

New Zealand white female rabbits injected with 6 X 10(7) to 5.5 X 10(8) CFU of Cryptococcus neoformans into the right carotid artery developed three types of clinically detectable eye lesions. These eye manifestations included: (1) iritis that had distinctive 1-3 mm white nodules; (2) white vitreous lesions with ill-defined borders, and (3) focal and diffusely infiltrative lesions of the optic nerve radiations. Most of the eye lesions appeared between 1 to 3 weeks after intracarotid injection and resolved almost completely by 10 weeks. Their clinical appearance resembled some of the eye lesions described from human cases. Predominantly a mononuclear leukocyte response with scattered cryptococcal organisms were seen in the iris and optic nerve lesions. Serial quantitative cultures from eye tissues, brain, and kidney indicated that the clearance of viable cryptococci was slowest from the eye tissues. Among the three diagnostic techniques (India Ink mount, culture, and cryptococcal polysaccharide antigen (CPA) determination), the CPA determination was the most sensitive test to detect C. neoformans in the aqueous and vitreous humors of clinically involved eyes. This rabbit model may be useful for further studies on hematogenous cryptococcal endophthalmitis.

Animals↗
PubMed1994

Prostatic sequestration of Cryptococcus neoformans in immunocompromised persons treated for cryptococcal meningoencephalitis.

We report a case of a patient with acquired immune deficiency syndrome who was successfully treated for cryptococcal meningoencephalitis with amphotericin B and 5-flucytosine. He died from other sequelae of acquired immune deficiency syndrome two years later. An autopsy revealed prominent cryptococcal prostatitis. Cryptococci were neither found in the central nervous system nor in other anatomic sites. The autopsy files yielded seven other cases of men with a history of cryptococcal meningoencephalitis. The possibility that the prostate sequesters Cryptococcus neoformans thereby contributing to systemic relapse is explored. The qualify as a sequestration, cyptococci must be cultured from the prostate, or from a midstream voided specimen after prostatic massage, and the prostate must be the only focus of infection.

AIDS-Related Opportunistic Infections↗
PubMed1997

Eosinophil-Cryptococcus neoformans interactions in vivo and in vitro.

Eosinophils are components of inflammatory responses to a variety of pathogens. Although a variety of beneficial and harmful functions have been ascribed to these cells, their role in protection against infectious agents remains uncertain. Previous studies have reported eosinophilic pneumonia in mice infected intratracheally with Cryptococcus neoformans. We confirmed this observation and studied the inflammatory response in the lung at day 14 by light and electron microscopy. Immunostaining for glucuronoxylomannan showed isolated cryptococci inside the eosinophilic cuffs. Eosinophils were found to be in close association with C. neoformans in vivo. Cryptococci were associated with eosinophils within eosinophilic perivascular cuffs, within granulomas, and lining the alveolar space. To further investigate this phenomenon in vitro, we isolated rat peritoneal eosinophils and studied cryptococcus-eosinophil interactions in the presence and absence of anti-capsular immunoglobulin G1 (IgG1) and IgE monoclonal antibody (MAb). Eosinophils phagocytosed C. neoformans only in the presence of specific antibody. Phagocytosis was rapid, and dense rings that appeared to consist of granule contents were formed around the organisms. Mast cells were observed to occasionally phagocytose C. neoformans in vitro in the presence of IgE MAb. Our observations suggest that eosinophils may be effector cells against C. neoformans.

Animals↗
PubMed2000

Differing requirement for inducible nitric oxide synthase activity in clearance of primary and secondary Cryptococcus neoformans infection.

The role of nitric oxide in resistance to cryptococcal infection was investigated. Mice deficient in inducible nitric oxide synthase (INOS) did not survive a primary intratracheal infection as did INOS-replete control mice. Despite adequate recruitment of host cells and generation of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha at the site of infection, INOS-deficient mice failed to clear yeast from their lungs by five weeks of infection, in contrast to wild-type mice. INOS-deficient mice also had higher yeast brain burdens than did control mice after a primary intracerebral infection. Therefore, generation of nitric oxide is required for resistance to primary cryptococcal infection. However, INOS-deficient mice vaccinated subcutaneously and rechallenged intravenously had lung and brain yeast burdens equivalent to those of vaccinated controls, and therefore expressed effective acquired immunity to Cryptococcus neoformans. Cells harvested from infected INOS-deficient mice by bronchoalveolar lavage acted as anti-cryptococcal effectors in vitro at an effector:target ratio of 100:1, provided IFN-gamma was present, but did not inhibit yeast proliferation at a 10:1 effector:target ratio as cells from wild-type mice did. Therefore, INOS activity is important for anti-cryptococcal function of effectors of immunity during the primary response, but not for the generation or expression of secondary immunity to C. neoformans.

Animals↗
PubMed1976

Amphotericin B and amphotericin B methyl ester ascorbate. I. Chemotherapeutic activity against Candida albicans, Cryptococcus neoformans, and Blastomyces dermatitidis in mice.

Amphotericin B methyl ester (AME) has been reported to possess in vitro antifungal activity similar to that of amphotericin B and to have less intrinsic toxicity in mice and dogs. For these reasons AME has been porposed as an alternative to amphotericin B in the therapy of deep mycoses. For comparison of the therapeutic efficacy of the two polyenes in laboratory animals before initiation of studies in humans, groups of mice were infected with Candida albicans, Cryptococcus neoformans, and Blastomyces dermatitidis. Treatment consisted of two or more doses of each drug given by the intravenous route. Concurrently, studies of subacute toxicity were conducted in the same species to permit calculation of therapeutic indices. These studies have shown that AME, as the ascorbate salt, is substantially less efficacious than amphotericin B (in colloidal dispersion with sodium deoxycholate) for treatment of the fungal infections and that amphotericin B had a higher therapeutic ratio for all infections studied than did AME.

Amphotericin B↗
PubMed2006

Cryptococcal parotid involvement: an uncommon localization of Cryptococcus neoformans.

We describe a cryptococcal infection localized in the parotid gland of an otherwise healthy 72-year-old woman. The patient presented with a painful, approximately 4.5 cm diameter mass in the anterior region of her right ear. Her symptoms were mild and uncharacteristic. The patient had previously fallen on her face in her garden, causing the loss and breakage of her dentures. Since the soil of the garden contained chicken droppings, it is quite likely that the oral prothesis became contaminated on contacting the soil. The fungus probably entered the parotid gland through the traumatization of the posterior lateral wall of her oral cavity by her broken denture. Numerous intra- and extracellular cryptococcal yeast cells were observed in both histopathological and mycological slide preparations. The yeastlike fungus was recovered in cultures inoculated with tissue collected through three biopsies of her parotid region. The isolates were identified as Cryptococcus neoformans by classical mycology methods and found to be susceptible, in vitro, to fluconazole, amphotericin B and flucytosine. Fluconazole treatment (400 mg/d, for 6 months) was started and the patients facial swelling resolved and the pain significantly reduced within 5 weeks of the initiation of treatment. While fungal infection of the parotid gland have been reported, to our knowledge, this is the first description of a non-disseminated primary parotid infection due to C. neoformans.

Aged↗
PubMed2006

Gib2, a novel Gbeta-like/RACK1 homolog, functions as a Gbeta subunit in cAMP signaling and is essential in Cryptococcus neoformans.

Canonical G proteins are heterotrimeric, consisting of alpha, beta, and gamma subunits. Despite multiple Galpha subunits functioning in fungi, only a single Gbeta subunit per species has been identified, suggesting that non-conventional G protein signaling exists in this diverse group of eukaryotic organisms. Using the Galpha subunit Gpa1 that functions in cAMP signaling as bait in a two-hybrid screen, we have identified a novel Gbeta-like/RACK1 protein homolog, Gib2, from the human pathogenic fungus Cryptococcus neoformans. Gib2 contains a seven WD-40 repeat motif and is predicted to form a seven-bladed beta propeller structure characteristic of beta transducins. Gib2 is also shown to interact, respectively, with two Ggamma subunit homologs, Gpg1 and Gpg2, similar to the conventional Gbeta subunit Gpb1. In contrast to Gpb1 whose overexpression promotes mating response, overproduction of Gib2 suppresses defects of gpa1 mutation in both melanization and capsule formation, the phenotypes regulated by cAMP signaling and associated with virulence. Furthermore, depletion of Gib2 by antisense suppression results in a severe growth defect, suggesting that Gib2 is essential. Finally, Gib2 is shown to also physically interact with a downstream target of Gpa1-cAMP signaling, Smg1, and the protein kinase C homolog Pkc1, indicating that Gib2 is also a multifunctional RACK1-like protein.

Amino Acid Sequence↗
PubMed2005

Carbonic anhydrase and CO2 sensing during Cryptococcus neoformans growth, differentiation, and virulence.

The gas carbon dioxide (CO2) plays a critical role in microbial and mammalian respiration, photosynthesis in algae and plants, chemoreception in insects, and even global warming . However, how CO2 is transported, sensed, and metabolized by microorganisms is largely not understood. For instance, CO2 is known to induce production of polysaccharide capsule virulence determinants in pathogenic bacteria and fungi via unknown mechanisms . Therefore, we studied CO2 actions in growth, differentiation, and virulence of the basidiomycetous human fungal pathogen Cryptococcus neoformans. The CAN2 gene encoding beta-carbonic anhydrase in C. neoformans was found to be essential for growth in environmental ambient conditions but dispensable for in vivo proliferation and virulence at the high CO2 levels in the host. The can2Delta mutant in vitro growth defect is largely attributable to defective fatty acid synthesis. CO2 was found to inhibit cell-cell fusion but not filamentation during sexual reproduction. The can2 mutation restored early mating events in high CO2 but not later steps (fruiting body formation, sporulation), indicating a major role for carbonic anhydrase and CO2/HCO3- in this developmental cascade leading to the production of infectious spores. Our studies illustrate diverse roles of an ancient enzyme class in enabling environmental survival of a ubiquitous human pathogen.

Base Sequence↗
PubMed2003

Superoxide dismutase influences the virulence of Cryptococcus neoformans by affecting growth within macrophages.

Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and molecular oxygen and has been shown to contribute to the virulence of many human-pathogenic bacteria through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. To examine the contribution of SOD to the pathogenesis of fungal infections, we cloned the Cu,Zn SOD-encoding gene (SOD1) from the human-pathogenic yeast Cryptococcus neoformans and made mutants via targeted disruption. The sod1 mutant strains had marked decreases in SOD activity and were strikingly more susceptible to reactive oxygen species in vitro. A sod1 mutant was significantly less virulent than the wild-type strain and two independent reconstituted strains, as measured by cumulative survival in the mouse inhalational model. In vitro studies established that the sod1 strain had attenuated growth compared to the growth of the wild type and a reconstituted strain inside macrophages producing reduced amounts of nitric oxide. These findings demonstrate that (i) the Cu,Zn SOD contributes to virulence but is not required for pathogenicity in C. neoformans; (ii) the decreased virulence of the sod1 strain may be due to increased susceptibility to oxygen radicals within macrophages; and (iii) other antioxidant defense systems in C. neoformans can compensate for the loss of the Cu,Zn SOD in vivo.

Animals↗
PubMed2000

A pre-existing infection by Mycobacterium avium subsp. avium modulates anti-Cryptococcus neoformans and anti-Candida albicans activities in human macrophages.

Mycobacterium avium is a facultative intracellular microorganism, able to survive and multiply within mammalian macrophages by circumventing antimicrobial mechanisms. In this study we hypothesize that pre-existing M. avium infection could result in macrophage superinfections by other microorganisms. We found that 24 h after ingestion of M. avium at a low multiplicity of infection, macrophages are unable to efficiently produce superoxide anions when over-stimulated with phorbol esters, and that the generation of oxidative burst is only partially restored 72 h after bacteria ingestion. We also demonstrate that intracellular killing of Cryptococcus neoformans is markedly impaired in human macrophages that have previously ingested M. avium (but not other bacteria such as Escherichia coli). This inhibitory effect is observed with live mycobacteria, but not when heat-inactivated bacteria are ingested. In contrast, when Candida albicans is given to macrophages instead of C. neoformans, an enhancement of intracellular killing is observed, suggesting that cytocidal mechanisms other than respiratory burst are involved in the anti- Candidacidal activity of macrophages.

Candida albicans↗
PubMed2006

Molecular analysis of 311 Cryptococcus neoformans isolates from a 30-month ECMM survey of cryptococcosis in Europe.

During a European Confederation of Medical Mycology (ECMM) prospective survey of cryptococcosis in Europe (from July 1997 to December 1999) 655 cases were reported from 17 countries; 565 of the completed questionnaires were evaluable. Cryptococcosis was associated with HIV infection in 77% of cases (range 57.5-94%). Assessment of the laboratory data highlighted the lack of defined standard procedures for the diagnosis of cryptococcosis: the antigen test was not usually used for screening, the disease was mainly recognised when meningitis occurred (65% of patients) and, with the exception of a few cases, the extent of the infection was not investigated. Cryptococcus neoformans was the etiological agent in all of the cases except for six caused by C. gattii and four by other Cryptococcus species. A total of 311 C. neoformans strains were serotyped by Crypto Check latex agglutination, genotyped by PCR-fingerprinting using the (GACA)4 oligonucleotide as a single primer, and their mating type was determined by PCR of the STE20 alleles. Serotype A was the most represented (51% of the isolates), followed by serotype D (30%) and serotype AD (19%). PCR-fingerprinting analysis significantly increased the percentage of hybrid strains to 30%, as 6% of the serotype A and 28% of the serotype D isolates were of the VN3 or VN4 hybrid genotype. In addition, the mating type determinations revealed the MATa serotype A allele in one haploid strain and 28 hybrids, and hybrid isolates with a single mating type (four Aalpha and two Dalpha) were also identified. This is the first prospective survey to be carried out in Europe which has attempted to investigate the epidemiology of cryptococcosis and the population structure of C. neoformans, and the results obtained thus far show the widespread involvement of AD hybrid strains in C. neoformans infections.

Adolescent↗