Urease as a virulence factor in experimental cryptococcosis.
ABSTRACT: Urease catalyzes the hydrolysis of urea to ammonia and carbamate and has been found to be an important pathogenic factor for certain bacteria. Cryptococcus neoformans is a significant human pathogenic fungus that produces large amounts of urease; thus we wanted to investigate the importance of urease in the pathogenesis of cryptococcosis. We cloned and sequenced the genomic locus containing the single-copy C. neoformans urease gene (URE1) and used this to disrupt the native URE1 in the serotype A strain H99. The ure1 mutant strains were found to have in vitro growth characteristics, phenoloxidase activity, and capsule size similar to those of the wild type. Comparison of a ure1 mutant with H99 after intracisternal inoculation into corticosteroid-treated rabbits revealed no significant differences in colony counts recovered from the cerebrospinal fluid. However, when these two strains were compared in both the murine intravenous and inhalational infection models, there were significant differences in survival. Mice infected with a ure1 strain lived longer than mice infected with H99 in both models. The ure1 strain was restored to urease positivity by complementation with URE1, and two resulting transformants were significantly more pathogenic than the ure1 strain. Our results suggest that urease activity is involved in the pathogenesis of cryptococcosis but that the importance may be species and/or infection site specific.
Project description:Cryptococcus neoformans is the most common cause of fungal meningoencephalitis in AIDS patients. Depletion of CD4 cells, such as occurs during advanced AIDS, is known to be a critical risk factor for developing cryptococcosis. However, the role of HIV-induced innate inflammation in susceptibility to cryptococcosis has not been evaluated. Thus, we sought to determine the role of Type I IFN induction in host defense against cryptococci by treatment of C. neoformans (H99) infected mice with poly-ICLC (pICLC), a dsRNA virus mimic. Unexpectedly, pICLC treatment greatly extended survival of infected mice and reduced fungal burdens in the brain. Protection from cryptococcosis by pICLC-induced Type I IFN was mediated by MDA5 rather than TLR3. PICLC treatment induced a large, rapid and sustained influx of neutrophils and Ly6Chigh monocytes into the lung while suppressing the development of eosinophilia. The pICLC-mediated protection against H99 was CD4 T cell dependent and analysis of CD4 T cell polyfunctionality showed a reduction in IL-5 producing CD4 T cells, marginal increases in Th1 cells and dramatic increases in ROR?t+ Th17 cells in pICLC treated mice. Moreover, the protective effect of pICLC against H99 was diminished in IFN? KO mice and by IL-17A neutralization with blocking mAbs. Furthermore, pICLC treatment also significantly extended survival of C. gattii infected mice with reduced fungal loads in the lungs. These data demonstrate that induction of type I IFN dramatically improves host resistance against the etiologic agents of cryptococcosis by beneficial alterations in both innate and adaptive immune responses.
Project description:Cryptococcosis is a fungal disease caused by multiple Cryptococcus serotypes; particularly C. neoformans (serotypes A and D) and C. gattii (serotypes B and C). To date, there is no clinically available vaccine to prevent cryptococcosis. Mice given an experimental pulmonary vaccination with a C. neoformans serotype A strain engineered to produce interferon-?, denoted H99?, are protected against a subsequent otherwise lethal experimental infection with C. neoformans serotype A. Thus, we determined the efficacy of immunization with C. neoformans strain H99? to elicit broad-spectrum protection in BALB/c mice against multiple disparate Cryptococcus serotypes. We observed significantly increased survival rates and significantly decreased pulmonary fungal burden in H99? immunized mice challenged with Cryptococcus serotypes A, B, or D compared to heat-killed H99? (HKH99?) immunized mice. Results indicated that prolonged protection against Cryptococcus serotypes B or D in H99? immunized mice was CD4+ T cell dependent and associated with the induction of predominantly Th1-type cytokine responses. Interestingly, immunization with H99? did not elicit greater protection against challenge with the Cryptococcus serotype C tested either due to low overall virulence of this strain or enhanced capacity of this strain to evade host immunity. Altogether, these studies provide "proof-of-concept" for the development of a cryptococcal vaccine that provides cross-protection against multiple disparate serotypes of Cryptococcus.
Project description:Degradation of purines to uric acid is generally conserved among organisms, however, the end product of uric acid degradation varies from species to species depending on the presence of active catabolic enzymes. In humans, most higher primates and birds, the urate oxidase gene is non-functional and hence uric acid is not further broken down. Uric acid in human blood plasma serves as an antioxidant and an immune enhancer; conversely, excessive amounts cause the common affliction gout. In contrast, uric acid is completely degraded to ammonia in most fungi. Currently, relatively little is known about uric acid catabolism in the fungal pathogen Cryptococcus neoformans even though this yeast is commonly isolated from uric acid-rich pigeon guano. In addition, uric acid utilization enhances the production of the cryptococcal virulence factors capsule and urease, and may potentially modulate the host immune response during infection. Based on these important observations, we employed both Agrobacterium-mediated insertional mutagenesis and bioinformatics to predict all the uric acid catabolic enzyme-encoding genes in the H99 genome. The candidate C. neoformans uric acid catabolic genes identified were named: URO1 (urate oxidase), URO2 (HIU hydrolase), URO3 (OHCU decarboxylase), DAL1 (allantoinase), DAL2,3,3 (allantoicase-ureidoglycolate hydrolase fusion protein), and URE1 (urease). All six ORFs were then deleted via homologous recombination; assaying of the deletion mutants' ability to assimilate uric acid and its pathway intermediates as the sole nitrogen source validated their enzymatic functions. While Uro1, Uro2, Uro3, Dal1 and Dal2,3,3 were demonstrated to be dispensable for virulence, the significance of using a modified animal model system of cryptococcosis for improved mimicking of human pathogenicity is discussed.
Project description:Cryptococcus neoformans var. grubii is the most frequent cause of AIDS-associated cryptococcosis worldwide, while Cryptococcus gattii usually infects immunocompetent people. To understand the mechanisms which cause differential susceptibility to these cryptococcal species in HIV infection, we established and characterized a model of cryptococcosis in CD4C/HIV(MutA) transgenic (Tg) mice expressing gene products of HIV-1 and developing an AIDS-like disease. Tg mice infected intranasally with C. neoformans var. grubii strain H99 or C23 consistently displayed reduced survival compared to non-Tg mice at three graded inocula, while shortened survival of Tg mice infected with C. gattii strain R265 or R272 was restricted to a single high inoculum. HIV-1 transgene expression selectively augmented systemic dissemination to the liver and spleen for strains H99 and C23 but not strains R265 and R272. Histopathologic examination of lungs of Tg mice revealed large numbers of widely scattered H99 cells, with a minimal inflammatory cell response, while in the non-Tg mice H99 was almost completely embedded within extensive mixed inflammatory cell infiltrates. In contrast to H99, R265 was dispersed throughout the lung parenchyma and failed to induce a strong inflammatory response in both Tg and non-Tg mice. HIV-1 transgene expression reduced pulmonary production of CCL2 and CCL5 after infection with H99 or R265, and production of these two chemokines was lower after infection with R265. These results indicate that an altered immune response in these Tg mice markedly enhances C. neoformans but not C. gattii infection. This model therefore provides a powerful new tool to further investigate the immunopathogenesis of cryptococcosis.
Project description:<h4>Unlabelled</h4>Urease in Cryptococcus neoformans plays an important role in fungal dissemination to the brain and causing meningoencephalitis. Although urea is not required for synthesis of apourease encoded by URE1, the available nitrogen source affected the expression of URE1 as well as the level of the enzyme activity. Activation of the apoenzyme requires three accessory proteins, Ure4, Ure6, and Ure7, which are homologs of the bacterial urease accessory proteins UreD, UreF, and UreG, respectively. A yeast two-hybrid assay showed positive interaction of Ure1 with the three accessory proteins encoded by URE4, URE6, and URE7. Metalloproteomic analysis of cryptococcal lysates using inductively coupled plasma mass spectrometry (ICP-MS) and a biochemical assay of urease activity showed that, as in many other organisms, urease is a metallocentric enzyme that requires nickel transported by Nic1 for its catalytic activity. The Ure7 accessory protein (bacterial UreG homolog) binds nickel likely via its conserved histidine-rich domain and appears to be responsible for the incorporation of Ni(2+) into the apourease. Although the cryptococcal genome lacks the bacterial UreE homolog, Ure7 appears to combine the functions of bacterial UreE and UreG, thus making this pathogen more similar to that seen with the plant system. Brain invasion by the ure1, ure7, and nic1 mutant strains that lack urease activity was significantly less effective in a mouse model. This indicated that an activated urease and not the Ure1 protein was responsible for enhancement of brain invasion and that the factors required for urease activation in C. neoformans resemble those of plants more than those of bacteria.<h4>Importance</h4>Cryptococcus neoformans is the major fungal agent of meningoencephalitis in humans. Although urease is an important factor for cryptococcal brain invasion, the enzyme activation system has not been studied. We show that urease is a nickel-requiring enzyme whose activity level is influenced by the type of available nitrogen source. C. neoformans contains all the bacterial urease accessory protein homologs and nickel transporters except UreE, a nickel chaperone. Cryptococcal Ure7 (a homolog of UreG) apparently functions as both the bacterial UreG and UreE in activating the Ure1 apoenzyme. The cryptococcal urease accessory proteins Ure4, Ure6, and Ure7 interacted with Ure1 in a yeast two-hybrid assay, and deletion of any one of these not only inactivated the enzyme but also reduced the efficacy of brain invasion. This is the first study showing a holistic picture of urease in fungi, clarifying that urease activity, and not Ure1 protein, contributes to pathogenesis in C. neoformans.
Project description:The human pathogenic fungus Cryptococcus neoformans has diverged from a common ancestor into three biologically distinct varieties or sibling species over the past 10-40 million years. During evolution of these divergent forms, serotype A C. neoformans var. grubii has emerged as the most virulent and cosmopolitan pathogenic clade. Therefore, understanding how serotype A C. neoformans is distinguished from less successful pathogenic serotypes will provide insights into the evolution of fungal virulence. Here we report that the structurally conserved Pbs2-Hog1 MAP kinase cascade has been specifically recruited as a global regulator to control morphological differentiation and virulence factors in the highly virulent serotype A H99 clinical isolate, but not in the laboratory-generated and less virulent serotype D strain JEC21. The mechanisms of Hog1 regulation are strikingly different between the two strains, and the phosphorylation kinetics and localization pattern of Hog1 are opposite in H99 compared with JEC21 and other yeasts. The unique Hog1 regulatory pattern observed in the H99 clinical isolate is widespread in serotype A strains and is also present in some clinical serotype D isolates. Serotype A hog1delta and pbs2delta mutants are attenuated in virulence, further underscoring the role of the Pbs2-Hog1 MAPK cascade in the pathogenesis of cryptococcosis.
Project description:Experimental pulmonary Cryptococcus neoformans infection in BALB/c mice is associated with polarized Th2-type cytokine production, alternative macrophage activation, and severe bronchopneumonia. In contrast, pulmonary infection with a C. neoformans strain that secretes IFN-?, H99?, elicits Th1-type cytokine production and classical macrophage activation. Additionally, mice infected with H99? resolve the acute infection and are subsequently protected against challenge with wild-type C. neoformans. The present study characterizes macrophage activation during the protective response to wild-type C. neoformans in mice previously immunized with H99?. We observed increased pulmonary Th1-type cytokine production in lung homogenates and classical macrophage activation as evidenced by enhanced expression of inducible NO synthase in the lungs of H99?-immunized mice compared with mice given a nonprotective immunization with heat-killed C. neoformans (HKCn). Furthermore, macrophages isolated from H99?-immunized mice on day 7 postchallenge and cultured in vitro were fungistatic against C. neoformans, whereas cryptococcal growth was uncontrolled within macrophages from HKCn-immunized mice. Th2-type cytokine production and induction of alternatively activated macrophages were also observed in lungs of HKCn-immunized mice during rechallenge. Gene expression arrays showed that classical macrophage activation during challenge infection in H99?-immunized mice was associated with induction of the transcription factor STAT1 and its downstream targets IFN regulatory factor-1, suppressor of cytokine signaling-1, CXCL9, and CXCL10. These studies demonstrate that protective responses to C. neoformans challenge in immunized mice include classical macrophage activation and enhanced macrophage fungistasis of C. neoformans yeasts. Finally, the classical activation phenotype of protective anticryptococcal macrophages is likely mediated via STAT1 signal transduction pathways.
Project description:Cryptococcus neoformans is a facultative intracellular pathogen and its interaction with macrophages is a key event determining the outcome of infection. Urease is a major virulence factor in C. neoformans but its role during macrophage interaction has not been characterized. Consequently, we analyzed the effect of urease on fungal-macrophage interaction using wild-type, urease-deficient and urease-complemented strains of C. neoformans. The frequency of non-lytic exocytosis events was reduced in the absence of urease. Urease-positive C. neoformans manifested reduced and delayed intracellular replication with fewer macrophages displaying phagolysosomal membrane permeabilization. The production of urease was associated with increased phagolysosomal pH, which in turn reduced growth of urease-positive C. neoformans inside macrophages. Interestingly, the ure1 mutant strain grew slower in fungal growth medium which was buffered to neutral pH (pH 7.4). Mice inoculated with macrophages carrying urease-deficient C. neoformans had lower fungal burden in the brain than mice infected with macrophages carrying wild-type strain. In contrast, the absence of urease did not affect survival of yeast when interacting with amoebae. Because of the inability of the urease deletion mutant to grow on urea as a sole nitrogen source, we hypothesize urease plays a nutritional role involved in nitrogen acquisition in the environment. Taken together, our data demonstrate that urease affects fitness within the mammalian phagosome, promoting non-lytic exocytosis while delaying intracellular replication and thus reducing phagolysosomal membrane damage, events that could facilitate cryptococcal dissemination when transported inside macrophages. This system provides an example where an enzyme involved in nutrient acquisition modulates virulence during mammalian infection.
Project description:The current studies evaluated the role of interleukin (IL)-17A in the induction of protective immunity against pulmonary cryptococcosis in mice. Protection against pulmonary infection with C. neoformans strain H99? was associated with increased IL-17A production. Signaling through the IFN-? receptor (R) was required for increased IL-17A production, however, a Th17-type cytokine profile was not observed. Neutrophils were found to be the predominant leukocytic source of IL-17A, rather than T cells, suggesting that the IL-17A produced was not part of a T cell-mediated Th17-type immune response. Depletion of IL-17A in mice during pulmonary infection with C. neoformans strain H99? resulted in an initial increase in pulmonary fungal burden, but had no effect on cryptococcal burden at later time points. Also, depletion of IL-17A did not affect the local production of other cytokines. IL-17RA?/? mice infected with C. neoformans strain H99? survived the primary infection as well as a secondary challenge with wild-type cryptococci. However, dissemination of the wild-type strain to the brain was noted in the surviving IL-17RA?/? mice. Altogether, our results suggested that IL-17A may be important for optimal protective immune responsiveness during pulmonary C. neoformans infection, but protective Th1-type immune responses are sufficient for protection against cryptococcal infection.
Project description:Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.