Characterization of the virulence of Cryptococcus neoformans strains in an insect model.
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ABSTRACT:
SUBMITTER: Bouklas T
PROVIDER: S-EPMC4826120 | biostudies-literature | 2015
REPOSITORIES: biostudies-literature
Publications
Characterization of the virulence of Cryptococcus neoformans strains in an insect model.
Bouklas Tejas T Diago-Navarro Elizabeth E Wang Xiaobo X Fenster Marc M Fries Bettina C BC
Virulence 20150912 8
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Project description:The basidiomycetous yeast Cryptococcus neoformans is an important human fungal pathogen. Two varieties, C. neoformans var. neoformans and C. neoformans var. gattii, have been identified. Both are heterothallic with two mating types, MATa and MATalpha. Some rare isolates are self-fertile and are considered occasional diploid or aneuploid strains. In the present study, 133 isolates, mostly from Italian patients, were investigated to detect the presence of diploid strains in the Igiene Università Milano culture collection. All of the diploid isolates were further investigated by different methods to elucidate their origins. Forty-nine diploid strains were identified by flow cytometry. PCR fingerprinting using the (GACA)(4) primer showed that the diploid state was associated with two specific genotypes identified as VN3 and VN4. Determination of mating type on V8 juice medium confirmed that the majority of the strains were sterile. PCR and dot blotting using the two pheromone genes (MFa and MFalpha) as probes identified 36 of the 49 diploid isolates as MATa/alpha. The results of pheromone gene sequencing showed that two allelic MFalpha genes exist and are distinct for serotypes A and D. In contrast, the MFa gene sequence was conserved in both serotype alleles. Amplification of serotype-specific STE20 alleles demonstrated that the diploid strains contained one mating locus inherited from a serotype A parent and one inherited from a serotype D parent. The present results suggest that diploid isolates may be common among the C. neoformans population and that in Italy and other European countries serotype A and D populations are not genetically isolated but are able to recombine by sexual reproduction.
| S-EPMC88460 | biostudies-literature
Project description:Cryptococcal meningitis (CM) causes high rates of HIV-related mortality, yet the Cryptococcus factors influencing patient outcome are not well understood. Pathogen-specific traits, such as the strain genotype and degree of antigen shedding, are associated with the clinical outcome, but the underlying biology remains elusive. In this study, we examined factors determining disease outcome in HIV-infected cryptococcal meningitis patients infected with Cryptococcus neoformans strains with the same multilocus sequence type (MLST). Both patient mortality and survival were observed during infections with the same sequence type. Disease outcome was not associated with the patient CD4 count. Patient mortality was associated with higher cryptococcal antigen levels, the cerebrospinal fluid (CSF) fungal burden by quantitative culture, and low CSF fungal clearance. The virulence of a subset of clinical strains with the same sequence type was analyzed using a mouse inhalation model of cryptococcosis. We showed a strong association between human and mouse mortality rates, demonstrating that the mouse inhalation model recapitulates human infection. Similar to human infection, the ability to multiply in vivo, demonstrated by a high fungal burden in lung and brain tissues, was associated with mouse mortality. Mouse survival time was not associated with single C. neoformans virulence factors in vitro or in vivo; rather, a trend in survival time correlated with a suite of traits. These observations show that MLST-derived genotype similarities between C. neoformans strains do not necessarily translate into similar virulence either in the mouse model or in human patients. In addition, our results show that in vitro assays do not fully reproduce in vivo conditions that influence C. neoformans virulence.
| S-EPMC6479045 | biostudies-literature
Project description:Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.
| S-EPMC387659 | biostudies-literature
Project description:Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis), where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH) and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work unravels (i) interesting physiological property of C. neoformans regarding its amino acid uptake system; (ii) an important aspect of virulence, which is the need for amino acid permeases during thermal and oxidative stress resistance and, hence, host invasion and colonization; and (iii) provides a convenient prototype for antifungal development, which are the amino acid permeases Aap4/Aap5 and their inhibitor.
| S-EPMC5047642 | biostudies-literature
Project description:Cryptococcus neoformans is an unconventional dimorphic fungus that can grow either as a yeast or in a filamentous form. To facilitate investigation of genetic factors important for its morphogenesis and pathogenicity, congenic a and ? strains for a filamentous form were constructed. XL280 (?) was selected as the background strain because of its robust ability to undergo the morphological transition from yeast to the filamentous form. The MATa allele from a sequenced strain JEC20 was introgressed into the XL280 background to generate the congenic a and ? pair strains. The resulting congenic strains were then used to test the impact of mating type on virulence. In both the inhalation and the intravenous infection models of murine cryptococcosis, the congenic a and ? strains displayed comparable levels of high virulence. The a-? coinfections displayed equivalent virulence to the individual a or ? infections in both animal models. Further analyses of the mating type distribution in a-? coinfected mice suggested no influence of a-? interactions on cryptococcal neurotropism, irrespective of the route of inoculation. Furthermore, deletion or overexpression of a known transcription factor, Znf2, in XL280 abolished or enhanced filamentation and biofilm formation, consistent with its established role. Overexpression of Znf2 in XL280 led to attenuation of virulence and a reduced abundance in the brain but not in other organs, suggesting that Znf2 might interfere with cryptococcal neurotropism upon extrapulmonary dissemination. In summary, the congenic strains provide a new resource for the exploration of the relationship in Cryptococcus between cellular morphology and pathogenesis.
| S-EPMC3697605 | biostudies-literature
Project description:In South Africa, Cryptococcus neoformans is the most common cause of adult meningitis. We performed multi locus sequence typing and fluconazole susceptibility testing of clinical C. neoformans isolates collected from 251 South African patients with cryptococcosis through national surveillance from 2005 to 2009. We examined the association between clinical characteristics of patients and genotype, and the effect of genotype on in-hospital mortality. We performed whole genome phylogenetic analysis of fifteen C. neoformans isolates with the molecular type VNB and tested their virulence in a Galleria mellonella model. Most isolates had the molecular type VNI (206/251, 82%), followed by VNII (25/251, 10%), VNB (15/251, 6%), and VNIV (5/251, 2%); 67 sequence types were identified. There were no differences in fluconazole minimum inhibitory concentration (MIC) values among molecular types and the majority of strains had low MIC values (MIC50 of 1 µg/mL and MIC90 of 4 µg/mL). Males were almost twice as likely of being infected with a non-VNI genotype (adjusted odds ratio [OR]: 1.65, 95% confidence interval [CI]: 0.25-10.99; p = 0.61). Compared to patients infected with a VNI genotype, those with a non-VNI genotype had a 50% reduced adjusted odds of dying in hospital (95% CI: 0.03-7.57; p = 0.62). However, for both these analyses, our estimates had wide confidence intervals spanning 1 with large p-values. Fifteen VNB strains were not as virulent in a G. mellonella larval model as the H99 reference strain. A majority of these VNB strains belonged to the VNBII clade and were very closely related by phylogenetic analysis.
| S-EPMC8146981 | biostudies-literature
Project description:UNLABELLED:While research has identified an important contribution for metals, such as iron, in microbial pathogenesis, the roles of other transition metals, such as copper, remain mostly unknown. Recent evidence points to a requirement for copper homeostasis in the virulence of Cryptococcus neoformans based on a role for a CUF1 copper regulatory factor in mouse models and in a human patient cohort. C. neoformans is an important fungal pathogen that results in an estimated 600,000 AIDS-related deaths yearly. In the present studies, we found that a C. neoformans mutant lacking the CUF1-dependent copper transporter, CTR4, grows normally in rich medium at 37°C but has reduced survival in macrophages and attenuated virulence in a mouse model. This reduced survival and virulence were traced to a growth defect under nutrient-restricted conditions. Expression studies using a full-length CTR4-fluorescent fusion reporter construct demonstrated robust expression in macrophages, brain, and lung, the latter shown by ex vivo fluorescent imaging. Inductively coupled mass spectroscopy (ICP-MS) was used to probe the copper quota of fungal cells grown in defined medium and recovered from brain, which suggested a role for a copper-protective function of CTR4 in combination with cell metallothioneins under copper-replete conditions. In summary, these data suggest a role for CTR4 in copper-related homeostasis and subsequently in fungal virulence. IMPORTANCE:Crytococcus neoformans is a significant global fungal pathogen, and copper homeostasis is a relatively unexplored aspect of microbial pathogenesis that could lead to novel therapeutics. Previous studies correlated expression levels of a Ctr4 copper transporter to development of meningoencephalitis in a patient cohort of solid-organ transplants, but a direct role for Ctr4 in mammalian pathogenesis has not been demonstrated. The present studies utilize a ?ctr4 mutant strain which revealed an important role for CTR4 in C. neoformans infections in mice and relate the gene product to homeostatic control of copper and growth under nutrient-restricted conditions. Robust expression levels of CTR4 during fungal infection were exploited to demonstrate expression and lung cryptococcal disease using ex vivo fluorescence imaging. In summary, these studies are the first to directly demonstrate a role for a copper transporter in fungal disease and provide an ex vivo imaging tool for further study of cryptococcal gene expression and pathogenesis.
| S-EPMC3518914 | biostudies-literature
Project description:Infection with Cryptococcus neoformans begins when desiccated yeast cells or spores are inhaled and lodge in the alveoli of the lungs. A subset of cryptococcal cells in the lungs differentiate into enlarged cells, referred to as titan cells. Titan cells can be as large as 50 to 100 μm in diameter and exhibit a number of features that may affect interactions with host immune defenses. To characterize the effect of titan cell formation on the host-pathogen interaction, we utilized a previously described C. neoformans mutant, the gpr4Δ gpr5Δ mutant, which has minimal titan cell production in vivo. The gpr4Δ gpr5Δ mutant strain had attenuated virulence, a lower CFU, and reduced dissemination compared to the wild-type strain. Titan cell production by the wild-type strain also resulted in increased eosinophil accumulation and decreased phagocytosis in the lungs compared to those with the gpr4Δ gpr5Δ mutant strain. Phagocytosed cryptococcal cells exhibited less viability than nonphagocytosed cells, which potentially explains the reduced cell survival and overall attenuation of virulence in the absence of titan cells. These data show that titan cell formation is a novel virulence factor in C. neoformans that promotes establishment of the initial pulmonary infection and plays a key role in disease progression.
| S-EPMC3486048 | biostudies-literature
Project description: Not available
| S-EPMC522596 | biostudies-literature
Project description:Cryptococcus neoformans serotypes A and D are responsible for the overwhelming majority of infections in patients with AIDS. The genetic relationship between the serotypes is poorly understood, but there are significant differences in the epidemiology and clinical presentation of serotype A and D infections. We evaluated the genetic relationship between reference C. neoformans strains belonging to serotypes A and D by analyzing their URA5 sequences and restriction fragment length polymorphisms (RFLPs) with the C. neoformans repetitive element 1 (CNRE-1) probe. The results were compared to those previously obtained for isolates from Brazil and New York City by the same typing methods, and dendrograms were generated. Serotype A and D strains produced distinct RFLP patterns consistent with their separation into two major clusters in the dendrogram generated on the basis of RFLP data. Similarly, serotype A and D strains clustered independently of the basis of the nucleotide sequences of their URA5 genes. Pairwise comparisons revealed average numbers of nucleotide differences within serotypes A and D of 3.0 +/- 1.7 and 7.2 +/- 3.4, respectively (P < 0.0001), and between serotypes A and D of 41.9 +/- 2.7. In summary, our results indicate phylogenetic differences between the two serotypes of C. neoformans var. neoformans and suggest that these serotypes could probably be considered different varieties of C. neoformans.
| S-EPMC105008 | biostudies-literature