Thermotolerance in the pathogen Cryptococcus neoformans is linked to antigen masking via mRNA decay-dependent reprogramming
Amanda L. M. BloomRichard M. JinJay LeipheimerJonathan BardDonald YergeauElizabeth A. WohlfertJohn C. Panepinto
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Abstract A common feature shared by systemic fungal pathogens of environmental origin, such as Cryptococcus neoformans , is their ability to adapt to mammalian core body temperature. In C. neoformans , this adaptation is accompanied by Ccr4-mediated decay of ribosomal protein mRNAs. Here we use the related, but thermo-intolerant species Cryptococcus amylolentus to demonstrate that this response contributes to host-temperature adaptation and pathogenicity of cryptococci. In a C. neoformans ccr4 Δ mutant, stabilized ribosomal protein mRNAs are retained in the translating pool, and stress-induced transcriptomic changes are reduced in comparison with the wild type strain, likely due to ineffective translation of transcription factors. In addition, the mutant displays increased exposure of cell wall glucans, and recognition by Dectin-1 results in increased phagocytosis by lung macrophages, linking mRNA decay to adaptation and immune evasion.Keywords:
Host adaptation
Reprogramming
Ribosomal protein
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.
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We report the first case of cryptococcosis due to Cryptococcus neoformans var. gattii in France. This Cambodian, non-AIDS patient had been living in France for 23 years when he developed cryptococcosis due to C. neoformans serotype B in 1985. It is possible that he was infected back in Asia or several months before, in Zaire, where he lived between 1981 and 1984. To our knowledge, this is the second case providing clinical evidence for reactivation of latent infection with C. neoformans. The mechanism is probably similar to that for C. neoformans var. neoformans.
Cryptococcus gattii
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Cryptococcus neoformans var. neoformans is the second most prevalent agent of cryptococcosis in central Europe. Infections mostly present with localized skin and disseminated infections. Previous studies did not find these presentations to be determined by the fungal genotype as detected by multilocus sequence typing (MLST). However, phenotypic fungal traits may impact clinical presentation. Here, we studied the growth and virulence factors of C. neoformans var. neoformans isolates from disseminated and localized infections and an environmental isolate. We used coincubation with Acanthamoeba castellanii and the Galleria mellonella infection model to identify phenotypic characteristics potentially associated with clinical presentation. Clinical isolates of C. neoformans var. neoformans present a substantial phenotypic variability. Median survival of G. mellonella varied between 6 and 14 days. C. neoformans var. neoformans isolates from disseminated infections showed stronger melanization and larger capsules. They demonstrated superior uptake into an amoeba and increased cytotoxicity for the amoeba. Differences of strains from localized and disseminated infections in coincubation with amoeba are in line with the importance of phagocytes in the pathogenesis of disseminated cryptococcosis. Phenotypic traits and non-vertebrate infection models may help understand the virulence potential of C. neoformans var. neoformans isolates.
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Multilocus sequence typing
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Cryptococcus gattii
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Abstract Vaccines are one of the greatest medical accomplishments to date, yet no fungal vaccines are currently available in humans mainly because opportunistic mycoses generally occur during immunodeficiencies necessary for vaccine protection. In previous studies, a live, attenuated Cryptococcus neoformans Δ sgl1 mutant accumulating sterylglucosides was found to be avirulent and protected mice from a subsequent lethal infection even in absence of CD4 + T cells, a condition most associated with cryptococcosis (e.g., HIV). Here, we tested three strategies of vaccination against cryptococcosis. First, in our preventative model, protection was achieved even after a 3-fold increase of the vaccination window. Second, because live C. neoformans Δ sgl1 -vaccinated mice challenged more than once with WT strain had a significant decrease in lung fungal burden, we tested C. neoformans Δ sgl1 as an immunotherapeutic. We found that therapeutic administrations of HK C. neoformans Δ sgl1 subsequent to WT challenge significantly improve the lung fungal burden. Similarly, therapeutic administration of HK C. neoformans Δ sgl1 post WT challenge resulted in 100% or 70% survival depending on the time of vaccine administration, suggesting that HK Δ sgl1 is a robust immunotherapeutic option. Third, we investigated a novel model of vaccination in preventing reactivation from lung granuloma using C. neoformans Δ gcs1 . Remarkably, we show that administration of HK Δ sgl1 prevents mice from reactivating Δ gcs1 upon inducing immunosuppression with corticosteroids or by depleting CD4 + T cells. Our results suggest that HK Δ sgl1 represents a clinically relevant, efficacious vaccine that confers robust host protection in three models of vaccination against cryptococcosis even during CD4-deficiency. Importance Cryptococcosis results in ∼180,000 global deaths per year in immunocompromised individuals. Current antifungal treatment options are potentially toxic, lacking in areas of need, and exhibit limited efficacy. In addition to these lackluster therapeutic options, no fungal vaccines are currently available for clinical use. Due to the increasing rate of immunocompromised individuals, there is a dire need for the development of improved antifungal therapeutics. Presently, we have demonstrated the high efficacy of a clinically relevant heat-killed mutant strain of Cryptococcus neoformans in inducing advantageous host protection in three models of vaccination against cryptococcosis during immunodeficiencies most associated with this disease.
Immunosuppression
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Background: Cryptococcosis is an infectious disease with worldwide distribution caused by Cryptococcus species. Cryptococcus neoformans and Cryptococcus gattii are the common species in human cryptococcal infection. Cryptococcus neoformans is a major cause of illness in people diagnosed with Human Immunodeficiency Virus (HIV), with an estimated 220,000 cases of cryptococcal meningitis occurring worldwide each year. This is a retrospective study on the frequency of cryptococcosis in different age groups and gender; as well as to identify the types of Cryptococcus species isolated in this population in University Malaya Medical Center from year 2013 to September 2019. Methods and materials: Data on cryptococcosis diagnosed in University Malaya Medical Center from year 2013 to September 2019 was extracted from the laboratory information system at the center. 59 newly diagnosed cryptococcosis cases were included in this study. Cryptococcus species were isolated from blood, cerebrospinal fluid, pus, body fluid, tissue and skin. Methods of identification of Cryptococcus species were via API 20C AUX, Vitek 2 and MALDI-TOF. Results: In this population of study, the frequency of cryptococcosis is 83.06% and 16.94% in males and females respectively. The frequency of Cryptococcus neoformans is 81.36%, with 83.33% comprising males (age 10–87) and 16.67% comprising females (age 33–81). The frequency of Cryptococcus neoformans var. grubii is 3.39% (2 males aged 33 and 53), Cryptococcus albidus is 3.39% (2 females aged 65 and 70), Cryptococcus uniguttulatus is 3.39% (two males aged 35 and 87) and Cryptococcus sp. is 3.39% (2 males aged 25 and 36). Cryptococcus gattii (34 year old male), Cryptococcus laurentii (71 year old male) and Cryptococcus humicola (59 years old male) are 1.70% in frequency each. Conclusion: In conclusion, the frequency of cryptococosis in this period of study is high in the male population with Cryptococcus neoformans as the commonest isolated species. Early diagnosis followed by treatment of cryptococcosis offers good to excellent prognosis for the patients. However, the prognosis varies in the immunocompromised. Targeted screening programmes should be carried out to detect early cryptococcal disease, especially in HIV-infected persons to reduce morbidity and mortality, and improve the overall quality of life.
Cryptococcus gattii
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Cryptococcus neoformans causes infection in individuals with defective T cell function, such as AIDS, as well as without underlying disease. It has been suggested that humoral as well as cellular immunity might play an important role in the immune response to C. neoformans infection. We have recently shown, using immunoblotting, that the 70-kD hsp family of C. neoformans was the major target molecule of the humoral response in murine pulmonary cryptococcosis. In this study we also used immunoblotting to define the antibody responses in the sera of 24 patients with pulmonary cryptococcosis: 21 proven and three suspected diagnoses. Anti-C. neoformans hsp70 antibody was detected in 16 of 24 (66.7%) patients with pulmonary cryptococcosis. Fourteen of 17 (82.3%) patients with high antigen titres (> or = 1:8) and two of seven (28.6%) patients with low titres (< or = 1:4) had detectable levels of anti-hsp70 antibody. Sera from patients positive for anti-hsp70 antibody showed high titres in the Eiken latex agglutination test for the detection of serum cryptococcal antigen. Our results indicate that the 70-kD hsp family from C. neoformans appears to be a major target molecule of the humoral response, not only in murine pulmonary cryptococcosis, but also in human patients with pulmonary cryptococcosis.
Latex fixation test
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