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1.
Med Mycol ; 61(10)2023 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-37818721

RESUMO

Cryptococcus neoformans is the primary causative agent of cryptococcosis. Since C. neoformans thrives in environments and its optimal growth temperature is 25-30°C, it needs to adapt to heat stress in order to cause infection in mammalian hosts. In this study, we aimed to investigate the role of an uncharacterized gene, CNAG_03308. Although the CNAG_03308 deletion strain grew as well as the parent strain KN99, it produced yeast cells with abnormal morphology at 37°C and failed to propagate at 39°C. Furthermore, the deletion strain exhibited slower growth at 37°C in the presence of congo red, which is a cell wall stressor. When cultured at 39°C, the deletion strain showed strong staining with fluorescent probes for cell wall chitin and chitosan, including FITC-labeled wheat germ agglutinin, Eosin Y, and calcofluor white. The transmission electron microscopy of the deletion strain revealed a thickened inner layer of the cell wall containing chitin and chitosan under heat stress. This cell-surface altered deletion strain induced dendritic cells to secrete more interleukin (IL)-6 and IL-23 than the control strains under heat stress. In a murine infection study, C57BL/6 mice infected with the deletion strain exhibited lower mortality and lower fungal burden in the lungs and brain compared to those infected with the control strains. Based on these findings, we concluded that CNAG_03308 gene is necessary for C. neoformans to adapt to heat stress both in vitro and in the host environment. Therefore, we designated the CNAG_03308 gene as TVF1, which stands for thermotolerance and virulence-related factor 1.


Cryptococcus neoformans is a fungal pathogen causing cryptococcosis, which requires thermotolerance to proliferate in the host environment. In the present study, we identified a novel gene, TVF1 (CNAG_03308), required for thermotolerance and virulence by reverse genetics approach.


Assuntos
Quitosana , Criptococose , Cryptococcus neoformans , Termotolerância , Animais , Camundongos , Cryptococcus neoformans/genética , Virulência , Camundongos Endogâmicos C57BL , Criptococose/microbiologia , Criptococose/veterinária , Quitina , Proteínas Fúngicas/genética , Mamíferos
2.
Microbiol Immunol ; 67(5): 211-223, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36786396

RESUMO

Cryptococcosis is a mycosis caused by Cryptococcus neoformans and C. gattii species complexes. Although this infection is potentially lethal, no prophylactic vaccine is yet commercially available, and the immune memory that enables prevention is still under investigation. These pathogens have a capsule layer for immune evasion and a sophisticated mechanism to advance the infection, and it is expected that these characteristics will make it difficult to develop prophylactic vaccines and to decipher the protective immunity. The current vaccine studies are focused on subunit, mRNA, DNA, and viral vector vaccines, with whole-cell vaccines also proving successful against cryptococcal infections. Cryptococcal whole-cell vaccines have been composed of highly immunostimulating strains with low-pathogenicity that are modified by genetic recombination technology. Examples include the whole-cell vaccines H99γ, sgl1∆, fbp1∆, znf2oe , cda1/2/3∆, cap59∆, and cap60∆. Some of these whole-cell vaccines were found to be highly effective in prolonging life and suppressing the fungal burden after an infection challenge in mice, and to be cross-reactive to C. neoformans, C. gattii, and other fungal pathogens. Furthermore, for some vaccines, the protective effect can be retained even in an immunocompromised host depleted of CD4+ T cells. These findings have provided new insights into protective immunity that should aid in vaccine development. In this review, we highlight the upsides and downsides of whole-cell vaccines against cryptococcosis.


Assuntos
Criptococose , Cryptococcus gattii , Cryptococcus neoformans , Vacinas , Animais , Camundongos , Criptococose/prevenção & controle , Criptococose/microbiologia , Linfócitos T
3.
Allergol Int ; 72(4): 530-536, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37482531

RESUMO

Cryptococcus neoformans and Cryptococcus gattii are pathogenic fungi that infect the human respiratory system and cause life-threatening pulmonary cryptococcosis. The immunopathology of cryptococcosis is completely different from that of other fungal allergies. In murine cryptococcal infection models, cryptococcal cells are usually injected via nasal or intratracheal routes. After the infection, the alveolar epithelial cells are impaired and release IL-33, an IL-1 family cytokine that functions as an alarmin. This cytokine detrimentally amplifies allergic responses, and also induces a protective immune response against parasitic infection. In the pulmonary cryptococcosis model, type-II alveolar epithelial cells are the major source of IL-33, and the alveolar epithelial cells, ILC2, and Th2 cells express the IL-33 receptor (ST2). In IL-33- or ST2-deficient mice, allergy-like immune responses are attenuated after the C. neoformans infection. The numbers of ILC2 and Th2 cells and the levels of type 2 cytokines, including IL-4, IL-5, and IL-13, are decreased in the mouse lungs in both models. In association with these changes, total blood IgE, bronchus mucus production, and the number of eosinophils are decreased. Conversely, lung neutrophils and M1-type macrophages are increased. These are protective immune subsets suppressing cryptococcal growth. As a result, the lung fungal burden of IL-33- and ST2-deficient mice is decreased post-infection, and both deficient mice show significantly improved mortality. This pathogenesis varies depending on the cryptococcal and murine strains used in the animal experiments. Here, we overview and discuss the itmmunopathology of the IL-33/ST2 axis in a murine lethal cryptococcal infection model.


Assuntos
Criptococose , Cryptococcus neoformans , Animais , Humanos , Camundongos , Criptococose/microbiologia , Criptococose/patologia , Citocinas , Modelos Animais de Doenças , Imunidade Inata , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-33 , Pulmão/patologia , Linfócitos
4.
Antimicrob Agents Chemother ; 66(3): e0208121, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35041512

RESUMO

Limited data are available on breakthrough fungemia, defined as fungemia that develops on administration of antifungal agents, in patients with hematological disorders. We reviewed the medical and microbiological records of adult patients with hematological diseases who had breakthrough fungemia between January 2008 and July 2019 at Toranomon Hospital and Toranomon Hospital Kajigaya in Japan. A total of 121 cases of breakthrough fungemia were identified. Of the 121 involved patients, 83, 11, 5, and 22 were receiving micafungin, voriconazole, itraconazole, and liposomal amphotericin B, respectively, when the breakthrough occurred. Of the 121 causative breakthrough fungal strains, 96 were Candida species, and the rest were 13 cases of Trichosporon species, 7 of Fusarium species, 2 of Rhodotorula mucilaginosa, and 1 each of Cryptococcus neoformans, Exophiala dermatitidis, and Magnusiomyces capitatus. The crude 14-day mortality rate of breakthrough fungemia was 36%. Significant independent factors associated with the crude 14-day mortality rate were age of ≥60 years (P = 0.011), chronic renal failure (P = 0.0087), septic shock (P < 0.0001), steroid administration (P = 0.0085), and liposomal amphotericin B breakthrough fungemia (P = 0.0011). An absolute neutrophil count of >500/µL was significantly more common in candidemia in the multivariate analysis (P = 0.0065), neutropenia and nonallogeneic hematopoietic stem cell transplants were significantly more common in Trichosporon fungemia (P = 0.036 and P = 0.033, respectively), and voriconazole breakthrough fungemia and neutropenia were significantly more common in Fusarium fungemia (P = 0.016 and P = 0.016, respectively). The epidemiological and clinical characteristics of breakthrough fungemia of patients with hematological disorders were demonstrated. Some useful factors to predict candidemia, Trichosporon fungemia, and Fusarium fungemia were identified.


Assuntos
Candidemia , Cryptococcus neoformans , Fungemia , Fusarium , Doenças Hematológicas , Trichosporon , Adulto , Antifúngicos/uso terapêutico , Candida , Candidemia/tratamento farmacológico , Fungemia/tratamento farmacológico , Fungemia/microbiologia , Doenças Hematológicas/complicações , Doenças Hematológicas/tratamento farmacológico , Humanos , Pessoa de Meia-Idade
5.
Eur J Immunol ; 51(9): 2281-2295, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33728652

RESUMO

Cryptococcus gattii is a capsular pathogenic fungus causing life-threatening cryptococcosis. Although the capsular polysaccharides (CPs) of C. gattii are considered as virulence factors, the physiological significance of CP biosynthesis and of CPs themselves is not fully understood, with many conflicting data reported. First, we demonstrated that CAP gene deletant of C. gattii completely lacked capsule layer and its virulence, and that the strain was susceptible to host-related factors including oxidizing, hypoxic, and hypotrophic conditions in vitro. Extracellular CPs recovered from culture supernatant bound specifically to C. gattii acapsular strains, not to other fungi and immune cells, and rendered them the immune escape effects. In fact, dendritic cells (DCs) did not efficiently uptake the CP-treated acapsular strains, which possessed no visible capsule layer, and a decreased amount of phosphorylated proteins and cytokine levels after the stimulation. DCs recognized C. gattii acapuslar cells via an immune receptor CD11b- and Syk-related pathway; however, CD11b did not bind to CP-treated acapsular cells. These results suggested that CPs support immune evasion by coating antigens on C. gattii and blocking the interaction between CD11b and C. gattii cells. Here, we describe the importance of CPs in pathogenicity and immune evasion mechanisms of C. gattii.


Assuntos
Antígeno CD11b/imunologia , Cryptococcus gattii/imunologia , Cápsulas Fúngicas/imunologia , Polissacarídeos Fúngicos/imunologia , Evasão da Resposta Imune/imunologia , Quinase Syk/metabolismo , Animais , Criptococose/imunologia , Cryptococcus gattii/genética , Cryptococcus gattii/patogenicidade , Citocinas/biossíntese , Células Dendríticas/imunologia , Feminino , Cápsulas Fúngicas/genética , Polissacarídeos Fúngicos/genética , Deleção de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Polissacarídeos/genética , Polissacarídeos/imunologia , Fatores de Virulência/imunologia
6.
Biol Pharm Bull ; 43(2): 230-239, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32009111

RESUMO

Cryptococcosis is a potentially lethal disease caused by fungal pathogens including Cryptococcus neoformans and Cryptococcus gattii species complex. These fungal pathogens live in the environment and are associated with certain tree species and bird droppings. This infectious disease is not contagious, and healthy individuals may contract cryptococcal infections by inhaling the airborne pathogens from the environment. Although cleaning a contaminated environment is a feasible approach to control environmental fungal pathogens, prophylactic immunization is also considered a promising method to regulate cryptococcal infections. We review the history of the development of cryptococcal vaccines, vaccine components, and the various forms of immune memory induced by cryptococcal vaccines.


Assuntos
Criptococose/terapia , Vacinas/uso terapêutico , Animais , Cryptococcus neoformans/imunologia , Modelos Animais de Doenças , Fatores Imunológicos , Memória Imunológica , Vacinação
7.
Med Mycol ; 57(8): 1046-1054, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668754

RESUMO

Vaccine-induced immune responses, including neutrophil, macrophage, and T-cell responses, ameliorate cryptococcosis caused by Cryptococcus gattii. However, whether neutrophils can exert fungicidal activity against C. gattii remains to be elucidated. Therefore, in this study, we investigated the neutrophil-mediated fungicidal effect against C. gattii R265 in vitro and compared it to the related fungal pathogen, Cryptococcus neoformans standard strain H99. We found that neutrophils recognized, phagocytosed, and killed C. gattii R265 in the presence of fresh mouse serum. This antifungal effect required phagocytosis and serine protease activity but not nicotinamide adenine dinucleotide phosphate oxidase activity. We also demonstrated that C. gattii R265 was more resistant to oxidative and nitrosative stress than C. neoformans H99. Together, these findings indicate that neutrophils can exert fungicidal activity against highly virulent C. gattii, at least under in vitro conditions.


Assuntos
Cryptococcus gattii/imunologia , Imunidade Celular , Neutrófilos/imunologia , Animais , Cryptococcus neoformans/imunologia , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Estresse Nitrosativo , Estresse Oxidativo , Fagocitose
8.
Med Mycol ; 56(3): 382-386, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28992282

RESUMO

The efficacy of recombinant interferon γ (rIFN-γ) for cryptococcal meningoencephalitis has been poorly understood. Compared to Cryptococcus gattii, rIFN-γ significantly improved the survival in experimental meningoencephalitis due to Cryptococcus neoformans. The number of phagocytic macrophages and the levels of inflammatory cytokines production for ex vivo co-incubation with C. neoformans were increased after rIFN-γ stimulation but not C. gattii. Intraspecies differences of phagocytosis by the rIFN-γ-activated macrophages might be associated to the severity of cryptococcal infection.


Assuntos
Interferon gama/uso terapêutico , Macrófagos/efeitos dos fármacos , Meningoencefalite/tratamento farmacológico , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Linhagem Celular , Contagem de Colônia Microbiana , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/patogenicidade , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/patogenicidade , Modelos Animais de Doenças , Feminino , Interferon gama/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Meningoencefalite/microbiologia , Meningoencefalite/mortalidade , Meningoencefalite/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/efeitos dos fármacos , Especificidade da Espécie , Taxa de Sobrevida , Virulência
9.
Infect Immun ; 83(4): 1577-86, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25644007

RESUMO

Cryptococcosis due to a highly virulent fungus, Cryptococcus gattii, emerged as an infectious disease on Vancouver Island in Canada and surrounding areas in 1999, causing deaths among immunocompetent individuals. Previous studies indicated that C. gattii strain R265 isolated from the Canadian outbreak had immune avoidance or immune suppression capabilities. However, protective immunity against C. gattii has not been identified. In this study, we used a gain-of-function approach to investigate the protective immunity against C. gattii infection using a dendritic cell (DC)-based vaccine. Bone marrow-derived dendritic cells (BMDCs) efficiently engulfed acapsular C. gattii (Δcap60 strain), which resulted in their expression of costimulatory molecules and inflammatory cytokines. This was not observed for BMDCs that were cultured with encapsulated strains. When Δcap60 strain-pulsed BMDCs were transferred to mice prior to intratracheal R265 infection, significant amelioration of pathology, fungal burden, and the survival rate resulted compared with those in controls. Multinucleated giant cells (MGCs) that engulfed fungal cells were significantly increased in the lungs of immunized mice. Interleukin 17A (IL-17A)-, gamma interferon (IFN-γ)-, and tumor necrosis factor alpha (TNF-α)-producing lymphocytes were significantly increased in the spleens and lungs of immunized mice. The protective effect of this DC vaccine was significantly reduced in IFN-γ knockout mice. These results demonstrated that an increase in cytokine-producing lymphocytes and the development of MGCs that engulfed fungal cells were associated with the protection against pulmonary infection with highly virulent C. gattii and suggested that IFN-γ may have been an important mediator for this vaccine-induced protection.


Assuntos
Criptococose/imunologia , Cryptococcus gattii/imunologia , Células Dendríticas/transplante , Cápsulas Fúngicas/imunologia , Vacinas Fúngicas/imunologia , Animais , Células da Medula Óssea/imunologia , Terapia Baseada em Transplante de Células e Tecidos , Criptococose/prevenção & controle , Células Dendríticas/imunologia , Cápsulas Fúngicas/genética , Células Gigantes/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interleucina-17/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Necrose Tumoral alfa/imunologia , Vacinação
10.
Genes Cells ; 19(6): 478-503, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24725256

RESUMO

DNA sequencing of the 5'-flanking region of the transcriptome effectively identifies transcription initiation sites and also aids in identifying unknown genes. This study describes a comprehensive polling of transcription start sites and an analysis of full-length complementary DNAs derived from the genome of the pathogenic fungus Candida glabrata. A comparison of the sequence reads derived from a cDNA library prepared from cells grown under different culture conditions against the reference genomic sequence of the Candida Genome Database (CGD: http://www.candidagenome.org/) revealed the expression of 4316 genes and their acknowledged transcription start sites (TSSs). In addition this analysis also predicted 59 new genes including 22 that showed no homology to the genome of Saccharomyces cerevisiae, a genetically close relative of C. glabrata. Furthermore, comparison of the 5'-untranslated regions (5'-UTRs) and core promoters of C. glabrata to those of S. cerevisiae showed various global similarities and differences among orthologous genes. Thus, the C. glabrata transcriptome can complement the annotation of the genome database and should provide new insights into the organization, regulation, and function of genes of this important human pathogen.


Assuntos
Candida glabrata/genética , Genoma Fúngico , Iniciação da Transcrição Genética , Regiões 5' não Traduzidas , Candida glabrata/patogenicidade , Perfilação da Expressão Gênica , Ontologia Genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Sítio de Iniciação de Transcrição
11.
J Infect Chemother ; 21(12): 831-6, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26477011

RESUMO

Among invasive fungal infections, cryptococcosis caused by inhalation of Cryptococcus neoformans or Cryptococcus gattii is particularly dangerous because it can disseminate to the central nervous system and cause life-threatening meningitis or meningoencephalitis. Previous reports described significant differences in the histopathological features of C. neoformans and C. gattii infection, such as greater pathogen proliferation and a limited macrophage response in mouse lung infected by C. gattii. To elucidate the difference in pathogenicity of these two Cryptococcus species, we investigated the interaction of C. neoformans and C. gattii with murine macrophages, the first line of host defense, by confocal laser microscopy. Only thin-capsulated, and not thick-capsulated C. neoformans and C. gattii were phagocytosed by macrophages. Preactivation with interferon-γ increased the phagocytic rate of thin-capsulated C. neoformans up to two-fold, but did not promote phagocytosis of thin-capsulated C. gattii. Lipopolysaccharide preactivation or Aspergillus fumigatus conidia co-incubation had no effect on internalization of thin-capsulated C. neoformans or C. gattii by macrophages. Phagocytosis of live thin-capsulated C. neoformans, but not that of live thin-capsulated C. gattii, induced interleukin-12 release from macrophages. However, phagocytosis of heat-killed or paraformaldehyde-fixed thin-capsulated C. neoformans did not increase IL-12 release, showing that the internalization of live yeast is important for initiating the immune response during C. neoformans-macrophage interactions. Our data suggest that macrophage response to C. gattii is limited compared with that to C. neoformans and that these results may partially explain the limited immune response and the greater pathogenicity of C. gattii.


Assuntos
Criptococose/tratamento farmacológico , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Interferon gama/farmacologia , Fagocitose/efeitos dos fármacos , Animais , Linhagem Celular , Criptococose/metabolismo , Criptococose/microbiologia , Interleucina-12/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/metabolismo , Pulmão/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos
12.
J Infect Dis ; 209(5): 799-810, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24096333

RESUMO

BACKGROUND: The commensal yeast Candida albicans is a major cause of invasive fungal infections. Despite treatment with antifungal agents, the mortality rate attributed to these types of infection is high. Although numerous cases have been reported regarding a poor outcome for patients with bacterial and C. albicans coinfection, the mechanisms by which the coinfecting bacteria exacerbate the C. albicans infection remain elusive. METHODS AND RESULTS: We evaluated how glycolipid-mediated activation of invariant natural killer T (iNKT) cells affects the clearance of C. albicans. Surprisingly, C. albicans-infected, glycolipid-treated mice exhibited significantly lower survival rates, increased fungal burden, and higher interleukin (IL)-6 production in the kidneys compared with control mice. Glycolipid-induced exacerbation of C. albicans infection was not observed in interferon-gamma knockout (IFN-γKO) mice. In the C. albicans-infected, glycolipid-treated mice, the number of neutrophils in the blood and bone marrow dramatically decreased in an IFN-γ-dependent manner. Furthermore, mice that were coinfected with C. albicans and nonfermentative gram-negative commensal bacteria exhibited increased fungal burden and inflammatory cytokine production in the kidneys that were dependent on IFN-γ and iNKT cells. CONCLUSIONS: Our results indicate that coinfecting commensal bacteria exacerbate C. albicans infection through IFN-γ produced, in part, by iNKT cells.


Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Coinfecção/imunologia , Glicolipídeos/imunologia , Interferon gama/imunologia , Células T Matadoras Naturais/imunologia , Animais , Bactérias/imunologia , Medula Óssea/imunologia , Medula Óssea/microbiologia , Medula Óssea/virologia , Candidíase/microbiologia , Candidíase/virologia , Coinfecção/microbiologia , Coinfecção/virologia , Interleucina-6/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/microbiologia , Células T Matadoras Naturais/virologia , Neutropenia/imunologia , Neutropenia/microbiologia , Neutropenia/virologia
13.
Mol Microbiol ; 88(2): 371-81, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23448689

RESUMO

During disseminated infection by the opportunistic pathogen Candida glabrata, uptake of sterols such as serum cholesterol may play a significant role during pathogenesis. The ATP-binding cassette transporter Aus1p is thought to function as a sterol importer and in this study, we show that uptake of exogenous sterols occurred under anaerobic conditions in wild-type cells of C. glabrata but not in AUS1-deleted mutant (aus1Δ) cells. In aerobic cultures, growth inhibition by fluconazole was prevented in the presence of serum, and AUS1 expression was upregulated. Uptake of sterol by azole treated cells required the presence of serum, and sterol alone did not reverse FLC inhibition of growth. However, if iron availability in the growth medium was limited by addition of the iron chelators ferrozine or apo-transferrin, growth of wild-type cells, but not aus1Δ cells, was rescued. In a mouse model of disseminated infection, the C. glabrata aus1Δ strain caused a significantly decreased kidney fungal burden than the wild-type strain or a strain in which AUS1 was restored. We conclude that sterol uptake in C. glabrata can occur in iron poor environment of host tissues and thus may contribute to C. glabrata pathogenesis.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Candida glabrata/metabolismo , Candida glabrata/patogenicidade , Regulação Fúngica da Expressão Gênica , Ferro/metabolismo , Esteróis/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Aerobiose , Anaerobiose , Animais , Candida glabrata/genética , Candidíase/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Rim/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Baço/microbiologia
14.
Intern Med ; 2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39231670

RESUMO

Histoplasmosis is caused by Histoplasma capsulatum and is prevalent in areas of the world where H. capsulatum is endemic. We herein report a patient diagnosed with HIV-1 who developed histoplasmosis from a non-H. capsulatum endemic area who experienced severe hemophagocytic syndrome due to a delayed diagnosis. The patient's symptoms emerged four years after residing in regions with a high histoplasmosis prevalence. The unrestricted administration of antifungal medication for oral candidiasis delayed the diagnosis because it improved the patient's condition. This case underscores the importance of prudent antifungal drug use in undiagnosed disseminated conditions and evaluating the travel history going back several years to facilitate a diagnosis.

15.
Med Mycol ; 51(4): 385-95, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23101887

RESUMO

Mannans are mannose polymers attached to cell wall proteins in all Candida species, including the pathogenic fungus Candida albicans. Mannans are sensed by pattern recognition receptors expressed on innate immune cells. However, the detailed structural patterns affecting immune sensing are not fully understood because mannans have a complex structure that includes α- and ß-mannosyl linkages. In this study, we focused on the ß-1,2-mannosides of N-linked mannan in C. albicans because this moiety is not present in the non-pathogenic yeast Saccharomyces cerevisiae. To investigate the impact of ß-1,2-mannosides on immune sensing, we constructed a C. albicans ∆mnn4/∆bmt1 double deletant. Thin-layer chromatography and nuclear magnetic resonance analyses revealed that the deletant lacked ß-1,2-mannosides in N-linked mannan. Mannans lacking the ß-1,2-mannosides induced the production of higher levels of inflammatory cytokines, including IL-6, IL-12p40 and TNF-α, in mice dendritic cells compared to wild-type mannan. Our data show that ß-1,2-mannosides in N-linked mannan reduce the production of inflammatory cytokines by dendritic cells.


Assuntos
Candida albicans/metabolismo , Citocinas/metabolismo , Células Dendríticas/imunologia , Mananas/imunologia , Oligossacarídeos/imunologia , Animais , Candida albicans/genética , Candida albicans/imunologia , Cromatografia em Camada Fina , Citocinas/análise , Células Dendríticas/efeitos dos fármacos , Humanos , Subunidade p40 da Interleucina-12/análise , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-6/análise , Interleucina-6/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Mananas/química , Mananas/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Deleção de Sequência , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo
16.
Genes Cells ; 16(1): 80-9, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21199190

RESUMO

Zn[2]-Cys[6] binuclear transcription factors Upc2p and Ecm22p regulate the expression of genes involved in ergosterol biosynthesis and exogenous sterol uptake in Saccharomyces cerevisiae. We identified two UPC2/ECM22 homologues in the pathogenic fungus Candida glabrata which we designated CgUPC2A and CgUPC2B. The contribution of these two genes to sterol homeostasis was investigated. Cells that lack CgUPC2A (upc2AΔ) exhibited enhanced susceptibility to the sterol biosynthesis inhibitors, fluconazole and lovastatin, whereas upc2BΔ-mutant cells were as susceptible to the drugs as wild-type cells. The growth of upc2AΔ cells was also severely attenuated under anaerobic conditions. Lovastatin treatment enhanced the expression of ergosterol biosynthetic genes, ERG2 and ERG3 in wild-type and upc2BΔ but not in upc2AΔ cells. Similarly, serum-induced expression of ERG2 and ERG3 was completely impaired in upc2AΔ cells but was unaffected in upc2BΔ cells, whereas serum-induced expression of the sterol transporter gene CgAUS1 was impaired in both upc2AΔ and upc2BΔ cells. These results suggest that in C. glabrata CgUPC2A but not in CgUPC2B is the main transcriptional regulator of the genes responsible for maintaining sterol homeostasis as well as susceptibility to sterol inhibitors.


Assuntos
Candida glabrata/metabolismo , Ergosterol/biossíntese , Ergosterol/genética , Fatores de Transcrição/fisiologia , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Candida glabrata/genética , Ergosterol/metabolismo , Fluconazol/metabolismo , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos/efeitos dos fármacos , Lovastatina/metabolismo , Lovastatina/farmacologia , Proteínas de Saccharomyces cerevisiae/genética , Esteróis/metabolismo , Transativadores/genética , Fatores de Transcrição/genética
17.
J Antimicrob Chemother ; 67(7): 1666-76, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22514266

RESUMO

OBJECTIVES: A mechanism for the acquisition of high-level echinocandin resistance in Candida glabrata was investigated. FKS mutants were constructed to: determine whether clinically significant micafungin resistance requires a hot-spot mutation in FKS1 and a premature stop codon in FKS2, as was observed in a clinical isolate; select for variants with reduced susceptibility and locate mutations in FKS genes; and assess the roles of FKS1 and FKS2. METHODS: A panel of FKS mutants was constructed using micafungin-susceptible parents by site-directed mutagenesis. Drug susceptibility, gene expression and glucan synthase activities were compared between mutants. Mutations acquired by selection were identified by DNA sequence analysis of FKS genes from selected variants. Single FKS deletants were constructed and their phenotypes examined. RESULTS: Introduction of the hot-spot mutation in FKS1 alone conferred an intermediate reduction in susceptibility, and the premature stop codon in FKS2 alone had no effect on susceptibility, while severely reduced susceptibility equivalent to that of the clinical isolate required both mutations. Exposure of susceptible strains to micafungin yielded variants with an intermediate reduction in susceptibility that possessed a hot-spot mutation in FKS1. Further exposure to micafungin yielded variants with severely reduced susceptibility that acquired various single mutations in FKS2. The phenotypes of Δfks1 and Δfks2 mutants indicate that the two FKS genes are functionally redundant, while deletion of both FKS1 and FKS2 conferred synthetic lethality. CONCLUSIONS: In the laboratory mutants of C. glabrata, clinically significant reduced susceptibility to micafungin required single nucleotide changes in both FKS1 and FKS2, and both genes encoded ß-1,3-glucan synthase catalytic subunits.


Assuntos
Antifúngicos/farmacologia , Candida glabrata/efeitos dos fármacos , Candida glabrata/enzimologia , Farmacorresistência Fúngica , Equinocandinas/farmacologia , Expressão Gênica , Glucosiltransferases/biossíntese , Lipopeptídeos/farmacologia , Candida glabrata/genética , Análise Mutacional de DNA , DNA Fúngico/química , DNA Fúngico/genética , Deleção de Genes , Glucosiltransferases/genética , Micafungina , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Seleção Genética , Análise de Sequência de DNA
18.
FEMS Yeast Res ; 12(6): 728-37, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22713118

RESUMO

We investigated the cellular function of Msi3p, belonging to the heat shock protein 70 family, in Candida albicans. The mutant strain tetMSI3 was generated, in which MSI3 was controlled by a tetracycline-repressive promoter, because there is evidence to suggest that MSI3 is an essential gene. We controlled the MSI3 expression level by doxycycline (DOX) and compared its phenotype with that of a control strain with the tetracycline-repressive promoter and a wild-type copy MSI3. The results indicated that MSI3 was essential for cell growth. In addition, all the tetMSI3-infected mice survived after DOX administration. Drug susceptibility tests indicated that repression of MSI3 expression resulted in hypersensitivity to fluconazole and conferred fungicidal activity to fluconazole. The expression levels of MSI3 and calcineurin-dependent genes were upregulated in response to fluconazole in the control strain. In tetMSI3, the upregulation of MSI3 was lost, and the expression level of the calcineurin-dependent genes was no longer elevated in response to fluconazole and was not affected by DOX, indicating that the upregulation of MSI3 expression was required for the induction of the calcineurin-dependent gene expression. These data suggest that Msi3p confers fluconazole tolerance by partially influencing the calcineurin signaling pathway and also other tolerance mechanisms.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Tolerância a Medicamentos , Fluconazol/farmacologia , Proteínas Fúngicas/metabolismo , Animais , Candidíase/microbiologia , Modelos Animais de Doenças , Doxiciclina/administração & dosagem , Doxiciclina/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Essenciais , Genes Fúngicos , Proteínas de Choque Térmico HSP70/genética , Camundongos , Recombinação Genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Análise de Sobrevida
19.
JAC Antimicrob Resist ; 4(1): dlac018, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35265841

RESUMO

Background: Candida glabrata is an emerging fungal pathogen in immune-compromised hosts. Previously undetected C. glabrata isolates were successfully recovered from clinical specimens by adding sterols to the growth medium. The clinical isolates are unable to synthesize ergosterol but can take up exogenous sterols under aerobic conditions. Objectives: This study characterizes the sterol-auxotrophic C. glabrata strains, examines the mutation(s) in sterol synthesis genes, characterizes the drug susceptibility and evaluates the virulence in a mouse infection model. Methods: Drug susceptibility of the C. glabrata strains was evaluated in a sterol-supplemented medium. The coding sequences of the sterol synthesis genes were analysed in six sterol-auxotrophic strains of C. glabrata. The fungal burden of mice infected with C. glabrata strain was determined. Results: The sterol-auxotrophic strains showed high-level resistance to both azoles and amphotericin B when sterols were supplied in the test medium. Additionally, the strains harbour missense mutations in either ERG1 or ERG7. Significant differences in fungal burden were not observed between the sterol-auxotrophic strain and the sterol-competent strain with the mice infection models. Conclusions: The sterol-auxotrophic C. glabrata strain investigated in this study seemed to maintain intact virulence, probably due to the supply of exogenous sterols from host organ(s). This suggests that exogenous sterol uptake develops antifungal resistance during infection.

20.
FEMS Yeast Res ; 11(5): 398-407, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21453325

RESUMO

It is known that cell wall remodeling and the salvaging pathway act to compensate for an impaired or a damaged cell wall. Lately, it has been indicated that this mechanism is partly required for resistance to the glucan synthesis inhibitor echinocandin. While cell wall remodeling has been described in mutants of glucan or mannan synthesis, it has not yet been reported in a chitin synthesis mutant. Here, we describe a novel cell wall remodeling and salvaging pathway in chitin synthesis mutants, Δchs3A and Δchs3B, of the pathogenic yeast Candida glabrata. Electron microscopic analysis revealed a thickened mannoprotein layer in Δchs3A cells and a thickened chitin-glucan layer of Δchs3B cells, and it indicated the hypothesis that mannan synthase and chitin-glucan synthase indemnify Δchs3A and Δchs3B cells, respectively. The double-mutant CHS3A and MNN10, encoding α-1,6-mannosyltransferase, showed synergistic stress sensitization, and the Δchs3B strain showed supersensitivity to echinocandins. Hence, these findings support the above hypothesis of remodeling. Furthermore, unlike Δchs3A cells, Δchs3B cells showed supersensitivity to calcineurin inhibitor FK506 and Tor1p kinase inhibitor rapamycin, indicating that the Δchs3B strain uses the calcineurin pathway and a Tor1p kinase for cell wall remodeling.


Assuntos
Candida glabrata/enzimologia , Parede Celular/metabolismo , Quitina Sintase/metabolismo , Sequência de Aminoácidos , Aminoglicosídeos/farmacologia , Calcineurina/metabolismo , Candida glabrata/efeitos dos fármacos , Candida glabrata/genética , Candida glabrata/fisiologia , Parede Celular/ultraestrutura , Quitina/antagonistas & inibidores , Quitina/metabolismo , Quitina Sintase/genética , Equinocandinas/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucanos/antagonistas & inibidores , Glucanos/metabolismo , Mananas/metabolismo , Glicoproteínas de Membrana/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Transdução de Sinais , Estresse Fisiológico
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