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1.
PLoS Biol ; 22(6): e3002682, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38843310

RESUMO

In exploring the evolutionary trajectories of both pathogenesis and karyotype dynamics in fungi, we conducted a large-scale comparative genomic analysis spanning the Cryptococcus genus, encompassing both global human fungal pathogens and nonpathogenic species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species, covering virtually all known diversity within these genera. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at preadaptive pathogenic potential, our analysis found evidence of gene gain (via horizontal gene transfer) and gene loss in pathogenic Cryptococcus species, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the 2 genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5, or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes showed reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Overall, our findings advance our understanding of genetic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.


Assuntos
Cromossomos Fúngicos , Cryptococcus , Evolução Molecular , Genoma Fúngico , Genômica , Cariótipo , Cryptococcus/genética , Cryptococcus/patogenicidade , Cryptococcus/classificação , Cromossomos Fúngicos/genética , Genômica/métodos , Filogenia , Sintenia , Centrômero/genética , Criptococose/microbiologia , Humanos
2.
Microbiol Spectr ; 12(7): e0390223, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38842310

RESUMO

Cryptococcus neoformans and Cryptococcus gattii are both known urease producers and have the potential to cause hyperammonemia. We hypothesized that the risk of hyperammonemia is increased by renal failure, burden of cryptococcal infection, and fungal strain characteristics. We performed a retrospective review of plasma ammonia levels in patients with cryptococcal infections. Risk factors for hyperammonemia were statistically compared between patients with and without hyperammonemia (>53 µmol/L). Cryptococcal cells from three patients included in the study were recovered from our biorepository. Strain characteristics including urease activity, ammonia production, growth curves, microscopy, melanin production, and M13 molecular typing were analyzed and compared with a wild-type (WT) C. neoformans strain. We included 29 patients, of whom 37.9% had hyperammonemia, 59% had disseminated cryptococcal infection (DCI), and 41% had isolated central nervous system infection. Thirty-eight percent of patients had renal failure and 28% had liver disease. Renal failure was associated with 4.4 times (95% confidence interval [CI] 1.5, 13.0) higher risk of hyperammonemia. This risk was higher in DCIs (RR 6.2, 95% CI 1.0, 40.2) versus isolated cryptococcal meningitis (RR 2.5, 95% CI, 0.40, 16.0). Liver disease and cryptococcal titers were not associated with hyperammonemia. C. neoformans from one patient with extreme hyperammonemia demonstrated a 4- to 5-fold increase in extracellular urease activity, slow growth, enlarged cell size phenotypes, and diminished virulence factors. Hyperammonemia was strongly associated with renal failure in individuals with DCI, surpassing associations with liver failure or cryptococcal titers. However, profound hyperammonemia in one patient was attributable to high levels of urease secretion unique to that cryptococcal strain. Prospective studies are crucial to exploring the significance of this association.IMPORTANCECryptococcus produces and secretes the urease enzyme to facilitate its colonization of the host. Urease breaks down urea into ammonia, overwhelming the liver's detoxification process and leading to hyperammonemia in some hosts. This underrecognized complication exacerbates organ dysfunction alongside the infection. Our study investigated this intricate relationship, uncovering a strong association between the development of hyperammonemia and renal failure in patients with cryptococcal infections, particularly those with disseminated infections. We also explore mechanisms underlying increased urease activity, specifically in strains associated with extreme hyperammonemia. Our discoveries provide a foundation for advancing research into cryptococcal metabolism and identifying therapeutic targets to enhance patient outcomes.


Assuntos
Criptococose , Cryptococcus gattii , Cryptococcus neoformans , Hiperamonemia , Urease , Humanos , Criptococose/microbiologia , Hiperamonemia/microbiologia , Hiperamonemia/etiologia , Feminino , Estudos Retrospectivos , Masculino , Pessoa de Meia-Idade , Urease/metabolismo , Adulto , Idoso , Amônia/metabolismo , Fatores de Risco , Insuficiência Renal/complicações , Insuficiência Renal/microbiologia , Idoso de 80 Anos ou mais
3.
Microbiol Spectr ; 12(7): e0341923, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38842336

RESUMO

Cryptococcus neoformans is a fungal pathogen responsible for >200,000 yearly cases with a mortality as high as 81%. This burden results, in part, from an incomplete understanding of its pathogenesis and ineffective antifungal treatments; hence, there is a pressing need to understand the biology and host interactions of this yeast to develop improved treatments. Protein palmitoylation is important for cryptococcal virulence, and we previously identified the substrates of its main palmitoyl transferase. One of them was encoded by the uncharacterized gene CNAG_02129. In the filamentous fungus Neurospora crassa, a homolog of this gene named hyphal anastomosis protein 13 plays a role in proper cellular communication and filament fusion. In Cryptococcus, cellular communication is essential during mating; therefore, we hypothesized that CNAG_02129, which we named hyphal anastomosis protein 1 (HAM1), may play a role in mating. We found that ham1Δ mutants produce more fusion products during mating, filament more robustly, and exhibit competitive fitness defects under mating and non-mating conditions. Additionally, we found several differences with the major virulence factor, the polysaccharide capsule, that may affect virulence, consistent with prior studies linking virulence to mating. We observed that ham1Δ mutants have decreased capsule attachment and transfer but exhibit higher amounts of exopolysaccharide shedding and biofilm production. Finally, HAM1 expression is significantly lower in mating media relative to non-mating conditions, consistent with it acting as a negative regulator of mating. Understanding the connection between mating and virulence in C. neoformans may open new avenues of investigation into ways to improve the treatment of this disease. IMPORTANCE: Fungal mating is a vital part of the lifecycle of the pathogenic yeast Cryptococcus neoformans. More than just ensuring the propagation of the species, mating allows for sexual reproduction to occur and generates genetic diversity as well as infectious propagules that can invade mammalian hosts. Despite its importance in the biology of this pathogen, we still do not know all of the major players regulating the mating process and if they are involved or impact its pathogenesis. Here, we identified a novel negative regulator of mating that also affects certain cellular characteristics known to be important for virulence. This gene, which we call HAM1, is widely conserved across the cryptococcal family as well as in many pathogenic fungal species. This study will open new avenues of exploration regarding the function of uncharacterized but conserved genes in a variety of pathogenic fungal species and specifically in serotype A of C. neoformans.


Assuntos
Criptococose , Cryptococcus neoformans , Proteínas Fúngicas , Fatores de Virulência , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/fisiologia , Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Virulência/genética , Criptococose/microbiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Genes Fúngicos Tipo Acasalamento/genética , Fenótipo , Regulação Fúngica da Expressão Gênica , Animais , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Camundongos
4.
Front Cell Infect Microbiol ; 14: 1392015, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38841113

RESUMO

Trehalose-6-phosphate synthase (TPS1) was identified as a virulence factor for Cryptococcus neoformans and a promising therapeutic target. This study reveals previously unknown roles of TPS1 in evasion of host defenses during pulmonary and disseminated phases of infection. In the pulmonary infection model, TPS1-deleted (tps1Δ) Cryptococci are rapidly cleared by mouse lungs whereas TPS1-sufficent WT (H99) and revertant (tps1Δ:TPS1) strains expand in the lungs and disseminate, causing 100% mortality. Rapid pulmonary clearance of tps1Δ mutant is T-cell independent and relies on its susceptibility to lung resident factors and innate immune factors, exemplified by tps1Δ but not H99 inhibition in a coculture with dispersed lung cells and its rapid clearance coinciding with innate leukocyte infiltration. In the disseminated model of infection, which bypasses initial lung-fungus interactions, tps1Δ strain remains highly attenuated. Specifically, tps1Δ mutant is unable to colonize the lungs from the bloodstream or expand in spleens but is capable of crossing into the brain, where it remains controlled even in the absence of T cells. In contrast, strains H99 and tps1Δ:TPS1 rapidly expand in all studied organs, leading to rapid death of the infected mice. Since the rapid pulmonary clearance of tps1Δ mutant resembles a response to acapsular strains, the effect of tps1 deletion on capsule formation in vitro and in vivo was examined. Tps1Δ cryptococci form capsules but with a substantially reduced size. In conclusion, TPS1 is an important virulence factor, allowing C. neoformans evasion of resident pulmonary and innate defense mechanisms, most likely via its role in cryptococcal capsule formation.


Assuntos
Criptococose , Cryptococcus neoformans , Modelos Animais de Doenças , Glucosiltransferases , Pulmão , Fatores de Virulência , Animais , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/genética , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/imunologia , Criptococose/microbiologia , Criptococose/imunologia , Camundongos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Interações Hospedeiro-Patógeno , Encéfalo/microbiologia , Baço/microbiologia , Feminino , Camundongos Endogâmicos C57BL , Imunidade Inata , Evasão da Resposta Imune , Deleção de Genes
5.
Artigo em Alemão | MEDLINE | ID: mdl-38925137

RESUMO

A 2-year-old Norwegian Forest cat was presented for evaluation of bilateral purulent nasal discharge and stertorous breathing. A computed tomography (CT) scan of the head revealed an intranasal mass of the left nasal cavity extending behind the tube openings and completely obstructing the nasopharynx. Rhinoscopy confirmed a pinkish, shiny mass. CT scan showed both compartments of the right middle ear filled with abnormal soft tissue attenuating material. There was no change in the bony outline of the middle ear. In the endoscopic examination, after endoscopically assisted tympanocentesis, this material in the accessible dorsolateral compartment proved to be classic polypous tissue in addition to highly viscous glue-like secretions. A secondary otitis media due to a drainage disorder was suspected.Using an endoscopic-interventional approach through the nostril, the nasopharyngeal mass was removed for histopathological examination, in order to restore the nasal airway, and to allow tube drainage. In contrast to cats with classical malignant nasal cavity masses, the cat showed several attachment points of the mass and multiple undulating elevations bilaterally in the nasopharyngeal mucosa.Cytological and histopathological examination identified the mass as a fungal granuloma in the context of a cryptococcus infection only rarely observed in Germany. Molecular genetic analysis confirmed an infection with Cryptococcus neoformans var. grubii.A single intranasal and nasopharyngeal endoscopic debridement resulted in a significant improvement of the clinical signs and a complete healing of the right middle ear (including the tympanic membrane) within 14 days, but not in a complete cure of the disease. The cat was therefore treated with oral itraconazole solution for several weeks.The case report shows that nasal cryptococcosis can also affect cats in Germany. Rhinoscopy reveals a nasopharyngeal mass with multiple attachment points, which is unusual for a neoplasia. In addition to the recommended removal of the mass, oral administration of systemic antimycotics is strongly advised.


Assuntos
Doenças do Gato , Criptococose , Animais , Gatos , Doenças do Gato/diagnóstico , Doenças do Gato/microbiologia , Doenças do Gato/patologia , Criptococose/veterinária , Criptococose/diagnóstico , Criptococose/microbiologia , Criptococose/patologia , Criptococose/tratamento farmacológico , Diagnóstico Diferencial , Neoplasias Nasofaríngeas/veterinária , Neoplasias Nasofaríngeas/diagnóstico , Alemanha , Tomografia Computadorizada por Raios X/veterinária , Doenças Nasofaríngeas/veterinária , Doenças Nasofaríngeas/diagnóstico , Doenças Nasofaríngeas/microbiologia , Doenças Nasofaríngeas/patologia
6.
Medicine (Baltimore) ; 103(26): e38671, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38941424

RESUMO

The aim of this study is to delineate the distinctive high-resolution computed tomography features of pulmonary cryptococcosis in non-HIV-infected patients. This retrospective analysis encompasses high-resolution computed tomography scans from 58 patients with histologically confirmed pulmonary cryptococcosis, focusing on the diagnostic challenges and the factors that lead to misdiagnosis. Analysis of computed tomography scans from these patients indicated that nodular or mass-like presentations were evident in 32 cases (55.2%), consolidation presentations in 7 cases (12.1%), and mixed presentations in 19 cases (32.8%). Lesions were predominantly located in the lower lobes of the lungs (40 cases, 69.0%) and in peripheral zones (55 cases, 94.8%). Notable radiographic signs included the presence of the burr sign in 55 cases (94.8%), lobulation sign in 53 cases (91.4%), halo sign in 53 cases (91.4%), and air bronchogram in 46 cases (79.0%). Moreover, 24 cases (41.4%) exhibited necrosis or cavitation, mediastinal lymphadenopathy was noted in 6 cases (10.3%), and pleural effusion was present in 5 cases (8.6%). Lesions were devoid of calcification. Pulmonary cryptococcosis ought to be contemplated in the differential diagnosis when computed tomography imaging exhibits patterns including, but not limited to, lower lobe and peripheral distribution, a broad base abutting the pleura, clustered growth with a propensity for fusion, air bronchogram within lesions, and peripheral halo sign.


Assuntos
Criptococose , Pneumopatias Fúngicas , Tomografia Computadorizada por Raios X , Humanos , Masculino , Feminino , Estudos Retrospectivos , Pessoa de Meia-Idade , Criptococose/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , Pneumopatias Fúngicas/diagnóstico por imagem , Adulto , Idoso , Pulmão/diagnóstico por imagem , Pulmão/patologia , Adulto Jovem
7.
Clin Lab ; 70(6)2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38868894

RESUMO

BACKGROUND: In July 2023, our hospital confirmed one case of lumbar spine infected complicated by Mycobacterium tuberculosis and Cryptococcus neoformans. The patient was admitted due to lower back pain for 1 year and a hard lump for 3 months. Symptoms and signs: Dressing can be seen fixed on the lower back, with severe bleeding. When the dressing is removed, a hard and protruding lump with a size of 6 cm x 8 cm, a sinus tract can be seen near the mass, with a slightly red wound and a sinus depth of about 3 cm. Light red fluid can be seen flowing out. There are no symptoms such as redness, swelling, or heat in the rest of the lower back, and the patient has no other underlying diseases or surgical history. METHODS: Lumbar magnetic resonance imaging and lumbar CT examination; Percutaneous puncture lumbar vertebral biopsy was performed, and the biopsy tissue was subjected to pathological examination, mNGS (metagenomic next-generation sequencing), and acid-fast staining; Extract pus from the lump for fungal culture and ink staining, and identify the fungi through MALDI-TOF MS. RESULTS: Bone destruction and bone marrow edema in the L5 vertebral body, compression of the spinal canal at the L5 vertebral body level; The pathological results of the biopsy tissue indicate granulomatous lesions. The acid-fast staining of the tissue is positive, and the mNGS of the tissue indicates infection with Mycobacterium tuberculosis. A single fungus was cultured from pus and identified by MALDI-TOF MS as Cryptococcus neoformans. Clinically, isoniazid 0.3 g ivgtt + rifampicin 0.45 g qd po + ethambutol 0.25 g qd po + pyrazinamide 0.75 g qd po + fluconazole 0.3 g qd po was administered for treatment. After 11 days, there was slight pain at the incision site, and the original symptoms were significantly relieved. The wound dressing was fixed in place, dry and without obvious exudation. Improved and discharged, followed up for 3 months with no recurrence of the lesion. CONCLUSIONS: mNGS is an effective identification technique that can be used to accurately diagnose suspected infection cases. MALDI-TOF MS has significant advantages over traditional detection methods in shortening detection time. This case achieved satisfactory treatment results for patients through a reasonable treatment plan, which is of great significance for exploring the diagnosis and treatment of similar disease infections.


Assuntos
Criptococose , Cryptococcus neoformans , Vértebras Lombares , Mycobacterium tuberculosis , Humanos , Cryptococcus neoformans/isolamento & purificação , Vértebras Lombares/microbiologia , Mycobacterium tuberculosis/isolamento & purificação , Criptococose/diagnóstico , Criptococose/microbiologia , Criptococose/tratamento farmacológico , Masculino , Tuberculose da Coluna Vertebral/diagnóstico , Tuberculose da Coluna Vertebral/microbiologia , Imageamento por Ressonância Magnética , Antituberculosos/uso terapêutico , Antituberculosos/administração & dosagem , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X
8.
Med Mycol ; 62(6)2024 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-38935902

RESUMO

Cryptococcosis causes a high burden of disease worldwide. This systematic review summarizes the literature on Cryptococcus neoformans and C. gattii infections to inform the World Health Organization's first Fungal Priority Pathogen List. PubMed and Web of Science were used to identify studies reporting on annual incidence, mortality, morbidity, antifungal resistance, preventability, and distribution/emergence in the past 10 years. Mortality rates due to C. neoformans were 41%-61%. Complications included acute renal impairment, raised intracranial pressure needing shunts, and blindness. There was moderate evidence of reduced susceptibility (MIC range 16-32 mg/l) of C. neoformans to fluconazole, itraconazole, ketoconazole, voriconazole, and amphotericin B. Cryptococcus gattii infections comprised 11%-33% of all cases of invasive cryptococcosis globally. The mortality rates were 10%-23% for central nervous system (CNS) and pulmonary infections, and ∼43% for bloodstream infections. Complications described included neurological sequelae (17%-27% in C. gattii infections) and immune reconstitution inflammatory syndrome. MICs were generally low for amphotericin B (MICs: 0.25-0.5 mg/l), 5-flucytosine (MIC range: 0.5-2 mg/l), itraconazole, posaconazole, and voriconazole (MIC range: 0.06-0.5 mg/l). There is a need for increased surveillance of disease phenotype and outcome, long-term disability, and drug susceptibility to inform robust estimates of disease burden.


Assuntos
Antifúngicos , Criptococose , Cryptococcus gattii , Cryptococcus neoformans , Farmacorresistência Fúngica , Organização Mundial da Saúde , Humanos , Criptococose/epidemiologia , Criptococose/microbiologia , Criptococose/mortalidade , Antifúngicos/uso terapêutico , Antifúngicos/farmacologia , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/isolamento & purificação , Testes de Sensibilidade Microbiana
10.
Front Immunol ; 15: 1397338, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38774865

RESUMO

Objectives: This manuscript undertakes a systematic examination of the research landscape concerning global Cryptococcus species and their dynamism with the host immune system spanning the past decade. It furnishes a detailed survey of leading knowledge institutions and critical focal points in this area, utilizing bibliometric analysis. Methods: VOSviewer and CiteSpace software platforms were employed to systematically analyze and graphically depict the relevant literature indexed in the WoSCC database over the preceding ten years. Results: In the interval between October 1, 2013, and October 1, 2023, a corpus of 795 publications was amassed. The primary research institutions involved in this study include Duke University, the University of Minnesota, and the University of Sydney. The leading trio of nations, in terms of publication volume, comprises the United States, China, and Brazil. Among the most prolific authors are Casadevall, Arturo; Wormley, Floyd L., Jr.; and Olszewski, Michal A., with the most highly cited author being Perfect, Jr. The most esteemed journal is Mbio, while Infection and Immunity commands the highest citation frequency, and the Journal of Clinical Microbiology boasts the most significant impact factor. Present research foci encompass the intricate interactions between Cryptococcus pathogenesis and host immunity, alongside immune mechanisms, complications, and immunotherapies. Conclusion: This represents the first exhaustive scholarly review and bibliometric scrutiny of the evolving landscapes in Cryptococcus research and its interactions with the host immune system. The analyses delineated herein provide insights into prevailing research foci and trajectories, thus furnishing critical directions for subsequent inquiries in this domain.


Assuntos
Bibliometria , Criptococose , Cryptococcus , Animais , Humanos , Criptococose/imunologia , Cryptococcus/imunologia , Interações Hospedeiro-Patógeno/imunologia , Sistema Imunitário/imunologia
11.
ACS Infect Dis ; 10(6): 2089-2100, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38819951

RESUMO

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, which poses a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semisynthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semisynthetic glycoconjugate vaccines contain an identical synthetic decasaccharide (M2 motif) antigen. This antigen is present in serotype A strains, which constitute 95% of the clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity toward M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced weakly opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). These findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. This antigen could serve as a component in a multivalent GXM motif vaccine.


Assuntos
Anticorpos Antifúngicos , Criptococose , Cryptococcus neoformans , Vacinas Fúngicas , Glicoconjugados , Vacinas Conjugadas , Cryptococcus neoformans/imunologia , Animais , Vacinas Fúngicas/imunologia , Camundongos , Criptococose/prevenção & controle , Criptococose/imunologia , Glicoconjugados/imunologia , Glicoconjugados/química , Vacinas Conjugadas/imunologia , Anticorpos Antifúngicos/imunologia , Feminino , Polissacarídeos/imunologia , Polissacarídeos/química , Camundongos Endogâmicos BALB C , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/química , Antígenos de Fungos/imunologia
12.
mBio ; 15(6): e0092024, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38742885

RESUMO

Cryptococcus neoformans causes cryptococcal meningoencephalitis, a disease that kills more than 180,000 people annually. Contributing to its success as a fungal pathogen is its cell wall surrounded by a capsule. When the cryptococcal cell wall is compromised, exposed pathogen-associated molecular pattern molecules (PAMPs) could trigger host recognition and initiate attack against this fungus. Thus, cell wall composition and structure are tightly regulated. The cryptococcal cell wall is unusual in that chitosan, the acetylated form of chitin, is predominant over chitin and is essential for virulence. Recently, it was shown that acidic pH weakens the cell wall and increases exposure of PAMPs partly due to decreased chitosan levels. However, the molecular mechanism responsible for the cell wall remodeling in acidic pH is unknown. In this study, by screening for genes involved in cryptococcal tolerance to high levels of CO2, we serendipitously discovered that the aspartyl peptidase May1 contributes to cryptococcal sensitivity to high levels of CO2 due to acidification of unbuffered media. Overexpression of MAY1 increases the cryptococcal cell size and elevates PAMP exposure, causing a hyper-inflammatory response in the host while MAY1 deletion does the opposite. We discovered that May1 weakens the cell wall and reduces the chitosan level, partly due to its involvement in the degradation of Chs3, the sole chitin synthase that supplies chitin to be converted to chitosan. Consistently, overexpression of CHS3 largely rescues the phenotype of MAY1oe in acidic media. Collectively, we demonstrate that May1 remodels the cryptococcal cell wall in acidic pH by reducing chitosan levels through its influence on Chs3. IMPORTANCE: The fungal cell wall is a dynamic structure, monitoring and responding to internal and external stimuli. It provides a formidable armor to the fungus. However, in a weakened state, the cell wall also triggers host immune attack when PAMPs, including glucan, chitin, and mannoproteins, are exposed. In this work, we found that the aspartyl peptidase May1 impairs the cell wall of Cryptococcus neoformans and increases the exposure of PAMPs in the acidic environment by reducing the chitosan level. Under acidic conditions, May1 is involved in the degradation of the chitin synthase Chs3, which supplies chitin to be deacetylated to chitosan. Consistently, the severe deficiency of chitosan in acidic pH can be rescued by overexpressing CHS3. These findings improve our understanding of cell wall remodeling and reveal a potential target to compromise the cell wall integrity in this important fungal pathogen.


Assuntos
Parede Celular , Cryptococcus neoformans , Proteínas Fúngicas , Cryptococcus neoformans/genética , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/patogenicidade , Parede Celular/metabolismo , Animais , Camundongos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/metabolismo , Concentração de Íons de Hidrogênio , Criptococose/microbiologia , Criptococose/patologia , Quitina/metabolismo , Virulência , Inflamação/microbiologia , Quitosana/metabolismo , Interações Hospedeiro-Patógeno
13.
mBio ; 15(6): e0060824, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38742909

RESUMO

Inositol tris/tetrakis phosphate kinases (IP3-4K) in the human fungal priority pathogens, Cryptococcus neoformans (CnArg1) and Candida albicans (CaIpk2), convey numerous virulence functions, yet it is not known whether the IP3-4K catalytic activity or a scaffolding role is responsible. We therefore generated a C. neoformans strain with a non-functional kinase, referred to as the dead-kinase (dk) CnArg1 strain (dkArg1). We verified that, although dkARG1 cDNA cloned from this strain produced a protein with the expected molecular weight, dkArg1 was catalytically inactive with no IP3-4K activity. Using recombinant CnArg1 and CaIpk2, we confirmed that, unlike the IP3-4K homologs in humans and Saccharomyces cerevisiae, CnArg1 and CaIpk2 do not phosphorylate the lipid-based substrate, phosphatidylinositol 4,5-bisphosphate, and therefore do not function as class I PI3Ks. Inositol polyphosphate profiling using capillary electrophoresis-electrospray ionization-mass spectrometry revealed that IP3 conversion is blocked in the dkArg1 and ARG1 deletion (Cnarg1Δ) strains and that 1-IP7 and a recently discovered isomer (4/6-IP7) are made by wild-type C. neoformans. Importantly, the dkArg1 and Cnarg1Δ strains had similar virulence defects, including suppressed growth at 37°C, melanization, capsule production, and phosphate starvation response, and were avirulent in an insect model, confirming that virulence is dependent on IP3-4K catalytic activity. Our data also implicate the dkArg1 scaffold in transcriptional regulation of arginine metabolism but via a different mechanism to S. cerevisiae since CnArg1 is dispensable for growth on different nitrogen sources. IP3-4K catalytic activity therefore plays a dominant role in fungal virulence, and IPK pathway function has diverged in fungal pathogens.IMPORTANCEThe World Health Organization has emphasized the urgent need for global action in tackling the high morbidity and mortality rates stemming from invasive fungal infections, which are exacerbated by the limited variety and compromised effectiveness of available drug classes. Fungal IP3-4K is a promising target for new therapy, as it is critical for promoting virulence of the human fungal priority pathogens, Cryptococcus neoformans and Candida albicans, and impacts numerous functions, including cell wall integrity. This contrasts to current therapies, which only target a single function. IP3-4K enzymes exert their effect through their inositol polyphosphate products or via the protein scaffold. Here, we confirm that the IP3-4K catalytic activity of CnArg1 promotes all virulence traits in C. neoformans that are attenuated by ARG1 deletion, reinforcing our ongoing efforts to find inositol polyphosphate effector proteins and to create inhibitors targeting the IP3-4K catalytic site, as a new antifungal drug class.


Assuntos
Cryptococcus neoformans , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/enzimologia , Virulência , Animais , Criptococose/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
14.
mSphere ; 9(6): e0028124, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38771036

RESUMO

The fungal pathogen Cryptococcus neoformans (C. neoformans) forms yeast cells of different sizes and morphological characteristics during infection. These features are usually not seen in standard laboratory in vitro conditions. Here, we describe in vivo cell morphologies when C. neoformans is grown in human plasma-like medium at 37°C, 5% CO2. We observed mixed-size populations of cells less than 1 µm up to 16.8 µm in cell diameter, increased capsule size, high chitin, and DNA content in larger cells. Our findings show that serum is not required for human plasma-like medium (HPLM)-induced C. neoformans cellular heterogeneity. Thus, this new method offers an opportunity to investigate factors of C. neoformans that mediate pathogenesis or host-pathogen interactions in a physiologically relevant setting.IMPORTANCEWe provide a description of new in vitro culture condition using the human plasma-like medium that supports the formation of the full range of in vivo cell morphologies of C. neoformans.


Assuntos
Criptococose , Cryptococcus neoformans , Meios de Cultura , Cryptococcus neoformans/citologia , Humanos , Meios de Cultura/química , Criptococose/microbiologia , Animais , Camundongos , Plasma/microbiologia , Interações Hospedeiro-Patógeno
15.
Indian J Med Microbiol ; 49: 100609, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38735642

RESUMO

We discuss a rare instance of cryptococcoma caused by Cryptococcus gattii in a 55-year-old woman initially treated for suspected COVID bronchopneumonia. The diagnosis posed a challenge due to vague symptoms and unclear imaging findings suggesting malignancy. Postoperative samples confirmed the presence of Cryptococcus gattii through culture of brain tissue and blood. Appropriate therapy was initiated, but despite treatment, it led to a fatal outcome. The case emphasizes the crucial role of microbiologist in early diagnosis of fungal infections of Central Nervous System. Additionally, the delayed diagnosis in immunocompetent individuals highlights the critical need for early recognition and intervention to mitigate potentially fatal outcomes.


Assuntos
Criptococose , Cryptococcus gattii , Glioblastoma , Humanos , Feminino , Pessoa de Meia-Idade , Cryptococcus gattii/isolamento & purificação , Criptococose/diagnóstico , Criptococose/microbiologia , Glioblastoma/diagnóstico , Diagnóstico Diferencial , Evolução Fatal , Encéfalo/patologia , Encéfalo/diagnóstico por imagem , Encéfalo/microbiologia , Neoplasias Encefálicas/diagnóstico , Antifúngicos/uso terapêutico , COVID-19/diagnóstico
16.
Front Cell Infect Microbiol ; 14: 1369301, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38774630

RESUMO

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.


Assuntos
Criptococose , Cryptococcus neoformans , Proteínas Fúngicas , Virulência , Animais , Feminino , Humanos , Camundongos , Parede Celular/metabolismo , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/genética , Cryptococcus neoformans/enzimologia , Modelos Animais de Doenças , Dano ao DNA , Cápsulas Fúngicas/metabolismo , Cápsulas Fúngicas/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Melaninas/metabolismo , Camundongos Endogâmicos BALB C , Estresse Oxidativo , Fosforilação , Sirolimo/farmacologia , Esporos Fúngicos/crescimento & desenvolvimento , Estresse Fisiológico
17.
Int Immunopharmacol ; 135: 112242, 2024 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-38772296

RESUMO

The emergence of Cryptococcus neoformans has posed an undeniable burden to many regions worldwide, with its strains mainly entering the lungs through the respiratory tract and spreading throughout the body. Limitations of drug regimens, such as high costs and limited options, have directed our attention toward the promising field of vaccine development. In this study, the subtractive proteomics approach was employed to select target proteins from databases that can accurately cover serotypes A and D of the Cryptococcus neoformans. Further, two multi-epitope vaccines consisting of T and B cell epitopes were demonstrated that they have good structural stability and could bind with immune receptor to induce desired immune responses in silico. After further evaluation, these vaccines show the potential for large-scale production and applicability to the majority of the population of the world. In summary, these two vaccines have been theoretically proven to combat Cryptococcus neoformans infections, awaiting further experimental validation of their actual protective effects.


Assuntos
Biologia Computacional , Criptococose , Cryptococcus neoformans , Epitopos de Linfócito B , Vacinas Fúngicas , Proteômica , Cryptococcus neoformans/imunologia , Vacinas Fúngicas/imunologia , Proteômica/métodos , Criptococose/imunologia , Criptococose/prevenção & controle , Humanos , Biologia Computacional/métodos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Animais , Antígenos de Fungos/imunologia , Proteínas Fúngicas/imunologia , Proteínas Fúngicas/química , Desenvolvimento de Vacinas , Imunoinformática
18.
Genetics ; 227(3)2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38752295

RESUMO

Cryptococcus neoformans is a fungal pathogen of the top critical priority recognized by the World Health Organization. This clinically important fungus also serves as a eukaryotic model organism. A variety of resources have been generated to facilitate investigation of the C. neoformans species complex, including congenic pairs, well-annotated genomes, genetic editing tools, and gene deletion sets. Here, we generated a set of strains with all major organelles fluorescently marked. We tested short organelle-specific targeting sequences and successfully labeled the following organelles by fusing the targeting sequences with a fluorescence protein: the plasma membrane, the nucleus, the peroxisome, and the mitochondrion. We used native cryptococcal Golgi and late endosomal proteins fused with a fluorescent protein to label these two organelles. These fluorescence markers were verified via colocalization using organelle-specific dyes. All the constructs for the fluorescent protein tags were integrated in an intergenic safe haven region. These organelle-marked strains were examined for growth and various phenotypes. We demonstrated that these tagged strains could be employed to track cryptococcal interaction with the host in phagocytosis assays. These strains also allowed us to discover remarkable differences in the dynamics of proteins targeted to different organelles during sexual reproduction. Additionally, we revealed that "dormant" spores transcribed and synthesized their own proteins and trafficked the proteins to the appropriate subcellular compartments, demonstrating that spores are metabolically active. We anticipate that these newly generated fluorescent markers will greatly facilitate further investigation of cryptococcal biology and pathogenesis.


Assuntos
Cryptococcus neoformans , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Animais , Criptococose/microbiologia , Fagocitose , Camundongos , Organelas/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética
19.
BMC Infect Dis ; 24(1): 533, 2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38802753

RESUMO

BACKGROUND: Hepatitis B virus (HBV) infection can cause liver failure, while individuals with Acquired Immunodeficiency Virus Disease (AIDS) are highly susceptible to various opportunistic infections, which can occur concurrently. The treatment process is further complicated by the potential occurrence of immune reconstitution inflammatory syndrome (IRIS), which presents significant challenges and contributes to elevated mortality rates. CASE PRESENTATION: The 50-year-old male with a history of chronic hepatitis B and untreated human immunodeficiency virus (HIV) infection presented to the hospital with a mild cough and expectoration, revealing multi-drug resistant pulmonary tuberculosis (MDR-PTB), which was confirmed by XpertMTB/RIF PCR testing and tuberculosis culture of bronchoalveolar lavage fluid (BALF). The patient was treated with a regimen consisting of linezolid, moxifloxacin, cycloserine, pyrazinamide, and ethambutol for tuberculosis, as well as a combination of bictegravir/tenofovir alafenamide/emtricitabine (BIC/TAF/FTC) for HBV and HIV viral suppression. After three months of treatment, the patient discontinued all medications, leading to hepatitis B virus reactivation and subsequent liver failure. During the subsequent treatment for AIDS, HBV, and drug-resistant tuberculosis, the patient developed disseminated cryptococcal disease. The patient's condition worsened during treatment with liposomal amphotericin B and fluconazole, which was ultimately attributed to IRIS. Fortunately, the patient achieved successful recovery after appropriate management. CONCLUSION: Enhancing medical compliance is crucial for AIDS patients, particularly those co-infected with HBV, to prevent HBV reactivation and subsequent liver failure. Furthermore, conducting a comprehensive assessment of potential infections in patients before resuming antiviral therapy is essential to prevent the occurrence of IRIS. Early intervention plays a pivotal role in improving survival rates.


Assuntos
Criptococose , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose Pulmonar , Humanos , Masculino , Pessoa de Meia-Idade , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Criptococose/complicações , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/complicações , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/complicações , Falência Hepática/virologia , Síndrome da Imunodeficiência Adquirida/complicações , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Hepatite B Crônica/complicações , Hepatite B Crônica/tratamento farmacológico , Coinfecção/tratamento farmacológico , Coinfecção/microbiologia , Coinfecção/virologia , Antituberculosos/uso terapêutico , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Infecções Oportunistas Relacionadas com a AIDS/microbiologia
20.
Methods Mol Biol ; 2775: 47-55, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38758310

RESUMO

In vivo models provide advantages to study the progression of disease and to identify potential biomarkers to detect and monitor infections. For the human fungal pathogen Cryptococcus neoformans, murine intranasal models aim to recapitulate natural infection from inhalation of desiccated fungal cells from the environment and permit monitoring of disease over time. In this chapter, we describe the establishment of a murine model for cryptococcosis and the subsequent collection of organs, tissues, and fluids for sampling. These samples may support novel diagnostic strategies and opportunities to monitor dissemination of the fungal cells throughout the host and propose new treatment options to combat disease.


Assuntos
Criptococose , Cryptococcus neoformans , Modelos Animais de Doenças , Animais , Cryptococcus neoformans/patogenicidade , Criptococose/microbiologia , Criptococose/diagnóstico , Camundongos , Manejo de Espécimes/métodos , Humanos
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