Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis

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.     

Brain Inositol Is a Novel Stimulator for Promoting Cryptococcus Penetration of the Blood-Brain Barrier

Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. The facts that human and animal brains contain abundant inositol and that Cryptococcus has a sophisticated system for the acquisition of inositol from the environment suggests that host inositol utilization may contribute to the development of cryptococcal meningitis. In this study, we found that inositol plays an important role in Cryptococcus traversal across the blood-brain barrier (BBB) both in an in vitro human BBB model and in in vivo animal models. The capacity of inositol to stimulate BBB crossing was dependent upon fungal inositol transporters, indicated by a 70% reduction in transmigration efficiency in mutant strains lacking two major inositol transporters, Itr1a and Itr3c. Upregulation of genes involved in the inositol catabolic pathway was evident in a microarray analysis following inositol treatment. In addition, inositol increased the production of hyaluronic acid in Cryptococcus cells, which is a ligand known to binding host CD44 receptor for their invasion. These studies suggest an inositol-dependent Cryptococcus traversal of the BBB, and support our hypothesis that utilization of host-derived inositol by Cryptococcus contributes to CNS infection.     

Cryptococcus Strains with Different Pathogenic Potentials Have Diverse Protein Secretomes

Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.     

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFNγ) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.     

Oropharyngeal Aspiration of Burkholderia mallei and Burkholderia pseudomallei in BALB/c Mice

Burkholderia mallei and Burkholderia pseudomallei are potentially lethal pathogens categorized as biothreat agents due, in part, to their ability to be disseminated via aerosol. There are no protective vaccines against these pathogens and treatment options are limited and cumbersome. Since disease severity is greatest when these agents are inhaled, efforts to develop pre- or post-exposure prophylaxis focus largely on inhalation models of infection. Here, we demonstrate a non-invasive and technically simple method for affecting the inhalational challenge of BALB/c mice with B. pseudomallei and B. mallei. In this model, two investigators utilized common laboratory tools such as forceps and a micropipette to conduct and characterize an effective and reproducible inhalational challenge of BALB/c mice with B. mallei and B. pseudomallei. Challenge by oropharyngeal aspiration resulted in acute disease. Additionally, 50% endpoints for B. pseudomallei K96243 and B. mallei ATCC 23344 were nearly identical to published aerosol challenge methods. Furthermore, the pathogens disseminated to all major organs typically targeted by these agents where they proliferated. The pro-inflammatory cytokine production in the proximal and peripheral fluids demonstrated a rapid and robust immune response comparable to previously described murine and human studies. These observations demonstrate that OA is a viable alternative to aerosol exposure.     

Tongue lesion due to Cryptococcus neoformans as the first finding in an HIV-positive patient

BackgroundCryptococcosis is a severe universally distributed mycosis which mainly affects immunocompromised hosts. This mycosis is caused by yeasts of two species complex of the genus Cryptococcus: Cryptococcus neoformans and Cryptococcus gattii. Meningeal cryptococcosis is the most frequent clinical presentation of this disseminated mycosis. The oral mucosa involvement is extremely unusual.Case reportWe present a case of cryptococcosis with an unusual clinical form. The patient was assisted because she had an ulcerated lesion on the lingual mucosa. Encapsulated yeasts compatible with Cryptococcus were found in microscopic exams of wet preparations from lingual ulcer clinical samples obtained for cytodiagnosis and mycological studies. Cryptococcus neoformans (C. neoformans var. grubii VNI) was isolated in culture. This patient did not know her condition of HIV seropositive before the appearance of the tongue lesion.ConclusionsThe involvement of the oral mucosa is uncommon in this fungal infection, but is important to include it in the differential diagnosis in HIV positive patients.     

An Acidic Microenvironment Increases NK Cell Killing of Cryptococcus neoformans and Cryptococcus gattii by Enhancing Perforin Degranulation

Cryptococcus gattii and Cryptococcus neoformans are encapsulated yeasts that can produce a solid tumor-like mass or cryptococcoma. Analogous to malignant tumors, the microenvironment deep within a cryptococcoma is acidic, which presents unique challenges to host defense. Analogous to malignant cells, NK cells kill Cryptococcus. Thus, as in tumor defense, NK cells must kill yeast cells across a gradient from physiologic pH to less than 6 in the center of the cryptococcoma. As acidic pH inhibits anti-tumor activities of NK cells, we sought to determine if there was a similar reduction in the anticryptococcal activity of NK cells. Surprisingly, we found that both primary human NK cells and the human NK cell line, YT, have preserved or even enhanced killing of Cryptococcus in acidic, compared to physiological, pH. Studies to explore the mechanism of enhanced killing revealed that acidic pH does not increase the effector to target ratio, binding of cytolytic cells to Cryptococcus, or the active perforin content in effector cells. By contrast, perforin degranulation was greater at acidic pH, and increased degranulation was preceded by enhanced ERK1/2 phosphorylation, which is essential for killing. Moreover, using a replication defective ras1 knockout strain of Cryptococcus increased degranulation occurred during more rapid replication of the organisms. Finally, NK cells were found intimately associated with C. gattii within the cryptococcoma of a fatal infection. These results suggest that NK cells have amplified signaling, degranulation, and greater killing at low pH and when the organisms are replicating quickly, which would help maintain microbicidal host defense despite an acidic microenvironment.     

Factors enforcing the species boundary between the human pathogens Cryptococcus neoformans and Cryptococcus deneoformans

Hybridization has resulted in the origin and variation in extant species, and hybrids continue to arise despite pre- and post-zygotic barriers that limit their formation and evolutionary success. One important system that maintains species boundaries in prokaryotes and eukaryotes is the mismatch repair pathway, which blocks recombination between divergent DNA sequences. Previous studies illuminated the role of the mismatch repair component Msh2 in blocking genetic recombination between divergent DNA during meiosis. Loss of Msh2 results in increased interspecific genetic recombination in bacterial and yeast models, and increased viability of progeny derived from yeast hybrid crosses. Hybrid isolates of two pathogenic fungal Cryptococcus species, Cryptococcus neoformans and Cryptococcus deneoformans, are isolated regularly from both clinical and environmental sources. In the present study, we sought to determine if loss of Msh2 would relax the species boundary between C. neoformans and C. deneoformans. We found that crosses between these two species in which both parents lack Msh2 produced hybrid progeny with increased viability and high levels of aneuploidy. Whole-genome sequencing revealed few instances of recombination among hybrid progeny and did not identify increased levels of recombination in progeny derived from parents lacking Msh2. Several hybrid progeny produced structures associated with sexual reproduction when incubated alone on nutrient-rich medium in light, a novel phenotype in Cryptococcus. These findings represent a unique, unexpected case where rendering the mismatch repair system defective did not result in increased meiotic recombination across a species boundary. This suggests that alternative pathways or other mismatch repair components limit meiotic recombination between homeologous DNA and enforce species boundaries in the basidiomycete Cryptococcus species.     

Successful treatment of Cryptococcus gattii neurocryptococcosis in a 5-year-old immunocompetent child from the French Guiana Amazon region

Compared to the incidence in adults, cryptococcosis is rare among children. We report a case of neurocryptococcosis due to Cryptococcus gattii in a five-year-old girl without identified risk factors living in French Guiana. Neurological surgery in combination with long-term antifungal treatment with amphotericin B and 5-flucytosine successfully resolved the cryptococcal infection. Subsequent molecular characterization of the Cryptococcus isolate revealed that the infection was caused by a C. gattii genotype AFLP6B/VGIIb strain.     

DNA Mutations Mediate Microevolution between Host-Adapted Forms of the Pathogenic Fungus Cryptococcus neoformans

The disease cryptococcosis, caused by the fungus Cryptococcus neoformans, is acquired directly from environmental exposure rather than transmitted person-to-person. One explanation for the pathogenicity of this species is that interactions with environmental predators select for virulence. However, co-incubation of C. neoformans with amoeba can cause a “switch” from the normal yeast morphology to a pseudohyphal form, enabling fungi to survive exposure to amoeba, yet conversely reducing virulence in mammalian models of cryptococcosis. Like other human pathogenic fungi, C. neoformans is capable of microevolutionary changes that influence the biology of the organism and outcome of the host-pathogen interaction. A yeast-pseudohyphal phenotypic switch also happens under in vitro conditions. Here, we demonstrate that this morphological switch, rather than being under epigenetic control, is controlled by DNA mutation since all pseudohyphal strains bear mutations within genes encoding components of the RAM pathway. High rates of isolation of pseudohyphal strains can be explained by the physical size of RAM pathway genes and a hypermutator phenotype of the strain used in phenotypic switching studies. Reversion to wild type yeast morphology in vitro or within a mammalian host can occur through different mechanisms, with one being counter-acting mutations. Infection of mice with RAM mutants reveals several outcomes: clearance of the infection, asymptomatic maintenance of the strains, or reversion to wild type forms and progression of disease. These findings demonstrate a key role of mutation events in microevolution to modulate the ability of a fungal pathogen to cause disease.     

Warfare and defense: The host response to Cryptococcus infection

Cryptococcus neoformans and Cryptococcus gatti are the etiological agents of cryptococcosis, a life-threatening mycosis affecting the central nervous system. Cryptococcal meningoencephalitis is the most fatal mycosis in AIDS patients, resulting almost 200 000 deaths annually. High cost, side effects and drug resistance are constant elements during treatment of cryptococcosis, encouraging the development of novel therapeutic strategies including immunomodulatory protocols. Thereby, to understand how the host responds to Cryptococcus is essential. In this review, we discuss the immune response against Cryptococcus and immunoevasion strategies.     

Ventriculoperitoneal shunt infection by Cryptococcus neoformans sensu stricto: Case report and literature review

BackgroundCryptococcal ventriculoperitoneal shunt infection is known to occur due to an underlying infection in the patient rather than by nosocomial transmission of Cryptococcus during shunt placement. A case of chronic hydrocephalus due to cryptococcal meningitis that was misdiagnosed as tuberculous meningitis is described.Case reportPatient details were extracted from charts and laboratory records. The identification of the isolate was confirmed by PCR-restriction fragment length polymorphism of the orotodine monophosphate pyrophosphorylase (URA5) gene. Antifungal susceptibility was determined using the CLSI M27-A3 broth microdilution method. Besides, a Medline search was performed to review all cases of Cryptococcus ventriculoperitoneal shunt infection. Cryptococcus neoformans sensu stricto (formerly Cryptococcus neoformans var. grubii), mating-type MATα was isolated from the cerebrospinal fluid and external ventricular drain tip. The isolate showed low minimum inhibitory concentrations for voriconazole (0.06 mg/l), fluconazole (8 mg/l), isavuconazole (<0.015 mg/l), posaconazole (<0.03 mg/l), amphotericin B (<0.06 mg/l) and 5-fluorocytosine (1 mg/l). The patient was treated with intravenous amphotericin B deoxycholate, but died of cardiopulmonary arrest on the fifteenth postoperative day.ConclusionsThis report underlines the need to rule out a Cryptococcus infection in those cases of chronic meningitis with hydrocephalus.     

Pigment Production on L-Tryptophan Medium by Cryptococcus gattii and Cryptococcus neoformans

In recent years strains previously grouped within Cryptococcus neoformans have been divided into two species C. neoformans and C. gattii, with Cryptococcus neoformans comprising serotypes A, D, and AD and C. gattii comprising serotypes B and C. Cryptococcus neoformans have also been subdivided into two varieties C. neoformans var. grubii, serotype A, and C. neoformans var. neoformans, serotype D. We analyzed the growth and pigment production characteristics of 139 strains of Cryptococcus spp. in L-tryptophan containing media. Nearly all strains of Cryptococcus, including each variety and serotype tested produced a pink water-soluble pigment (molecular weight of 535.2 Da) from L-tryptophan. Consequently, the partial separation of the species was based on whether the pink pigment was secreted into the medium (extracellular) or retained as an intracellular pigment. On L-tryptophan medium C. neoformans var. grubii and serotype AD produced a pink extracellular pigment. In contrast, for C. gattii, the pink pigment was localized intracellularly and masked by heavy production of brown pigments. Pigment production by C. neoformans var. neoformans was variable with some strains producing the pink extracellular pigment and others retained the pink pigment intracellularly. The pink intracellular pigment produced by strains of C. neoformans var. neoformans was masked by production of brown pigments. Cryptococcus laccase mutants failed to produce pigments from L-tryptophan. This is the first report that the enzyme laccase is involved in tryptophan metabolism. Prior to this report Cryptococcus laccase produced melanin or melanin like-pigments from heterocyclic compounds that contained ortho or para diphenols, diaminobenzenes and aminophenol compounds. The pigments produced from L-tryptophan were not melanin.     

Cytoplasmic inorganic polyphosphate participates in the heavy metal tolerance of Cryptococcus humicola

The basidiomycetous yeast Cryptococcus humicola was shown to be tolerant to manganese, cobalt, nickel, zinc, lanthanum, and cadmium cations at a concentration of 2.5 mmol/L, which is toxic for many yeasts. The basidiomycetous yeast Cryptococcus terreus was sensitive to all these ions and did not grow at the above concentration. In the presence of heavy metal cations, С. humicola, as opposed to C. terreus, was characterized by the higher content of acid-soluble inorganic polyphosphates. In vivo 4′,6′-diamino-2-phenylindole dihydrochloride staining revealed polyphosphate accumulation in the cell wall and cytoplasmic inclusions of С. humicola in the presence of heavy metals. In C. terreus, polyphosphates in the presence of heavy metals accumulate mainly in vacuoles, which results in morphological changes in these organelles and, probably, disturbance of their function. The role of polyphosphate accumulation and cellular localization as factors of heavy metal tolerance of Cryptococcus humicola is discussed.     

Native trees of the Northeast Argentine: Natural hosts of the Cryptococcus neoformans–Cryptococcus gattii species complex

BackgroundIn Argentina, information about epidemiology and environmental distribution of Cryptococcus is scarce. The city of Resistencia borders with Brazil and Paraguay where this fungus is endemic. All these supported the need to investigate the ecology of the genus and the epidemiology of cryptococcosis in this area.AimsThe aim was to investigate the presence of species of Cryptococcus neoformans–Cryptococcus gattii complex and their genotypes in trees of the city of Resistencia.MethodsOne hundred and five trees were sampled by swabbing technique. The isolates were identified using conventional and commercial methods and genotyped by PCR-RFLP (Restriction Fragment Length Polymorphism).ResultsCryptococcus was found in 7 out of the total trees. 6 out of 7 Cryptococcus isolates were identified as C. neoformans and one as C. gattii. C. gattii was isolated from Grevillea robusta. C. neoformans strains were isolated from Tabebuia avellanedae and Peltophorum dubium. Genotyping showed that all C. neoformans belonged to the VNI type and C. gattii belonged to the VGI type.ConclusionsThis represents the first study on the ecology of Cryptococcus spp. associated to trees from northeastern Argentina, and the first report describing Grevillea robusta as a host of members of this fungal genus. Another finding is the isolation of C. neoformans from Tabebuia avellanedae and Peltophorum dubium, both tree species native to northeastern Argentina.     

De novo GTP Biosynthesis Is Critical for Virulence of the Fungal Pathogen Cryptococcus neoformans

We have investigated the potential of the GTP synthesis pathways as chemotherapeutic targets in the human pathogen Cryptococcus neoformans, a common cause of fatal fungal meningoencephalitis. We find that de novo GTP biosynthesis, but not the alternate salvage pathway, is critical to cryptococcal dissemination and survival in vivo. Loss of inosine monophosphate dehydrogenase (IMPDH) in the de novo pathway results in slow growth and virulence factor defects, while loss of the cognate phosphoribosyltransferase in the salvage pathway yielded no phenotypes. Further, the Cryptococcus species complex displays variable sensitivity to the IMPDH inhibitor mycophenolic acid, and we uncover a rare drug-resistant subtype of C. gattii that suggests an adaptive response to microbial IMPDH inhibitors in its environmental niche. We report the structural and functional characterization of IMPDH from Cryptococcus, revealing insights into the basis for drug resistance and suggesting strategies for the development of fungal-specific inhibitors. The crystal structure reveals the position of the IMPDH moveable flap and catalytic arginine in the open conformation for the first time, plus unique, exploitable differences in the highly conserved active site. Treatment with mycophenolic acid led to significantly increased survival times in a nematode model, validating de novo GTP biosynthesis as an antifungal target in Cryptococcus.     

Innate host defenses against <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, can cause life-threatening infections of the central nervous system in immunocompromised and immunocompetent individuals. Cryptococcal meningoencephalitis is the most common disseminated fungal infection in AIDS patients, and remains the third most common invasive fungal infection among organ transplant recipients. The administration of highly active antiretroviral therapy (HAART) has resulted in a decrease in the number of cases of AIDS-related cryptococcosis in developed countries, but in developing countries where HAART is not readily available, Cryptococcus is still a major concern. Therefore, there is an urgent need for the development of novel therapies and/or vaccines to combat cryptococcosis. Understanding the protective immune responses against Cryptococcus is critical for development of vaccines and immunotherapies to combat cryptococcosis. Consequently, this review focuses on our current knowledge of protective immune responses to C. neoformans, with an emphasis on innate immune responses.     

Killed Saccharomyces cerevisiae Protects Against Lethal Challenge of Cryptococcus grubii

Heat-killed Saccharomyces cerevisiae (HKY) vaccination protects mice against aspergillosis, coccidioidomycosis, mucormycosis, or candidiasis. We studied HKY protection against murine cryptococcosis. Once weekly subcutaneous HKY doses (S, 6 × 10⁷; 2S, 1.2 × 10⁸; 3S, 2.4 × 10⁸) began 28 (×3), 35 (×4), or 42 (×6) days prior to intravenous Cryptococcus grubii infection. Survival through 28 days, and CFU in the organs of survivors, were compared to saline-vaccinated controls. In the initial experiment, S, S×4, or 2S reduced brain CFU; liver or spleen CFU was reduced by S×4 or 2S. In a more lethal second experiment, 2S×6, 2S, or 3S×4 improved survival, and HKY regimens reduced CFU in the brain, liver, or spleen, with 2S×6, 2S, or 3S×4 most efficacious. Dose size appears more important than the number of doses: Regimens >S were superior, and 2S and 2S×6 were equivalent. 2S and 3S were equivalent, suggesting doses >2S do not provide additional protection. HKY protects against Cryptococcus, supporting components of HKY as a basis for the development of a panfungal vaccine.