Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus.

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.

A conceptual framework for nomenclatural stability and validity of medically important fungi: a proposed global consensus guideline for fungal name changes supported by ABP, ASM, CLSI, ECMM, ESCMID-EFISG, EUCAST-AFST, FDLC, IDSA, ISHAM, MMSA, and MSGERC.

The rapid pace of name changes of medically important fungi is creating challenges for clinical laboratories and clinicians involved in patient care. We describe two sources of name change which have different drivers, at the species versus the genus level. Some suggestions are made here to reduce the number of name changes. We urge taxonomists to provide diagnostic markers of taxonomic novelties. Given the instability of phylogenetic trees due to variable taxon sampling, we advocate to maintain genera at the largest possible size. Reporting of identified species in complexes or series should where possible comprise both the name of the overarching species and that of the molecular sibling, often cryptic species. Because the use of different names for the same species will be unavoidable for many years to come, an open access online database of the names of all medically important fungi, with proper nomenclatural designation and synonymy, is essential. We further recommend that while taxonomic discovery continues, the adaptation of new name changes by clinical laboratories and clinicians be reviewed routinely by a standing committee for validation and stability over time, with reference to an open access database, wherein reasons for changes are listed in a transparent way.

Cryptococcus neoformans requires the TVF1 gene for thermotolerance and virulence.

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.

Neutralizing GM-CSF autoantibodies in pulmonary alveolar proteinosis, cryptococcal meningitis and severe nocardiosis.

BACKGROUND: Anti GM-CSF autoantibodies (aAb) have been related to acquired pulmonary alveolar proteinosis (PAP) and described in cases of severe infections such as cryptococcosis and nocardiosis in previously healthy subjects. Whether there are different anti-GM-CSF autoantibodies corresponding to these phenotypes is unclear. Therefore, we examined anti-GM-CSF autoantibodies to determine whether amount or neutralizing activity could distinguish between groups. METHODS: Plasma samples gathered in the National Institute of Health from patients with anti GM-CSF aAb and either PAP (n = 15), cryptococcal meningitis (n = 15), severe nocardiosis (n = 5) or overlapping phenotypes (n = 6) were compared. The relative amount of aAb was assessed using a particle-based approach, reported as a mouse monoclonal anti-human GM-CSF as standard curve and expressed in an arbitrary Mouse Monoclonal Antibody Unit (MMAU). The neutralizing activity of the plasma was assessed by inhibition of GM-CSF-induced intracellular phospho-STAT5 (pSTAT5) in monocytes. RESULTS: Anti-GM-CSF aAb relative amounts were higher in PAP patients compared to those with cryptococcosis (mean 495 ± 464 MMAU vs 197 ± 159 MMAU, p = 0.02); there was no difference with patients with nocardiosis (430 ± 493 MMAU) nor between the two types of infections. The dilution of plasma resulting in 50% inhibition of GM-CSF-induced pSTAT5 (approximate IC50) did not vary appreciably across groups of patients (1.6 ± 3.1%, 3.9 ± 6% and 1.8 ± 2.2% in PAP patients, cryptococcosis and nocardiosis patients, respectively). Nor was the concentration of GM-CSF necessary to induce 50% of maximal GM-CSF-induced pSTAT5 in the presence of 10 MMAU of anti-GM-CSF aAb (EC50). When studying longitudinal samples from patients with PAP or disseminated nocardiosis, the neutralizing effect of anti-GM-CSF aAb was relatively constant over time despite targeted treatments and variations in aAb levels. CONCLUSIONS: Despite different clinical manifestations, anti-GM-CSF antibodies were similar across PAP, cryptococcosis and nocardiosis. Underlying host genetics and functional analyses may help further differentiate the biology of these conditions.

Antifungal Susceptibility Profiles of Olorofim (Formerly F901318) and Currently Available Systemic Antifungals against Mold and Yeast Phases of Talaromyces marneffei.

In vitro antifungal susceptibility profiling of 32 clinical and environmental Talaromyces marneffei isolates recovered from southern China was performed against olorofim and 7 other systemic antifungals, including amphotericin B, 5-flucytosine, posaconazole, voriconazole, caspofungin, and terbinafine, using CLSI methodology. In comparison, olorofim was the most active antifungal agent against both mold and yeast phases of all tested Talaromyces marneffei isolates, exhibiting an MIC range, MIC50, and MIC90 of 0.0005 to 0.002 µg/ml, 0.0005 µg/ml, and 0.0005 µg/ml, respectively.

Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment.

Cryptococcus neoformans and Cryptococcus gattii species complexes are the etiologic agents of cryptococcosis. We have deciphered the roles of three ABC transporters, Afr1, Afr2, and Mdr1, in the representative strains of the two species, C. neoformans H99 and C. gattii R265. Deletion of AFR1 in H99 and R265 drastically reduced the levels of resistance to three xenobiotics and three triazoles, suggesting that Afr1 is the major drug efflux pump in both strains. Fluconazole susceptibility was not affected when AFR2 or MDR1 was deleted in both strains. However, when these genes were deleted in combination with AFR1, a minor additive effect in susceptibility toward several drugs was observed. Deletion of all three genes in both strains caused further increases in susceptibility toward fluconazole and itraconazole, suggesting that Afr2 and Mdr1 augment Afr1 function in pumping these triazoles. Intracellular accumulation of Nile Red significantly increased in afr1Δ mutants of both strains, but rhodamine 6G accumulation increased only in the mdr1Δ mutant of H99. Thus, the three efflux pumps play different roles in the two strains when exposed to different azoles and xenobiotics. AFR1 and AFR2 expression was upregulated in H99 and R265 when treated with fluconazole. However, MDR1 expression was upregulated only in R265 under the same conditions. We screened a library of transcription factor mutants and identified several mutants that manifested either altered fluconazole sensitivity or an increase in the frequency of fluconazole heteroresistance. Gene expression analysis suggests that the three efflux pumps are regulated independently by different transcription factors in response to fluconazole exposure.

Cryptococcus gattii Capsule Blocks Surface Recognition Required for Dendritic Cell Maturation Independent of Internalization and Antigen Processing.

Cryptococcus gattii is an emerging fungal pathogen on the west coast of Canada and the United States that causes a potentially fatal infection in otherwise healthy individuals. In previous investigations of the mechanisms by which C. gattii might subvert cell-mediated immunity, we found that C. gattii failed to induce dendritic cell (DC) maturation, leading to defective T cell responses. However, the virulence factor and the mechanisms of evasion of DC maturation remain unknown. The cryptococcal polysaccharide capsule is a leading candidate because of its antiphagocytic properties. Consequently, we asked if the capsule of C. gattii was involved in evasion of DC maturation. We constructed an acapsular strain of C. gattii through CAP59 gene deletion by homologous integration. Encapsulated C. gattii failed to induce human monocyte-derived DC maturation and T cell proliferation, whereas the acapsular mutant induced both processes. Surprisingly, encapsulation impaired DC maturation independent of its effect on phagocytosis. Indeed, DC maturation required extracellular receptor signaling that was dependent on TNF-α and p38 MAPK, but not ERK activation, and the cryptococcal capsule blocked this extracellular recognition. Although the capsule impaired phagocytosis that led to pH-dependent serine-, threonine-, and cysteine-sensitive protease-dependent Ag processing, it was insufficient to impair T cell responses. In summary, C. gattii affects two independent processes, leading to DC maturation and Ag processing. The polysaccharide capsule masked extracellular detection and reduced phagocytosis that was required for DC maturation and Ag processing, respectively. However, the T cell response was fully restored by inducing DC maturation.

Fundamental niche prediction of the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii in Europe.

Fundamental niche prediction of Cryptococcus neoformans and Cryptococcus gattii in Europe is an important tool to understand where these pathogenic yeasts have a high probability to survive in the environment and therefore to identify the areas with high risk of infection. In this study, occurrence data for C. neoformans and C. gattii were compared by MaxEnt software with several bioclimatic conditions as well as with soil characteristics and land use. The results showed that C. gattii distribution can be predicted with high probability along the Mediterranean coast. The analysis of variables showed that its distribution is limited by low temperatures during the coldest season, and by heavy precipitations in the driest season. C. neoformans var. grubii is able to colonize the same areas of C. gattii but is more tolerant to cold winter temperatures and summer precipitations. In contrast, the C. neoformans var. neoformans map was completely different. The best conditions for its survival were displayed in sub-continental areas and not along the Mediterranean coasts. In conclusion, we produced for the first time detailed prediction maps of the species and varieties of the C. neoformans and C. gattii species complex in Europe and Mediterranean area.

Type I IFN Induction via Poly-ICLC Protects Mice against Cryptococcosis.

Cryptococcus neoformans is the most common cause of fungal meningoencephalitis in AIDS patients. Depletion of CD4 cells, such as occurs during advanced AIDS, is known to be a critical risk factor for developing cryptococcosis. However, the role of HIV-induced innate inflammation in susceptibility to cryptococcosis has not been evaluated. Thus, we sought to determine the role of Type I IFN induction in host defense against cryptococci by treatment of C. neoformans (H99) infected mice with poly-ICLC (pICLC), a dsRNA virus mimic. Unexpectedly, pICLC treatment greatly extended survival of infected mice and reduced fungal burdens in the brain. Protection from cryptococcosis by pICLC-induced Type I IFN was mediated by MDA5 rather than TLR3. PICLC treatment induced a large, rapid and sustained influx of neutrophils and Ly6Chigh monocytes into the lung while suppressing the development of eosinophilia. The pICLC-mediated protection against H99 was CD4 T cell dependent and analysis of CD4 T cell polyfunctionality showed a reduction in IL-5 producing CD4 T cells, marginal increases in Th1 cells and dramatic increases in RORγt+ Th17 cells in pICLC treated mice. Moreover, the protective effect of pICLC against H99 was diminished in IFNγ KO mice and by IL-17A neutralization with blocking mAbs. Furthermore, pICLC treatment also significantly extended survival of C. gattii infected mice with reduced fungal loads in the lungs. These data demonstrate that induction of type I IFN dramatically improves host resistance against the etiologic agents of cryptococcosis by beneficial alterations in both innate and adaptive immune responses.

Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus's Role in Virulence.

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.

Molecular mechanisms of hypoxic responses via unique roles of Ras1, Cdc24 and Ptp3 in a human fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans encounters a low oxygen environment when it enters the human host. Here, we show that the conserved Ras1 (a small GTPase) and Cdc24 (the guanine nucleotide exchange factor for Cdc42) play an essential role in cryptococcal growth in hypoxia. Suppressor studies indicate that PTP3 functions epistatically downstream of both RAS1 and CDC24 in regulating hypoxic growth. Ptp3 shares sequence similarity to the family of phosphotyrosine-specific protein phosphatases and the ptp3Δ strain failed to grow in 1% O2. We demonstrate that RAS1, CDC24 and PTP3 function in parallel to regulate thermal tolerance but RAS1 and CDC24 function linearly in regulating hypoxic growth while CDC24 and PTP3 reside in compensatory pathways. The ras1Δ and cdc24Δ strains ceased to grow at 1% O2 and became enlarged but viable single cells. Actin polarization in these cells, however, was normal for up to eight hours after transferring to hypoxic conditions. Double deletions of the genes encoding Rho GTPase Cdc42 and Cdc420, but not of the genes encoding Rac1 and Rac2, caused a slight growth retardation in hypoxia. Furthermore, growth in hypoxia was not affected by the deletion of several central genes functioning in the pathways of cAMP, Hog1, or the two-component like phosphorylation system that are critical in the cryptococcal response to osmotic and genotoxic stresses. Interestingly, although deletion of HOG1 rescued the hypoxic growth defect of ras1Δ, cdc24Δ, and ptp3Δ, Hog1 was not hyperphosphorylated in these three mutants in hypoxic conditions. RNA sequencing analysis indicated that RAS1, CDC24 and PTP3 acted upon the expression of genes involved in ergosterol biosynthesis, chromosome organization, RNA processing and protein translation. Moreover, growth of the wild-type strain under low oxygen conditions was affected by sub-inhibitory concentrations of the compounds that inhibit these biological processes, demonstrating the importance of these biological processes in the cryptococcal hypoxia response.

Surface structure characterization of Aspergillus fumigatus conidia mutated in the melanin synthesis pathway and their human cellular immune response.

In Aspergillus fumigatus, the conidial surface contains dihydroxynaphthalene (DHN)-melanin. Six-clustered gene products have been identified that mediate sequential catalysis of DHN-melanin biosynthesis. Melanin thus produced is known to be a virulence factor, protecting the fungus from the host defense mechanisms. In the present study, individual deletion of the genes involved in the initial three steps of melanin biosynthesis resulted in an altered conidial surface with masked surface rodlet layer, leaky cell wall allowing the deposition of proteins on the cell surface and exposing the otherwise-masked cell wall polysaccharides at the surface. Melanin as such was immunologically inert; however, deletion mutant conidia with modified surfaces could activate human dendritic cells and the subsequent cytokine production in contrast to the wild-type conidia. Cell surface defects were rectified in the conidia mutated in downstream melanin biosynthetic pathway, and maximum immune inertness was observed upon synthesis of vermelone onward. These observations suggest that although melanin as such is an immunologically inert material, it confers virulence by facilitating proper formation of the A. fumigatus conidial surface.

Genetic relatedness versus biological compatibility between Aspergillus fumigatus and related species.

Aspergillus section Fumigati contains 12 clinically relevant species. Among these Aspergillus species, A. fumigatus is the most frequent agent of invasive aspergillosis, followed by A. lentulus and A. viridinutans. Genealogical concordance and mating experiments were performed to examine the relationship between phylogenetic distance and mating success in these three heterothallic species. Analyses of 19 isolates from section Fumigati revealed the presence of three previously unrecognized species within the broadly circumscribed species A. viridinutans. A single mating type was found in the new species Aspergillus pseudofelis and Aspergillus pseudoviridinutans, but in Aspergillus parafelis, both mating types were present. Reciprocal interspecific pairings of all species in the study showed that the only successful crosses occurred with the MAT1-2 isolates of both A. parafelis and A. pseudofelis. The MAT1-2 isolate of A. parafelis was fertile when paired with the MAT1-1 isolates of A. fumigatus, A. viridinutans, A. felis, A. pseudoviridinutans, and A. wyomingensis but was not fertile with the MAT1-1 isolate of A. lentulus. The MAT1-2 isolates of A. pseudofelis were fertile when paired with the MAT1-1 isolate of A. felis but not with any of the other species. The general infertility in the interspecies crossings suggests that genetically unrelated species are also biologically incompatible, with the MAT1-2 isolates of A. parafelis and A. pseudofelis being the exception. Our findings underscore the importance of genealogical concordance analysis for species circumscription, as well as for accurate species identification, since misidentification of morphologically similar pathogens with differences in innate drug resistance may be of grave consequences for disease management.

Chloroquine modulates the fungal immune response in phagocytic cells from patients with chronic granulomatous disease.

Invasive aspergillosis is a major threat to patients with chronic granulomatous disease (CGD). Fungal pathogenesis is the result of a diminished antifungal capacity and dysregulated inflammation. A deficient NADPH-oxidase complex results in defective phagolysosomal alkalization. To investigate the contribution of defective pH regulation in phagocytes among patients with CGD during fungal pathogenesis, we evaluated the effect of the acidotropic, antimalarial drug chloroquine (CQ) on the antifungal capacity of polymorphonuclear cells (PMNs) and on the inflammatory response of peripheral blood mononuclear cells (PBMCs). Chloroquine exerted a direct pH-dependent antifungal effect on Aspergillus fumigatus and Aspergillus nidulans; it increased the antifungal activity of PMNs from patients with CGD at a significantly lower concentration, compared with the concentration for PMNs from healthy individuals; and decreased the hyperinflammatory state of PBMCs from patients with CGD, as observed by decreased tumor necrosis factor α and interleukin 1ß release. Chloroquine targets both limbs of fungal pathogenesis and might be of great value in the clearance of invasive aspergillosis in patients with CGD.

Determination of Ploidy Levels and Nuclear DNA Content in Cryptococcus neoformans by Flow Cytometry: Drawbacks with Variability.

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus.

Mucor germinans, a novel dimorphic species resembling Paracoccidioides in a clinical sample: questions on ecological strategy.

Dimorphism is known among the etiologic agents of endemic mycoses as well as in filamentous Mucorales. Under appropriate thermal conditions, mononuclear yeast forms alternate with multi-nucleate hyphae. Here, we describe a dimorphic mucoralean fungus obtained from the sputum of a patient with Burkitt lymphoma and ongoing graft-versus-host reactions. The fungus is described as Mucor germinans sp. nov. Laboratory studies were performed to simulate temperature-dependent dimorphism, with two environmental strains Mucor circinelloides and Mucor kunryangriensis as controls. Both strains could be induced to form multinucleate arthrospores and subsequent yeast-like cells in vitro. Multilateral yeast cells emerge in all three Mucor species at elevated temperatures. This morphological transformation appears to occur at body temperature since the yeast-like cells were observed in the lungs of our immunocompromised patient. The microscopic appearance of the yeast-like cells in the clinical samples is easily confused with that of Paracoccidioides. The ecological role of yeast forms in Mucorales is discussed.IMPORTANCEMucormycosis is a devastating disease with high morbidity and mortality in susceptible patients. Accurate diagnosis is required for timely clinical management since antifungal susceptibility differs between species. Irregular hyphal elements are usually taken as the hallmark of mucormycosis, but here, we show that some species may also produce yeast-like cells, potentially being mistaken for Candida or Paracoccidioides. We demonstrate that the dimorphic transition is common in Mucor species and can be driven by many factors. The multi-nucleate yeast-like cells provide an effective parameter to distinguish mucoralean infections from similar yeast-like species in clinical samples.

Cryptococcus neoformans activates RhoGTPase proteins followed by protein kinase C, focal adhesion kinase, and ezrin to promote traversal across the blood-brain barrier.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Previous studies have demonstrated that Cryptococcus binding and invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for transmigration across the blood-brain barrier. However, the molecular mechanism involved in the cryptococcal blood-brain barrier traversal is poorly understood. In this study we examined the signaling events in HBMEC during interaction with C. neoformans. Analysis with inhibitors revealed that cryptococcal association, invasion, and transmigration require host actin cytoskeleton rearrangement. Rho pulldown assays revealed that Cryptococcus induces activation of three members of RhoGTPases, e.g. RhoA, Rac1, and Cdc42, and their activations are required for cryptococcal transmigration across the HBMEC monolayer. Western blot analysis showed that Cryptococcus also induces phosphorylation of focal adhesion kinase (FAK), ezrin, and protein kinase C α (PKCα), all of which are involved in the rearrangement of host actin cytoskeleton. Down-regulation of FAK, ezrin, or PKCα by shRNA knockdown, dominant-negative transfection, or inhibitors significantly reduces cryptococcal ability to traverse the HBMEC monolayer, indicating their positive role in cryptococcal transmigration. In addition, activation of RhoGTPases is the upstream event for phosphorylation of FAK, ezrin, and PKCα during C. neoformans-HBMEC interaction. Taken together, our findings demonstrate that C. neoformans activates RhoGTPases and subsequently FAK, ezrin, and PKCα to promote their traversal across the HBMEC monolayer, which is the critical step for cryptococcal brain infection and development of meningitis.

Hyaluronic acid receptor CD44 deficiency is associated with decreased Cryptococcus neoformans brain infection.

Cryptococcus neoformans is a pathogenic yeast that can invade the brain and cause meningoencephalitis. Our previous in vitro studies suggested that the interaction between C. neoformans hyaluronic acid and human brain endothelial CD44 could be the initial step of brain invasion. In this report, we used a CD44 knock-out (KO or CD44(-/-)) mouse model to explore the importance of CD44 in C. neoformans brain invasion. Our results showed that C. neoformans-infected CD44 KO mice survived longer than the infected wild-type mice. Consistent with our in vitro results, the brain and cerebrospinal fluid fungal burden was reduced in CD44-deficient mice. Histopathological studies showed smaller and fewer cystic lesions in the brains of CD44 KO mice. Interestingly, the cystic lesions contained C. neoformans cells embedded within their polysaccharide capsule and were surrounded by host glial cells. We also found that a secondary hyaluronic acid receptor, RHAMM (receptor of hyaluronan-mediated motility), was present in the CD44 KO mice. Importantly, our studies demonstrated an in vivo blocking effect of simvastatin. These results suggest that the CD44 and RHAMM receptors function on membrane lipid rafts during invasion and that simvastatin may have a potential therapeutic role in C. neoformans infections of the brain.