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.     

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.     

Identification of Genes from the Fungal Pathogen Cryptococcus neoformans Related to Transmigration into the Central Nervous System

Background

A mouse brain transmigration assessment (MBTA) was created to investigate the central nervous system (CNS) pathogenesis of cryptococcal meningoencephalitis.

Methodology/Principal Findings

Two cryptococcal mutants were identified from a pool of 109 pre-selected mutants that were signature-tagged with the nourseothricin acetyltransferase (NAT) resistance cassette. These two mutants displayed abnormal transmigration into the central nervous system. One mutant displaying decreased transmigration contains a null mutation in the putative FNX1 gene, whereas the other mutant possessing a null mutation in the putative RUB1 gene exhibited increased transmigration into the brain. Two macrophage adhesion-defective mutants in the pool, 12F1 and 3C9, showed reduced phagocytosis by macrophages, but displayed no defects in CNS entry suggesting that transit within macrophages (the “Trojan horse” model of CNS entry) is not the primary mechanism for C. neoformans migration into the CNS in this MBTA.

Conclusions/Significance

This research design provides a new strategy for genetic impact studies on how Cryptococcus passes through the blood-brain barrier (BBB), and the specific isolated mutants in this assay support a transcellular mechanism of CNS entry.     

Microbiological,Epidemiological, and Clinical Characteristics and Outcomes of Patients with Cryptococcosis in Taiwan, 1997–2010

Background

Among members of Cryptococcus neoformans- Cryptococcus gattii species complex, C. neoformans is distributed worldwide whereas C. gattii is considered to be more prevalent in the subtropics and tropics including Taiwan. This nationwide study was undertaken to determine the distribution of genotypes, clinical characteristics and outcomes of 219 patients with proven cryptococcosis at 20 hospitals representative of all geographic areas in Taiwan during 1997–2010.

Methods and Findings

Of 219 isolates analyzed, C. neoformans accounted for 210 isolates (95.9%); nine isolates were C. gattii (4.1%). The predominant genotype was VNI (206 isolates). The other genotypes included VNII (4 isolates), VGI (3 isolates) and VGII (6 isolates). Antifungal minimal inhibition concentrations higher than epidemiologic cutoff values (ECVs) were found in nine VNI isolates (7 for amphotericin B). HIV infection was the most common underlying condition (54/219, 24.6%). Among HIV-negative patients, liver diseases (HBV carrier or cirrhosis) were common (30.2%) and 15.4% did not have any underlying condition. Meningoencephalitis was the most common presentation (58.9%), followed by pulmonary infection (19.6%) and “others” (predominantly cryptococcemia) (18.7%). The independent risk factors for 10-week mortality, by multivariate analysis, were cirrhosis of liver (P = 0.014) and CSF cryptococcal antigen titer ≥512 (P = 0.020). All except one of 54 HIV-infected patients were infected by VNI genotype (98.1%). Of the 13 isolates of genotypes other than VNI, 12 (92.3%) were isolated from HIV-negative patients. HIV-infected patients compared to HIV-negative patients were more likely to have meningoencephalitis and serum cryptococcal antigen ≥1∶512. Patients infected with C. gattii compared to C. neoformans were younger, more likely to have meningoencephalitis (100% vs. 57%), reside in Central Taiwan (56% vs. 31%), and higher 10-week crude mortality (44.4% vs. 22.2%).

Conclusions

Cryptococcus neoformans in Taiwan, more prevalent than C. gatii, has a predominant VNI genotype. Isolates with antifungal MIC higher than ECVs were rare.     

Role of IL-17A on resolution of pulmonary C. neoformans infection

The current studies evaluated the role of interleukin (IL)-17A in the induction of protective immunity against pulmonary cryptococcosis in mice. Protection against pulmonary infection with C. neoformans strain H99γ was associated with increased IL-17A production. Signaling through the IFN-γ receptor (R) was required for increased IL-17A production, however, a Th17-type cytokine profile was not observed. Neutrophils were found to be the predominant leukocytic source of IL-17A, rather than T cells, suggesting that the IL-17A produced was not part of a T cell-mediated Th17-type immune response. Depletion of IL-17A in mice during pulmonary infection with C. neoformans strain H99γ resulted in an initial increase in pulmonary fungal burden, but had no effect on cryptococcal burden at later time points. Also, depletion of IL-17A did not affect the local production of other cytokines. IL-17RA^−/− mice infected with C. neoformans strain H99γ survived the primary infection as well as a secondary challenge with wild-type cryptococci. However, dissemination of the wild-type strain to the brain was noted in the surviving IL-17RA^−/− mice. Altogether, our results suggested that IL-17A may be important for optimal protective immune responsiveness during pulmonary C. neoformans infection, but protective Th1-type immune responses are sufficient for protection against cryptococcal infection.     

Cryptococcus neoformans phospholipase B1 activates host cell Rac1 for traversal across the blood-brain barrier

Cryptococcus neoformans penetration into the central nervous system (CNS) requires traversal of the blood–brain barrier that is composed of a single layer of human brain microvascular endothelial cells (HBMEC), but the underlying mechanisms of C. neoformans traversal remain incompletely understood. C. neoformans transcytosis of HBMEC monolayer involves rearrangements of the host cell actin cytoskeleton and small GTP‐binding Rho family proteins such as Rac1 are shown to regulate host cell actin cytoskeleton. We, therefore, examined whether C. neoformans traversal of the blood–brain barrier involves host Rac1. While the levels of activated Rac1 (GTP‐Rac1) in HBMEC increased significantly upon incubation with C. neoformans strains, pharmacological inhibition and down‐modulation of Rac1 significantly decreased C. neoformans transcytosis of HBMEC monolayer. Also, Rac1 inhibition was efficient in preventing C. neoformans penetration into the brain. In addition, C. neoformans phospholipase B1 (Plb1) was shown to contribute to activating host cell Rac1, andSTAT3 was observed to associate with GTP‐Rac1 in HBMEC that were incubated with C. neoformans strain but not with its Δplb1 mutant. These findings demonstrate for the first time that C. neoformans Plb1 aids fungal traversal across the blood–brain barrier by activating host cell Rac1 and its association with STAT3, and suggest that pharmacological intervention of host–microbial interaction contributing to traversal of the blood–brain barrier may prevent C. neoformans penetration into the brain.     

The highly conserved 5' untranslated region as an effective target towards the inhibition of Enterovirus 71 replication by unmodified and appropriate 2'-modified siRNAs

Background

Cryptococcus neoformans has a predilection for central nervous system infection. C. neoformans traversal of the blood brain barrier, composed of human brain microvascular endothelial cells (HBMEC), is the crucial step in brain infection. However, the molecular mechanism of the interaction between Cryptococcus neoformans and HBMEC, relevant to its brain invasion, is still largely unknown.

Methods

In this report, we explored several cellular and molecular events involving the membrane lipid rafts and caveolin-1 (Cav1) of HBMEC during C. neoformans infection. Immunofluorescence microscopy was used to examine the roles of Cav1. The knockdown of Cav1 by the siRNA treatment was performed. Phosphorylation of Cav1 relevant to its invasion functions was investigated.

Results

We found that the host receptor CD44 colocalized with Cav1 on the plasma membrane, and knockdown of Cav1 significantly reduced the fungal ability to invade HBMEC. Although the CD44 molecules were still present, HBMEC membrane organization was distorted by Cav1 knockdown. Concomitantly, knockdown of Cav1 significantly reduced the fungal crossing of the HBMEC monolayer in vitro. Upon C. neoformans engagement, host Cav1 was phosphorylated in a CD44-dependent manner. This phosphorylation was diminished by filipin, a disrupter of lipid raft structure. Furthermore, the phosphorylated Cav1 at the lipid raft migrated inward to the perinuclear localization. Interestingly, the phospho-Cav1 formed a thread-like structure and colocalized with actin filaments but not with the microtubule network.

Conclusion

These data support that C. neoformans internalization into HBMEC is a lipid raft/caveolae-dependent endocytic process where the actin cytoskeleton is involved, and the Cav1 plays an essential role in C. neoformans traversal of the blood-brain barrier.     

A proteomic‐based approach for the identification of immunodominant Cryptococcus neoformans proteins

Cryptococcus neoformans is an opportunistic fungal pathogen that can cause life‐threatening meningoencephalitis in immune compromised patients. Previous, studies in our laboratory have shown that prior exposure to an IFN‐γ‐producing C. neoformans strain (H99γ) elicits protective immunity against a second pulmonary C. neoformans challenge. Here, we characterized the antibody response produced in mice protected against experimental pulmonary C. neoformans infection compared to nonprotected mice. Moreover, we evaluated the efficacy of using serum antibody from protected mice to detect immunodominant C. neoformans proteins. Protected mice were shown to produce significantly more C. neoformans‐specific antibodies following a second experimental pulmonary cryptococcal challenge compared to nonprotected mice. Immunoblot analysis of C. neoformans proteins resolved by 2‐DE using serum from nonprotected mice failed to show any reactivity. In contrast, serum from protected mice was reactive with several cryptococcal protein spots. Analysis of these spots by capillary HPLC‐ESI‐MS/MS identified several cryptococcal proteins shown to be associated with the pathogenesis of cryptococcosis. Our studies demonstrate that mice immunized with C. neoformans strain H99γ produce antibodies that are immune reactive against specific cryptococcal proteins that may provide a basis for the development of immune based therapies that induce protective anticryptococcal immune responses.     

Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion

Background

The fungal pathogen Cryptococcus neoformans is a leading cause of illness and death in persons with predisposing factors, including: malignancies, solid organ transplants, and corticosteroid use. C. neoformans is ubiquitous in the environment and enters into the lungs via inhalation, where it can disseminate through the bloodstream and penetrate the central nervous system (CNS), resulting in a difficult to treat and often-fatal infection of the brain, called meningoencephalitis. Plasminogen is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by cancer cells during metastasis and several pathogenic species of bacteria have been found to manipulate the host plasminogen system to facilitate invasion of tissues during infection by modifying the activation of this process through the binding of plasminogen at their surface.

Methodology

The invasion of the brain and the central nervous system by penetration of the protective blood-brain barrier is a prerequisite to the establishment of meningoencephalitis by the opportunistic fungal pathogen C. neoformans. In this study, we examined the ability of C. neoformans to subvert the host plasminogen system to facilitate tissue barrier invasion. Through a combination of biochemical, cell biology, and proteomic approaches, we have shown that C. neoformans utilizes the host plasminogen system to cross tissue barriers, providing support for the hypothesis that plasminogen-binding may contribute to the invasion of the blood-brain barrier by penetration of the brain endothelial cells and underlying matrix. In addition, we have identified the cell wall-associated proteins that serve as plasminogen receptors and characterized both the plasminogen-binding and plasmin-activation potential for this significant human pathogen.

Conclusions

The results of this study provide evidence for the cooperative role of multiple virulence determinants in C. neoformans pathogenesis and suggest new avenues for the development of anti-infective agents in the prevention of fungal tissue invasion.     

Ultrastructural study ofCryptococcus neoformans by quick-freezing and deep-etching method

The three-dimensional ultrastructure ofCryptococcus neoformans was studied by quick-freezing and deep-etching (QF-DE) method.C. neoformans, strain CDC551, was cultured on agar. The viable yeast cells (10⁷ cells) were inoculated into each mouse from the tail vein. Three weeks after the inoculation, the brains of the mice were perfused with fixatives, quickly frozen, freeze-fractured, deeply etched and rotary shadowed with platinum and carbon. In addition, the viable cells ofC. neoformans on agar were picked up and quickly frozen, and replica membranes were prepared as described above. The ultrastructure ofC. neoformans was three-dimensionally demonstrated by the QF-DE method. The capsule was composed of fine meshworks of microfibrils (10–13 nm in diameter), which were directly attached to the cell walls. The capsule of the in vivo yeasts (yeast cells in the brain lesion) was thicker than that of the in vitro yeasts (yeast cells on agar culture). At the outer part of the cell wall, a particle-accumulating layer was observed. This layer in vivo was thicker than that in vitro. Occasionally, the yeast cells were ingested by phagocytes in the mouse brain. Although the cytoplasm of such yeast cells was destroyed, the capsular meshworks were well preserved. The ultrastructure of the capsule was the same both in cultured and phagocytized yeasts in the cystic lesions of the brains. This lack of morphological changes of the capsular meshworks suggests that they are resistant to the digestion by phagocytes. This stability of capsular structures may provide one of the important pathogenic factors in cystic lesions byC. neoformans.     

Cryptococcus neoformans Hyperfilamentous Strain Is Hypervirulent in a Murine Model of Cryptococcal Meningoencephalitis

Cryptococcus neoformans is a human fungal pathogen that causes lethal infections of the lung and central nervous system in immunocompromised individuals. C. neoformans has a defined bipolar sexual life cycle with a and α mating types. During the sexual cycle, which can occur between cells of opposite mating types (bisexual reproduction) or cells of one mating type (unisexual reproduction), a dimorphic transition from yeast to hyphal growth occurs. Hyphal development and meiosis generate abundant spores that, following inhalation, penetrate deep into the lung to enter the alveoli, germinate, and establish a pulmonary infection growing as budding yeast cells. Unisexual reproduction has been directly observed only in the Cryptococcus var. neoformans (serotype D) lineage under laboratory conditions. However, hyphal development has been previously associated with reduced virulence and the serotype D lineage exhibits limited pathogenicity in the murine model. In this study we show that the serotype D hyperfilamentous strain XL280α is hypervirulent in an animal model. It can grow inside the lung of the host, establish a pulmonary infection, and then disseminate to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans, and reveal the virulence potential of serotype D as broader and more dynamic than previously appreciated.     

L3T4(CD4)-, Lyt-2(CD8)- and Mac-1(CD11b)-phenotypic leukocytes in murine cryptococcal meningoencephalitis

An immunohistological study of L3T4(CD4)+ and LYT-2(CD8)+ lymphocytes, Mac-1(CD11b)+ monocytes and granulocytes in experimental murine cryptococcal meningoencephalitis was conducted. To assess the concomitant inflammatory reaction in an extracerebral site, livers were examined in parallel. Mice were infected i.v. withCryptococcus neoformans, group A/D, and organs were examined immunohistologically for CD4-, CD8- and monocyteand granulocyte-specific CD11b-phenotypic leukocytes over a period of 60 days. Intracerebrally, agglomerations of cryptococci formed pseudocysts that were surrounded by CD4+ and CD8+ lymphocytes at the end of the second week post-infection, followed by the invasion of monocytes and granulocytes into the lesions. After the fourth week post-infection, most of the invaded lesions were transformed into glious scars. Meningitis was usually marked and showed a homogenous distribution of CD4-, CD8- and CD11b-phenotypic cells, with a predominance of monocytes and CD4+ lymphocytes. Inflammatory infiltrates in the liver were found already 4 days post-infection. CD4+ lymphocytes and monocytes were distributed homogenously in the infiltrates, with a lower number of CD8+ lymphocytes being located rather in the periphery of the infiltrates. Comparing leukocyte kinetics in brain and liver, an important observation was the delayed immigration of immune cells at the intracerebral cryptococcal lesions as compared with the liver, and the different migration patterns of T-lymphocyte subgroups and macrophages. These results suggest that there are differential leukocyte migration patterns in the liver and brain following disseminated cryptococcosis. The immunological aspects of the observed leukocyte kinetics are discussed.     

Cholesterol and sphingomyelin are critical for Fcγ receptor–mediated phagocytosis of Cryptococcus neoformans by macrophages

Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores by aiding in clearance but can also potentially serve as a niche for their dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high-order plasma membrane domains enriched in cholesterol and sphingomyelin [SM]) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages'' ability to phagocytose C. neoformans. We found that cholesterol or SM depletion resulted in significantly deficient immunoglobulin G (IgG)-mediated phagocytosis of fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency, whereas a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable SM (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis, whereas the raft-inhibiting conformation (cis-AzoSM) significantly but reversibly blunted phagocytosis. We observed that the effect on phagocytosis may be facilitated by Fcγ receptor (FcγR) function, whereby IgG immune complexes crosslink to FcγRIII, resulting in tyrosine phosphorylation of FcR γ-subunit (FcRγ), an important accessory protein in the FcγR signaling cascade. Correspondingly, cholesterol or SM depletion resulted in decreased FcRγ phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcRγ phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating FcγRIII-mediated phagocytosis of C. neoformans.     

Insights into the Mechanisms of Protective Immunity against Cryptococcus neoformans Infection Using a Mouse Model of Pulmonary Cryptococcosis

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening pneumonia and meningoencephalitis in immune compromised individuals. Previous studies have shown that immunization of BALB/c mice with an IFN-γ-producing C. neoformans strain, H99γ, results in complete protection against a second pulmonary challenge with an otherwise lethal cryptococcal strain. The current study evaluated local anamnestic cell-mediated immune responses against pulmonary cryptococcosis in mice immunized with C. neoformans strain H99γ compared to mice immunized with heat-killed C. neoformans (HKC.n.). Mice immunized with C. neoformans strain H99γ had significantly reduced pulmonary fungal burden post-secondary challenge compared to mice immunized with HKC.n. Protection against pulmonary cryptococcosis was associated with increased pulmonary granulomatous formation and leukocyte infiltration followed by a rapid resolution of pulmonary inflammation, which protected the lungs from severe allergic bronchopulmonary mycosis (ABPM)-pathology that developed in the lungs of mice immunized with HKC.n. Pulmonary challenge of interleukin (IL)-4 receptor, IL-12p40, IL-12p35, IFN-γ, T cell and B cell deficient mice with C. neoformans strain H99γ demonstrated a requirement for Th1-type T cell-mediated immunity, but not B cell-mediated immunity, for the induction of H99γ-mediated protective immune responses against pulmonary C. neoformans infection. CD4⁺ T cells, CD11c⁺ cells, and Gr-1⁺ cells were increased in both proportion and absolute number in protected mice. In addition, significantly increased production of Th1-type/pro-inflammatory cytokines and chemokines, and conversely, reduced Th2-type cytokine production was observed in the lungs of protected mice. Interestingly, protection was not associated with increased production of cytokines IFN-γ or TNF-α in lungs of protected mice. In conclusion, immunization with C. neoformans strain H99γ results in the development of protective anti-cryptococcal immune responses that may be measured and subsequently used in the development of immune-based therapies to combat pulmonary cryptococcosis.     

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.     

Cryptococcus neoformans meningoencephalitis in a patient with polyarteritis nodosa

Case of 59-year-old male with chronic obstructive pulmonary disease and a number of comorbidities, who has developed meningoencephalitis caused by Cryptococcus neoformans var. grubii with polyarteritis nodosa diagnosed during hospitalization, was presented. Before evidence of meningoencephalitis, the patient was being treated with ketoconazole and low doses of fluconazole (200 mg/day) for alleged candidiasis. The dosage was increased (800 mg/day) following laboratory diagnosis of C. neoformans based on positive latex agglutination test and biochemical identification of encapsulated yeast isolated from the blood and CSF. Later, the yeast identification was confirmed by sequencing analysis. Owing to inadequate clinical response, fluconazole therapy was switched to voriconazole (400 mg/day) and later to intravenous amphotericin B (1.0 mg/kg per day). Despite of a temporary stabilization and improvement, which correlated with decline of cryptococcal antigen titers (from 1:1024 to 1:8), after 6 weeks, the patient’s underlying condition deteriorated due to severe pancolitis and serious nosocomial bacterial infections. The patient died of multiorgan failure several days later. Our case demonstrates a possible connection between the development of life-threatening cryptococcosis and an autoimmune vasculitis disease and emphasizes that the outcome of the management of cryptococcal meningoencephalitis is highly dependent on early diagnosis, adequate treatment, including dosage, and last but not least control of underlying disease and risk factors.     

Role for Chitin and Chitooligomers in the Capsular Architecture of Cryptococcus neoformans

Molecules composed of β-1,4-linked N-acetylglucosamine (GlcNAc) and deacetylated glucosamine units play key roles as surface constituents of the human pathogenic fungus Cryptococcus neoformans. GlcNAc is the monomeric unit of chitin and chitooligomers, which participate in the connection of capsular polysaccharides to the cryptococcal cell wall. In the present study, we evaluated the role of GlcNAc-containing structures in the assembly of the cryptococcal capsule. The in vivo expression of chitooligomers in C. neoformans varied depending on the infected tissue, as inferred from the differential reactivity of yeast forms to the wheat germ agglutinin (WGA) in infected brain and lungs of rats. Chromatographic and dynamic light-scattering analyses demonstrated that glucuronoxylomannan (GXM), the major cryptococcal capsular component, interacts with chitin and chitooligomers. When added to C. neoformans cultures, chitooligomers formed soluble complexes with GXM and interfered in capsular assembly, as manifested by aberrant capsules with defective connections with the cell wall and no reactivity with a monoclonal antibody to GXM. Cultivation of C. neoformans in the presence of an inhibitor of glucosamine 6-phosphate synthase resulted in altered expression of cell wall chitin. These cells formed capsules that were loosely connected to the cryptococcal wall and contained fibers with decreased diameters and altered monosaccharide composition. These results contribute to our understanding of the role played by chitin and chitooligosaccharides on the cryptococcal capsular structure, broadening the functional activities attributed to GlcNAc-containing structures in this biological system.Cryptococcus neoformans is the etiologic agent of cryptococcosis, a disease still characterized by high morbidity and mortality despite antifungal therapy (3). Pathogenic species belonging to the Cryptococcus genus also include Cryptococcus gattii, which causes disease mostly in immunocompetent individuals (24). A unique characteristic of Cryptococcus species is the presence of a polysaccharide capsule, which is essential for virulence (7-9, 19, 25, 33).C. neoformans has a complex cell surface. The thick fungal cell wall is composed of polysaccharides (29), pigments (11), lipids (35), and proteins (36). External to the cryptococcal cell wall, capsular polysaccharides form a capsule (19). Seemingly, the assembly of the surface envelope of C. neoformans requires the interaction of cell wall components with capsular elements. Some of the cryptococcal cell wall-capsule connectors have been identified, including the structural polysaccharide α-1,3-glucan and chitooligomers (29, 30, 32).Chitin-like molecules in fungi are polymerized by chitin synthases, which use cytoplasmic pools of UDP-GlcNAc (N-acetylglucosamine) to form β-1,4-linked oligosaccharides and large polymers. In C. neoformans, the final cellular site of chitin accumulation is the cell wall. The polysaccharide is also used for chitosan synthesis through enzymatic deacetylation (1). Eight putative cryptococcal chitin synthase genes and three regulator proteins have been identified (2). The chitin synthase Chs3 and regulator Csr2 may form a complex with chitin deacetylases for conversion of chitin to chitosan (1). Key early events in the synthesis of chitin/chitosan require the activity of glucosamine 6-phosphate synthase, which promotes the glutamine-dependent amination of fructose 6-phosphate to form glucosamine 6-phosphate, a substrate used for UDP-GlcNAc synthesis (23).In a previous study, we demonstrated that β-1,4-linked GlcNAc oligomers, which are specifically recognized by the wheat germ agglutinin (WGA), form bridge-like connections between the cell wall and the capsule of C. neoformans (32). In fact, other reports indicate that molecules composed of GlcNAc or its deacetylated derivative play key roles in C. neoformans structural biology. For example, mutations in the genes responsible for the expression of chitin synthase 3 or of the biosynthetic regulator Csr2p caused the loss of the ability to retain the virulence-related pigment melanin in the cell wall (1, 2). These cells were also defective in the synthesis of chitosan, which has also been demonstrated to regulate the retention of cell wall melanin (1). Treatment of C. neoformans acapsular mutants with chitinase affected the incorporation of capsular components into the cell wall (32). Considering that melanin and capsular components are crucial for virulence, these results strongly suggest that GlcNAc-derived molecules are key components of the C. neoformans cell surface. The expression of GlcNAc-containing molecules is likely to be modulated during infection since chitinase expression by host cells is induced during lung cryptococcosis (37).In this study, we used β-1,4-linked GlcNAc oligomers and an inhibitor of UDP-GlcNAc synthesis to evaluate the role played by GlcNAc-containing molecules in the surface architecture of C. neoformans. The results point to a direct relationship between the expression of GlcNAc-containing molecules and capsular assembly, indicating that chitin and chitooligomers are required for capsule organization in C. neoformans.