Role of the Apt1 Protein in Polysaccharide Secretion by Cryptococcus neoformans

Flippases are key regulators of membrane asymmetry and secretory mechanisms. Vesicular polysaccharide secretion is essential for the pathogenic mechanisms of Cryptococcus neoformans. On the basis of the observations that flippases are required for polysaccharide secretion in plants and the putative Apt1 flippase is required for cryptococcal virulence, we analyzed the role of this enzyme in polysaccharide release by C. neoformans, using a previously characterized apt1Δ mutant. Mutant and wild-type (WT) cells shared important phenotypic characteristics, including capsule morphology and dimensions, glucuronoxylomannan (GXM) composition, molecular size, and serological properties. The apt1Δ mutant, however, produced extracellular vesicles (EVs) with a lower GXM content and different size distribution in comparison with those of WT cells. Our data also suggested a defective intracellular GXM synthesis in mutant cells, in addition to changes in the architecture of the Golgi apparatus. These findings were correlated with diminished GXM production during in vitro growth, macrophage infection, and lung colonization. This phenotype was associated with decreased survival of the mutant in the lungs of infected mice, reduced induction of interleukin-6 (IL-6) cytokine levels, and inefficacy in colonization of the brain. Taken together, our results indicate that the lack of APT1 caused defects in both GXM synthesis and vesicular export to the extracellular milieu by C. neoformans via processes that are apparently related to the pathogenic mechanisms used by this fungus during animal infection.     

Parallel beta-helix proteins required for accurate capsule polysaccharide synthesis and virulence in the yeast Cryptococcus neoformans

The principal capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans consists of an alpha-1,3-linked mannose backbone decorated with a repeating pattern of glucuronyl and xylosyl side groups. This structure is critical for virulence, yet little is known about how the polymer, called glucuronoxylomannan (GXM), is faithfully synthesized and assembled. We have generated deletions in two genes encoding predicted parallel beta-helix repeat proteins, which we have designated PBX1 and PBX2. Deletion of either gene results in a dry-colony morphology, clumpy cells, and decreased capsule integrity. Two-dimensional nuclear magnetic resonance spectroscopy of purified GXM from the mutants indicated that both the wild-type GXM structure and novel, aberrant linkages were present. Carbohydrate composition and linkage analysis determined that these aberrant structures are correlated with the incorporation of terminal glucose residues that are not found in wild-type capsule polysaccharide. We conclude that Pbx1 and Pbx2 are required for the fidelity of GXM synthesis and may be involved in editing incorrectly added glucose residues. PBX1 and PBX2 knockout mutants showed severely attenuated virulence in a murine inhalation model of cryptococcosis. Unlike acapsular strains, these mutant strains induced delayed symptoms of cryptococcosis, though the infected animals eventually contained the infection and recovered.     

Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity

The polysaccharide capsule surrounding Cryptococcus neoformans comprises manose, xylose and glucuronic acid, of which mannose is the major constituent. The GDP-mannose biosynthesis pathway is highly conserved in fungi and consists of three key enzymes: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP). The MAN1 gene, encoding for the PMI enzyme, was isolated and sequenced from C. neoformans, and a disruption of the MAN1 gene was generated. One MAN1 disruption mutant, man1, which showed poor capsule formation, reduced polysaccharide secretion and morphological abnormalities, was chosen for virulence studies. In both the rabbit and the mouse models of invasive cryptococcosis, man1 was shown to be severely impaired in its virulence, with complete elimination of the yeast from the host. A reconstituted strain of man1 was constructed using gene replacement at the native locus. The wild-type and reconstituted strains were significantly more virulent than the knock-out mutant in both animal models. Our findings reveal that PMI activity is essential for the survival of C. neoformans in the host. The fact that the man1 mutant was not pathogenic suggests that blocking mannose synthesis could be fungicidal in the mammalian host and thus an excellent target for antifungal drug development.     

Direct inhibition of T-cell responses by the Cryptococcus capsular polysaccharide glucuronoxylomannan

The major virulence factor of the pathogenic fungi Cryptococcus neoformans and C. gattii is the capsule. Glucuronoxylomannan (GXM), the major component of the capsule, is a high-molecular-weight polysaccharide that is shed during cryptococcosis and can persist in patients after successful antifungal therapy. Due to the importance of T cells in the anticryptococcal response, we studied the effect of GXM on the ability of dendritic cells (DCs) to initiate a T-cell response. GXM inhibited the activation of cryptococcal mannoprotein-specific hybridoma T cells and the proliferation of OVA-specific OT-II T cells when murine bone marrow-derived DCs were used as antigen-presenting cells. Inhibition of OT-II T-cell proliferation was observed when either OVA protein or OVA323-339 peptide was used as antigen, indicating GXM did not merely prevent antigen uptake or processing. We found that DCs internalize GXM progressively over time; however, the suppressive effect did not require DCs, as GXM directly inhibited T-cell proliferation induced by anti-CD3 antibody, concanavalin A, or phorbol-12-myristate-13-acetate/ionomycin. Analysis of T-cell viability revealed that the reduced proliferation in the presence of GXM was not the result of increased cell death. GXM isolated from each of the four major cryptococcal serotypes inhibited the proliferation of human peripheral blood mononuclear cells stimulated with tetanus toxoid. Thus, we have defined a new mechanism by which GXM can impart virulence: direct inhibition of T-cell proliferation. In patients with cryptococcosis, this could impair optimal cell-mediated immune responses, thereby contributing to the persistence of cryptococcal infections.     

Monomerization and ER Relocalization of GRASP Is a Requisite for Unconventional Secretion of CFTR

Induction of endoplasmic reticulum (ER)‐to‐Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)‐mediated, Golgi‐independent unconventional cell‐surface trafficking of the folding‐deficient ΔF508‐cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation‐dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508‐CFTR, such as induction of ER‐to‐Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C‐terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation‐mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation‐inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER‐retained ΔF508‐CFTR and mediates the ER stress‐induced unconventional secretion pathway.      

Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport

The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment.     

Unconventional secretion of Acb1 is mediated by autophagosomes

Starving Dictyostelium discoideum cells secrete AcbA, an acyl coenzyme A–binding protein (ACBP) that lacks a conventional signal sequence for entering the endoplasmic reticulum (ER). Secretion of AcbA in D. discoideum requires the Golgi-associated protein GRASP. In this study, we report that starvation-induced secretion of Acb1, the Saccharomyces cerevisiae ACBP orthologue, also requires GRASP (Grh1). This highlights the conserved function of GRASP in unconventional secretion. Although genes required for ER to Golgi or Golgi to cell surface transport are not required for Acb1 secretion in yeast, this process involves autophagy genes and the plasma membrane t-SNARE, Sso1. Inhibiting transport to vacuoles does not affect Acb1 secretion. In sum, our experiments reveal a unique secretory pathway where autophagosomes containing Acb1 evade fusion with the vacuole to prevent cargo degradation. We propose that these autophagosome intermediates fuse with recycling endosomes instead to form multivesicular body carriers that then fuse with the plasma membrane to release cargo.     

Is unconventional secretion inhibited during cell division by Cdk1 activity?

A process of unconventional secretion that is dependent on the Golgi stacking protein GRASP and multiple components of the autophagy machinery has recently been documented for several cytoplasmic and membrane protein. Classical secretion via the exocytic pathway is inhibited during cell division in animal cells, as key membrane compartments, particularly the Golgi, are disassembled and fragmented. The question as to whether unconventional secretion is likewise inhibited during mitosis has not been explored. This mode of secretion supposedly bypasses the Golgi. However, GRASP and Vps34 (a key autophagy protein) are both substrates of the cell cycle regulating cyclin‐dependent kinase 1 (Cdk1), and their activities are apparently inhibited by Cdk1 phosphorylation. Is unconventional secretion therefore similarly inhibited during cell division like conventional secretion? The story may yet turn out to be more complicated. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Isolation and characterization of capsule structure mutant strains of Cryptococcus neoformans

The capsule of Cryptococcus neoformans is the most obvious virulence factor of this pathogenic yeast. The main capsule constituents are glucuronoxylomannans (GXM). Although several studies have focused on GXM composition and structure, very little is known about their genetics. To elucidate the relationship between the capsule structure and the pathophysiology of the cryptococcosis, genetic screening for mutant strains producing a structurally modified capsule was set up. Using monoclonal antibodies specific for different capsule sugar epitopes, we isolated strains with different mutated capsule structures (Cas mutants). According to their reactivities with various monoclonal antibodies, the mutants were classified into six groups (Cas1 to Cas6). One Cas2 mutant was used to clone the corresponding gene by complementation. This gene (USX1) encodes the previously identified UDP-xylose synthase. We demonstrated that it is necessary for both capsule xylosylation and C. neoformans virulence.     

Cas1p is a membrane protein necessary for the O-acetylation of the Cryptococcus neoformans capsular polysaccharide

The capsule is certainly the most obvious virulence factor for Cryptococcus neoformans. The main capsule constituents are glucuronoxylomannans (GXM). Several studies have focused on the structure and chemistry of the GXM component of the capsule, yet little is known about the genetic basis of the capsule construction. Using a monoclonal antibody specific to a sugar epitope, we isolated a capsule-structure mutant strain and cloned by complementation a gene named CAS1 that codes for a putative membrane protein. Although no sequence homology was found with any known protein in the different databases, protein analysis using the PROPSEARCH software classified Cas1p as a putative glycosyltransferase. Cas1p is a well-conserved evolutionary protein, as we identified one orthologue in the human genome, one in the drosophila genome and four in the plant Arabidopsis thaliana genome. Analysis of the capsule structure after CAS1 deletion showed that it is required for GXM O-acetylation.     

Cryptococcal xylosyltransferase 1 (Cxt1p) from Cryptococcus neoformans plays a direct role in the synthesis of capsule polysaccharides

The opportunistic yeast Cryptococcus neoformans causes serious disease in humans and expresses a prominent polysaccharide capsule that is required for its virulence. Little is known about how this capsule is synthesized. We previously identified a beta1,2-xylosyltransferase (Cxt1p) with in vitro enzymatic activity appropriate for involvement in capsule synthesis. Here, we investigate C. neoformans strains in which the corresponding gene has been deleted (cxt1Delta). Loss of CXT1 does not affect in vitro growth of the mutant cells or the general morphology of their capsules. However, NMR structural analysis of the two main capsule polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), showed that both were missing beta1,2-xylose residues. There was an approximately 30% reduction in the abundance of this residue in GXM in mutant compared with wild-type strains, and mutant GalXM was almost completely devoid of beta1,2-linked xylose. The GalXM from the mutant strain was also missing a beta1,3-linked xylose residue. Furthermore, deletion of CXT1 led to attenuation of cryptococcal growth in a mouse model of infection, suggesting that the affected xylose residues are important for normal host-pathogen interactions. Cxt1p is the first glycosyltransferase with a defined role in C. neoformans capsule biosynthesis, and cxt1Delta is the only strain identified to date with structural alterations of the capsule polysaccharide GalXM.     

The Golgi-associated protein GRASP is required for unconventional protein secretion during development

During Dictyostelium development, prespore cells secrete acyl-CoA binding protein (AcbA). Upon release, AcbA is processed to generate a peptide called spore differentiation factor-2 (SDF-2), which triggers terminal differentiation of spore cells. We have found that cells lacking Golgi reassembly stacking protein (GRASP), a protein attached peripherally to the cytoplasmic surface of Golgi membranes, fail to secrete AcbA and, thus, produce inviable spores. Surprisingly, AcbA lacks a signal sequence and is not secreted via the conventional secretory pathway (endoplasmic reticulum-Golgi-cell surface). GRASP is not required for conventional protein secretion, growth, and the viability of vegetative cells. Our findings reveal a physiological role of GRASP and provide a means to understand unconventional secretion and its role in development.     

Glucuronoxylomannan of Cryptococcus neoformans exacerbates in vitro yeast cell growth by interleukin 10-dependent inhibition of CD4+ T lymphocyte responses

Glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is the most important virulence factor of this fungus. We analyzed the molecular events related to protective immune responses against a non-encapsulated strain of C. neoformans, mediated by murine splenic CD4(+) T lymphocytes in vitro, and the impact of GXM addition upon these events. Both the lymphoproliferation of CD4(+) T cells and the control of fungus growth were dependent on B7 co-stimulation. Addition of GXM did not modify CD4(+) T cell proliferation, but exacerbated infection in cultures obtained from normal and infected hosts. GXM enhanced the secretion of IL-10 and IL-4, while it reduced the production of pro-inflammatory cytokines TNF-alpha and IFN-gamma. The blockade of IL-10 activity with neutralizing antibodies increased TNF-alpha production and reduced yeast cell growth. The findings suggest that GXM exacerbates infection by down-regulating cell-mediated protective immune response and that IL-10 is implicated in yeast evasion.     

UDP-glucose dehydrogenase plays multiple roles in the biology of the pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic fungus surrounded by an elaborate polysaccharide capsule that is strictly required for its virulence in humans and other mammals. Nearly half of the sugar residues in the capsule are derived from UDP-glucuronic acid or its metabolites. To examine the role of these nucleotide sugars in C. neoformans, the gene encoding UDP-glucose dehydrogenase was disrupted. Mass spectrometry analysis of nucleotide sugar pools showed that the resulting mutant lacked both UDP-glucuronic acid and its downstream product, UDP-xylose, thus confirming the effect of the knockout and indicating that an alternate pathway for UDP-glucuronic acid production was not used. The mutant was dramatically affected by the lack of specific sugar donors, demonstrating altered cell integrity, temperature sensitivity, lack of growth in an animal model of cryptococcosis, and morphological defects. Additionally, the polysaccharide capsule could not be detected on the mutant cells, although the possibility remains that abbreviated forms of capsule components are made, possibly without proper surface display. The capsule defect is largely independent of the other observed changes, as cells that are acapsular because of mutations in other genes show lack of virulence but do not exhibit alterations in cell integrity, temperature sensitivity, or cellular morphology. All of the observed alterations were reversed by correction of the gene disruption.     

An alpha-1,3-mannosyltransferase of Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic fungus, distinguished by an elaborate polysaccharide capsule that is essential for its virulence. As part of an effort to understand the biosynthesis of this important structure, we initiated purification of an alpha-1,3-mannosyltransferase with appropriate specificity for a role in building the main capsule polysaccharide, glucuronoxylomannan. A pool of proteins that was 5,000-fold enriched in this activity included several polypeptides, which acted potentially as the catalytic protein. These were analyzed using sequence information and double-stranded RNA interference. Interference that targeted a sequence corresponding to part of a 46 kDa protein in the enriched fraction abolished the activity of interest and reduced the capsule on the affected cells. This gene was cloned and expressed in active form in Saccharomyces cerevisiae to confirm function, and was termed CMT1, for cryptococcal mannosyltransferase 1. CMT1 has no confirmed homologs in GenBank other than CAP59, a cryptococcal gene encoding a protein of unknown function that is required for capsule synthesis and virulence. The Cmt1p protein also co-purifies with a homolog of CAP64, a gene whose product has similarly been implicated in capsule synthesis and virulence. A strain disrupted in CMT1 was generated in C. neoformans; this had no effect on virulence in an animal model of cryptococcosis.     

Polysaccharides, mimotopes and vaccines for fungal and encapsulated pathogens.

Vaccination is a rational alternative to treatment for Cryptococcus neoformans infections, as these infections are currently intractable in immunocompromised (including HIV-infected) individuals. Vaccines composed of the cryptococcal capsular polysaccharide glucuronoxylomannan (GXM), the key C. neoformans virulence factor, elicit protective antibodies in mice, although deleterious antibodies can also be induced. By contrast, polysaccharides are poor immunogens in HIV-infected humans and others with B-cell defects. Peptide mimotopes of GXM can induce protective immunity to C. neoformans in mice, however, our knowledge of the mechanisms of mimotope-induced protection is incomplete and further work is needed if polysaccharide- or mimotope-based vaccines are to be used to manage C. neoformans infection.     

Ste50 adaptor protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway

The mitogen-activated protein kinase (MAPK) pathways control diverse cellular functions in pathogenic fungi, including sexual differentiation, stress response, and maintenance of cell wall integrity. Here we characterized a Cryptococcus neoformans gene, which is homologous to the yeast Ste50 that is known to play an important role in mating pheromone response and stress response as an adaptor protein to the Ste11 MAPK kinase kinase in Saccharomyces cerevisiae. The C. neoformans Ste50 was not involved in any of the stress responses or virulence factor production (capsule and melanin) that are controlled by the HOG and Ras/cAMP signaling pathways. However, Ste50 was required for mating in both serotype A and serotype D C. neoformans strains. The ste50Δ mutant was completely defective in cell-cell fusion and mating pheromone production. Double mutation of the STE50 gene blocked increased production of pheromone and the hyper-filamentation phenotype of cells deleted of the CRG1 gene, which encodes the RGS protein that negatively regulates pheromone responsive G-protein signaling via the MAPK pathway. Regardless of the presence of the basidiomycota-specific SH3 domains of Ste50 that are known to be required for full virulence of Ustilago maydis, Ste50 was dispensable for virulence of C. neoformans in a murine model of cryptococcosis. In conclusion, the Ste50 adaptor protein controls sexual differentiation of C. neoformans via the pheromone-responsive MAPK pathway but is not required for virulence.     

A cryptococcal capsular polysaccharide mimotope prolongs the survival of mice with Cryptococcus neoformans infection

Defined Abs to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been shown to be protective against experimental cryptococcosis. This suggests that if a vaccine could induce similar Abs it might protect against infection. However, the potential use of a GXM-based vaccine has been limited by evidence that GXM is a poor immunogen that can induce nonprotective and deleterious, as well as protective, Abs, and that the nature of GXM oligosaccharide epitopes that can elicit a protective response is unknown. In this study, we investigated whether a peptide surrogate for a GXM epitope could induce an Ab response to GXM in mice. The immunogenicity of peptide-protein conjugates produced by linking a peptide mimetic of GXM, P13, to either BSA, P13-BSA, or tetanus toxoid, P13-tetanus toxoid, was examined in BALB/c and CBA/n mice that received four s.c. injections of the conjugates at 14- to 30-day intervals. All mice immunized with conjugate produced IgM and IgG to P13 and GXM. Challenge of conjugate-immunized mice with C. neoformans revealed longer survival and lower serum GXM levels than control mice. These results indicate that 1) P13 is a GXM mimotope and 2) that it induced a protective response against C. neoformans in mice. P13 is the first reported mimotope of a C. neoformans Ag. Therefore, the P13 conjugates are vaccine candidates for C. neoformans and their efficacy in this study suggests that peptide mimotopes selected by protective Abs deserve further consideration as vaccine candidates for encapsulated pathogens.     

Glucuronoxylomannan-mediated interaction of Cryptococcus neoformans with human alveolar cells results in fungal internalization and host cell damage

Infection by Cryptococcus neoformans begins with inhalation of infectious propagules. Fungi reach the lung tissue and interact with epithelial cells in a crucial but poorly understood process. In this study, the interaction of C. neoformans with the human alveolar epithelial cell lineage A549 was investigated, focusing on the relevance of the capsular polysaccharide in this process. The association of encapsulated strains with A549 cells was significantly inhibited by a monoclonal antibody to glucuronoxylomannan (GXM), a major component of the cryptococcal capsule. A purified preparation of GXM produced similar results, suggesting the occurrence of surface receptors for this polysaccharide on the surface of alveolar cells. A549 cells were in fact able to bind soluble GXM, as confirmed by indirect immunofluorescence analysis using the anti-polysaccharide antibody. C. neoformans is internalized after GXM-mediated interaction with A549 cells in a process that culminates with death of host cells. Our results suggest that C. neoformans can use GXM for attachment to alveolar epithelia, allowing the fungus to reach the intracellular environment and damage host cells through still uncharacterized mechanisms.