Phospholipids trigger Cryptococcus neoformans capsular enlargement during interactions with amoebae and macrophages

A remarkable aspect of the interaction of Cryptococcus neoformans with mammalian hosts is a consistent increase in capsule volume. Given that many aspects of the interaction of C. neoformans with macrophages are also observed with amoebae, we hypothesized that the capsule enlargement phenomenon also had a protozoan parallel. Incubation of C. neoformans with Acanthamoeba castellanii resulted in C. neoformans capsular enlargement. The phenomenon required contact between fungal and protozoan cells but did not require amoeba viability. Analysis of amoebae extracts showed that the likely stimuli for capsule enlargement were protozoan polar lipids. Extracts from macrophages and mammalian serum also triggered cryptococcal capsular enlargement. C. neoformans capsule enlargement required expression of fungal phospholipase B, but not phospholipase C. Purified phospholipids, in particular, phosphatidylcholine, and derived molecules triggered capsular enlargement with the subsequent formation of giant cells. These results implicate phospholipids as a trigger for both C. neoformans capsule enlargement in vivo and exopolysaccharide production. The observation that the incubation of C. neoformans with phospholipids led to the formation of giant cells provides the means to generate these enigmatic cells in vitro. Protozoan- or mammalian-derived polar lipids could represent a danger signal for C. neoformans that triggers capsular enlargement as a non-specific defense mechanism against potential predatory cells. Hence, phospholipids are the first host-derived molecules identified to trigger capsular enlargement. The parallels apparent in the capsular response of C. neoformans to both amoebae and macrophages provide additional support for the notion that certain aspects of cryptococcal virulence emerged as a consequence of environmental interactions with other microorganisms such as protists.     

Eicosanoids mediate Galleria mellonella cellular immune response to viral infection

Nodulation is the predominant insect cellular immune response to bacterial and fungal infections and it can also be induced by some viral infections. Treating seventh instar larvae of greater wax moth Galleria mellonella with Bovine herpes simplex virus-1 (BHSV-1) induced nodulation reactions in a dose-dependent manner. Because eicosanoids mediate nodulation reactions to bacterial and fungal infection, we hypothesized that eicosanoids also mediate nodulation reactions to viral challenge. To test this idea, we injected G. mellonella larvae with indomethacin, a nonsteroidal anti-inflammatory drug immediately prior to intrahemocoelic injection of BHSV-1. Relative to vehicle-treated controls, indomethacin-treated larvae produced significantly reduced numbers of nodules following viral infection (down from approximately 190 nodules/larva to <50 nodules/larva). In addition to injection treatments, increasing dietary indomethacin dosages (from 0.01% to 1%) were associated with decreasing nodulation (by 10-fold) and phenoloxidase activity (by 3-fold) reactions to BHSV-1 injection. We infer from these findings that cyclooxygenase products, prostaglandins, mediate nodulation response to viral infection in G. mellonella.     

The capsular dynamics of Cryptococcus neoformans

Cryptococcus neoformans is a soil-dwelling fungus that causes life-threatening illness in immunocompromised individuals and latently infects many healthy individuals. C. neoformans, unlike other human pathogenic fungi, is surrounded by a polysaccharide capsule that is essential for survival and enables C. neoformans to thwart the mammalian immune system. The capsule is a dynamic structure that undergoes changes in size and rearranges during budding. Here, the latest information and unresolved questions regarding capsule synthesis, structure, assembly, growth and rearrangements are discussed along with the concept that self-assembly is important in capsular dynamics.     

Changes in Galleria mellonella apolipophorin III level during Pseudomonas aeruginosa infection

The level of apoLp-III in fat body, hemocytes and plasma from Galleria mellonella larvae infected with Pseudomonas aeruginosa was studied. It was found that the amount of 18kDa protein present in fat body and hemocytes decreased progressively with time after infection. In the case of plasma, an increase in apoLp-III content was observed during the first 19h after infection and then decreased significantly after prolonged infection time. The decreased level of apoLp-III in plasma 24h after infection was accompanied by the appearance of smaller than 18kDa immunoreactive polypeptides. Four intermediate forms with molecular mass of, respectively, 15, 13.3, 12 and 9.5kDa were detectable. The size of polypeptides detected in experiments performed in vivo is comparable with the degradation products of apoLp-III produced by serine protease IV in vitro. In addition, the total proteolytic activity of plasma increased progressively during infection time. The results of our studies suggest that a significant part of total proteolytical activity in the plasma of infected G. mellonella larvae can be attributed to proteases produced by P. aeruginosa during pathogenesis. We discuss the possibility that protease IV of P. aeruginosa is responsible for apoLp-III degradation in vivo.     

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.     

FITC-lectin avidity of Cryptococcus neoformans cell wall and capsular components

Flow cytometry and confocal microscopy were used to quantify and visualize FITC-lectin binding to cell-surface carbohydrate ligands of log and stationary phase acapsular and capsular Cryptococcus neoformans strains. Cell populations demonstrated marked avidity for terminal α-linked mannose and glucose specific FITC-Con A, mannose specific FITC-GNL, as well as N-acetylglucosamine specific FITC-WGA. Exposure to other FITC-lectins specific for mannose, fucose and N-acetylgalactosamine resulted in little cell-surface fluorescence. The nature of cell-surface carbohydrates was investigated further by measurement of the fluorescence from surfaces of log and stationary phase cell populations after exposing them to increasing concentrations of FITC-Con A and FITC-WGA. Cell fluorescence increased significantly with small increases in FITC-Con A and FITC-WGA concentrations attaining reproducible maxima. Measurements of this nature supported calculation of the lectin binding determinants EC 50, Hn, Fmax and relative Bmax values. EC50 values indicated that the yeast-cell surfaces had greatest affinity for FITC-WGA, however, relative Bmax values indicated that greater numbers of Con A binding sites were present on these same cell surfaces. Hn values suggested a co-operative lectin-carbohydrate ligand interaction. Imaging of FITC-Con A and FITC-WGA cell-surface fluorescence by confocal microscopy demonstrated marked localization of both lectins to cell surfaces associated with cell division and maturation, indicative of dynamic carbohydrate ligand exposure and masking. Some fluorescence was associated with entrapment of FITC-Con A by capsular components, but FITC-Con A and FITC-WGA readily penetrated the capsule matrix to bind to the same cell surfaces labelled in acapsular cells.     

Non-specific immunosuppression by Cryptococcus neoformans infection

Cryptococcus neoformans-infected animals were found to be immunosuppressed when tested by a variety of assays for immune competence. Primary humoral immune responses and delayed-type hypersensitivity reactions to sheep erythrocytes were suppressed in animals which had been infected for two weeks. Lymphocyte proliferation (LP) assays to sRBC stroma were also significantly diminished at two weeks of infection. Spleen cells of infected mice suppressed the LP response of sRBC immunized, normal mice in vitro. At least a part of the suppression could be attributed to a nylon wool non-adherent cell. Suppressor cells continued to be present in spleen cell suspensions following treatment with anti-T cell serum or anti-immunoglobulin and complement. When infected spleen cells were separated by adherence to plastic, both the adherent and non-adherent fractions exhibited suppressive activity. Incubation of infected spleen cells in tissue culture for 48 hr resulted in the elaboration of soluble immunosuppreessive factors into the tissue culture medium. These data indicated that immune suppression in cryptococcosis can occur as a result of infection with Cryptococcus neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells in the spleens of infected mice.     

Chemical characterization of capsular polysaccharide from Cryptococcus neoformans serotype A-D

During a study of serotyping of Cryptococcus neoformans, we found that the type strain of C. neoformans (CBS 132) was serotype A-D. This strain agglutinated with both factor 7 serum (specific for serotype A) and factor 8 serum (specific for serotype D) in our serotyping system. Therefore, we investigated the chemical structure of the antigenic capsular polysaccharide of this strain. The soluble capsular polysaccharide was obtained from the culture supernatant fluid by precipitation with ethanol. Column chromatography of the polysaccharide on DEAE-cellulose yielded three fractions (F-1 to F-3). The major antigenic activity was found in the F-3 fraction. The results obtained by methylation analysis, controlled Smith degradation-methylation analysis, partial acid hydrolysis, and other structural studies of F-3 polysaccharide indicated that the polysaccharide contains mannose, xylose, and glucuronic acid at a ratio of 7:2:2, and has a backbone of alpha (1-3)-linked D-mannopyranoside residues with a single branch of beta (1-2)-xylose and glucuronic acid. The ratio of mannose residues with or without a branch in the F-3 polysaccharide was 4:3 and its molecular weight calculated from the average of the degree of polymerization was 46,500 daltons. These results indicate that the chemical structure of the capsular polysaccharide of serotype A-D is very similar to those from serotypes A and D, suggesting that small differences in the molar ratio and pattern of linkage of monosaccharides in the branch of the polysaccharides of the three serotypes may be responsible for their different specificities.     

UDP glucuronate decarboxylase and synthesis of capsular polysaccharide in Cryptococcus neoformans

UDP glucuronate decarboxylase activity was comparable in encapsulated and non-encapsulated strains of Cryptococcus neoformans, required NAD (K_a = 0.2 mM), and was inhibited by NADH (K_i = 0.1 mM) and UDP xylose.     

Cryptococcus neoformans capsule regrowth experiments reveal dynamics of enlargement and architecture

The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded complete characterization. Cryptococcal polysaccharides are known to either remain attached to the cell as capsular polysaccharides (CPSs) or to be shed into the extracellular space as exopolysaccharides (EPSs). While many studies have examined the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and isolated capsular material. We show that sonication or Glucanex enzyme cocktail digestion yields soluble CPS preparations, while use of a French pressure cell press or Glucanex digestion followed by cell disruption removed the capsule and produced cell wall–associated polysaccharide aggregates that we call “capsule ghosts”, implying an inherent organization that allows the CPS to exist independent of the cell wall surface. Since sonication and Glucanex digestion were noncytotoxic, it was also possible to observe the cryptococcal cells rebuilding their capsule, revealing the presence of reducing end glycans throughout the capsule. Finally, analysis of dimethyl sulfoxide-extracted and sonicated CPS preparations revealed the conservation of previously identified glucuronoxylomannan motifs only in the sonicated CPS. Together, these observations provide new insights into capsule architecture and synthesis, consistent with a model in which the capsule is assembled from the cell wall outward using smaller polymers, which are then compiled into larger ones.     

Biological correlates of capsular (quellung) reactions of Cryptococcus neoformans

The capsular swelling or quellung reaction was reported almost 100 years ago and described the effect of Abs on the appearance of microbial capsules. Despite widespread use to assess Ab binding to capsules, relatively little is known as to the mechanism of this effect or its biological consequences. The fungus Cryptococcus neoformans is an attractive system to study capsule reactions because it has a large polysaccharide capsule that is readily visible by light microscopy. When viewed by differential interference contrast microscopy, binding of mAb to C. neoformans cells produced two distinct capsular reactions that depended on the Ab epitope specificity and the yeast serotype. In the first pattern, termed "rim," the capsule appears transparent with a highly refractive outer edge. In the second pattern, termed "puffy," the capsule appears opaque and lacks a highly refractive outer rim. mAbs that bind with a rim pattern suppress the overall rate of C3 deposition on the yeast via the classical and alternative complement pathways. In contrast, mAbs that bind with a puffy pattern do not affect C3 deposition. Protective and nonprotective IgM mAbs produce rim and puffy patterns, respectively. These results indicate that: 1) capsule reactions are a consequence of Ab-induced changes in capsular refractive index; 2) the type of capsule reaction depends on the Ab specificity; and 3) Ab-induced changes in refractive index correlate with biological activities important for host defense against C. neoformans. Our results provide the first evidence associating distinct capsule reaction patterns with Ab biological activity.     

Cryptococcus neoformans capsular glucuronoxylomannan induces expression of fas ligand in macrophages

The major component of capsular material of Cryptococcus neoformans is glucuronoxylomannnan (GXM), a polysaccharide that exhibits potent immunosuppressive properties in vitro and in vivo. The results reported here show that 1) soluble purified GXM induces a prompt, long-lasting, and potent up-regulation of Fas ligand (FasL) on macrophages, 2) the up-regulation of FasL is related to induced synthesis and increased mobilization to the cellular surface, 3) this effect is largely mediated by interaction between GXM and TLR4, 4) FasL up-regulation occurs exclusively in GXM-loaded macrophages, 5) macrophages that show up-regulation of FasL induce apoptosis of activated T cells expressing Fas and Jurkat cells that constitutively express Fas, and 6) anti-Fas Abs rescue T cells from apoptosis induced by GXM. Collectively our results reveal novel aspects of the immunoregulatory properties of GXM and suggest that this nontoxic soluble compound could be used to dampen the immune response, to promote or accelerate the death receptor, and to fix FasL expression in a TLR/ligand-dependent manner. In the present study, we delineate potential new therapeutic applications for GXM that exploit death receptors as key molecular targets in regulating cell-mediated cytotoxicity, immune homeostasis, and the immunopathology of diseases.     

Self-aggregation of Cryptococcus neoformans capsular glucuronoxylomannan is dependent on divalent cations

The capsular components of the human pathogen Cryptococcus neoformans are transported to the extracellular space and then used for capsule enlargement by distal growth. It is not clear, however, how the glucuronoxylomannan (GXM) fibers are incorporated into the capsule. In the present study, we show that concentration of C. neoformans culture supernatants by ultrafiltration results in the formation of highly viscous films containing pure polysaccharide, providing a novel, nondenaturing, and extremely rapid method to isolate extracellular GXM. The weight-averaged molecular mass of GXM in the film, determined using multiangle laser light scattering, was ninefold smaller than that of GXM purified from culture supernatants by differential precipitation with cetyl trimethyl ammonium bromide (CTAB). Polysaccharides obtained either by ultrafiltration or by CTAB-mediated precipitation showed different reactivities with GXM-specific monoclonal antibodies. Viscosity analysis associated with inductively coupled plasma mass spectrometry and measurements of zeta potential in the presence of different ions implied that polysaccharide aggregation was a consequence of the interaction between the carboxyl groups of glucuronic acid and divalent cations. Consistent with this observation, capsule enlargement in living C. neoformans cells was influenced by Ca(2+) in the culture medium. These results suggest that capsular assembly in C. neoformans results from divalent cation-mediated self-aggregation of extracellularly accumulated GXM molecules.     

Ecdysteroid titres during larval life and metamorphosis of Galleria mellonella

The ecdysteroid titre in Galleria larvae normally follows either a short larval cycle, which is characterized by a single peak on day 2, or a long metamorphic cycle, which includes a small peak on day 6 and a large one on day 7. Intermediate cycles and moult induction with reduced ecdysteroid concentrations occur under experimental conditions. The cycles and peaks are initiated and, until the brain critical period, driven by prothoracicotropic hormone (PTTH), while juvenile hormone (JH) titre determines which type of cycle will be activated. In larval development JH stimulates PTTH release but the metamorphic programme of neuroendocrine regulations involves blocking PTTH production by JH. This hormone then causes an interruption of the ecdysteroid cycle and extension of the last larval instar.     

Phagocytic activity and encapsulation rate of Galleria mellonella larval haemocytes during bacterial infection by Bacillus thuringiensis

The bacterium Bacillus thuringiensis (Bt) is a pathogen of many insect species and is actively used in biocontrol. After the peroral inoculation of Galleria mellonella by the Bt in 5% sublethal concentration (LC₅), a 1.5-fold increase in the phagocytic activity of infected larvae has been registered on the second and third days after the inoculation. With the increase of Bt-inoculum amount to 15% of sublethal concentration (LC₁₅), a further increase of the phagocytic activity and enhanced encapsulation rates in the haemolymph of infected larvae has been observed. The enhanced cellular immunity during the bacteriosis seems to have resulted from the destruction of midgut epithelium cells followed by the subsequent exposure of gut content to lymph factors activating the immune system of haemocoel.