Insights into mechanisms of antibody-mediated immunity from studies with Cryptococcus neoformans

At first glance Cryptococcus neoformans appears an unlikely microbe to provide a new understanding of mechanisms of antibody-mediated immunity (AMI), because it is a facultative intracellular fungal pathogen for which the role of naturally acquired AMI in host defense is uncertain. However, numerous studies have now established that certain antibodies (Abs) against C. neoformans are protective in certain hosts. Studies with Abs to C. neoformans have provided new insights into AMI and generated new precedents with implications for other pathogens. The following concepts have emerged: 1) susceptibility to C. neoformans may be related to qualitative and quantitative aspects of the Ab response; 2) protective monoclonal Abs can be generated against pathogens even when the role of humoral immunity is uncertain; 3) Abs to C. neoformans mediate protection by immunomodulatory effects, thereby linking Ab efficacy to the overall host immune response; 4) Ab efficacy is critically dependent on fine specificity, which in turn is affected by immunoglobulin variable region usage, somatic mutation and constant region usage; 5) the efficacy of passive Ab therapy is a function of Ab dose and infecting innoculum, with lack of efficacy at the extremes of Ab concentration; 6) Ab-mediated toxicity resulting from antigen-Ab complex-induced release of platelet activating factor is isotype dependent. Observations with C. neoformans have stimulated a reappraisal of the role of humoral immunity for other pathogens and highlighted the limitations in current methods of assessing the role of Ab in host defense.     

Mechanism for the isotype dependence of antibody-mediated toxicity in Cryptococcus neoformans-infected mice

Ab-based therapies have undergone a renaissance in recent years, but infusion-related reactions are a significant clinical problem. Administration of certain mAbs to Swiss Webster mice infected with Cryptococcus neoformans can result in acute lethal toxicity (ALT) characterized by cardiovascular collapse. The ability of a mAb to produce ALT is isotype dependent and occurs with IgG1 but not IgG3. To investigate this phenomenon, we measured spleen and liver cytokine responses and platelet-activating factor (PAF) content in mice given C. neoformans glucuronoxylomannan (GXM) followed by specific Ab of IgG1 or IgG3 isotype. We found no evidence to suggest that the differences in IgG1 and IgG3 toxicity were due to differences in chemokine or cytokine response. In contrast, liver and spleen tissue PAF content was significantly greater in mice IgG1. Furthermore, our results show differences in the response to IgG1- and IgG3-GXM complexes regarding: 1) macrophage-inflammatory protein-1alpha and monocyte chemoattractant protein-1 regulation, 2) splenic and hepatic PAF content, and 3) hepatic PAF content in infected mice. IgG1-associated ALT appears to be the result of greater production of PAF in response to IgG1-GXM complex formation. The results are consistent with the view that IgG1 and IgG3 interact with different Fc receptors. Our findings strongly suggest that the mechanism for Ab-mediated ALT is different from the cytokine release syndrome described after administration of other therapeutic mAbs.     

An innate immune system cell is a major determinant of species-related susceptibility differences to fungal pneumonia

Rats and mice are considered resistant and susceptible hosts, respectively, for experimental cryptococcosis. For both species, alveolar macrophages (AM) are central components of the host response to pulmonary Cryptococcus neoformans infection. We explored the role of AM in three strains of mice and three strains of rats during cryptococcal infection by comparing the outcome of infection after macrophage depletion using liposomal clodronate. AM depletion was associated with enhancement and amelioration of disease in rats and mice, respectively, as measured by lung fungal burden. The apparent protective role for AM in rats correlated with enhanced anti-cryptococcal activity as measured by phagocytic activity, oxidative burst, lysozyme secretion, and ability to limit intracellular growth of C. neoformans. Furthermore, rat AM were more resistant to lysis in association with intracellular infection. In summary, differences in AM function in rats and mice suggest an explanation for the species differences in susceptibility to C. neoformans based on the inherent efficacy of a central effector cell of the innate immune system.     

Radiological studies reveal radial differences in the architecture of the polysaccharide capsule of Cryptococcus neoformans

The polysaccharide capsule of the pathogenic fungus Cryptococcus neoformans is an important virulence factor, but relatively little is known about its architecture. We applied a combination of radiological, chemical, and serological methods to investigate the structure of this polysaccharide capsule. Exposure of C. neoformans cells to gamma radiation, dimethyl sulfoxide, or radiolabeled monoclonal antibody removed a significant part of the capsule. Short intervals of gamma irradiation removed the outer portion of the cryptococcal capsule without killing cells, which could subsequently repair their capsules. Survival analysis of irradiated wild-type, acapsular mutant, and complemented mutant strains demonstrated that the capsule contributed to radioprotection and had a linear attenuation coefficient higher than that of lead. The capsule portions remaining after dimethyl sulfoxide or gamma radiation treatment were comparable in size, 65 to 66 microm3, and retained immunoreactivity for a monoclonal antibody to glucuronoxylomannan. Simultaneous or sequential treatment of the cells with dimethyl sulfoxide and radiation removed the remaining capsule so that it was not visible by light microscopy. The capsule could be protected against radiation by either of the free radical scavengers ascorbic acid and sorbitol. Sugar composition analysis of polysaccharide removed from the outer and inner parts of the capsule revealed significant differences in glucuronic acid and xylose molar ratios, implying differences in the chemical structure of the constituent polysaccharides. Our results provide compelling evidence for the existence of two zones in the C. neoformans capsule that differ in susceptibility to dimethyl sulfoxide and radiation and, possibly, in packing and composition.     

Cryptococcus neoformans CAP59 (or Cap59p) is involved in the extracellular trafficking of capsular glucuronoxylomannan

Several genes are essential for Cryptococcus neoformans capsule synthesis, but their functions are unknown. We examined the localization of glucuronoxylomannan (GXM) in strain B-3501 and in cap59 mutants B-4131 and C536. Wild-type strain B-3501 showed a visible capsule by India ink staining and immunofluorescence with anticapsular monoclonal antibodies (MAbs) 12A1 and 18B7. B-4131, a mutant containing a missense mutation in CAP59, showed no capsule by India ink staining but revealed the presence of capsular polysaccharide on the cell surface by immunofluorescence. The cap59 gene deletion mutant (C536), however, did not show a capsule by either India ink staining or immunofluorescence. Analysis of cell lysates for GXM by enzyme-linked immunosorbent assay revealed GXM in C536 samples. Furthermore, the epitopes recognized by MAbs 12A1, 2D10, 13F1, and 18B7 were each detected in the cytoplasm of all strains by immunogold electron microscopy, although there were differences in location consistent with differences in epitope synthesis and/or transport. In addition, the cells of B-3501 and B-4131, but not those of the cap59 deletant, assimilated raffinose or urea. Hence, the missense mutation of CAP59 in B-4131 partially hampered the trafficking of GXM but allowed the secretion of enzymes involved in hydrolysis of raffinose or urea. Furthermore, the cell diameter and volume for strain C536 are higher than those for strain B-3501 or B-4131 and may suggest the accumulation of cellular material in the cytoplasm. Our results suggest that CAP59 is involved in capsule synthesis by participating in the process of GXM (polysaccharide) export.     

Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans

The human pathogenic fungus Cryptococcus neoformans secretes a phospholipase enzyme that demonstrates phospholipase B (PLB), lysophospholipase hydrolase and lysophospholipase transacylase activities. This enzyme has been postulated to be a cryptococcal virulence factor. We cloned a phospholipase-encoding gene (PLB1) from C. neoformans and constructed plb1 mutants using targeted gene disruption. All three enzyme activities were markedly reduced in the mutants compared with the wild-type parent. The plb1 strains did not have any defects in the known cryptococcal virulence phenotypes of growth at 37 degrees C, capsule formation, laccase activity and urease activity. The plb1 strains were reconstituted using the wild-type locus and this resulted in restoration of all extracellular PLB activities. In vivo testing demonstrated that the plb1 strain was significantly less virulent than the control strains in both the mouse inhalational model and the rabbit meningitis model. We also found that the plb1 strain exhibited a growth defect in a macrophage-like cell line. These data demonstrate that secretory phospholipase is a virulence factor for C. neoformans.     

Mutagenic properties of a nitrofuran, 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), in lacI transgenic mice

The in vivo mutagenic properties of a 5-nitrofuran, the 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), already well known in bacteria, was evaluated in lacI transgenic mice (Big Blue). The mutation frequency was determined in various organs of i.p. — treated mice and the nature of induced mutations was determined for the target organs in which mutation induction was significant. It was found that R7000 is mutagenic in mice, although, on the basis of the number of induced mutants per unit mass in comparison with other known mutagenic chemicals, R7000 appears to be considerably less mutagenic in mice than in bacteria. The most affected organs, small intestine, caecum and colon organs belong to the digestive apparatus. The distribution of R7000-induced mutations in the lacI gene recovered from small intestine of transgenic mice was very similar to that which had been found in E. coli. The difference between mouse and E. coli in the R7000 induced mutational spectra are mainly in the proportion of single base frameshifts versus base substitutions. Since R7000 induced mutations seemed to arise in the population of stem cells and that the stem cells are important for carcinogenesis, our results are compatible with a possible carcinogenic effect of R7000 and other nitrofurans.     

Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans

The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans‐cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow‐derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C. albicans EV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850 nm. Two predominant seroreactive proteins (27 kDa and 37 kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C. albicans EV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin‐layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)‐12, transforming growth factor‐beta (TGF‐β) and IL‐10. Similarly, EV‐treated DC produced IL‐12p40, IL‐10 and tumour necrosis factor‐alpha. In addition, EV treatment induced the up‐regulation of CD86 and major histocompatibility complex class‐II (MHC‐II). Inoculation of G. mellonella larvae with EV followed by challenge with C. albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C. albicans EV were immunologically active and could potentially interfere with the host responses in the setting of invasive candidiasis.     

Aging‐related anatomical and biochemical changes in lymphatic collectors impair lymph transport,fluid homeostasis,and pathogen clearance

The role of lymphatic vessels is to transport fluid, soluble molecules, and immune cells to the draining lymph nodes. Here, we analyze how the aging process affects the functionality of the lymphatic collectors and the dynamics of lymph flow. Ultrastructural, biochemical, and proteomic analysis indicates a loss of matrix proteins, and smooth muscle cells in aged collectors resulting in a decrease in contraction frequency, systolic lymph flow velocity, and pumping activity, as measured in vivo in lymphatic collectors. Functionally, this impairment also translated into a reduced ability for in vivo bacterial transport as determined by time‐lapse microscopy. Ultrastructural and proteomic analysis also indicates a decrease in the thickness of the endothelial cell glycocalyx and loss of gap junction proteins in aged lymph collectors. Redox proteomic analysis mapped an aging‐related increase in the glycation and carboxylation of lymphatic's endothelial cell and matrix proteins. Functionally, these modifications translate into apparent hyperpermeability of the lymphatics with pathogen escaping from the collectors into the surrounding tissue and a decreased ability to control tissue fluid homeostasis. Altogether, our data provide a mechanistic analysis of how the anatomical and biochemical changes, occurring in aged lymphatic vessels, compromise lymph flow, tissue fluid homeostasis, and pathogen transport.     

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

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