Michael K. Mansour

Involvement of CD14, Toll-Like Receptors 2 and 4, and MyD88 in the Host Response to the Fungal Pathogen Cryptococcus neoformans In Vivo

ABSTRACT The major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14. In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C. neoformans. MyD88−/− mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with live C. neoformans. CD14−/− mice had reduced survival when infected i.v., while TLR2−/− mice died significantly earlier after i.n. infection. Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v. or i.n. challenge with C. neoformans. The course of pulmonary cryptococcosis was studied in more detail in the CD14−/−, TLR2−/−, and MyD88−/− mice. MyD88−/− mice infected i.n. had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did. Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs of C. neoformans-infected knockout mice compared with wild-type mice. Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups. These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88−/− mice correlating with increased numbers of lung CFU and serum and lung GXM levels.

Optimal T Cell Responses to Cryptococcus neoformans Mannoprotein Are Dependent on Recognition of Conjugated Carbohydrates by Mannose Receptors

Cryptococcosis is a leading cause of death among individuals with compromised T cell function. Soluble Cryptococcus neoformans mannoproteins (MP) have emerged as promising vaccine candidates due to their capacity to elicit delayed-type hypersensitivity and Th type 1-like cytokines, both critical to the clearance of this pathogenic yeast. In this study, the mechanisms responsible for the potent immunostimulatory properties of MP were explored. Using Chinese hamster ovary cells expressing human macrophage mannose receptor (MMR), we determined that MP is a MMR ligand. Functionally, competitive blockade of multilectin mannose receptors (MR) on APCs diminished MP-dependent stimulation of primary T cells from immunized mice and the MP-reactive CD4+ T cell hybridoma, P1D6, by 72 and 99%, respectively. Removal of O-linked saccharides from MP by β-elimination inhibited MP-dependent stimulation of P1D6 and primary T cells by 89 and 90%, respectively. In addition, MP-dependent stimulation of P1D6 was abrogated after digestion with proteinase K, suggesting the protein core of MP contributed the antigenic moiety presented by APC. Stimulation of P1D6 by MP also was abolished using APC obtained from invariant chain-deficient mice, demonstrating Ag presentation was MHC class II restricted. Our data suggest that MP is a ligand for the MMR and that T cell stimulation is functionally inhibited either by competitive blockade of MR or by removal of carbohydrate residues critical for recognition. The demonstration that efficient T cell responses to MP require recognition of terminal mannose groups by MMR provides both a molecular basis for the immunogenicity of cryptococcal MP and support for vaccination strategies that target MR.

Molecular characterization of a mannoprotein with homology to chitin deacetylases that stimulates T cell responses to Cryptococcus neoformans

The fungus Cryptococcus neoformans is a major cause of morbidity and mortality in patients with impaired CD4+ T cell function, particularly those with AIDS. To identify cryptococcal antigens that could serve as vaccine candidates by stimulating T cell responses, C. neoformans-reactive CD4+ T cell hybridomas were generated by immunization of C57BL/6 mice and fusion of splenocytes with thymoma cells. The antigen that stimulated one of the hybridomas, designated P1D6, to produce IL-2 was purified to homogeneity by sequential anion exchange chromatography, hydrophobic interaction chromatography, and SDS/PAGE. Based on its apparent molecular mass of 98 kDa and mannosylation, the antigen of interest was named MP98. MP98 was N terminal-sequenced, and the gene encoding the protein was cloned and sequenced. Recombinant MP98, expressed in Saccharomyces cerevisiae, stimulated P1D6 to produce IL-2. Analysis of the derived 458-aa sequence of MP98 reveals an N-terminal cleavable signal sequence, a polysaccharide deacetylase domain found in fungal chitin deacetylases, and a serine/threonine-rich C-terminal region. Overall, there were 103 serine/threonine residues serving as potential O-linked glycosylation sites as well as 12 possible N-linked glycosylation sites. Thus, a C. neoformans mannoprotein has been characterized that stimulates T cell responses and has molecular properties of a chitin deacetylase.

Cryptococcus neoformans glycoantigens are captured by multiple lectin receptors and presented by dendritic cells.

Cell-mediated immune responses to glycoantigens have been largely uncharacterized. Protective T cell responses to the pathogenic yeast Cryptococcus neoformans are dependent on heavily mannosylated Ags termed mannoproteins. In the work presented, the innate immune response to mannoprotein was determined. Purified murine splenic dendritic cells (DC), B cells, and macrophages were used to stimulate mannoprotein-specific T cells. Only DC were capable of any measurable stimulation. Depletion of DC resulted in the abrogation of the T cell response. Human and murine DC rapidly captured fluorescent-labeled mannoprotein by a mannose receptor-mediated process. Using transfected cell lines, the type II C-type lectin receptor DC-specific ICAM-3-grabbing nonintegrin (CD209) was determined to have affinity for mannoprotein. Taken together with prior work demonstrating that mannoprotein was captured by the macrophage mannose receptor (CD206), these data suggest that multiple mannose receptors on DC recognize mannoprotein. Pulsing experiments demonstrated that DC captured sufficient mannoprotein over 2 h to account for 50% of total stimulation. Capture appeared dependent on mannose receptors, as competitive mannosylated inhibitors and calcium chelators each interfered with T cell stimulation. By confocal microscopy, intracellular mannoprotein trafficked to an endo-lysosomal compartment in DC, and at later time points extended into tubules in a similar fashion to the degradation marker DQ-OVA. Mannoprotein colocalized intracellularly with CD206 and CD209. These data suggest that DC provide the crucial link between innate and adaptive immune responses to C. neoformans via a process that is dependent upon the efficient uptake of mannoprotein by mannose receptors.

Interactions of fungi with phagocytes.

Recent advances have broadened our knowledge of the unique role that dendritic cells, macrophages and neutrophils play in protecting the host against fungal infections and the mechanisms by which fungal pathogens attempt to subvert phagocytic defenses. In this article, the interplay between phagocytes and fungi is reviewed.

A Model Vaccine Exploiting Fungal Mannosylation to Increase Antigen Immunogenicity

Ag mannosylation represents a promising strategy to augment vaccine immunogenicity by targeting Ag to mannose receptors (MRs) on dendritic cells. Because fungi naturally mannosylate proteins, we hypothesized that Ags engineered in fungi would have an enhanced capacity to stimulate T cell responses. Using the model Ag OVA, we generated proteins that differentially expressed N- and O-linked mannosylation in the yeast Pichia pastoris and compared them to their unglycosylated counterparts produced in Escherichia coli. We found that yeast-derived OVA proteins containing N-linkages, extensive O-linkages, or both were more potent than the unmannosylated Ags at inducing OVA-specific CD4+ T cell proliferation. This elevated response to fungal Ags was inhibited by mannan, suggesting involvement of MRs. However, the macrophage MR (CD206) was not essential, because macrophage MR-deficient dendritic cells were fully competent in presenting yeast-derived OVA Ags. Thus, the use of fungal glycosylation to provide N-linked and/or extensive O-linked mannosylation increased the capacity of the model Ag OVA to stimulate Ag-specific T cell responses in an MR-dependent manner. These data have implications for vaccine design by providing proof of principle that yeast-derived mannosylation can enhance immunogenicity.

Purification and Characterization of a Second Immunoreactive Mannoprotein from Cryptococcus neoformans That Stimulates T-Cell Responses

ABSTRACT Although T-cell responses are known to be critical for effective host defenses against the fungal pathogen Cryptococcus neoformans, the antigens that stimulate protective responses are poorly characterized but are thought to be comprised, at least in part, of mannoproteins. Recently, we created a panel of murine CD4+-T-cell hybridomas that react with C. neoformans antigens. A mannoprotein antigen, MP98, that stimulated one of the hybridomas was purified, and the gene encoding MP98 was cloned. In the present study, the cryptococcal antigen, MP88, that stimulated a second T-cell hybridoma, X5A3, to secrete interleukin-2 was characterized. MP88 was purified from supernatants of glass bead-disrupted C. neoformans by anion-exchange and hydrophobic interaction chromatography. A single band with an apparent molecular mass of 88 kDa was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to partial internal amino acid sequencing. The gene encoding MP88 was cloned and sequenced. MP88 features a C-terminal serine/threonine-rich region, which presumably serves as a site for extensive O glycosylation, followed by a putative glycosylphosphatidylinositol anchor site. A search of C. neoformans genomic databases revealed that MP88 shares this feature with at least 11 other genes, including MP98. The mannoprotein nature of MP88 was established based upon the capacity of (i) the mannoprotein fraction of C. neoformans supernatants to stimulate X5A3 and (ii) mannosylated ligands to competitively inhibit this stimulation. Thus, a second cryptococcal mannoprotein has been identified which stimulates T-cell responses and is a vaccine candidate.

Acute and long-term outcome of narrowed saphenous venous grafts treated by endoluminal stenting and directional atherectomy

Angioplasty of the narrowed saphenous vein bypass grafts remains a difficult challenge. Over a 37-month period at this institution, 119 of 176 interventions (68%) on saphenous vein grafts (average age 8.3 years from bypass surgery to graft intervention) were performed using either directional coronary atherectomy (n = 35) or Palmaz-Schatz intracoronary stents (n = 84), representing 37% of all stents and 15% of all atherectomies during the study period, respectively. Of the 57 saphenous vein graft lesions treated with conventional balloon angioplasty during this period, 49 (86%) had 1 or more contraindications to stenting or directional atherectomy (thrombus, total occlusion, reference vessel less than 3 mm in diameter). The acute success rate was 99% for stents (1 failure to dilate) and 94% for directional atherectomy (2 failures to cross the lesion with the atherectomy device). Lumen diameter increased from 0.9 to 3.6 mm (reference vessel 3.6) for stents, and from 0.9 to 3.5 mm (reference 3.8) for atherectomy (for all comparisons, p = not significant), with no major complications (abrupt or subabrupt closure, emergent coronary bypass surgery, death, or Q-wave myocardial infarctions). During the same time period 50 of 57 vein grafts (88%) rejected for stenting or atherectomy were dilated successfully by conventional balloon angioplasty, with 3 patients (5%) requiring emergent coronary bypass surgery. Angiographic follow-up was available for 50 of 64 eligible patients (78%).(ABSTRACT TRUNCATED AT 250 WORDS)

KRE5 Gene Null Mutant Strains of Candida albicans Are Avirulent and Have Altered Cell Wall Composition and Hypha Formation Properties

ABSTRACT The UDP-glucose:glycoprotein glucosyltransferase (UGGT) is an endoplasmic reticulum sensor for quality control of glycoprotein folding. Saccharomyces cerevisiae is the only eukaryotic organism so far described lacking UGGT-mediated transient reglucosylation of N-linked oligosaccharides. The only gene in S. cerevisiae with similarity to those encoding UGGTs is KRE5. S. cerevisiae KRE5 deletion strains show severely reduced levels of cell wall β-1,6-glucan polymer, aberrant morphology, and extremely compromised growth or lethality, depending on the strain background. Deletion of both alleles of the Candida albicans KRE5 gene gives rise to viable cells that are larger than those of the wild type (WT), tend to aggregate, have enlarged vacuoles, and show major cell wall defects. C. albicans kre5/kre5 mutants have significantly reduced levels of β-1,6-glucan and more chitin and β-1,3-glucan and less mannoprotein than the WT. The remaining β-1,6-glucan, about 20% of WT levels, exhibits a β-1,6-endoglucanase digestion pattern, including a branch point-to-linear stretch ratio identical to that of WT strains, suggesting that Kre5p is not a β-1,6-glucan synthase. C. albicans KRE5 is a functional homologue of S. cerevisiae KRE5; it partially complements both the growth defect and reduced cell wall β-1,6-glucan content of S. cerevisiae kre5 viable mutants. C. albicans kre5/kre5 homozygous mutant strains are unable to form hyphae in several solid and liquid media, even in the presence of serum, a potent inducer of the dimorphic transition. Surprisingly the mutants do form hyphae in the presence of N-acetylglucosamine. Finally, C. albicans KRE5 homozygous mutant strains exhibit a 50% reduction in adhesion to human epithelial cells and are completely avirulent in a mouse model of systemic infection.

Protective Efficacy of Antigenic Fractions in Mouse Models of Cryptococcosis

ABSTRACT Infections due to the encapsulated fungus Cryptococcus neoformans are a significant cause of morbidity and mortality in patients with impaired T-cell function, particularly those with AIDS. Presumably then, T-cell responses to cryptococcal antigens are critical for protection against this ubiquitous fungus. To test the protective efficacy of these antigens as vaccine candidates, secreted cryptococcal antigens were separated by concanavalin A affinity chromatography into adherent (mannoprotein [MP]) and nonadherent (flowthrough [FT]) fractions, and the fractions were tested in murine models of disseminated cryptococcosis. Compared with adjuvant alone, C57BL/6 mice that received two inoculations of MP and FT exhibited prolonged survival and reduced brain and kidney fungal loads following intravenous challenge with C. neoformans strain B3501. MP-immunized animals had increased brain levels of tumor necrosis factor alpha, gamma interferon, and interleukin-2. Histopathologic examination revealed that compared with organs from mice that received only adjuvant, MP-immunized mice were able to recruit a stronger cellular infiltrate in brain, kidney, and liver in response to cryptococcal infection. Conjugated O-linked glycans were necessary for optimal MP-mediated protection, because chemical O deglycosylation reduced the protective efficacy of MP immunization. FT and MP immunization protected B-cell-deficient, but not T-cell-deficient mice, suggesting that protection was T-cell mediated. CBA/J mice also benefited from immunization with FT and MP, although the benefits were more modest than those seen with C57BL/6 mice. Thus, both MP and FT fractions of C. neoformans contain components that protect mice from disseminated cryptococcosis, and this protection appears to be T-cell mediated.