Histoplasma Capsulatum -(1,3)-Glucan Blocks Innate Immune Recognition By
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Histoplasma capsulatum ␣-(1,3)-glucan blocks innate immune recognition by the -glucan receptor Chad A. Rappleye*, Linda Groppe Eissenberg, and William E. Goldman† Department of Molecular Microbiology, Washington University, St. Louis, MO 63110 Edited by Emil C. Gotschlich, The Rockefeller University, New York, NY, and approved November 30, 2006 (received for review November 6, 2006) Successful infection by fungal pathogens depends on subversion of inhaled into the lung germinate into yeast, and this conversion is host immune mechanisms that detect conserved cell wall compo- absolutely required for pathogenicity (3, 4), suggesting that yeast nents such as -glucans. A less common polysaccharide, ␣-(1,3)- phase-specific characteristics include determinants important for glucan, is a cell wall constituent of most fungal respiratory patho- virulence. gens and has been correlated with pathogenicity or linked directly One such yeast phase-specific component is ␣-(1,3)-glucan, a to virulence. However, the precise mechanism by which ␣-(1,3)- homopolymer of glucose with ␣-glycosidic linkages, which has glucan promotes fungal virulence is unknown. Here, we show that been linked to fungal virulence. The cell walls of most medically ␣-(1,3)-glucan is present in the outermost layer of the Histoplasma important fungi contain ␣-(1,3)-glucan, and reduction in ␣-(1,3)- capsulatum yeast cell wall and contributes to pathogenesis by glucan, through either laboratory passage of Histoplasma, Blas- concealing immunostimulatory -glucans from detection by host tomyces, and Paracoccidioides (5–7) or genetic loss of ␣-(1,3)- phagocytic cells. Production of proinflammatory TNF␣ by phago- glucan synthase (AGS1)inHistoplasma (8), has no effect on in cytes was suppressed either by the presence of the ␣-(1,3)-glucan vitro growth but severely attenuates virulence in murine respi- layer on yeast cells or by RNA interference based depletion of the ratory infection models. However, the mechanism by which host -glucan receptor dectin-1. Thus, we have functionally de- ␣-(1,3)-glucan facilitates the pathogenesis of dimorphic fungi has fined key molecular components influencing the initial host–patho- not been determined. We present evidence showing that His- gen interaction in histoplasmosis and have revealed an important toplasma cell wall ␣-(1,3)-glucan blocks host PRR recognition of mechanism by which H. capsulatum thwarts the host immune the fungal PAMP -glucan, enabling Histoplasma yeast to avoid system. Furthermore, we propose that the degree of this evasion detection as a fungal invader. contributes to the difference in pathogenic potential between dimorphic fungal pathogens and opportunistic fungi. Results Histoplasma ␣-(1,3)-Glucan Production Alters the Exposed Yeast Cell cell wall ͉ fungal pathogenesis ͉ virulence factor ͉ dectin-1 ͉ macrophage Surface. As one of the first fungal structures sampled by the host immune surveillance system, the Histoplasma yeast cell wall is he mammalian innate immune system detects invading mi- likely organized to influence the initial host–pathogen interac- Tcrobial pathogens in part by recognition of pathogen- tion. Respiratory infection by Histoplasma occurs by inhalation associated molecular patterns (PAMPs) and utilizes this infor- of mold-produced conidia that germinate into the parasitic yeast mation to tailor an appropriate immune response. The form on exposure to mammalian body temperatures. Although corresponding host molecules used, termed pattern-recognition mold-form Histoplasma entirely lacks ␣-(1,3)-glucan, germina- receptors (PRRs), detect molecules that characterize broad tion of Histoplasma conidia at 37°C into yeast cells initiates classes of microbes. Many of these PRRs are highly expressed on production of this polysaccharide, which we detected only on the front-line immune cells, particularly macrophages and dendritic emergent yeast cell wall and not the conidial structure (Fig. 1 cells. For fungi, the polysaccharide-rich cell wall is a major A–C). This finding suggests that the immediate synthesis of source of PAMPs, and it comprises the initial structure sampled ␣-(1,3)-glucan is required specifically for pulmonary infection by cells of the immune system. Identified PRRs important for the and yeast survival. detection of fungal surface components include the Toll-like The cell walls of wild-type Histoplasma yeast contain primarily receptors TLR2 and TLR4, the collectins SP-A and SP-D, three polysaccharides: chitin, -glucans [-(1,3)- and -(1, 6)- pentraxin-3, the CR3 integrin, and C-type lectins, all of which linked], and ␣-(1,3)-glucan (9–12). Immunofluorescence local- appear to detect fungal-associated carbohydrates (1). Recogni- ization in cross-sections of wild-type yeast cells with ␣-(1,3)- and tion of fungal-surface polysaccharides initiates immediate re- -(1,3)-glucan-specific antibodies showed a nonhomogeneous sponses such as phagocytosis, production of antimicrobial com- spatial distribution (Fig. 2 A–C). Although some overlap exists pounds, and induction of proinflammatory cytokines that between ␣-(1,3)-glucan and -(1,3)-glucan, merging the images activate and recruit other immune effector cells. Recent work on suggests that the cell wall is somewhat layered, with ␣-(1,3)- the nonclassical C-type lectin, dectin-1, has defined its substrate glucan being more external (Fig. 2 C and D). This layered to be oligomers of -(1,3)-glucan (2), a constituent of the cell wall of all fungi and a potent immunostimulatory molecule that induces TNF␣ production by macrophages. Author contributions: C.A.R., L.G.E., and W.E.G. designed research; C.A.R. and L.G.E. performed research; C.A.R. and L.G.E. analyzed data; and C.A.R. and W.E.G. wrote the Successful pathogens must therefore have mechanisms to avoid paper. or counteract detection by host PRRs. Such mechanisms are likely The authors declare no conflict of interest. to distinguish primary pathogens that cause disease in normal hosts This article is a PNAS direct submission. from opportunistic pathogens that require suppression of the host Abbreviations: PAMP, pathogen-associated molecular pattern; PRR, pattern-recognition immune system. The primary pathogenic fungi include Histoplasma receptor; shRNA, short-hairpin RNA; TLR, Toll-like receptor. capsulatum, Paracoccidioides brasiliensis, Blastomyces dermatitidis, *Present address: Department of Microbiology, Ohio State University, Columbus, OH Coccidioides spp., and Cryptococcus neoformans. With the excep- 43210. tion of Cryptococcus, each of these species is ‘‘dimorphic,’’ growing †To whom correspondence should be addressed at: Department of Molecular Microbiol- as a saprophytic mold form at ambient temperatures (i.e., the soil ogy, Campus Box 8230, Washington University School of Medicine, St. Louis, MO 63110. environment) and a parasitic yeast (or spherule) form at mamma- E-mail: [email protected]. lian body temperatures. Mold-produced conidia (or spores) that are © 2007 by The National Academy of Sciences of the USA 1366–1370 ͉ PNAS ͉ January 23, 2007 ͉ vol. 104 ͉ no. 4 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609848104 Downloaded by guest on September 24, 2021 Fig. 1. ␣-(1,3)-Glucan is produced during the transition from conidia to yeast. Histoplasma conidia were incubated for 18 h at 37°C in F-12 medium containing 10% FBS and visualized by direct interference contrast microscopy (DIC) (A) and immunofluorescence microscopy (B) after staining with an antibody specific for ␣-(1,3)-glucan. Arrowheads indicate germinating conidia with emergent yeast buds that have a smoother cell surface. (C) Merged DIC and immunofluorescence images show that ␣-(1,3)-glucan is present only around the yeast buds and not the conidial structure. organization supports earlier biochemical analyses that used serial ␣-glucanase and -glucanase treatments to quantify cell wall polysaccharides as well as ultrastructural studies in which alkali treatment of isolated cell walls removed an outer fibrillar pattern (9, 11). To confirm this spatial organization, ␣-(1,3)- glucan and -(1,3)-glucan were localized by immunoelectron microscopy of Histoplasma yeast (Fig. 2E). Quantitation of the gold particle labeling corroborates the immunofluorescence localization patterns, with Ϸ50% of ␣-(1,3)-glucan being located more distal to the yeast cell membrane than -(1,3)-glucan (Fig. 2 F and G). This organization suggests that the ␣-(1,3)-glucan layer may conceal the underlying -glucans from detection by the Fig. 3. The ␣-(1,3)-glucan layer blocks recognition of Histoplasma yeast by host. dectin-1.(A) Live Histoplasma yeasts lacking ␣-(1,3)-glucan [ags1(⌬)] readily bind to dectin-1-expressing 3T3 cells, whereas yeasts possessing ␣-(1,3)-glucan ␣-(1,3)-Glucan Prevents Recognition of Histoplasma Yeast by dectin-1. [AGS1(ϩ)] do not bind. Binding was measured by coincubation of Histoplasma Because dectin-1 functions as the primary -glucan receptor on yeast with confluent monolayers of 3T3 cells or dectin-1-expressing 3T3 cells host cells, we tested whether ␣-(1,3)-glucan could block dectin-1 followed by quantitation of bound yeasts with the fungal fluorescent stain Uvitex 3BSA. (B and C) Binding of ags1(⌬) yeast cells depends on dectin-1 expression. (B) Merged phase-contrast and GFP fluorescence images show that only 3T3 cells expressing dectin-1 (marked by cotransfection of a gfp expres- sion plasmid) bind ags1(⌬) yeast. (C) Immunofluorescence labeling of cells with an antibody to dectin-1 shows enrichment of dectin-1 protein at sites of contact