Evidence for Proinflammatory β-1,6 Glucans in the Pneumocystis carinii Cell Wall

Abstract

Inflammation is a major cause of respiratory impairment during Pneumocystis pneumonia. Studies support a significant role for cell wall ␤-glucans in stimulating inflammatory responses. Fungal ␤-glucans are comprised of D-glucose homopolymers containing ␤-1,3-linked glucose backbones with ␤-1,6-linked glucose side chains. Prior studies in Pneumocystis carinii have characterized ␤-1,3 glucan components of the organism. However, recent investigations in other organisms support important roles for ␤-1,6 glucans, predominantly in mediating host cellular activation. Accordingly, we sought to characterize ␤-1,6 glucans in the cell wall of Pneumocystis and to establish their activity in lung cell inflammation. Immune staining revealed specific ␤-1,6 localization in P. carinii cyst walls. Homology-based cloning facilitated characterization of a functional P. carinii kre6 (Pckre6) ␤-1,6 glucan synthase in Pneumocystis that, when expressed in kre6-deficient Saccharomyces cerevisiae, restored cell wall stability. Recently synthesized ␤-1,6 glucan synthase inhibitors decreased the ability of isolated P. carinii preparations to generate ␤-1,6 carbohydrate. In addition, isolated ␤-1,6 glucan fractions from Pneumocystis elicited vigorous tumor necrosis factor alpha (TNF-␣) responses from macrophages. These inflammatory responses were significantly dampened by inhibition of host cell plasma membrane microdomain function. Together, these studies indicate that ␤-1,6 glucans are present in the P. carinii cell wall and contribute to lung cell inflammatory activation during infection. P neumocystis organisms are opportunistic fungi that produce significant morbidity and mortality in immunocompromised hosts, with infection-related fatalities ranging between 10% and 45% (1). Pneumocystis jirovecii is the species known to infect humans, while Pneumocystis carinii represents the parallel species studied widely in rodents (2). Pneumocystis pneumonia remains a significant cause of mortality during AIDS, despite highly active antiretroviral therapy (3-5). Severe Pneumocystis pneumonia is characterized by intense lung inflammation involving CD8 ϩ cells and neutrophils, impairing gas exchange (6-9).The P. carinii cell walls contain abundant ␤-glucan molecules (10). Fungal cell wall ␤-glucans are homopolymers of D-glucose consisting of ␤-1,3 core chains with variable numbers of ␤-1,6 glucose side chains. The variable inflammatory activities of different glucan preparations have been postulated to be related to the relative amounts and configurations of these two major structures (␤-1,3 versus ␤-1,6) (11). Almost all of the initial studies in fungi have largely focused on unfractionated glucans (10). In fact, all prior studies in P. carinii previously utilized only unfractionated ␤-1,3/␤-1,6 glucans. Interestingly, recent investigations in Saccharomyces cerevisiae indicate major roles for ␤-1,6 glucans in strongly mediating cellular activation and inflammation (11). Our investigations of unfractionated P. carinii ␤-glucans indicate that innate ...

Characterization of N-Acetylglucosamine Biosynthesis in Pneumocystis species. A New Potential Target for Therapy

“…Populations of Pc were isolated from chronically infected rat lungs by homogenization and filtration through 10-mm filters, as we previously described (3,18). Unless otherwise noted, the remaining reagents were obtained from Sigma-Aldrich (St. Louis, MO).…”

“…The cell surface of the pathogenic fungal species represented by Pneumocystis organisms have not been fully elucidated, but are known to contain b-1,3 and b-1,6 glucans, and the major surface mannoserich glycoproteins, variously termed major surface glycoprotein (MSG) or glycoprotein A (gpA) (3)(4)(5). We and others have shown that the Pneumocystis species contain b-glucan synthetases that can be inhibited by specific b-glucan inhibitors, such as the echinocandins, pneumocandins, and newly generated compounds (3,4,6,7).…”

“…We and others have shown that the Pneumocystis species contain b-glucan synthetases that can be inhibited by specific b-glucan inhibitors, such as the echinocandins, pneumocandins, and newly generated compounds (3,4,6,7). A number of these drugs are already implemented as antifungal therapies against pathogens (8).…”

“…For example, over the past 20 years, only a handful of studies have searched for GlcNAc residues in Pneumocystis organisms. These included classic biochemical methods for determination of this moiety in the organism via GlcNAc-binding/-labeling experiments using antibody staining techniques, as well as incorporation of 3 H-glucosamine into the P. carinii (Pc) cell walls, supportive of GlcNAc deposition (12)(13)(14). More recently, an intriguing study by Rapaka and colleagues (15) suggested that mice infected with P. murina (Pm) could generate increased levels of IgM antibodies against fungal carbohydrates, including GlcNAc-containing carbohydrates.…”

The Life Cycle Stages of Pneumocystis murina Have Opposing Effects on the Immune Response to This Opportunistic Fungal Pathogen

“…Cysts are ascuslike structures that consist of multiple nuclei surrounded by a fungal cell wall. ␤-1,3-Glucan and ␤-1,6-glucan serve as the structural components of the cyst wall (15,16). Trophic forms do not express ␤-glucan (15).…”