Role of Sulfated Glycans in Adherence of the Microsporidian Encephalitozoon intestinalis to Host Cells In Vitro

Abstract

Microsporidia are obligate intracellular opportunistic protists that infect a wide variety of animals, including humans, via environmentally resistant spores. Infection requires that spores be in close proximity to host cells so that the hollow polar tube can pierce the cell membrane and inject the spore contents into the cell cytoplasm. Like other eukaryotic microbes, microsporidia may use specific mechanisms for adherence in order to achieve target cell proximity and increase the likelihood of successful infection. Our data show that Encephalitozoon intestinalis exploits sulfated glycans such as the cell surface glycosaminoglycans (GAGs) in selection of and attachment to host cells. When exogenous sulfated glycans are used as inhibitors in spore adherence assays, E. intestinalis spore adherence is reduced by as much as 88%. However, there is no inhibition when nonsulfated glycans are used, suggesting that E. intestinalis spores utilize sulfated host cell glycans in adherence. These studies were confirmed by exposure of host cells to xylopyranoside, which limits host cell surface GAGs, and sodium chlorate, which decreases surface sulfation. Spore adherence studies with CHO mutant cell lines that are deficient in either surface GAGs or surface heparan sulfate also confirmed the necessity of sulfated glycans. Furthermore, when spore adherence is inhibited, host cell infection is reduced, indicating a direct association between spore adherence and infectivity. These data show that E. intestinalis specifically adheres to target cells by way of sulfated host cell surface GAGs and that this mechanism serves to enhance infectivity.

Interaction and Assembly of Two Novel Proteins in the Spore Wall of the Microsporidian Species Nosema bombycis and Their Roles in Adherence to and Infection of Host Cells

“…The procedures for these assays were similar to those previously reported, with some changes (20,24,27). For the antibody-blocking assay, the anti-SWP9 rabbit antibody (5 g/ml), anti-SWP7 rabbit antibody (5 g/ml), and negativecontrol rabbit antibody (5 g/ml) were used to block N. bombycis spores (2.8 ϫ 10 6 ) for 2 h at 28°C prior to placing the spores onto BmE cell monolayers (1 ϫ 10 5 cells).…”

“…For N. bombycis, only two exosporal proteins (21)(22)(23), three endosporal proteins (21,24,25), and a BAR-2 spore wall protein (26) have been characterized. In addition, several spore wall proteins that adhere to host cells via a heparin-binding motif (HBM) and interact with sulfated glycosaminoglycans (GAGs) of the host cell surface have been identified (20,24,27,28). In recent years, several studies have found that the spores of N. bombycis not only infect the Bombyx mori ovarian cell line BmN-SWU1, but also adhere to and infect the B. mori embryonic cell line BmE-SWU1.…”

The microsporidian polar tube: A highly specialised invasion organelle

“…These modifications may be important in adherence of the spore wall to mucin or to host cells during passage of the spores in the gastrointestinal tract, facilitating invasion. It has, for example, been demonstrated that exogenous glycosaminoglycans can decrease the adherence of spores to a host cell monolayer (Hayman et al, 2005).…”

Expression and characterization of a novel spore wall protein from Nosema bombycis

“…Adherence of N. bombycis spores and infection were measured as previously described (Hayman et al, 2005;Wu et al, 2009). Approximately 1×10 6 cells with 2 ml growth medium in each dish were allowed to grow at 27°C for 16 h. Recombinant proteins (0.1-10 µg/ml), as well as the control sample (negative serum), were incubated with 5.0×10…”

“…The spore wall, which consists of an electron-dense, proteinaceous outer layer (exospore), an electron-lucent inner layer (endospore) composed of chitin and protein, and a plasma membrane (Vavra et al, 1999), provides structural rigidity and protects the mature spore from the outer environment. The spore wall proteins (SWPs) of microsporidia may play a role in recognition by the host during the invasion process (Hayman et al, 2005;Southern et al, 2006), and the interactions between the SWPs and the host plasma membrane mediate the microsporidian spores being phagocytized by the host cell (Couzinet et al, 2000;Franzen et al, 2005).…”