Application of mass spectrometry to elucidate the pathophysiology of Encephalitozoon cuniculi infection in rabbits.

Encephalitozoon cuniculi is a microsporidian species which can induce subclinical to serious disease in mammals including rabbits, a definitive natural host. The pathophysiology of infection has not been comprehensively elucidated. In this exploratory study, we utilized two mass spectrometry approaches: first, the analysis of the humoral response by profiling the microsporidian antigens as revealed by Western blot screening, and second, implementing the iTRAQ®-labeling protocol to focus on the changes within the host proteome during infection. Seven E. cuniculi proteins were identified at one-dimensional gel regions where specific seropositive reaction was observed by Western blot, including polar tube protein 3, polar tube protein 2, and for the first time reported: heat shock related 70kDa protein, polysaccharide deacetylase domain-containing protein, zinc finger protein, spore wall and anchoring disk complex protein EnP1, and translation elongation factor 1 alpha. In addition, there was a significant increase of nine host proteins in blood samples from E. cuniculi-diseased rabbits in comparison with non-diseased control subjects undergoing various inflammatory processes. This included serum paraoxonase, alpha-1-antiproteinase F precursor and alpha-1-antiproteinase S-1 which have presumptive catalytic activity likely related to infection control, and cystatin fetuin-B-type, an enzyme regulator that has been poorly studied to date. Notably, 11 proteins were found to be statistically increased in rabbits with neurological versus renal clinical presentation of E. cuniculi infection. Overall, this novel analysis based on mass spectrometry has provided new insights on the inflammatory and humoral responses during E. cuniculi infection in rabbits.

B-1 cell decreases susceptibility to encephalitozoonosis in mice.

Encephalitozoon cuniculi is an opportunist intracellular pathogen of mammals. The adaptive immune response is essential to eliminate E. cuniculi, but evidence is mounting that the response initiated by the innate immune response may ultimately define whether or not the parasite can survive. B-1 cells may act as antigen-presenting cells or differentiate into phagocytes, playing different roles in many infection models. However, the role of these cells in the dynamics of Encephalitozoon sp. infections is still unknown. To investigate the role of B-1 cells in E. cuniculi infection, BALB/c and BALB/c XID (B-1 cells deficient) mice were infected with E. cuniculi spores. Cytometric analyses of peritoneal cells showed that B-1 cells and macrophages increased significantly in infected BALB/c mice compared to uninfected controls. Despite the increase in the number of CD4+ and CD8+ lymphocytes in XID mice, these animals were more susceptible to infection as evidenced histologically with more prominent inflammatory lesions and parasite burden. Pro-inflammatory cytokines increased in both infected BALB/c and BALB/c XID mice. To confirm B-1 cells role in encephalitozoonosis, we adoptively transferred B-1 cells to BALB/c XID mice and this group showed few symptoms and microscopic lesions, associated with an increased in cytokines. Together, these results suggest that B-1 cells may increase the resistance of BALB/c mice to encephalitozoonosis, evidencing for the first time the important role of B-1 lymphocytes in the control of microsporidia infection.

Encephalitozoon microsporidia modulates p53-mediated apoptosis in infected cells.

Microsporidia are intracellular obligate parasites which have recently been found to be related to fungi. They have a unique extrusion apparatus that is able to inject the sporoplasm directly into the target cell without using receptors. Encephalitozoon microsporidia are a source of morbidity and mortality in humans. It has been suggested that microsporidia may modulate the host cell cycle and apoptosis. We report here that caspase-3 cleavage is inhibited at different times of Vero cell infection by Encephalitozoon microsporidia and that the phosphorylation and translocation of p53 to the nucleus, previous steps for the activation of this protein, do not occur after infection of Vero cells. Consequently, the transcriptional function of p53 is impaired during the infection cycle as demonstrated by luciferase reporter assays. Thus, to our knowledge, for the first time it is shown that an intracellular parasite may be able to multiply in the host cell without activating the p53 apoptotic pathway of that cell. However, changes in the expression of Bcl-2 or Bax levels were not observed.

Functional characterization of a putative aquaporin from Encephalitozoon cuniculi, a microsporidia pathogenic to humans.

The microsporidia are a group of obligate intracellular parasitic protists that have been implicated as both human and veterinary pathogens. The infectious process of these organisms is believed to be dependent upon the rapid influx of water into spores, presumably via aquaporins (AQPs), transmembrane channels that facilitate osmosis. An AQP-like sequence of the microsporidium Encephalitozoon cuniculi (EcAQP), when cloned and expressed in oocytes of Xenopus laevis, rendered these oocytes highly permeable to water. No permeability to the solutes glycerol or urea was observed. Pre-treatment of EcAQP-expressing oocytes with HgCl(2) failed to inhibit their osmotic permeability, as predicted from EcAQP's lack of mercury-sensitive cysteine residues near the NPA motifs which line the AQP aqueous pore. EcAQP exhibits sequence identity to AQP A of Dictyostelium discoideum (26%) and human AQP 2 (24%). Further study of AQPs in microsporidia and their potential inhibitors may yield novel therapeutic agents for microsporidian infections.

Role of sulfated glycans in adherence of the microsporidian Encephalitozoon intestinalis to host cells in vitro.

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.