C-227. Detection of Microsporidia Related to Encephalitozoon intestinalis in the Non-Small Cell Lung Cancer Cell Line A549

P. O. Gaboton, M. Dao;
Univ. of South Florida, Tampa, FL.

Background: Mebendazole (MBZ), a bendizimidazole antihelminthic and antimicrosporidial drug, was reported to have an anti-mitotic activity on the A549 cells (non-small cell lung cancer cell line) in in vitro and in vivo studies (Mukhopadhyay et al.; Sasaki et al., 2002). The present study has focused on investigating whether the A549 cells were infected with microsporidia. Methods: A novel chitin staining method for observation of fungi and protozoa in the light microscope was used to stain the A549 cells obtained from ATCC. Upon detection, microsporidian spores were isolated by treating the culture with the chaotropic agent guanidine hydrochloride (6M), followed by centrifugation. DNA was extracted from the spores and used as a template for PCR analysis using pan specific primers for Encephalitozoon species (E. cuniculi, E. hellem and E. intestinalis), and species-specific primers. The amplicon obtained was analyzed by electrophoresis in 2% agarose gel in parallel with those obtained with known E. species used as controls. Immunological identity was performed by immunodot and western immunoblot analysis using a pan-specific rabbit antibody Results: Presence of microsporidia was detected by the chitin staining method. PCR analysis of the spores obtained from the A549 cells showed positive amplification by the pan-specific primers, and by the E. intestinalis-specific primers. A positive reaction was obtained by immunodot, while western immunoblot analysis showed that the spores from the A549 had an extra antigen band as compared to E. intestinalis. Conclusion: The A549 cells were confirmed to be infected with microsporidia related to E. intestinalis. Sequence analysis of the small rRNA gene is underway to determine whether the extra antigen band was due to a difference in gene sequence or gene expression as compared to E. intestinalis. Detection of microsporidia in the A549 cells suggested that the killing of these protozoans by mebendazole might play a role in the death of these cells since microsporidiosis was reported to block host cell apoptosis, and hence supported the need for further investigation of microsporidiosis in cancer patients for treatment with mebendazole.