Comparison of protein-free defined media, and effect of l-cysteine and ascorbic acid supplementation on viability of axenic Entamoeba histolytica

Abstract

Entamoeba histolytica is the etiologic agent for amoebiasis. The excretory-secretory (ES) products of the trophozoites contain virulence factors and antigens useful for diagnostic applications. Contaminants from serum supplements and dead trophozoites impede analysis of ES. Therefore, a protein-free medium that can sustain maximum viability of E. histolytica trophozoites for the longest time duration will enable collection of contaminant-free and higher yield of ES products. In the present study, we compared the efficacy of four types of media in maintaining ≥ 95% trophozoite viability namely Roswell Memorial Park Institute (RPMI-1640), Dulbecco's Modified Eagle Medium (DMEM), phosphate-buffered saline for amoeba (PBS-A), and Hank's balanced salt solution (HBSS). Concurrently, the effect of adding L: -cysteine and ascorbic acid (C&A) to each medium on the parasite viability was also compared. DMEM and RPMI 1640 showed higher viabilities as compared to PBS-A and HBSS. Only RPMI 1640 showed no statistical difference with the control medium for the first 4 h, however the ≥ 95% viability was only maintained for the first 2 h. The other protein-free media showed differences from the serum- and vitamin-free TYI-S-33 control media even after 1 h of incubation. When supplemented with C&A, all media were found to sustain higher trophozoite viabilities than those without the supplements. HBSS-C&A, DMEM-C&A, and RPMI 1640-C&A demonstrated no difference (P>0.05) in parasite viabilities when compared with the control medium throughout the 8-h incubation period. DMEM-C&A showed an eightfold increment in time duration of sustaining ≥ 95% parasite viability, i.e. 8 h, as compared to DMEM alone. Both RPMI 1640-C&A and HBSS-C&A revealed fourfold and threefold increments (i.e., 8 and 6 h, respectively), whereas PBS-A-C&A showed only one fold improvement (i.e., 2 h) as compared to the respective media without C&A. Thus, C&A-supplemented DMEM or RPMI are recommended for collection of ES products.

Proteome analysis of excretory-secretory proteins of Entamoeba histolytica HM1:IMSS via LC–ESI–MS/MS and LC–MALDI–TOF/TOF

“…The protein-free media (RPMI-C-A) used in this experiment has previously been shown to support 95% viability of E. histolytica trophozoites for up to 8 h [21]. Nevertheless, there may still be a small number of dead E. histolytica trophozoites that were lysed and released proteins into the medium.…”

“…The supernatant was then pooled and centrifuged at 10,000× g for 5 min at 4 °C and filtered through 0.2 µm filter (Sartorius Stedim, Germany). Subsequently, the supernatant was concentrated 1000 times using a spin filter with 5 kDa molecular-weight cut off (Vivapsin, Sartorius), and a cocktail of protease inhibitors (Roche, Germany) was added [20]. Protein samples and RPMI-CA medium (concentrated 100X, as control) were reduced with 0.284 M β-mercaptoethanol by boiling for 5 min.…”

Analysis of Entamoeba histolytica Excretory-Secretory Antigen and Identification of a New Potential Diagnostic Marker

“…In addition, iodoacetamide was added to the RPMI-C-A medium containing the ESA upon its collection in order to protect the protein from degradation by proteases (3,13,15). Although we have previously optimized the growth conditions of E. histolytica trophozoites so that ϳ 95% of the parasites were alive when ESA was collected (20), nevertheless it is still possible that some of the antigenic bands may have come from ruptured trophozoites.…”

“…Subsequently, the cell density was determined via the trypan blue exclusion method. Trophozoites were seeded into a culture tube 80% filled with RPMI-C-A medium at a cell density of 0.8 ϫ 10 6 cells per ml and incubated at 36°C for 6 h. Using this method, we have previously shown that ϳ95% trophozoite viability can be maintained throughout the incubation period (20). Upon completion, culture tubes were subjected to centrifugation at 22 ϫ g for 2 min at 4°C.…”

“…Cocktail protease inhibitor (Roche Diagnostic, Germany) was then added to the concentrated ESA. The protein concentration of the ESA was estimated using a Bradford protein assay (10,15,20).…”