Observations made from recent studies using in-vivo rodent models as well as clinical reports of Blastocystis infections suggest that a variation in virulence might exist between different genotypes of Blastocystis (Hussein et al., 2008b; Kaneda et al., 2001). Blastocystis cysteine proteases have been reported in in-vitro studies to cleave human secretory IgA (Puthia et al., 2005) as well as induce pro-inflammatory cytokine IL-8 production (Puthia et al., 2008a) suggesting that these are potential virulence factors, however nothing is known about relative protease activity
between Blastocystis subtypes and within isolates of the same subtype. In this study, we report for the first time that there is a quantitative difference between cysteine proteases activity of isolates belonging to two different subtypes. We also observed that in addition to an inter-subtype variation, a quantitative intra-subtype protease activity difference also exists between isolate B and E of subtype-7.
Extensive size variations have been reported in Blastocystis laboratory cultures (Dunn et al., 1989) and stool specimens (MacPherson and MacQueen, 1994). The significance of this variation is unknown. Cell size variations within yeast microbial cultures may arise from asymmetrical division of daughter cells and is a natural phenomenon, which becomes pronounced in the presence of stress (Rupes, 2002).
As soon as the insulting agent is removed, order is restored leading to a reduction of
and Tyers, 2004). Studies on yeast suggest that nutrient deficiency increase cell size variations in a given colony. New daughter cells in such cultures grow more than mother cells to acquire the “critical size” before division, hence prolonging the cell cycle (Rupes, 2002). Cytometric analysis of Blastocystis cultures in our study suggests that ST-7 isolate cultures exhibit more pronounced asymmetrical cell division compared to rodent isolate cultures. ST-7 isolates also have significantly prolonged generation time. In light of our understanding of cell size homeostasis in other organisms, a simple explanation for extensive cell size distribution in
Blastocystis cultures could be that current in-vitro growth conditions are not optimal for the ST-7 isolates. ST-7 subtypes may grow optimally at 40°C instead of 37°C, as is the case for the ST-7 protozoan flagellate Histomonas meleagridis (van der Heijden and Landman, 2008). However, when in-vitro time course experiments were repeated after more than 30 days and including multiple subculture cycles between the replicate experiments, an almost exact pattern of fluctuation over time in cell size distribution was observed for each isolate, suggesting that these
fluctuations are stable over time. Hence, an extensive analysis of the Blastocystis cell cycle is needed to better understand the functional roles of Blastocystis size
variation.
Cysteine proteases play a crucial role in the cell cycle progression of eukaryotic cells (Concha et al., 2005) including protozoans (Gantt et al., 1998; Sharma et al., 1996;
Ward et al., 1997). Day-to-day variation in protease activity was reported for E.
histolytica (Reed et al., 1989) and culture age-dependent variation in total protease
activity was also previously observed for Blastocystis isolate B (Sio et al., 2006). In this study we observed protease activity variations in cultures of isolates E, WR-1 and S-1. Inhibition of cysteine protease activity by iodoacetamide revealed that this variation is predominantly due to fluctuations in cysteine proteases. Interestingly this fluctuation coincides with age-dependent variation in culture cell size
distribution. Cysteine protease activity and cell size distribution of Blastocystis isolates peak simultaneously at 24-h post-incubation. The only exception was isolate E for which cysteine proteases activity peaked at 48 h but cell size distribution peaked at 24 h. This suggests that, unlike the other isolates, the production of cellular proteases is not tightly coupled to the attainment of maximum cell size, and may be associated with the longer generation time of this isolate, which in turn might have influenced the rate at which proteases accumulate in the cell. Biological properties of different Blastocystis isolates change over time. This poses a particular difficulty for comparative studies. Based on the current findings we will use only 24 h old cultures of all four isolates in the following experiments, since the size of all the isolates and cysteine proteases activity of all but one isolate (isolatet E) peak at this time point.
Pathogenic strains of E. histolytica show higher cysteine protease activity per cell compared to non-pathogenic strains (Reed et al., 1989). We observed that the larger ST-7 Blastocystis isolates showed almost twice as much cysteine protease activity compared to smaller rodent isolates. Interestingly rodent and not ST-7 subtypes are
2005; Puthia et al., 2008b) and in-vivo experimental models (Hussein et al., 2008b) as well as epidemiological studies (Kaneda et al., 2001) associate subtype-4 (rodent isolates) with gastrointestinal symptoms. A possibility for this apparent discrepancy could be qualitative differences between cysteine proteases of the two subtypes.
Interestingly, a G. intestinalis isolate from a symptomatic patient was shown to have lower cysteine protease activity than isolates from asymptomatic carriers (Coradi and Guimarães, 2006). It has been reported that G. intestinalis and E. histolytica possess qualitative inter-strain variations in cysteine protease activity and that these variations contribute to differences in pathogenicity (Davis et al., 2007;
DuBois et al., 2006; Guimarães et al., 2003; Hirata et al., 2007). For Blastocystis, rodent isolates may express specific cysteine proteases which are potentially injurious to the gastrointestinal epithelium. Future studies should include a qualitative analysis of cysteine proteases of pathogenic and non-pathogenic Blastocystis subtypes.