Despite recent advances in our understanding of the parasite, several pertinent questions concerning the clinical significance of Blastocystis remain unanswered.
Asymptomatic carriage (Stensvold et al., 2009b), as well as eradication of infections (Stensvold et al., 2010) have troubled both scientists and clinicians alike (Tan et al., 2010). Lack of an appropriate infection model is the major limitation in addressing these controversies. The aim of this study is to address clinically relevant questions concerning Blastocystis infections in light of recent developments in the areas of Blastocystis cell biology, genotyping, epidemiology, antibiotic susceptibility and host-pathogen interaction modeling. Using in-vitro approaches, the study will identify for the first time a metronidazole-resistant strain of Blastocystis capable of evading host immunity, and causing intestinal epithelial pathology. Based on these observations we will also address clinical controversies concerning the parasite pathogenic potential and evaluate alternative and adjunctive treatment options against Blastocystis infections. Several studies in recent years suggest that
asymptomatic carriage (Stensvold et al., 2009b) as well as inconsistent outcomes of antiparasitic treatment (Stensvold et al., 2010) might be due to subtype-dependent variations in Blastocystis pathobiology, but direct evidence of these variation are lacking. Therefore, this study will also evaluate the relevance of subtyping in predicting the severity as well as treatment outcomes of Blastocystis infections.
Very little is known about Blastocystis virulence factors. Cysteine proteases are suggested to be potential virulence factors of the parasite (Puthia et al., 2008a;
Puthia et al., 2005; Tan et al., 2010). As we have discussed in the previous Chapter, they play a significant role in activation of host epithelial NF-κB and up-regulation of pro-inflammatory cytokine IL-8 (Puthia et al., 2008a) as well as degradation of human secretory IgAs (Puthia et al., 2005). This study will provide further evidence on the importance of cysteine proteases in the parasite pathobiology. A comparison of the activity of these proteases between parasite isolates of two clinically relevant zoonotic subtypes ST-4 and ST-7 will also be studied. We will also study differences between the cell size and generation times of these subtypes. This information will provide the stepping stone for more detailed analysis of parasite pathogenecity, immune evasion and antibiotic resistance in this study.
Chronic carriage, both symptomatic and asymptomatic, is a common observation in Blastocystis infections (Boorom et al., 2008; Gupta and Parsi, 2006; Kaneda et al., 2000; Pasqui et al., 2004; Vogelberg et al., 2010). The tug of war between host antimicrobial defense and microbial immune evasion mechanisms determine the effective colonization of gut lumen by non-invasive pathogens. Since Blastocystis is an opportunistic pathogen (Kurniawan et al., 2009; Tan et al., 2009), competence of the host immune response plays an important role in the ability of the parasite to colonize human gut. In this study, we will explore the susceptibility of ST-4 and ST-7 isolates to nitrosative stress, a possible host anti-Blastocystis defense mechanism.
Furthermore, using differentiated and polarized Caco-2 epithelial cells as a host- pathogen interaction model, we will also study the suppression of the host
Due to a lack of appropriate infection models, Blastocystis pathogenic mechanisms are difficult to decipher. We will use the Caco-2 host-epithelial interaction model to study the role of Blastocystis cysteine proteases in the modulation of the epithelial cytoskeleton and tight junctions. We will also study whether these alteration lead to an increase in epithelial permeability, exposing subepithelial tissue to luminal contents. Moreover, we will identify epithelial pathways modulated by Blastocystis leading to epithelial barrier compromise. Although several studies, including this one, elaborate on the pthogenecity of Blastocystis, but asymtpomatic carriage of the parasite continues to cast doubts over its pathogenic potential. Using the Caco-2 model, we will also study a strain-to-strain variation between pathogenecity of Blastocystis ST-4 and ST-7 isolates. Identification of virulent and avirulent strains of the parasite might provide an explanation for inconsistencies in the clinical outcome of Blastocystis infections.
Another controversy concerning the pathogenic potential of Blastocystis is
treatment failure. It is often argued that, since anti-Blastocystis treatment does not lead to resolution of intestinal symptoms, Blastocystis might not be the etiological agent and the symptoms might be caused by some undiagnosed pathogen. Giardia intestinaliswas recognized as a clinically significant organism only after it became possible to effectivelyeliminate it from the gut (Markell, 1995). Although antibiotic resistance is suspected by some as a reason for treatment failure in Blastocystis infections (Stensvold et al., 2008), a lack of adequate drug susceptibility assays limits our understanding in this area. In this study we will develop high-throughput drug susceptibility assays for Blastocystis to study antibiotic susceptibility and
resistance in isolates of Blastocystis ST-4 and ST-7. The assays will be useful in evaluating the susceptibility of Blastocystis strains to a panel of drugs, compounds and host-derived anti-microbial molecules. Importantly, this method will allow us to identify metronidazole-resistant strains of Blastocystis.
Pathogenic strains of Blastocystis that are resistant to antibiotics pose a significant threat. We will utilize the knowledge acquired by this study regarding the
pathogenic pathway of a metronidazole-resistant isolate of Blastocystis to evaluate the therapeutic potential of a few novel treatment options. In particular, we will study the inhibition of host HMG-CoA-reductase and Rho Kinase by Simvastatin and Fasudil, respectively, as possible alternative or adjunctive treatment options against drug resistant intestinal infections.
Hence, the specific objectives of this study are:
1. To characterize biological differences between two clinically relevant zoonotic Blastocystis subtypes by:
a. Studying the differences in cysteine protease activity, generation time and cell size between ST-4 and ST-7 isolates of Blastocystis.
b. Developing high throughput in-vitro drug susceptibility assays for Blastocystis in order to identify metronidazole resistant isolates and study variations in susceptibility of ST-4 and ST-7 isolates of the
a. Studying the inhibition of epithelial nitric oxide production by Blastocystis as a survival and immune evasion mechanism.
b. Studying the effect of Blastocystis cysteine proteases on intestinal epithelial permeability and transepithelial resistance.
3. To characterize the signaling pathways involved in host cell pathobiology and to exploit this knowledge for the development of novel treatment strategies by:
a. Identifying molecular pathways modulated by Blastocystis to induce tight junction and cytoskeletal rearrangements leading to epithelial barrier dysfunction.
b. Studying the protective effect (therapeutic potential) of epithelial Rho kinase and HMG CoA reductase inhibition by Fasudil and Simvastatin, respectively, against epithelial barrier dysfunction induced by
metronidazole resistant Blastocystis.
CHAPTER 2: