Abstract
Toxoplasma is a significant opportunistic pathogen in AIDS, and bradyzoite differentiation is the critical step in the pathogenesis of chronic infection. Bradyzoite development has an apparent tropism for cells and tissues of the central nervous system, suggesting the need for a specific molecular environment in the host cell, but it is unknown whether this environment is parasite directed or the result of molecular features specific to the host cell itself. We have determined that a trisubstituted pyrrole acts directly on human and murine host cells to slow tachyzoite replication and induce bradyzoite-specific gene expression in type II and III strain parasites but not type I strains. New mRNA synthesis in the host cell was required and indicates that novel host transcripts encode signals that were able to induce parasite development. We have applied multivariate microarray analyses to identify and correlate host gene expression with specific parasite phenotypes. Human cell division autoantigen-1 (CDA1) was identified in this analysis, and small interfering RNA knockdown of this gene demonstrated that CDA1 expression causes the inhibition of parasite replication that leads subsequently to the induction of bradyzoite differentiation. Overexpression of CDA1 alone was able to slow parasite growth and induce the expression of bradyzoite-specific proteins, and thus these results demonstrate that changes in host cell transcription can directly influence the molecular environment to enable bradyzoite development. Investigation of host biochemical pathways with respect to variation in strain type response will help provide an understanding of the link(s) between the molecular environment in the host cell and parasite development.
Highlights
Toxoplasma gondii infects a multitude of warm-blooded hosts where tachyzoite and bradyzoite life stages form in various tissues [1]
To harvest tachyzoites, infected human foreskin fibroblast (HFF) monolayers were scraped from culture flasks, needle-passed, and filtered using a 3-lm nucleopore membrane to separate parasites from host cell debris
Parasite bradyzoite-specific antigen 1 (BAG1) expression was evaluated by IFA in HFF cells cultured in eightwell slides as previously described [2,8]
Summary
Toxoplasma gondii infects a multitude of warm-blooded hosts where tachyzoite and bradyzoite life stages form in various tissues [1]. By identifying those host mRNAs whose levels were altered in predictable ways with respect to the treatment regimens to induce parasite BAG1 expression, we were able to reduce the relevant number of potential host cell mRNAs. In Table 1, the range of host mRNAs from successive queries of the database drops from 37,000 possible genes on the array to 79 when the analyses combine Compound 1 induction with recovery for 6 or 12 h prior to infection, with the noninductive effects of DRB and 506126, and with the antagonism of Compound 1 by DRB and SB202190. Parasite development in HFF cells cultured in alkaline pH is only effective when maintained for long periods; the single transfection of CDA1 siRNAs was much less effective when parasites were continually exposed to either Compound 1 or alkaline media (pH 8.2)
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