ABSTRACT
Cryptosporidium is an enigmatic apicomplexan protist pathogen. It has completely lost its ability to synthesize
purines and pyrimidines and must rely completely on salvage from the host in order to replicate its genome and
express its genes. Whether it is correlation or causation, Cryptosporidium has evolved to have among the
smallest genome sequences in the phylum at 9.12 Mb and 3930 protein-encoding genes, with 4843 genes total.
Recently we identified polycistronic transcription in C. parvum. 423 protein-encoding genes are observed in 201
polycistronic transcripts in sporozoites. These polycistronic transcripts have been confirmed with multiple
alternative approaches including RT-PCR confirmation of full transcripts containing as many as 4 genes. They
are not read-through artifacts. Polycistronic transcription has never been reported in the Apicomplexa until now.
Cryptosporidium is an early branching apicomplexan most closely related to gregarines. It is a major
cause of diarrhea in both industrialized and developing nations, especially in the immunocompromised and
infants in their first year of life. Transcriptome analyses have shown that nearly all genes in Cryptosporidium
overlap in their untranslated regions regardless of the strand on which they are encoded. Thus, single-molecule
long-read sequencing is necessary to determine transcript boundaries. Transcriptome analyses also revealed
that antisense coding and non-coding RNA genes are rampant. Cryptosporidium is clearly expressing genes
from both DNA strands at many loci, especially in polycistronic transcripts. Interestingly, we often, but not always,
observe polycistronic transcriptional units and one or more of their internal monocistronic transcripts at the same
time. Do the internal monocistronic RNAs represent independent transcriptional units, or might they be
processed from the larger polycistronic transcripts? We are curious about this new form of gene regulation in the
Apicomplexa. To address gene regulation involving polycistronic transcripts, we will examine both transcriptional
and translational aspects of gene regulation using in vitro cell and organoid culture as appropriate. Together with
collaborators, we will also generate a polycistronic transfection construct that will be useful for the functional
dissection of transcription and translation of polycistronic gene regulation. There is significant new gene
regulation and post-transcriptional regulation biology to be learned for these important pathogens that can also
be used for many purposes, including to better inform the design and integration location of transgenes in the
community’s quest to develop better genetic tools for dissection of Cryptosporidium infection biology.