Project Grant R21AI180871

GENE REGULATION IN CRYPTOSPORIDIUM - 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.