CAMPYLOBACTER JEJUNI IS A FOODBORNE BACTERIAL PATHOGEN THAT CAUSES DISEASE IN HUMANS AND ACCOUNTS FOR NEARLY 800,000 CASES OF FOODBORNE ILLNESS ANNUALLY WITHIN THE UNITED STATES, PRIMARILY DUE TO TRANSMISSION VIA RAW POULTRY PRODUCTS. ADDITIONALLY, C. JEJUNI CAUSES GREATER THAN 90% OF CAMPYLOBACTER RELATED SHEEP ABORTIONS WITHIN THE UNITED STATES; THESE CASES OF SHEEP ABORTION ARE PRIMARILY DUE TO C. JEJUNI SHEEP ABORTION (SA) CLONE. THERE IS A FUNDAMENTAL KNOWLEDGE GAP PERTAINING TO HOW THE SA CLONE CAUSES DISEASE WITHIN THE OVINE HOST AND WHAT KEY FACTORS CONTRIBUTE TO DISEASE PROGRESSION. SMALL REGULATORY RNAS (SRNAS) HAVE RECENTLY BEEN IDENTIFIED IN BACTERIA AND HAVE BEEN DEMONSTRATED TO INTERACT WITH AND REGULATE TRANSCRIBED MESSENGER RNAS (MRNAS), WHICH ARE PRODUCED BY THE CELL TO GENERATE PROTEIN, ULTIMATELY AFFECTING CELLULAR FUNCTION. THE PRIMARY GOAL OF THIS STUDY IS TO UNDERSTAND THE REGULATORY INTERACTIONS OF TWO SRNAS, CJNC110 AND CJNC140, WHICH ARE EXPRESSED BY THE C. JEJUNI SA CLONE, TO FURTHER OUR UNDERSTANDING OF HOW THIS IMPORTANT PATHOGEN CAUSES DISEASE. AN IMPROVED UNDERSTANDING OF CJNC110 AND CJNC140 MRNA AND PROTEIN MOLECULAR INTERACTIONS WILL LAY THE FOUNDATION FOR FUTURE PHENOTYPE STUDIES AND ANIMAL MODEL STUDIES INCLUDING OVINE AND POULTRY HOSTS, TO BETTER UNDERSTAND COLONIZATION AND PATHOBIOLOGY OF DISEASE. UNDERSTANDING THE INTERACTIONS OF THESE SRNAS WITH THEIR TARGET MRNAS AND PROTEINS WILL PROVIDE KEY KNOWLEDGE ABOUT CELLULAR FUNCTIONS UTILIZED BY THIS BACTERIAL PATHOGEN WITHIN ANIMAL HOSTS. IMPROVED UNDERSTANDING OF THE REGULATORY INTERACTIONS OF SRNAS UTILIZED BY C. JEJUNI WILL THEN PROVIDE POTENTIAL DRUG TARGETS; TO DECREASE THE BURDEN OF C. JEJUNI ANIMAL DISEASE AND POULTRY TRANSMISSION. AS A SECONDARY GOAL, MOLECULAR TOOLS WILL BE DEVELOPED DURING THIS STUDY THAT CAN BE UTILIZED TO CHARACTERIZE REGULATORY INTERACTIONS OF OTHER C. JEJUNI SRNAS AND PROVIDE A TOOL FOR CAMPYLOBACTER RESEARCHERS AND RESEARCHERS STUDYING RELATED ORGANISMS. BY IDENTIFYING THE GENE-PRODUCTS REGULATED BY SRNAS CJNC110 AND CJNC140, WE CAN DIRECTLY BENEFIT BOTH HUMAN AND ANIMAL HEALTH.DURING STUDY 1.1, WE WILL USE ADVANCED GENE SEQUENCING TECHNIQUES TO STUDY CHANGES IN GENE EXPRESSION OF THE SA CLONE FOLLOWING INACTIVATION OF SRNAS CJNC110 AND CJNC140. RNA TRANSCRIBED BY THE CELL WILL BE SEQUENCED AND CELLS LACKING SMALL RNA (CJNC110 AND CJNC140) EXPRESSION WILL BE COMPARED TO CELLS THAT STILL EXPRESS THE SRNAS TO REVEAL POTENTIAL REGULATORY TARGETS. DURING STUDY 1.2, WE WILL USE ADVANCED MOLECULAR TAGGING TECHNIQUES FOLLOWED BY AFFINITY PULL-DOWN ASSAYS TO CAPTURE SRNA-MRNA TARGETS OF BOTH CJNC110 AND CJNC140. SPECIFICALLY, SRNAS CJNC110 AND CJNC140 WILL BE LINKED TO A TAG TO CAPTURE REGULATORY TARGETS OF EACH SRNA; THESE CAPTURED TARGETS WILL THEN BE SEQUENCED TO REVEAL MRNA BINDING PARTNERS OF SRNAS CJNC110 AND CJNC140. DURING STUDIES 2.1 AND 2.2, WE WILL EXTRACT AND SEQUENCE CELLULAR PROTEINS TO IDENTIFY PROTEINS WITHIN THE REGULATORY NETWORK OF BOTH CJNC110 AND CJNC140. FOR STUDY 2.1, PROTEIN EXPRESSION WILL BE ANALYZED BY COMPARING CELLS LACKING SRNAS CJNC110 AND CJNC140 TO CELLS THAT STILL EXPRESS THE SRNA TO REVEAL PROTEIN EXPRESSION CHANGES. FOR STUDY 2.2, SRNAS CJNC140 AND CJNC110 WILL AGAIN BE LINKED TO A MOLECULAR TAG TO CAPTURE PROTEIN REGULATORY TARGETS OF EACH SRNA; THESE CAPTURED TARGETS WILL THEN BE SEQUENCED TO REVEAL PROTEIN BINDING PARTNERS OF SRNAS CJNC110 AND CJNC140. THE COLLECTIVE RESULTS OF STUDIES 1.1-1.2 AND 2.1-2.2 WILL ALLOW FOR THE, SRNA REGULATORY NETWORKS OF BOTH CJNC110 AND CJNC140 TO BE DECIPHERED, WHICH FULLY ALIGNS WITH THE PRIMARY GOAL OF THE PROPOSED STUDY.