Novel Effector Protein Functions Encoded By T3ss Positive V. Cholerae

Vibrio cholerae causes the severe diarrheal disease cholera that is endemic in much of Asia, Africa, and SouthAmerica, and has recently been reintroduced into Haiti. The species is highly diverse, although only O1 orO139 serogroup strains cause epidemic disease. However, increasing sporadic disease has been reported inendemic areas, and is caused by strains belonging to non-O1/non-O139 serogroups that present a publichealth threat both in developed and industrialized nations, including the United States. Unlike pathogenic O1and O139 strains, the vast majority of pathogenic non-O1/non-O139 strains do not carry the well characterizedvirulence factors for colonization (TCP) and toxin production (CT), and the virulence mechanisms used bythese strains are not well understood. Our study of pathogenic non-O1/non-O139 serogroup strains beganwith genomic sequencing of the clinically isolated O39 serogroup strain, AM-19226, which revealed a TypeThree Secretion System (T3SS) that is conserved among other V. cholerae isolates. Our subsequentexperiments identified 13 effector proteins that are translocated into the eukaryotic cell by the T3SS apparatus,and two transcriptional regulators encoded within the T3SS genomic island. We hypothesize that a subset ofeffectors and a T3SS encoded non-effector protein play important roles in colonization, and that other, uniqueV. cholerae effectors cooperate to disrupt host cell signaling to result in the diarrheal response that arises fromcell-cell junction disruption, ionic transport imbalance and/or cellular stress responses. We propose to usecomplimentary in vitro and in vivo approaches to identify and characterize the mechanism of colonization andthe host cell proteins and pathways targeted by V. cholerae effector proteins. Initial studies will define theminimum set of T3SS encoded genes necessary for colonization and define their roles in adherence to hostcells. Effector protein analysis will focus on a total of five proteins and their roles in different stages ofinfection/disease: VopZZ, VopX, VopM, VopF, and VopK. VopZZ and VopM are critical for colonization, andVops F and M have functions associated with cytoskeletal remodeling, which will be investigated in our studies.VopsX and K are unique to the V. cholerae T3SS, VopsX and K. We will use our experience the S. cerevisiaemodel system to discover host cell proteins that are the targets of effector activity and direct our studies inmammalian cells. We will also examine the host cell response to AM-19226 infection in vitro using mammalianco-culture and expression models. The co-culture assay will be used as a tool to dissect how effectors interactwith mammalian signal transduction pathways during infection to disrupt homeostasis, whereas a viral-basedexpression system will be used to biochemically identify targets of effector protein activity and the response ofmammalian cells to effector expression. We expect our results to reveal the molecular mechanisms of TCP/CTindependent pathogenesis in the subset of non-O1/non-O139 strains that encode a T3SS.