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Type III secretion systems (T3SSs) form a needle-like complex that translocates specialized "effector proteins" into host cells to disrupt normal host-cell physiology. T3SSs are important virulence factors in food-borne pathogens including Yersinia, Salmonella and E. coli O157:H7. <P> Vibrio parahaemolyticus, a seafood-borne pathogen, harbors two phylogenetically distinct T3SSs, T3SS1 and T3SS2, that use effector proteins to induce host-cell death and enterotoxicity, respectively. Effector proteins are thought to encode a secretion signal (export sequence), chaperone-binding domain, and a catalytic domain, and we have hypothesized that an export sequence is required for secretion through the T3SS, but both an export sequence and chaperone-binding domain are required for T3SS specificity and translocation of effector proteins. <P> Our long-term goal is to use V. parahaemolyticus as a model system to better understand the regulation of the T3SS as a means to identify alternative control and therapeutic strategies for Vibrio as well as other food-borne pathogens. <P> We intend to accomplish this through the following specific aims: (1) use bait-and-capture experiments to identify chaperone proteins that bind T3SS1- and T3SS2-specific effector proteins; (2) identify the export sequence and chaperone-binding domain for these effector proteins; and (3) use homologous recombination to both remove and exchange these signaling motifs to directly assess secretion, specificity and translocation of effector proteins. <P> We expect to identify novel chaperone proteins and determine effector protein export sequences within the first year of study, and to identify the chaperone-binding domains and assess the roles of the signaling motifs by the end of the second year. We expect that this work will culminate in the publication of 1-2 peer-reviewed papers in reputable scientific journals. <P> Additionally, during the period of this study I will assist with teaching the laboratory section of the General Bacteriology course taught by Dr. Devendra Shah to veterinary students at Washington State University in the fall semester of 2011 and 2012. Here I will contribute to the training of DVM students in laboratory techniques and diagnostic testing as they relate to clinical disease in animals. We will also recruit two summer students for the duration of the fellowship to work with me directly. We will meet weekly to discuss research efforts and to help the summer students develop and refine one-page concept and objective papers while they learn basic molecular lab skills such as PCR, cell culture and cloning. I will be responsible for the day-to-day training of the students while they are a part of my research efforts. <P> The goal of this additional training is to develop important skills in training/mentoring students on how to develop projects, describe them in writing, and execute well-designed experiments.
NON-TECHNICAL SUMMARY: <BR> The type III secretion system (T3SS) is a bacterial structure that forms a needle-like complex used to transport specialized "effector proteins" into cells to cause or exacerbate disease. The T3SS is an important virulence factor in many food-borne pathogens including Salmonella and E. coli O157:H7. Vibrio parahaemolyticus, a seafood-borne pathogen, harbors two distinct T3SSs, T3SS1 and T3SS2, that use effector proteins to cause cell death and gastroenteritis, respectively. V. parahaemolyticus is estimated by the Centers for Disease Control and Prevention (CDC) to cause over 8000 infections annually. Consumption of raw or undercooked shellfish is the primary risk factor and infection results in symptoms including watery diarrhea, abdominal cramps, vomiting, headaches, and more serious infections that may result in death. V. parahaemolyticus has a worldwide distribution with increasing incidence since 1996 that is expected to increase further with rising ocean temperatures associated with global warming. Many studies of the V. parahaemolyticus T3SSs have focused on the roles of the T3SSs and select effector proteins in pathogenesis, while relatively little work has examined the molecular signals responsible for effector protein trafficking through the T3SS. Our studies aim to determine the roles of the signals involved in regulating effector transport through the T3SSs in V. parahaemolyticus. Through this work we will gain insight into the nature of the interactions between effectors and the T3SS, which may offer unique targets for therapeutic intervention both in Vibrio and other organisms harboring a T3SS.
<P> APPROACH:<BR> To identify effector specific chaperones, T3SS1 and T3SS2-specific effectors will be polyhistidine(His)-tagged and expressed in V. parahaemolyticus followed by cellular lysis. His-tagged proteins bound to their cognate chaperones will be isolated by passage through a nickel column and putative chaperones will be isolated and analyzed by mass spectrometry. To confirm binding, putative chaperones will be hemagglutinin(HA)-tagged and expressed concurrently with His-tagged effector proteins. Cells will then be lysed and chaperone-effector complexes will be isolated by passage through an anti-HA column. Lysate and eluted proteins will be immunoblotted to detect the presence of both the HA-tagged chaperone and His-tagged effector protein following elution. To identify the export sequence, a series of effector protein truncations containing a C-terminal epitope tag comprising the Bordetella pertussis adenylate cyclase toxin (CyaA) will be generated in V. parahaemolyticus using homologous recombination. Following T3SS induction, the cell-free supernatant will be immunoblotted for presence of the CyaA-tagged protein constructs. Truncation to the point at which secretion is eliminated will identify the functional components of the export sequence. To identify the chaperone-binding domain, HA-tagged chaperones will be bound to an anti-HA column. A series of CyaA-tagged truncated effectors will then be applied to the anti-HA column. Protein complexes will be eluted and immunoblotted for presence of the CyaA-tag to determine the functional limits of the effector's chaperone-binding domain. To determine the role of the export sequence and chaperone-binding domain on secretion and T3SS specificity, four constructs will be created using homologous recombination: wild-type, export sequence deletion, chaperone-binding domain deletion, and catalytic domain deletion, each incorporating a C-terminal CyaA-tag. These constructs will be generated in wild-type strain NY-4 and strains with deletions of T3SS1, T3SS2 and T3SS1/2 and expressed under T3SS inducing conditions. Cell-free supernatant for each strain containing each construct will be probed to detect their secretion. To assess the involvement of these signal motifs on effector translocation, the four constructs in wild-type NY-4 will be incubated with HeLa cells. Translocation of CyaA into eukaryotic cells results in conversion of ATP to cAMP, which is measured using a commercial enzyme immunoassay. The signals responsible for effector protein specificity will be demonstrated by signal exchange between T3SS1- and T3SS2-specific effectors. Effectors containing exchanged signals will be expressed under T3SS inducing conditions in wild-type strain NY-4 and T3SS1, T3SS2 and T3SS1/2 mutants and immunoblotted to assess redirection of effector transport. Assessment of instruction in Dr. Shah's Bacteriology course will be performed by student evaluations, interactions with students and other course instructors and evaluation by Dr. Shah. Assessment of summer students will be determined by weekly meetings, interactions with Dr. Call and myself and evaluation of their laboratory techniques and writing.