Host detection of Vibrio cholerae-induced actin damage

Project Summary/Abstract Vibrio cholerae is a water-borne bacterial pathogen that causes the non-inflammatory diarrheal disease cholera. Millions of cholera cases each year imperil vulnerable populations, especially in areas of humanitarian crisis and regions of great poverty, highlighting an urgent need to expand our understanding of V. cholerae pathogenesis. One of the unique aspects of V. cholerae is its secreted Multifunctional-Autoprocessing Repeats-in-Toxin (MARTX) that contributes to the establishment and persistence of infection. After secretion, MARTX forms a pore in eukaryotic cell membranes to translocate its effector cassette into the host cytoplasm. There, the cysteine protease domain of MARTX releases multiple effectors that disrupt host signaling, cell structure, and endocytic trafficking. This proposal focuses on one effector called the actin crosslinking domain (ACD), which introduces iso-peptide bonds between globular-actin monomers leading to filamentous-actin dissociation and cytoskeletal collapse. ACD was recently found to promote activation of the mitogen-activated kinases (MAPK) and lead to the secretion of pro-inflammatory interleukins; however, how cells detect actin damage is unknown. This proposal seeks to determine the host signaling response to ACD and characterize its role in V. cholerae infection. Elucidating this signaling response will provide insights into both V. cholerae pathogenesis and cellular response to actin depolymerization; therefore, the goals of this project closely align with the mission of NIAID to better understand infectious diseases. Aim 1 seeks to determine the signaling downstream of actin crosslinking that leads to MAPK activation using inhibition and genetic knockdown strategies. Aim 2 seeks to define the impact of ACD on V. cholerae colonization and bacterial-stimulated host signaling using an adult mouse colonization model. The training received while completing the proposed work will be fantastic preparation for the career goal of the trainee to lead a lab studying host-pathogen interactions. Through this proposal, the trainee will gain: 1) expertise in defining host signaling responses to bacterial toxins with techniques such as retroviral transfection, siRNA knockdown, cell line development, and western blot; 2) skills in mouse infection experimentation to assess in vivo pathogenesis and signaling; and 3) other skills needed for academic research such as science communication, grant writing, and manuscript generation. Feinberg School of Medicine at Northwestern University provides an excellent environment to complete the proposed scientific aims and achieve build towards the career goals of this trainee. Dr. Karla Satchell sponsors this proposal as an expert in MARTX toxins and host responses. Her mentorship will provide the trainee thorough expertise in bacterial toxins and host-pathogen interactions. Also, weekly bacteriology journal club provides frequent opportunities for the trainee to present their science and new literature. Finally, the department and university have elite core facilities with expert consultants in techniques such as proteomics, mass spectrometry, imaging, and advanced sequencing.