PROJECT SUMMARY Vibrio cholerae, the causative agent of the diarrheal disease cholera, forms biofilm aggregates in the host intestinal crypts that are critical to disease pathogenesis. One factor that affects V. cholerae virulence and biofilm formation is the sensing of nitric oxide (NO), an important mediator of host immune defense. Intestinal infection with V. cholerae causes upregulation of inducible NO synthase in the intestinal epithelium and in intestinal macrophages. This in turn leads to elevated NO levels in the intestinal lumen where V. cholerae resides. A better understanding of the mechanisms involved in V. cholerae's response to NO is important, since common therapeutics used in cholera treatment alter host production of NO. V. cholerae can sense and respond to NO through two major NO- sensing proteins: Heme nitric oxide/oxygen binding protein Fig. 1. Overview of the project and the collaborative team. (H-NOX) and NO-sensing protein (NosP). Intriguingly, H- NOX is thought to promote biofilm formation through indirect inhibition of a phosphodiesterase (PDE), while NosP is thought to favor biofilm dissolution by inhibition of the quorum sensing histidine kinase (HK) VpsS. How the two V. cholerae NO-sensors (H-NOX and NosP) in combination affect biofilm formation and virulence during infection of the host intestinal epithelium is unclear. Addressing the complex interplay between host NO production, V. cholerae NO-sensing, NO signaling pathways, biofilm formation, and the resulting host response requires complementary expertise in mucosal immunobiology, bacterial signaling pathways, and biofilms. We have assembled a multi-disciplinary team of mid-career PIs from three INBRE states to undertake this work. The focus of our project on NO-sensing and biofilm formation in V. cholerae will enable all three PIs to apply their expertise to a new area of investigation while developing a highly synergistic collaborative project. Dr. Tseng at the University of Nevada Las Vegas (UNLV) will contribute biofilm expertise that she has gained in studies on Pseudomonas aeruginosa. Dr. Bimczok at Montana State University (MSU) will contribute her expertise in organoid-immune cell co-culture models of infection that she has gained in her research on gastric H. pylori infection. Dr. Yukl at New Mexico State University (NMSU) will contribute his expertise on bacterial NO sensing systems and extend his work to define relevant signaling pathways downstream of the NO sensors (Fig. 1). For this team development project, we aim to, first, establish and validate our experimental model systems, second, generate preliminary data, and third, strengthen our collaborative interactions. With our proposed experiments, we will (1) assess in vitro biofilm formation phenotypes of V. cholerae NO-sensing mutants (Tseng); (2) elucidate how sensing of epithelial and macrophage-derived NO by V. cholerae impacts biofilm formation and virulence in the context of host cells (Bimczok); and (3) define H-NOX and NosP signaling pathways in planktonic cultures and biofilms of V. cholerae (Yukl). The proposed studies include discovery- focused omics approaches (i.e., bacterial and host RNASeq, bacterial proteomics and phosphoproteomics). This strategy will enable us to identify potential target molecules and pathways in both bacteria and host cells that we can further explore in hypothesis-driven follow-up studies and proposals. The data to be generated will enable us to publish a collaborative manuscript and will position us for a successful multi-PI R01 or a Collaborative Program RM1 application. The three PIs are committed to sharing their expertise across the groups, which will benefit both the project and the breadth of research in all three laboratories. The proposed tri-state collaboration is expected to generate unique insight into mechanisms of V. cholerae pathogenesis from the perspective of both pathogen and host. In addition, the project will provide excellent opportunities for training the biomedical workforce at the undergraduate, graduate, and postdoctoral levels. Overall, the proposed work is highly suitable for funding opportunity NOT-GM-24-001 for Team Science Development Projects. Our project and its future directions are a clear fit to three out of four thematic focus areas for New Mexico INBRE, specifically Pathogens, Cell and Organism, and Structure and Function of Biomolecules. It also furthers the stated goals of the New Mexico INBRE parent award, which are to support multi-disciplinary collaborative research, build and enhance the biomedical research base through faculty development, and provide student-focused training and research experiences. Finally, the proteomics and transcriptomics experiments proposed herein will leverage the relevant core facilities at NV and MT INBRE and the IDeA National Resource for Quantitative Proteomics.

PROJECT NARRATIVE Nitric oxide (NO) is an important signaling molecule that mediates interactions between the host and bacterial pathogens. Vibrio cholerae, the causative agent of cholera, expresses two putative NO-sensor proteins that are likely important for virulence and biofilm formation within the host, although this has never been evaluated largely due to the lack of appropriate animal models for cholera disease. This collaborative project will use a novel human intestinal organoid model as well as in vitro biofilm characterization and advanced proteomics to determine NO sensor function and mechanism in V. cholerae with implications for the development of improved cholera treatments.