PROJECT ABSTRACT/SUMMARY Bacterial virulence often relies on the ability of pathogens to secrete effectors that facilitate diverse processes such as biofilm formation and immune evasion. Gram-negative bacteria have evolved to encode secretion systems to transport proteins across the cell envelope, thereby facilitating pathogenesis. The canonical type VIII secretion system (T8SS), which is found in Escherichia coli and Salmonella spp., employs the outer membrane (OM) pore CsgG to secrete curli subunits across the OM where extensive networks of curli amyloid fibers are formed, serving as a scaffold for biofilm formation. A system closely related to the T8SS is the holdfast anchor of Caulobacter crescentus, which is a complex consisting of the CsgG homolog, HfaB, and two proteins secreted via HfaB to the bacterial surface. The complex anchors holdfast polysaccharide to the cell, facilitating biofilm formation. Therefore, the T8SS and a related system function in biofilm formation through distinct processes. We recently identified a broadly conserved three-gene operon in Gram-negative bacteria that encodes a CsgG/HfaB homolog. Notably, this operon is found in numerous pathogens that do not produce curli/holdfast, implying a divergent role for CsgG homologs. To investigate the function of this operon, we first employed Acinetobacter baumannii as a model. Intriguingly, we found that mutation of the operon results in the absence of surface-associated poly-N-acetylglucosamine (PNAG), the major polysaccharide component of Acinetobacter biofilms. Accordingly, mutants are attenuated in biofilm formation. We next extended our study of the operon to another Gram-negative pathogen, Pseudomonas aeruginosa. Similarly, mutation resulted in decreased Congo red binding, indicative of a reduction in biofilm-associated polysaccharides (e.g., Pel and Psl). Accordingly, biofilm formation was significantly attenuated. Based on this data and the known function of HfaB, we hypothesize that this three-gene operon encodes a novel variant of the T8SS that is widely distributed in Gram-negative pathogens and plays a role in attachment of biofilm-associated polysaccharides to the cell surface. To test this, the following aims are proposed; In Aim 1, molecular approaches will be used to characterize the localization of and interactions between components of the proposed T8SS variant in A. baumannii and P. aeruginosa, as well as determine its structure. In Aim 2, we will assess the role of the T8SS in anchoring polysaccharides to the membrane in A. baumannii and P. aeruginosa. Additionally, we will identify effectors of the putative T8SS variant in both bacteria. In Aim 3, using A. baumannii as a model, we will determine the role of the putative T8SS in virulence using the clinically-relevant pneumonia and catheter- associated UTI (CAUTI) murine infection models. In all, these Aims will assess the function(s) of a novel secretion system involved in key aspects of pathogenesis in...