PROJECT SUMMARY Systemic Lupus Erythematosus (SLE) is an autoimmune disease with a complex pathogenesis, in which environmental triggers act in individuals with a genetic susceptibility to break immunological self-tolerance. Urinary tract infections (UTI) are among the most common infections in patients with SLE. We have recently reported that a subset of female patients with SLE, who have asymptomatic persistent bacteriuria, show higher levels of pro-inflammatory markers and disease flares. These patients have high levels of anti-dsDNA antibodies (Abs) that correlate with high levels of Abs against curli/DNA, a bacterial amyloid complexed with bacterial DNA. Curli/DNA is produced by Gram-negative bacteria, like UroPathogenic Escherichia coli (UPEC), a frequent cause of bacteriuria and UTI. Translational and epidemiological studies indicate that patients with SLE are frequently exposed to microbial products and suggest that bacterial infections may promote SLE in predisposed individuals, but the underlying mechanisms remain unknown. We have also reported that curli/DNA can accelerate lupus onset in lupus-prone mice and activate immune cells to secrete type I Interferons (IFNs), a cytokine important in lupus pathogenesis. We hypothesize that recurrent mucosal infections, like UTI, which remain often untreated because subclinical in women, may provide a repetitive immune stimulus in individuals genetically susceptible to autoimmunity and trigger lupus onset or flares. The objective of this R03 application is to establish a mouse model of UTI in lupus-prone mice that can be used to study how recurrent or chronic bacteriuria contribute to lupus disease. In Aim 1. we will establish a model of recurrent/chronic UTls in lupus prone mice by using a lab strain of UPEC, which express curli amyloid, and adapting the conditions of inoculation previously established in WT mice. We will study the ability of bacteria to establish biofilms in the bladder, whether they become systemic and activate the immune system in lupus prone mice vs WT mice. We will analyze the induction of autoantibodies as markers of onset of autoimmunity. Comparing a strain of UPEC that expresses curli amyloid and its curli-deficient mutant, we can determine the role of curli in UTI. In Aim 2. we will test three clinical UPEC isolates from patients with SLE and persistent bacteriuria to establish an infection relevant for patients living with SLE. The successful completion of this project will provide a protocol that lupus experts can use to study the mechanisms for the role of bacterial mucosal infections, like the subclinical UTls occurring in patients living with SLE, in the pathogenesis of autoimmune diseases. It may highlight curli amyloid as pivotal stimulus in infection-induced autoimmunity. Finally, it will generate a tool to test novel therapeutic approaches in lupus through the control of subacute infections and the re-establishment of the correct urinary microbiome.