Formation and Pathogenicity of Cytotoxic Curli Intermediates

Abstract

The first observations of biofilms were made by the “father of microbiology” Antonie van Leeuwenhoek in the 17th century. The number of publications on biofilms has grown exponentially in the last 20 years, highlighting the medical relevance of the field. The complexity of the bacterial biofilm as well as its variability across species provides a continual channel for discovery. While all biofilms differ, there are some components that remain standard such as proteins, polysaccharides and DNA allowing for linkages between seemingly distinct biofilms. Biofilm-associated infections account for more than 65% of all infections implicating the need for understanding the stages of biofilm formation and development. Our lab focuses on the amyloid component of the biofilm and has identified that curli and extracellular DNA (eDNA) complex irreversibly within the biofilms of Salmonella enterica serovar Typhimurium and Escherichia coli. Here, we investigate the formation and pathogenicity of cytotoxic curli intermediates previously unidentified in the in vitro biofilm. The identification of multiple curli conformations within the biofilm biogenesis aides in the understanding of amyloid kinetics in the enteric biofilm. Together, these studies provide a link between biofilm-associated infections and autoimmune responses in the host. In these studies, we planned to isolate curli from different stages in biofilm development to observe their differences both structurally and through their interactions. We identified turbulence has a significant impact on the formation of mature biofilm. We were able to isolate an intermediate form of curli through increased turbulence during biofilm growth. There has never been an intermediate form of curli isolated before our studies due to the high efficiency of the nucleation-precipitation process of curli fiber formation. From this isolation, we characterized these intermediates in comparison to the mature curli complexes. We observed that higher turbulence leads to lesser biofilm formation by sedimentation assay and crystal violet staining. Additionally, we investigated the expression of the curli forming genes csgBA via flow cytometry analysis which indicated that csgBA was preferentially expressed under low turbulence conditions. When investigating the curli conformations isolated from the biofilm, we found that intermediate complexes incorporated less thioflavin T (ThT) indicating lesser amyloid content. We also differentiated the mature and intermediate curli aggregate populations using multiple microscopy techniques. Under confocal microscopy, intermediate fibers seldom measured larger than 100 µm, while mature curli did. Electron dense regions were observed under transmission electron microscopy in the mature curli indicating high fibrillization and compact structure of these aggregates, not seen in the intermediates. Due to known interactions of curli with eDNA in the biofilm, next we investigated the DNA content of the complexes. We hypothesized that the mature structured complexes would have greater DNA content supporting the maturation of the fibrils and the structural compaction. Indeed, we found more DNA could be extracted from the mature curli fibers. Interestingly, we increased the fibrillization of intermediates upon addition of exogenous genomic DNA suggesting DNA incorporation was necessary for the formation of the mature fibrillar aggregates. Intermediates of amyloid β are found to be more cytotoxic than the mature form of the amyloid. For this reason, we hypothesized that curli intermediates could also be cytotoxic. After treating bone marrow-derived macrophages with mature and intermediate curli complexes, we observed that the intermediate aggregates were significantly more cytotoxic to immune cells than mature aggregates. Together, this data implicates a role for the cytotoxic intermediate form of curli in the pathogenesis of Salmonella as well as other enteric bacteria. Curli complexes have been previously described as a novel pathogen associated molecular pattern (PAMP) by their ability to activate numerous receptors in immune cells. The host immune response to curli complexes has been elucidated in our lab. First, binding to Toll-like receptor-2 (TLR2) begins with recognition of the conserved cross-β sheet secondary structure. Mutations disrupting this structure are shown to abrogate immune cell recognition and signaling. For this reason, we next investigated the pathogenicity of the intermediates discovered and characterized above. As cytotoxic oligomers exist for human amyloids, we aimed isolate an earlier form of the intermediate curli and investigate the ability of these conformations to activate host immune responses. As mature curli has been reported to induce anti-dsDNA antibodies in murine models of systemic lupus erythematosus (SLE), we anticipated differences in the autoimmune response to these different curli aggregates as well. First, we isolated and characterized an early form of intermediates isolated at 24 hours which were smaller in size and incorporated less DNA within their complexes than the aforementioned intermediates. Further investigations into the structure of these early intermediates described altered secondary structure by circular dichroism. The lack of fully formed secondary structure in the early intermediates we hypothesized to decrease the ability of these complexes to interact with immune receptors as mature curli. Indeed, we saw decreased response in pro-inflammatory cytokine production as well as type I IFN production. This lack of type I IFN production, lead us to investigate the autoantibody response to the early intermediates. When treating both wild-type and autoimmune-prone mice with curli complexes, early autoantibodies responses were dependent on the DNA content of the complex. However, after continual treatment for 10 weeks, intermediate complexes produced levels of anti-dsDNA similar to that of the mature curli treatment. In addition to anti-dsDNA antibodies, for the first time, other anti-nuclear antibodies, such as anti-C1q and anti-nucleosome, were produced in response to these treatments as well. Finally, chronic exposure to curli complexes led to significant histopathological changes including synovial proliferation and periosteal resorption, in the joints of mice autoimmune-prone mice. Together, this data identified that chronic exposure to curli induced autoimmune sequelae which is thought to be transient in genetically healthy individuals, but leads to joint inflammation in individuals whom are genetically predisposed to autoimmunity. In summary, these data significantly broaden the knowledge of curli amyloid formation during biofilm biogenesis in vitro through the identification of previously unidentified cytotoxic intermediate conformations of curli. Additionally, our work forwards the fields of both autoimmunity and biofilm-associated infection research by providing evidence of the direct impact that chronic exposure to the biofilm has on the host, both transient and long-lasting in those whom are pre-disposed to autoimmunity.