The human microbiota is the community of microorganisms that live upon or within their human host. The microbiota consists of various microorganisms including bacteria, fungi, viruses, and archaea; the gut microbiota is comprised mostly of bacteria. Many bacterial species within the gut microbiome grow as biofilms, which are multicellular communities embedded in an extracellular matrix. Studies have shown that the relative abundances of bacterial species, and therefore biofilms and bacterial byproducts, change during progression of a variety of human diseases including gastrointestinal, autoimmune, neurodegenerative, and cancer. Studies have shown the location and proximity of the biofilms within the gastrointestinal tract might impact disease outcome. Gram-negative enteric bacteria secrete the amyloid curli, which makes up as much as 85% of the extracellular matrix of enteric biofilms. Curli mediates cell-cell attachment and attachment to various surfaces including extracellular matrix components such as fibronectin and laminin. Structurally, curli is strikingly similar to pathological and immunomodulatory human amyloids such as amyloid-β, which has been implicated in Alzheimer's disease, α-synuclein, which is involved in Parkinson's disease, and serum amyloid A, which is secreted during the acute phase of inflammation. The immune system recognizes both bacterial amyloid curli and human amyloids utilizing the same receptors, so curli also induces inflammation. Moreover, recent work indicates that curli can participate in the self-assembly process of pathological human amyloids. Curli is found within biofilms of commensal enteric bacteria as well as invasive pathogens; therefore, evidence suggests that curli contributes to complex human diseases. In this review, we summarize the recent findings on how bacterial biofilms containing curli participate in the pathological and immunological processes in gastrointestinal diseases, systemic autoimmune diseases, and neurodegenerative diseases.

Recent studies provided important insight into our understanding of the role of c-di-GMP signaling and the regulation of enteric biofilms. Despite an improved understanding of how c-di-GMP signaling regulates S. Typhimurium biofilms, the processes that affect the intracellular c-di-GMP levels and the formation of multicellular communities in vivo during infections remain unknown.

Deposition of human amyloids is associated with complex human diseases such as Alzheimer’s and Parkinson’s. Amyloid proteins are also produced by bacteria. The bacterial amyloid curli, found in the extracellular matrix of both commensal and pathogenic enteric bacterial biofilms, forms complexes with extracellular DNA, and recognition of these complexes by the host immune system may initiate an autoimmune response. Here, we isolated early intermediate, intermediate, and mature curli fibrils that form throughout the biofilm development and investigated the structural and pathogenic properties of each. Early intermediate aggregates were smaller than intermediate and mature curli fibrils, and circular dichroism, tryptophan, and thioflavin T analyses confirmed the establishment of a beta-sheet secondary structure as the curli conformations matured. Intermediate and mature curli fibrils were more immune stimulatory than early intermediate fibrils in vitro. The intermediate curli was cytotoxic to macrophages independent of Toll-like receptor 2. Mature curli fibrils had the highest DNA content and induced the highest levels of Isg15 expression and TNFα production in macrophages. In mice, mature curli fibrils induced the highest levels of anti-double-stranded DNA autoantibodies. The levels of autoantibodies were higher in autoimmune-prone NZBWxF/1 mice than wild-type C57BL/6 mice. Chronic exposure to all curli forms led to significant histopathological changes and synovial proliferation in the joints of autoimmune-prone mice; mature curli was the most detrimental. In conclusion, curli fibrils, generated during biofilm formation, cause pathogenic autoimmune responses that are stronger when curli complexes contain higher levels of DNA and in mice predisposed to autoimmunity.

A hexanucleotide GGGGCC repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal degeneration. Accurate determination and quantitation of the repeat length is critical in both clinical and research settings. However, because of the complexity of the C9orf72 expansion with high GC content, large size of repeats, and high rate of insertions/deletions (indels) and sequence variations in the flanking regions, molecular genetic analysis of the locus is challenging. To improve the performance characteristics for clinical testing, we evaluated a commercially available long-read C9orf72 PCR assay for research use only, AmplideX PCR/CE C9orf72 assay (AmplideX-C9), and compared its performance with our existing laboratory-developed C9orf72 expansion procedure. Overall, in comparison to the laboratory-developed C9orf72 expansion procedure, AmplideX-C9 demonstrated a more efficient workflow, greater PCR efficiency for sizing of repeat expansions, and improved peak amplitude with lower DNA input and higher analytic sensitivity. This, in turn, permitted detection of indels in the 3' downstream of the repeat expansion region in expanded alleles, showed a higher success rate with formalin-fixed, paraffin-embedded tissue specimens, and facilitated the assessment of repeat mosaicism. In summary, AmplideX-C9 will not only help to improve clinical testing for C9orf72-associated amyotrophic lateral sclerosis and frontotemporal degeneration but will also be a valuable research tool to better characterize the complexity of expansions and study the effects of indels/sequence variations in the flanking region.

The bacterial amyloid curli, produced by Enterobacteriales including Salmonella species and Escherichia coli, is implicated in the pathogenesis of several complex autoimmune diseases. Curli binds to extracellular DNA, and these complexes drive autoimmunity via production of anti-double-stranded DNA autoantibodies. Here, we investigated immune activation by phenol-soluble modulins (PSMs), the amyloid proteins expressed by Staphylococcus species. We confirmed the amyloid nature of PSMs expressed by S. aureus using a novel specific amyloid stain, (E,E)-1-fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy) styrylbenzene (FSB). Direct interaction of one of the S. aureus PSMs, PSMα3, with oligonucleotides promotes fibrillization of PSM amyloids and complex formation with bacterial DNA. Finally, utilizing a mouse model with an implanted mesh-associated S. aureus biofilm, we demonstrated that exposure to S. aureus biofilms for six weeks caused anti-double-stranded DNA autoantibody production in a PSM-dependent manner. Taken together, these results highlight how the presence of PSM-DNA complexes in S. aureus biofilms can induce autoimmune responses, and suggest an explanation for how bacterial infections trigger autoimmunity.