Lactate promotes the biofilm-to-invasive-planktonic transition in Salmonella enterica serovar Typhimurium via the de novo purine pathway.

Salmonella enterica serovar Typhimurium (S. Typhimurium) infection triggers an inflammatory response that changes the concentration of metabolites in the gut impacting the luminal environment. Some of these environmental adjustments are conducive to S. Typhimurium growth, such as the increased concentrations of nitrate and tetrathionate or the reduced levels of Clostridia-produced butyrate. We recently demonstrated that S. Typhimurium can form biofilms within the host environment and respond to nitrate as a signaling molecule, enabling it to transition between sessile and planktonic states. To investigate whether S. Typhimurium utilizes additional metabolites to regulate its behavior, our study delved into the impact of inflammatory metabolites on biofilm formation. The results revealed that lactate, the most prevalent metabolite in the inflammatory environment, impedes biofilm development by reducing intracellular c-di-GMP levels, suppressing the expression of curli and cellulose, and increasing the expression of flagellar genes. A transcriptomic analysis determined that the expression of the de novo purine pathway increases during high lactate conditions, and a transposon mutagenesis genetic screen identified that PurA and PurG, in particular, play a significant role in the inhibition of curli expression and biofilm formation. Lactate also increases the transcription of the type III secretion system genes involved in tissue invasion. Finally, we show that the pyruvate-modulated two-component system BtsSR is activated in the presence of high lactate, which suggests that lactate-derived pyruvate activates BtsSR system after being exported from the cytosol. All these findings propose that lactate is an important inflammatory metabolite used by S. Typhimurium to transition from a biofilm to a motile state and fine-tune its virulence.IMPORTANCEWhen colonizing the gut, Salmonella enterica serovar Typhimurium (S. Typhimurium) adopts a dynamic lifestyle that alternates between a virulent planktonic state and a multicellular biofilm state. The coexistence of biofilm formers and planktonic S. Typhimurium in the gut suggests the presence of regulatory mechanisms that control planktonic-to-sessile transition. The signals triggering the transition of S. Typhimurium between these two lifestyles are not fully explored. In this work, we demonstrated that in the presence of lactate, the most dominant host-derived metabolite in the inflamed gut, there is a reduction of c-di-GMP in S. Typhimurium, which subsequently inhibits biofilm formation and induces the expression of its invasion machinery, motility genes, and de novo purine metabolic pathway genes. Furthermore, high levels of lactate activate the BtsSR two-component system. Collectively, this work presents new insights toward the comprehension of host metabolism and gut microenvironment roles in the regulation of S. Typhimurium biology during infection.

Assembly of ordered DNA-curli fibril complexes during Salmonella biofilm formation correlates with strengths of the type I interferon and autoimmune responses.

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.

Bacterial amyloid curli activates the host unfolded protein response via IRE1α in the presence of HLA-B27.

Salmonella enterica serovar Typhimurium (STm) causes gastroenteritis and can progress to reactive arthritis (ReA). STm forms biofilms in the gut that secrete the amyloid curli, which we previously demonstrated can trigger autoimmunity in mice. HLA-B27 is a genetic risk factor for ReA; activation of the unfolded protein response (UPR) due to HLA-B27 misfolding is thought to play a critical role in ReA pathogenesis. To determine whether curli exacerbates HLA-B27-induced UPR, bone marrow-derived macrophages (BMDMs) isolated from HLA-B27 transgenic (tg) mice were used. BMDMs treated with purified curli exhibited elevated UPR compared to C57BL/6, and curli-induced IL-6 was reduced by pre-treating macrophages with inhibitors of the IRE1α branch of the UPR. In BMDMs, intracellular curli colocalized with GRP78, a regulator of the UPR. In vivo, acute infection with wild-type STm increased UPR markers in the ceca of HLA-B27tg mice compared to C57BL/6. STm biofilms that contain curli were visible in the lumen of cecal tissue sections. Furthermore, curli was associated with macrophages in the lamina propria, colocalizing with GRP78. Together, these results suggest that UPR plays a role in the curli-induced inflammatory response, especially in the presence of HLA-B27, a possible mechanistic link between STm infection and genetic susceptibility to ReA.

Systemic exposure to bacterial amyloid curli alters the gut mucosal immune response and the microbiome, exacerbating Salmonella-induced arthritis.

The Salmonella biofilm-associated amyloid protein, curli, is a dominant instigator of systemic inflammation and autoimmune responses following Salmonella infection. Systemic curli injections or infection of mice with Salmonella Typhimurium induce the major features of reactive arthritis, an autoimmune disorder associated with Salmonella infection in humans. In this study, we investigated the link between inflammation and microbiota in exacerbating autoimmunity. We studied C57BL/6 mice from two sources, Taconic Farms and Jackson Labs. Mice from Taconic Farms have been reported to have higher basal levels of the inflammatory cytokine IL - 17 than do mice from Jackson Labs due to the differences in their microbiota. When we systemically injected mice with purified curli, we observed a significant increase in diversity in the microbiota of Jackson Labs mice but not in that of the Taconic mice. In Jackson Labs, mice, the most striking effect was the expansion of Prevotellaceae. Furthermore, there were increases in the relative abundance of the family Akkermansiaceae and decreases in families Clostridiaceae and Muribaculaceae in Jackson Labs mice. Curli treatment led to significantly aggravated immune responses in the Taconic mice compared to Jackson Labs counterparts. Expression and production of IL - 1ß, a cytokine known to promote IL - 17 production, as well as expression of Tnfa increased in the gut mucosa of Taconic mice in the first 24 hours after curli injections, which correlated with significant increases in the number of neutrophils and macrophages in the mesenteric lymph nodes. A significant increase in the expression of Ccl3 in colon and cecum of Taconic mice injected with curli was detected. Taconic mice injected with curli also had elevated levels of inflammation in their knees. Overall, our data suggest that autoimmune responses to bacterial ligands, such as curli, are amplified in individuals with a microbiome that promote inflammation.

Phenol-Soluble Modulins From Staphylococcus aureus Biofilms Form Complexes With DNA to Drive Autoimmunity.

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.

Microbiome or Infections: Amyloid-Containing Biofilms as a Trigger for Complex Human Diseases.

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-ß, 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.

Nitrate Is an Environmental Cue in the Gut for Salmonella enterica Serovar Typhimurium Biofilm Dispersal through Curli Repression and Flagellum Activation via Cyclic-di-GMP Signaling.

Curli, a major component of the bacterial biofilms in the intestinal tract, activates pattern recognition receptors and triggers joint inflammation after infection with Salmonella enterica serovar Typhimurium. The factors that allow S. Typhimurium to disperse from biofilms and invade the epithelium to establish a successful infection during acute inflammation remain unknown. Here, we studied S. Typhimurium biofilms in vitro and in vivo to understand how the inflammatory environment regulates the switch between multicellular and motile S. Typhimurium in the gut. We discovered that nitrate generated by the host is an environmental cue that induces S. Typhimurium to disperse from the biofilm. Nitrate represses production of an important biofilm component, curli, and activates flagella via the modulation of intracellular cyclic-di-GMP levels. We conclude that nitrate plays a central role in pathogen fitness by regulating the sessile-to-motile lifestyle switch during infection. IMPORTANCE 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. Here, we show that nitrate generated in the intestinal lumen during infection with S. Typhimurium is an important regulator of biofilm formation in vivo.

Validation of a Long-Read PCR Assay for Sensitive Detection and Sizing of C9orf72 Hexanucleotide Repeat Expansions.

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.