Microbial warfare: B. subtilis antagonizes E. coli biofilm formation.

Biofilm formation helps bacteria to survive environmental challenges. Biofilm development often involves multiple genetic pathways that can be regulated by external signals. Diego Serra and his team (Cordisco et al.) explore how Bacillus subtilis can antagonize Escherichia coli macrocolony biofilm formation via the metabolite bacillaene.

Oral versus intravenous acetaminophen for perioperative pain management in adult patients undergoing non-cardiac surgery: A quantile segmented regression analysis.

STUDY OBJECTIVE: Determine whether preferential use of perioperative enteral acetaminophen is associated with changes in perioperative pain, narcotic administration, or time to meeting criteria for post anesthesia care unit (PACU) discharge, compared to preferential parenteral administration. DESIGN: Retrospective Cohort with quantile segmented regression analysis. Groups determined by date of surgery, one year pre-initiative and one year post-initiative. SETTING: Operating room and PACU of a tertiary academic medical center. PATIENTS: Adult (age > 18 years), ASA status 1-5, non-pregnant patients undergoing non-cardiac surgery of less than six hours duration admitted to the PACU postoperatively. INTERVENTIONS: A multidisciplinary initiative to preferentially utilize enteral over parenteral acetaminophen. MEASUREMENTS: The primary outcome was narcotic consumption in the PACU. Secondary outcomes were intraoperative narcotic administration, pain score on PACU admission and discharge, and time to meeting criteria for PACU discharge. RESULTS: 24,701 patients were included in the analysis; 12,379 had surgery prior to the initiative and 12,322 after. Enteral acetaminophen administration increased preoperatively from 13.49% to 26.84%, and postoperatively from 43.16% to 51.45%, while intraoperative parenteral APAP use dropped from 43.23% to 6.81%. Quantile Segmented regression analysis after adjusting for period (pre versus postintervention), day, age, gender, inpatient status, and ASA class demonstrated a decrease in adjusted median perioperative acetaminophen dose (-175 mg P < 0.001), with no significant difference in level change of intraoperative or PACU narcotic administration. There was no significant difference in median time to meet criteria for PACU discharge, though there was a significant change in the slope, (-0.36, p = 0.007.) Median pain scores measured on a standard 0-10 numeric rating scale at PACU admission did not change, while median pain scores at PACU discharge decreased slightly (-0.24 p < 0.001). There was no change in the probability of PONV. CONCLUSION: In adult patients undergoing non-cardiac surgery of <6 h duration, preferential use of enteral rather than parenteral acetaminophen is associated with non-inferior outcomes in narcotic requirements, pain scores, time to PACU discharge, and probability of PONV when compared with routine parenteral administration. Further studies are needed to validate these findings.

Cell surface-localized CsgF condensate is a gatekeeper in bacterial curli subunit secretion.

Curli are functional amyloids present on the outer membrane of E. coli. CsgF is required for the proper assembly of curli. Here, we found that the CsgF phase separates in vitro and that the ability of CsgF variants to phase-separate is tightly correlated with CsgF function during curli biogenesis. Substitution of phenylalanine residues in the CsgF N-terminus both reduced the propensity of CsgF to phase-separate and impaired curli assembly. Exogenous addition of purified CsgF complemented csgF - cells. This exogenous addition assay was used to assess the ability of CsgF variants to complement csgF ‒ cells. CsgF on the cell surface modulated the secretion of CsgA, the curli major subunit, to the cell surface. We also found that the CsgB nucleator protein can form SDS-insoluble aggregates within the dynamic CsgF condensate. We propose that these multicomponent CsgF-B condensates form a nucleation-competent complex that templates CsgA amyloid formation on the cell surface.

Electrostatic interactions mediate the nucleation and growth of a bacterial functional amyloid.

Bacterial biofilm formation can have severe impacts on human and environmental health. Enteric bacteria produce functional amyloid fibers called curli that aid in biofilm formation and host colonization. CsgA is the major proteinaceous component of curli amyloid fibers and is conserved in many gram-negative enteric bacteria. The CsgA amyloid core consists of five imperfect repeats (R1-R5). R2, R3, and R4 have aspartic acid (D) and glycine (G) residues that serve as "gatekeeper" residues by modulating the intrinsic aggregation propensity of CsgA. Here, using mutagenesis, salt-mediated charge screening, and by varying pH conditions, we show that the ability of CsgA variants to nucleate and form amyloid fibers is dictated by the charge state of the gatekeeper residues. We report that in Citrobacter youngae CsgA, certain arginine (R) and lysine (K) residues also act as gatekeeper residues. A mechanism of gatekeeping is proposed wherein R and K residues electrostatically interact with negatively charged D residues, tempering CsgA fiber formation.

Mechanistic insights into accelerated α-synuclein aggregation mediated by human microbiome-associated functional amyloids.

The gut microbiome has been shown to have key implications in the pathogenesis of Parkinson's disease (PD). The Escherichia coli functional amyloid CsgA is known to accelerate α-synuclein aggregation in vitro and induce PD symptoms in mice. However, the mechanism governing CsgA-mediated acceleration of α-synuclein aggregation is unclear. Here, we show that CsgA can form stable homodimeric species that correlate with faster α-synuclein amyloid aggregation. Furthermore, we identify and characterize new CsgA homologs encoded by bacteria present in the human microbiome. These CsgA homologs display diverse aggregation kinetics, and they differ in their ability to modulate α-synuclein aggregation. Remarkably, we demonstrate that slowing down CsgA aggregation leads to an increased acceleration of α-synuclein aggregation, suggesting that the intrinsic amyloidogenicity of gut bacterial CsgA homologs affects their ability to accelerate α-synuclein aggregation. Finally, we identify a complex between CsgA and α-synuclein that functions as a platform to accelerate α-synuclein aggregation. Taken together, our work reveals complex interplay between bacterial amyloids and α-synuclein that better informs our understanding of PD causation.

Bacterial Amyloid Curli Associated Gut Epithelial Neuroendocrine Activation Predominantly Observed in Alzheimer's Disease Mice with Central Amyloid-ß Pathology.

BACKGROUND: Substantial evidence from recent research suggests an influential and underappreciated force in Alzheimer's disease (AD) pathogenesis: the pathological signals originate from outside the brain. Pathogenic bacteria produce amyloid-like proteins "curli" that form biofilms and show functional similarities to human amyloid-ß (Aß). These proteins may contribute to neurological disease progression via signaling cascade from the gut to the brain. OBJECTIVE: We propose that curli causes neuroendocrine activation from the gut to brain that promotes central Aß pathology. METHODS: PGP9.5 and TLR2 levels in response to curli in the lumen of Tg2576 AD mice were analyzed by immunohistochemical and qRT-PCR analysis. Western blot and human 3D in vitro enteroids culture systems were also used. 16S rRNA gene sequencing was used to investigate bacterial dysbiosis. RESULTS: We found significant increase in bacterial-amyloid curli with elevated TLR2 at the mRNA level in the pre- and symptomatic Tg-AD gut compared to littermate WT controls. This data associates with increased gram-positive bacterial colonization in the ileum of the symptomatic AD mice. We found fundamental evidence for vagus nerve activation in response to bacterial curli. Neuroendocrine marker PGP9.5 was significantly elevated in the gut epithelium of symptomatic AD mice, and this was colocalized with increased TLR2 expression. Enteroids, 3D-human ileal mini-gut monolayer in vitro model system also revealed increase levels of TLR2 upon stimulation with purified bacterial curli fibrils. CONCLUSION: These findings reveal the importance of pathological changes within the gut-vagus-brain signaling in response to luminal bacterial amyloid that might play a vital role in central Aß pathogenesis seen in the AD brain.

Half a century of amyloids: past, present and future.

Amyloid diseases are global epidemics with profound health, social and economic implications and yet remain without a cure. This dire situation calls for research into the origin and pathological manifestations of amyloidosis to stimulate continued development of new therapeutics. In basic science and engineering, the cross-ß architecture has been a constant thread underlying the structural characteristics of pathological and functional amyloids, and realizing that amyloid structures can be both pathological and functional in nature has fuelled innovations in artificial amyloids, whose use today ranges from water purification to 3D printing. At the conclusion of a half century since Eanes and Glenner's seminal study of amyloids in humans, this review commemorates the occasion by documenting the major milestones in amyloid research to date, from the perspectives of structural biology, biophysics, medicine, microbiology, engineering and nanotechnology. We also discuss new challenges and opportunities to drive this interdisciplinary field moving forward.

Tuning Functional Amyloid Formation Through Disulfide Engineering.

Many organisms produce "functional" amyloid fibers, which are stable protein polymers that serve many roles in cellular biology. Certain Enterobacteriaceae assemble functional amyloid fibers called curli that are the main protein component of the biofilm extracellular matrix. CsgA is the major protein subunit of curli and will rapidly adopt the polymeric amyloid conformation in vitro. The rapid and irreversible nature of CsgA amyloid formation makes it challenging to study in vitro. Here, we engineered CsgA so that amyloid formation could be tuned to the redox state of the protein. A double cysteine variant of CsgA called CsgACC was created and characterized for its ability to form amyloid. When kept under oxidizing conditions, CsgACC did not adopt a ß-sheet rich structure or form detectable amyloid-like aggregates. Oxidized CsgACC remained in a soluble, non-amyloid state for at least 90 days. The addition of reducing agents to CsgACC resulted in amyloid formation within hours. The amyloid fibers formed by CsgACC were indistinguishable from the fibers made by CsgA WT. When measured by thioflavin T fluorescence the amyloid formation by CsgACC in the reduced form displayed the same lag, fast, and plateau phases as CsgA WT. Amyloid formation by CsgACC could be halted by the addition of oxidizing agents. Therefore, CsgACC serves as a proof-of-concept for capitalizing on the convertible nature of disulfide bonds to control the aggregation of amyloidogenic proteins.

Bacterial functional amyloids: Order from disorder.

The discovery of intrinsic disorderness in proteins and peptide regions has given a new and useful insight into the working of biological systems. Due to enormous plasticity and heterogeneity, intrinsically disordered proteins or regions in proteins can perform myriad of functions. The flexibility in disordered proteins allows them to undergo conformation transition to form homopolymers of proteins called amyloids. Amyloids are highly structured protein aggregates associated with many neurodegenerative diseases. However, amyloids have gained much appreciation in recent years due to their functional roles. A functional amyloid fiber called curli is assembled on the bacterial cell surface as a part of the extracellular matrix during biofilm formation. The extracellular matrix that encases cells in a biofilm protects the cells and provides resistance against many environmental stresses. Several of the Csg (curli specific genes) proteins that are required for curli amyloid assembly are predicted to be intrinsically disordered. Therefore, curli amyloid formation is highly orchestrated so that these intrinsically disordered proteins do not inappropriately aggregate at the wrong time or place. The curli proteins are compartmentalized and there are chaperone-like proteins that prevent inappropriate aggregation and allow the controlled assembly of curli amyloids. Here we review the biogenesis of curli amyloids and the role that intrinsically disordered proteins play in the process.

Curli Biogenesis: Bacterial Amyloid Assembly by the Type VIII Secretion Pathway.

In 1989, Normark and coworkers reported on fibrous surface structures called curli on strains of Escherichia coli that were suspected of causing bovine mastitis. Subsequent work by many groups has revealed an elegant and highly regulated curli biogenesis pathway also referred to as the type VIII secretion system. Curli biogenesis is governed by two divergently transcribed operons, csgBAC and csgDEFG. The csgBAC operon encodes the structural subunits of curli, CsgA and CsgB, along with a chaperone-like protein, CsgC. The csgDEFG operon encodes the accessory proteins required for efficient transcription, secretion, and assembly of the curli fiber. CsgA and CsgB are secreted as largely unstructured proteins and transition to ß-rich structures that aggregate into regular fibers at the cell surface. Since both of these proteins have been shown to be amyloidogenic in nature, the correct spatiotemporal synthesis of the curli fiber is of paramount importance for proper functioning and viability. Gram-negative bacteria have evolved an elegant machinery for the safe handling, secretion, and extracellular assembly of these amyloidogenic proteins.

Physical Determinants of Amyloid Assembly in Biofilm Formation.

A wide range of bacterial pathogens have been shown to form biofilms, which significantly increase their resistance to environmental stresses, such as antibiotics, and are thus of central importance in the context of bacterial diseases. One of the major structural components of these bacterial biofilms are amyloid fibrils, yet the mechanism of fibril assembly and its importance for biofilm formation are currently not fully understood. By studying fibril formation in vitro, in a model system of two common but unrelated biofilm-forming proteins, FapC from Pseudomonas fluorescens and CsgA from Escherichia coli, we found that the two proteins have a common aggregation mechanism. In both systems, fibril formation proceeds via nucleated growth of linear fibrils exhibiting similar measured rates of elongation, with negligible fibril self-replication. These similarities between two unrelated systems suggest that convergent evolution plays a key role in tuning the assembly kinetics of functional amyloid fibrils and indicates that only a narrow window of mechanisms and assembly rates allows for successful biofilm formation. Thus, the amyloid assembly reaction is likely to represent a means for controlling biofilm formation, both by the organism and by possible inhibitory drugs.IMPORTANCE Biofilms are generated by bacteria, embedded in the formed extracellular matrix. The biofilm's function is to improve the survival of a bacterial colony through, for example, increased resistance to antibiotics or other environmental stresses. Proteins secreted by the bacteria act as a major structural component of this extracellular matrix, as they self-assemble into highly stable amyloid fibrils, making the biofilm very difficult to degrade by physical and chemical means once formed. By studying the self-assembly mechanism of the fibrils from their monomeric precursors in two unrelated bacteria, our experimental and theoretical approaches shed light on the mechanism of functional amyloid assembly in the context of biofilm formation. Our results suggest that fibril formation may be a rate-limiting step in biofilm formation, which in turn has implications on the protein self-assembly reaction as a target for potential antibiotic drugs.

Discovery of New Genes Involved in Curli Production by a Uropathogenic Escherichia coli Strain from the Highly Virulent O45:K1:H7 Lineage.

Curli are bacterial surface-associated amyloid fibers that bind to the dye Congo red (CR) and facilitate uropathogenic Escherichia coli (UPEC) biofilm formation and protection against host innate defenses. Here we sequenced the genome of the curli-producing UPEC pyelonephritis strain MS7163 and showed it belongs to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. MS7163 produced curli at human physiological temperature, and this correlated with biofilm growth, resistance of sessile cells to the human cationic peptide cathelicidin, and enhanced colonization of the mouse bladder. We devised a forward genetic screen using CR staining as a proxy for curli production and identified 41 genes that were required for optimal CR binding, of which 19 genes were essential for curli synthesis. Ten of these genes were novel or poorly characterized with respect to curli synthesis and included genes involved in purine de novo biosynthesis, a regulator that controls the Rcs phosphorelay system, and a novel repressor of curli production (referred to as rcpA). The involvement of these genes in curli production was confirmed by the construction of defined mutants and their complementation. The mutants did not express the curli major subunit CsgA and failed to produce curli based on CR binding. Mutation of purF (the first gene in the purine biosynthesis pathway) and rcpA also led to attenuated colonization of the mouse bladder. Overall, this work has provided new insight into the regulation of curli and the role of these amyloid fibers in UPEC biofilm formation and pathogenesis.IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains are the most common cause of urinary tract infection, a disease increasingly associated with escalating antibiotic resistance. UPEC strains possess multiple surface-associated factors that enable their colonization of the urinary tract, including fimbriae, curli, and autotransporters. Curli are extracellular amyloid fibers that enhance UPEC virulence and promote biofilm formation. Here we examined the function and regulation of curli in a UPEC pyelonephritis strain belonging to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. Curli expression at human physiological temperature led to increased biofilm formation, resistance of sessile cells to the human cationic peptide LL-37, and enhanced bladder colonization. Using a comprehensive genetic screen, we identified multiple genes involved in curli production, including several that were novel or poorly characterized with respect to curli synthesis. In total, this study demonstrates an important role for curli as a UPEC virulence factor that promotes biofilm formation, resistance, and pathogenesis.

Amyloid by Design: Intrinsic Regulation of Microbial Amyloid Assembly.

The term amyloid has historically been used to describe fibrillar aggregates formed as the result of protein misfolding and that are associated with a range of diseases broadly termed amyloidoses. The discovery of "functional amyloids" expanded the amyloid umbrella to encompass aggregates structurally similar to disease-associated amyloids but that engage in a variety of biologically useful tasks without incurring toxicity. The mechanisms by which functional amyloid systems ensure nontoxic assembly has provided insights into potential therapeutic strategies for treating amyloidoses. Some of the most-studied functional amyloids are ones produced by bacteria. Curli amyloids are extracellular fibers made by enteric bacteria that function to encase and protect bacterial communities during biofilm formation. Here we review recent studies highlighting microbial functional amyloid assembly systems that are tailored to enable the assembly of non-toxic amyloid aggregates.

Structure-Function Analysis of the Curli Accessory Protein CsgE Defines Surfaces Essential for Coordinating Amyloid Fiber Formation.

Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed primarily of the major structural subunit CsgA. The CsgE chaperone facilitates the secretion of CsgA through CsgG by forming a cap at the base of the nonameric CsgG outer membrane pore. We elucidated a series of finely tuned nonpolar and charge-charge interactions that facilitate the oligomerization of CsgE and its ability to transport unfolded CsgA to CsgG for translocation. CsgE oligomerization in vitro is temperature dependent and is disrupted by mutations in the W48 and F79 residues. Using nuclear magnetic resonance (NMR), we identified two regions of CsgE involved in the CsgE-CsgA interaction: a head comprising a positively charged patch centered around R47 and a stem comprising a negatively charged patch containing E31 and E85. Negatively charged residues in the intrinsically disordered N- and C-terminal "tails" were not implicated in this interaction. Head and stem residues were mutated and interrogated using in vivo measurements of curli production and in vitro amyloid polymerization assays. The R47 head residue of CsgE is required for stabilization of CsgA- and CsgE-mediated curli fiber formation. Mutation of the E31 and E85 stem residues to positively charged side chains decreased CsgE-mediated curli fiber formation but increased CsgE-mediated stabilization of CsgA. No single-amino-acid substitutions in the head, stem, or tail regions affected the ability of CsgE to cap the CsgG pore as determined by a bile salt sensitivity assay. These mechanistic insights into the directed assembly of functional amyloids in extracellular biofilms elucidate possible targets for biofilm-associated bacterial infections.IMPORTANCE Curli represent a class of functional amyloid fibers produced by Escherichia coli and other Gram-negative bacteria that serve as protein scaffolds in the extracellular biofilm matrix. Despite the lack of sequence conservation among different amyloidogenic proteins, the structural and biophysical properties of functional amyloids such as curli closely resemble those of amyloids associated with several common neurodegenerative diseases. These parallels are underscored by the observation that certain proteins and chemicals can prevent amyloid formation by the major curli subunit CsgA and by alpha-synuclein, the amyloid-forming protein found in Lewy bodies during Parkinson's disease. CsgA subunits are targeted to the CsgG outer membrane pore by CsgE prior to secretion and assembly into fibers. Here, we use biophysical, biochemical, and genetic approaches to elucidate a mechanistic understanding of CsgE function in curli biogenesis.

Bacterial Amyloids.

Many bacteria can assemble functional amyloid fibers on their cell surface. Most bacterial amyloids contribute to biofilm or other community behaviors where cells interact with a surface or with other cells. Bacterial amyloids, like all functional amyloids, share structural and biochemical properties with disease-associated eukaryotic amyloids. The general ability of amyloids to bind specific dyes, like Congo red and Thioflavin T, and their resistance to denaturation have provided useful tools for scoring and quantifying bacterial amyloid formation. Here, we present basic approaches to study bacterial amyloids by focusing on the well-studied curli amyloid fibers expressed by Enterobacteriaceae. These methods exploit the specific tinctorial and biophysical properties of amyloids. The methods described here are straightforward and can be easily applied by any modern molecular biology lab for the study of other bacterial amyloids.

Thiol Starvation Induces Redox-Mediated Dysregulation of Escherichia coli Biofilm Components.

A hallmark of bacterial biofilms is the production of an extracellular matrix (ECM) that encases and protects the community from environmental stressors. Biofilm formation is an integral portion of the uropathogenic Escherichia coli (UPEC) life cycle. Approximately 2% of UPEC isolates are cysteine auxotrophs. Here, we investigated how cysteine homeostasis impacted UPEC UTI89 strain biofilm formation and, specifically, the production of the ECM components curli and cellulose. Cysteine auxotrophs produced less cellulose and slightly more curli compared to wild-type (WT) strains, and cysteine auxotrophs formed smooth, nonrugose colonies. Cellulose production was restored in cysteine auxotrophs when YfiR was inactivated. YfiR is a redox-sensitive regulator of the diguanylate cyclase, YfiN. The production of curli, a temperature-regulated appendage, was independent of temperature in UTI89 cysteine auxotrophs. In a screen of UPEC isolates, we found that ∼60% of UPEC cysteine auxotrophs produced curli at 37°C, but only ∼2% of cysteine prototrophic UPEC isolates produced curli at 37°C. Interestingly, sublethal concentrations of amdinocillin and trimethoprim-sulfamethoxazole inhibited curli production, whereas strains auxotrophic for cysteine continued to produce curli even in the presence of amdinocillin and trimethoprim-sulfamethoxazole. The dysregulation of ECM components and resistance to amdinocillin in cysteine auxotrophs may be linked to hyperoxidation, since the addition of exogenous cysteine or glutathione restored WT biofilm phenotypes to mutants unable to produce cysteine and glutathione.IMPORTANCE Uropathogenic Escherichia coli (UPEC) bacteria are the predominant causative agent of urinary tract infections (UTIs). UTIs account for billions of dollars of financial burden annually to the health care industry in the United States. Biofilms are an important aspect of the UPEC pathogenesis cascade and for the establishment of chronic infections. Approximately 2% of UPEC isolates from UTIs are cysteine auxotrophs, yet there is relatively little known about the biofilm formation of UPEC cysteine auxotrophs. Here we show that cysteine auxotrophs have dysregulated biofilm components due to a change in the redox state of the periplasm. Additionally, we show the relationship between cysteine auxotrophs, biofilms, and antibiotics frequently used to treat UTIs.

The role of microbial amyloid in neurodegeneration.

It has become apparent that the intestinal microbiota orchestrates important aspects of our metabolism, immunity, and development. Recent work has demonstrated that the microbiota also influences brain function in healthy and diseased individuals. Of great interest are reports that intestinal bacteria play a role in the pathogenic cascade of both Parkinson and Alzheimer diseases. These neurodegenerative disorders both involve misfolding of endogenous proteins that spreads from one region of the body to another in a manner analogous to prions. The mechanisms of how the microbiota influences or is correlated with disease require elaboration. Microbial proteins or metabolites may influence neurodegeneration through the promotion of amyloid formation by human proteins or by enhancing inflammatory responses to endogenous neuronal amyloids. We review the current knowledge concerning bacterial amyloids and their potential to influence cerebral amyloid aggregation and neuroinflammation. We propose the term "mapranosis" to describe the process of microbiota-associated proteopathy and neuroinflammation. The study of amyloid proteins made by the microbiota and their influence on health and disease is in its infancy. This is a promising area for therapeutic intervention because there are many ways to alter our microbial partners and their products, including amyloid proteins.

Inhibition of curli assembly and Escherichia coli biofilm formation by the human systemic amyloid precursor transthyretin.

During biofilm formation, Escherichia coli and other Enterobacteriaceae produce an extracellular matrix consisting of curli amyloid fibers and cellulose. The precursor of curli fibers is the amyloidogenic protein CsgA. The human systemic amyloid precursor protein transthyretin (TTR) is known to inhibit amyloid-ß (Aß) aggregation in vitro and suppress the Alzheimer's-like phenotypes in a transgenic mouse model of Aß deposition. We hypothesized that TTR might have broad antiamyloid activity because the biophysical properties of amyloids are largely conserved across species and kingdoms. Here, we report that both human WT tetrameric TTR (WT-TTR) and its engineered nontetramer-forming monomer (M-TTR, F87M/L110M) inhibit CsgA amyloid formation in vitro, with M-TTR being the more efficient inhibitor. Preincubation of WT-TTR with small molecules that occupy the T4 binding site eliminated the inhibitory capacity of the tetramer; however, they did not significantly compromise the ability of M-TTR to inhibit CsgA amyloidogenesis. TTR also inhibited amyloid-dependent biofilm formation in two different bacterial species with no apparent bactericidal or bacteriostatic effects. These discoveries suggest that TTR is an effective antibiofilm agent that could potentiate antibiotic efficacy in infections associated with significant biofilm formation.

The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of Escherichia coli.

Curli amyloid fibers are the major protein component of the extracellular matrix produced by Enterobacteriaceae during biofilm formation. Curli are required for proper biofilm development and environmental persistence by Escherichia coli. Here, we present a complete and vetted genetic analysis of functional amyloid fiber biogenesis. The Keio collection of single gene deletions was screened on Congo red indicator plates to identify E. coli mutants that had defective amyloid production. We discovered that more than three hundred gene products modulated curli production. These genes were involved in fundamental cellular processes such as regulation, environmental sensing, respiration, metabolism, cell envelope biogenesis, transport, and protein turnover. The alternative sigma factors, σS and σE, had opposing roles in curli production. Mutations that induced the σE or Cpx stress response systems had reduced curli production, while mutant strains with increased σS levels had increased curli production. Mutations in metabolic pathways, including gluconeogenesis and the biosynthesis of lipopolysaccharide (LPS), produced less curli. Regulation of the master biofilm regulator, CsgD, was diverse, and the screen revealed several proteins and small RNAs (sRNA) that regulate csgD messenger RNA (mRNA) levels. Using previously published studies, we found minimal overlap between the genes affecting curli biogenesis and genes known to impact swimming or swarming motility, underlying the distinction between motile and sessile lifestyles. Collectively, the diversity and number of elements required suggest curli production is part of a highly regulated and complex developmental pathway in E. coli.