Plasminogen, absorbed by Escherichia coli expressing curli or by Salmonella enteritidis expressing thin aggregative fimbriae, can be activated by simultaneously captured tissue-type plasminogen activator (t-PA)

Curli are fimbrial structures expressed by Escherichia coli that specifically interact with matrix proteins such as fibronectin and laminin. Similar structures are also expressed by Salmonella enteritidis and have been denoted thin aggregative fimbriae. Bacteria expressing curli and thin aggregative fimbriae were found to bind radiolabelled plasminogen as well as the tissue-type plasminogen activator (t-PA). By contrast, E. coli carrying a gene locus with an insertionally inactivated chromosomal curlin subunit were unable to bind the two human proteins. The purified subunit polypeptides of curli and thin aggregative fimbriae bound plasminogen and t-PA with high affinity (1 × 10⁸ to 2 × 10⁸ M^-1). The binding of plasminogen and t-PA to curli-expressing E. coli was only partially inhibited by fibronectin and laminin. Plasminogen absorbed from human plasma by curli-expressing E. coli was readily converted to plasmin by t-PA; both plasmin and t-PA were functionally active when bound to the bacteria. A simultaneous binding of fibrinolytic proteins and matrix proteins to fimbriae of E. coli and S. enteritidis could provide these pathogens with both adhesive and invasive properties.     

Promiscuous Cross-seeding between Bacterial Amyloids Promotes Interspecies Biofilms

Amyloids are highly aggregated proteinaceous fibers historically associated with neurodegenerative conditions including Alzheimers, Parkinsons, and prion-based encephalopathies. Polymerization of amyloidogenic proteins into ordered fibers can be accelerated by preformed amyloid aggregates derived from the same protein in a process called seeding. Seeding of disease-associated amyloids and prions is highly specific and cross-seeding is usually limited or prevented. Here we describe the first study on the cross-seeding potential of bacterial functional amyloids. Curli are produced on the surface of many Gram-negative bacteria where they facilitate surface attachment and biofilm development. Curli fibers are composed of the major subunit CsgA and the nucleator CsgB, which templates CsgA into fibers. Our results showed that curli subunit homologs from Escherichia coli, Salmonella typhimurium LT2, and Citrobacter koseri were able to cross-seed in vitro. The polymerization of Escherichia coli CsgA was also accelerated by fibers derived from a distant homolog in Shewanella oneidensis that shares less than 30% identity in primary sequence. Cross-seeding of curli proteins was also observed in mixed colony biofilms with E. coli and S. typhimurium. CsgA was secreted from E. coli csgB− mutants assembled into fibers on adjacent S. typhimurium that presented CsgB on its surfaces. Similarly, CsgA was secreted by S. typhimurium csgB− mutants formed curli on CsgB-presenting E. coli. This interspecies curli assembly enhanced bacterial attachment to agar surfaces and supported pellicle biofilm formation. Collectively, this work suggests that the seeding specificity among curli homologs is relaxed and that heterogeneous curli fibers can facilitate multispecies biofilm development.     

Characterization of the binding of diarrheagenic strains of E. coli to plant surfaces and the role of curli in the interaction of the bacteria with alfalfa sprouts

Diarrheagenic Escherichia coli were able to bind to plant surfaces, including alfalfa sprouts and open seed coats, and tomato and Arabidopsis thaliana seedlings incubated in water. The characteristics of the binding differed with the bacterial strain examined. Laboratory K12 strains of E. coli failed to show significant binding to any of the plant surfaces examined, suggesting that some of the genes present and expressed in pathogenic strains and absent or unexpressed in K12 strains may be required for binding to plants. When a plasmid carrying the mlrA gene (a positive regulator of curli biosynthesis) or a plasmid carrying the operons that encode the synthesis of curli (csgA-G) was introduced into K12 strains, the bacteria acquired the ability to bind to sprouts. CsgA mutants of an avian pathogenic E. coli and an O157:H7 strain showed no reduction in their ability to bind to sprouts. Thus, the production of curli appears to be sufficient to allow K12 strains to bind, but curli are not necessary for the binding of pathogenic strains, suggesting that pathogenic strains may have more than one mechanism for binding to plant surfaces.     

Spatial Clustering of the Curlin Secretion Lipoprotein Requires Curli Fiber Assembly

Gram-negative bacteria assemble functional amyloid surface fibers called curli. CsgB nucleates the major curli subunit protein, CsgA, into a self-propagating amyloid fiber on the cell surface. The CsgG lipoprotein is sufficient for curlin transport across the outer membrane and is hypothesized to be the central molecule of the curli fiber secretion and assembly complex. We tested the hypothesis that the curli secretion protein, CsgG, was restricted to certain areas of the cell to promote the interaction of CsgA and CsgB during curli assembly. Here, electron microscopic analysis of curli-producing strains showed that relatively few cells in the population contacted curli fibers and that curli emanated from spatially discrete points on the cell surface. Microscopic analysis revealed that CsgG was surface exposed and spatially clustered around curli fibers. CsgG localization to the outer membrane and exposure of the surface domain were not dependent on any other csg-encoded protein, but the clustering of CsgG required the csg-encoded proteins CsgE, CsgF, CsgA, and CsgB. CsgG formed stable oligomers in all the csg mutant strains, but these oligomers were distinct from the CsgG complexes assembled in wild-type cells. Finally, we found that efficient fiber assembly was required for the spatial clustering of CsgG. These results suggest a new model where curli fiber formation is spatially coordinated with the CsgG assembly apparatus.     

The influence of curli, a MHC-I-binding bacterial surface structure, on macrophage-T cell interactions

Escherichia coli express thin surface fimbriae called curli which bind soluble matrix proteins and major histocompatibility complex (MHC)-I molecules. The present study addressed the ability of purified curli or curliated E. coli to influence peptide presentation on MHC-I, T cell proliferation and bacterial uptake by macrophages. In vitro studies with curli-proficient E. coli YMel and the isogenic curli-deficient strain YMel-1, both expressing the model antigen Crl-OVA, showed that curli expression by E. coli does not appear to influence the efficiency by which the bacteria are processed by murine macrophages for OVA(257-264) presentation on K(b). Furthermore, curli expression by E. coli did not influence the binding of exogenously added OVA(257-264) peptide to K(b) on the surface of prefixed macrophages. In addition, neither curliated nor non-curliated heat-killed bacteria influenced proliferation of either murine or human T cells stimulated with anti-CD3. Finally, curliated E. coli adhered to and were internalized by macrophages from C57BL/6 and MHC-I-deficient TAP1(-/-) mice equally well. Together these studies show that curli expression by E. coli does not appear to influence phagocytic processing of bacteria expressing Crl-OVA for OVA(257-264)/K(b) presentation, the binding of exogenously added OVA(257-264) to K(b) or T cell proliferation. In addition, although curli expression by E. coli enhances bacterial interaction with macrophages, curli interaction with MHC-I does not significantly contribute to this adherence.     

Curli biogenesis: Order out of disorder

Many bacteria assemble extracellular amyloid fibers on their cell surface. Secretion of proteins across membranes and the assembly of complex macromolecular structures must be highly coordinated to avoid the accumulation of potentially toxic intracellular protein aggregates. Extracellular amyloid fiber assembly poses an even greater threat to cellular health due to the highly aggregative nature of amyloids and the inherent toxicity of amyloid assembly intermediates. Therefore, temporal and spatial control of amyloid protein secretion is paramount. The biogenesis and assembly of the extracellular bacterial amyloid curli is an ideal system for studying how bacteria cope with the many challenges of controlled and ordered amyloid assembly. Here, we review the recent progress in the curli field that has made curli biogenesis one of the best-understood functional amyloid assembly pathways. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

大肠杆菌卷曲菌毛的生物合成和致病性

大肠杆菌卷曲菌毛是其菌体表面的一种含纤维素样蛋白质附着器官,出现在大肠杆菌生理和病理过程中。卷曲菌毛可以通过黏附等作用介导大肠杆菌侵袭宿主;作为一种细菌淀粉样蛋白,卷曲菌毛有可能引起淀粉样蛋白相关疾病;卷曲菌毛可以诱导宿主炎症因子水平升高,引起脓毒血症;卷曲菌毛可以和纤维素等一起构成菌外基质,参与生物膜的形成。我们简要综述了大肠杆菌卷曲菌毛的生物合成、生物学功能和致病性。     

Assembly of human contact phase proteins and release of bradykinin at the surface of curli-expressing Escherichia coli

Previous work has demonstrated that most strains of the human pathogen Streptococcus pyogenes bind kininogens through M protein, a fibrous surface protein and virulence determinant. Here we find that strains of several other pathogenic bacterial species, both Gram-positive and Gram-negative, isolated from patients with sepsis, also bind kininogens, especially H-kininogen (HK). The most pronounced interaction was seen between HK and Escherichia coli. Among clinical isolates of E. coli, the majority of the entero-haemorrhagic, enterotoxigenic, and sepsis strains, but none of the enteroinvasive and enteropathogenic strains, bound HK. Binding of HK to E. coli correlated with the expression of curli, another fibrous bacterial surface protein, and the binding of HK to purified curli was specific, saturable, and of high affinity; Ka = 9 107M-1. Other contact phase proteins such as factor XI, factor XII, and prekallikrein bound to curliated E. coli, but not to an isogenic curli-deficient mutant strain, suggesting that contact phase activation may occur at the surface of curliated bacteria. Kininogens are also precursor molecules of the vasoactive kinins. When incubated with human plasma, curli-expressing bacteria absorbed HK. Addition of purified plasma kallikrein to the HK-loaded bacteria resulted in a rapid and efficient release of bradykinin from surface-bound HK. The assembly of contact phase factors at the surface of pathogenic bacteria and the release of the potent proinflammatory and vasoactive peptide bradykinin, should have a major impact on the host-microbe relationship and may contribute to bacterial pathogencity and virulence.     

Variation in the ratio of curli and phosphoethanolamine cellulose associated with biofilm architecture and properties

Bacterial biofilms are communities of bacteria entangled in a self‐produced extracellular matrix (ECM). Escherichia coli direct the assembly of two insoluble biopolymers, curli amyloid fibers, and phosphoethanolamine (pEtN) cellulose, to build remarkable biofilm architectures. Intense curiosity surrounds how bacteria harness these amyloid‐polysaccharide composites to build biofilms, and how these biopolymers function to benefit bacterial communities. Defining ECM composition involving insoluble polymeric assemblies poses unique challenges to analysis and, thus, to comparing strains with quantitative ECM molecular correlates. In this work, we present results from a sum‐of‐the‐parts ¹³C solid‐state nuclear magnetic resonance (NMR) analysis to define the curli‐to‐pEtN cellulose ratio in the isolated ECM of the E. coli laboratory K12 strain, AR3110. We compare and contrast the compositional analysis and comprehensive biofilm phenotypes for AR3110 and a well‐studied clinical isolate, UTI89. The ECM isolated from AR3110 contains approximately twice the amount of pEtN cellulose relative to curli content as UTI89, revealing plasticity in matrix assembly principles among strains. The two parent strains and a panel of relevant gene mutants were investigated in three biofilm models, examining: (a) macrocolonies on agar, (b) pellicles at the liquid‐air interface, and (c) biomass accumulation on plastic. We describe the influence of curli, cellulose, and the pEtN modification on biofilm phenotypes with power in the direct comparison of these strains. The results suggest that curli more strongly influence adhesion, while pEtN cellulose drives cohesion. Their individual and combined influence depends on both the biofilm modality (agar, pellicle, or plastic‐associated) and the strain itself.     

Fibronectin binding to a Streptococcus pyogenes strain.

In previous studies, Staphylococcus aureus has been shown to bind fibronectin (P. Kuusela, Nature (London) 276:718-720, 1978), an interaction that may be important in bacterial attachment and opsonization. Recently some strains of streptococci of serological groups A, C, and G were also found to bind fibronectin. The binding to one selected strain of Streptococcus pyogenes has been characterized here. The binding of [125I]fibronectin to streptococcal cells resembles that to staphylococcal cells and was found to be time dependent, functionally irreversible, and specific in the sense that unlabeled proteins other than fibronectin did not block binding. Bacteria incubated with proteases largely lost their ability to bind fibronectin, and material released from the streptococci by a brief trypsin digestion contained active fibronectin receptors. This material inhibited the binding of [125I]fibronectin to the streptococci. The inhibitory activity was adsorbed on a column of fibronectin-Sepharose but not on a column of unsubstituted Sepharose 4B or egg albumin Sepharose. The receptor appeared to be a protein nature since the inhibitory activity of the trypsinate was destroyed by papain and was not absorbed on a column containing monoclonal antibodies directed against lipoteichoic acid bound to protein A-Sepharose. Binding sites in fibronectin for streptococci and staphylococci, respectively, were localized by analyzing the ability of isolated fragments to inhibit [125I]fibronectin binding to bacteria and by adsorbing 125I-labeled tryptic fragments with staphylococcal and streptococcal cells. Both species of bacteria appeared to preferentially bind a fragment (Mr = approximately 25,000) originating from the N-terminal region of the protein. In addition, streptococci also bound a slightly smaller fragment (Mr = approximately 23,000). Fibronectin receptors solubilized from either streptococci or staphylococci inhibited the binding of fibronectin to both species of bacteria.     

Sequence determinants of bacterial amyloid formation

Amyloids are proteinaceous fibers commonly associated with neurodegenerative diseases and prion-based encephalopathies. Many different polypeptides can form amyloid fibers, leading to the suggestion that amyloid is a primitive main chain-dominated structure. A growing body of evidence suggests that amino acid side chains dramatically influence amyloid formation. The specific role fulfilled by side chains in amyloid formation, especially in vivo, remains poorly understood. Here, we determined the role of internally conserved polar and aromatic residues in promoting amyloidogenesis of the functional amyloid protein CsgA, which is the major protein component of curli fibers assembled by enteric bacteria such as Escherichia coli and Salmonella spp. In vivo CsgA polymerization into an amyloid fiber requires the CsgB nucleator protein. The CsgA amyloid core region is composed of five repeating units, defined by regularly spaced Ser, Gln and Asn residues. The results of a comprehensive alanine scan mutagenesis screen showed that Gln and Asn residues at positions 49, 54, 139 and 144 were critical for curli assembly. Alanine substitution of Q49 or N144 impeded the ability of CsgA to respond to CsgB-mediated heteronucleation, and the ability of CsgA to self-polymerize in vitro. However, CsgA proteins harboring these mutations were still seeded by preformed wild-type CsgA fibers in vitro. This suggests that CsgA-fibril-mediated seeding and CsgB-mediated heteronucleation have distinguishable mechanisms. Remarkably, Gln residues at positions 49 and 139 could not be replaced by Asn residues without interfering with curli assembly, suggesting that the side chain requirements were especially stringent at these positions. This analysis demonstrates that bacterial amyloid formation is driven by specific side chain contacts, and provides a clear illustration of the essential roles of specific side chains in promoting amyloid formation.     

Curli Functional Amyloid Systems Are Phylogenetically Widespread and Display Large Diversity in Operon and Protein Structure

Escherichia coli and a few other members of the Enterobacteriales can produce functional amyloids known as curli. These extracellular fibrils are involved in biofilm formation and studies have shown that they may act as virulence factors during infections. It is not known whether curli fibrils are restricted to the Enterobacteriales or if they are phylogenetically widespread. The growing number of genome-sequenced bacteria spanning many phylogenetic groups allows a reliable bioinformatic investigation of the phylogenetic diversity of the curli system. Here we show that the curli system is phylogenetically much more widespread than initially assumed, spanning at least four phyla. Curli fibrils may consequently be encountered frequently in environmental as well as pathogenic biofilms, which was supported by identification of curli genes in public metagenomes from a diverse range of habitats. Identification and comparison of curli subunit (CsgA/B) homologs show that these proteins allow a high degree of freedom in their primary protein structure, although a modular structure of tightly spaced repeat regions containing conserved glutamine, asparagine and glycine residues has to be preserved. In addition, a high degree of variability within the operon structure of curli subunits between bacterial taxa suggests that the curli fibrils might have evolved to fulfill specific functions. Variations in the genetic organization of curli genes are also seen among different bacterial genera. This suggests that some genera may utilize alternative regulatory pathways for curli expression. Comparison of phylogenetic trees of Csg proteins and the 16S rRNA genes of the corresponding bacteria showed remarkably similar overall topography, suggesting that horizontal gene transfer is a minor player in the spreading of the curli system.     

Entry of OpaA+ gonococci into HEp-2 cells requires concerted action of glycosaminoglycans, fibronectin and integrin receptors

Heparan sulphate proteoglycans are increasingly implicated as eukaryotic cell surface receptors for bacterial pathogens. Here, we report that Neisseria gonorrhoeae adheres to proteoglycan receptors on HEp-2 epithelial cells but that internalization of the bacterium by this cell type requires the serum glycoprotein fibronectin. Fibronectin was shown to bind specifically to gonococci producing the OpaA adhesin. Binding assays with fibronectin fragments located the bacterial binding site near the N-terminal end of the molecule. However, none of the tested fibronectin fragments supported gonococcal entry into the eukaryotic cells; a 120 kDa fragment carrying the cell adhesion domain with the amino acid sequence RGD even inhibited the fibronectin-mediated uptake of MS11-OpaA. This inhibition could be mimicked by an RGD-containing hexapeptide and by α5β1 integrin-specific antibodies, suggesting that interaction of the central region of fibronectin with integrin receptors facilitated bacterial uptake. Fibronectin was unable to promote gonococcal entry into HEp-2 cells that had been treated with the enzyme heparinase III, which degrades the glycosaminoglycan side-chains of proteoglycan receptors. On the basis of these results, we propose a novel cellular uptake pathway for bacteria, which involves the binding of the pathogen to glycosaminoglycans that, in turn, act as co-receptors facilitating fibronectin-mediated bacterial uptake through integrin receptors. In this scenario, fibronectin would act as a molecular bridge linking the Opa–proteoglycan complex with host cell integrin receptors.     

Curli provide the template for understanding controlled amyloid propagation

The uncontrolled formation of amyloid fibers is the hallmark of more than twenty human diseases. In contrast to disease-associated amyloids, which are the products of protein misfolding, E. coli assembles functional amyloid fibers called curli on its surface using an elegant biogenesis machine. Composed of a major subunit, CsgA, and a minor subunit, CsgB, curli play important roles in host cell adhesion, long-term survival and other bacterial community behaviors. Assembly of curli fibers is a template-directed conversion process where membrane-tethered CsgB initiates CsgA polymerization. The CsgA amyloid core is composed of five imperfect repeating units. In a series of in vivo and in vitro experiments, we determined the sequence and structural determinants that guide the initiation and propagation of CsgA polymers. The CsgA N- and C-terminal repeating units govern its polymerization and responsiveness to CsgB. Specifically, conserved glutamine and asparagine residues present in the CsgA N- and C-terminal repeating units are required for CsgB-mediated nucleation and efficient self-assembly.Key words: amyloid, nucleation, polymerization, curli, sequence determinants     

The Bacterial Amyloid Curli Is Associated with Urinary Source Bloodstream Infection

Urinary tract infections are the most common cause of E. coli bloodstream infections (BSI) but the mechanism of bloodstream invasion is poorly understood. Some clinical isolates have been observed to shield themselves with extracellular amyloid fibers called curli at physiologic temperature. We hypothesize that curli fiber assembly at 37°C promotes bacteremic progression by urinary E. coli strains. Curli expression by cultured E. coli isolates from bacteriuric patients in the presence and absence of bacteremia were compared using Western blotting following amyloid fiber disruption with hexafluoroisopropanol. At 37°C, urinary isolates from bacteremic patients were more likely to express curli than those from non-bacteremic patients [16/22 (73%) vs. 7/21 (33%); p = 0.01]. No significant difference in curli expression was observed at 30°C [86% (19/22) vs. 76% (16/21); p = 0.5]. Isolates were clonally diverse between patients, indicating that this phenotype is distributed across multiple lineages. Most same-patient urine and blood isolates were highly related, consistent with direct invasion of urinary bacteria into the bloodstream. 37°C curli expression was associated with bacteremic progression of urinary E. coli isolates in this population. These findings suggest new future diagnostic and virulence-targeting therapeutic approaches.     

Congo Red Interactions with Curli-Producing E. coli and Native Curli Amyloid Fibers

Microorganisms produce functional amyloids that can be examined and manipulated in vivo and in vitro. Escherichia coli assemble extracellular adhesive amyloid fibers termed curli that mediate adhesion and promote biofilm formation. We have characterized the dye binding properties of the hallmark amyloid dye, Congo red, with curliated E. coli and with isolated curli fibers. Congo red binds to curliated whole cells, does not inhibit growth, and can be used to comparatively quantify whole-cell curliation. Using Surface Plasmon Resonance, we measured the binding and dissociation kinetics of Congo red to curli. Furthermore, we determined that the binding of Congo red to curli is pH-dependent and that histidine residues in the CsgA protein do not influence Congo red binding. Our results on E. coli strain MC4100, the most commonly employed strain for studies of E. coli amyloid biogenesis, provide a starting point from which to compare the influence of Congo red binding in other E. coli strains and amyloid-producing organisms.     

Curli provide the template for understanding controlled amyloid propagation

The uncontrolled formation of amyloid fibers is the hallmark of more than twenty human diseases. In contrast to disease-associated amyloids, which are the products of protein misfolding, E. coli assembles functional amyloid fibers called curli on its surface using an elegant biogenesis machine. Composed of a major subunit, CsgA, and a minor subunit, CsgB, curli play important roles in host cell adhesion, long-term survival and other bacterial community behaviors. Assembly of curli fibers is a template-directed conversion process where membrane-tethered CsgB initiates CsgA polymerization. The CsgA amyloid core is composed of five imperfect repeating units. In a series of in vivo and in vitro experiments, we determined the sequence and structural determinants that guide the initiation and propagation of CsgA polymers. The CsgA N- and C-terminal repeating units govern its polymerization and responsiveness to CsgB. Specifically, conserved glutamine and asparagine residues present in the CsgA N- and C-terminal repeating units are required for CsgB-mediated nucleation and efficient self-assembly.     

The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells

Group A streptococci (GAS) specifically attach to and internalize into human epithelial host cells. In some GAS isolates, fibronectin-binding proteins were identified as being responsible for these virulence traits. In the present study, the previously identified global negative regulator Nra was shown to control the binding of soluble fibronectin probably via regulation of protein F2 and/or SfbII expression in the serotype M49 strain 591. According to results from a conventional invasion assay based on the recovery of viable intracellular bacteria, the increased fibronectin binding did not affect bacterial adherence to HEp-2 epithelial cells, but was associated with a reduction in the internalization rates. However, when examined by confocal and electron microscopy techniques, the nra-mutant bacteria were shown to exhibit higher adherence and internalization rates than the corresponding wild type. The mutant bacteria escaped from the phagocytic vacuoles much faster, promoting consistent morphological changes which resulted in severe host cell damage. The apoptotic and lytic processes observed in nra-mutant infected host cells were correlated with an increased expression of the genes encoding superantigen SpeA, the cysteine protease SpeB, and streptolysin S in the nra-mutant bacteria. Adherence and internalization rates of a nra/speB-double mutant at wild-type levels indicated that the altered speB expression in the nra mutant contributed to the observed changes in both processes. The Nra-dependent effects on bacterial virulence were confined to infections carried out with stationary growth phase bacteria. In conclusion, the obtained results demonstrated that the global GAS regulator Nra modulates virulence genes, which are involved in host cell damage. Thus, by helping to achieve a critical balance of virulence factor expression that avoids the injury of target cells, Nra may facilitate GAS persistence in a safe intracellular niche.