Structural basis for mechanical force regulation of the adhesin FimH via finger trap-like beta sheet twisting.

The Escherichia coli fimbrial adhesive protein, FimH, mediates shear-dependent binding to mannosylated surfaces via force-enhanced allosteric catch bonds, but the underlying structural mechanism was previously unknown. Here we present the crystal structure of FimH incorporated into the multiprotein fimbrial tip, where the anchoring (pilin) domain of FimH interacts with the mannose-binding (lectin) domain and causes a twist in the beta sandwich fold of the latter. This loosens the mannose-binding pocket on the opposite end of the lectin domain, resulting in an inactive low-affinity state of the adhesin. The autoinhibition effect of the pilin domain is removed by application of tensile force across the bond, which separates the domains and causes the lectin domain to untwist and clamp tightly around the ligand like a finger-trap toy. Thus, beta sandwich domains, which are common in multidomain proteins exposed to tensile force in vivo, can undergo drastic allosteric changes and be subjected to mechanical regulation.

Structural insights into a cooperative switch between one and two FimH bacterial adhesins binding pauci- and high-mannose type N-glycan receptors.

The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1-Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4'-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity.

Rate limiting step of the allosteric activation of the bacterial adhesin FimH investigated by molecular dynamics simulations.

The bacterial adhesin FimH is a model for the study of protein allostery because its structure has been resolved in multiple configurations, including the active and the inactive state. FimH consists of a pilin domain (PD) that anchors it to the rest of the fimbria and an allosterically regulated lectin domain (LD) that binds mannose on the surface of infected cells. Under normal conditions, the two domains are docked to each other and LD binds mannose weakly. However, in the presence of tensile force generated by shear the domains separate and conformational changes propagate across LD resulting in a stronger bond to mannose. Recently, the crystallographic structure of a variant of FimH has been resolved, called FimH FocH , where PD contains 10 mutations near the inter-domain interface. Although the X-ray structures of FimH and FimH FocH are almost identical, experimental evidence shows that FimH FocH is activated even in the absence of shear. Here, molecular dynamics simulations combined with the Jarzynski equality were used to investigate the discrepancy between the crystallographic structures and the functional assays. The results indicate that the free energy barrier of the unbinding process between LD and PD is drastically reduced in FimH FocH . Rupture of inter-domain hydrogen bonds involving R166 constitutes a rate limiting step of the domain separation process and occurs more readily in FimH FocH than FimH. In conclusion, the mutations in FimH FocH shift the equilibrium toward an equal occupancy of bound and unbound states for LD and PD by reducing a rate limiting step.

adhesiomeR: a tool for Escherichia coli adhesin classification and analysis.

Adhesins are crucial factors in the virulence of bacterial pathogens such as Escherichia coli. However, to date no resources have been dedicated to the detailed analysis of E. coli adhesins. Here, we provide adhesiomeR software that enables characterization of the complete adhesin repertoire, termed the adhesiome. AdhesiomeR incorporates the most comprehensive database of E. coli adhesins and facilitates an extensive analysis of adhesiome. We demonstrate that adhesiomeR achieves 98% accuracy when compared with experimental analyses. Based on analysis of 15,000 E. coli genomes, we define novel adhesiome profiles and clusters, providing a nomenclature for a unified comparison of E. coli adhesiomes.

Investigation of an AIDA-I based expression system for display of various affinity proteins on Escherichia coli.

Autotransporters constitute a large family of natural proteins that are essential for delivering many types of proteins and peptides across the outer membrane in Gram-negative bacteria. In biotechnology, autotransporters have been explored for display of recombinant proteins and peptides on the surface of Escherichia coli and have potential as tools for directed evolution of affinity proteins. Here, we investigate conditions for AIDA-I autotransporter-mediated display of recombinant proteins. A new expression vector was designed and engineered for this purpose, and a panel of proteins from different affinity-protein classes were subcloned to the vector, followed by evaluation of expression, surface display and functionality. Surface expression was explored in ten different E. coli strains together with assessment of transformation efficiencies. Furthermore, the most promising strain and expression vector combination was used in mock library selections for evaluation of magnetic-assisted cell sortings (MACS). The results demonstrated dramatically different performances depending on the type of affinity protein and choice of E. coli strain. The optimized MACS protocol showed efficient enrichment, and thus potential for the new AIDA-I display system to be used in methods for directed evolution of affinity proteins.

Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli.

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.

FdeC expression regulates motility and adhesion of the avian pathogenic Escherichia coli strain IMT5155.

Adaptation of avian pathogenic E. coli (APEC) to changing host environments including virulence factors expression is vital for disease progression. FdeC is an autotransporter adhesin that plays a role in uropathogenic Escherichia coli (UPEC) adhesion to epithelial cells. Expression of fdeC is known to be regulated by environmental conditions in UPEC and Shiga toxin-producing E. coli (STEC). The observation in a previous study that an APEC strain IMT5155 in which the fdeC gene was disrupted by a transposon insertion resulted in elevated adhesion to chicken intestinal cells prompted us to further explore the role of fdeC in infection. We found that the fdeC gene prevalence and FdeC variant prevalence differed between APEC and nonpathogenic E. coli genomes. Expression of the fdeC gene was induced at host body temperature, an infection relevant condition. Disruption of fdeC resulted in greater adhesion to CHIC-8E11 cells and increased motility at 42 °C compared to wild type (WT) and higher expression of multiple transporter proteins that increased inorganic ion export. Increased motility may be related to increased inorganic ion export since this resulted in downregulation of YbjN, a protein known to supress motility. Inactivation of fdeC in APEC strain IMT5155 resulted in a weaker immune response in chickens compared to WT in experimental infections. Our findings suggest that FdeC is upregulated in the host and contributes to interactions with the host by down-modulating motility during colonization. A thorough understanding of the regulation and function of FdeC could provide novel insights into E. coli pathogenesis.

Handover mechanism of the growing pilus by the bacterial outer-membrane usher FimD.

Pathogenic bacteria such as Escherichia coli assemble surface structures termed pili, or fimbriae, to mediate binding to host-cell receptors1. Type 1 pili are assembled via the conserved chaperone-usher pathway2-5. The outer-membrane usher FimD recruits pilus subunits bound by the chaperone FimC via the periplasmic N-terminal domain of the usher. Subunit translocation through the ß-barrel channel of the usher occurs at the two C-terminal domains (which we label CTD1 and CTD2) of this protein. How the chaperone-subunit complex bound to the N-terminal domain is handed over to the C-terminal domains, as well as the timing of subunit polymerization into the growing pilus, have previously been unclear. Here we use cryo-electron microscopy to capture a pilus assembly intermediate (FimD-FimC-FimF-FimG-FimH) in a conformation in which FimD is in the process of handing over the chaperone-bound end of the growing pilus to the C-terminal domains. In this structure, FimF has already polymerized with FimG, and the N-terminal domain of FimD swings over to bind CTD2; the N-terminal domain maintains contact with FimC-FimF, while at the same time permitting access to the C-terminal domains. FimD has an intrinsically disordered N-terminal tail that precedes the N-terminal domain. This N-terminal tail folds into a helical motif upon recruiting the FimC-subunit complex, but reorganizes into a loop to bind CTD2 during handover. Because both the N-terminal and C-terminal domains of FimD are bound to the end of the growing pilus, the structure further suggests a mechanism for stabilizing the assembly intermediate to prevent the pilus fibre diffusing away during the incorporation of thousands of subunits.

Impact of an alpha helix and a cysteine-cysteine disulfide bond on the resistance of bacterial adhesion pili to stress.

Escherichia coli express adhesion pili that mediate attachment to host cell surfaces and are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow forces, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that class 1a pili of uropathogenic E. coli (UPEC), as well as class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC class 5 pili. Examining structural and steered molecular dynamics simulation data, we find that this difference in class 1 pili subunit behavior originates from an α-helical motif that can unfold when exposed to force. A disulfide bond cross-linking ß-strands in class 1 pili stabilizes subunits, allowing them to tolerate higher forces than class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche, since resident bacteria are exposed to stronger, more transient drag forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of class 5 ETEC pili.

Distribution of papA and papG Variants among Escherichia coli Genotypes: Association with Major Extraintestinal Pathogenic Lineages.

The pyelonephritis-associated fimbria (P fimbria) is one of the most recognized adhesion determinants of extraintestinal pathogenic Escherichia coli strains (ExPECs). Twelve variants have been described for the gene encoding the P fimbria major structural subunit PapA and three variants for the gene encoding the adhesin subunit PapG. However, their distribution among the ExPEC diversity has not been comprehensively addressed. A complete landscape of that distribution might be valuable for delineating basic studies about the pathogenicity mechanisms of ExPECs and following up on the evolution of ExPEC lineages, particularly those most epidemiologically relevant. Therefore, we performed a massive descriptive study to detect the papA and papG variants along different E. coli genotypes represented by genomic sequences contained in the NCBI Assembly Refseq database. The most common papA variants were F11, F10, F48, F16, F12, and F7-2, which were found in significant association with the most relevant ExPEC genotypes, the phylogroups B2 and D, and the sequence types ST95, ST131, ST127, ST69, ST12, and ST73. On the other hand, the papGII variant was by far the most common followed by papGIII, and both were also found to have a significant association with common ExPEC genotypes. We noticed the presence of genomes, mainly belonging to the sequence type ST12, harboring two or three papA variants and two papG variants. Furthermore, the most common papA and papG variants were also detected in records representing strains isolated from humans and animals such as poultry, bovine, and dogs, supporting previous hypotheses of potential cross-transmission. Finally, we characterized a set of 17 genomes from Chilean uropathogenic E. coli strains and found that ST12 and ST73 were the predominant sequence types. Variants F7-1, F7-2, F8, F9, F11, F13, F14, F16, and F48 were detected for papA, and papGII and papGIII variants were detected for papG. Significant associations with the sequence types observed in the analysis of genomes contained in the NCBI Assembly Refseq database were also found in this collection in 16 of 19 cases for papA variants and 7 of 9 cases for the papG variants. This comprehensive characterization might support future basic studies about P fimbria-mediated ExPEC adherence and future typing or epidemiological studies to monitor the evolution of ExPECs producing P fimbria.

Binding site plasticity regulation of the FimH catch-bond mechanism.

The bacterial fimbrial adhesin FimH is a remarkable and well-studied catch-bond protein found at the tip of E. coli type 1 pili, which allows pathogenic strains involved in urinary tract infections to bind high-mannose glycans exposed on human epithelia. The catch-bond behavior of FimH, where the strength of the interaction increases when a force is applied to separate the two partners, enables the bacteria to resist clearance when they are subjected to shear forces induced by urine flow. Two decades of experimental studies performed at the single-molecule level, as well as x-ray crystallography and modeling studies, have led to a consensus picture whereby force separates the binding domain from an inhibitor domain, effectively triggering an allosteric conformational change in the former. This force-induced allostery is thought to be responsible for an increased binding affinity at the core of the catch-bond mechanism. However, some important questions remain, the most challenging one being that the crystal structures corresponding to these two allosteric states show almost superimposable binding site geometries, which questions the molecular origin for the large difference in affinity. Using molecular dynamics with a combination of enhanced-sampling techniques, we demonstrate that the static picture provided by the crystal structures conceals a variety of binding site conformations that have a key impact on the apparent affinity. Crucially, the respective populations in each of these conformations are very different between the two allosteric states of the binding domain, which can then be related to experimental affinity measurements. We also evidence a previously unappreciated but important effect: in addition to the well-established role of the force as an allosteric regulator via domain separation, application of force tends to directly favor the high-affinity binding site conformations. We hypothesize that this additional "local" catch-bond effect could delay unbinding between the bacteria and the host cell before the "global" allosteric transition occurs, as well as stabilizing the complex even more once in the high-affinity allosteric state.

A host receptor enables type 1 pilus-mediated pathogenesis of Escherichia coli pyelonephritis.

Type 1 pili have long been considered the major virulence factor enabling colonization of the urinary bladder by uropathogenic Escherichia coli (UPEC). The molecular pathogenesis of pyelonephritis is less well characterized, due to previous limitations in preclinical modeling of kidney infection. Here, we demonstrate in a recently developed mouse model that beyond bladder infection, type 1 pili also are critical for establishment of ascending pyelonephritis. Bacterial mutants lacking the type 1 pilus adhesin (FimH) were unable to establish kidney infection in male C3H/HeN mice. We developed an in vitro model of FimH-dependent UPEC binding to renal collecting duct cells, and performed a CRISPR screen in these cells, identifying desmoglein-2 as a primary renal epithelial receptor for FimH. The mannosylated extracellular domain of human DSG2 bound directly to the lectin domain of FimH in vitro, and introduction of a mutation in the FimH mannose-binding pocket abolished binding to DSG2. In infected C3H/HeN mice, type 1-piliated UPEC and Dsg2 were co-localized within collecting ducts, and administration of mannoside FIM1033, a potent small-molecule inhibitor of FimH, significantly attenuated bacterial loads in pyelonephritis. Our results broaden the biological importance of FimH, specify the first renal FimH receptor, and indicate that FimH-targeted therapeutics will also have application in pyelonephritis.

Toggle switch residues control allosteric transitions in bacterial adhesins by participating in a concerted repacking of the protein core.

Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an 'inactive' low-affinity state (LAS) to an 'active' high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH's ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function.

Design of a chimeric protein composed of FimH, FyuA and CNF-1 virulence factors from uropathogenic Escherichia coli and evaluation its biological activity and immunogenicity in vitro and in vivo.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most prevalent bacterial infections in humans. Antibiotic resistance among UPEC isolates is increasing, and designing an effective vaccine can prevent or reduce these infections. FimH adhesin, iron scavenger receptor FyuA, and cytotoxic necrotizing factor -1 (CNF-1) are among the most important virulence factors of UPEC strains. Thus, a novel multi-epitope protein composed of FimH, FyuA, and CNF-1 was designed to evaluate its biological activity and immunogenicity in vitro and in vivo, respectively. The final vaccine design had seven domains, including the N-terminal domain of FimH, four domains of FyuA, and two domains of CNF-1, as determined by immunoinformatics analysis. The results of tertiary structure prediction showed that the chimeric protein had a C-score of -0.25 and Z-score of -1.94. Molecular docking indicated that thirty six ligand residues of the chimeric protein interacted with 53 receptor residues of TLR-4 by hydrogen bonds and hydrophobic interactions. Analysis of protein expression by SDS-PAGE showed an approximately 44 kDa band with different concentrations of IPTG which were confirmed by Western blot. According to ELISA results, the level of IL-8 produced by stimulated Ht29 cells with the chimeric protein was significantly higher than the stimulated Ht29 cells with CNF-1 alone and un-stimulated Ht29 cells. Rabbits subcutaneously immunized with the chimeric protein admixed with Freund adjuvant induced higher level of serum IgG on day 14 after the first vaccination than control rabbits. Furthermore, the booster dose of the chimeric protein significantly enhanced the IgG levels as compared to day 14 and also controls. As, the chimeric protein has suitable B-cell epitopes and MHC-I and MHC-II binding epitopes to stimulate humoral and cellular immunity, it could be a promising vaccine candidate against UTIs caused by UPEC. Evaluating the multi-epitope protein in inducing humoral and cellular immune responses, as well as protection, is ongoing in the mice models.

Multivalent calix[4]arene-based mannosylated dendrons as new FimH ligands and inhibitors.

We report the synthesis and biological characterization of a novel class of multivalent glycoconjugates as hit compounds for the design of new antiadhesive therapies against urogenital tract infections (UTIs) caused by uropathogenic E. coli strains (UPEC). The first step of UTIs is the molecular recognition of high mannose N-glycan expressed on the surface of urothelial cells by the bacterial lectin FimH, allowing the pathogen adhesion required for mammalian cell invasion. The inhibition of FimH-mediated interactions is thus a validated strategy for the treatment of UTIs. To this purpose, we designed and synthesized d-mannose multivalent dendrons supported on a calixarene core introducing a significant structural change from a previously described family of dendrimers bearing the same dendrons units on a flexible pentaerythritol scaffold core. The new molecular architecture increased the inhibitory potency against FimH-mediated adhesion processes by about 16 times, as assessed by yeast agglutination assay. Moreover, the direct molecular interaction of the new compounds with FimH protein was assessed by on-cell NMR experiments acquired in the presence of UPEC cells.

Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually. Despite effective antibiotic therapy, 30-50% of patients experience recurrent UTIs. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.

Screening of antigenic epitopes related to the adhesion of the avian Escherichia coli Type 1 Fimbrial Agglutinin Domain.

BACKGROUND: Avian Escherichia coli (E.coli) type 1 fimbriae adhere to avian tracheal epithelial cells through the FimH protein. However, the adhesion-related antigen is still unknown. The purpose of this study was to analyze the antigenicity of the type 1 fimbrial FimH protein of wild-type avian E. coli, screen antigen epitopes, and prepare monoclonal antibodies (mAbs) that can block the adhesion of avian E. coli. RESULTS: In this study, the nucleic acid homologies of MG2 (O11), TS12 (O18), and YR5 (O78) with K12 were 97.7%, 99.6%, and 97.7%, respectively, and the amino acid sequence similarity reached 98.7%, 99.3%, and 98.0%, respectively. The epitopes and hydrophilicities of the FimH proteins of these three strains were similar. The more obvious lectin domain epitopes were located at FimH protein positions 111-124 and 154-162. The mAbs 7C2 and 7D8 against these two epitopes were prepared. An adhesion inhibition test showed that 7C2 and 7D8 blocked bacterial adhesion to avian tracheal epithelial cells. The mAb 7C2 against the 111-124 epitope inhibited O78 strain adhesion by 93%, and the mAb 7D8 against the 154-162 epitope inhibited O78 strain adhesion by 49%, indicating that these two epitopes are closely related to the adhesion of type 1 fimbriae. However, only the 111-124 epitope-recognizing mAb 7C2 inhibited bacterial agglutination of erythrocytes, indicating that host cell receptor binding and erythrocyte agglutination are not mediated by the same spatial locations within the FimH protein. CONCLUSIONS: The results demonstrate that the mAbs 7C2 and 7D8 against FimH protein positions 111-124 and 154-162 could inhibit the adhesion of E.coli to the chicken trachea.

Genome-Wide Analysis of Antigen 43 (Ag43) Variants: New Insights in Their Diversity, Distribution and Prevalence in Bacteria.

Antigen 43 (Ag43) expression induces aggregation and biofilm formation that has consequences for bacterial colonisation and infection. Ag43 is secreted through the Type 5 subtype "a" secretion system (T5aSS) and is a prototypical member of the family of self-associating autotransporters (SAATs). As a T5aSS protein, Ag43 has a modular architecture comprised of (i) a signal peptide, (ii) a passenger domain that can be subdivided into three subdomains (SL, EJ, and BL), (iii) an autochaperone (AC) domain, and (iv) an outer membrane translocator. The cell-surface SL subdomain is directly involved in the "Velcro-handshake" mechanism resulting in bacterial autoaggregation. Ag43 is considered to have a ubiquitous distribution in E. coli genomes and many strains harbour multiple agn43 genes. However, recent phylogenetic analyses indicated the existence of four distinct Ag43 classes exhibiting different propensities for autoaggregation and interactions. Given the knowledge of the diversity and distribution of Ag43 in E. coli genomes is incomplete, we have performed a thorough in silico investigation across bacterial genomes. Our comprehensive analyses indicate that Ag43 passenger domains cluster in six phylogenetic classes associated with different SL subdomains. The diversity of Ag43 passenger domains is a result of the association of the SL subtypes with two different EJ-BL-AC modules. We reveal that agn43 is almost exclusively present among bacterial species of the Enterobacteriaceae family and essentially in the Escherichia genus (99.6%) but that it is not ubiquitous in E. coli. The gene is typically present as a single copy but up to five copies of agn43 with different combinations of classes can be observed. The presence of agn43 as well as its different classes appeared to differ between Escherichia phylogroups. Strikingly, agn43 is present in 90% of E. coli from E phylogroup. Our results shed light on Ag43 diversity and provide a rational framework for investigating its role in E. coli ecophysiology and physiopathology.

Revisiting the functions of periplasmic chaperones in the quality control of the autotransporter Ag43 using a phenotypically homogeneous Escherichia coli strain.

Antigen 43 is a surface-displayed autotransporter protein that mediates bacterial self-association and pathogenicity. The quality control factors that facilitate Ag43 crossing the periplasm and inserting into the outer membrane remain enigmatic, mostly because Ag43 is phase variable and associated with heterologous phenotypes, which obscures the mutational effects of potential quality control factors. Here, we describe a screening method that allowed us to isolate a subpopulation of Escherichia coli that consistently displays an Ag43-mediated autoaggregation phenotype. Based on this subpopulation, we analyzed how disruptions of known periplasmic chaperones affect Ag43 biogenesis. We found that only the disruption of surA reduced Ag43 levels and abolished the autoaggregation phenotype of cells, but surA disruption did not affect the phase-variable expression of agn43. Using purified proteins, we showed that SurA effectively protected the ß-barrel domain of Ag43 from aggregation. In contrast, the previously reported Ag43 biogenesis factor OsmY showed weak chaperoning effects on Ag43 only in the absence of SurA. Our results shed light on the roles of different periplasmic chaperones in Ag43 biogenesis and provide a methodology applicable to the study of other phase-variable proteins.

A shear stress micromodel of urinary tract infection by the Escherichia coli producing Dr adhesin.

In this study, we established a dynamic micromodel of urinary tract infection to analyze the impact of UT-segment-specific urinary outflow on the persistence of E. coli colonization. We found that the adherence of Dr+ E. coli to bladder T24 transitional cells and type IV collagen is maximal at lowest shear stress and is reduced by any increase in flow velocity. The analyzed adherence was effective in the whole spectrum of physiological shear stress and was almost irreversible over the entire range of generated shear force. Once Dr+ E. coli bound to host cells or collagen, they did not detach even in the presence of elevated shear stress or of chloramphenicol, a competitive inhibitor of binding. Investigating the role of epithelial surface architecture, we showed that the presence of budding cells-a model microarchitectural obstacle-promotes colonization of the urinary tract by E. coli. We report a previously undescribed phenomenon of epithelial cell "rolling-shedding" colonization, in which the detached epithelial cells reattach to the underlying cell line through a layer of adherent Dr+ E. coli. This rolling-shedding colonization progressed continuously due to "refilling" induced by the flow-perturbing obstacle. The shear stress of fluid containing free-floating bacteria fueled the rolling, while providing an uninterrupted supply of new bacteria to be trapped by the rolling cell. The progressive rolling allows for transfer of briefly attached bacteria onto the underlying monolayer in a repeating cascading event.