The evolution of group-level pathogenic traits

A group-selection model for the evolutionary origin of phase-variation in E. coli is proposed. Populations of commensal strains of E. coli populating mammalian hosts modulate its immune defenses through population-level control of the expression of fimbriae. At any time only a proportion of the population expresses these cell-surface adhesins. Collectively they elicit a host-based nutrient release if the fimbriae expression is low. Too high levels of fimbriation would provoke an inflammatory response and thus intolerable conditions for the cells. The optimal level of fimbriation is a group property and its evolution is difficult to explain by naive individual selection scenarios. This article presents a computational model to simulate the evolution of fimbriae. The two main conclusions of this contribution are: (i) the evolution of this group property requires the population to be partitioned into weakly interacting sub-populations. (ii) Given certain scenarios evolution consistently under-performs, in the sense that it does not find the optimal level of fimbriation.     

Type 1 fimbriae deliver an LPS- and TLR4-dependent activation signal to CD14-negative cells

Fimbriae target bacteria to different mucosal surfaces and enhance the inflammatory response at these sites. Inflammation may be triggered by the fimbriae themselves or by fimbriae-dependent delivery of other host activating molecules such as lipopolysaccharide (LPS). Although LPS activates systemic inflammation through the CD14 and Toll-like receptor 4 (TLR4) pathways, mechanisms of epithelial cell activation by LPS are not well understood. These cells lack CD14 receptors and are unresponsive to pure LPS, but fimbriated Escherichia coli overcome this refractoriness and trigger epithelial cytokine responses. We now show that type 1 fimbriae can present an LPS- and TLR4-dependent signal to the CD14-negative epithelial cells. Human uroepithelial cells were shown to express TLR4, and type 1 fimbriated E. coli strains triggered an LPS-dependent response in those cells. A similar LPS- and fimbriae-dependent response was observed in the urinary tract of TLR4-proficient mice, but not in TLR4-defective mice. The moderate inflammatory response in the TLR4-defective mice was fimbriae dependent but LPS independent. The results demonstrate that type 1 fimbriae present LPS to CD14-negative cells and that the TLR4 genotype determines this response despite the absence of CD14 on the target cells. The results illustrate how the host "sees" LPS and other microbial products not as purified molecules but as complexes, and that fimbriae determine the molecular context in which LPS is presented to host cells.     

Modeling fimbriae mediated parasite-host interactions

Type 1 fimbriae are a known virulence factor in a number of pathogenic enterobacteriaceae, including Salmonella, Shigella and E. coli. Yet, they are also expressed by some commensal strains, notably of E. coli. One hypothesis of the role of fimbriae in commensals is that they evoke a small but tolerable host immune response in order to have the host release sialic acid, which is a valuable nutrient. Genetic evidence suggests that sialic acid down-regulates fimbriation. This has been believed to enable the cells to reduce virulence when the host response is increasing, thus avoiding a full activation of host defenses. In this article we assess the plausibility of this hypothesis using mathematical models. Our models lead us to two main conclusions: A slight activation of host defenses is only possible with a carefully tuned set of parameters, whereas under a wide range of parameters and assumptions, the model predicts the host defenses to be activated to at least half their potential in response to fimbriation. Secondly, the fact that fimbriation is suppressed by sialic acid seems irrelevant for the global qualitative properties.     

Analysis of immunostimulatory activity of Porphyromonas gingivalis fimbriae conferred by Toll-like receptor 2

Bacterial fimbriae are an important pathogenic factor. It has been demonstrated that fimbrial protein encoded by fimA gene (FimA fimbriae) of Porphyromonas gingivalis not only contributes to the abilities of bacterial adhesion and invasion to host cells, but also strongly stimulates host innate immune responses. However, FimA fimbriae separated from P. gingivalis ATCC 33277 using a gentle procedure showed very weak proinflammatory activity compared with previous reports. Therefore, in the present study, biological characteristics of FimA fimbriae were further analyzed in terms of proinflammatory activity in macrophages. Macrophages differentiated from THP-1 cells were stimulated with native, heat-denatured, or either proteinase- or lipoprotein lipase-treated FimA fimbriae of P. gingivalis ATCC 33277. Stimulating activities of these FimA fimbriae were evaluated by TNF-α-inducing activity in the macrophages. To clarify the mode of action of FimA fimbriae, anti-Toll-like receptor (TLR) 2 blocking antibody was added prior to stimulation. Weak stimulatory activity of native FimA fimbriae was enhanced by heat treatment and low-dose proteinase K treatment. Higher dose of proteinase K treatment abrogated this up-regulation. The activity of treated FimA fimbriae was suppressed by anti-TLR2 antibody, and more substantially by lipoprotein lipase treatment. These results suggest that lipoproteins or lipopeptides associated with FimA fimbriae could at least in part account for signaling via TLR2 and subsequent TNF-α production in macrophages.     

Orientational control is an efficient control mechanism for phase switching in the E. coli fim system

The fim system in E. coli controls the expression of type-1 fimbriae. These are hair-like structures that can be used to attach to host cells. Fimbriation is controlled by a mechanism called "orientational control." We present two families of models for orientational control to understand the details of how it works. We find that the main benefits of orientational control are that (i) it allows rapid adjustment of fimbriation levels in response to a change of environmental conditions while (ii) keeping the overall frequencies with which a cell switches between the fimbriate state and the afimbriate state low. The main reason for the efficiency of orientational control in regulation of fimbriation levels is that it keeps the system far from its steady state.     

Precise and rapid assessment of Escherichia coli adherence to vaginal epithelial cells by flow cytometry

BACKGROUND: In the pathogenesis of Escherichia coli urinary tract infections (UTIs) in women, infecting bacteria adhere to vaginal and periurethral epithelial cells prior to ascending to the bladder and causing infection. Complex interactions among specific bacterial adhesins and various host factors appear to influence adherence of E. coli to mucosal surfaces such as the urogenital epithelium. To conduct population-based studies assessing host epithelial cell determinants that influence bacterial attachment, a method of measuring bacterial adherence utilizing clinically derived epithelial cell samples is needed. METHODS: We developed and standardized an efficient, accurate, high-throughput method for analyzing the adherence of uropathogenic E. coli to clinical samples containing a large number of exfoliated vaginal epithelial cells (VEC). Three wild-type E. coli strains isolated from women with UTI (IA2 expressing pap-encoded, class II fimbriae only; F24 expressing pap-encoded, class II and type 1 fimbriae; and F20, without pap-encoded or type I fimbriae) were transformed with gfpmut3, encoding green fluorescent protein, incubated with VECs, and analyzed by flow cytometry. RESULTS: Enumeration of the binding of each E. coli strain to 10,000 VECs showed reproducible, highly significant strain-dependent differences in adherence to VECs. Differential analysis of the relative contributions of type 1 pili and P fimbrial-mediated binding to the adherence phenotype was performed. It demonstrated that IA2 binding was dependent entirely on P fimbriae, whereas F24 binding was dependent on both P and type 1 fimbriae. CONCLUSIONS: This method has great potential for use in high-throughput analyses of clinically derived epithelial cell samples and will be valuable in population-based investigations of host-parasite interactions in UTI utilizing VECs collected from specific patient groups.     

The role of E. coli adhesiveness in the pathogenesis and clinical course of urinary tract infections]

An infection with E. coli is the most frequent cause of the urinary infections in childhood. Virulence depends on several factors out of which a principal role is played by the adhesion of bacteria to the urinary tract epithelium. Such a property have E. coli strains with adherence mannose-positive fimbriae of type P with antigens recognizing and binding glycolipid receptors on epithelial cells in the urinary tract. Children with such infections owe their "sensitivity+" (10% of the population) to genetically determined large number o receptors binding E. coli strains. Incidence and clinical course of the urinary tract infections have been analysed in the group of 184 children. Moreover, sequelae of the urinary tract infections with E. coli have been analysed in dependence on E. coli strain characteristics, i.e. presence or absence of adherent fimbriae from cases of cystitis and significant asymptomatic bacteriuria. Considering pathogenesis of the urinary tract infections as the result of interactions between bacteria and host, antigenic properties of adherent fimbriae might be used for preparation of a vaccine preventing such infections.     

Fimbrial adhesins: similarities and variations in structure and biogenesis

Abstract Fimbriae are wiry (2 to 4 nm diam.) or rod-shaped (6 to 8 nm diam.), fibre-like structures on the surfaces of bacteria which mediate attachment to host cells. Much has been learned in recent years about the biogenesis, structure and regulation of expression of these adhesive organelles in Gram-negative bacteria. Analyses of the genetic determinants encoding the biogenesis of fimbriae has revealed that the adhesive interaction of fimbriae can be mediated by major subunits (CFA/I and CS1 fimbriae) or minor subunits (P, S, and type 1 fimbriae), with the adhesin being located either at the tip of the fimbria or along the length of the fimbrial shaft. Minor subunits can also act as adapters, anchors, initiators or elongators. Post-translational glycosylation of the type 4 pilins of Neisseria gonorrhoeae, Neisseria meningitidis and Pseudomonas aeruginosa has been demonstrated. The structures of the PapD chaperone of Escherichia coli and of N. gonorrhoeae type 4 fimbrin have been resolved at 2.0–2.6 Å. Rod-shaped fimbriae should not be thought of as being rigid inflexible structures but rather as dynamic structures which can undergo transition from a helicoidal to a fibrillar conformation to provide a degree of elasticity and plasticity to the fimbriae so that they can resist shear forces, rather like a bungee cord. At least four mechanisms have been identified in the assembly of fimbriae from fimbrin subunits, namely the chaperone-usher pathway (e.g., P-fimbriae of uropathogenic E. coli ), the general secretion assembly pathway (e.g., type 4 fimbriae or N -methylphenylalanine fimbriae of P. aeruginosa , the extracellular nucleation-precipitation pathway (e.g., curli of E. coli ) and the CFA/I, CS1 and CS2 fimbrial pathway.     

Expression of a cloned 987P adhesion-antigen fimbrial determinant in Escherichia coli K-12 strain HB101

The properties of three independent enterotoxigenic Escherichia coli isolates known to express 987P adhesion fimbriae in a manner subject to phase variation were examined. Phase variation could not be correlated with any major changes in the plasmid DNA content of these strains or with readily detectable changes in any other tested phenotypic markers. The 987P genetic determinant from one of these strains, E. coli 987, was cloned into the non-fimbriated E. coli K-12 strains HB101, and expressed, using the cosmid vector system. 987P fimbriae produced by cells harbouring these recombinant plasmids (987P+ phenotype) could not be distinguished from 987P fimbriae produced by strain 987. Expression of 987P fimbriae from some recombinant plasmids was unstable but none of the recombinants exhibited the phase variation phenotype displayed by the parental strain. One recombinant plasmid, pPM200, contained an insert of strain 987 DNA of ca. 33 kb. The HB101[pPM200] displayed a rather stable 987P+ phenotype, but this was not true for several hosts, since pPM200 acquired approx. 20-kb deletions following transformations of E. coli K-12 strains other than HB101. The deletions mapped to the same region of pPM200 irrespective of the host strain transformed. Cells harbouring the deleted plasmids did not express 987P fimbriae (987P- phenotype).     

Electron microscopic study of the interaction of nephritogenic strains of Escherichia coli carrying D-mannose-resistant fimbriae with the cells of a human kidney line

The electron-microscopic study revealed that nephritogenic E. coli having L-mannose-resistant fimbriae contacted with subcultured human renal cells due to the interaction of fimbriae with microvilli and, less frequently, with the cytoplasmic membrane surrounding the main part of the cell, as well as with the electron-opaque fibrillar material in the intercellular space. The possibility of very close interaction was demonstrated; in some cases this interaction was so close that the outlines of bacterial and epitheloid cells followed each other, the invagination of the external membrane of the host cell being sometimes observed. The expediency of using ruthenium red for detecting fimbriae in morphological studies was shown. The cytopathogenic effect observed in this study and developing by the end of the 5-hour period from the moment of the inoculation of the monolayer with E. coli was manifested by the swelling of mitochondria accompanied by the partial ruptures of cristae, the widening of channels in the endoplasmic reticulum, the appearance of the secondary lysosomes and the increase of their number.     

Quantitative differences in adhesiveness of type 1 fimbriated Escherichia coli due to structural differences in fimH genes.

Type 1 fimbriae are heteropolymeric surface organelles responsible for the D-mannose-sensitive (MS) adhesion of Escherichia coli. We recently reported that variation of receptor specificity of type 1 fimbriae can result solely from minor alterations in the structure of the gene for the FimH adhesin subunit. To further study the relationship between allelic variation of the fimH gene and adhesive properties of type 1 fimbriae, the fimH genes from five additional strains were cloned and used to complement the FimH deletion in E. coli KB18. When the parental and recombinant strains were tested for adhesion to immobilized mannan, a wide quantitative range in the ability of bacteria to adhere was noted. The differences in adhesion do not appear to be due to differences in the levels of fimbriation or relative levels of incorporation of FimH, because these parameters were similar in low-adhesion and high-adhesion strains. The nucleotide sequence for each of the fimH genes was determined. Analysis of deduced FimH sequences allowed identification of two sequence homology groups, based on the presence of Asn-70 and Ser-78 or Ser-70 and Asn-78 residues. The consensus sequences for each group conferred very low adhesion activity, and this low-adhesion phenotype predominated among a group of 43 fecal isolates. Strains isolated from a different host niche, the urinary tract, expressed type 1 fimbriae that conferred an increased level of adhesion. The results presented here strongly suggest that the quantitative variations in MS adhesion are due primarily to structural differences in the FimH adhesin. The observed differences in MS adhesion among populations of E. coli isolated from different host niches call attention to the possibility that phenotypic variants of FimH may play a functional role in populations dynamics.     

Toxoplasma gondii: chimeric Dr fimbriae as a recombinant vaccine against toxoplasmosis

Toxoplasma gondii is responsible for fetopathy in farm animals and humans and severe disease in immunocompromised individuals (i.e. AIDS patients). Effective vaccines, inducing protective and long-lasting immunity to this global parasite, are still desired. In the work, we evaluated the immunogenic and immunoprotective activity of Escherichia coli chimeric Dr fimbriae bearing selected antigenic epitopes of three T. gondii antigens (SAG1, GRA1 and MAG1), in comparison with conventional recombinant antigens obtained in E. coli expression system. Our data demonstrate a very high protective efficacy of recombinant antigens supplemented with Freund's adjuvants, whereas chimeric Dr fimbriae as a vaccine proved non-protective. The recombinant antigen vaccine induced a strong specific antibody response and prevented the brain cysts formation by 89%. The results are promising and should be confirmed in further study on farm animals by use of less aggressive than Freund's adjuvant preparations.     

Carbohydrates as future anti-adhesion drugs for infectious diseases

Adhesion of pathogenic organisms to host tissues is the prerequisite for the initiation of the majority of infectious diseases. In many systems, it is mediated by lectins present on the surface of the infectious organism that bind to complementary carbohydrates on the surface of the host tissues. Lectin-deficient mutants often lack the ability to initiate infection. The bacterial lectins are typically in the form of elongated submicroscopic multi-subunit protein appendages, known as fimbriae (or pili). The best characterized of these are the mannose-specific type 1 fimbriae, the galabiose-specific P fimbriae and the N-acetylglucosamine-specific fimbriae of Escherichia coli. Soluble carbohydrates recognized by the bacterial surface lectins block the adhesion of the bacteria to animal cells in vitro. Aromatic alpha-mannosides are potent inhibitors of type 1 fimbriated E. coli, being up to 1000 times more active than MealphaMan, with affinities in the nanomolar range. This is due to the presence of a hydrophobic region next to the monosaccharide-binding site of the fimbriae, as recently demonstrated by X-ray studies. Polyvalent saccharides (e.g., neoglycoproteins or dendrimers) are also powerful inhibitors of bacterial adhesion in vitro. Very significantly, lectin-inhibitory saccharides have been shown to protect mice, rabbits, calves and monkeys against experimental infection by lectin-carrying bacteria. Since anti-adhesive agents do not act by killing or arresting the growth of the pathogens, it is very likely that strains resistant to such agents will emerge at a markedly lower rate than of strains that are resistant to antibiotics. Suitable sugars also inhibit the binding to cells of carbohydrate-specific toxins, among them those of Shigella dysenteriae Type 1, and of the homologous Verotoxins of E. coli, specific for galabiose. Appropriately designed polyvalent ligands are up to six orders of magnitude stronger inhibitors of toxin binding in vitro than the monovalent ones, and they protect mice against the Shigella toxin. The above data provide clear proof for the feasibility of anti-adhesion therapy of infectious diseases, although this has not yet been successful in humans. All in all, however, there is little doubt that inhibitors of microbial lectins will in the near future join the arsenal of drugs for therapy of infectious diseases.     

FimH-mediated Escherichia coli K1 invasion of human brain microvascular endothelial cells

Adhesion to brain microvascular endothelial cells, which constitute the blood-brain barrier is considered important in Escherichia coli K1 bacterial penetration into the central nervous system. Type 1 fimbriae are known to mediate bacterial interactions with human brain microvascular endothelial cells (HBMEC). Here, we demonstrate that type 1 fimbriae, specifically FimH adhesin is not only an adhesive organelle that provides bacteria with a foothold on brain endothelial cells but also triggers signalling events that promote E. coli K1 invasion in HBMEC. This is shown by our demonstrations that exogenous FimH increases cytosolic-free-calcium levels as well as activates RhoA. Using purified recombinant mannose-recognition domain of FimH, we identified a glycosylphosphatidylinositol-anchored receptor, CD48, as a putative HBMEC receptor for FimH. Furthermore, E. coli K1 binding to and invasion of HBMEC were blocked by CD48 antibody. Taken together, these findings indicate that FimH induces host cell signalling cascades that are involved in E. coli K1 invasion of HBMEC and CD48 is a putative HBMEC receptor for FimH.     

Characterization of the type 3 fimbriae with different MrkD adhesins: possible role of the MrkD containing an RGD motif

Four novel mrkD alleles namely mrkD(V1), mrkD(V2), mrkD(V3), and mrkD(V4) were identified in seventeen Klebsiella pneumoniae meningitis strains using PCR-RFLP and sequence determination. Comparative analysis revealed a most variable region containing an RGD motif in the receptor domain of MrkD(V3). In order to determine if the sequence confers the K. pneumoniae mrkD(V3) the highest level of the fimbrial activity, a type 3 fimbriae display system was constructed in Escherichia coli. The E. coli JM109[pmrkABCD(V3)F] displaying meshwork-like fimbriae also had the most fimbrial activity, supporting a possible role of the varied sequences. In a dose-dependent manner, the GRGDSP hexapeptide appeared to inhibit the adhesion of the E. coli JM109[pmrkABCD(V3)F] to HCT-8, an ileocecal epithelial cell line. In addition, the adhesion activity was reduced by the addition of anti-alpha5beta1 integrin monoclonal antibody, indicating that the RGD containing region in MrkD(V3) is responsible for the binding of type 3 fimbriae to integrin.     

Chaperone-usher fimbriae in a diverse selection of Gallibacterium genomes

Background

Fimbriae are bacterial cell surface organelles involved in the pathogenesis of many bacterial species, including Gallibacterium anatis, in which a F17-like fimbriae of the chaperone-usher (CU) family was recently shown to be an important virulence factor and vaccine candidate. To reveal the distribution and variability of CU fimbriae 22 genomes of the avian host-restricted bacteria Gallibacterium spp. were investigated. Fimbrial clusters were classified using phylogeny-based and conserved domain (CD) distribution-based approaches. To characterize the fimbriae in depth evolutionary analysis and in vitro expression of the most prevalent fimbrial clusters was performed.

Results

Overall 48 CU fimbriae were identified in the genomes of the examined Gallibacterium isolates. All fimbriae were assigned to γ4 clade of the CU fimbriae of Gram-negative bacteria and were organized in four-gene clusters encoding a putative major fimbrial subunit, a chaperone, an usher and a fimbrial adhesin. Five fimbrial clusters (Flf-Flf4) and eight conserved domain groups were defined to accommodate the identified fimbriae. Although, the number of different fimbrial clusters in individual Gallibacterium genomes was low, there was substantial amino acid sequence variability in the major fimbrial subunit and the adhesin proteins. The distribution of CDs among fimbrial clusters, analysis of their flanking regions, and evolutionary comparison of the strains revealed that Gallibacterium fimbrial clusters likely underwent evolutionary divergence resulting in highly host adapted and antigenically variable fimbriae. In vitro, only the fimbrial subunit FlfA was expressed in most Gallibacterium strains encoding this protein. The absence or scarce expression of the two other common fimbrial subunits (Flf1A and Flf3A) indicates that their expression may require other in vitro or in vivo conditions.

Conclusions

This is the first approach establishing a systematic fimbria classification system within Gallibacterium spp., which indicates a species-wide distribution of γ₄ CU fimbriae among a diverse collection of Gallibacterium isolates. The expression of only one out of up to three fimbriae present in the individual genomes in vitro suggests that fimbriae expression in Gallibacterium is highly regulated. This information is important for future attempts to understand the role of Gallibacterium fimbriae in pathogenesis, and may prove useful for improved control of Gallibacterium infections in chickens.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1093) contains supplementary material, which is available to authorized users.     

Escherichia coli P fimbriae utilize the Toll-like receptor 4 pathway for cell activation

Fimbriae mediate bacterial attachment to host cells and provide a mechanism for tissue attack. They activate a host response by delivery of microbial products such as lipopolysaccharide (LPS) or through direct fimbriae-dependent signalling mechanisms. By coupling to glycosphingolipid (GSL) receptors, P fimbriae trigger cytokine responses in CD14 negative host cells. Here we show that P fimbriae utilize the Toll-like receptor 4 (TLR4)-dependent pathway to trigger mucosal inflammation. Escherichia coli strains expressing P fimbriae as their only virulence factor stimulated chemokine and neutrophil responses in the urinary tract of TLR4 proficient mice, but TLR4 defective mice failed to respond to infection. Mucosal cells were CD14 negative but expressed several TLR species including TLR4, and TLR4 protein was detected. Infection with P fimbriated bacteria stimulated an increase in TLR4 mRNA levels. The activation signal did not involve the LPS-CD14 pathway and was independent of lipid A myristoylation, as shown by mutational inactivation of the msbB gene. Co-staining experiments revealed that TLR4 and the GSL receptors for P fimbriae co-localized in the cell membrane. The results demonstrate that P fimbriae activate epithelial cells by means of a TLR4-dependent signalling pathway, and suggest that GSL receptors for P fimbriae can recruit TLR4 as co-receptors.     

Cloning and characterization of the gene cluster encoding type 3 (MR/K) fimbriae of Klebsiella pneumoniae

Abstract The gene cluster encoding the type 3 fimbriae of a Klebsiella pneumoniae isolate was cloned using the cosmid-cloning technique. Escherichia coli transformants, expressing type 3 fimbriae, were selected by reactivity with a monoclonal antibody directed against an epitope of the purified type 3 fimbriae. The phenotypic expression of type 3 fimbriae by transformants possessing the parental plasmid was dependent upon the host strain used. However, subcloning of this plasmid resulted in the construction of a chimeric molecule which imparted a stable phenotype regardless of the host strain. In addition, subcloning of the parental recombinant plasmid suggested that the minimal size of DNA necessary for production and expression of fimbriae was approximately 5.5 kb.     

Fragmentation of Escherichia coli type 1 fimbriae exposes cryptic D-mannose-binding sites.

Cells of the gram-negative bacterium Escherichia coli are able to attach to various host cells by means of a mannose-specific adhesin associated with type 1 fimbriae. Here we show that fragmentation of type 1 fimbriae by freezing and thawing results in increased mannose-binding activity as demonstrated by increased hemagglutination, increased stimulation of human lymphocyte proliferation, and increased binding of the mannose-containing enzyme horseradish peroxidase. Increased activity in all three assays was mannose sensitive and was not exhibited by FimH- mutant type 1 fimbriae lacking the adhesin. Scatchard analysis of the data from peroxidase binding assays showed that unfrozen and frozen fimbriae contain binding sites displaying two classes of affinity. Frozen and thawed fimbriae expressed an increase in the number of high-affinity binding sites. These results show that fragmentation of the fimbrial structure exposes cryptic mannose-binding activity associated with type 1 fimbriae, presumably that of internally located adhesin molecules. Our data support earlier observations that adhesin moieties of type 1 fimbriae are located both at the tips and at intervals along the length of the fimbriae. In addition, our data suggest that only the adhesin moieties that are located at the fimbrial tips are functional in binding mannose. Adhesins located along the length of the fimbriae have their mannose-binding activity buried within the fimbrial structure and hence are not functional. We propose an updated model for the structure of type 1 fimbriae that is in agreement with the above observations.