High-risk Escherichia coli clones that cause neonatal meningitis and association with recrudescent infection.

Neonatal meningitis is a devastating disease associated with high mortality and neurological sequelae. Escherichia coli is the second most common cause of neonatal meningitis in full-term infants (herein NMEC) and the most common cause of meningitis in preterm neonates. Here, we investigated the genomic relatedness of a collection of 58 NMEC isolates spanning 1974-2020 and isolated from seven different geographic regions. We show NMEC are comprised of diverse sequence types (STs), with ST95 (34.5%) and ST1193 (15.5%) the most common. No single virulence gene profile was conserved in all isolates; however, genes encoding fimbrial adhesins, iron acquisition systems, the K1 capsule, and O antigen types O18, O75, and O2 were most prevalent. Antibiotic resistance genes occurred infrequently in our collection. We also monitored the infection dynamics in three patients that suffered recrudescent invasive infection caused by the original infecting isolate despite appropriate antibiotic treatment based on antibiogram profile and resistance genotype. These patients exhibited severe gut dysbiosis. In one patient, the causative NMEC isolate was also detected in the fecal flora at the time of the second infection episode and after treatment. Thus, although antibiotics are the standard of care for NMEC treatment, our data suggest that failure to eliminate the causative NMEC that resides intestinally can lead to the existence of a refractory reservoir that may seed recrudescent infection.

Penicillin binding protein 3 of Staphylococcus aureus NCTC 8325-4 binds and activates human plasminogen.

BACKGROUND: Staphylococcus aureus is a versatile pathogen expressing a number of virulence-associated adhesive molecules. In a previous study, we generated in a secretion-competent Escherichia coli strain a library of random FLAG-tag positive (FTP) polypeptides of S. aureus. To identify adhesive proteins and gain additional knowledge on putative virulence factors of S. aureus, we here screened the FTP library against human serum proteins. FINDINGS: Staphylococcus aureus NCTC 8325-4, origin of the FTP library, adhered to immobilized plasminogen in vitro. In an enzyme-linked immunoassay a C-terminal part of penicillin binding protein 3 (PBP3), included in the FTP library, bound to immobilized plasminogen. We expressed and purified full-length PBP3 and its C-terminal fragments as recombinant proteins. In a time-resolved fluorometry-based assay the PBP3 polypeptides bound to immobilized plasminogen. The polypeptides enhanced formation of plasmin from plasminogen as analyzed by cleavage of a chromogenic plasmin substrate. CONCLUSIONS: The present findings, although preliminary, demonstrate reliably that S. aureus NCTC 8325-4 adheres to immobilized plasminogen in vitro and that the adhesion may be mediated by a C-terminal fragment of the PBP3 protein. The full length PBP3 and the penicillin binding C-terminal domain of PBP3 expressed as recombinant proteins bound plasminogen and activated plasminogen to plasmin. These phenomena were inhibited by the lysine analogue ε-aminocaproic acid suggesting that the binding is mediated by lysine residues. A detailed molecular description of surface molecules enhancing the virulence of S. aureus will aid in understanding of its pathogenicity and help in design of antibacterial drugs in the future.

Fermentation and dry fractionation increase bioactivity of cloudberry (Rubus chamaemorus).

Phenolic composition and bioactivity of cloudberry was modified by bioprocessing, and highly bioactive fractions were produced by dry fractionation of the press cake. During fermentation polymeric ellagitannins were partly degraded into ellagic acid derivatives. Phenolic compounds were differentially distributed in seed coarse and fine fractions after dry fractionation process. Tannins concentrated in fine fraction, and flavonol derivatives were mainly found in coarse fraction. Ellagic acid derivatives were equally distributed between the dry fractions. Fermentation and dry fractionation increased statistically significantly anti-adhesion and anti-inflammatory activity of cloudberry. The seed fine fraction showed significant inhibition of P fimbria-mediated haemagglutination assay of uropathogenic Escherichia coli. The seed coarse fraction significantly reduced NO and IL-6 production and iNOS expression in activated macrophages. Fermentation did not affect antimicrobial activity, but slight increase in activity was detected in dry fractions. The results indicate the potential of cloudberry in pharma or health food applications.

Comparative genomics of Lactobacillus crispatus suggests novel mechanisms for the competitive exclusion of Gardnerella vaginalis.

BACKGROUND: Lactobacillus crispatus is a ubiquitous micro-organism encountered in a wide range of host-associated habitats. It can be recovered from the gastrointestinal tract of animals and it is a common constituent of the vaginal microbiota of humans. Moreover, L. crispatus can contribute to the urogenital health of the host through competitive exclusion and the production of antimicrobial agents. In order to investigate the genetic diversity of this important urogenital species, we performed a comparative genomic analysis of L. crispatus. RESULTS: Utilizing the completed genome sequence of a strain ST1 and the draft genome sequences of nine other L. crispatus isolates, we defined the scale and scope of the pan- and core genomic potential of L. crispatus. Our comparative analysis identified 1,224 and 2,705 ortholog groups present in all or only some of the ten strains, respectively. Based on mathematical modeling, sequencing of additional L. crispatus isolates would result in the identification of new genes and functions, whereas the conserved core of the ten strains was a good representation of the final L. crispatus core genome, estimated to level at about 1,116 ortholog groups. Importantly, the current core was observed to encode bacterial components potentially promoting urogenital health. Using antibody fragments specific for one of the conserved L. crispatus adhesins, we demonstrated that the L. crispatus core proteins have a potential to reduce the ability of Gardnerella vaginalis to adhere to epithelial cells. These findings thereby suggest that L. crispatus core proteins could protect the vagina from G. vaginalis and bacterial vaginosis. CONCLUSIONS: Our pan-genome analysis provides insights into the intraspecific genome variability and the collective molecular mechanisms of the species L. crispatus. Using this approach, we described the differences and similarities between the genomes and identified features likely to be important for urogenital health. Notably, the conserved genetic backbone of L. crispatus accounted for close to 60% of the ortholog groups of an average L. crispatus strain and included factors for the competitive exclusion of G. vaginalis, providing an explanation on how this urogenital species could improve vaginal health.

Cytotoxic response persists in subjects treated for tuberculosis decades ago.

BACKGROUND: Data exploring the potential use of effector molecules produced by cytotoxic T lymphocytes (CTLs) in the immunodiagnostics of tuberculosis (TB) are scarce. The present study focused a) to gain an insight into the discriminatory power of CTLs in patients with acute pulmonary or extra-pulmonary TB, or latent tuberculosis infection (LTBI); and b) to evaluate the influence of various anti-TB therapeutic schemes on the immunological profiles of residual CTLs. METHODS: Immunological signatures of antigen-specific CTLs were explored in patients with active pulmonary and extra-pulmonary TB, LTBI and in those treated for TB decades ago by using ELISPOT, intracellular flow cytometry and extracellular CD107a detection. RESULTS: No difference was seen between active TB, LTBI or any of those treated for TB in the ELISPOT analysis of antigen-specific Granzyme B (GrB), Perforin (Prf) and interferon-gamma (IFN-γ) producing lymphocytes, the FACS analysis of the intracellular expression of IFN-γ, or the surface expression of CD107a degranulation factor of both CD8+ and CD4+ antigen-specific T cell subsets. The effector memory (TEM) phenotype proved predominant in the surface marker profiling both in active TB and LTBI. The proportion of the CD107a degranulation factor proved higher in the central memory (TCM) than in the other cell subsets in all the study groups. Interestingly, functionally and phenotypically similar CTLs profiles were observed in active TB, LTBI and in all the three groups treated for TB. CONCLUSION: The phenotypic and functional profiling of CTLs has a limited potential in the immunodiagnostics of active TB. Antigen-specific CTLs persist in patients treated for TB decades ago regardless of the efficacy of implemented and completed anti-TB therapy.

Fibrinolytic and coagulative activities of Yersinia pestis.

The outer membrane protease Pla belongs to the omptin protease family spread by horizontal gene transfer into Gram-negative bacteria that infect animals or plants. Pla has adapted to support the life style of the plague bacterium Yersinia pestis. Pla has a ß-barrel fold with 10 membrane-spanning ß strands and five surface loops, and the barrel surface contains bound lipopolysaccharide (LPS) that is critical for the conformation and the activity of Pla. The biological activity of Pla is influenced by the structure of the surface loops around the active site groove and by temperature-induced LPS modifications. Several of the putative virulence-related functions documented for Pla in vitro address control of the human hemostatic system, i.e., coagulation and fibrinolysis. Pla activates human plasminogen to the serine protease plasmin and activates the physiological plasminogen activator urokinase. Pla also inactivates the protease inhibitors alpha-2-antiplasmin and plasminogen activator inhibitor 1 (PAI-1) and prevents the activation of thrombin-activatable fibrinolysis inhibitor (TAFI). These functions enhance uncontrolled fibrinolysis which is thought to improve Y. pestis dissemination and survival in the mammalian host, and lowered fibrin(ogen) deposition has indeed been observed in mice infected with Pla-positive Y. pestis. However, Pla also inactivates an anticoagulant, the tissue factor (TF) pathway inhibitor, which should increase fibrin formation and clotting. Thus, Pla and Y. pestis have complex interactions with the hemostatic system. Y. pestis modifies its LPS upon transfer to the mammalian host and we hypothesize that the contrasting biological activities of Pla in coagulation and fibrinolysis are influenced by LPS changes during infection.

Phylogenetic group-associated differences in regulation of the common colonization factor Mat fimbria in Escherichia coli.

Heterogeneity of cell population is a key component behind the evolutionary success of Escherichia coli. The heterogeneity supports species adaptation and mainly results from lateral gene transfer. Adaptation may also involve genomic alterations that affect regulation of conserved genes. Here we analysed regulation of the mat (or ecp) genes that encode a conserved fimbrial adhesin of E. coli. We found that the differential and temperature-sensitive expression control of the mat operon is dependent on mat promoter polymorphism and closely linked to phylogenetic grouping of E. coli. In the mat promoter lineage favouring fimbriae expression, the mat operon-encoded regulator MatA forms a positive feedback loop that overcomes the repression by H-NS and stabilizes the fimbrillin mRNA under low growth temperature, acidic pH or elevated levels of acetate. The study exemplifies phylogenetic group-associated expression of a highly common surface organelle in E. coli.

The role of the bacterial flagellum in adhesion and virulence.

The bacterial flagellum is a complex apparatus assembled of more than 20 different proteins. The flagellar basal body traverses the cell wall, whereas the curved hook connects the basal body to the whip-like flagellar filament that protrudes several µm from the bacterial cell. The flagellum has traditionally been regarded only as a motility organelle, but more recently it has become evident that flagella have a number of other biological functions. The major subunit, flagellin or FliC, of the flagellum plays a well-documented role in innate immunity and as a dominant antigen of the adaptive immune response. Importantly, flagella have also been reported to function as adhesins. Whole flagella have been indicated as significant in bacterial adhesion to and invasion into host cells. In various pathogens, e.g., Escherichia coli, Pseudomonas aeruginosa and Clostridium difficile, flagellin and/or the distally located flagellar cap protein have been reported to function as adhesins. Recently, FliC of Shiga-toxigenic E. coli was shown to be involved in cellular invasion via lipid rafts. Here, we examine the latest or most important findings regarding flagellar adhesive and invasive properties, especially focusing on the flagellum as a potential virulence factor.

The response regulator RcsB activates expression of Mat fimbriae in meningitic Escherichia coli.

The common colonization factor of Escherichia coli, the Mat (also termed ECP) fimbria, functions to advance biofilm formation on inert surfaces as well as bacterial adherence to epithelial cells and subsequent colonization. We used global mini-Tn5 transposon mutagenesis to identify novel regulators of biofilm formation by the meningitic E. coli isolate IHE 3034. Of the 4,418 transformants, we found 17 that were impaired in biofilm formation. Most of these mutants were affected in lipopolysaccharide synthesis and were reduced in growth but not in Mat fimbria expression. In contrast, two mutants grew well but did not express Mat fimbria. The insertions in these two mutants were located at different sites of the rcsB gene, which encodes a DNA-binding response regulator of the Rcs response regulon. The mutations abrogated temperature-dependent biofilm formation by IHE 3034, and the phenotype correlated with loss of mat expression. The defect in biofilm formation in the rcsB mutant was reversed upon complementation with rcsB as well as by overexpression of structural mat genes but not by overexpression of the fimbria-specific activator gene matA. Monitoring of the mat operon promoter activity with chromosomal reporter fusions showed that the RcsB protein and an RcsAB box in the mat regulatory region, but not RcsC, RcsD, AckA, and Pta, are essential for initiation of mat transcription. Gel retardation assays showed that RcsB specifically binds to the mat promoter DNA, which enables its function in promoting biofilm formation by E. coli.

Identification of a high-molecular-mass Lactobacillus epithelium adhesin (LEA) of Lactobacillus crispatus ST1 that binds to stratified squamous epithelium.

Lactobacilli belong to the normal gastrointestinal and genital tract microbiota of human and animal hosts. Adhesion is important for bacterial colonization; however, only a few Lactobacillus adhesins have been identified so far. We studied extracted surface proteins from an adhesive Lactobacillus crispatus strain, ST1, which efficiently colonizes the chicken alimentary tract, for their binding to tissue sections of the chicken crop, and identified a novel high-molecular-mass repetitive surface protein that shows specific binding to stratified squamous epithelium. The adhesin binds to both crop epithelium and epithelial cells from human vagina, and was named Lactobacillus epithelium adhesin (LEA). Expression of LEA is strain-specific among L. crispatus strains and corresponds directly to in vitro bacterial adhesion ability. The partial sequence of the lea gene predicts that the LEA protein carries an N-terminal YSIRK signal sequence and a C-terminal LPxTG anchoring motif, as well as a highly repetitive region harbouring 82 aa long repeats with non-identical sequences that show similarity to Lactobacillus Rib/alpha-like repeats. LEA-mediated epithelial adherence may improve bacterial colonization in the chicken crop and the human vagina, which are the natural environments for L. crispatus.

The fimbriae activator MatA switches off motility in Escherichia coli by repression of the flagellar master operon flhDC.

Flagella provide advantages to Escherichia coli by facilitating taxis towards nutrients and away from unfavourable niches. On the other hand, flagellation is an energy sink to the bacterial cell, and flagella also stimulate host innate inflammatory responses against infecting bacteria. The flagellar assembly pathway is ordered and under a complex regulatory circuit that involves three classes of temporally regulated promoters as well as the flagellar master regulator FlhD(4)C(2). We report here that transcription of the flhDC operon from the class 1 promoter is under negative regulation by MatA, a key activator of the common mat (or ecp) fimbria operon that enhances biofilm formation by E. coli. Ectopic expression of MatA completely precluded motility and flagellar synthesis in the meningitis-associated E. coli isolate IHE 3034. Northern blotting, analysis of chromosomal promoter-lacZ fusions and electrophoretic mobility shift assays revealed an interaction between MatA and the flhDC promoter region that apparently repressed flagellum biosynthesis. However, inactivation of matA in the chromosome of IHE 3034 had only a minor effect on flagellation, which underlines the complexity of regulatory signals that promote flagellation in E. coli. We propose that the opposite regulatory actions of MatA on mat and on flhDC promoters advance the adaptation of E. coli from a planktonic to an adhesive lifestyle.

Adhesive polypeptides of Staphylococcus aureus identified using a novel secretion library technique in Escherichia coli.

BACKGROUND: Bacterial adhesive proteins, called adhesins, are frequently the decisive factor in initiation of a bacterial infection. Characterization of such molecules is crucial for the understanding of bacterial pathogenesis, design of vaccines and development of antibacterial drugs. Because adhesins are frequently difficult to express, their characterization has often been hampered. Alternative expression methods developed for the analysis of adhesins, e.g. surface display techniques, suffer from various drawbacks and reports on high-level extracellular secretion of heterologous proteins in Gram-negative bacteria are scarce. These expression techniques are currently a field of active research. The purpose of the current study was to construct a convenient, new technique for identification of unknown bacterial adhesive polypeptides directly from the growth medium of the Escherichia coli host and to identify novel proteinaceous adhesins of the model organism Staphylococcus aureus. RESULTS: Randomly fragmented chromosomal DNA of S. aureus was cloned into a unique restriction site of our expression vector, which facilitates secretion of foreign FLAG-tagged polypeptides into the growth medium of E. coli ΔfliCΔfliD, to generate a library of 1663 clones expressing FLAG-tagged polypeptides. Sequence and bioinformatics analyses showed that in our example, the library covered approximately 32% of the S. aureus proteome. Polypeptides from the growth medium of the library clones were screened for binding to a selection of S. aureus target molecules and adhesive fragments of known staphylococcal adhesins (e.g coagulase and fibronectin-binding protein A) as well as polypeptides of novel function (e.g. a universal stress protein and phosphoribosylamino-imidazole carboxylase ATPase subunit) were detected. The results were further validated using purified His-tagged recombinant proteins of the corresponding fragments in enzyme-linked immunoassay and surface plasmon resonance analysis. CONCLUSIONS: A new technique for identification of unknown bacterial adhesive polypeptides was constructed. Application of the method on S. aureus allowed us to identify three known adhesins and in addition, five new polypeptides binding to human plasma and extracellular matrix proteins. The method, here used on S. aureus, is convenient due to the use of soluble proteins from the growth medium and can in principle be applied to any bacterial species of interest.

Lingonberry (Vaccinium vitis-idaea) and European cranberry (Vaccinium microcarpon) proanthocyanidins: isolation, identification, and bioactivities.

European, small-fruited cranberries (Vaccinium microcarpon) and lingonberries (Vaccinium vitis-idaea) were characterized for their phenolic compounds and tested for antioxidant, antimicrobial, antiadhesive, and antiinflammatory effects. The main phenolic compounds in both lingonberries and cranberries were proanthocyanidins comprising 63-71% of the total phenolic content, but anthocyanins, hydroxycinnamic acids, hydroxybenzoic acids, and flavonols were also found. Proanthocyanidins are polymeric phenolic compounds consisting mainly of catechin, epicatechin, gallocatechin, and epigallocatechin units. In the present study, proanthocyanidins were divided into three groups: dimers and trimers, oligomers (mDP 4-10), and polymers (mDP > 10). Catechin, epicatechin, A-type dimers and trimers were found to be the terminal units of isolated proanthocyanidin fractions. Inhibitions of lipid oxidation in liposomes were over 70% and in emulsions over 85%, and in most cases the oligomeric or polymeric fraction was the most effective. Polymeric proanthocyanidin extracts of lingonberries and cranberries were strongly antimicrobial against Staphylococcus aureus, whereas they had no effect on other bacterial strains such as Salmonella enterica sv. Typhimurium, Lactobacillus rhamnosus and Escherichia coli. Polymeric fraction of cranberries and oligomeric fractions of both lingonberries and cranberries showed an inhibitory effect on hemagglutination of E. coli, which expresses the M hemagglutin. Cranberry phenolic extract inhibited LPS-induced NO production in a dose-dependent manner, but it had no major effect on iNOS of COX-2 expression. At a concentration of 100 µg/mL cranberry phenolic extract inhibited LPS-induced IL-6, IL-1ß and TNF-α production. Lingonberry phenolics had no significant effect on IL-1ß production but inhibited IL-6 and TNF-α production at a concentration of 100 µg/mL similarly to cranberry phenolic extract. In conclusion the phenolics, notably proanthocyanidins (oligomers and polymers), in both lingonberries and cranberries exert multiple bioactivities that may be exploited in food development.

Molecular adaptation of a plant-bacterium outer membrane protease towards plague virulence factor Pla.

BACKGROUND: Omptins are a family of outer membrane proteases that have spread by horizontal gene transfer in Gram-negative bacteria that infect vertebrates or plants. Despite structural similarity, the molecular functions of omptins differ in a manner that reflects the life style of their host bacteria. To simulate the molecular adaptation of omptins, we applied site-specific mutagenesis to make Epo of the plant pathogenic Erwinia pyrifoliae exhibit virulence-associated functions of its close homolog, the plasminogen activator Pla of Yersinia pestis. We addressed three virulence-associated functions exhibited by Pla, i.e., proteolytic activation of plasminogen, proteolytic degradation of serine protease inhibitors, and invasion into human cells. RESULTS: Pla and Epo expressed in Escherichia coli are both functional endopeptidases and cleave human serine protease inhibitors, but Epo failed to activate plasminogen and to mediate invasion into a human endothelial-like cell line. Swapping of ten amino acid residues at two surface loops of Pla and Epo introduced plasminogen activation capacity in Epo and inactivated the function in Pla. We also compared the structure of Pla and the modeled structure of Epo to analyze the structural variations that could rationalize the different proteolytic activities. Epo-expressing bacteria managed to invade human cells only after all extramembranous residues that differ between Pla and Epo and the first transmembrane ß-strand had been changed. CONCLUSIONS: We describe molecular adaptation of a protease from an environmental setting towards a virulence factor detrimental for humans. Our results stress the evolvability of bacterial ß-barrel surface structures and the environment as a source of progenitor virulence molecules of human pathogens.

Two distinct regions in the model protein Peb1 are critical for its heterologous transport out of Escherichia coli.

BACKGROUND: Escherichia coli is frequently the first-choice host organism in expression of heterologous recombinant proteins in basic research as well as in production of commercial, therapeutic polypeptides. Especially the secretion of proteins into the culture medium of E. coli is advantageous compared to intracellular production due to the ease in recovery of the recombinant protein. Since E. coli naturally is a poor secretor of proteins, a few strategies for optimization of extracellular secretion have been described. We have previously reported efficient secretion of the diagnostically interesting model protein Peb1 of Campylobacter jejuni into the growth medium of Escherichia coli strain MKS12 (ΔfliCfliD). To generate a more detailed understanding of the molecular mechanisms behind this interesting heterologous secretion system with biotechnological implications, we here analyzed further the transport of Peb1 in the E. coli host. RESULTS: When mature Peb1 was expressed without its SecA-YEG-dependent signal sequence and without the putative signal peptidase II recognition sequence in E. coli MKS111ΔHBB lacking the flagellar secretion complex, the protein was found in the periplasm and growth medium which indicated a flagellum-independent translocation. We assessed the Peb1 secretion proficiency by an exhaustive search for transport-affecting regions using a transposition-based scanning mutagenesis strategy. Strikingly, insertion mutagenesis of only two segments, called TAR1 (residues 42 and 43) and TAR2 (residues 173 to 180), prevented Peb1 secretion individually. We confirmed the importance of TAR regions by subsequent site-specific mutagenesis and verified that the secretion deficiency of Peb1 mutants was not due to insolubility or aggregation of the proteins in the cytoplasm. We found by cell fractionation that the mutant proteins were present in the periplasm as well as in the cytoplasm of MKS12. Hence, mutagenesis of TAR regions did not affect export of Peb1 across the cytoplasmic membrane, whereas its export over the outer membrane was markedly impaired. CONCLUSIONS: We propose that the localization of the model protein Peb1 in the growth medium of E. coli is due to active secretion by a still unknown pathway of E. coli. The secretion apparently is a two-step process involving a periplasmic step and the TAR regions.

Rowanberry phenolics: compositional analysis and bioactivities.

Berries contain a large variety of different phenolic compounds such as anthocyanins, flavonols, tannins, and phenolic acids. Due to variation in the nature and content of the phenolic compounds, the antioxidant effect and other bioactivities of berry phenolics are strongly dependent on the berry raw material as the activities differ between the different phenolic constituents. In the present study, wild rowanberries ( Sorbus aucuparia ) and four cultivated sweet rowanberries, Burka, Granatnaja, Titan, and Zoltaja, were characterized for their phenolic composition and screened for antioxidant, antimicrobial, and antiadhesive activities. The HPLC and LC-MS analyses of phenolic composition revealed that the main phenolic constituents were caffeoylquinic acids, varying from 56 to 80% total phenolics. The cultivated species contained less caffeoylquinic acids and more anthocyanins (up to 28.5%). The phenolics derived from wild rowanberries were significantly effective at inhibiting lipid oxidation both in liposomes and in emulsions, especially when assessed by inhibition of the formation of hexanal (86-97% inhibition depending on concentration). The increase in anthocyanin content in the cultivated species did not result in significantly increased antioxidant activity. Both wild and cultivated rowanberry phenolics exhibited a bacteriostatic effect toward Staphylococcus aureus . In addition, the phenolic extract from Zoltaja was weakly inhibitory toward Salmonella sv. Typhimurium, whereas both Zoltaja- and Granatnaja-derived phenolics retarded Escherichia coli growth. The phenolic extracts of wild rowanberries and Burka showed an inhibitory effect on hemagglutination of E. coli HB101 (pRR7), which expresses the M hemagglutinin. It can be concluded that cultivation of rowanberries resulted in increased anthocyanin content, but this did not diminish their bioactivity in comparison to wild rowanberries rich in caffeoylquinic acids.

Mat fimbriae promote biofilm formation by meningitis-associated Escherichia coli.

The mat (or ecp) fimbrial operon is ubiquitous and conserved in Escherichia coli, but its functions remain poorly described. In routine growth media newborn meningitis isolates of E. coli express the meningitis-associated and temperature-regulated (Mat) fimbria, also termed E. coli common pilus (ECP), at 20 degrees C, and here we show that the six-gene (matABCDEF)-encoded Mat fimbria is needed for temperature-dependent biofilm formation on abiotic surfaces. The matBCDEF deletion mutant of meningitis E. coli IHE 3034 was defective in an early stage of biofilm development and consequently unable to establish a detectable biofilm, contrasting with IHE 3034 derivatives deleted for flagella, type 1 fimbriae or S-fimbriae, which retained the wild-type biofilm phenotype. Furthermore, induced production of Mat fimbriae from expression plasmids enabled biofilm-deficient E. coli K-12 cells to form biofilm at 20 degrees C. No biofilm was detected with IHE 3034 or MG1655 strains grown at 37 degrees C. The surface expression of Mat fimbriae and the frequency of Mat-positive cells in the IHE 3034 population from 20 degrees C were high and remained unaltered during the transition from planktonic to biofilm growth and within the matured biofilm community. Considering the prevalence of the highly conserved mat locus in E. coli genomes, we hypothesize that Mat fimbria-mediated biofilm formation is an ancestral characteristic of E. coli.

Genome sequence of Lactobacillus crispatus ST1.

Lactobacillus crispatus is a common member of the beneficial microbiota present in the vertebrate gastrointestinal and human genitourinary tracts. Here, we report the genome sequence of L. crispatus ST1, a chicken isolate displaying strong adherence to vaginal epithelial cells.

The SfaXII protein from newborn meningitis E. coli is involved in regulation of motility and type 1 fimbriae expression.

The genomes of pathogenic Escherichia coli may contain several different fimbrial operons. How bacteria regulate and coordinate the choice of fimbrial expression under different circumstances remains largely unanswered. In this report we have investigated the role of the sfaX(II) gene associated to the Sfa(II) fimbrial determinant in the E. coli isolate IHE3034. sfaX(II) belongs to a subfamily of genes, the 17k Da genes, located near different fimbrial operons in uropathogenic and newborn meningitis E. coli (NMEC) strains. Using the NMEC isolate IHE3034 and non-pathogenic E. coli strains we found that the sfaX(II) gene had an inhibitory effect on type 1 fimbriae expression. Down-regulation of type 1 fimbriae was exerted at transcriptional level both by inhibiting expression from the fimA promoter and by reducing the frequency of OFF-to-ON switching. The effect of sfaX(II) on expression of the recombinase FimB that catalyzes OFF-to-ON switching might explain the described reduction in percentage of ON cells. Moreover, expression of the sfaX(II) gene strongly influenced motility and flagella production of the NMEC isolate IHE3034. We propose that the sfaX(II) gene, and presumably other members in the 17 kDa gene family, may play a role in the control of virulence related gene expression in pathogenic E. coli.

The single substitution I259T, conserved in the plasminogen activator Pla of pandemic Yersinia pestis branches, enhances fibrinolytic activity.

The outer membrane plasminogen activator Pla of Yersinia pestis is a central virulence factor in plague. The primary structure of the Pla beta-barrel is conserved in Y. pestis biovars Antiqua, Medievalis, and Orientalis, which are associated with pandemics of plague. The Pla molecule of the ancestral Y. pestis lineages Microtus and Angola carries the single amino acid change T259I located in surface loop 5 of the beta-barrel. Recombinant Y. pestis KIM D34 or Escherichia coli XL1 expressing Pla T259I was impaired in fibrinolysis and in plasminogen activation. Lack of detectable generation of the catalytic light chain of plasmin and inactivation of plasmin enzymatic activity by the Pla T259I construct indicated that Microtus Pla cleaved the plasminogen molecule more unspecifically than did common Pla. The isoform pattern of the Pla T259I molecule was different from that of the common Pla molecule. Microtus Pla was more efficient than wild-type Pla in alpha(2)-antiplasmin inactivation. Pla of Y. pestis and PgtE of Salmonella enterica have evolved from the same omptin ancestor, and their comparison showed that PgtE was poor in plasminogen activation but exhibited efficient antiprotease inactivation. The substitution (259)IIDKT/TIDKN in PgtE, constructed to mimic the L5 region in Pla, altered proteolysis in favor of plasmin formation, whereas the reverse substitution (259)TIDKN/IIDKT in Pla altered proteolysis in favor of alpha(2)-antiplasmin inactivation. The results suggest that Microtus Pla represents an ancestral form of Pla that has evolved into a more efficient plasminogen activator in the pandemic Y. pestis lineages.