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PubMedDate not supplied

Type 1 fimbriae of Escherichia coli: genetic regulation, morphogenesis, and role in pathogenesis.

Over the last several years a number of aspects related to the molecular biology of the type 1 fimbriae of Escherichia coli have been investigated with use of a variety of approaches, including molecular genetics, electron microscopy, and cell biology. With the help of these analytic tools, new insights have emerged. The on-and-off genetic regulation of expression, referred to as phase variation, has been found to be regulated at the level of transcription by the inversion of a 314-base-pair segment of DNA. Morphogenesis of the organelles, which depends on the assembly of processed subunits of 17,000-dalton mass, has been found to proceed from the addition of new subunits at the base, rather than at the tip, of the organelle. Although the precise role of type 1 fimbriae in the pathogenesis of E. coli infections is still somewhat obscure, new data indicate that they significantly potentiate the uptake of nutrients from and the delivery of toxins to eukaryotic cells.

Animals↗
PubMed2001

Coordinate gene regulation by fimbriae-induced signal transduction.

Fimbriae are thread-like polymers displayed in large amounts on the bacterial surface and used by many pathogens to attach to receptors on host tissue surfaces. Fimbriae contain disulfide bridges, contrary to many Escherichia coli surface proteins produced in bulk amounts. Here we investigate whether fimbriae expression can affect expression of other genes. Analysis of gene expression in two E.coli strains, differing in the fim locus, indicated the flu gene to be affected. The flu gene encodes the antigen 43 (Ag43) surface protein, specifically involved in bacterial aggregation, and microcolony and biofilm formation. Ag43 production is repressed by the global regulator OxyR, which monitors the cell's thiol-disulfide status. Only the thiol form of OxyR represses Ag43 production. We demonstrate that production of several different disulfide-containing fimbriae results in the abolition of Ag43 production. No effect was observed in an oxyR mutant. We conclude that fimbriae expression per se constitutes a signal transduction mechanism that affects a number of unrelated genes via the thiol-disulfide status of OxyR. Thus, phase variation in fimbrial expression is coordinated with the expression of other disease- and colonization-related genes.

Adhesins, Bacterial↗
PubMed2001

Cpx signaling pathway monitors biogenesis and affects assembly and expression of P pili.

P pili are important virulence factors in uropathogenic Escherichia coli. The Cpx two-component signal transduction system controls a stress response and is activated by misfolded proteins in the periplasm. We have discovered new functions for the Cpx pathway, indicating that it may play a critical role in pathogenesis. P pili are assembled via the chaperone/usher pathway. Subunits that go 'OFF-pathway' during pilus biogenesis generate a signal. This signal is derived from the misfolding and aggregation of subunits that failed to come into contact with the chaperone in the periplasm. In response, Cpx not only controls the stress response, but also controls genes necessary for pilus biogenesis, and is involved in regulating the phase variation of pap expression and, potentially, the expression of a panoply of other virulence factors. This study demonstrates how the prototypic chaperone/usher pathway is intricately linked and dependent upon a signal transduction system.

Bacterial Proteins↗
PubMed2002

Mutations in polI but not mutSLH destabilize Haemophilus influenzae tetranucleotide repeats.

Haemophilus influenzae (Hi), an obligate upper respiratory tract commensal/pathogen, uses phase variation (PV) to adapt to host environment changes. Switching occurs by slippage of nucleotide repeats (microsatellites) within genes coding for virulence molecules. Most such microsatellites in Hi are tetranucleotide repeats, but an exception is the dinucleotide repeats in the pilin locus. To investigate the effects on PV rates of mutations in genes for mismatch repair (MMR), insertion/deletion mutations of mutS, mutL, mutH, dam, polI, uvrD, mfd and recA were constructed in Hi strain Rd. Only inactivation of polI destabilized tetranucleotide (5'AGTC) repeat tracts of chromosomally located reporter constructs, whereas inactivation of mutS, but not polI, destabilized dinucleotide (5'AT) repeats. Deletions of repeats were predominant in polI mutants, which we propose are due to end-joining occurring without DNA polymerization during polI-deficient Okazaki fragment processing. The high prevalence of tetranucleotides mediating PV is an exceptional feature of the Hi genome. The refractoriness to MMR of hypermutation in Hi tetranucleotides facilitates adaptive switching without the deleterious increase in global mutation rates that accompanies a mutator genotype.

Adenosine Triphosphatases↗
PubMed1983

The role of pili and capsule in the pathogenesis of neonatal infection with Escherichia coli K1.

The role of pili and capsule was studied in neonatal infection with Escherichia coli K1. E coli strains were selectively cultured into three phases: mannose-sensitive (MS) piliated, non-mannose-sensitive (NMS) piliated, and nonpiliated. A high percentage of neonatal rats fed each phase of K1 strains developed bacteremia; there was no bacteremia with non-K1 strains or an acapsular mutant of K1 strain C94 (C94K-). Oral cavity colonization was noted in nearly 100% of rats fed K1 strains, non-K1 strains, or C94K-, regardless of the phase of piliation at feeding. Only MS piliated bacteria were found on oral cavity culture, indicating a rapid shift of NMS piliated and nonpiliated bacteria to the MS piliated phase. Conversely, only nonpiliated bacteria were found on blood culture when neonatal rats were fed piliated bacteria. Colonization of ileal epithelium was not observed. Thus, in vivo phase variation may be important in colonization and bacteremia with E coli K1.

Adhesiveness↗
PubMed1987

Expression of P, type-1, and type-1C fimbriae of Escherichia coli in the urine of patients with acute urinary tract infection.

In vivo expression, in human urine, of P, type-1, and type-1C fimbriae by Escherichia coli was analyzed by indirect immunofluorescence. Urine samples from 20 patients with acute urinary tract infection, as well as cultures of the corresponding E. coli isolates, were immunostained with three polyclonal antisera to P fimbriae, antiserum to type-1 fimbriae, monoclonal antibody specific for type-1C fimbriae, and fluorochrome-conjugated second antibodies. P fimbriae were found in 17 urine samples and in 18 of the isolated strains. Type-1-fimbriate bacteria were detected in only nine urine samples, although 18 of the isolated strains expressed type-1 fimbriae after growth in vitro. Four strains possessed type-1C fimbriae; only two expressed type-1C fimbriae in urine. The bacterial populations in urine were heterogeneous, and in each positive staining, only a fraction of the bacterial cells were reactive. The results show that E. coli P fimbriae are expressed and are subject to phase variation in vivo during acute urinary tract infection.

Acute Disease↗
PubMed1994

Modulation of Escherichia coli type 1 fimbrial expression and adherence to uroepithelial cells following exposure of logarithmic phase cells to quinolones at subinhibitory concentrations.

Quinolone antibiotics at sub-inhibitory concentrations have been shown to antagonize the adherence of Escherichia coli to urinary tract epithelium. This may be due either to reduced expression or to alterations to the structure of the fimbriae which mediate adherence. While E. coli cells in the stationary growth phase have previously been used to investigate quinolone-induced inhibition of adherence, the present study has demonstrated that bacteria in the logarithmic phase also produce type 1 fimbriae and that the adherence of these organisms is reduced following exposure to various quinolones. In all experiments, cells in the logarithmic phase were incubated for 3 h in the presence of ciprofloxacin, enoxacin, CI-960 or PD131628 at a concentration equivalent to 0.5 x MIC. An in-vitro adherence assay which used acid-washed uroepithelial cells and a type 1-fimbriated strain of E. coli showed reductions in adherence of 47%, 72% and 95% after exposure to enoxacin, ciprofloxacin and PD131628, respectively. The effects of ciprofloxacin, enoxacin, CI-960 and PD131628 on two phase variation controlling genes, fimB and fimE, and the main structural gene, fimA, were evaluated by quantifying beta-galactosidase production encoded by chromosomally-located fim::lacZ fusions. All four quinolones tested caused reductions in beta-galactosidase production by a fimA::lacZ fusion strain, but did not significantly affect production of this enzyme by fimB::lacZ and fimE::lacZ fusion strains; these agents also led to decreases in wild-type beta-galactosidase production. Amplification of the invertible element after exposure to enoxacin at 0.25, 0.5 or 1 x MIC revealed no changes in orientation distribution compared with the antibiotic-free control. In addition, a fluorescence assay specific for type 1 fimbriae showed only 23%, 21%, 25% and 11% reductions in the surface expression of the structural subunit after incubation in the presence of ciprofloxacin, enoxacin, CI-960 and PD131628, respectively, at 0.5 x MIC.

4-Quinolones↗
PubMed2006

Antimicrobial susceptibility of Bartonella henselae using Etest methodology.

OBJECTIVES: Bartonella henselae is a fastidious slow growing pathogen which is seldom cultured in the laboratory. Previous descriptions of antimicrobial susceptibility have been largely limited to feline isolates and/or laboratory reference strains, with no accounting for genotypic or phenotypic diversity. METHODS: An optimal method of antimicrobial susceptibility testing by Etest was established to compare the antimicrobial susceptibilities of 12 different isolates of B. henselae, 5 human and 7 feline, which have previously been well characterized by 16S rRNA sequencing, multi-locus sequence typing (MLST), phase variation and passage number. RESULTS: No difference in susceptibility could be attributed to differences in genotype, source of the isolate or passage number. Where comparisons were drawn with previously published results, these were found to be concordant. CONCLUSIONS: We conclude that antibiotic susceptibility can be determined by a simple Etest method for B. henselae isolates. This method is reproducible among diverse strains, and is sufficiently predictable that generalizations can be confidently made about optimal antibiotic choices.

Angiomatosis, Bacillary↗
PubMed2005

Destabilization of tetranucleotide repeats in Haemophilus influenzae mutants lacking RnaseHI or the Klenow domain of PolI.

A feature of Haemophilus influenzae genomes is the presence of several loci containing tracts of six or more identical tetranucleotide repeat units. These repeat tracts are unstable and mediate high frequency, reversible alterations in the expression of surface antigens. This process, termed phase variation (PV), enables H.influenzae to rapidly adapt to fluctuations in the host environment. Perturbation of lagging strand DNA synthesis is known to destabilize simple sequence repeats in yeast and Escherichia coli. By using a chromosomally located reporter construct, we demonstrated that the mutation of an H.influenzae rnhA (encoding RnaseHI) homologue increases the mutation rates of tetranucleotide repeats approximately 3-fold. Additionally, deletion of the Klenow domain of DNA polymerase I (PolI) resulted in a approximately 35-fold increase in tetranucleotide repeat-mediated PV rates. Deletion of the PolI 5'>3' exonuclease domain appears to be lethal. The phenotypes of these mutants suggest that delayed or mutagenic Okazaki fragment processing destabilizes H.influenzae tetranucleotide repeat tracts.

Cell Proliferation↗
PubMed2000

Bacterial otitis media: pathogenetic considerations.

A variety of exciting and important new observations regarding the pathogenesis of nontypable H. influenzae infection have been made in the past decade. The interactions between mucin and OMPs show a high degree of specificity. Multiple adhesins have been identified on the bacterial surface. Colonization of the upper respiratory tract is a dynamic process. Immunodominant, antigenically heterogeneous OMPs are the targets of strain-specific immune responses, accounting in part for the recurrent nature of OM in otitis-prone children. The LOS of nontypable H. influenzae displays a remarkable degree of antigenic and phase variation and may be involved in molecular mimicry of host antigens. Finally nontypable H. influenzae not only lives on the mucosal surface but also clearly has been demonstrated to enter epithelial cells and remain viable in intracellular and intercellular locations in the human upper respiratory tract. These areas of investigation have important implications in understanding the pathogenesis of OM. Elucidating mechanisms of pathogenesis will be important in guiding development of novel ways to prevent OM.

Child↗
PubMed1997

Characterization of smooth and rough morphotypes of Peptostreptococcus micros.

Isolation of the smooth (Sm) morphotype of Peptostreptococcus micros, a suspected oral pathogen, is sometimes accompanied by isolation of a rough (Rg) morphotype of P. micros. The Rg type readily changes to a Sm-like variant (RgSm) in broth culture. Sm and Rg isolates and RgSm variants were compared to determine whether these three types are the result of phase variation. The RgSm variants resembled the Sm morphotype in colony morphology; furthermore, the Sm type and the RgSm type did not have the fibrillar surface structures characteristic of the Rg type, and the Sm and RgSm types were more hydrophobic than the Rg type. However, when we compared the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of whole-cell proteins, serotyping data, pyrolysis mass spectrometry data, 16S ribosomal DNA sequences, and hemolytic activities, the RgSm variants and the Rg isolates were very similar and were clearly distinct from the Sm isolates. These results suggest that the Rg and RgSm types form a cluster distinct from the Sm type and thus provide evidence that P. micros can be differentiated into two groups, one consisting of the Sm type and the other consisting of the Rg and RgSm types.

Base Sequence↗
PubMed1985

Genomic analysis of phase I and II Coxiella burnetii with restriction endonucleases.

Restriction endonuclease-digested DNAs from several isolates of phase I and phase II Coxiella burnetii were compared using agarose gel electrophoresis and soft-laser scanning densitometry. Our results demonstrate that the two phases are, as previously assumed, alternative phases of the same organism. Although the restriction endonuclease digestion revealed genetic differences between clonal isolates of phase I and phase II C. burnetii Nine Mile strain, these differences do not appear to be related to antigenic phase variation. However, analyses of the fragment patterns generated by restriction enzyme digestion suggest potential grouping of the different isolates.

Coxiella↗
PubMed2001

Comparative whole-genome analyses reveal over 100 putative phase-variable genes in the pathogenic Neisseria spp.

Previously, a complete genome analysis of Neisseria meningitidis strain MC58 revealed the largest repertoire of putative phase-variable genes described in any species to date. Initial comparisons with two incomplete Neisseria spp. genome sequences available at that time revealed differences in the repeats associated with these genes in the form of polymorphisms, the absence of the potentially unstable elements in some alleles, and in the repertoire of the genes that were present. Analyses of the complete genomes of N. meningitidis strain Z2491 and Neisseria gonorrhoeae strain FA1090 have been performed and are combined with a comprehensive comparative analysis between the three available complete genome sequences. This has increased the sensitivity of these searches and provided additional contextual information that facilitates the interpretation of the functional consequences of repeat instability. This analysis identified: (i) 68 phase-variable gene candidates in N. meningitidis strain Z2491, rather than the 27 previously reported; (ii) 83 candidates in N. gonorrhoeae strain FA1090; and (iii) 82 candidates in N. meningitidis strain MC58, including an additional 19 identified through cross-comparisons with the other two strains. In addition to the 18 members of the opa gene family, a repertoire of 119 putative phase-variable genes is described, indicating a huge potential for diversification mediated by this mechanism of gene switching in these species that is central to their interactions with the host and environmental transitions. Eighty-two of these are either known (14) or strong (68) candidates for phase variation, which together with the opa genes make a total of 100 identified genes. The repertoires of the genes identified in this analysis diverge from the different species groupings, indicating horizontal exchange that significantly affects the species and strain complements of these genes.

Bacterial Proteins↗
PubMed1991

Virulence of transparent and opaque colony types of Neisseria gonorrhoeae for the genital tract of mice.

The virulence of transparent (Tr) and opaque (Op) colony types of Neisseria gonorrhoeae in the genital tract of female mice was evaluated at two stages of oestrous. Isogenic pairs of Tr and Op variants were isolated from N. gonorrhoeae strain 57-120. Both variants exhibited a T2 morphology, but only the Op variant possessed protein II (P.II) in outer-membrane fractions. When administered by intravaginal inoculation Op gonococci were highly infective only for mice in late pro-oestrous, whereas Tr gonococci were virulent for mice at both late pro-oestrous and dioestrous. Gonococci recovered from the uterus were of both Tr and Op phenotypes in equal proportions when mice were infected at dioestrous with Tr cells. In contrast, greater than 90% of recovered colonies were of Op phenotype when mice were infected at late pro-oestrous with either Op or Tr cells. These results indicate that the virulence of gonococci for the genital tract of female mice differs from that for the chicken embryo. Furthermore, gonococcal survival in the female genital tract might be attributable to phase variation from Tr to Op phenotypes.

Animals↗
PubMed1975

Cell-envelope proteins of Bordetella pertussis.

Cell-envelope polypeptides of eight phase-I and five phase-IV strains of Bordetella pertussis were compared by SDS-polyacrylamide gel electrophoresis. All phase-I strains gave a strikingly similar but complex pattern of protein bands, which did not appear to vary with known differences in heat-labile agglutinogens. Phase-IV strains gave the same pattern as phase-I strains, except that one band was missing and another was either much reduced or absent. Envelopes from phase-I strains grown in Hornibrook medium rich in Mg-2+ ions to produce "antigenically-modulated" C-mode cells gave a pattern of bands indistinguishable from phase-IV strains. A phase-IV strain grown in the high-Mg-2+ medium gave the same pattern of bands as when grown in unmodified Hornibrook medium. We suggest that the two polypeptide bands that show changes may be responsible for one or more of the immunological or physiopathological activities that are lost during phase variation and antigenic modulation in B. pertussis.

Allergens↗
PubMed1995

Reversible expression of motility and flagella in Clostridium chauvoei and their relationship to virulence.

Clostridium chauvoei strain Okinawa produced spontaneous non-motile variants at an unusually high rate (approx. 10(-4) per generation) under normal conditions without mutagen. Revertants of non-motile variants were detected at a rate of approximately 10(-3). Biochemically, every variant corresponded well with the parental strain. By transmission electron microscopy, three of nine non-motile variants of strain Okinawa were found to be flagellate, while the other six were found to be aflagellate. These phenotypes were confirmed by Western blot analysis using monoclonal antibodies directed against the flagella of C. chauvoei. Moreover, the parental flagellate strain and non-motile flagellate variants were significantly more virulent in mice than non-motile, aflagellate variants. Our results demonstrated that phase variation in motility and flagellation occurs in C. chauvoei, and that the flagella are associated with the full expression of virulence.

Animals↗
PubMed2005

Regulation of type 1 fimbriae synthesis and biofilm formation by the transcriptional regulator LrhA of Escherichia coli.

Type 1 fimbriae of Escherichia coli facilitate attachment to the host mucosa and promote biofilm formation on abiotic surfaces. The transcriptional regulator LrhA, which is known as a repressor of flagellar, motility and chemotaxis genes, regulates biofilm formation and expression of type 1 fimbriae. Whole-genome expression profiling revealed that inactivation of lrhA results in an increased expression of structural components of type 1 fimbriae. In vitro, LrhA bound to the promoter regions of the two fim recombinases (FimB and FimE) that catalyse the inversion of the fimA promoter, and to the invertible element itself. Translational lacZ fusions with these genes and quantification of fimE transcript levels by real-time PCR showed that LrhA influences type 1 fimbrial phase variation, primarily via activation of FimE, which is required for the ON-to-OFF transition of the fim switch. Enhanced type 1 fimbrial expression as a result of lrhA disruption was confirmed by mannose-sensitive agglutination of yeast cells. Biofilm formation was stimulated by lrhA inactivation and completely suppressed upon LrhA overproduction. The effects of LrhA on biofilm formation were exerted via the changed levels of surface molecules, most probably both flagella and type 1 fimbriae. Together, the data show a role for LrhA as a repressor of type 1 fimbrial expression, and thus as a regulator of the initial stages of biofilm development and, presumably, bacterial adherence to epithelial host cells also.

Animals↗
PubMed2004

Propagation in and scattering from a matched metamaterial having a zero index of refraction.

Planar metamaterials that exhibit a zero index of refraction have been realized experimentally by several research groups. Their existence stimulated the present investigation, which details the properties of a passive, dispersive metamaterial that is matched to free space and has an index of refraction equal to zero. Thus, unlike previous zero-index investigations, both the permittivity and permeability are zero here at a specified frequency. One-, two-, and three-dimensional source problems are treated analytically. The one- and two-dimensional source problem results are confirmed numerically with finite difference time domain (FDTD) simulations. The FDTD simulator is also used to treat the corresponding one- and two-dimensional scattering problems. It is shown that in both the source and scattering configurations the electromagnetic fields in a matched zero-index medium take on a static character in space, yet remain dynamic in time, in such a manner that the underlying physics remains associated with propagating fields. Zero phase variation at various points in the zero-index medium is demonstrated once steady-state conditions are obtained. These behaviors are used to illustrate why a zero-index metamaterial, such as a zero-index electromagnetic band-gap structured medium, significantly narrows the far-field pattern associated with an antenna located within it. They are also used to show how a matched zero-index slab could be used to transform curved wave fronts into planar ones.

Journal Article↗