Comparative sequence analysis of the plasmid-encoded regulator of enteropathogenic Escherichia coli Strains.

Enteropathogenic Escherichia coli (EPEC) strains that carry the EPEC adherence factor (EAF) plasmid were screened for the presence of different EAF sequences, including those of the plasmid-encoded regulator (per). Considerable variation in gene content of EAF plasmids from different strains was seen. However, bfpA, the gene encoding the structural subunit for the bundle-forming pilus, bundlin, and per genes were found in 96.8% of strains. Sequence analysis of the per operon and its promoter region from 15 representative strains revealed that it is highly conserved. Most of the variation occurs in the 5' two-thirds of the perA gene. In contrast, the C-terminal portion of the predicted PerA protein that contains the DNA-binding helix-turn-helix motif is 100% conserved in all strains that possess a full-length gene. In a minority of strains including the O119:H2 and canine isolates and in a subset of O128:H2 and O142:H6 strains, frameshift mutations in perA leading to premature truncation and consequent inactivation of the gene were identified. Cloned perA, -B, and -C genes from these strains, unlike those from strains with a functional operon, failed to activate the LEE1 operon and bfpA transcriptional fusions or to complement a per mutant in reference strain E2348/69. Furthermore, O119, O128, and canine strains that carry inactive per operons were deficient in virulence protein expression. The context in which the perABC operon occurs on the EAF plasmid varies. The sequence upstream of the per promoter region in EPEC reference strains E2348/69 and B171-8 was present in strains belonging to most serogroups. In a subset of O119:H2, O128:H2, and O142:H6 strains and in the canine isolate, this sequence was replaced by an IS1294-homologous sequence.

EspG, a novel type III system-secreted protein from enteropathogenic Escherichia coli with similarities to VirA of Shigella flexneri.

The function of the rorf2 gene located on the locus of enterocyte effacement (LEE) pathogenicity island of enteropathogenic Escherichia coli (EPEC) has not been described. We report that rorf2 encodes a novel protein, named EspG, which is secreted by the type III secretory system and which is translocated into host epithelial cells. EspG is homologous with Shigella flexneri protein VirA, and the cloned espG (rorf2) gene can rescue invasion in a Shigella virA mutant, indicating that these proteins are functionally equivalent in Shigella. An EPEC espG mutant had no apparent defects in in vitro assays of virulence phenotypes, but a rabbit diarrheagenic E. coli strain carrying a mutant espG showed diminished intestinal colonization and yet diarrheal attack rates similar to those of the wild type. A second EspG homolog, Orf3, is encoded on the EspC pathogenicity islet. The cloned orf3 gene could also rescue invasion in a Shigella virA mutant, but an EPEC espG orf3 double mutant was not diminished in any tested in vitro assays for EPEC virulence factors. Our results indicate that EspG plays an accessory but as yet undefined role in EPEC virulence that may involve intestinal colonization.

espC pathogenicity island of enteropathogenic Escherichia coli encodes an enterotoxin.

At least five proteins are secreted extracellularly by enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea in developing countries. However only one, EspC, is known to be secreted independently of the type III secretion apparatus encoded by genes located within the 35.6-kb locus of enterocyte effacement pathogenicity island. EspC is a member of the autotransporter family of proteins, and the secreted portion of the molecule is 110 kDa. Here we determine that the espC gene is located within a second EPEC pathogenicity island at 60 min on the chromosome of E. coli. We also show that EspC is an enterotoxin, indicated by rises in short-circuit current and potential difference in rat jejunal tissue mounted in Ussing chambers. In addition, preincubation with antiserum against the homologous Pet enterotoxin of enteroaggregative E. coli eliminated EspC enterotoxin activity. Like the EAF plasmid, the espC pathogenicity island was found only in a subset of EPEC, suggesting that EspC may play a role as an accessory virulence factor in some but not all EPEC strains.

Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by Ler.

Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing lesions (AE) on epithelial cells. The genes involved in the formation of the AE lesions are contained within a pathogenicity island named the locus of enterocyte effacement (LEE). The LEE comprises 41 open reading frames organized in five major operons: LEE1, LEE2, LEE3, LEE4 and tir. The first gene of the LEE1 operon encodes a transcription activator of the other LEE operons that is called the LEE-encoded regulator (Ler). The LEE2 and LEE3 operons are divergently transcribed with overlapping -10 promoter regions, and gene fusion studies have shown that they are both activated by Ler. Deletion analysis, using lacZ reporter fusions, of the LEE2 and LEE3 promoters demonstrated that deletions extending closer to the LEE2 transcription start site than -247 bp lead to loss of activation by Ler, whereas only 70 bp upstream of the LEE3 transcription start site is required for Ler-mediated activation. We have purified Ler as a His-tagged protein and used it to perform DNA-binding assays with LEE2 and LEE3. We observed that Ler bound to a DNA fragment containing the -300 to +1 region of LEE2; however, it failed to bind to a DNA fragment containing the -300 to +1 region of LEE3, suggesting that Ler activates both operons by only binding to the regulatory region upstream of LEE2. The Ler-activatable LEE3:lacZ fusions extended to what would be -246 bp of the LEE2 operon. A lacZ fusion from the -300 to +1 region of LEE3 failed to be activated by Ler, consistent with our hypothesis that Ler activates the expression of LEE2 and LEE3 by binding to a region located downstream of the LEE3 transcription start site. DNase I footprinting revealed that Ler protected a region of 121 bp upstream of LEE2. Purified Ler mutated in the coiled-coil domain was unable to activate transcription and to bind to the LEE2 regulatory region. These data indicate that Ler may bind as a multimer to LEE2 and activate both divergent operons by a novel mechanism potentially involving changes in the DNA structure.

The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli.

Regulation of virulence gene expression in enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is incompletely understood. In EPEC, the plasmid-encoded regulator Per is required for maximal expression of proteins encoded on the locus of enterocyte effacement (LEE), and a LEE-encoded regulator (Ler) is part of the Per-mediated regulatory cascade upregulating the LEE2, LEE3, and LEE4 promoters. We now report that Ler is essential for the expression of multiple LEE-located genes in both EPEC and EHEC, including those encoding the type III secretion pathway, the secreted Esp proteins, Tir, and intimin. Ler is therefore central to the process of attaching and effacing (AE) lesion formation. Ler also regulates the expression of LEE-located genes not required for AE-lesion formation, including rorf2, orf10, rorf10, orf19, and espF, indicating that Ler regulates additional virulence properties. In addition, Ler regulates the expression of proteins encoded outside the LEE that are not essential for AE lesion formation, including TagA in EHEC and EspC in EPEC. delta ler mutants of both EPEC and EHEC show altered adherence to epithelial cells and express novel fimbriae. Ler is therefore a global regulator of virulence gene expression in EPEC and EHEC.

Proton magnetic resonance spectroscopy in Kennedy syndrome.

OBJECTIVE: To seek regional metabolite abnormalities in patients with Kennedy disease (KD) using proton magnetic resonance spectroscopy. DESIGN: Nine patients with KD showing the typical phenotype without clinical signs of upper motor neuron involvement were compared with 17 male, age-matched, healthy control subjects. Relative metabolite concentrations for N-acetyl (NA) groups, choline-containing groups (Cho), phosphocreatine (Cr), and lactate (Lac) were determined in the brainstem and the motor region. RESULTS: Pathologic Lac signals suggesting impaired energy metabolism were absent in patients and controls. In the brainstem area, patients with KD showed a significant reduction in the NA/Cho metabolite ratio (P = .01). In the motor region, NA/Cho (P = .04) and NA/Cr (P = .03) ratios were significantly reduced. The reduction of the NA/Cho ratio in the motor region mainly resulted from decreased metabolite ratios in 3 patients. Changes in metabolite ratios did not correlate with the number of trinucleotide cytosine-adenine-guanine repeats from leukocytes. Because of the relatively small sample size due to the rarity of KD, these results should be considered preliminary. CONCLUSIONS: Spectroscopic data fail to provide further evidence for altered energy metabolism in KD. Metabolite changes in the brainstem indicate a reduction of the neuronal marker NA or elevated Cho. These findings may reflect neuronal loss or gliosis consistent with the known pathologic features. In a subset of patients, altered metabolite ratios best explained by neuronal loss suggest subclinical involvement of the motor region. The extent of metabolite changes does not correlate with the trinucleotide repeat length.

Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli.

Enterohemorrhagic Escherichia coli O157:H7 and enteropathogenic E. coli cause a characteristic histopathology in intestinal cells known as attaching and effacing. The attaching and effacing lesion is encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, which encodes a type III secretion system, the intimin intestinal colonization factor, and the translocated intimin receptor protein that is translocated from the bacterium to the host epithelial cells. Using lacZ reporter gene fusions, we show that expression of the LEE operons encoding the type III secretion system, translocated intimin receptor, and intimin is regulated by quorum sensing in both enterohemorrhagic E. coli and enteropathogenic E. coli. The luxS gene recently shown to be responsible for production of autoinducer in the Vibrio harveyi and E. coli quorum-sensing systems is responsible for regulation of the LEE operons, as shown by the mutation and complementation of the luxS gene. Regulation of intestinal colonization factors by quorum sensing could play an important role in the pathogenesis of disease caused by these organisms. These results suggest that intestinal colonization by E. coli O157:H7, which has an unusually low infectious dose, could be induced by quorum sensing of signals produced by nonpathogenic E. coli of the normal intestinal flora.

[CADASIL. Clinical aspects, neuroradiology, genetics and diagnosis]. / CADASIL. Klinik, Neuroradiologie, Genetik und Diagnose.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary cerebral vasculopathy progressing to subcortical dementia, caused by multiple lacunar infarcts and ischemic white matter degeneration. Migraine with aura, epileptic seizures and affective disorders are frequent additional symptoms of CADASIL. The causative mutations of the Notch3 gene are located on chromosome 19p13.1. There is complete penetrance of this disorder, although individual expression of symptoms may vary. Manifestation of CADASIL is usually in the 3rd decade, but some individuals remain asymptomatic close to the age of 60. MRI displays a marked leukoencephalopathy in affected individuals as early as in the age of 20. Frontal and subcortical hypoperfusion in demented individuals was demonstrated by SPECT-studies. The prevalence of CADASIL is still not known. To date there is no causative therapy.

Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli.

The locus of enterocyte effacement of enteropathogenic Escherichia coli encodes a type III secretion system, an outer membrane protein adhesin (intimin, the product of eae ) and Tir, a translocated protein that becomes a host cell receptor for intimin. Many type III secreted proteins require chaperones, which function to stabilize proteins, prevent inappropriate protein-protein interactions and aid in secretion. An open reading frame located between tir and eae, previously named orfU, was predicted to encode a protein with partial similarity to the Yersinia SycH chaperone. We examined the potential of the orfU gene product to serve as a chaperone for Tir. The orfU gene encoded a 15 kDa cytoplasmic protein that specifically interacted with Tir as demonstrated by the yeast two-hybrid assay, column binding and coimmunoprecipitation experiments. An orfU mutant was defective in attaching-effacing lesion formation and Tir secretion, but was unaffected in expression of other virulence factors. OrfU appeared to stabilize Tir levels in the cytoplasm, but was not absolutely necessary for secretion of Tir. Based upon the physical similarities, phenotypic characteristics and the demonstrated interaction with Tir, orfU is redesignated as cesT for the chaperone for E. coli secretion of T ir.

The Per regulon of enteropathogenic Escherichia coli : identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler).

Enteropathogenic Escherichia coli (EPEC) is the prototype organism of a group of pathogenic Gram-negative bacteria that cause attaching and effacing (AE) intestinal lesions. All EPEC genes necessary for the AE phenotype are encoded within a 35.6 kb pathogenicity island termed the locus of enterocyte effacement (LEE). The LEE encodes 41 predicted open reading frames (ORFs), including components of a type III secretion apparatus and secreted molecules involved in the disruption of the host cell cytoskeleton. To initiate our studies on regulation of genes within the LEE, we determined the genetic organization of the LEE, defining transcriptional units and mapping transcriptional start points. We found that components of the type III secretion system are transcribed from three polycistronic operons designated LEE1, LEE2 and LEE3. The secreted Esp molecules are part of a fourth polycistronic operon designated LEE4. Using reporter gene fusion assays, we found that the previously described plasmid-encoded regulator (Per) activated operons LEE1, LEE2 and LEE3, and modestly increased the expression of LEE4 in EPEC. Using single-copy lacZ fusions in K-12-derived strains, we determined that Per only directly activated the LEE1:lacZ fusion, and did not directly activate the other operons. Orf1 of the LEE1 operon activated the expression of single-copy LEE2:lacZ and LEE3:lacZ fusions in trans and modestly increased the expression of LEE4:lacZ in K-12 strains. Orf1 was therefore designated Ler, for LEE-encoded regulator. Thus, the four polycistronic operons of the LEE that encode type III secretion components and secreted molecules are now included in the Per regulon, where Ler participates in this novel regulatory cascade in EPEC.

SPECT study of a German CADASIL family: a phenotype with migraine and progressive dementia only.

OBJECTIVE: We describe the clinical, molecular, genetic, MRI, and SPECT features of a German family with autosomal dominant migraine and dementia, mapping to the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) locus. We studied the correlation of cerebral blood flow, MRI, and cognitive function. BACKGROUND: CADASIL is a small-vessel disease of the brain mapped to chromosome 19p13.1. Mutations of the Notch3 gene cause this disorder. Most phenotypes are characterized by transient ischemic attacks (TIAs) and lacunar strokes leading to dementia. Migraine is frequent. A single photon emission computed tomographic (SPECT) study of this disorder has not yet been published. METHODS: We studied 13 individuals clinically and performed neuroimaging studies with MRI and SPECT. RESULTS: Genetic analysis strongly supported linkage to the CADASIL locus, and the disease haplotype was found in six individuals. Analysis by single-strand confirmation polymorphism did not identify Notch3 mutations. All affected individuals had MRI white matter hyperintensities and four individuals had additional basal ganglial signal abnormalities. Four affected individuals had migraine, two of whom had slowly progressive dementia. TIAs, stroke, and focal neurologic signs were absent. Cerebral blood flow reduction in SPECT studies of affected individuals matched with MRI signal abnormalities. Cognitive impairment was linked to signal abnormalities and hypoperfusion in the basal ganglia. Demented patients had a pattern of frontal, temporal, and basal ganglial hypoperfusion. CONCLUSIONS: We describe a CADASIL phenotype that is characterized by the absence of focal neurologic symptoms and present the first SPECT study of this disorder.

The Neisseria gonorrhoeae gene aniA encodes an inducible nitrite reductase.

The aniA gene of Neisseria gonorrhoeae encodes an outer membrane lipoprotein which is strongly induced when gonococci are grown anaerobically in vitro in the presence of nitrite. Database searches with the amino acid sequence derived from the aniA structural gene revealed significant homologies to copper-containing nitrite reductases from several denitrifying bacteria. We constructed an insertional mutation in the aniA locus of strain MS11 by allelic replacement, to determine whether this locus was necessary for growth in oxygen-depleted environments, and to demonstrate that AniA was indeed a nitrite reductase. The mutant was severely impaired in its ability to grow micro-aerophilically in the presence of nitrite, and we observed a loss in viability over several hours of incubation. No measurable nitrite reductase activity was detected in the aniA mutant strain, and activity in the strain with a wild-type locus was inducible. Finally, we report investigations to determine whether AniA protein is involved in gonococcal pathogenesis.

Transcriptional regulation of pilC2 in Neisseria gonorrhoeae: response to oxygen availability and evidence for growth-phase regulation in Escherichia coli.

The type-4 pilus of Neisseria gonorrhoeae is a dominant surface antigen which facilitates adhesion to host target cells, an essential event in gonococcal infection. pilC2 encodes a 110-kDa protein involved in pilus assembly, pilus-mediated adherence to human epithelial cells in culture and natural competence for DNA transformation. Luciferase activity directed from a chromosomal pilC2::luxAB transcriptional fusion was reduced approximately 4-fold when cells were grown anaerobically. We observed a concomitant reduction in gonococcal piliation by electron microscopy and a reduction in the ability to adhere to ME-180 human epithelial cells when bacteria were grown in the absence of oxygen. Furthermore, we present evidence for growth-phase regulation of the gonococcal pilC2 gene in Escherichia coli, and show that all sequences necessary for growth-phase regulation are contained on a 121-bp pilC2 fragment. Expression from the minimal pilC2 fragment fused to lacZ in single-copy in E. coli was induced 2-fold when cells entered stationary phase. Surprisingly, induction does not require rpoS, the gene, which encodes the starvation-induced sigma factor RpoS. In summary, we have demonstrated that pilC2 is both positively and negatively regulated at the level of transcription. This regulation is most probably relevant to physiological conditions within the human host which influence gonococcal infections.

Two different Escherichia coli proP promoters respond to osmotic and growth phase signals.

proP of Escherichia coli encodes an active transport system for proline and glycine betaine which is activated by both hyperosmolarity and amino acid-limited growth. proP DNA sequences far upstream from the translational start site are strongly homologous to the promoter of proU, an operon that specifies another osmoregulated glycine betaine transport system. Mutation and deletion analysis of proP and primer extension experiments established that this promoter, P1, was responsible for proP's strong expression in minimal medium and its response to osmotic signals. When cells were grown in complex medium, expression from a proP-lacZ fusion was induced three- to fourfold as growth slowed and cells entered stationary phase. Stationary-phase induction was dependent on rpoS, which encodes a stationary-phase sigma factor. Deletion of 158 bp of the untranslated leader sequence between P1 and the proP structural gene abolished rpoS-dependent stationary-phase regulation. Transcription initiation detected by primer extension within this region was absent in an rpoS mutant. proP is therefore a member of the growing class of sigma S-dependent genes which respond to both stationary-phase and hyperosmolarity signals.

The Escherichia coli proU promoter element and its contribution to osmotically signaled transcription activation.

The proU operon of Escherichia coli encodes a high-affinity glycine betaine transport system which is osmotically inducible and enables the organism to recover from the deleterious effects of hyperosmotic shock. Regulation occurs at the transcriptional level. KMnO4 footprinting showed that the preponderance of transcription initiated at a single primary promoter region and that proU transcription activation did not occur differentially at alternate promoters in response to various levels of salt shock. Mutational analysis confirmed the location of the primary promoter and identified an extended -10 region required for promoter activity. Specific nucleotides within the spacer, between position -10 and position -35, were important for maximal expression, but every mutant which retained transcriptional activity remained responsive to osmotic signals. A chromosomal 90-bp minimal promoter fragment fused to lacZ was not significantly osmotically inducible. However, transcription from this fragment was resistant to inhibition by salt shock. A mutation in osmZ, which encodes the DNA-binding protein H-NS, derepressed wild-type proU expression by sevenfold but did not alter expression from the minimal promoter. The current data support a model in which the role of the proU promoter is to function efficiently at high ionic strength while other cis-acting elements receive and respond to the osmotic signal.

Exposure to Psoroptes sp. mites is common among bighorn sheep (Ovis canadensis) populations in California.

Sera (n = 806) from 50 populations of bighorn sheep (Ovis canadensis) in California (USA) were evaluated for antibodies to Psoroptes sp. mites using a kinetic enzyme-linked immunosorbent assay (ELISA). Test values for each sample were determined to be either positive or negative at each of two ELISA cutoff values that provided either 100% sensitivity (low cutoff) or 100% specificity (high cutoff), respectively. One hundred sixty-eight (20.8%) sera were seropositive at the low cutoff value, and 87 (10.8%) of these sera also were seropositive at the high cutoff value. Eleven populations were designated as scabies-suspect and 25 populations were designated as scabies-positive because they had at least one seropositive animal at the low and the high cutoff values, respectively. Based on these results, exposure to Psoroptes sp. mites appeared to be widely distributed among bighorn sheep populations from 1980 to 1990 and infested animals may have been present prior to 1980.

Kinetic ELISA for detection of antibodies to Psoroptes sp. (Acari: Psoroptidae) in bighorn sheep (Ovis canadensis).

A kinetic enzyme-linked immunosorbent assay (ELISA) was developed using antigenic extracts prepared from Psoroptes cuniculi mites and sera from 37 Psoroptes sp.-infested and 43 uninfested bighorn sheep (Ovis canadensis). Serial dilutions of these serum samples, representing 3 bighorn sheep subspecies and 9 geographic areas, gave parallel responses when plotted as log dilution versus log kinetic rate. Therefore, all 80 samples were run at a single dilution (1:100) and positive/negative cutoff values were established as the mean kinetic rate of all negative sera plus either 2, 3, or 4 standard deviations. The resulting ELISA was highly reproducible and accurate with sensitivities and specificities of 100% and 97.7%, 94.6% and 97.7%, and 94.6% and 100%, respectively. This immunoassay will be useful for prospective and retrospective studies assessing the distribution and prevalence of Psoroptes sp. infestations in bighorn sheep.