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1.
Harry L.T. Mobley Karen G. Jarvis John P. Elwood Donna I. Whittle C. Virginia Lockatell Robert G. Russell David E. Johnson Michael S. Donnenberg John W. Warren 《Molecular microbiology》1993,10(1):143-155
Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap+, sfa+, pil+, hly+ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were sub-cloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (NalR) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA+ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or α Gal(1–4)β Gal-coated latex beads. CBA mice (N =100) were challenged transurethrally with 105, 106, 107, or 109 cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uro-pathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model. 相似文献
2.
Lisa Beltz Opendra Narayan Robert J. Adams Stephen J. Noga Albert D. Donnenberg 《Journal of medical primatology》1991,20(4):144-151
Rhesus progenitor-enriched BM was exposed overnight to SIV and cultured in a limiting dilution assay where the potential for progenitor interaction with lymphocytes or macrophages was low. Virus was consistently isolated late in culture, detection being aided by coculture with CEM174 lymphoblasts. Although infected cells had reduced clonogenic activity, colonies were indistinguishable from those derived from uninfected BM with respect to proliferative potential, morphology, and longevity in culture. Primate immunodeficiency viruses, therefore, may infect immature BM populations, directly affecting hematopoietic activity. 相似文献
3.
The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs. 相似文献
4.
A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus 总被引:12,自引:3,他引:9
Kelly D. Stone Hong-Zhong Zhang Lisa K. Carlson Michael S. Donnenberg 《Molecular microbiology》1996,20(2):325-337
Enteropathogenic Escherichia coli (EPEC) adhere to epithelial cells in microcolonies, a pattern termed localized adherence (LA). LA is dependent upon the presence of 50–70MDa plasmids, termed EPEC adherence factor (EAF) plasmids. Expression of an EAF plasm id-encoded type IV fimbria, the bundle-forming pilus (BFP), is associated with the LA phenotype. TnphoA insertions in bfpA, the gene encoding the major structural subunit of the BFP, abolish LA. While bfpA::TnphoA mutants cannot be complemented for LA by plasmids carrying the bfpA gene alone in trans, this work shows that they can be complemented by plasmids carrying the bfpA gene, as well as approximately 10kb of downstream sequence, suggesting that such mutations have polar effects on downstream genes. The identification and characterization of a cluster of 13 genes immediately downstream of bfpA are described. The introduction into a laboratory Escherichia coli strain of a plasmid containing these 14 bfp gene cluster genes, along with pJPN14, a plasmid containing another fragment derived from the EAF plasmid, confers LA ability and BFP biogenesis. However, when a mutation is introduced into the last gene of the bfp cluster, neither LA nor BFP biogenesis is conferred. This work also provides evidence to show that the fragment cloned in pJPN14 encodes a factor(s) which results in increased levels of the pilin protein. Finally, it is shown that expression of the 14 genes in the bfp cluster from an IPTG-inducible promoter, in the absence of pJPN14, is sufficient to reconstitute BFP biogenesis in a laboratory E. cob strain, but is insufficient for LA. This is the first report demonstrating the reconstitution of a type IV pilus in a laboratory E. coli strain with a defined set of genes. The 8FP system should prove to be a useful model for studying the molecular mechanisms of type IV pilus biogenesis. 相似文献
5.
6.
Adhesion of enteropathogenic Escherichia coli to host cells 总被引:9,自引:1,他引:8
Enteropathogenic Escherichia coli (EPEC) adhere to the intestinal mucosa and to tissue culture cells in a distinctive fashion, destroying microvilli, altering the cytoskeleton and attaching intimately to the host cell membrane in a manner termed the attaching and effacing effect. Typical EPEC strains also form three-dimensional microcolonies in a pattern termed localized adherence. Attaching and effacing, and in particular intimate attachment requires an outer membrane adhesin called intimin, which binds to the translocated intimin receptor, Tir. Tir is produced by the bacteria and delivered to the host cell via a type III secretion system. In addition to this well-established adhesin-receptor pair, numerous other adhesin interactions between EPEC and host cells have been described including those between intimin and cellular receptors and those involving a bundle-forming pilus and flagella and unknown receptors. Much additional work is needed before a full understanding of EPEC adhesion to host cells comes to light. 相似文献
7.
Michael S. Donnenberg Tracy H. Hazen Tamer H. Farag Sandra Panchalingam Martin Antonio Anowar Hossain Inacio Mandomando John Benjamin Ochieng Thandavarayan Ramamurthy Boubou Tamboura Anita Zaidi Myron M. Levine Karen Kotloff David A. Rasko James P. Nataro 《PLoS neglected tropical diseases》2015,9(5)
Background
Typical enteropathogenic Escherichia coli (tEPEC) strains were associated with mortality in the Global Enteric Multicenter Study (GEMS). Genetic differences in tEPEC strains could underlie some of the variability in clinical outcome.Methods
We produced draft genome sequences of all available tEPEC strains from GEMS lethal infections (LIs) and of closely matched EPEC strains from GEMS subjects with non-lethal symptomatic infections (NSIs) and asymptomatic infections (AIs) to identify gene clusters (potential protein encoding sequences sharing ≥90% nucleotide sequence identity) associated with lethality.Results
Among 14,412 gene clusters identified, the presence or absence of 392 was associated with clinical outcome. As expected, more gene clusters were associated with LI versus AI than LI versus NSI. The gene clusters more prevalent in strains from LI than those from NSI and AI included those encoding proteins involved in O-antigen biogenesis, while clusters encoding type 3 secretion effectors EspJ and OspB were among those more prevalent in strains from non-lethal infections. One gene cluster encoding a variant of an NleG ubiquitin ligase was associated with LI versus AI, while two other nleG clusters had the opposite association. Similar associations were found for two nleG gene clusters in an additional, larger sample of NSI and AI GEMS strains.Conclusions
Particular genes are associated with lethal tEPEC infections. Further study of these factors holds potential to unravel the mechanisms underlying severe disease and to prevent adverse outcomes. 相似文献8.
Ramboarina S Fernandes PJ Daniell S Islam S Simpson P Frankel G Booy F Donnenberg MS Matthews S 《The Journal of biological chemistry》2005,280(48):40252-40260
Bundle-forming pili (BFP) are essential for the full virulence of enteropathogenic Escherichia coli (EPEC) because they are required for localized adherence to epithelial cells and auto-aggregation. We report the high resolution structure of bundlin, the monomer of BFP, solved by NMR. The structure reveals a new variation in the topology of type IVb pilins with significant differences in the composition and relative orientation of elements of secondary structure. In addition, the structural parameters of native BFP filaments were determined by electron microscopy after negative staining. The solution structure of bundlin was assembled according to these helical parameters to provide a plausible atomic resolution model for the BFP filament. We show that EPEC and Vibriocholerae type IVb pili display distinct differences in their monomer subunits consistent with data showing that bundlin and TcpA cannot complement each other, but assemble into filaments with similar helical organization. 相似文献
9.
Lieberman JA Frost NA Hoppert M Fernandes PJ Vogt SL Raivio TL Blanpied TA Donnenberg MS 《Journal of bacteriology》2012,194(7):1646-1658
Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis. 相似文献
10.
Romney M. Humphries Michael S. Donnenberg Jonathan Strecker Elena Kitova John S. Klassen Lina Cui Thomas P. Griener George L. Mulvey Glen D. Armstrong 《Molecular microbiology》2009,72(4):859-868
Bundle-forming pili (BFP) promote the adherence of typical enteropathogenic Escherichia coli (EPEC) to human intestinal epithelial cells. BFP are polymers of bundlin and nine bundlin alleles have been identified in EPEC isolated from diverse sources. These alleles are divided into two main groups, α and β, based on their amino acid sequences. Alpha bundlins are also N -acetyllactosamine- (LacNAc) specific lectins and bind to HEp-2 cells, whereas β bundlins do not display these characteristics. The four surface-exposed regions of amino acid sequence heterogeneity between α and β bundlin were therefore investigated as potential LacNAc-specific carbohydrate-binding domains in a bundlin. Mutation of one of these domains, 137-GENNI-141, in α1 bundlin to that of β bundlin (136-SPDST-140) resulted in BFP that no longer bound to LacNAc or HEp-2 cells. Conversely, mutating the β3 bundlin gene to encode the α bundlin sequence at this domain resulted in the gain of HEp-2 cell adherence. The importance of this domain in carbohydrate binding is supported by the finding that introducing the mutation GENNI→GENNT altered the α1 bundlin carbohydrate-binding specificity from LacNAc to the Lewis X glycan sequence. 相似文献