scholarly journals Role of Decay-Accelerating Factor Domains and Anchorage in Internalization of Dr-Fimbriated Escherichia coli

2000 ◽  
Vol 68 (3) ◽  
pp. 1391-1399 ◽  
Author(s):  
R. Selvarangan ◽  
P. Goluszko ◽  
V. Popov ◽  
J. Singhal ◽  
T. Pham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Interaction Model ◽  
Electron Microscopic ◽  
Decay Accelerating Factor ◽  
Glycosylphosphatidylinositol Anchor ◽  
E Coli ◽  
Short Consensus Repeat

ABSTRACT Dr-fimbriated Escherichia coli capable of invading epithelial cells recognizes human decay-accelerating factor (DAF) as its cellular receptor. The role of extracellular domains and the glycosylphosphatidylinositol anchor of DAF in the process of internalization of Dr+ E. coli was characterized in a cell-cell interaction model. Binding of Dr+ E. coli to the short consensus repeat 3 domain of DAF expressed by Chinese hamster ovary cells was critical for internalization to occur. Deletion of short consensus repeat 3 domain or replacement of Ser165 by Leu in this domain, or the use of a monoclonal antibody to this region abolished internalization. Replacing the glycosylphosphatidylinositol anchor of DAF with the transmembrane anchor of membrane cofactor protein or HLA-B44 resulted in abolition or reduction of internalization respectively. Cells expressing glycosylphosphatidylinositol-anchored DAF but not the transmembrane-anchored DAF internalized Dr+ E. coli through a glycolipid pathway, since the former cells were more sensitive to inhibition by methyl-β-cyclodextrin, a sterol-chelating agent. Electron microscopic studies revealed that the intracellular vacuoles containing the internalized Dr+ E. coli were morphologically distinct between the anchor variants of DAF. The cells expressing glycosylphosphatidylinositol-anchored DAF contained a single bacterium in tight-fitting vacuoles, while the cells expressing transmembrane-anchored DAF contained multiple (two or three) bacteria in spacious phagosomes. This finding suggests that distinct postendocytic events operate in the cells expressing anchor variants of DAF. We provide direct evidence for the DAF-mediated internalization of Dr+ E. coli and demonstrate the significance of the glycosylphosphatidylinositol anchor, which determines the ability and efficiency of the internalization event.

scholarly journals Macrophage Class A Scavenger Receptor-Mediated Phagocytosis of Escherichia coli: Role of Cell Heterogeneity, Microbial Strain, and Culture Conditions In Vitro

2000 ◽  
Vol 68 (4) ◽  
pp. 1953-1963 ◽  
Author(s):  
Leanne Peiser ◽  
Peter J. Gough ◽  
Tatsuhiko Kodama ◽  
Siamon Gordon
Keyword(s):  
Escherichia Coli ◽  
Host Defense ◽  
Bacterial Strain ◽  
Chinese Hamster Ovary Cells ◽  
Culture Conditions ◽  
E Coli ◽  
Class A

ABSTRACT Macrophage class A scavenger receptors (SR-AI and SR-AII) contribute to host defense by binding polyanionic ligands such as lipopolysaccharide and lipoteichoic acid. SR-A knockout (SR-A−/−) mice are more susceptible to endotoxic shock and Listeria monocytogenes infection in vivo, possibly due to decreased clearance of lipopolysaccharide and microorganisms, respectively. We have used flow cytometry to analyze the role of SR-A and other scavenger-like receptors in phagocytosis of bacteria in vitro. Chinese hamster ovary cells stably transfected with human SR-A bound Escherichia coli and Staphylococcus aureus but ingested few organisms. Primary human monocyte-derived macrophages (Mφ) bound and ingested E. coli more efficiently, and this was partially but selectively blocked by the general SR inhibitor, poly(I). A specific and selective role for SR-A was shown, since bone marrow culture-derived Mφ from SR-A−/− mice ingested fewer E. coli organisms than did wild-type cells, while uptake of antibody-opsonized E. coli was unaffected. SR-A-dependent uptake of E. colivaried with the bacterial strain; ingestion of DH5α and K1 by SR-A−/− Mφ was reduced by 30 to 60% and 70 to 75%, respectively. Phagocytosis and endocytosis via SR-A were markedly down-modulated when Mφ were plated on serum-coated tissue culture plastic compared to bacteriologic plastic, where cell adhesion is mediated by SR-A and CR3, respectively. This paper demonstrates that SR-A can bind and ingest bacteria directly, consistent with a role in host defense in vivo, and highlights the importance of the source of the Mφ, bacterial strain, and culture conditions on receptor function in vitro.

scholarly journals Human Diffusely Adhering Escherichia coli Expressing Afa/Dr Adhesins That Use Human CD55 (Decay-Accelerating Factor) as a Receptor Does Not Bind the Rodent and Pig Analogues of CD55

2004 ◽  
Vol 72 (8) ◽  
pp. 4859-4863 ◽  
Author(s):  
Sylvie Hudault ◽  
O. Brad Spiller ◽  
B. Paul Morgan ◽  
Alain L. Servin
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Intestinal Cells ◽  
Gastrointestinal Infections ◽  
Decay Accelerating Factor ◽  
E Coli ◽  
Empty Vector ◽  
Human Intestinal Cells

ABSTRACT Afa/Dr diffusely adhering Escherichia coli (DAEC) bacteria that are responsible for recurrent urinary tract and gastrointestinal infections recognized as a receptor the glycosylphosphatidylinositol (GPI)-anchored protein decay-accelerating factor (DAF; CD55) at the brush border of cultured human intestinal cells. Results show that Afa/Dr DAEC C1845 bacteria were poorly associated with the mucosa of the gastrointestinal tract of infected mice. We conducted experiments with Chinese hamster ovary (CHO) cells stably transfected with mouse (GPI or transmembrane forms), pig, or human CD55 or mouse Crry cDNAs or transfected with empty vector pDR2EF1α. Recombinant E. coli AAEC185 bacteria expressing Dr or F1845 adhesins bound strongly to CHO cells expressing human CD55 but not to the CHO cells expressing mouse (transmembrane and GPI anchored), rat, or pig CD55 or mouse Crry. Positive clustering of CD55 around Dr-positive bacteria was observed in human CD55-expressing CHO cells but not around the rarely adhering Dr-positive bacteria randomly distributed at the cell surface of CHO cells expressing mouse, rat, or pig CD55.

scholarly journals Increased Escherichia coli enterotoxin detection after concentrating culture supernatants: possible new enterotoxin detectable in dogs but not in infant mice

1978 ◽  
Vol 8 (6) ◽  
pp. 700-703
Author(s):  
D R Nalin ◽  
M M Levine ◽  
C R Young ◽  
E J Bergquist ◽  
J C McLaughlin
Keyword(s):  
Escherichia Coli ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Negative Control ◽  
Intestinal Loop ◽  
Fluid Accumulation ◽  
Ovary Cells ◽  
Infant Mouse ◽  
E Coli ◽  
Culture Supernatants

The heat-stable enterotoxin (ST) of Escherichia coli can be detected by infant mouse or dog intestinal loop tests. These tests differ in that the dog assay uses concentrated culture supernatants and is based on measurements of net intestinal absorption, whereas the mouse test uses unconcentrated supernatants and depends on gross fluid accumulation. To compare the relative sensitivities of these assays, culture supernatants of randomly selected E. coli isolates from 34 Bangalee diarrhea patients were tested for ST in dog loops and infant mice. Supernatants were also tested for heat-labile enterotoxin (LT) in dog loops, Y-1 adrenal cells, and Chinese hamster ovary cells. E. coli supernatants that produced positive responses for both ST and LT in the dog loop assay (ST+/LT+) also produced positive responses when tested for ST in infant mice and for LT in cell lines. Supernatants of strains negative for ST and LT in dog loop (ST-/LT) were also negative in other assays. Of 10 strains positive for just ST in the dog loop test (ST+/LT-), only 5 were ST positive in the standard infant mouse test. Supernatants of the other five strains (dog loop positive, mouse test negative) were then concentrated 100-fold and retested in mice. Three of these five gave consistently positive results after concentration, and two were only intermittently positive. Concentrated supernatants of negative control strains (ST-/LT-) were all negative in mice. The dog assay detects more strains producing ST than the infant mouse test. The infant mouse test, which detects only gross fluid accumulation, failed to detect approximately half of the 10 strains which produced ST alone (ST+/LT-; P = 0.025). Concentrating supernatants for the mouse assay increases sensitivity for detection of ST, but certain E. coli strains produce a variety of ST to which infant mice do not respond.

scholarly journals Assay method for Vibrio cholerae and Escherichia coli enterotoxins by automated counting of floating chinese hamster ovary cells in culture medium

1978 ◽  
Vol 7 (5) ◽  
pp. 479-485
Author(s):  
R T Nozawa ◽  
T Yokota ◽  
S Kuwahara
Keyword(s):  
Escherichia Coli ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Culture Medium ◽  
Culture Media ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Assay Method ◽  
Ovary Cells ◽  
E Coli

As Chinese hamster ovary (CHO) cells on plastic proliferate, many cells float off into the medium instead of piling up after they form a monolayer. Fewer cells were floating in the medium when CHO cells were incubated with cholera toxin at a concentration as low as 10 pg/ml. The toxin increased the adhesiveness of the cells forming confluent monolayers so that the floating cells accumulated on the adherent monolayers. On the basis of this finding, a simple, quantitative assay method for cholera and Escherichia coli enterotoxins was devised by cultivating CHO cells in a Linbro multidish and counting the cells in the medium with a Coulter Counter. The method was sensitive enough to detect toxins in 100- to 200-fold-diluted culture media of toxigenic E. coli strains. Little or no activity was detected by this method in the culture medium of nontoxigenic E. coli.

scholarly journals Structure-Function Analysis of Decay-Accelerating Factor: Identification of Residues Important for Binding of the Escherichia coli Dr Adhesin and Complement Regulation

2002 ◽  
Vol 70 (8) ◽  
pp. 4485-4493 ◽  
Author(s):  
Rafia J. Hasan ◽  
Edyta Pawelczyk ◽  
Petri T. Urvil ◽  
Mathura S. Venkatarajan ◽  
Pawel Goluszko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Function Analysis ◽  
Chinese Hamster Ovary Cells ◽  
Regulatory Protein ◽  
Chinese Hamster ◽  
Homology Model ◽  
Inhibitory Protein ◽  
Decay Accelerating Factor ◽  
Complement Regulation

ABSTRACT Decay-accelerating factor (DAF), a complement regulatory protein, also serves as a receptor for Dr adhesin-bearing Escherichia coli. The repeat three of DAF was shown to be important in Dr adhesin binding and complement regulation. However, Dr adhesins do not bind to red blood cells with the rare polymorphism of DAF, designated Dr(a−); these cells contain a point mutation (Ser165-Leu) in DAF repeat three. In addition, monoclonal antibody IH4 specific against repeat three was shown to block both Dr adhesin binding and complement regulatory functions of DAF. Therefore, to identify residues important in binding of Dr adhesin and IH4 and in regulating complement, we mutated 11 amino acids—predominantly those in close proximity to Ser165 to alanine—and expressed these mutations in Chinese hamster ovary cells. To map the mutations, we built a homology model of repeat three based on the poxvirus complement inhibitory protein, using the EXDIS, DIAMOD, and FANTOM programs. We show that perhaps Ser155, and not Ser165, is the key amino acid that interacts with the Dr adhesin and amino acids Gly159, Tyr160, and Leu162 and also aids in binding Dr adhesin. The IH4 binding epitope contains residues Phe148, Ser155, and L171. Residues Phe123 and Phe148 at the interface of repeat 2-3, and also Phe154 in the repeat three cavity, were important for complement regulation. Our results show that residues affecting the tested functions are located on the same loop (148 to 171), at the same surface of repeat three, and that the Dr adhesin-binding and complement regulatory epitopes of DAF appear to be distinct and are ≈20 Å apart.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals Distribution of Lactoferrin Is Related with Dynamics of Neutrophils in Bacterial Infected Mice Intestine

Molecules ◽  
2020 ◽  
Vol 25 (7) ◽  
pp. 1496 ◽  
Author(s):  
Li Liang ◽  
Zhen-Jie Wang ◽  
Guang Ye ◽  
Xue-You Tang ◽  
Yuan-Yuan Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Mucosal Immunity ◽  
Mrna Levels ◽  
Iron Binding ◽  
E Coli ◽  
New Knowledge ◽  
Activated Neutrophils ◽  
Binding Glycoprotein

Lactoferrin (Lf) is a conserved iron-binding glycoprotein with antimicrobial activity, which is present in secretions that recover mucosal sites regarded as portals of invaded pathogens. Although numerous studies have focused on exogenous Lf, little is known about its expression of endogenous Lf upon bacterial infection. In this study, we investigated the distribution of Lf in mice intestine during Escherichia coli (E. coli) K88 infection. PCR and immunohistology staining showed that mRNA levels of Lf significantly increased in duodenum, ileum and colon, but extremely decreased in jejunum at 8 h and 24 h after infection. Meanwhile, endogenous Lf was mostly located in the lamina propria of intestine villi, while Lf receptor (LfR) was in the crypts. It suggested that endogenous Lf-LfR interaction might not be implicated in the antibacterial process. In addition, it was interesting to find that the infiltration of neutrophils into intestine tissues was changed similarly to Lf expression. It indicated that the variations of Lf expression were rather due to an equilibrium between the recruitment of neutrophils and degranulation of activated neutrophils. Thus, this new knowledge will pave the way to a more effective understanding of the role of Lf in intestinal mucosal immunity.

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