An in vitro system to study interactions between bacteria and epithelial cells at the molecular level

This paper describes an experimental system to study interactions between porcine enterotoxigenic Escherichia coli (ETEC) and porcine intestinal epithelial cells in vitro at the molecular level. Radiolabelled bacteria or bacterial membrane fractions were incubated with brush borders prepared from purified epithelial cells, which were then washed repeatedly. The bacterial components removed by washing or retained by the brush borders were analysed to determine their composition and source. For this it was necessary to develop a minimal medium in which attachment factors of porcine ETEC could be radiolabelled. Furthermore, an improved method for the isolation of porcine intestinal epithelial cells was developed, since other procedures did not yield sufficiently pure preparations. The resulting method was rapid and yielded large quantities of viable epithelial cells, free from crypt cells and contaminating intestinal contents. Finally, we adapted existing procedures to isolate brush borders from these epithelial cells with special emphasis on the removal of nuclear and cytosolic material and on the isolation of morphologically intact brush borders. Using this system, mixtures of bacterial cytoplasmic and outer membranes were incubated with brush borders. Cytoplasmic membranes were easily removed by washing, while the outer membranes were not.     

The interaction of the K88 antigen with porcine intestinal epithelial cell brush borders

The interaction of 125I-labelled K88 antigen with brush borders of the epithelial cells of the pig small intestine has been studied. The iodinated antigen bound avidly to the brush borders prepared from adhesive (receptor-positive) pigs even after pretreatment of the brush borders with formaldehyde, whereas the brush borders from non-adhesive (receptor-negative) pigs failed to bind the antigen under these conditions. Treatment with glutaraldehyde rapidly destroyed the ability of both types of brush border to bind the K88 antigen. Studies on the binding of antigen to brush borders revealed the presence of high affinity receptors, but the non-linearity of the Scatchard plot could be explained by cooperative-like interactions, which view was supported by dissociation experiments. Rapid dissociation only in the presence of unlabelled K88 antigen suggested the existence of receptor site interactions of the negatively cooperative type. Attempts to inhibit the binding of 125I-labelled K88 with simple monosaccharides and oligosaccharides suggested that the binding of antigen to brush borders involves complex interactions and that galactosyl residues may be important.     

Studies on the brush border membrane of the mouse duodenum

Summary Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

A study on the isolation of intestinal brush borders in saline

Summary Attempts to remove brush borders from rat intestinal mucosal scrapings in a saline medium are described. Variation of the pH of the medium showed that at about pH 6.5, morphologically intact columnar epithelial cells could be isolated and a suitable method for preparing whole cells is suggested. When the pH was raised to 8.5, the brush borders were liberated. Studies by electron microscopy revealed that a considerable amount of granular cytoplasmic material remained attached to the terminal web. This attached material could be removed, however, by adding EDTA to a suspension of the brush borders.The supernatants and the brush borders sedimented by centrifugation were investigated for contaminants from other regions of the cell. In general, the levels of contamination were higher in the brush borders isolated in saline than when isolated in the presence of EDTA. Phospholipid: cholesterol ratios for brush borders isolated in saline varied from 1.121 to 1.211, whereas in whole cells the ratio was about 3.51.The results are discussed in relation to previous studies.     

In vivo antibody-mediated modulation of aminopeptidase A in mouse proximal tubular epithelial cells.

Aminopeptidase A (APA) is one of the many renal hydrolases. In mouse kidney, APA is predominantly expressed on the brush borders and sparsely on the basolateral membranes of proximal tubular epithelial cells. However, when large amounts of monoclonal antibodies (MAbs) against APA were injected into mice, we observed strong binding of the MAbs to the basolateral membranes, whereas the MAbs bound only transiently to the brush borders of the proximal tubular epithelial cells. In parallel, APA itself disappeared from the brush borders by both endocytosis and shedding, whereas it was increasingly expressed on the basolateral sides. Using ultrastructural immunohistology, we found no evidence for transcellular transport of endocytosed APA to the basolateral side of the proximal tubular epithelial cells. The absence of transcellular transport was confirmed by experiments in which we used a low dose of the MAbs. Such a low dose did not result in binding of the MAbs to the brush borders and had no effect on the presence of APA in the brush borders of the proximal tubular epithelial cells. In these experiments we still could observe binding of the MAbs to the basolateral membranes in parallel with the local appearance of APA. In addition, treatment of mice with chlorpromazine, a calmodulin antagonist that interferes with cytoskeletal function, largely inhibited the MAb-induced modulation of APA. Our studies suggest that injection of MAbs to APA specifically interrupts the normal intracellular traffic of this enzyme in proximal tubular epithelial cells. This intracellular transport is dependent on the action of cytoskeletal proteins.     

Studies on the brush border membrane of the mouse duodenum. I. Membrane isolation and analysis of protein components.

Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

Identification and characterization of two huge protein components of the brush border cytoskeleton: evidence for a cellular isoform of titin

Two extremely high molecular weight proteins were found to be components of the intestinal epithelial cell brush border cytoskeleton. The largest brush border protein, designated T-protein, migrated on SDS gels as a doublet of polypeptides with molecular weights similar to muscle titin T I and T II. The other large brush border protein, designated N-protein, was found to have a polypeptide molecular weight similar to muscle nebulin. In Western analysis, a polyclonal antibody raised against brush border T-protein reacted specifically with T-protein in isolated brush borders and cross-reacted with titin in pectoralis and cardiac muscle samples. T-protein was distinguished from the muscle titins by an anti-cardiac titin mAb. A polyclonal antibody raised against N-protein was specific for N-protein in brush borders and cross-reacted with nothing in pectoralis muscle. Immunolocalization in cryosections of intestinal epithelia and SDS-PAGE analysis of fractionated brush borders revealed that both T-protein and N-protein are concentrated distinctly in the brush border terminal web region subjacent to the microvilli, but absent from the microvilli. EM of rotary-replicated T-protein samples revealed many of the molecules to be long (912 +/- 40 nm) and fibrous with a globular head on one end. In some of the molecules, the head domain appeared to be extended in a fibrous conformation yielding T-protein up to 1,700-nm long. The brush border N-protein was found as long polymers with a repeating structural unit of approximately 450 nm. Our findings indicate that brush border T-protein is a cellular isoform of titin and suggest that both T-protein and N-protein play structural roles in the brush border terminal web.     

Ca++-calmodulin-dependent phosphorylation of myosin, and its role in brush border contraction in vitro

We have reinvestigated the effects of Ca++ and ATP on brush borders isolated from intestinal epithelial cells. At 37 degrees C, Ca++ (1 microM) and ATP cause a dramatic contraction of brush border terminal webs, not a retraction of microvilli as previously reported (M. S. Mooseker, 1976, J. Cell Biol. 71:417-433). Terminal web contraction, which occurs over the course of 1-5 min at 37 degrees C, actively constricts brush borders at the level of their zonula adherens. Contraction requires ATP, is stimulated by Ca++ (1 microM), and occurs in both membrane-intact and demembranated brush borders. Ca++ - dependent-solation of microvillus cores requires a concentration of Ca++ slightly greater (10 microM) than that required for contraction. Under conditions in which brush borders contract, many proteins in the isolated brush borders become phosphorylated. However, the phosphorylation of only one of the brush border proteins, the 20,000 dalton (20-kdalton) light chain of brush border myosin (BBMLC20), is stimulated by Ca++. At 37 degrees C, BBMLC20 phosphorylation correlates directly with brush border contraction. Furthermore, both BBMLC20 phosphorylation and brush border contraction are inhibited by trifluoperazine, an anti-psychotic phenothiazine that inhibits calmodulin activity. These results indicate that Ca++ regulates brush border contractility in vitro by stimulating cytoskeleton-associated, Ca++- and calmodulin-dependent brush border myosin light chain kinase.     

Localization of renal and intestinal trehalase with immunofluorescence- and enzyme-labeled antibody techniques

The localization of trehalase with fluorescein isothiocyanate-conjugated and peroxidase-conjugated antibody techniques was examined. Antiserum against purified rabbit renal trehalase was produced against guinea pigs. Anti-renal trehalase immunoglobulin (Ig)G was isolated from the serum and used for the immunohistochemical localization of intestinal and renal trehalases. Specific fluorescence and peroxidase staining were observed in the brush borders of proximal tubules and of intestinal epithelial cells. These results are in good agreement with the biochemical results. Thus, it is concluded that trehalase is specifically localized in the renal and intestinal brush borders. Sections of rabbit intestine and of rabbit kidney treated with anti-rabbit renal trehalase IgG were observed to have a specific fluorescence at the brush borders. Sections of rat intestine treated with the same antibody, however, showed no specific fluorescence at the brush borders. From these results, it is strongly suggested that renal trehalase and intestinal trehalase from the rabbit have common antigenic determinants and that these differ from those in rat intestinal trehalase.     

Characterization and localization of myosin in the brush border of intestinal epithelial cells

The brush border of intestinal epithelial cells consists of a tightly packed array of microvilli, each of which contains a core of actin filaments. It has been postulated that microvillar movements are mediated by myosin interactions in the terminal web with the basal ends of these actin cores (Mooseker, M.S. 1976. J. Cell. Biol. 71:417-433). We report here that two predictions of this model are correct: (a) The brush border contains myosin, and (b) myosin is located in the terminal web. Myosin is isolated in 70 percent purity by solubilization of Triton-treated brush borders in 0.6 M KI, and separation of the components by gel filtration. Most of the remaining contaminants can be removed by precipitation of the myosin at low ionic strength. This yield is approximately 1 mg of myosin/30 mg of solubilized brush border protein. The molecule consists of three subunits with molecular weights of 200,000, 19,000, and 17,000 daltons in a 1:1:1 M ratio. At low ionic strength, the myosin forms small, bipolar filaments with dimensions of 300 X 11nm, that are similar to filaments seen previously in the terminal web of isolated brush borders. Like that of other vertebrate, nonmuscle myosins, the ATPase activity of isolated brush border myosin in 0.6 M KCI is highest with EDTA (1 μmol P(i)/mg-min; 37 degrees C), intermediate with Ca++ (0.4 μmol P(i)/mg-min), and low with Mg++ (0.01 μmol P(i)/mg-min). Actin does not stimulate the Mg-ATPase activity of the isolated enzyme. Antibodies against the rod fragment of human platelet myosin cross-react by immunodiffusion with brush border myosin. Staining of isolated mouse or chicken brush borders with rhodamine-antimyosin demonstrates that myosin is localized exclusively in the terminal web.     

大肠杆菌F4在3个品种猪中的黏附模式

大肠杆菌F4是引起仔猪断奶前腹泻的一种主要细菌,F4黏附于小肠上皮细胞是其致病的前提。小肠上皮细胞的F4受体是由常染色体上的基因编码的,如果无受体,仔猪表现为大肠杆菌抗性。为了研究黏附的遗传机制,本实验利用大白、长白、松辽黑猪的小肠刷状缘细胞与F4ab、F4ac、F4ad进行离体黏附实验,结果发现3品种(系)猪之间黏附情况存在显著差别(P<0.01),松辽黑猪以非黏附型为主,而长白猪中黏附型比例较高,在同一品种猪内,3种菌株的黏附比例在松辽黑猪内和大白猪内有极显著差异,但在长白猪中无显著差异。从3种细菌与刷状缘的黏附模式来看,F4ab、F4ac和F4ad分别有3种不同的受体,它们可能是由3个不同的基因座编码的。     

Discrete subcellular localization of a cytoplasmic and a mitochondrial isozyme of creatine kinase in intestinal epithelial cells

Two isozymes of creatine kinase have been purified differentially from mitochondrial and cytoplasmic subfractions of intestinal epithelial cells. These intestinal epithelial cell creatine kinases were indistinguishable from the cytoplasmic (B-CK) and mitochondrial (Mi-CK) creatine kinase isozymes of brain when compared by SDS-PAGE, cellulose polyacetate electrophoresis, and peptide mapping. In intestinal epithelial cells, immunolocalization of the Mi-CK isozyme indicates that it is associated with long, thin mitochondria, which are excluded from the brush border at the apical end of each cell. In contrast, immunolocalization of the B-CK isozyme indicates that it is concentrated distinctly in the brush border terminal web domain. Although absent from the microvilli, B-CK also is distributed diffusely throughout the cytoplasm. Terminal web localization of B-CK was maintained in glycerol-permeabilized cells and in isolated brush borders, indicating that B-CK binds to the brush border structure. The abundance and localization of the mitochondrial and cytoplasmic creatine kinase isozymes suggest that they are part of a system that temporally and/or spatially buffers dynamic energy requirements of intestinal epithelial cells.     

The visualization of actin filament polarity in thin sections. Evidence for the uniform polarity of membrane-associated filaments

We have developed an improved method for visualizing actin filament polarity in thin sections. Myosin subfragment-1 (S-1)-decorated actin filaments display a dramatically enhanced arrowhead configuration when fixed in a medium which contains 0.2 % tannic acid. With the exception of brush borders from intestinal epithelial cells, the arrowhead periodicity of decorated filaments in a variety of nonmuscle cells is similar to that in isolated myofibrils. The periodicity of decorated filaments in brush borders is significantly smaller. Actin filaments which attach to membranes display a clear, uniform polarity, with the S-1 arrowheads pointing away from the plasma membrane, while those which comprise the stress fibers of myoblasts and CHO cells have antiparallel polarities. These observations are consistent with a sliding filament mechanism of cell motility.     

Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells

The association of actin filaments with membranes is now recognized as an important parameter in the motility of nonmuscle cells. We have investigated the organization of one of the most extensive and highly ordered actin filament-membrane complexes in nature, the brush border of intestinal epithelial cells. Through the analysis of isolated, demembranated brush borders decorated with the myosin subfragment, S1, we have determined that all the microvillar actin filaments have the same polarity. The S1 arrowhead complexes point away from the site of attachment of actin filaments at the apical tip of the microvillar membrane. In addition to the end-on attachment of actin filaments at the tip of the microvillus, these filaments are also connected to the plasma membrane all along their lengths by periodic (33 nm) cross bridges. These bridges were best observed in isolated brush borders incubated in high concentrations of Mg++. Their visibility is attributed to the induction of actin paracrystals in the filament bundles of the microvilli. Finally, we present evidence for the presence of myosinlike filaments in the terminal web region of the brush border. A model for the functional organization of actin and myosin in the brush border is presented.     

Identification of the microvillar 110-kDa calmodulin complex (myosin-1) in kidney.

The epithelial layer lining the proximal convoluted tubule of mammalian kidney contains a brush border of numerous microvilli. These microvilli appear in structure to be very similar to the microvilli on epithelial cells of the small intestine. Microvilli found in both the small intestine and the proximal convoluted tubules in kidney have a core bundle of actin filaments bundled by the accessory proteins villin and fimbrin. Along the length of intestinal microvilli, lateral links can be observed to connect the core bundle of actin filaments to the membrane. These cross-bridges are comprised of a 110-kDa calmodulin complex which belongs to a class of single-headed myosin molecules, collectively referred to as myosin-1. We now report that an analogous calmodulin-binding polypeptide of 105 kDa has been identified in rat kidney cortex. The 105-kDa polypeptide is preferentially found in purified kidney brush borders, can be extracted with ATP, and co-elutes with calmodulin on gel filtration and anion exchange chromatography. Fractions containing the 105-kDa polypeptide exhibit a modest ATPase activity in buffer containing CaCl2. The partially purified 105-kDa polypeptide will bind iodinated calmodulin and will sediment with F-actin in buffer containing ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or Ca2+. The addition of ATP partially reverses this association with F-actin. These results indicate that myosin-1, in addition to its presence in intestinal brush borders, is present in the brush border of kidney. We also provide preliminary evidence to indicate that the 105-kDa polypeptide is not restricted to tissues possessing a brush border.     

Purification of microvilli and an analysis of the protein components of the microfilament core bundle

A fast and convenient method for the purification of microvilli from chicken intestinal brush borders is described. The microvilli appear morphologically very similar to those found on intact brush borders. Removal of the microvillus membrane from the microvilli by Triton X-100 treatment reveals compact bundles of microfilaments with small regularly spaced projections along their length. SDS-polyacrylamide gel analysis of the protein components of the brush border, the microvilli and the microvillus core bundles shows that little or no tropomyosin, myosin or filamin is found in the microvillus, whereas polypeptide chains with mobilities characteristic for these proteins are present in the whole brush border. The majority of the microvillus core protein is actin, and the other major protein present has a polypeptide molecular weight of 95 000. Total actin from both brush borders and microvilli, characterized by isoelectric focussing analysis, contained about 40% β actin and 60% γ actin. The presence of both the β and γ cytoplasmic actins in the highly ordered parallel arrays of microfilaments of the microvilli is discussed in light of hypotheses for different functional roles of these two actin species.     

Subcellular localization of (Na+ + K+)-activated ATPase in the brush border membrane of the mucosal cell of the rat small intestine.

The localization of (Na+ + K+)-activated ATPase was investigated in isolated brush borders of rat small intestinal mucosa. The purity of the fractions has been checked by morphological and enzymatic criteria. The brush borders were found to contain a significant quantity of (Na+ + K+)-activated ATPase. Separation of isolated brush borders into their substructures suggests that (Na+ + K+)-activated ATPase is localized deeper within the brush border region than invertase. These findings are discussed in relation to active monosaccharide transport in the intestine.     

Reactivation of intestinal epithelial cell brush border motility: ATP- dependent contraction via a terminal web contractile ring

Various models have been put forward suggesting ways in which brush borders from intestinal epithelial cells may be motile. Experiments documenting putative brush border motility have been performed on isolated brush borders and have generated models suggesting microvillar retraction or microvillar rootlet interactions. The reported Ca++ ATP- induced retraction of microvilli has been shown, instead, to be microvillar dissolution in response to Ca++ and not active brush border motility. I report here studies on the reactivation of motility in intact sheets of isolated intestinal epithelium. Whole epithelial sheets were glycerinated, which leaves the brush border and intercellular junctions intact, and then treated with ATP, PPi, ITP, ADP, GTP, or delta S-ATP. Analysis by video enhanced differential interference-contrast microscopy and thin-section transmission electron microscopy reveals contractions in the terminal web region causing microvilli to be fanned apart in response to ATP and delta S-ATP but not in response to ADP, PPi, ITP, or GTP. Electron microscopy reveals that the contractions occur at the level of the intermediate junction in a circumferential constriction which can pull cells completely apart. This constriction occurs in a location occupied by an actin- containing circumferential band of filaments, as demonstrated by S-1 binding, which completely encircles the terminal web at the level of the intermediate junction. Upon contraction, this band becomes denser and thicker. Since myosin, alpha-actinin and tropomyosin, in addition to actin, have been localized to this region of the terminal web, it is proposed that the intestinal epithelial cell can be motile via a circumferential terminal web contractile ring analogous to the contractile ring of dividing cells.     

Cryptosporidium cuniculus in the rabbit (Oryctolagus cuniculus)

Cryptosporidium cuniculus was identified by light and electron microscopy in two apparently healthy rabbits. Organisms were firmly attached at the brush borders of intestinal epithelial cells. Cellular alterations were minimal, consisting of elongation and shortening of microvilli adjacent to the attachment sites of organisms. Various developmental stages of cryptosporidium were identified by electron microscopy.     

Histone H1 proteins act as receptors for the 987P fimbriae of enterotoxigenic Escherichia coli

The tip adhesin FasG of the 987P fimbriae of enterotoxigenic Escherichia coli mediates two distinct adhesive interactions with brush border molecules of the intestinal epithelial cells of neonatal piglets. First, FasG attaches strongly to sulfatide with hydroxylated fatty acyl chains. This interaction involves lysine 117 and other lysine residues of FasG. Second, FasG recognizes specific intestinal brush border proteins that migrate on a sodium-dodecyl sulfate-polyacrylamide gel like a distinct set of 32-35-kDa proteins, as shown by ligand blotting assays. The protein sequence of high performance liquid chromatography-purified tryptic fragments of the major protein band matched sequences of human and murine histone H1 proteins. Porcine histone H1 proteins isolated from piglet intestinal epithelial cells demonstrated the same SDS-PAGE migration pattern and 987P binding properties as the 987P-specific protein receptors from porcine intestinal brush borders. Binding was dose-dependent and shown to be specific in adhesion inhibition and gel migration shift assays. Moreover, mapping of the histone H1 binding domain suggested that it is located in their lysine-rich C-terminal domains. Histone H1 molecules were visualized on the microvilli of intestinal epithelial cells by immunohistochemistry and electron microscopy. Taken together these results indicated that the intestinal protein receptors for 987P are histone H1 proteins. It is suggested that histones are released into the intestinal lumen by the high turnover of the intestinal epithelium. Their strong cationic properties can explain their association with the negatively charged brush border surfaces. There, the histone H1 molecules stabilize the sulfatide-fimbriae interaction by simultaneously binding to the membrane and to 987P.