Role of intestinal surfactant-like particles as a potential reservoir of uropathogenic Escherichia coli.

The binding of uropathogenic Escherichia coli is mediated at the tips of pili by the PapG adhesin, which recognizes the Galalpha(1-4)Gal disaccharide on the uroepithelial surface. These receptors have been identified unequivocally in the human and murine urinary tracts but not in intestinal epithelium, yet uropathogenic E. coli strains are commonly found in normal colonic microflora. The gastrointestinal tract from duodenum to rectum elaborates a phospholipid-rich membrane particle with surfactant-like properties. In these studies, we report that purified murine particles contain a receptor recognized by the class I PapG adhesin because: (1) PapD-PapG complexes and class I pili bound to surfactant-like particles in a solid-phase assay, whereas binding was not detected in microvillous membranes derived from the same tissues, (2) purified PapD-PapG complex bound to a glycolipid receptor detectable in lipid extracts from the particles, and (3) soluble Galalpha(1-4)Gal inhibited the adhesin by 72% from binding to surfactant-like particles. The Galalpha(1-4)Gal receptor present in the intestinal surfactant-like particle which overlies the intestinal mucosa could provide one means to establish an intestinal habitat for uropathogenic E. coli.

Binding of pili from uropathogenic Escherichia coli to membranes secreted by human colonocytes and enterocytes.

PapG adhesins mediate the binding of uropathogenic Escherichia coli. Although receptors for these adhesins have not been demonstrated in intestinal epithelia, the colonic microflora includes strains of uropathogenic E. coli. We now report that surfactant-like particles secreted by the human intestine contain receptors for PapG adhesins and may provide an intestinal habitat for uropathogenic bacteria.

Caco-2 cells express a combination of colonocyte and enterocyte phenotypes.

Caco-2 cells are derived from a human colonic adenocarcinoma, but differentiate into small intestinal-like cells after confluence. While this enterocytic differentiation has been well studied, the presumed parallel loss of colonocyte function has not been as thoroughly examined. To follow the phenotype for both tissues, Western blots were performed using antisera recognizing liver/bone/kidney alkaline phosphatase and surfactant-like particle proteins found in normal human colon, along with antisera against the small bowel representatives of the same proteins. Antisera against proteins enriched in either enterocytes (alpha1-antitrypsin) or colonocytes (surfactant protein A) were also evaluated. Alkaline phosphatase activity increased from 3 to 18 days post-confluence. Activity at 3 days post-confluence derived substantially from both isomers. Thereafter, the colonic (liver/bone/kidney) isomer declined to low levels as the content of the enterocytic isomer rose. A similar pattern was found with colonic (decreasing expression) and enterocytic (increasing expression) surfactant-like particle proteins. In particular, the content of larger enterocytic particle proteins (97 and 116 kDa) increased with time in culture. Expression of alpha1-antitrypsin increased early and remained high, whereas surfactant protein A generally declined after the third day post-confluency. In summary, undifferentiated Caco-2 cells express very low levels of proteins characteristic of either colonocytes or enterocytes. Immediately after confluence, they expressed proteins characteristic of both cell types. Thereafter, the content of colonocyte-specific proteins decreased, whereas those specific for the enterocyte increased. The timing and degree of this phenotypic switch have implications for the interpretation of experiments using Caco-2 cells as a model of small intestinal function.

Isolation and characterization of surfactant-like particles in rat and human colon.

The source of a phospholipid-rich layer recovered from the surface of the mammalian colon has been obscure. This report describes the isolation of a low-density membrane from the surface of rat and human colons (d = 1.07-1.08 g/ml), with a low cholesterol-to-phospholipid ratio and phosphatidylcholine as its major phospholipid. Electron microscopy shows unilamellar and partially coiled membranes. Compared with microvillous membranes isolated from underlying mucosa, this extracellular membrane is enriched for tissue-unspecific alkaline phosphatase and surfactant protein A. It does not contain small intestinal marker proteins (intestinal alkaline phosphatase and sucrase-isomaltase). The human membrane contains only traces of the colonic microvillous membrane marker, carcinoembryonic antigen. Antiserum against the rat colonic membrane does not recognize colonic microvillous membrane or small intestinal surfactant-like particle proteins. Antiserum against human colonic membrane identifies one protein in the surfactant-like particle from the adjacent small intestine and two proteins in the colonic microvillous membrane. These data show that the colonocyte microvillous membrane is covered by another membrane with a different protein composition. Enrichment for surfactant protein A suggests that this colonic membrane is another example of a surfactant-like particle sharing proteins with pulmonary surfactant.

Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. I. Replication of DNA containing an alteration in position 1 of the 30-nucleotide icosahedral bacteriophage origin.

The influence of a C----G transversion at position 1 of the 30-base pair replication origin of bacteriophage phi X174 replicative form I DNA (phi X RFI) was examined in the RF----single-stranded circular DNA replication pathway catalyzed by the combined action of the purified phi X A protein, the Escherichia coli DNA polymerase III holoenzyme, rep helicase, and single-stranded DNA binding protein (Eisenberg, S., Scott, J.F., and Kornberg, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1594-1597; Reinberg, D., Zipursky, S.L., and Hurwitz, J. (1981) J. Biol. Chem. 256, 13143-13151). RFI DNA containing this transversion was cleaved to RFII by the phi X A protein as effectively as DNA containing the wild-type origin. The altered duplex DNA, however, supported replication at a slower rate (3- to 4-fold) than the wild-type DNA due to a defect in the termination and reinitiation reactions catalyzed by the phi X A protein. This defect resulted in the accumulation of DNA products containing long single strands covalently joined to the mutant DNA. These single strands were susceptible to nuclease S1 and exonuclease VII attack. The defect in the template DNA containing C----G transversion was not corrected when this mutant origin was placed on the same strand with a wild-type origin. This double-origin DNA was also replicated poorly and led to the accumulation of large products, in contrast to the products formed with RFI DNA containing two wild-type 30-base pair replication origins on the same strand.

Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. II. Effects of DNA replication of mutations in the 30-nucleotide icosahedral bacteriophage origin.

phi X174 viral strand circular DNA can be synthesized in vitro from phi X174 replicative form I (RFI) DNA in the presence of the phi X A protein, the Escherichia coli DNA polymerase III elongation system, the E. coli rep helicase, and the E. coli single-stranded DNA binding protein. M13mp9 or pBR322 RFI DNAs containing a 30-base pair sequence found at the phi X origin of replication supported phi X A protein synthesis as well as the phi X template, giving rise to a net molar excess of deoxynucleotide incorporation. In this paper, we show that mutations in positions 1-3 of the 30-nucleotide origin replicated at a lower efficiency than plasmids containing the wild-type origin, because of a deficiency in the reinitiation reaction. Mutations in positions 4-7, upstream of the phi X A protein cleavage site, failed to support replication because of their inability to support nicking. An origin containing a mutation at the residue to which the phi X A protein is covalently linked to the DNA was an active template that supported a net molar excess of incorporation. Mutations at the 3' end of the origin region, retaining only the first 21-25 nucleotides of the 30-base pair origin, failed to support replication because of impaired binding of the phi X A protein to the template and consequently poor nicking. A construct bearing the first 28 nucleotides of the origin supported wild-type replication, as did a plasmid containing a 28-mer origin with a point mutation at position 26, but this latter construct also appeared to be partially deficient in phi X A protein binding activity. These results are consistent with the presence of a phi X A protein binding domain at the 3' end of the origin.

Studies on the role of the phi X174 gene A protein in phi X174 viral strand synthesis. III. Replication of DNA containing two viral replication origins.

Supercoiled plasmid bearing two wild-type phi X origin sequences on the same strand supported the phi X A protein-dependent in vitro formation of two smaller single-stranded circles, the lengths of which were equivalent to the distance between the two origins. Additional double origin plasmids were utilized to determine whether origins defective in the initial nicking event (initiation) could support circularization (termination). In all cases tested, the presence of a mutant origin on the same strand with a wild-type origin affected the level of replication in a manner consistent with the previously determined activity of the mutant origin. When a functional mutant origin was present on the same strand as a wild-type origin, the efficiency of replication and the DNA products formed were almost identical to those of the plasmid containing two wild-type origins. Plasmid DNA bearing both a wild-type origin and a mutant origin that did not support phi X A protein binding or nicking activity, on the other hand, supported efficient DNA synthesis of only full-length circular products, indicating that the origin defective for initiation was incapable of supporting termination. In contrast, the presence of a wild-type origin and an origin that did bind the phi X A protein but was not cleaved resulted in a marked decrease in DNA synthesis along with the production of only full-length products. This suggests that the phi X A protein stalls when it encounters a sequence to which it can bind but cannot cleave. Replication of double origin plasmids containing one functional phi X origin on each strand of the supercoiled DNA was also examined. With such templates, synthesis from the wild-type origin predominated, indicating preferential cleavage of the intact origin sequence. Replication of such substrates also produced a number of aberrant structures, the properties of which suggested that interstrand exchange of the phi X A protein had occurred.

Effect of phi X C protein on leading strand DNA synthesis in the phi X174 replication pathway.

The influence of the bacteriophage phi X174 (phi X) C protein on the replication of bacteriophage phi X174 DNA has been examined. This small viral protein, which is required for the packaging of phi X DNA into proheads, inhibits leading strand DNA synthesis. The inhibitory effect of the phi X C protein requires a DNA template bearing an intact 30-base pair (bp) phi X origin of DNA replication that is the target site recognized by the phi X A protein. Removal of nucleotides from the 3' end of this 30-bp conserved origin sequence prevents the inhibitory effects of the phi X C protein. Leading strand replication of supercoiled DNA substrates containing the wild-type phi X replication origin results in the production of single-stranded circular DNA as well as the formation of small amounts of multimeric and sigma structures. These aberrant products are formed when the termination and reinitiation steps of the replication pathway reactions are skipped as the replication fork moves through the origin sequence. Replication carried out in the presence of the phi X C protein leads to a marked decrease in these aberrant structures. While the exact mechanism of action of the phi X C protein is not clear, the results presented here suggest that the phi X C protein slows the movement of the replication fork through the 30-bp origin sequence, thereby increasing the fidelity of the termination and reinitiation reactions. In keeping with the requirement for the phi X C protein for efficient packaging of progeny phi X DNA into proheads, the phi X C protein-mediated inhibition of leading strand synthesis is reversed by the addition of proteins essential for phi X bacteriophage formation. Incubation of plasmid DNA substrates bearing mutant 30 base pair phi X origin sequences in the complete packaging system results in the in vitro packaging and production of infectious particles in a manner consistent with the replication activity of the origin under study.

The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity.

The DNA helicase activity associated with purified simian virus 40 (SV40) large tumor (T) antigen has been examined. A variety of DNA substrates were used to characterize this ATP-dependent activity. Linear single-stranded M13 DNA containing short duplex regions at both ends was used to show that SV40 T antigen helicase displaced the short, annealed fragment by unwinding in a 3' to 5' direction. Three different partial duplex structures consisting of 71-, 343-, and 851-nucleotide long fragments annealed to M13 single-stranded circular DNA were used to show that SV40 T antigen can readily unwind short and long duplex regions with almost equal facility. ATP and MgCl2 were required for this reaction. With the exception of GTP, dGTP, and CTP, the other common nucleoside triphosphates substituted for ATP with varied efficiency, while adenosine 5'-O-(thiotriphosphate) was inactive. The T antigen helicase activity was also examined using completely duplex DNA fragments (approximately 300 base pairs) with or without the SV40 origin sequence as substrates. In reactions containing small amounts (0.6 ng) of DNA, the ATP-dependent unwinding of duplex DNA fragments occurred with no dependence on the origin sequence. This reaction was stimulated 5- to 6-fold by the addition of the Escherichia coli single-stranded DNA-binding protein. When competitor DNA was added so that the ratio of SV40 T antigen to DNA was reduced 1000-fold, only DNA fragments containing a functional SV40 origin of replication were unwound. This reaction was dependent on ATP, MgCl2, and a DNA-binding protein, and was stimulated by inorganic phosphate or creatine phosphate. The origin sequence requirements for the unwinding reaction were the same as those for replication (the 64-base pair sequence present at T antigen binding site 2). Thus, under specified conditions, only duplex DNA fragments containing an intact SV40 core origin were unwound. In contrast, unwinding of partially duplex segments of DNA flanked by single-stranded regions can occur with no sequence specificity.