Factors affecting expression of the Escherichia coli pilus K99.

An enzyme-linked antibody centrifuge assay for the detection of Escherichia coli pilus K99 was developed and shown to be a specific and quantitative assay for the detection of cell-bound K99. The data presented demonstrate the usefulness of the assay as a diagnostic tool. Using the assay, several factors that affect expression of K99 were investigated. Expression of K99 was dependent upon the degree of aeration provided: nonaerated bacteria produced little or no K99, whereas aerated bacteria produced large amounts of K99. K99 also appeared to be produced only by logarithmically growing cells, whereas there was a demonstrable decline in the amount of K99 per cell during stationary phase. Glucose was shown to repress K99 expression. At 0.5% glucose, K99 expression was highly repressed. Glucose-mediated repression could be overcome by the addition of cyclic adenosine 3',5'-monophosphate. Several other carbon sources also inhibited K99 expression, including pyruvate, arabinose, and lactose; glycerol was stimulatory.     

Effect of sodium acetate on expression of K99 pili by Escherichia coli

Sodium acetate suppressed K99 production in Escherichia coli strains cultured on a minimal medium, as determined by seroagglutination and enzyme-linked immunosorbent assay. The greatest suppression occurred when the medium contained both sodium acetate and glucose. Glucose alone did not suppress K99 production.     

K99 surface antigen of Escherichia coli: antigenic characterization.

K99 prepared by acid precipitation hemagglutinated guinea pig erythrocytes, whereas K99 prepared by chromatography on diethylaminoethyl-Sephadex did not. K99 purified by either procedure hemagglutinated horse erythrocytes. K99 prepared by acid precipitation contained a second antigen not presnet in the K99 prepared by chromatography on diethylaminoethyl-Sephadex. This antigen could be detected by immunoprecipitation with some, but not all, sera prepared against K99-positive Escherichia coli strains. It was assumed that this second antigen is not K99 and is responsible for the guinea pig erythrocyte hemagglutination reaction. Furthermore, the second antigen has an isoelectric point of 4.2, which has been reported by Morris and co-workers to be the isoelectric point of K99.     

F41 pili as protective antigens of enterotoxigenic Escherichia coli that produce F41, K99, or both pilus antigens.

Pigs suckling dams that have been vaccinated with pilus antigen are protected against challenge with enterotoxigenic Escherichia coli (ETEC) strains that express the same pilus antigen. However, some ETEC strains express more than one pilus antigen. Pregnant swine were vaccinated either with E. coli HB101 that harbored a recombinant plasmid coding for F41 expression (F41+) or with the HB101 parent strain that carries the pHC79 vector (F41-). Suckling pigs born to vaccinated dams were challenged with ETEC that expressed either K99, F41, or both pilus antigens. Production of F41 in vivo was demonstrated by immunofluorescence assay of sections of ileum and by seroconversion against F41 antigen by pigs challenged with F41+ and K99+ F41+ ETEC strains. The F41+ vaccine protected against challenge with an F41+ ETEC strain. In contrast, F41+ vaccination did not protect against challenge with K99+ or K99+ F41+ ETEC strains. The F41- vaccine did not protect against challenge with any strain used. The results indicate that K99+ F41+ ETEC strains produce F41 antigen in the small intestine during disease and that F41+ vaccination can be a protective antigen if the challenge strain expresses only F41 antigen, but that F41+ vaccination may not protect against strains that produce both K99 and F41 antigens.     

Analysis of a naturally occurring K99+ enterotoxigenic Escherichia coli strain that fails to produce K99.

A spontaneously occurring field isolate of enterotoxigenic Escherichia coli that was genotypically K99+ but phenotypically K99- was analyzed for the reason that it did not express K99. The defect, which was cis active, was located within an area 5' to the first gene required for K99 biogenesis and was the result of the deletion of a single base pair.     

Ganglioside epitope recognized by K99 fimbriae from enterotoxigenic Escherichia coli.

The receptor structure recognized by Escherichia coli possessing K99 fimbriae and by isolated K99 fimbrial fractions was examined by using an equine erythrocyte hemagglutination inhibition test. Both K99-positive organisms (strain B41) and fimbrial preparations reacted with N-glycolylneuraminyl-lactosyl-ceramide (NeuGcLacCer) purified from equine erythrocytes with very high potency. Fimbrial preparations were 253 times more potent than intact organisms, indicating that isolated fimbriae more precisely recognize the structure of NeuGcLacCer than do fimbriae located on the bacterial cell wall. Structurally, the N-glycolyl group of the sialic acid was shown to be essential because substitution of the N-acetyl group for the N-glycolyl group caused the reactivity to completely disappear. The substitution of the O-acetyl group for the C4 hydroxyl group of the sialic acid (4-O-Ac-NeuGcLacCer) also diminished the reactivity by about 500 times, indicating that the fine structure of NeuGc is necessary for recognition. N-Glycolylneuraminyl-neolactotetraosyl-ceramide (NeuGcnLc4Cer) and N-glycolylneuraminyl-neolactohexaosyl-ceramide (NeuGcnLc6Cer), both of which have identical disaccharides at the nonreducing terminal and longer carbohydrate chains, showed reduced reactivity, indicating that the ceramide of NeuGcLacCer is also involved in the recognition. Indeed, NeuGcLac oligosaccharides altered by cleavage of the ceramide or the terminal sialic acid (NeuGc) showed dramatically reduced reactivities. Ten other E. coli strains (isolated from diseased calves) and two strains (isolated from diseased piglets) which possessed the same K99 antigen and various O antigens were used for the recognition test. The results obtained were similar to those mentioned above.     

K99 surface antigen of Escherichia coli: purification and partial characterization.

K99, a presumed colonizing factor of enterotoxigenic Escherichia coli of calf origin, has been purified. K99 was removed from K99+ bacteria by salt extraction and subsequently purified by ammonium sulfate precipitation and column chromatography on diethylaminoethyl-Sephadex. The purified material was homogenous in size, having an s20,w of 13 to 15 S. It was composed of two subunits: a major component with a molecular weight of 22,500 and a minor component of 29,500. When observed in the electron microscope, K99 appeared to be rod-shaped, with a strong tendency for self-aggregation. At concentrations where aggregation was minimized, individual rods were observed with diameters of 8.4 nm and mean lengths of 130 nm. Based on the subunit structure, exterior location, and rod-like shape of K99, it is concluded that K99 is a pilus or pilus-like structure. Chemically, K99 is composed primarily of protein and has an isoelectric point of greater than 10. Purified K99 did not hemagglutinate guinea pig erythrocytes.     

Cloning and expression in Escherichia coli of LKP pilus genes from a nontypeable Haemophilus influenzae strain.

Nontypeable Haemophilus influenzae HF0295, isolated by aspiration from the middle ear of a patient with otitis media, expresses long, thick, and hemagglutinating pili of a single serotype (LKP1) on its surface. An intact pilus vaccine consisting of the LKP1 serotype protected chinchillas against experimental otitis media (C. C. Brinton, Jr., M. J. Carter, D. B. Derber, S. Kar, J. A. Kramarik, A. C. C. To, S. C. M. To, and S. W. Wood, Pediatr. Infect. Dis. J. 8:554-561, 1989; R. B. Karasic, D. J. Beste, S. C. M. To, W. J. Doyle, S. W. Wood, M. J. Carter, A. C. C. To, K. Tanpowpong, C. D. Bluestone, and C. C. Brinton, Jr., Pediatr. Infect. Dis. J. 8:562-565, 1989). The genes encoding LKP1 pili were cloned from a genomic library of the clinical strain as a 12.5-kilobase insert on a plasmid vector and inserted into Escherichia coli K-12. Transposon mutagenesis and deletion constructs mapped the pilus-coding region within a 7-kilobase region of insert DNA. The recombinant bacteria were found by electron microscopy to express pili morphologically similar to LKP1 pili. Purified pilus rods from the recombinant and its parental strain were composed of a single detectable protein with an apparent molecular weight of 27,500. Antibodies raised against LKP1 pili purified from H. influenzae immunologically reacted with pili from the recombinant bacteria. Pili from both strains also adhered to human erythrocytes and buccal cells with the same specificity.     

Relationship of type 1 pilus expression in Escherichia coli to ascending urinary tract infections in mice.

The role of type 1 pili and P adhesins during the in vivo growth of Escherichia coli inoculated into the urethras of BALB/c mice was studied. Strains which produced type 1 pili when grown in broth but lost this trait when grown on agar (regulated variants) were tested. Broth-grown organisms colonized the bladder of every animal tested, with counts of 10(3) to 10(4) viable organisms recovered from bladder homogenates. Agar-grown organisms gave lower rates of infection and the number of viable organisms recovered from bladders was significantly reduced. The degree of inoculum piliation influenced bladder colonization in a direct way: as piliation increased, the number of bacteria recovered from bladders also increased. After intraurethral inoculation, all of the bladders and 44% of the kidneys were colonized on day 1, and by day 5, 94% of the bladders and 16% of the kidneys were positive. Hemagglutination titers remained high for the bladder isolates, but the organisms colonizing the kidneys became significantly less piliated with time. Bacteriuria was unrelated to bladder or renal colonization. Strains that demonstrated random phase variation of type 1 pili during growth on agar produced similar colonizations of the urinary tract with broth- and agar-grown inocula. Strains that produced only P adhesins were less effective in colonizing the urinary tract than were type 1 piliated organisms. Other strains which did not produce pili only minimally colonized the bladder. The results suggest that type 1 pili play an essential role in ascending infections of the urinary tract.     

Expression of K99 adhesion antigen controlled by the Escherichia coli tryptophan operon promoter.

The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 [O101:K99] has been cloned as a 7.0-kilobase BamHI-generated DNA fragment into the vector pBR322 by us and others (J. D. A. van Embden, F. K. de Graaf, L. M. Schouls, and J. S. Teppma, Infect. Immun. 29:1125-1133, 1980). Cells harboring one such construction, known as pK99-64, are capable of expressing K99 antigen on the cell surface. We replaced the natural promoter sequence for the gene encoding the K99 pilus subunit with a strong, inducible exogenous promoter, the E. coli tryptophan (trp) operon promoter, to construct the plasmid pBR-TrpK99. E. coli cells harboring pBR-TrpK99 or a similar construction in the plasmid pDR540, known as pKO-TrpK99, upon induction with 3-beta-indoleacrylic acid, produced about fourfold more K99 antigen than did cells bearing pK99-64 with the natural promoter. Expression of the pilus antigen was found to be under control of the tryptophan promoter. Plasmid instability was encountered, however, in cells bearing pKO-TrpK99 when the trp promoter was derepressed. Introduction of the aminoglycoside 3'-phosphotransferase gene of transposable element Tn5 into pKO-TrpK99 to generate pKON-TrpK99 effectively stabilized the plasmid in cells grown under identical conditions in medium containing kanamycin.     

Occurrence of K99 antigen on Escherichia coli isolated from pigs and colonization of pig ileum by K99+ enterotoxigenic E. coli from calves and pigs.

Several strains of enterotoxigenic Escherichia coli (ETEC) isolated from pigs were found to have an antigen (K99) previously reported only on strains of calf and lamb origin and which facilitates intestinal colonization in the latter two species. Several human ETEC were also tested for K99; however, none were positive. Each of four K99-positive ETEC strains of calf origin and one of pig origin produced K99 in pig ileum in vivo, adhered to villous epithelium in pig ileum, colonized pig ileum, and caused profuse diarrhea in newborn pigs. In contrast to the K99-positive strains above, four K99-negative ETEC from humans and chickens and one K99-positive ETEC from a calf either did not colonize pig ileum or did so inconsistently. When the K99-negative strains did colonize, they had little or no tendency to adhere to intestinal villi. These results are consistent with the hypothesis that K99 facilitates adhesion to and colonization of pig ileum by some ETEC.     

Monoclonal antibodies specific for the bundle-forming pilus of enteropathogenic Escherichia coli.

The bundle-forming pilus (BFP) produced by enteropathogenic Escherichia coli (EPEC) is associated with the presence of a large EPEC adherence factor plasmid and the formation of localized adherence clusters on tissue culture cells. Three mouse monoclonal antibodies (ICA2, ICA3, and ICA4) were produced against BFP purified from EPEC B171 (O111:NM). These monoclonal antibodies reacted in immunoblots with different epitopes of the 19.5-kDa bundlin subunit of BFP of heterologous EPEC. These reagents could serve as diagnostic tools for the identification of EPEC as well as for studying the role of BfpA in the interaction of EPEC with eukaryotic cells.     

Purification, characterization, and partial covalent structure of Escherichia coli adhesive antigen K99.

The adhesive antigen K99 of enterotoxigenic Escherichia coli strains of calf origin was isolated and purified. The K99 fimbriae were removed from the cells by heat treatment, concentrated by precipitation with ammonium sulfate, and purified by gel filtration on Sepharose CL-4B and treatment with deoxycholate. The purified K99 antigen was composed of protein subunits with a molecular weight of 18,500 and had an isoelectric point of 9.5. The N-terminal amino acid sequence, as well as the composition of the C-terminal part of the K99 protein subunits, was determined.     

Identification of enteropathogenic Escherichia coli by PCR-based detection of the bundle-forming pilus gene.

A rapid and simple method of detecting enteropathogenic Escherichia coli (EPEC) was developed. The procedure is based on amplifying by the PCR method a 326-bp region of the bundle-forming pilus gene of EPEC. The oligonucleotide DNA primers used in this procedure did not amplify DNA of any other bacterial enteropathogens tested. The procedure was 100% specific for EPEC strains that exhibit a characteristic pattern of attachment (localized adherence) to HeLa cells.     

Monoclonal antibody enzyme-linked immunosorbent assay for identification of K99-positive Escherichia coli isolates from calves.

For Escherichia coli to produce diarrhea in animals it must possess the ability to attach to the epithelial cells of the intestine and to produce enterotoxins. Tests developed to differentiate pathogenic from nonpathogenic E. coli have relied on detection of adherence structures called pili or detection of the toxins. We utilized a monoclonal antibody to K99 pili in an enzyme-linked immunosorbent assay to detect the presence of K99 pili in E. coli isolated from calves. Twenty-three E. coli isolates that were known to be stable toxin positive were all shown to produce K99 pili. A 100% correlation also was shown between the presence of K99 antigen and production of stable toxin by E. coli isolates. Of the 251 isolates, 245 were negative by K99 enzyme-linked immunosorbent assay and stable toxin assay. The other six were positive on both tests. The enzyme-linked immunosorbent assay also was shown to be specific for K99 pili by antibody-blocking assays. The number of E. coli necessary for detecting K99 pili by enzyme-linked immunosorbent assay was determined to be 3.5 X 10(5) bacteria per ml.     

Improved minca medium for the detection of K99 antigen in calf enterotoxigenic strains of Escherichia coli.

The K99 antigen of Escherichia coli can be detected more readily in cultures grown on Minca medium at 37 degrees C for 6 to 8 h or grown on Minca plus 1% Iso VitaleX for 20 to 24 h.     

Passive immunity in calf diarrhea: vaccination with K99 antigen of enterotoxigenic Escherichia coli and rotavirus.

Twenty-four pregnant cows were vaccinated intramuscularly with K99 extract from enterotoxigenic Escherichia coli and inactivated rotavirus as follows: six cows were injected with 2 ml of oil-adjuvanted vaccine; six cows were injected with 0.5 ml of oil-adjuvanted vaccine; six cows were injected with 4 ml of aluminum hydroxide-adjuvanted vaccine twice with a four-week interval; and six cows were unvaccinated as controls. Calves born to these cows were challenged with enterotoxigenic E. coli at 6 to 18 h after birth. Serum and milk antibodies to K99 and rotavirus in cows vaccinated with either dose of oil vaccine were significantly increased until at least 28 days after calving. In cows vaccinated with alhydrogel vaccine, there was a significant K99 antibody increase in serum and in colostrum but not in milk and a significant rotavirus antibody increase only in colostrum. Five of six calves born to unvaccinated cows developed enterotoxic colibacillosis after challenge, and all excreted the challenge strain of enterotoxigenic E. coli. None of the 18 calves in the three vaccinated groups developed clinical colibacillosis, and fecal excretion of the challenge organism was reduced. A combined enterotoxigenic E. coli-rotavirus vaccine may prove useful in preventing some outbreaks of calf diarrhea.     

Purification and characterization of a receptor for the 987P pilus of Escherichia coli.

A receptor-containing fraction that caused visible aggregation of piliated Escherichia coli strain 987 bacteria was released into solution when brush borders from adult rabbit small intestines were stored. The receptor-containing fraction was separated into more than 50 bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 4 of which contained carbohydrate as demonstrated by staining with periodic acid-Schiff. Receptor activity in the receptor-containing fraction was associated with a low-molecular-weight (less than 14,000) glycoprotein as measured by Western blots. Extraction of brush borders with chloroform-methanol-water (4:8:3) resulted in increased pilus 987P receptor activity in the aqueous extract. Gel filtration chromatography of sodium dodecyl sulfate-solubilized aqueous extract yielded a fraction with 987P receptor activity that contained only the low-molecular-weight glycoprotein. The purified 987P receptor contained 81% carbohydrate and 19% amino acids by weight. Isoelectric focusing separated the 987P receptor receptor into 3 peaks corresponding to isoelectric points of 2.2, 3.8, and 4.1. Each of the peaks contained 987P receptor activity. The 987P receptor activity was sensitive to periodate oxidation and to digestion with pronase. The strain 987-agglutinating activity of the receptor was inhibited by amino sugars and their N-acetylated derivatives, by compounds having a free amino group, by high concentrations of salt, and by lectins that recognize D-galactose or L-fucose.     

In vivo emergence of enterotoxigenic Escherichia coli variants lacking genes for K99 fimbriae and heat-stable enterotoxin.

Neonatal pigs were inoculated with porcine enterotoxigenic Escherichia coli 431, which carries genes for K99 fimbriae and STaP enterotoxin. Colonies of strain 431 were recovered from feces of pigs for up to 17 days after inoculation and tested for hybridization with gene probes for K99 and STaP. Variants of strain 431 that did not hybridize with the probes were considered to have lost the genes. Variants were recovered from 10 of 13 suckling pigs that survived the infection. Only 0.4% of the isolates recovered during the first 2 days after inoculation were variants. Of the isolates recovered 3 to 5 days after inoculation, 20 to 36% were variants. Variant colonies were detected more frequently among pigs in some litters than in others. The litter with the highest number of variant-shedding pigs had the dam with the highest titer of K99 antibody in her colostrum. Variants also occurred in colostrum-deprived, artificially reared pigs. However, the number of variants detected was lower and they occurred later in the course of the infection in colostrum-deprived pigs than in suckling pigs. More variants were detected and they were detected earlier in colostrum-deprived pigs fed anti-K99 monoclonal antibody than in controls fed anti-K88 monoclonal antibody. Loss of STaP appeared to be secondary to loss of K99 in that some variants lacked only K99 (K99- STaP+) and some lacked both genes (K99- STaP-), but none was of the K99+ STaP- type. Our results confirmed reports of gene loss from enterotoxigenic E. coli during infection. They are consistent with the hypothesis that variants emerge under in vivo selection pressure of K99 antibody and with the speculation that gene loss may be an important component of protection in vaccinated populations. However, the emergence of variants did not appear to play a major role in the recovery of individual pigs from clinical disease.