Expression in Escherichia coli K-12 of the 76,000-dalton iron-regulated outer membrane protein of Shigella flexneri confers sensitivity to cloacin DF13 in the absence of Shigella O antigen.

One of the chromosomal segments associated with virulence in Shigella flexneri encodes the production of aerobactin and the synthesis of an iron-regulated 76-kilodalton outer membrane protein believed to be the ferric-aerobactin receptor. However, S. flexneri expressing this putative aerobactin receptor, which is slightly larger than that encoded by pColV, is insensitive to the killing action of cloacin DF13, a bacteriocin which binds to other aerobactin receptor proteins and kills the cells. In this paper we show that the conjugal transfer of DNA encoding the iron-regulated 76-kilodalton protein from S. flexneri to Escherichia coli K-12 conferred cloacin DF13 sensitivity on the recipients. However, E. coli K-12 which had also inherited genes specifying Shigella O-antigen biosynthesis remained cloacin insensitive. The data suggest that it is unwise to use cloacin DF13 sensitivity alone to screen transconjugants or clinical isolates for the expression of aerobactin receptor proteins.     

Molecular cloning and expression of Neisseria meningitidis class 1 outer membrane protein in Escherichia coli K-12

A genomic library of meningococcal DNA from a clinical isolate of Neisseria meningitidis was constructed in the expression vector lambda gt11. Outer membrane complex was prepared from the same strain and used to immunize rabbits to raise polyclonal anti-outer membrane complex serum. The amplified library was probed with this polyclonal serum, and seven expressing recombinants were isolated; further investigations indicated these to be identical. The expressed meningococcal gene in these recombinants was fused to vector beta-galactosidase and shown to encode epitopes present on the 42-kilodalton class 1 outer membrane protein. Estimation of the size of the recombinant fusion protein suggests that up to 40 kilodaltons of protein-coding sequence is present. The lambda gt11 recombinant contains a 3.4-kilobase DNA insert, which has been recloned into a plasmid and characterized by restriction endonuclease analysis. A restriction fragment from the insert, representing the protein-coding region hybridizes to a single 2.2-kilobase XbaI fragment from the homologous strain and to similar-sized XbaI fragments in other strains of meningococci, expressing antigenically distinct class 1 proteins.     

A 101-kilodalton heme-binding protein associated with congo red binding and virulence of Shigella flexneri and enteroinvasive Escherichia coli strains.

The ability of Shigella flexneri to bind Congo red or hemin is associated with virulence. A 101-kilodalton (kDa) protein responsible for this phenotype (Crb+) in S. flexneri was identified by a tetramethylbenzidine staining procedure which detects heme-protein complexes in polyacrylamide gels. Labeling of cell-surface polypeptides with 125I revealed that the 101-kDa heme-binding protein is expressed on the cell surface. Expression of the protein was regulated by growth temperature and was found to be encoded by the large virulence plasmid of S. flexneri. Deletion mutants and a Tn5 insertion mutant which were negative for Congo red binding (Crb-) did not express the 101-kDa protein. Enteroinvasive Escherichia coli strains that were Crb+, and whose plasmids shared homology with the S. flexneri virulence plasmid, also expressed the 101-kDa protein. Expression of the protein in S. flexneri and enteroinvasive E. coli correlated with the presence of a 9.2-kilobase EcoRI fragment of these plasmids.     

ColV plasmid-specific aerobactin synthesis by invasive strains of Escherichia coli.

Certain strains of Escherichia coli associated with bacteremia of humans and domestic animals harbor plasmids that promote efficient iron uptake. The mechanism, which is an important component of the virulence of invasive strains, is independent of the enterobactin system for iron uptake. Plasmid-specified siderophore was assayed by its ability to support the growth of a chelator-deficient mutant in conditions of iron deprivation. The chelator, which was chemically determined to be a hydroxamate compound, was identical on the basis of field desorption mass spectrometry with aerobactin, a siderophore synthesized by Aerobacter aerogenes. In conditions of iron stress, aerobactin is secreted into the culture medium of plasmid-bearing E. coli strains. Reconstruction experiments involving a chelator-deficient mutant growing with exogenous chelator suggested that association of a small fraction of the total siderophore synthesized with cellular material is due to transient binding of aerobactin to membrane receptors during active bacterial growth.     

Genetic Transfer of Shigella flexneri Antigens to Escherichia coli K-12

The genes controlling synthesis of Shigella flexneri group- and type-specific antigens were transferred to Escherichia coli K-12 recipients by conjugation with an S. flexneri Hfr. After mating E. coli with an Hfr strain of S. flexneri 2a and selecting for his⁺ recombinants, a high proportion of the E. coli hybrids agglutinated in S. flexneri grouping serum. None of these hybrids expressed S. flexneri type-specific antigen II. When an E. coli his⁺ hybrid possessing the S. flexneri group antigen was remated with the same Hfr with selection for pro⁺ hybrids, a high proportion now expressed the type-specific antigen as well as the previously inherited group antigen. If such crosses were performed in reverse order (i.e., pro⁺ followed by his⁺ selection), a different pattern of serological behavior was observed. None of the pro⁺ hybrids showed the type-specific antigen. Subsequent mating for his⁺ resulted in hybrids with both the group- and type-specific antigens. These results show that genes controlling the synthesis of S. flexneri group antigen (linked to the his locus) and type-specific antigen (linked to the pro locus) are widely separated on the chromosome. Expression of the type-specific antigen II depends on the presence of the group antigen.     

Isolation and properties of complement-resistant strains of Escherichia coli K-12.

Several strains that were resistant to the bactericidal action of antibody and complement were isolated from Escherichia coli K-12 W3110/SM by selecting them through the medium containing antiserum and complement. They can be agglutinated by antiserum against the parent strain and showed similar immune adherence reactivity to the parent when sensitized with this antiserum. Few differences were found in the compositions of phospholipids and proteins between both inner and outer membranes of these strains and those of the parent. However, there were fewer short-chain and more long-chain fatty acids in these strains than in the parent. It was also found that unsaturated fatty acide decreased and saturated and cyclopropanoic acids increased in phosphatidylethanolamine and phosphatidylglycerol in both inner and outer membranes of one of these strains when compared with those from the parent. Therefore, the resistance of these strains to the complement-mediated bactericidal action was considered to be due to the rigidity of their membrane structures which might repel the insertion of membrane-attack complement complex C5b-9, although they could fix the earlier complement components up to the step of the formation of C4b,2a,3b complex enzyme.     

Competition between congenic Escherichia coli K-12 strains in vivo.

The ability of Escherichia coli to colonize the large bowels of animals is related to many factors inherent to the intestinal environment and the bacterium. The use of germfree mice eliminates the competition between E. coli and the other microflora and allows most E. coli strains to colonize. We found that E. coli K-12 strains differing in chromosomal antibiotic resistance could monoassociate in germfree mice in large numbers. However, when two or more strains were in competition with each other, we detected quantitative differences in the abilities of the strains to colonize. The order of colonizing ability was as follows: nalidixic acid resistance greater than streptomycin resistance greater than rifampin resistance. We also found that a nalidixic acid-resistant strain bearing plasmid pBR322 colonized less efficiently and at lower levels when in competition with the nalidixic acid-resistant strain. Studies of the membrane proteins of the various strains indicated that changes in membrane proteins occurred concomitantly with altered resistance to antimicrobial agents. These results suggest that chromosomally linked alterations in antimicrobial sensitivity may also reflect changes in membrane proteins and a decreased ability to colonize mammalian intestines in otherwise isogenic bacterial strains.     

Passive immunization with antibodies against iron-regulated outer membrane proteins protects turkeys from Escherichia coli septicemia.

Escherichia coli septicemia is a common disease of young poultry and several species of mammals. Rabbit antiserum was prepared against iron-regulated outer membrane proteins of E. coli. Eighteen-day-old turkeys were passively immunized with antiserum and challenged by air sac inoculation of 1 X 10(6) to 2 X 10(6) CFU of E. coli O78:K80:H9. Turkeys injected with normal rabbit serum or saline solution before challenge served as controls. Fatalities (8 of 51 turkeys inoculated) occurred only in groups given saline solution or normal rabbit serum. The remaining turkeys were necropsied 96 h after challenge. Passive immunization with antiserum significantly (P less than 0.05) reduced the frequency of bacteremia at 96 h after challenge, the frequency of recovery of E. coli from air sacs, and the severity of gross lesions in inoculated birds as compared with birds given normal rabbit serum or saline solution.     

Enterotoxin and cytotoxin production by enteroinvasive Escherichia coli.

It has long been suspected that besides their ability to invade enterocytes, enteroinvasive Escherichia coli (EIEC) strains have the ability to elaborate an enterotoxin. We tested 35 EIEC strains for cytotoxins and 9 (1 per serogroup) for enterotoxins. All 35 strains exhibited low levels of Vero cell cytotoxins that are immunologically and genetically distinct from Shiga-like toxin I or II of enterohemorrhagic E. coli. Sterile supernatants and cell lysates of two EIEC strains were tested in rabbit ileal loops, and both stimulated moderate fluid accumulation (circa 0.5 ml/cm) without tissue damage; secretory activity was confirmed in Ussing chambers, where these two strains and the seven others tested significantly increased short circuit current without altering tissue conductance. Curing the 140-MDa invasiveness plasmid from an EIEC strain did not diminish enterotoxin production. Culture in minimal Fe2+ medium is necessary to detect expression of the enterotoxin which is circa 68 to 80 kDa in size and is distinct from the EIEC cytotoxin.     

Presence and expression of aerobactin genes in virulent avian strains of Escherichia coli.

Virulent and nonvirulent isolates of avian Escherichia coli were tested for the presence of aerobactin genes by colony hybridization with a specific gene probe constructed from plasmid pABN1 (A. Bindereif and J. B. Neilands, J. Bacteriol. 153:1111-1113, 1983). Positive hybridization with the gene probe was highly correlated with virulence, as measured by the 50% lethal dose of the strains for chicks. Evidence for the expression of aerobactin genes in the virulent strains was obtained by demonstrating their susceptibility to cloacin DF13, which binds to the same receptor that binds aerobactin, and their ability to produce aerobactin, as revealed by cross-feeding the E. coli mutant WO987 (aroB fepA iuc iut+), which is unable to synthesize but capable of taking up aerobactin. We suggest that the production of aerobactin is involved in the virulence of avian septicemic E. coli.     

Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains.

We have employed a molecular genetic approach to characterize the nature of enteroinvasive Escherichia coli (EIEC) enterotoxic activity, as previously observed in Ussing chambers (A. Fasano, B.A. Kay, R.G. Russell, D.R. Maneval, Jr., and M.M. Levine, Infect. Immun. 58:3717-3723, 1990). The screening of TnphoA mutants of EIEC yielded a single insertion mutant which had significantly reduced levels of enterotoxic activity in the Ussing chamber assay. DNA flanking the insertion was used as a probe to screen for EIEC cosmid clones which conferred secretogenic activity. Such screening resulted in the identification of two overlapping cosmid clones which elicited significant changes in mucosal short-circuit current (Isc). Subcloning and nucleotide sequence analysis of a DNA fragment from one of the cosmid clones led to the identification of a single open reading frame which conferred this enterotoxic activity. By DNA hybridization, this gene (designated sen for shigella enterotoxin) was found in 75% of EIEC strains and 83% of Shigella strains and was localized to the inv plasmid of Shigella flexneri 2457T. By PCR, a sen gene with 99.7% nucleotide identity was cloned and sequenced from 2457T. A deletion in the EIEC sen gene was constructed by allelic exchange, resulting in significantly lower rises in Isc than were elicited by the wild-type parent; however, significant enterotoxic activity remained in the sen deletion mutant. To purify the Sen protein, the gene was cloned into the multiple cloning site of the expression vector pKK223-3. Purification of the sen gene product yielded a protein with a molecular mass of 63 kDa which elicited rises in Isc in the Ussing chamber. We believe that the sen gene product may constitute all or part of a novel enterotoxin in EIEC and Shigella spp.     

Oral vaccination of monkeys with an invasive Escherichia coli K-12 hybrid expressing Shigella flexneri 2a somatic antigen.

A living oral vaccine, designed to protect against Shigella flexneri 2a infections, was constructed by using Escherichia coli K-12 as a carrier strain. The hybrid strain, designated EC104, contained both chromosomal and plasmid genes from S. flexneri donor strains. In addition to expressing the S. flexneri 2a somatic antigen, it had inherited the property of epithelial-cell invasion. After the oral administration to rhesus monkeys, EC104 was isolated from the feces for up to 3 days, but by day 4 all stool cultures were negative. The serum antibody response against S. flexneri 2a somatic antigen was variable, but the vaccine conferred significant protection against an oral challenge with virulent S. flexneri 2a.     

Naturally occurring antibodies in human sera that react with the iron-regulated outer membrane proteins of Escherichia coli

Sera from normal healthy human adults and infants, as well as sera from mice, rabbits, and guinea pigs, were examined by immunoblotting for naturally occurring antibodies reacting with outer membrane proteins of two Escherichia coli strains, O111 and O18. Some individuals had antibodies reacting very strongly with the iron-regulated outer membrane proteins, including the ferric-enterochelin receptor protein (Mr, 81,000), as well as with ompA. However, sera from infants contained predominantly antibodies to ompA; antibodies recognizing the iron-regulated outer membrane proteins were either absent or barely detectable. In human serum the antibodies were mainly of the immunoglobulin G class. No serotype-specific antibodies to the lipopolysaccharide of E. coli O111 or O18 were found in the sera tested.     

Modified enzyme-linked immunosorbent assay for detecting enteroinvasive Escherichia coli and virulent Shigella strains.

Immune sera were produced in rabbits with living cells of an enteroinvasive O143 strain of Escherichia coli. To remove O and K antibodies, sera were absorbed with an avirulent derivative of the same strain. In the enzyme-linked immunosorbent assay, absorbed sera reacted specifically with only virulent Shigella strains and enteroinvasive E. coli strains of different geographical origin, regardless of species or serogroups. The investigation of 83 strains indicated complete agreement between enzyme-linked immunosorbent assay results and those of the keratoconjunctivitis test. It is assumed that the absorbed immune sera reacted with a possible virulence marker antigen. This inexpensive and simple method provides an alternative to other virulence tests. It has a definite advantage for screening large number of isolates within 24 h.     

Lack of Shiga-like cytotoxin production by enteroinvasive Escherichia coli.

Enteroinvasive Escherichia coli has not been extensively studied for cytotoxin production. We evaluated 30 well-characterized enteroinvasive E. coli strains of all the known invasive serogroups from several geographic regions for their ability to produce Shiga-like cytotoxic activity assayed in a HeLa cell system. None of these strains produced cytotoxic activity that was neutralizable with antibody to Shiga-like toxin I or II.     

Alterations in the pathogenicity of Escherichia coli K-12 after transfer of plasmid and chromosomal genes from Shigella flexneri.

A 140-megadalton plasmid (pWR110), which has previously been associated with virulence in Shigella flexneri, was transferred to Escherichia coli K-12. Segments of S. flexneri chromosomal material were then transferred to the plamid-bearing K-12 strains. The virulence of these transconjugant hybrids was assessed in the HeLa cell model, in ligated rabbit ileal loops, or in the Sereny test. A K-12 strain which harbored only pWR110 invaded HeLa cells, produced minimal lesions in the rabbit ileal mucosa, and was negative in the Sereny test. Plasmid-containing K-12 hybrids which had incorporated various shigella chromosomal regions gave differential reactions in the rabbit ileal loops and in the Sereny test. Analysis of these transconjugants indicated that three regions were linked with virulent phenotypes. These included the his region (when the genes responsible for O-antigen synthesis were cotransferred) and the kcp locus (linked to the lac-gal region). Either of these chromosomal regions was sufficient to allow invasion of the rabbit ileal mucosa. In addition to both of these regions, another shigella chromosomal segment linked to the arg and mtl loci was necessary for fluid production in the rabbit ileal loop and for a positive Sereny reaction. Thus, derivatives of an E. coli K-12 strain, constructed by the stepwise conjugal transfer of a large plasmid and three chromosomal segments from S. flexneri, appeared to contain the necessary determinants for full pathogenicity in a variety of laboratory models.     

Fusion of genes encoding Escherichia coli heat-stable enterotoxin and outer membrane protein OmpC.

The OmpC outer membrane protein of Escherichia coli was used as a carrier molecule for the nonimmunogenic heat-stable enterotoxin STa. Two fragments of different lengths of the gene encoding STa were fused in vitro to the 3' terminus of the truncated ompC gene. The resulting OmpC-STa hybrid proteins could be detected by L-[35S]cysteine labeling, and they were processed and thus exported. All synthesized hybrid protein remained cell bound and was found by fractionation mainly in the periplasm. Immunoblot analysis showed that the hybrid proteins reacted in vitro both with anti-OmpC and anti-STa antibodies, and immunization of rabbits evoked an antibody response to either of these proteins.     

Characterization of enteroinvasive Escherichia coli strains isolated from children with diarrhea in Chile.

Analysis of stool samples from 912 cases of diarrhea among Chilean infants and 1,112 controls resulted in the isolation of 17 enteroinvasive Escherichia coli (EIEC) strains from diarrhea cases (1.9%) and 3 EIEC from the asymptomatic controls (0.3%). Biochemical analysis of the 20 isolates showed variability among them. However, the majority were lysine decarboxylase negative and nonmotile and utilized sodium acetate. The strains belonged to the O groups 28ac, 124, 143, or 144 or were untypable with the antisera used. Most of them had conjugative plasmids which mediated multiple antibiotic resistance. There was a strong correlation in this group of strains between a positive Sereny test, the presence of a plasmid of 120 megadaltons, and hybridization with the invasiveness probe, an HindIII fragment derived from the plasmid pPS15A. The isolates had a wide range of plasmid profiles. Bioassays and colony and Southern hybridization tests with iron uptake DNA probes indicated that 80% of the EIEC strains produced aerobactin and expressed its receptor, the genes for which are known to be chromosomally located.     

Characterization of virulence plasmids and plasmid-associated outer membrane proteins in Shigella flexneri, Shigella sonnei, and Escherichia coli.

The 140-megadalton plasmids of Shigella flexneri serotypes 1, 3, and 5, in addition to the 120-megadalton plasmid of Shigella sonnei, are associated with virulence. The present study showed that a 140-megadalton plasmid is also associated with virulence in Escherichia coli. When these plasmids were cleaved with EcoRI or BamHI restriction endonucleases, considerable homology was evident in plasmids from S. sonnei strains, whereas only a few common fragments were observed among the S. flexneri and enteroinvasive E. coli plasmids. Nitrocellulose filter hybridization demonstrated that, despite variations in restriction sites, all these plasmids shared a considerable complement of homologous sequences. Minicell-producing strains were obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Transmission electron microscopy of infected HeLa cells showed that minicells from invasive strains retained the invasive phenotype. Sixteen polypeptides were labeled when S. flexneri 5 minicells were incubated with [35S]methionine. Fourteen of these plasmid-coded polypeptides were associated with the outer membrane in invasive strains of S. flexneri 5, and nine polypeptides of similar molecular weight were labeled in the outer membrane of invasive strains of S. flexneri 3, S. sonnei, and E. coli. Seven of the S. flexneri 5 polypeptides were not labeled in a noninvasive strain which had sustained a large deletion in the virulence-associated plasmid, and none were labeled in minicells which no longer harbored this plasmid.