Non-toxigenic mutants of "Bordetella pertussis".
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Animals↗
Biomedical subjects
S Falkow
Publications and source records attributed to S Falkow.
AFA-I, a cloned afimbrial X-type adhesin from a human pyelonephritic Escherichia coli strain. Purification and chemical, functional and serologic characterization.
AFA-I, a mannose-resistant, P-independent, X-binding afimbrial Escherichia coli adhesin was purified from a recombinant strain and chemically, functionally and serologically characterized. AFA-I exists on the bacterial surface and free as a macromolecular aggregate in the supernatant of spent culture medium. It is composed of a single, repeating 16-kDa polypeptide subunit. The AFA-I protein amino acid composition is remarkable for the presence of 22% non-polar hydrophobic residues and 2.5-3.0 cysteines per subunit. Since AFA-I travels as a monomer in sodium dodecyl sulfate/polyacrylamide gel electrophoresis under non-reducing conditions, no disulfide bonds exist between subunits and at least one free sulfhydryl per subunit is available. The AFA-I N-terminal amino acid sequence residues 1-24 was unrelated to E. coli fimbrial sequences; however, the N-terminus of AFA-I and GV-12, another E. coli afimbrial protein, was asparagine. HB101 (pIL 14), the AFA-I recombinant strain, agglutinated only human and gorilla erythrocytes, indicating a preference for receptor molecules on the red cells of man and the anthropoid apes. AFA-I did not bind glycophorin A or sialyl glycosides and is therefore distinct from the E. coli X-binding adhesins with M and S specificity. The AFA-I receptor was found to be abundant and diffusely distributed on HeLa tissue culture monolayer cell surfaces by indirect fluorescent microscopy. Anti-AFA-I sera bound AFA-I in Western blots of 4 out of 16 X-binding E. coli urine isolates. They did not bind MS or P pili. AFA-I may be exemplary of an adhesin class significant for the pathogenesis of human urinary tract infections.
Adhesins, Escherichia coli↗
Gal-Gal binding and hemolysin phenotypes and genotypes associated with uropathogenic Escherichia coli.
To determine whether uropathogenic strains of Escherichia coli exhibit a distinctive constellation of phenotypes, we examined 44 urinary isolates from women with radiologically normal urinary tracts and pyelonephritis, cystitis, or asymptomatic bacteriuria and 73 fecal isolates from healthy control subjects. The strains were characterized by their O serogroup, by their binding specificity (as determined by adhesins), and by their production of hemolysin and colicin V. In addition, the strains were assessed for homologous gene sequences by means of DNA-hybridization probes prepared from cistrons that encode hemolysin and the Gal-Gal binding adhesin--two determinants of virulence, which cause tissue injury and promote bacterial colonization of uroepithelia, respectively. In contrast to most isolates from normal feces and from the urine of patients with asymptomatic bacteriuria, pyelonephritis strains belong to a small number of O serogroups; all express the Gal--Gal binding adhesin and 75 per cent are hemolytic. A gene probe for the Gal--Gal binding adhesin, derived from the chromosome of one strain from a patient with pyelonephritis, hybridized with the DNA of all other pyelonephritis strains. The probe for the hemolysin gene hybridized with DNA from all other hemolytic strains. These data indicate that most cases of pyelonephritis are due to a small number of pathogenic clones that express critical determinants of virulence, and that the nucleotide sequences for hemolysin and the Gal--Gal binding adhesin in heterologous strains share homology. We are tempted to speculate that the gene products of these shared regions of the genome might form the basis for a vaccine against pyelonephritis.
Adhesiveness↗
Genetic analysis of bacterial virulence determinants in Bordetella pertussis and the pathogenetic Yersinia.
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Animals↗
Pertussis toxin is required for pertussis vaccine encephalopathy.
A mouse model for encephalopathy induced by pertussis immunization has been described; it has features that closely resemble some of the severe reactions, including seizures and a shock-like state leading to death, occasionally seen after administration of Bordetella pertussis (whooping cough) vaccine. Susceptibility to encephalopathy maps to genes of the major histocompatibility complex and correlates as well with the genetic regulation of the level of antibody response to bovine serum albumin. In this study we have investigated which bacterial determinant is responsible for the encephalopathy. Two lines of evidence implicate pertussis toxin as the active bacterial component. Single-site mutants of B. pertussis with single affected virulence factors were tested. A mutant that produces a defective pertussis toxin had greatly diminished capacity to induce encephalopathy, whereas a hemolysin- and adenylate-cyclase-deficient avirulent mutant had the same activity in the mouse model as a virulent strain. Purified pertussis toxin plus bovine serum albumin was tested and found to induce the lethal encephalopathy, demonstrating that the toxin was the critical constituent of B. pertussis responsible for encephalopathy.
Adenylate Cyclase Toxin↗
Transfer of the virulence plasmid of Yersinia pestis to Yersinia pseudotuberculosis.
Transposon Tn5 insertion derivatives of the virulence plasmid pYV019 of Yersinia pestis were transferred by P1 transduction into a plasmid-free strain of Y. pseudotuberculosis. One of these plasmid derivatives conferred virulence upon the Y. pseudotuberculosis strain. This strain had the ability to express temperature-inducible plasmid-coded outer membrane proteins and was also found to be Ca2+ dependent.
Animals↗
Nucleotide sequence of pilA, the gene encoding the structural component of type 1 pili in Escherichia coli.
The pilA gene of Escherichia coli J96 encoding pilin, the structural component of type 1 pili, was sequenced and found to specify a polypeptide 159 amino acids long preceded by a 23-amino-acid signal peptide. As determined from the DNA sequence, the mature peptide lacked tryptophane and methionine, two amino acids previously shown to be lacking in type 1 pili from E. coli. Also, the amino-terminal sequence of amino acids inferred from the DNA sequence corresponded to earlier 20-amino-acid amino-terminal sequences determined by protein sequencing. In addition, piliation was abolished after a mutation was introduced into the pilA coding region in vitro. A possible site for initiation of transcription and a possible site encoding translation initiation were suggested 85 and 7 base pairs, respectively, from the pilA start codon. There appeared to be scant DNA sequence homology and scant amino acid sequence homology between type 1 pilin and other pilin species isolated from uropathogenic and enteropathogenic E. coli.
Amino Acid Sequence↗
Frequency among Enterobacteriaceae of the DNA sequences encoding type 1 pili.
Type 1 pili, characterized by mannose-inhibitable agglutination of fowl or guinea pig erythrocytes, have been found throughout the family Enterobacteriaceae. A radiolabeled probe was prepared from a restriction endonuclease-digested fragment of the Escherichia coli pil operon and used to detect homologous DNA sequences in 236 bacteria representing 11 genera of Enterobacteriaceae. Only isolates identified as E. coli or Shigella spp. exhibited homology. In contrast, mannose-sensitive hemagglutination was observed in nine genera. Probe DNA did not hybridize to plasmid DNA, indicating a chromosomal location for the pil operon. Analysis of restriction nuclease-digested whole-cell DNA from 60 E. coli and two Shigella sp. isolates indicated that internal sequences were conserved in most strains, but that changes in flanking sequences in the chromosome were common.
DNA Restriction Enzymes↗
Genetic organization of the afimbrial adhesin operon and nucleotide sequence from a uropathogenic Escherichia coli gene encoding an afimbrial adhesin.
The uropathogenic Escherichia coli KS52 strain expresses a mannose-resistant hemagglutinin AFA-I, which recognizes a human erythrocyte site distinct from the alpha-digalactoside glycosphingolipid receptor common to uropathogenic E. coli strains specifying a P adhesin. A 6.7-kilobase chromosomal DNA fragment was cloned from KS52 into pBR322 and was shown to be necessary for host cell mannose-resistant hemagglutination expression and uroepithelial cell adherence (Labigne-Roussel et al., Infect. Immun. 46:251-259, 1984). The genetic organization of the 6.7-kilobase DNA fragment was investigated by generating derivative plasmids, and the polypeptides encoded by those plasmids in isolated minicells were analyzed on polyacrylamide gel. The 6.7-kilobase insert expresses five polypeptides of molecular mass 13,000, 16,000, 18,500, 30,000, and 100,000 daltons encoded, respectively, by the afaA, afaE, afaD, afaB, and afaC genes. The five genes were localized and were shown to belong to the same transcriptional unit. The afaB, afaC, and afaE gene products are required for mannose-resistant hemagglutination expression, whereas mutations in or deletions of either afaA or afaD do not modify host mannose-resistant hemagglutination expression. The afaE gene was identified as the structural gene encoding hemagglutinin. The gene has been sequenced; it encodes a 152-residue protein containing a typical 21-residue procaryote signal sequence and a 131-residue mature polypeptide, the AFA-I adhesin.
Adhesiveness↗
Cloning and sequencing of a Moraxella bovis pilin gene.
Moraxella bovis pili have been shown to play a major role in both infectivity and protective immunity of bovine infectious keratoconjunctivitis. Sonicated M. bovis DNA from the piliated strain EPP63 was inserted into the vector lambda gt11 with EcoRI linkers. Recombinant phage were screened with an oligonucleotide probe based on the amino-terminal portion of the DNA sequence of a Neisseria gonorrhoeae pilin gene. Two candidate phages produced a protein that comigrated with EPP63 beta pilin in sodium dodecyl sulfate-polyacrylamide gels and bound anti-pilus antisera. The 1.9-kilobase insert from one of these, lambda gt11M182, was subcloned in both orientations into pBR322, forming the plasmids pMxB7 and pMxB9, both of which produced beta pilin, as did pMxB12, a HindIII deletion derivative of pMxB7. In HB101(pMxB12), the M. bovis pilin protein was shown to be primarily localized in the inner membrane. The entire 939-base-pair insert of pMxB12 was sequenced, revealing a ribosome binding site just upstream of the coding region and an AT-rich region further upstream containing some potential RNA polymerase recognition sites. The translation of the sequence predicts a six-amino-acid leader sequence preceding the phenylalanine that begins the mature protein. Codon usage analysis of the M. bovis beta pilin gene revealed greater use of the CUA codon for leucine than usual for a well-expressed Escherichia coli gene. Comparisons of the M. bovis EPP63 beta pilin protein sequence with other pilin gene sequences are presented.
Amino Acid Sequence↗
Two modes of control of pilA, the gene encoding type 1 pilin in Escherichia coli.
Type 1 piliation in Escherichia coli is subject to metastable regulation at the transcriptional level (B. I. Eisenstein, Science 214:337-339, 1981). However, the genes controlling in this fashion are not known. We present evidence that the pilA gene, encoding the structural subunit of type 1 pili, is subject to metastable transcriptional regulation. A pilA'-lacZ fusion, constructed in vitro on a recombinant plasmid, was used in conjunction with a recBC sbcB mutant of E. coli K-12 to introduce the fusion into the chromosomal region encoding Pil. This fusion was found to be subject to metastable transcriptional control. The rate of switching from the Lac+ to the Lac- phenotype was 4 X 10(-4) per cell per generation and 6.2 X 10(-4) in the opposite direction. A ca. 10-fold difference in beta-galactosidase activity was observed between phenotypically "ON" (Lac+) and "OFF" (Lac-) populations. P1 transduction experiments showed that the element determining the ON or OFF phenotype was tightly linked to pilA. In addition to the metastable regulation of pilA, a second type of transcriptional regulation was effected by the product of a gene, hyp, adjacent to pilA. By using a recombinant plasmid containing just a pilA'-lacZ fusion and the putative pilA promoter, we found that a lesion in hyp conferred a beta-galactosidase activity about fivefold higher than that of a strain possessing the parental hyp gene. Mutants constructed to have a pilA'-lacZ fusion and a hyp::Tn5-132 mutation in the chromosome exhibited a frequency of switching from Lac+ to Lac- and vice versa indistinguishable from that of the parental strain. However, in the ON mode, hyp::Tn5-132 mutants showed a twofold-higher beta-galactosidase activity. Thus, hyp does not appear to affect metastable variation but does affect the level of transcription of the pilA gene in the ON (transcribed) mode.
Bacterial Outer Membrane Proteins↗
Molecular basis of Escherichia coli colonization of the upper urinary tract in BALB/c mice. Gal-Gal pili immunization prevents Escherichia coli pyelonephritis in the BALB/c mouse model of human pyelonephritis.
Most human pyelonephritis Escherichia coli isolates express both mannose (MS)- and globoside (Gal-Gal)-binding pili. An ascending E. coli urinary tract infection model was established in the 16-wk-old female BALB/c mouse to compare the pathogenic significance of MS and Gal-Gal pili and their efficacy as vaccines for the prevention of pyelonephritis. The distribution and density of pilus receptor compounds in urogenital tissues and as soluble compounds in urine were determined with antibodies to the synthetic receptor analogues, alpha D-Gal(1----4) beta D-Gal and alpha D-Man(1----2) alpha D-Man. Both carbohydrates were detected in vagina, bladder, ureter, and renal pelvis epithelium and in collecting duct and tubular cells. A pilus receptor compound also was detected in urine. It competitively inhibited the binding capacity of MS pili and was found to be physically, chemically, and immunologically related to Tamm-Horsfall uromucoid. Infectivity and invasiveness were quantitatively and histologically characterized for four E. coli strains: J96, a human pyelonephritis strain that expresses both MS and Gal-Gal pili; two recombinant strains prepared from J96 chromosomal DNA encoding MS pili or Gal-Gal pili; and the nonpiliated K12 recipient. Intravesicular administration of J96 (10(6) colony-forming units [CFU]) resulted in renal colonization and invasion in each of nine mice. The Gal-Gal clone (10(6) CFU) colonized the kidneys in each of 10 mice but did not invade. In contrast, the MS clone (10(6) CFU) did not colonize renal epithelium or invade. This effect was superceded when larger doses (greater than or equal to 10(10) CFU) of the MS clone were administered in volumes that cause acute vesicoureteric reflux. The efficacy was determined of vaccines composed of pure MS or Gal-Gal pili or the lipopolysaccharide containing O somatic antigen of the challenge strain, J96. The Gal-Gal pilus vaccine blocked renal colonization in 19 of 22 mice and renal invasion in 10 of 11 mice. Gal-Gal pili may be useful immunogens for the prevention of pyelonephritis in anatomically normal urinary tracts.
Animals↗
Genetic studies of the molecular basis of whooping cough.
The etiologic agent of whooping cough, Bordetella pertussis, synthesizes several biochemically and biologically active factors believed to contribute to the disease process. They include filamentous hemagglutinin (FHA), pertussis toxin (PTX), adenylate cyclase toxin (AC), and hemolysin (HLY). We have used a genetic approach to evaluate the contribution of these factors to the virulence of B. pertussis. A series of B. pertussis mutants prepared by transposon Tn5 insertion mutagenesis were characterized and determined to be specifically deficient in the elaboration of these factors. The wild type strain and the mutants were tested for their ability to produce a lethal infection in the classical infant mouse intranasal infection model. The dose of organisms required to cause lethal infection in 50% of the animals (LD50) for the wild type strain was 2 X 10(3) bacteria. A mutant deficient in production of the proposed adherence factor, FHA, was equivalent in virulence to the wild type strain (BP353, LD50 = 8 X 10(3)). In contrast, mutants deficient in production of PTX alone (BP357, LD50 = 5 X 10(6)), AC and HLY (BP348, LD50 greater than 3 X 10(7)), or all known toxins and virulence factors (BP347, LD50 greater than 3 X 10(7)) were severely impaired in their ability to cause a lethal infection. Our data indicate the hemolysin and adenylate cyclase as well as pertussis toxin are important virulence factors and provide the basis for a working model of the molecular basis of the disease caused by Bordetella pertussis.
Adenylate Cyclase Toxin↗
Murine model for pertussis vaccine encephalopathy: role of the major histocompatibility complex; antibody to albumin and to Bordetella pertussis and pertussis toxin.
A mouse model for pertussis immunization encephalopathy has been described with features that closely resemble the severe adverse reactions occasionally seen after pertussis vaccine administration,m including seizures and a shock-like state leading to death. These reactions are produced with nearly one hundred percent efficiency provided that the mice immunized with Bordetella pertussis have 1) the appropriate major histocompatibility (H-2) genotype, 2) have been sensitized to bovine serum albumin (BSA), and 3) that the injected B. pertussis contained sufficient amounts of pertussis toxin. Antibody titres were measured in mice with haplotypes H-2d.s.k. that are highly susceptible to encephalopathy as well as in H-2b mice, that are totally resistant. Mice with H-2d.s.k. haplotypes were high responders to BSA, while H-2b (B10) mice were non-responders to BSA. Both H-2d and H-2b mice responded well to B. pertussis. Encephalopathy was induced in resistant H-2b mice with B. pertussis and passively administered anti-BSA antiserum, but not with B. pertussis and anti-(T,G)-A--L antibody. This indicated that B. pertussis and anti-BSA were absolutely required for development of encephalopathy. Encephalopathy could be induced in mice decomplemented with cobra venom factor and given BSA and B. pertussis. Several single-site mutants of B. pertussis affecting single virulence factors were induced with transposon Tn5. One of these mutants, BP357, deficient in pertussis toxin production, had a greatly reduced encephalopathic potential in the mouse model compared to the virulent strain BP 338, or to BP348, an adenylate cyclase and hemolysin double mutant, or to BP 349, a hemolysin mutant.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenylate Cyclase Toxin↗
Hepatic Hodgkin's disease simulating cholestatic hepatitis with liver failure.
Fulminant liver failure is an unusual complication that may be seen in patients with hepatic infiltration by non-Hodgkin's lymphomas and leukemias. In such cases, hepatomegaly, marked abnormalities of serum liver function tests, and lactic acidosis may simulate severe viral, alcoholic, or drug hepatitis. We describe the clinical presentation of severe cholestatic hepatitis with liver failure in a patient who was found to have widespread liver involvement by Hodgkin's disease at autopsy. The major histologic findings included extensive portal and periportal Hodgkin's infiltrates, associated loss of periportal liver cells, and variable damage to small bile ducts.
Aged↗
Mutations in E coli cistrons affecting adhesion to human cells do not abolish Pap pili fiber formation.
A chromosomal DNA fragment which mediates Pap (pili associated with pyelonephritis) pili formation, mannose-resistant hemagglutination ( MRHA ) and binding to uroepithelial cells has been isolated from the uropathogenic Escherichia coli clinical isolate J96 , and genetically studied. Analysis of polypeptides expressed by the Pap DNA led to detection of a number of polypeptides ranging in mol. wt. from 13 000 to 81 000 daltons. The gene order and transcriptional orientation for four of the corresponding cistrons was: 13 000 ( papB ) 19 500 ( papA , structural gene for the Pap pilus subunit), 81 000 ( papC ) and 28 500 ( papD ). Analyses of a lacZ- papA gene fusion located a promoter upstream from papA within the cloned DNA. Transposon Tn5 insertions in any of these four cistrons decreased or eliminated Pap pili formation. A number of transposon Tn5 mutations were identified in a region distal to papD that expressed normal levels of the papA protein on the cell surface in the form of recognizable pili structures but did not agglutinate human erythrocytes or adhere to uroepithelial cells. This region expressed polypeptides of 15 000, 24 000, 26 000 and 35 000 daltons. This finding shows that Pap pili formation and binding properties can be genetically dissociated.
Antigens, Bacterial↗
A simple, sensitive, and specific assay for the heat-stable enterotoxin of Escherichia coli.
A simple assay for the heat-stable enterotoxin (ST) of Escherichia coli was developed on the basis of ST activation of guanylate cyclase in membranes from the intestinal mucosa of mice. ST activated guanylate cyclase in mucosal membranes in a linear fashion over a 50-fold range of toxin concentrations with Mg++-guanosine 5'-triphosphate as substrate. Activation of guanylate cyclase was detectable at concentrations of ST that were five- to 10-fold lower than those resulting in increases in the ratio of gut weight to carcass weight of mice. This assay was used to quantify ST in crude and purified samples from culture filtrates of wild-type strains and recombinant strains of E coli containing the gene for ST. Activation of guanylate cyclase was specific for ST; purified cholera toxin and E coli heat-labile enterotoxin did not activate guanylate cyclase. Thus, this assay for ST is sensitive, specific, and will facilitate rapid analysis of samples for quantification of ST.
Animals↗
Pertussis toxin and extracytoplasmic adenylate cyclase as virulence factors of Bordetella pertussis.
A wild-type strain of Bordetella pertussis and a series of transposon Tn5-induced mutants deficient in the production of various factors believed to play a role in pertussis (whooping cough) were tested for virulence in infant mice. The 50% lethal dose of the wild-type strain in these animals was 2 X 10(3) bacteria. A mutant deficient in the production of the filamentous hemagglutinin was almost as virulent as the wild type. Avirulent phase mutants (i.e., deficient in the production of all toxins), a pertussis-toxin mutant, and a double mutant deficient in both hemolysin and adenylate cyclase were severely impaired in the ability to cause pertussis. These data underscore the role of pertussis toxin in the disease and provide the first direct evidence that adenylate cyclase and hemolysin are important in the pathogenesis of the infection.
Adenylate Cyclase Toxin↗