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E C Gotschlich

Publications and source records attributed to E C Gotschlich.

At least 19 recordsLinked to original sources
PubMed1992

Neisseria meningitidis encodes an FK506-inhibitable rotamase.

Eukaryotic peptidyl-prolyl cis-trans isomerases (rotamases) fall into two classes, the cyclophilins inhibited by cyclosporin A and the FK506-binding proteins inhibited by the macrolide antibiotic FK506. In prokaryotes homologs of cyclophilins have been identified and found to have rotamase activity. Sequence similarities have been noted between FK506-binding proteins and gene products in a number of bacterial species, but whether these bacterial proteins have rotamase activity is not known. Using the polymerase chain reaction, we have cloned and sequenced a homolog of an FK506-binding protein from Neisseria meningitidis and expressed the gene product as a fusion protein with maltose-binding protein. The fusion protein was purified by affinity chromatography. By measuring the rate of chymotrypsin cleavage of the substrate succinyl-Ala-Ala-Pro-Phe p-nitroanilide, we found that the fusion protein had rotamase activity comparable to that of human FK506-binding protein. This rotamase activity was inhibited by FK506.

Amino Acid Sequence↗
PubMed1992

Gonococcal porin vaccine evaluation: comparison of Por proteosomes, liposomes, and blebs isolated from rmp deletion mutants.

The gonococcal major outer membrane protein, Por (protein I), is a potential vaccine candidate. A large-scale animal trial compared the immunogenicity of Por inserted in liposomes (mimicking its in vivo structure) with previously used vaccines containing Por. Por was purified from Rmp (protein III)-negative gonococcal mutants and made into five different formulations: proteosomes, proteosomes absorbed to alum, liposomes, gonococcal membrane blebs, and gonococcal membrane blebs absorbed to alum. Por liposomes induced the greatest amount of Por antibodies; proteosomes and both preparations of blebs induced minimal amounts of Por antibodies. Proteosomes absorbed to alum induced slightly lower amounts of Por antibodies than did liposomes, especially when protein IA was used. Whole-organism absorption studies revealed that a significantly greater percentage of liposome-induced Por antibodies recognized exposed portions of the protein than did proteosome- or proteosome/alum-induced antibodies.

Adjuvants, Immunologic↗
PubMed1992

Gonococcal lipooligosaccharide sialylation prevents complement-dependent killing by immune sera.

Previous investigators have demonstrated that a sialic acid residue is added to the terminal galactose moiety of gonococcal lipooligosaccharide (LOS) when incubated with 5'-CMP-N-acetylneuraminic acid. When this in vitro sialylation occurs, gonococci become resistant to the bactericidal activity of normal human serum. This is believed to result because the added sialic acid residue blocks the binding of bactericidal anti-LOS antibodies present in normal human serum. We extend these studies by demonstrating that sialylated gonococci also become resistant to the bactericidal effect of immune sera containing antibodies that recognize exposed components of the outer membrane besides LOS. Prevention of antibody binding to the organism was not the cause, since the same percentage of bactericidal antibodies to the major outer membrane protein, Protein I, can be absorbed with sialylated organisms as with wild-type organisms. In addition, gonococcal sialylation prevents opsonophagocytosis by antigonococcal antisera. The negative effect of sialic acid on the complement pathway might be the reason for the findings in this study.

Antigens, Bacterial↗
PubMed1992

Elimination of the vitamin B12 uptake or synthesis pathway does not diminish the virulence of Escherichia coli K1 or Salmonella typhimurium in three model systems.

The role of iron in infection is of great importance and is well understood. During infection, both the host and the pathogen go through many complicated changes to regulate iron levels. Iron and vitamin B12 share certain features. For example, Escherichia coli has similar transport systems for both nutrients, and binding proteins for both are located in gastric juice, liver, saliva, granulocytes, and milk. It is because of such parallels between iron and B12 that we have explored the role of B12 in virulence. A btuB::Tn10 insertion which disrupts the gene encoding the vitamin B12 receptor from E. coli K-12 was P1 transduced into a virulent E. coli K1 strain. In both an infant-rat model and a chicken embryo model, no difference in virulence between the wild-type and the mutant strains was found. Strains of Salmonella typhimurium with mutations in the cobalamin synthesis pathway (Cob) and in btuB were used in a mouse model of virulence. Mutation of the Cob locus or of btuB does not decrease virulence. Interestingly, the inability to synthesize vitamin B12 actually increases virulence compared with the wild type in the S. typhimurium model. This effect is independent of the B12 intake of the mice.

Animals↗
PubMed1991

Outer membrane protein A (OmpA) contributes to serum resistance and pathogenicity of Escherichia coli K-1.

We examined whether outer membrane protein A (OmpA) contributes to gram-negative pathogenesis by determining the effect of mutagenesis of ompA in a virulent Escherichia coli K-1 isolate. An OmpA mutant was generated by insertion of the transposon TnphoA, which was genetically modified to increase the efficiency of its delivery by conjugation. The mutant was less virulent than its parent strain in two models of E. coli K-1 infection. Equal inocula of the OmpA+ and OmpA- strains fed to neonatal rats resulted in a sevenfold-greater incidence of bacteremia at 72 h from the OmpA+ strain. The lethal effect of the OmpA- mutant was significantly less than that of the OmpA+ parent strain when inoculated onto the chorioallantoic membrane of 10-day embryonated chick eggs. There was, however, no difference between strains in growth characteristics under physiologic conditions, either in rat serum or in unembryonated chick eggs. In the presence of a 10-day chick embryo, there was a 10-fold increase in the survival and growth of the OmpA+ strain. Correction of the mutation in ompA with an E. coli K-12 ompA gene restored a level of virulence equivalent to that of the parent strain. The ompA mutant was more sensitive to the bactericidal effect of pooled human serum by the classical pathway of complement activation. These results suggest that OmpA contributes to E. coli K-1 pathogenesis by a mechanism which may involve increased serum resistance.

Animals↗
PubMed1991

High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor.

Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5'-MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GGMECC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.

Conjugation, Genetic↗
PubMed1990

Surface array protein of Campylobacter fetus. Cloning and gene structure.

The high molecular mass (97-149 kDa) surface array proteins (SAP) of Campylobacter fetus are critical to virulence. We created a bank of 160,000 random 1.0-6.5-kilobase (kb) chromosomal DNA fragments of C. fetus strain 84-32 (23D) using lambda gt11. Screening this bank in Escherichia coli Y1090 with antibody raised against purified SAP permitted isolation and purification of a clone with a 4.0-kb insert. Subcloning this insert in the E. coli vector, pUC9, permitted expression of a protein of approximately 100 kDa, not fused with beta-galactoside or inducible by isopropyl-beta-D-thiogalactopyranoside. Digestion with restriction endonucleases, and construction of deletion mutations indicated that the gene extended over 2.8 kb, proceeding toward the start of the beta-galactosidase gene. Taking advantage of a unique PstI site at 1.7 kb, we subcloned PstI-EcoRI fragments in both orientations into M13 vectors, then generated and sequenced 48 deletion mutants. In the 3974-base insert, an open reading frame, beginning at nucleotide 24 and terminating at 2825, was found to encode a 933-amino acid polypeptide having a calculated molecular mass of 96,758 daltons. The first 20 amino acids exactly match those determined from amino-terminal sequencing, indicating that this protein is secreted without a leader sequence. The deduced amino acid composition matches that of the purified SAP. We identified a ribosomal binding site 9 bases upstream, and a putative transcription terminator (delta G = -12.4) 21 bases downstream. There is partial homology of primary and secondary structure with five other bacterial S-layer proteins.

Amino Acid Sequence↗
PubMed1989

The construction and characterization of Neisseria gonorrhoeae lacking protein III in its outer membrane.

Protein III (PIII) is a highly conserved, antigenically stable gonococcal outer membrane protein that is closely associated with the major outer membrane protein, protein I (PI). We have previously reported the cloning of the PIII gene. This gene was inserted into the Eco RI site of the runaway plasmid pMOB45. The beta-lactamase (beta la) Bam HI restriction fragment from the gonococcal plasmid pFA3 was inserted at the Xba I site in the PIII gene. The plasmid construct was Hae III methylated and the PIII/beta la insert was excised with Eco RI and used to transform gonococcal strain F62. One beta la+, ampicillin-resistant transformant was isolated and designated 2D. A Western blot of 2D whole cell lysate was probed with affinity-purified polyclonal PIII antisera. No PIII reactivity was detected. Southern blot analysis was performed on F62 and 2D chromosomal DNA that were cut with Eco RI or Cla I. A PIII DNA probe hybridized with fragments 2.2 kb larger in strain 2D than strain F62. This corresponds to the size of the beta la insert. A beta la-specific probe hybridized with the same 2D restriction fragments as above, but did not react with any F62 fragments, confirming that homologous recombination had occurred. There were minimal phenotypic changes between 2D and its parent strain, F62. Chromosomal DNA from 2D was able to transform gonococcal strains F62, UU1, and Pgh 3-2, rendering these PIII-. 2D and other PIII- transformants can now be used to study the role of PIII in gonococcal physiology, metabolism, membrane structure, and pathogenesis. Moreover, we now have organisms from which we can purify gonococcal proteins without PIII contamination.

Ampicillin Resistance↗
PubMed1989

The virulence-associated gonococcal H.8 gene encodes 14 tandemly repeated pentapeptides.

H.8 is a virulence-associated, surface-exposed immunogenic macromolecule composed of lipid and protein, common to Neisseria gonorrhoeae and Neisseria meningitidis. The H.8 DNA sequence predicted a 6.9 kD peptide comprising 14 tandemly repeated pentameric sequences. Ten were identical: Pro, Ala, Ala, Glu, Ala. Also predicted was a lipoprotein leader consensus sequence which probably specified acylation since the Escherichia coli-expressed protein was tightly associated with lipid. Lipid appeared to contribute significantly to H.8 antigen's electrophoretic mobility. This is the first description of a prokaryotic outer membrane protein composed solely of tandem repeats. Furthermore, DNA encoding this repeat appears to have been duplicated and translocated into another neisserial gene encoding an azurin.

Amino Acid Sequence↗
PubMed1989

Sequence of the structural gene (rmpM) for the class 4 outer membrane protein of Neisseria meningitidis, homology of the protein to gonococcal protein III and Escherichia coli OmpA, and construction of meningococcal strains that lack class 4 protein.

The structural gene (rmpM) of the class 4 outer membrane protein of Neisseria meningitidis has been cloned and sequenced. The derived amino acid sequence reveals a 218-amino-acid protein following a 22-amino-acid signal peptide. The protein shows 94.2% homology with protein III of Neisseria gonorrhoeae and shares its two potential disulfide loops. The protein also shares limited homology with Escherichia coli OmpA. N. gonorrhoeae protein III has been shown to elicit blocking antibodies that prevent the killing of serum-resistant strains by immune sera (P. A. Rice, H. E. Vayo, M. R. Tam, and M. S. Blake, J. Exp. Med. 164:1735-1748, 1986). The very close homology of meningococcal class 4 protein with gonococcal protein III suggests that meningococcal outer membrane preparations containing class 4 protein may similarly stimulate blocking antibodies. In order to investigate the role of the class 4 protein in the pathogenesis of meningococcal infection, we have used an erythromycin resistance gene in developing two meningococcal strains that lack class 4 protein.

Amino Acid Sequence↗
PubMed1989

Cloning and characterization of the structural gene for the class 2 protein of Neisseria meningitidis.

The class 2 protein of Neisseria meningitidis is the major outer membrane protein and a porin. A lambda gt11 bank of meningococcal chromosomal DNA was screened with monoclonal antibodies against gonococcal protein IB that cross-react with meningococcal class 2 protein. Three independent immunoreactive clones were isolated. DNA sequence analysis indicated that these clones included regions encoding the N-terminal portion of the class 2 protein. An oligonucleotide of 21 bases that was complementary to this sequence was synthesized and used as a probe for a second screening of the lambda gt11 bank. One of the positive clones isolated contained the complete gene, including the ribosome binding site, but lacked the promoter region. On the basis of the DNA sequence, a protein of 360 amino acids was predicted. Comparison of the predicted protein sequence with that of gonococcal protein I showed little homology in six regions constituting 29% of the total amino acids, while the remainder of the coding frame showed 81% homology of amino acid residues. The DNA homology in the immediate 5' and 3' noncoding sequences was very striking. Following the putative transcription terminator, the 3' DNA sequence contained a complex pattern of direct and inverted repeats having some similarity to the 3' sequence of the gonococcal protein IB gene and very close homology to a sequence located in the pilS1 region (R. Haas and T. F. Meyer, Cell 44:107, 1986), an area of the gonococcal genome containing silent pilin genes.

Amino Acid Sequence↗
PubMed1988

Characterization and specificity of antibodies to protein I of Neisseria gonorrhoeae produced by injection with various protein I-adjuvant preparations.

A major goal of gonococcal research is the development of a gonorrheal vaccine. A vaccine candidate is the major outer membrane protein (PI) of the gonococcus, which has limited antigenic variability. Two main subtypes, PIA and PIB, and nine main serotypes have been described. To avoid raising anti-protein III (PIII)-blocking antibodies and limit potential lipooligosaccharide toxicity, PI was chromatographically isolated with minimal PIII contamination (less than 1%) from Pgh 3-2 (PIB), a serum-sensitive gonococcal strain and UU1 (PIA), a serum-resistant gonococcal strain. Alum was used as an adjuvant and the antibodies raised in rabbits did not agglutinate the organisms, were not opsonic, and bactericidal titers were not increased. To present PI in a form mimicking its in vivo disposition, it was inserted into liposomes. The resulting antisera did agglutinate the organism and contained opsonic and bactericidal activity greater than the preimmune sera or alum-generated sera. The PIB liposome antisera also had higher ELISA titers to a synthetic peptide equivalent to an exposed portion of PIB and a higher percentage of antibodies absorbed by whole organisms than the PIB alum antisera. We speculate that when PI is presented in liposomes, the antibodies raised are mainly to surface-exposed epitopes of the protein as opposed to when PI is presented absorbed to alum, where the antibodies are produced mainly to buried epitopes.

Adjuvants, Immunologic↗