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R Morona

Publications and source records attributed to R Morona.

54 records · Page 3Linked to original sources
PubMed1991

Characterization and immunogenicity of EX880, a Salmonella typhi Ty21a-based clone which produces Vibrio cholerae O antigen.

EX645 is a derivative of Salmonella typhi Ty21a which carries a plasmid specifying production of Vibrio cholerae O antigen. When cultured with exogenous galactose to overcome the galE defect of the vector, EX645 also synthesizes S. typhi O antigen, and this can result in the masking of the shorter V. cholerae O antigen on the bacterial surface. To determine whether the potential for such masking at least partly underlies the inconsistency of anti-V. cholerae responses elicited by EX645, a derivative of this strain has been isolated, characterized, and tested for immunogenicity in human volunteers. EX880 has an rfb defect which prevents synthesis of S. typhi O antigen, and consequently V. cholerae O antigen is still detectable on the surface of the clone following growth in the presence of galactose. Compared with EX645, EX880 more consistently elicited significant rises in serum bactericidal antibody levels, although individual responses within a cohort still varied widely.

Antibodies, Bacterial↗
PubMed1990

Regions of the cloned Vibrio cholerae rfb genes needed to determine the Ogawa form of the O-antigen.

The O-antigen of the lipopolysaccharides of Vibrio cholerae 01 can exist in two forms termed Inaba and Ogawa. We used a complementation system to demonstrate that the Ogawa phenotype is dominant over the Inaba phenotype. By using a set of deletions affecting the Ogawa rfb genes, we identified two regions which are needed to confer the Ogawa phenotype. In vitro mutagenesis of the cloned Ogawa rfb genes resulted in the isolation of variants with the Inaba phenotype. The results are interpreted with respect to previous studies demonstrating interconversion between the two forms of the V. cholerae O-antigen.

Antigens, Bacterial↗
PubMed1990

Surface co-expression of Vibrio cholerae and Salmonella typhi O-antigens on Ty21a clone EX210.

In an attempt to construct a bivalent, live, oral cholera-typhoid vaccine, genes specifying the biosynthesis of Vibrio cholerae O-antigen have been transferred into a modified version of the attenuated, oral typhoid vaccine strain Salmonella typhi Ty21a. The present report investigates the production of V. cholerae and S. typhi O-antigens by one such clone, EX210. When cultured without galactose supplementation EX210 produces surface O-antigen of V. cholerae type, as detected by haemagglutination-inhibition and bactericidal assays, and by immuno-electron microscopy. However, the protective efficacy of Ty21a depends upon growth in the presence of exogenous galactose and under these conditions only S. typhi O-antigen is detectable on the surface of EX210. Subsequent experiments revealed that the proportion of polysaccharide of S. typhi type is dependent upon the level of galactose supplementation, and identified a limiting sugar concentration which results in surface co-expression of both O-antigens. Visualization of the two polysaccharides on silver-stained polyacrylamide gels indicates that S. typhi O-antigen subunits are polymerized into longer sidechains, suggesting that at higher galactose concentrations their predominance results in a masking of the shorter V. cholerae O-antigen.

Animals↗
PubMed1990

Safety, immunogenicity, and efficacy against cholera challenge in humans of a typhoid-cholera hybrid vaccine derived from Salmonella typhi Ty21a.

A live oral vaccine consisting of attenuated Salmonella typhi Ty21a expressing Vibrio cholerae O1 Inaba lipopolysaccharide (LPS) O antigen was constructed and tested in volunteers for safety, immunogenicity, and efficacy. Fourteen adults ingested three doses of 10(10) viable organisms with buffer. One month later, 8 vaccinees and 13 unimmunized controls were challenged with 10(6) pathogenic V. cholerae O1 E1 T or Inaba organisms. No significant adverse reactions to vaccination were observed. All volunteers had significant rises in serum immunoglobulin G (IgG) antibody to S. typhi LPS. Only 2 (14%) of 14 had significant rises in serum IgA or IgG antibody to Inaba LPS, and 5 (36%) of 14 had fourfold rises in vibriocidal antibody. In the challenge study, diarrhea occurred in 13 of 13 controls and 6 of 8 vaccinees (vaccine efficacy, 25%; P = 0.13). The vaccine significantly reduced the severity of the clinical illness (P less than 0.05) and caused decreased excretion of challenge vibrios (P less than 0.05). Although the typhoid-cholera hybrid vaccine did not provide significant protection overall against experimental cholera, this study demonstrates the importance of antibody to V. cholerae O antigen in ameliorating clinical illness and illustrates the use of an S. typhi carrier vaccine strain expressing a foreign antigen.

Antibodies, Bacterial↗
PubMed1988

Towards a live oral vaccine against enterotoxigenic Escherichia coli of swine.

A live oral vaccine has been developed against scouring induced in piglets by enterotoxigenic Escherichia coli (ETEC). An attenuated strain of Salmonella typhimurium, G30, has been used as a vector for plasmids encoding the production of the fimbrial colonization factors of porcine ETEC. Initial studies with clones expressing K88 or K99 fimbriae have shown them to be well tolerated when administered orally in very high doses. The clones elicited serum, colostral and milk antibodies to the fimbrial antigens, and a challenge trial indicated that such responses were sufficient to ensure the passive transfer of protective immunity to suckling piglets. The possible advantages of this approach are discussed.

Administration, Oral↗
PubMed1988

A galE via (Vi antigen-negative) mutant of Salmonella typhi Ty2 retains virulence in humans.

We have recently described the construction of a galE derivative of Salmonella typhi Ty2 (Ty2H1) which had a 0.4-kilobase deletion in the galE gene and was sensitive to galactose-induced lysis when cultured with greater than or equal to 0.06 mM galactose (D. M. Hone, R. Morona, S. Attridge, and J. Hackett, J. Infect. Dis. 156:167-174, 1987). We now report the selection of a rifampin-resistant, via derivative of Ty2H1, EX462. Compared with the Ty2 parent strain, EX462 was serum sensitive and highly attenuated in the mouse mucin virulence assay. When four human volunteers ingested 7 X 10(8) viable EX462, two became ill and developed a typhoidlike disease with fever and bacteremia. Blood isolates from these individuals were indistinguishable from the vaccine strain by a variety of criteria. We concluded that, even in a via background, the galE mutation was not attenuating for S. typhi in humans.

Animals↗
PubMed1987

A physical map of the chromosomal region determining O-antigen biosynthesis in Vibrio cholerae O1.

We have previously described the cosmid cloning of the genes determining the biosynthesis of the Inaba and Ogawa O-antigens of the lipopolysaccharides of Vibrio cholerae O1 (Manning et al., 1986). By Southern hybridization analysis of chromosomal and cosmid DNA, and heteroduplex analysis between the clones we have been able to precisely define the region of contiguous chromosomal DNA in the vicinity of the O-antigen-encoding region. These data and comparison of end points of clones and of deletion derivatives demonstrate that at least 16 kb of a 19-kb SstI fragment is required to encode O-antigen biosynthesis. Expression of O-antigen is independent of the orientation of this SstI fragment with respect to cloning vectors suggesting that its regulatory region has been cloned intact. No detectable differences were observed in the restriction patterns of the Inaba and Ogawa coding regions implying that only minor changes are involved when serotype conversion (Inaba to Ogawa or vice versa) occurs. Bhaskaran [Ind. J. Med. Res. 47 (1959) 253-260] originally defined this region associated with O-antigen biosynthesis oag; however, to be consistent with other organisms [Hitchcock et al., J. Bacteriol. 166 (1986) 699-705], it is suggested this be changed to rfb.

Antigens, Bacterial↗
PubMed1987

Construction of defined galE mutants of Salmonella for use as vaccines.

We describe the molecular cloning of the gal operon of Salmonella typhimurium LT2 and the localization of the gal promoter and the genes galE, galT, and galK. The order of the genes and the direction of transcription was the same as in Escherichia coli K12. A restriction enzyme map of the operon was obtained, and the approximate termini of the gal genes were located by using transposon insertion mutagenesis and minicell analysis. We constructed a plasmid that contained a defined 0.4-kilobase deletion in the galE but still expressed galT and galK activities from the gal promoter. This galE deletion was recombined into the chromosomal gal operons of S. typhimurium and Salmonella typhi Ty2. The resulting strains were vigorous, nonreverting galE mutants that were sensitive to galactose-induced lysis at 0.2 mM galactose. The S. typhimurium galE derivatives were avirulent and protective in mice.

Animals↗
PubMed1986

New locus (ttr) in Escherichia coli K-12 affecting sensitivity to bacteriophage T2 and growth on oleate as the sole carbon source.

The nature of resistance to phage T2 in Escherichia coli K-12 was investigated by analyzing a known phage T2-resistant mutant and by isolating new T2-resistant mutants. It was found that mutational alterations at two loci, ompF (encoding the outer membrane protein OmpF) and ttr (T-two resistance), are needed to give full resistance to phage T2. A ttr::Tn10 mutation was isolated and was mapped between aroC and dsdA, where the fadL gene (required for long-chain fatty acid transport) is located. The receptor affected by ttr was the major receptor used by phage T2 and was located in the outer membrane. Phage T2 was thus able to use two outer membrane proteins as receptors. All strains having a ttr::Tn10 allele and most of the independently isolated phage T2-resistant mutants were unable to grow on oleate as the sole carbon and energy source, i.e., they had the phenotype of fadL mutants. The gene fadL is known to encode an inner membrane protein. The most likely explanation is that fadL and ttr are in an operon and that ttr encodes an outer membrane protein which functions in translocating long-chain fatty acids across the outer membrane and also as a receptor for phage T2.

Bacterial Outer Membrane Proteins↗
PubMed1985

Demonstration of a bacteriophage receptor site on the Escherichia coli K12 outer-membrane protein OmpC by the use of a protease.

The Escherichia coli K12 outer-membrane proteins OmpA, OmpC, OmpF, PhoE, and LamB (all of transmembrane nature) can serve as phage receptors. We have shown previously that one OmpA-specific phage, Ox2, can give rise to the host range mutants Ox2h10 and Ox2h12, with the latter being derived from the former [Morona, R. & Henning, U. (1984) J. Bacteriol. 159, 579-582]. Unlike Ox2, both host range phages can use the OmpA and OmpC proteins as receptors and Ox2h12 is better adapted to the OmpC protein than Ox2h10. In a search for the site(s) of OmpC protein involved in phage recognition, it was found that proteinase K is able to cleave all of the proteins mentioned above. OmpC protein (Mr = 38306) could be cleaved from outside the cell by proteinase K resulting in two fragments of Mr approximately equal to 21000 and Mr approximately equal to 17500. The use of OmpC-PhoE hybrid proteins allowed us to assign the approximately equal to 21000-Mr fragment to the CO2H-terminal moiety of the protein. Proteinase K treatment of intact cells abolished their activity to neutralize the OmpC-specific phage Tulb and reduced this ability towards phage Ox2h12. The OmpA, OmpF, PhoE and LamB proteins were cleaved by the protease not in intact cells but only when acting on cell envelopes. The sizes of the OmpC protein fragments and the results obtained with the hybrid proteins very strongly suggest that the protein is cleaved from outside the cell at a region involving amino acid residues 150-178 of the 346-residue protein, which shows homology to two regions of the OmpA protein which are involved in its phage receptor site (loc. cit.). These areas also exhibit some homology to a region of the LamB protein which is thought to be part of this protein's receptor site [Charbit et al. (1984) J. Mol. Biol. 175, 395-401]. This suggests that there is a common denominator for proteinaceous phage receptor site because the LamB-specific phage lambda and phage Tulb are of completely different nature. We conclude that the region of the OmpC protein in question is cell-surface-exposed and acts as a phage receptor site.

Bacterial Outer Membrane Proteins↗
PubMed1985

The nature of ompA mutants of Escherichia coli K12 exhibiting temperature-sensitive bacteriophage resistance.

A class of ompA mutants of Escherichia coli, exhibiting temperature-sensitive resistance towards phages using the OmpA protein as receptor, was analysed. The mutants produce detectable levels of the protein at 42 degrees C but not at 30 degrees C (Manning and Reeves 1976). They were found to have a deletion (one isolate) or insertions (three isolates) upstream of the coding part of the ompA gene. Several previously characterized mutants possessing insertions or a deletion in the non-translated 5' area of the gene also exhibited a similar temperature-sensitive phage resistance. This cold-sensitive phenotype is explained in terms of the recent discovery that the stability of ompA mRNA is regulated by the rate of cell growth (Nilsson et al. 1984).

Bacterial Outer Membrane Proteins↗
PubMed1985

Bacteriophage receptor area of outer membrane protein OmpA of Escherichia coli K-12.

A number of T-even-like bacteriophages use the outer membrane protein OmpA of Escherichia coli as a receptor. We had previously analyzed a series of ompA mutants which are resistant to such phages and which still produce the OmpA protein (R. Morona, M. Klose, and U. Henning, J. Bacteriol. 159:570-578, 1984). Mutational alterations were found near or at residues 70, 110 and 154. Based on these and other results a model was proposed showing the amino-terminal half of the 325-residue protein crossing the outer membrane repeatedly and being cell surface exposed near residues 25, 70, 110, and 154. We characterized, by DNA sequence analysis, an additional 14 independently isolated phage-resistant ompA mutants which still synthesize the protein. Six of the mutants had alterations identical to the ones described before. The other eight mutants possessed seven new alterations: Ile-24----Asn, Gly-28----Val, deletion of Glu-68, Gly-70----Cys, Ser-108----Phe, Ser-108----Pro, and Gly-154----Asp (two isolates). Only the latter alteration resulted in a conjugation-deficient phenotype. The substitutions at Ile-24 and Gly-28 confirmed the expectation that this area of the protein also participates in its phage receptor region. It is unlikely that still other such sites of the protein are involved in the binding of phage, and it appears that the phage receptor area of the protein has now been characterized completely.

Amino Acid Sequence↗
PubMed1984

Escherichia coli K-12 outer membrane protein (OmpA) as a bacteriophage receptor: analysis of mutant genes expressing altered proteins.

The outer membrane protein OmpA of Escherichia coli K-12 serves as a receptor for a number of T-even-like phages. We have isolated a series of ompA mutants which are resistant to such phages but which still produce the OmpA protein. None of the mutants was able to either irreversibly or reversibly bind the phage with which they had been selected. Also, the OmpA protein is required for the action of colicins K and L and for the stabilization of mating aggregates in conjugation. Conjugal proficiency was unaltered in all cases. Various degrees of colicin resistance was found; however, the resistance pattern did not correlate with the phage resistance pattern. DNA sequence analyses revealed that, in the mutants, the 325-residue OmpA protein had suffered the following alterations: Gly-65----Asp, Gly-65----Arg, Glu-68----Gly, Glu-68----Lys (two isolates), Gly-70----Asp (four isolates), Gly-70----Val, Ala-Asp-Thr-Lys-107----Ala-Lys (caused by a 6-base-pair deletion), Val-110----Asp, and Gly-154----Ser. These mutants exhibited a complex pattern of resistance-sensitivity to 14 different OmpA-specific phages, suggesting that they recognize different areas of the protein. In addition to the three clusters of mutational alterations around residues 68, 110, and 154, a site around residue 25 has been predicted to be involved in conjugation and in binding of a phage and a bacteriocin (R. Freudl, and S. T. Cole, Eur. J. Biochem, 134:497-502, 1983; G. Braun and S. T. Cole, Mol. Gen. Genet, in press). These four areas are regularly spaced, being about 40 residues apart from each other. A model is suggested in which the OmpA polypeptide repeatedly traverses the outer membrane in cross-beta structure, exposing the four areas to the outside.

Amino Acid Sequence↗
PubMed1984

Host range mutants of bacteriophage Ox2 can use two different outer membrane proteins of Escherichia coli K-12 as receptors.

The Escherichia coli K-12 outer membrane protein OmpA functions as the receptor for bacteriophage Ox2. We isolated a host range mutant of this phage which was able to grow on an Ox2-resistant ompA mutant producing an altered OmpA protein. From this mutant, Ox2h5, a second-step host range mutant was recovered which formed turbid plaques on a strain completely lacking the OmpA protein. From one of these mutants, Ox2h10, a third-step host range mutant, Ox2h12, was isolated which formed clear plaques on a strain missing the OmpA protein. Ox2h10 and Ox2h12 apparently were able to use both outer membrane proteins OmpA and OmpC as receptors. Whereas there two proteins are very different with respect to primary structures and functions, the OmpC protein is very closely related to another outer membrane protein, OmpF, which was not recognized by Ox2h10 or Ox2h12. An examination of the OmpC amino acid sequence, in the regions where it differs from that of OmpF, revealed that one region shares considerable homology with a region of the OmpA protein which most likely is required for phage Ox2 receptor activity.

Alleles↗
PubMed1983

Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli.

We used the cloned tolC gene to identify, locate, and purify its gene product. Strains carrying pPR13 or pPR42 overproduced a cell envelope protein (molecular weight, 52,000). A protein of the same molecular weight was identified in radioactively labeled minicells carrying pPR13; this protein was absent in pPR11-carrying minicells. This protein was the tolC gene product, since pPR11 differed from pPR13 in having a Tn10 insertion in the tolC gene. The protein seen in cell envelopes of whole cells (TolC protein) was found to exist in an aggregated state in the outer membrane; under conditions in which OmpC and OmpF were peptidoglycan associated, TolC protein was not likewise associated. Using these properties, we purified the TolC protein and determined the sequence of twelve amino acids from the amino-terminal end. The location of the TolC protein in the outer membrane was consistent with the proposed function for the tolC gene product as a processing protein in the outer membrane.

Amino Acid Sequence↗
PubMed1982

The tolC locus of Escherichia coli affects the expression of three major outer membrane proteins.

tolC mutants, which are resistant to colicin E1 and also highly sensitive to detergents and dyes, were shown to lack the OmpF outer membrane protein. There was little effect on transcription as judged by the use of an ompF-lac operon fusion strain, and the tolC effect was probably due to a post-transcriptional effect. The NmpC protein and protein 2 were also tolC dependent.

Bacterial Outer Membrane Proteins↗
PubMed1981

Molecular cloning of the tolC locus of Escherichia coli K-12 with the use of transposon Tn10.

We have cloned the tolC gene of E. coli K-12 into pSF2124 by using transposon Tn10 as the marker to first isolate the relevant DNA fragment. The gene is on a 10.5 kb EcoRI fragment, and Tn5 insertion mutagenesis locates the gene near one end of this EcoRI fragment. An EcoRI-PstI fragment has been subcloned into pBR322 to facilitate further analysis of the gene.

Cloning, Molecular↗