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J S Kroll

Publications and source records attributed to J S Kroll.

At least 37 records · Page 2Linked to original sources
PubMed1997

Development of a Haemophilus two-dimensional protein database.

Members of the Haemophilus genus are responsible for various human infections including respiratory infections and meningitis. The complete nucleotide sequence of the Rd strain of Haemophilus influenzae has been reported and represents a valuable resource to investigate gene expression within this bacterial group. We described previously the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to characterise the proteins of Haemophilus influenzae (Cash et al., Electrophoresis 1995, 16, 135-148). We have extended these data with comparative studies of the proteins from other members of the Haemophilus genus (specifically H. parainfluenzae, H. haemolyticus and H. parahaemolyticus) to identify homologous proteins and, by extension, the genes encoding them, among these bacteria. The proteins extracted from each of these bacterial isolates were compared by coelectrophoresis to the 2-D protein profile of the reference nontypable strain of H. influenzae (HI-64443) used as the basis for the 2-D protein database. A composite reference 2-D protein profile of HI-64443 was derived from three independent analyses of the soluble bacterial proteins. Between 21% and 37% of the HI-64443 proteins from the reference 2-D protein profile comigrated with proteins in the other isolates from the Haemophilus genus. This compared with 62% and 64% comigration when HI-64443 was compared with the Eagan and Rd strains of H. influenzae, respectively. The 2-D protein profile of the Rd strain of H. influenzae was compared to that of HI-64443 by coelectrophoresis; 64% of the proteins detected for the Rd strain comigrated with proteins found for HI-64443 when analysed in parallel. The capacity of 2-D PAGE to investigate global interactions of gene expression was applied to the analysis of superoxide dismutase (SOD) expression in H. influenzae strain Eagan. A "knock-out" mutant in the sodA gene which encodes [Mn]-SOD was characterised with respect to protein synthesis compared to the parental isolate. From these analyses, the primary product of sodA was provisionally identified as a protein with a molecular mass of 25500 Da and an estimated pI of 6.55. Quantitative changes in the expression of two other proteins in the SOD mutant were detected by comparison with the parental isolate. These data are discussed in relation to the development of a 2-D protein database for H. influenzae and related bacteria to investigate genome homologies and gene expression.

Bacterial Proteins↗
PubMed1997

Bacterial copper- and zinc-cofactored superoxide dismutase contributes to the pathogenesis of systemic salmonellosis.

Copper/zinc-cofactored superoxide dismutase ([Cu,Zn]-SOD) has been found in the periplasm of many bacterial species but its biological function is unknown. Here we report the cloning and characterization of sodC, encoding [Cu,Zn]-SOD, from Salmonella typhimurium. The predicted protein sequence shows only 58% identity to Escherichia coil SodC, and from this its chromosomal location and its immediate proximity to a phage gene, sodC, in Salmonella is speculated to have been acquired by bacteriophage-mediated horizontal transfer from an unknown donor. A sodC mutant of S. typhimurium was unimpaired on aerobic growth in rich medium but showed enhanced sensitivity in vitro to the microbicidal action of superoxide. S. typhimurium, S. choleraesuis and S. dublin sodC mutants showed reduced lethality in a mouse model of oral infection and persisted in significantly lower numbers in livers and spleens after intraperitoneal infection, suggesting that [Cu,Zn]-SOD plays a role in pathogenicity, protecting Salmonella against oxygen radical-mediated host defences. There was, however, no observable difference compared with wild type in the interaction of sodC mutants with porcine pleural, mouse peritoneal or J774 macrophages in vitro, perhaps reflecting the hierarchical capacity of different macrophage lines to kill Salmonella, the most efficient overwhelming the proposed protective effect of periplasmic SOD.

Amino Acid Sequence↗
PubMed1997

Distribution, cloning, characterisation and mutagenesis of sodC, the gene encoding copper/zinc superoxide dismutase, a potential determinant of virulence, in Haemophilus ducreyi.

The sodC gene encoding the periplasmic enzyme copper/zinc superoxide dismutase (CuZnSOD) has been cloned from Haemophilus ducreyi, the causative agent of the genital ulcer disease, chancroid. Examination of a collection of diverse strains indicates that it is present throughout the species. Cloned sodC has been expressed in E. coli and shown to encode active enzyme. Insertional mutagenesis was used to construct a non-functional version of the gene. This has been transferred into the chromosome of the parent H. ducreyi strain by electroporation and homologous recombination, in preparation for studies of the role of this enzyme in the interactive biology of the organism with its host, perhaps in protecting bacteria from superoxide radicals and their reactive progeny generated by neutrophils in the context of host defence.

Amino Acid Sequence↗
PubMed1997

Role of bacterial Mn-cofactored superoxide dismutase in oxidative stress responses, nasopharyngeal colonization, and sustained bacteremia caused by Haemophilus influenzae type b.

Haemophilus influenzae type b, a causative agent of bacterial sepsis and meningitis in young children, contains a single superoxide dismutase (SOD), a cytoplasmic MnSOD. To study the role of this enzyme, a chromosomal sodA::lacZ mutant (M-2) was constructed. M-2 had an increased sensitivity towards oxygen and the redox-active agent paraquat. A 3.4-fold increase in sodA-lacZ expression was found in M-2 grown with oxygen supply rates between 3 and 36 mmol of O2/liter/h. In similar experiments with the wild type, assaying SodA activity, a 3.1-fold increase was found. Both the wild type and M-2 grew best at the lowest oxygen supply rate tested, consistent with the notion that H. influenzae prefers a more anaerobic environment. In the infant rat model of infection, the ability of M-2 to colonize the nasopharynx was found to be impaired, but its ability to cause invasive disease was unaffected. This suggests that after invasion, the growth disadvantage imposed by a SodA- phenotype is not limiting.

Animals↗
PubMed1996

Occurrence of [copper, zinc]-cofactored superoxide dismutase in Pasteurella haemolytica and its serotype distribution.

Fifty-two ovine strains of Pasteurella haemolytica and P. trehalosi representing serotypes 1-16 were examined for the presence of [copper, zinc]superoxide dismutase DNA sequences. This was done using a combination of polymerase chain reaction with degenerate primers based on the sequence of the [Cu,Zn]superoxide dismutase gene (sodC) in related species and Southern hybridization using a fragment of sodC from P. haemolytica A2 serotype as a probe. Both detection methods identified a fragment of the sodC gene in 9/9 strains of P. haemolytica serotype 2 examined and in 5/8 strains of serotype 7. No evidence of this gene was found in any other serotype of P. haemolytica or in any P. trehalosi serotype. Comparison of DNA sequence showed near identity between sodC from the A2 and A7 serotypes of P. haemolytica and substantial similarity (70%) to sodC previously sequenced in P. multocida, Haemophilus parainfluenzae and H. influenzae. Analysis by gel electrophoresis of the superoxide dismutase activity present in cell lysates showed that one or more superoxide dismutase is present in all serotypes. However, cyanide-inhibitable activity, corresponding to [Cu,Zn]superoxide dismutase, was detected only in those strains of serotypes A2 and A7 which showed evidence of the sodC gene fragment.

Amino Acid Sequence↗
PubMed1996

Identification of sodC encoding periplasmic [Cu,Zn]-superoxide dismutase in Salmonella.

sodC, encoding [Cu,Zn]-cofactored superoxide dismutase, once thought to be virtually confined to eukaryotes, has now been described in many Gram-negative pathogens that have their primary niche of colonization in the upper respiratory tract. Their role in host-parasite interactive biology is unknown. We here show that members of the major human and animal enteric pathogenic species Salmonella harbour a version of sodC most closely resembling that found in Brucella abortus. The enzyme it encodes is a novel candidate determinant of virulence in Salmonella, an intracellular pathogen potentially exposed to toxic oxygen free radicals within its intracellular niche.

Blotting, Southern↗
PubMed1996

Haemophilus influenzae: capsule vaccine and capsulation genetics.

In 1931, Dr Margaret Pittman reported her discovery that Haemophilus influenzae strains responsible for meningitis had a polysaccharide capsule, and that one capsular type, serotype b, was responsible for nearly all cases. Diverse programmes of research aimed at understanding and exploiting this seminal observation culminated, in the 1980s, in the introduction of a purified type b polysaccharide vaccine to protect children against this terrible disease. Subsequent improvements in vaccine immunogenicity have translated into impressive efficacy and the suggestion that, were all children to be immunized, a major cause of life-threatening childhood infection might be vanquished.

Animals↗
PubMed1996

Cloning and molecular characterization of Cu,Zn superoxide dismutase from Actinobacillus pleuropneumoniae.

Copper-zinc superoxide dismutases (Cu,Zn SODs), until recently considered very unusual in bacteria, are now being found in a wide range of gram-negative bacterial species. Here we report the cloning and characterization of sodC, encoding Cu,Zn SOD in Actinobacillus pleuropneumoniae, a major pathogen of pigs and the causative organism of porcine pleuropneumonia. sodC was shown to lie on a monocistronic operon, at the chromosomal locus between the genes asd (encoding aspartate semialdehyde dehydrogenase) and recF. The primary gene product was shown to have an N-terminal peptide extension functioning as a leader peptide, so that the mature Actinobacillus enzyme, like other bacterial examples, is directed to the periplasm, where it is appropriately located to dismutate exogenously generated superoxide. While the role of these secreted bacterial SODs is unknown, we speculate that in A. pleuropneumoniae the enzyme may confer survival advantage by accelerating dismutation of superoxide derived from neutrophils, a central host defense response in the course of porcine infection.

Actinobacillus pleuropneumoniae↗
PubMed1995

Bacterial [Cu,Zn]-superoxide dismutase: phylogenetically distinct from the eukaryotic enzyme, and not so rare after all!

Copper- and zinc-containing superoxide dismutases ([Cu,Zn]-SODs) are generally considered almost exclusively eukaryotic enzymes, protecting the cytosol and extracellular compartments of higher organisms from damage by oxygen free-radicals. The recent description of a few examples of bacterial forms of the enzyme, located in the periplasm of different Gram-negative micro-organisms, prompted a re-evaluation of this general perception. A PCR-based approach has been developed and used successfully to identify bacterial genes encoding [Cu,Zn]-SOD in a wide range of important human and animal pathogens-members of the Haemophilus, Actinobacillus and Pasteurella (HAP) group, and Neisseria meningitidis. Comparison of [Cu,Zn]-SOD peptide sequences found in Haemophilus ducreyi, Actinobacillus pleuropneumoniae, Actinobacillus actinomycetemcomitans, Pasteurella multocida, and N. meningitidis with previously described bacterial proteins and examples of eukaryotic [Cu,Zn]-SOD has shown that the bacterial proteins constitute a distinct family apparently widely separated in evolutionary terms from the eukaryotic examples. The widespread occurrence of [Cu,Zn]-SOD in the periplasm of bacterial pathogens, appropriately located to dismute exogenously derived superoxide radical anions, suggests that this enzyme may play a role in the interactive biology of organisms with their hosts and so contribute to their capacity to cause disease.

Actinobacillus↗
PubMed1995

Region II of the Haemophilus influenzae type be capsulation locus is involved in serotype-specific polysaccharide synthesis.

The central (serotype-specific) Region II of the Haemophilus influenzae Type b capsulation locus cap is 8.3 kb long and contains a cluster of four genes. We show that these genes, designated orf1 to orf4, are involved in the biosynthetic steps required for the formation of the Type b capsular polysaccharide and that orf1 probably encodes a CDP-ribitolpyrophosphorylase. We present evidence that growth of polysaccharide chains takes place through the alternating addition of single sugar nucleotides.

Amino Acid Sequence↗
PubMed1994

Natural genetic transfer of a putative virulence-enhancing mutation to Haemophilus influenzae type a.

Haemophilus influenzae strains of serotype a very rarely cause life-threatening infections. Examination of strains from the Gambia, West Africa, that caused septicemia, meningitis, or both revealed that a clone has emerged that carries a DNA deletion previously identified only in type b strains that is hypothesized to contribute to the special virulence of that serotype. This clone appears to have arisen by transfer of DNA between type a and type b strains, a transformation event that has happened more than once, as shown by the discovery Kenya, East Africa, of a clonally distinct type a strain bearing the identical deletion. The implications for the emergence of clinically important non-type b strains of H. influenzae are obvious.

Amino Acid Sequence↗
PubMed1994

PCR for capsular typing of Haemophilus influenzae.

A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine.

Bacterial Capsules↗
PubMed1993

HIV-specific cytotoxic T-cell activity in an HIV-exposed but uninfected infant.

The factors necessary for protective immunity against HIV-1 are unknown. Important information about these factors should come from study of people at high risk of HIV infection who have not apparently become infected. Among these are the estimated 60-85% of children who may be exposed in utero or perinatally to HIV-1 but do not become infected. We observed the transient appearance of HIV-specific cytotoxic T-lymphocyte (CTL) activity in a baby born to HIV-1-infected parents, in whom all standard markers of infection remained negative. These findings suggest that HIV-specific CTLs may be a marker for recently exposed, but uninfected, individuals.

CD4-CD8 Ratio↗
PubMed1993

An ancestral mutation enhancing the fitness and increasing the virulence of Haemophilus influenzae type b.

Capsulate Haemophilus influenzae is a major cause of septicemia and meningitis in children. Virtually all invasive strains have a type b polysaccharide capsule and belong to division I of the two phylogenetic divisions into which the H. influenzae population segregates. In this study 18 isolates, collected from all over the world and representative of the whole population of division I type b strains, have been shown to be the progeny of a common ancestor in which a founder mutation occurred, the deletion of one of two copies of the gene bexA. BexA is essential for exporting capsular polysaccharide to the bacterial surface, and a single copy of its gene lies at the center of an otherwise duplicated capsulation locus. Deletion of the other copy has had the paradoxical effect of enhancing pathogenicity, through increasing the potential for amplification of capsule biosynthetic genes and capsule production.

ATP-Binding Cassette Transporters↗
PubMed1993

Molecular and genetic characterization of superoxide dismutase in Haemophilus influenzae type b.

Oxygen free radicals present a serious potential threat to microbial survival, through their ability to inflict indiscriminate damage on proteins and DNA. Superoxide dismutase (SOD, EC 1.15.1.1), among other oxygen-metabolizing enzymes, is essential to prevent these toxic molecules from accumulating in the bacterial cytosol during aerobic metabolism. The gene sodA, encoding manganese-containing SOD ([Mn]-SOD), has been cloned from a virulent strain of Haemophilus influenzae type b using degenerate oligonucleotides encoding regions of the gene conserved across different bacterial species. The gene product has been identified as [MN]-SOD by its similarity at key amino acid residues to known examples of the enzyme, by expression of enzymatically active protein from cloned DNA expressed in Escherichia coli, and by demonstration that an in-frame deletion in the gene abolishes this activity. In contrast to the situation in E. coli, this [Mn]-SOD is the only active SOD detected in H. influenzae. In further contrast to E. coli, [Mn]-SOD gene expression in H. influenzae has been found to be only partially repressed under anaerobic conditions. When expressed in E. coli the gene is regulated by Fur and Fnr, and the promoter region, identified experimentally, has been found to contain nucleotide sequence motifs similar to the Fur- and Fnr-binding sequences of E. coli, suggesting the involvement of analogues of these aerobiosis-responsive activators in H. influenzae gene expression.

Aerobiosis↗
PubMed1992

Palindromic Haemophilus DNA uptake sequences in presumed transcriptional terminators from H. influenzae and H. parainfluenzae.

We have found palindromic pairs of near matches to the 11-bp Haemophilus DNA uptake motif shortly after the stop codons of three Haemophilus genes. Short runs of thymidylate residues follow the stem-loop structures thus defined. This organization suggests that, in H. influenzae, the uptake motif may be preferentially incorporated into gene termination signals, as has been proposed for Neisseria gonorrhoeae.

Base Sequence↗