PubMed Health⌕ Search

Biomedical subjects

F Heffron

Publications and source records attributed to F Heffron.

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

Contribution of horizontal gene transfer and deletion events to development of distinctive patterns of fimbrial operons during evolution of Salmonella serotypes.

Only certain serotypes of Salmonella represent 99% of all human clinical isolates. We determined whether the phylogenetic distribution of fimbrial operons would account for the host adaptations observed for Salmonella serotypes. We found that three fimbrial operons, fim, lpf, and agf, were present in a lineage ancestral to Salmonella. While the fim and agf fimbrial operons were highly conserved among all Salmonella serotypes, sequence analysis suggested that the lpf operon was lost from many distantly related lineages. As a consequence, the distribution of the lpf operon cannot be explained easily and may be a consequence of positive and negative selection in different hosts for the presence of these genes. Two other fimbrial operons, sef and pef, each entered two distantly related Salmonella lineages and each is present only in a small number of serotypes. These results show that horizontal gene transfer and deletion events have created unique combinations of fimbrial operons among Salmonella serotypes. The presence of sef and pef correlated with serotypes frequently isolated from common domesticated animals.

Chromosomes, Bacterial↗
PubMed1997

Rapid detection of Salmonella enterica with primers specific for iroB.

The iroB gene of Salmonella enterica is absent from the chromosome of the related organism Escherichia coli. We determined the distribution of this gene among 150 bacterial isolates, representing 51 serotypes of different Salmonella species and subspecies and 8 other bacterial species which are frequent contaminants during routine enrichment procedures by Southern hybridization. An iroB-specific DNA probe detected homologous sequences in all strains of S. enterica, including serotypes of S. enterica subsp. enterica (I), salamae (II), diarizonae (IIIb), and houtenae (IV). No hybridization signal was obtained with strains of Salmonella bongori or other bacterial species. In contrast, hybridization with a DNA probe specific for purD, a purine biosynthesis gene, detected homologs in all bacterial species tested. Primers specific for iroB were used to amplify this gene from 197 bacterial isolates by PCR. The iroB gene could be PCR amplified from S. enterica subsp. enterica (I), salamae (II), diarizonae (IIIb), houtenae (IV), arizonae (IIIa), and indica (VI), but not from S. bongori or other bacterial species. Thus, PCR amplification of iroB can be used to distinguish between S. enterica and other bacterial species, including S. bongori. A combination of preenrichment in buffered peptone water supplemented with ferrioxamine E and amplification of iroB by magnetic immuno-PCR allowed detection of S. enterica in albumen within 24 h. In conclusion, PCR amplification of iroB is a new sensitive and selective method which has the potential to rapidly detect S. enterica serotypes.

Bacterial Typing Techniques↗
PubMed1997

Fimbrial adhesins of Salmonella typhimurium. Role in bacterial interactions with epithelial cells.

S. typhimurium initiates infection of its mammalian host by attachment to mucosal surfaces in the intestine and subsequent invasion of epithelial cells. To date, three S. typhimurium fimbrial operons, fim, lpf and pef, have been characterized. This analysis suggests that fimbrial adhesins fulfill multiple functions during the initial phase of an infection. In addition to their role in colonization of the small intestine, adhesins contribute to the tissue tropism for Peyer's patches, which is characteristic for Salmonella infections. Furthermore, by mediating the initial contact to epithelial cells, fimbrial adhesins appear to be necessary for invasion and possibly for elicitation of an inflammatory response. Thus, fimbriae are important virulence factors of S. typhimurium and their future analysis promises to yield fascinating new insights into host-parasite interactions of this pathogen.

Adhesins, Bacterial↗
PubMed1996

Identification of a new iron regulated locus of Salmonella typhi.

In order to identify genes belonging to the Fur regulon of Salmonella typhi which are absent from Escherichia coli K-12, a plasmid gene bank consisting of 4000 independent clones was screened for Fur regulated promoters using the Fur titration assay (FURTA). DNA probes generated from FURTA positive plasmids were then used for hybridization with chromosomal DNA from S. typhi, Salmonella typhimurium and E. coli. Using these techniques we identified an iron regulated locus present in S. typhi and S. typhimurium but not in E. coli. Further cloning and nucleotide sequence analysis identified two open reading frames, termed iroBC, organized in a typical operon structure. The genes iroBC were located at 4 and 57 centisomes on the physical maps of Salmonella typhi and S. typhimurium, respectively. This region of the S. typhimurium chromosome contains a large DNA loop which is absent from the corresponding area of the E. coli chromosome. Finally, we developed a new method for generation of single copy transcriptional fusions. A suicide vector was constructed, which allows for the generation of chromosomal fusions to the promoterless E. coli lacZYA genes. By integration of this construct at the iro locus we could establish iron responsive expression of iroBC.

Bacterial Proteins↗
PubMed1996

Macrophage killing is an essential virulence mechanism of Salmonella typhimurium.

Phagocytic cells are a critical line of defense against infection. The ability of a pathogen to survive and even replicate within phagocytic cells is a potent method of evading the defense mechanisms of the host. A number of pathogens survive within macrophages after phagocytosis and this contributes to their virulence. Salmonella is one of these pathogens. Here we report that 6-14 hr after Salmonella enters the macrophage and replicates, it resides in large vacuoles and causes the destruction of these cells. Furthermore, we identified four independently isolated MudJ-lacZ insertion mutants that no longer cause the formation of these vacuoles or kill the macrophages. All four insertions were located in the ompR/envZ regulon. These findings suggest that killing and escape from macrophages may be as important steps in Salmonella pathogenesis as are survival and replication in these host cells.

Animals↗
PubMed1996

The lpf fimbrial operon mediates adhesion of Salmonella typhimurium to murine Peyer's patches.

We investigated the role of the Salmonella typhimurium fimbrial operon formed by the genes lpfABCDE in infection of mice. A mutant in lpfC, the gene encoding the fimbrial outer membrane usher, had an approximately 5-fold increased 50% lethal dose when administered orally to mice. When mice were infected with a mixture of the lpfC mutant and isogenic wild-type S. typhimurium, the lpfC mutant was recovered in lower numbers from Peyer's patches, mesenteric lymph nodes, liver, and spleen. In an organ culture model using murine intestinal loops, lpfC mutants were shown to be associated in lower numbers than wild-type bacteria with Peyer's patches but not with villous intestine. The defect of the lpfC mutant in adhesion to Peyer's patches could be complemented by introducing lpfABCDE on a cosmid. Similarly, heterologous expression of the Salmonella lpf operon in Escherichia coli resulted in an increased adhesion to histological thin sections of Peyer's patch lymph follicles. Electron microscopic analysis of histological sections taken from Peyer's patches after intragastric infection of mice showed that, in contrast to the S. typhimurium wild type, the isogenic lpfC mutant did not destroy M cells of the follicle-associated epithelium. These data show that the Salmonella lpf operon is involved in adhesion to murine Peyer's patches.

Animals↗
PubMed1996

Induction of SIV capsid-specific CTL and mucosal sIgA in mice immunized with a recombinant S. typhimurium aroA mutant.

We have developed a new expression system based on the E. coli groEL promoter. The suicide vector constructed (called APC vector) allows simultaneous attenuation of a Salmonella strain by disruption of the coding sequence for aroA and stable integration of a gene into the bacterial chromosome. High-level expression of antigen is achieved after Salmonella is taken up by macrophages, a major antigen processing cell of the host. The chloramphenicol acetyltransferase (CAT) and the simian immunodeficiency virus capsid (p27gag) genes were cloned downstream of the groEL promoter and expressed within S. typhimurium. By measuring CAT activity, we showed that the groEL promoter was up-regulated during infection of the J774 macrophage line. The immune response to SIV capsid was assessed in Balb/c mice given one oral dose of vaccine. A local mucosal secretory IgA response against SIV capsid was detected but no systemic antibody response to the same antigen. A systemic CTL response was detected as early as 28 days to as late as 70 days post-immunization. CTL activity was MHC restricted (H-2d) and was mediated by CD3+, CD8+, CD4- T-lymphocytes. These results indicate that with only one oral dose of recombinant Salmonella using the APC vector, a systemic CTL response and a mucosal secretory response against the SIV capsid antigen are elicited in a mouse model.

Animals↗
PubMed1996

The pef fimbrial operon of Salmonella typhimurium mediates adhesion to murine small intestine and is necessary for fluid accumulation in the infant mouse.

We investigated the role of the pef operon, containing the genes for plasmid-encoded (PE) fimbriae of Salmonella typhimurium, in adhesion to the murine small intestine. In an organ culture model, a mutant of S. typhimurium carrying a tetracycline resistance cassette inserted in pefC was found to be associated in lower numbers with murine small intestine than the wild-type. Similarly, heterologous expression of PE fimbriae in Escherichia coli increased the bacterial numbers recovered from the intestine in the organ culture model. Adhesion to villous intestine mediated by PE fimbriae was further demonstrated by binding of an E. coli strain expressing PE fimbriae to thin sections of mouse small intestine. The contribution of pef-mediated adhesion on fluid accumulation was investigated in infant mice. Intragastric injection of S. typhimurium 14028 and SR-11 caused fluid accumulation in infant mice. In contrast, pefC mutants of S. typhimurium 14028 and SR-11 were negative in the infant mouse assay. Introduction of a plasmid containing pefBACD and orf5, the first five genes of the pef operon, into the pefC mutant complemented for fluid accumulation in the infant mouse assay. However, heterologous expression of PE fimbriae in E. coli did not result in fluid accumulation in the infant mouse, suggesting that factors other than fimbriae are involved in causing fluid accumulation.

Animals↗
PubMed1996

Contribution of TonB- and Feo-mediated iron uptake to growth of Salmonella typhimurium in the mouse.

We examined the role of iron(II) and iron(III) uptake, mediated by FeoB and TonB, respectively, in infection of the mouse by Salmonella typhimurium. The S. typhimurium feoB gene, encoding a homolog of an Escherichia coli cytoplasmic membrane iron(II) permease, was cloned, and a mutant was generated by allelic exchange. In addition, an S. typhimurium tonB mutant was constructed. Together these two mutations inactivate all known iron uptake systems of S. typhimurium. We examined the abilities of these mutants to grow in vitro and in different compartments of the host. Mutants in feoB were outcompeted by the wild type during mixed colonization of the mouse intestine, but the feoB mutation did not attenuate S. typhimurium for oral or intraperitoneal infection of mice. The tonB mutation attenuated S. typhimurium for infection of mice by the intragastric route but not the intraperitoneal route, and the mutant was recovered in lower numbers from the Peyer's patches and mesenteric lymph nodes than the wild type. These results indicate that TonB-mediated iron uptake contributes to colonization of the Peyer's patches and mesenteric lymph nodes but not the liver and spleen of the mouse. The tonB feoB double mutant, given intraperitoneally, was able to infect the liver and spleen at wild-type doses, indicating that additional iron acquisition systems are used during growth at systemic sites of infection.

Animals↗
PubMed1996

Contribution of fimbrial operons to attachment to and invasion of epithelial cell lines by Salmonella typhimurium.

The role of the Salmonella typhimurium fimbrial operons, lpf, fim, and pef, in adhesion to and invasion of epithelial cell lines was investigated. An S. typhimurium lpfC mutant was unable to adhere to or to invade HEp-2 cells, while an S. typhimurium fim deletion mutant did not attach to or enter HeLa cells. These results suggest that adhesion is a prerequisite for invasion and that distinct fimbrial adhesins select different target cells for invasion by S. typhimurium.

Adhesins, Bacterial↗
PubMed1995

The Neisseria meningitidis haemoglobin receptor: its role in iron utilization and virulence.

The Neisseria meningitidis haemoglobin receptor gene, hmbR, was cloned by complementation in a porphyrin-requiring Escherichia coli mutant. hmbR encodes an 89.5 kDa outer membrane protein which shares amino acid homology with the TonB-dependent receptors of Gram-negative bacteria. HmbR had the highest similarity to Neisseria transferrin and lactoferrin receptors. The utilization of haemoglobin as an iron source required internalization of the haemin moiety by the cell. The mechanism of haemin internalization via the haemoglobin receptor was TonB-dependent in E. coli. A N. meningitidis hmbR mutant was unable to use haemoglobin but could still use haemin as a sole iron source. The existence of a second N. meningitidis receptor gene, specific for haemin, was shown by the isolation of cosmids which did not hybridize with the hmbR probe, but which were able to complement an E. coli hemA aroB mutant on haemin-supplemented plates. The N. meningitidis hmbR mutant was attenuated in an infant rat model for meningococcal infection, indicating that haemoglobin utilization is important for N. meningitidis virulence.

Aldehyde Oxidoreductases↗
PubMed1995

Role of Salmonella typhimurium Mn-superoxide dismutase (SodA) in protection against early killing by J774 macrophages.

The Salmonella typhimurium gene for Mn-cofactored superoxide dismutase (sodA) was cloned by complementation of an Escherichia coli sodA sodB mutant for growth on minimal medium. Sequence analysis revealed an open reading frame of 618 bp encoding a polypeptide with 97% identity to E. coli SodA. A S. typhimurium sodA mutant was created by allelic exchange and tested for the ability to survive in the murine macrophage-like cell line J774. Growth of bacteria under iron-limiting conditions, inactivation of the Fur repressor, or expression of sodA from a plasmid resulted in increased resistance to early killing by J774 cells, which was abolished in the sodA mutant. These results suggest that resistance to the early oxygen-dependent microbicidal mechanisms of phagocytes involves the SodA gene product. The S. typhimurium sodA mutant was not significantly attenuated in mice, however, which suggests that resistance to early oxygen-dependent microbicidal mechanisms in vivo may play only a minor role in Salmonella pathogenesis.

Amino Acid Sequence↗
PubMed1995

Fur regulon of Salmonella typhimurium: identification of new iron-regulated genes.

In order to identify genes belonging to the Fur regulon of Salmonella typhimurium, a bank of 10,000 independent S. typhimurium MudJ insertion mutants was screened for lacZ fusions regulated by the iron response regulator Fur. In parallel, a plasmid gene bank of S. typhimurium consisting of 10,000 independent clones was screened for Fur-regulated promoters or iron binding proteins by the Fur titration assay (FURTA). Fur-regulated MudJ insertions and Fur-regulated promoters were mapped. In addition, iron-regulated promoter activities of transcriptional fusions from MudJ insertions and FURTA-positive clones were quantified. The nucleotide sequences of 11 FURTA-positive plasmids and of short fragments of DNA flanking three MudJ insertions were determined. By these methods we identified 14 Fur-regulated genes of S. typhimurium. For 11 of these genes, Fur-regulated homologs have been described in Escherichia coli or Yersinia enterocolitica, including fhuA,fhuB,fepA,fes,fepD,p43,entB,fur ,foxA,hemP, and fhuE. In addition, we identified three genes with homologs in other bacteria which have not previously been shown to be Fur regulated.

Amino Acid Sequence↗
PubMed1995

Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster.

A fragment of the Salmonella typhimurium ethanolamine utilization operon was cloned and characterized. The 6.3-kb nucleotide sequence encoded six complete open reading frames, termed cchA, cchB, eutE, eutJ, eutG, and eutH. In addition, the nucleotide sequences of two incomplete open reading frames, termed eutX and eutI, were also determined. Comparison of the deduced amino acid sequences and entries in the GenBank database indicated that eutI encodes a phosphate acetyltransferase-like enzyme. The deduced amino acid sequences of the EutE and EutG proteins revealed a significant degree of homology with the Escherichia coli alcohol dehydrogenase AdhE sequence. Mutations in eutE or eutG completely abolished the ability of mutants to utilize ethanolamine as a carbon source and reduced the ability to utilize ethanolamine as a nitrogen source. The product of eutE is most probably an acetaldehyde dehydrogenase catalyzing the conversion of acetaldehyde into acetyl coenzyme A. The product of the eutG gene, an uncommon iron-containing alcohol dehydrogenase, may protect the cell from unconverted acetaldehyde by converting it into an alcohol. The deduced amino acid sequence of cchA resembles that of carboxysome shell proteins from Thiobacillus neapolitanus and Synechococcus sp. as well as that of the PduA product from S. typhimurium. CchA and CchB proteins may be involved in the formation of an intracellular microcompartment responsible for the metabolism of ethanolamine. The hydrophobic protein encoded by the eutH gene possesses some characteristics of bacterial permeases and might therefore be involved in the transport of ethanolamine. Ethanolamine-utilization mutants were slightly attenuated in a mouse model of S. typhimurium infection, indicating that ethanolamine may be an important source of nitrogen and carbon for S. typhimurium in vivo.

Alcohol Dehydrogenase↗
PubMed1995

Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.

A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences.

Amino Acid Sequence↗
PubMed1994

The effects of two acylcoenzyme A: cholesterol acyltransferase (ACAT) inhibitors, cyclandelate and a non-hydrolysable ether analogue, benzyl3,3,5-trimethylcyclohexanol on low density lipoprotein metabolism in macrophages and hepatocytes.

Cyclandelate (3,3,5-trimethylcyclohexanylmandelate) caused a dose-dependent decrease in the metabolism of radioiodinated low density lipoprotein [125I-LDL] by J774 mouse macrophages. This was probably an indirect effect due to the inhibition of cholesterol esterification by the cells rather than a direct one on the interaction of LDL with its receptor, since no inhibition was seen in cells which had been cholesterol-depleted by prior incubation with lipoprotein-depleted serum for 48 hr. Cyclandelate also inhibited immediately de novo synthesis of cholesterol from [1-14C]acetate in J774 cells, suggesting a direct action of the drug on an enzyme of the cholesterol biosynthetic pathway. The drug was an efficient inhibitor of hamster and rat intestinal acylcoenzyme A: cholesterol acyltransferase (ACAT) activity in vitro with an IC50 of 20 microM. Addition of cyclandelate to the diet of meal-fed rats caused a marked inhibition of the rate of appearance of dietary [4-14C]cholesterol in the plasma. A nonhydrolysable ether analogue of cyclandelate, benzyl3,3,5-trimethylcyclohexanol, was prepared to compare hepatic and extrahepatic actions of the two molecules. The analogue inhibited cholesterol esterification in J774 cells, transformed human macrophages U937 and human umbilical vein endothelial cells with an IC50 of 20 microM and had effects similar to those of cyclandelate on 125I-LDL metabolism in J774 cells. Differences between the analogue and cyclandelate were seen in hepatocytes and hepatic microsomal fractions, where preincubation with the analogue inhibited cholesterol esterification in both systems while cyclandelate had no inhibitory action in either. Consequently, preincubation of rat hepatocytes with benzyl3,3,5-trimethylcyclohexanol for 17 hr caused a marked decrease in the binding of 125I-LDL to the cells, whereas binding to cells preincubated with cyclandelate was the same as to control cells.

Acyl Coenzyme A↗
PubMed1994

A cytolysin encoded by Salmonella is required for survival within macrophages.

A Salmonella gene encoding a cytolysin has been identified by screening for hemolysis on blood agar. DNA sequence analyses together with genetic mapping in Salmonella suggest that it is unrelated to other toxins or hemolysins. The gene (slyA) is present in every strain of Salmonella examined, in Shigella, and in enteroinvasive Escherichia coli but not in other Enterobacteriaceae. SlyA (salmolysin) purified from a derivative of the original clone has hemolytic and cytolytic activity and has a molecular weight predicted by the DNA sequence. The median lethal dose and infection kinetics in mice suggest that the toxin is required for virulence and facilitates Salmonella survival within mouse peritoneal macrophages.

Amino Acid Sequence↗