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Biomedical subjects

A J Lax

Publications and source records attributed to A J Lax.

At least 19 recordsLinked to original sources
PubMed2001

Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice.

Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.

Amino Acid Substitution↗
PubMed2001

Pasteurella multocida toxin: the mitogenic toxin that stimulates signalling cascades to regulate growth and differentiation.

Pasteurella multocida toxin (PMT) is an unusual toxin that acts as a mitogen by stimulating various intracellular signalling cascades. Pathways downstream of the G-protein Gq and also downstream of the Rho proteins are activated. Thus PMT action stimulates phospholipase C leading to activation of protein kinase C, an increase in inositol phosphates, and a rise in intracellular calcium. Rho activation of the Rho kinase leads to cytoskeletal reorganisation, tyrosine phosphorylation of the focal adhesion kinase, and activation of the Src proto-oncogene. In addition, signalling through the Ras-MAP kinase signalling pathway is also initiated. PMT is an intracellularly acting toxin, and functional domains that carry out different aspects of its function have been described. The intracellular target of the toxin is currently not known. PMT also acts to inhibit differentiation, in particular of bone cells, where it prevents the formation of mineralised bone nodules in vitro. The toxin is the causative agent of a porcine disease that is characterised by bone resorption. Injection of very low doses of toxin leads to proliferative effects, but at higher doses is lethal. The possible effect of PMT-induced perturbation of signal transduction pathways is discussed.

3T3 Cells↗
PubMed2001

Cytotoxic necrotizing factor from Escherichia coli induces RhoA-dependent expression of the cyclooxygenase-2 Gene.

Cytotoxic necrotizing factor 1 (CNF) is a toxin produced by some isolates of Escherichia coli that cause extraintestinal infections. CNF can initiate signaling pathways that are mediated by the Rho family of small GTPases through a covalent modification that results in constitutive activation. In addition to regulating the assembly of actin stress fibers and focal adhesion complexes, RhoA can also regulate gene expression at the level of transcription. Here we demonstrate for the first time, by using a luciferase-based reporter system, that the transcription of cyclooxygenase-2 (COX-2) is strongly upregulated in NIH 3T3 fibroblasts treated with CNF and that this effect is dependent upon the activation of RhoA by the toxin. Subsequent protein tyrosine phosphorylation events modulate the induction, but the transcription signal is not mediated by Rho-associated kinase (p160/ROCK) and so must rely upon another effector that is activated by RhoA. CNF therefore induces COX-2 expression via a RhoA-dependent signaling pathway that diverges from the pathway that regulates cytoskeletal rearrangements in response to RhoA activation.

3T3 Cells↗
PubMed2001

Localization of functional domains of the mitogenic toxin of Pasteurella multocida.

The locations of the catalytic and receptor-binding domains of the Pasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed alpha/beta domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

3T3 Cells↗
PubMed2001

Escherichia coli cytotoxic necrotizing factor and Pasteurella multocida toxin induce focal adhesion kinase autophosphorylation and Src association.

Cytotoxic necrotizing factor 1 and Pasteurella multocida toxin induced dose- and time-dependent increases in focal adhesion kinase (FAK) Tyr397 phosphorylation in Swiss 3T3 cells. FAK autophosphorylation was sensitive to inhibitors of p160/ROCK and coincided with the formation of stable complexes between FAK and Src family members.

3T3 Cells↗
PubMed2000

The pathological effect of the Bordetella dermonecrotic toxin in mice.

The effect of dermonecrotic toxin (DNT) expression of Bordetella bronchiseptica was studied in mice by comparing the pathology induced by a wild type strain with that induced by an isogenic DNT- strain in which part of the structural gene has been replaced by an antibiotic resistance cassette. While extracts of strain B58 proved toxic in intravenously inoculated mice, similar extracts from strain B58GP had lost toxic activity. The parent (B58) and the mutant (B58GP) strains of B. bronchiseptica each possessed comparable virulence for mice. These findings confirmed that DNT production was successfully abolished in strain B58GP while other virulence characteristics required for pathogenicity in mice remained intact, at a comparable level to the parent strain. Turbinate atrophy was observed in mice infected with the DNT+ strain, but not in those infected with the DNT- strain. This indicates that DNT is the cause of turbinate atrophy in the mice and not other factors produced by phase I strains of B. bronchiseptica. B. bronchiseptica DNT showed a lienotoxic effect (lymphocyte depletion and a reduction in the intensity of extramedullar haemocytopoieis) that is considered to adversely alter the immune function of the host animal. In mice infected with strain B58GP, catarrhal pneumonia with characteristic lympho-histiocytic peribronchial and perivascular infiltration was noticed. In mice infected with strain B58, large necrotic areas were seen surrounded by an inflammatory reaction. The DNT appears to directly damage lung tissues, at least in mice. DNT production seems to enhance the establishment of B. bronchiseptica in the lungs, presumably by reducing the local resistance and causing severe local damage to the lung tissues.

Animals↗
PubMed1999

Reduced pH causes structural changes in the potent mitogenic toxin of Pasteurella multocida.

Pasteurella multocida toxin is a potent mitogen that is believed to act intracellularly. On transverse urea gradient gels at pH 8.0 the toxin displayed one major unfolding transition at 4 M urea. However, at pH 6.1 the unfolding transition took place at 3.5 M urea. Circular dichroism spectra also indicated that a structural change took place at acidic pH. In addition it was found that the toxin that had been denatured in 8 M urea refolded in solution with a high recovery of biological activity. These findings are discussed in terms of the likely domain structure of the P. multocida toxin.

3T3 Cells↗
PubMed1998

Pasteurella multocida toxin stimulates bone resorption by osteoclasts via interaction with osteoblasts.

In this study we used an in vitro assay system with osteoblast and osteoclast co-cultures to assess the effect of purified recombinant Pasteurella multocida toxin on bone resorption. Resorption was measured by the release of a telopeptide breakdown product of type I collagen. It was found that P. multocida did not stimulate bone resorption by osteoclasts directly and also did not stimulate bone breakdown via the release of collagenase or other proteases from osteoblasts. During co-culture of osteoblasts and osteoclasts, with cell-cell contact prevented, P. multocida toxin produced no significant effect. Osteoblast-conditioned media gave a biphasic effect; low concentrations of P. multocida toxin stimulated bone resorption, whereas 100 ng/ml inhibited resorption by osteoclasts. However, when both cell types were co-cultured with cell-cell contact permitted, P. multocida toxin induced a large concentration-dependent increase in bone resorption over a 7-day period. This suggested that P. multocida toxin causes bone breakdown via an osteoblast-dependent mechanism and that a membrane-bound receptor may be involved.

Animals↗
PubMed1998

Activity of the mitogenic Pasteurella multocida toxin requires an essential C-terminal residue.

Pasteurella multocida toxin (PMT) is a potent mitogen that also affects bone resorption. PMT acts intracellularly and is therefore postulated to have several domains involved in different aspects of its function. The toxin contains eight cysteine residues. Mutants with individual substitutions for each of these residues were constructed, and the effects of these on the biological activity of the toxin were determined by cultured-cell assays. Only the most C-terminal of the eight cysteines (C1165) was essential for full activity, although mutation of the cysteine residue at position 1159 caused a slight but reproducible loss of potency. In animal challenge experiments, mutant toxin (C1165S) was not toxic to piglets, even at doses exceeding a lethal dose of active PMT 1, 000-fold. The mutant and wild-type toxins displayed identical purification characteristics, similar susceptibility to proteolytic digestion, and circular dichroism profiles, which indicated that no gross structural changes had taken place. The function of the essential C1165 residue is not yet known, although its most likely role is an enzymatic one at or near the catalytic center of the toxin.

3T3 Cells↗
PubMed1997

Identification of restriction barriers in Pasteurella multocida.

Several naturally occurring antibiotic resistance plasmids were isolated from Pasteurella multocida type D strains. One plasmid, pPM1, was used to study transfer of DNA among P. multocida strains, and could be transferred into Escherichia coli and some P. multocida isolates. However, pPM1 could only be transferred into the toxigenic P. multocida LFB3 at very low frequency. Plasmid recovered from the electrotransformants could be transferred to LFB3 at high frequency. These plasmid DNAs were resistant to PstI, and sensitive to DpnI digestion. Sensitivity to DpnI was common to all the P. multocida DNAs, but resistance to PstI was confined to LFB3. Plasmid pPM1 treated with PstI methylase was able to transform LFB3 at an increased frequency compared to unmethylated DNA, suggesting that LFB3 has a restriction system which cleaves at or near PstI sites.

DNA, Bacterial↗
PubMed1997

Cytotoxic necrotizing factor 1 from Escherichia coli and dermonecrotic toxin from Bordetella bronchiseptica induce p21(rho)-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 cells.

Treatment of Swiss 3T3 cells with cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli and dermonecrotic toxin (DNT) from Bordetella bronchiseptica, which directly target and activate p21(rho), stimulated tyrosine phosphorylation of focal adhesion kinase (p125(fak)) and paxillin. Tyrosine phosphorylation induced by CNF1 and DNT occurred after a pronounced lag period (2 h), and was blocked by either lysosomotrophic agents or incubation at 22 degrees C. CNF1 and DNT stimulated tyrosine phosphorylation of p125(fak) and paxillin, actin stress fiber formation, and focal adhesion assembly with similar kinetics. Cytochalasin D and high concentrations of platelet-derived growth factor disrupted the actin cytoskeleton and completely inhibited CNF1 and DNT induced tyrosine phosphorylation. Microinjection of Clostridium botulinum C3 exoenzyme which ADP-ribosylates and inactivates p21(rho) function, prevented tyrosine phosphorylation of focal adhesion proteins in response to either CNF1 or DNT. In addition, our results demonstrated that CNF1 and DNT do not induce protein kinase C activation, inositol phosphate formation, and Ca2+ mobilization. Moreover, CNF1 and DNT stimulated DNA synthesis without activation of p42(mapk) and p44(mapk) providing additional evidence for a novel p21(rho)-dependent signaling pathway that leads to entry into the S phase of the cell cycle in Swiss 3T3.

3T3 Cells↗
PubMed1996

Constitutive expression of Pasteurella multocida toxin.

The expression of the Pasteurella multocida toxin (PMT) gene toxA was investigated. Growth in vitro at 30 degrees C or added iron caused less than 4-fold repression of toxA expression. The putative repressor TxaR was expressed in Escherichia coli but deletion and frameshift mutations abolishing TxaR production had no effect on toxA expression. Naturally occurring non-toxigenic mutants which contained the toxA gene had no large rearrangements near toxA or changes in toxA promoter structure. Thus PMT is constitutively expressed and is only regulated in a minor way.

Bacterial Proteins↗
PubMed1996

Pasteurella multocida toxin, a potent intracellularly acting mitogen, induces p125FAK and paxillin tyrosine phosphorylation, actin stress fiber formation, and focal contact assembly in Swiss 3T3 cells.

Treatment of Swiss 3T3 cells with recombinant Pasteurella multocida toxin (rPMT), a potent intracellularly acting mitogen, stimulated tyrosine phosphorylation of multiple substrates including bands of M(r) 110,000-130,000 and M(r) 70,000-80,000. Tyrosine phosphorylation induced by rPMT occurred after a pronounced lag period (1 h) and was blocked by either lysosomotrophic agents or incubation at 22 degrees C. Focal adhesion kinase (p125FAK) and paxillin are prominent substrates for rPMT-stimulated tyrosine phosphorylation. Tyrosine phosphorylation by rPMT could be dissociated from both protein kinase C activation and the mobilization of calcium from intracellular stores. rPMT stimulated striking actin stress fiber formation and focal adhesion assembly in Swiss 3T3 cells. Cytochalasin D, which disrupts the actin cytoskeleton, completely inhibited rPMT-induced tyrosine phosphorylation. In addition, tyrosine phosphorylation of p125FAK and paxillin in response to rPMT was completely abolished when cells were subsequently treated with platelet-derived growth factor at a concentration (30 ng/ml) that disrupted the actin cytoskeleton. Our results demonstrate for the first time that rPMT, a bacterial toxin, induces tyrosine phosphorylation of p125FAK and paxillin and promotes actin stress fiber formation and focal adhesion assembly in Swiss 3T3 cells.

3T3 Cells↗
PubMed1996

The Salmonella dublin virulence plasmid does not modulate early T-cell responses in mice.

The virulence plasmid in Salmonella dublin mediates systemic infection in mice and cattle. The role of gamma delta T cells or hepatic extrathymic T cells has recently been reported to be important in the control of the early stage of Salmonella choleraesuis infections of mice. Here, we report on T-cell responses in conventional mice after challenge with a virulent strain of S. dublin carrying a virulence plasmid or with a strain cured of the plasmid. Over a period of 4 days postinfection, when both strains could be compared, similar changes in alpha beta and gamma delta T-cell subsets in peritoneal cavities, livers, and spleens were recorded, demonstrating no clear role of the virulence plasmid in modulation of early T-cell responses. To investigate further the role of the virulence plasmid in pathogenesis, the growth of the plasmid-cured strain was assessed in SCID, SCID bg, and irradiated mice. During the first 6 days after infection, there was no statistically difference in the net growth of Salmonella cells in the livers and spleens of SCID and SCID bg mice compared with conventional BALB/mice. This observation excludes a key role for a T- or B-cell-mediated immune response in controlling the initial growth of the plasmid-cured S. dublin strain. Thereafter, the immunocompromised mice were no longer able to control infection, although SCID mice were more efficient at controlling net bacterial multiplication than SCID bg mice, potentially implicating NK cells in the control of infection in SCID mice. The early control of net bacterial multiplication in the spleens and livers of BALB/c mice was ablated by whole-body X-irradiation. Both wild-type and plasmid-cured strains multiplied significantly more rapidly in irradiated than in conventional BALB/c mice. However, the numbers of wild-type bacterial still increased more rapidly than in the numbers of the cured strains. These results are consistent with a role of the S. dublin virulence plasmid in promoting in vivo growth of Salmonella cells.

Animals↗
PubMed1996

Cloning, expression, and molecular characterization of the dermonecrotic toxin gene of Bordetella spp.

A cosmid library of random fragments of Bordetella bronchiseptica genomic DNA was prepared and screened with oligonucleotides designed from the sequence of the B. pertussis dermonecrotic toxin (DNT) gene. Two cosmid clones which apparently contained the complete B. bronchiseptica DNT gene were identified, but they did not express the toxin. A 5-kb fragment containing the DNT gene was subcloned from one of the cosmid clones onto a high-copy-number plasmid, and this resulted in low-level expression of the toxin. The expression level was increased by deletion of a small region upstream of the coding sequence. Assays for biological activity, including the infant mouse dermonecrosis assay, confirmed that the product of the cloned gene was DNT. The complete sequence of the B. bronchiseptica DNT gene was determined and was more than 99% homologous to the DNT gene of B. pertussis. A putative purine nucleotide-binding motif was shown to be important for toxic activity. Extracts containing the recombinant or the native toxin induced DNA synthesis in Swiss 3T3 cells but inhibited cell division leading to binucleation.

3T3 Cells↗
PubMed1996

Pasteurella multocida toxin is a mitogen for bone cells in primary culture.

The effect of recombinant Pasteurella multocida toxin (PMT) on primary cultures of embryonic chick bone-derived osteoblastic cells was investigated. It was found that PMT was a potent mitogen for primary derived chicken osteoblasts. The toxin stimulated DNA synthesis and cell proliferation in quiescent osteoblasts at the first passage and accelerated cell growth in subconfluent cultures. Cell viability was not affected by PMT, even at relatively high concentrations. Osteoblast numbers increased in a dose-dependent manner in response to PMT. Intracellular inositol phosphates were elevated in response to PMT, but no elevation in cyclic AMP (cAMP) levels was evident. Indeed, PMT inhibited cAMP elevation in osteoblasts in response to cholera toxin at a stage before other PMT-mediated events take place. In addition to increased cell turnover, PMT down-regulated the expression of several markers of osteoblast differentiation. Both alkaline phosphatase and type I collagen were reduced, but osteonectin was not affected. The in vitro deposition of mineral in cultures of primary osteoblasts and osteoblast-like osteosarcoma cells was also inhibited by the presence of PMT. This suggests that PMT interferes with differentiation at a preosteoblastic stage.

Animals↗
PubMed1996

The Salmonella virulence plasmid enhances Salmonella-induced lysis of macrophages and influences inflammatory responses.

The Salmonella dublin virulence plasmid mediates systemic infection in mice and cattle. Here, we analyze the interaction between wild-type and plasmid-cured Salmonella strains with phagocytes in vitro and in vivo. The intracellular recovery of S. dublin from murine peritoneal and bovine alveolar macrophages cultured in the presence of gentamicin in vitro was not related to virulence plasmid carriage. However, the virulence plasmid increased the lytic activity of S. dublin, Salmonella typhimurium, and Salmonella choleraesuis for resident or activated mouse peritoneal macrophages. Lysis was not mediated by spv genes and was abolished by cytochalasin D treatment. Peritoneal and splenic macrophages were isolated from mice 4 days after intraperitoneal infection with wild-type or plasmid-cured S. dublin strains. The wild-type strain was recovered in significantly higher numbers than the plasmid-cured strain. However, the intracellular killing rates of such cells cultured in vitro for both S. dublin strains were not significantly different. Four days after infection, there was a lower increase of phagocyte numbers in the peritoneal cavities and spleens of mice infected with the wild-type strain compared with the plasmid-cured strain. The virulence plasmid influenced the survival of macrophages in vitro following infection in vivo as assessed by microscopy. Cells from mice infected with the plasmid-cured strain survived better than those from mice infected with the wild-type strain. This is the first report demonstrating an effect of the virulence plasmid on the interaction of Salmonella strains with macrophages. Plasmid-mediated macrophage dysfunction could influence the recruitment and/or the activation of phagocytic cells and consequently the net growth of Salmonella strains during infection.

Animals↗