PubMed Health⌕ Search

Biomedical subjects

M P Jackson

Publications and source records attributed to M P Jackson.

At least 19 recordsLinked to original sources
PubMed1998

Inhibition of prokaryotic translation by the Shiga toxin enzymatic subunit.

Shiga toxin, produced by Shigella dysenteriae serotype 1, is a member of the large family of ribosome-inactivating proteins (RIPs) which are primarily produced by plants. All RIPs are rRNA N-glycosidases which inactivate ribosomes through the removal of a specific adenine residue from the well-conserved aminoacyl-tRNA-accepting loop of rRNA. As a type II RIP, STX is believed to have little effect on prokaryotic ribosomes. However, we have demonstrated that over-expression of the STX enzymatic (A1) polypeptide which lacks a signal sequence caused a reduced rate of growth of its Escherichia coli host. Over-expression of the same StxA1 polypeptide with a catalytic site substitution had no effect on the growth of E. coli. In addition, purified StxA1 was an inhibitor of prokaryotic protein synthesis as assessed using an in vitro transcription and translation assay. The specific activity of StxA1 was significantly higher than ricin, which is another type II RIP, with both eukaryotic and prokaryotic translation systems.

Bacterial Proteins↗
PubMed1997

Investigation of ribosome binding by the Shiga toxin A1 subunit, using competition and site-directed mutagenesis.

The enzymatic subunit of Shiga toxin (StxA1) is a member of the ribosome-inactivating protein (RIP) family, which includes the ricin A chain as well as other examples of plant toxins. StxA1 catalytically depurinates a well-conserved GAGA tetra-loop of 28S rRNA which lies in the acceptor site of eukaryotic ribosomes. The specific activities of native StxA1, as well as mutated forms of the enzyme with substitutions in catalytic site residues, were measured by an in vitro translation assay. Electroporation was developed as an alternative method for the delivery of purified A1 polypeptides into Vero cells. Site-directed mutagenesis coupled with N-bromosuccinimide modification indicated that the sole tryptophan residue of StxA1 is required for binding it to the 28S rRNA backbone. Northern analysis established that the catalytic site substitutions reduced enzymatic activity by specifically interfering with the capacity of StxA1 to depurinate 28S rRNA. Ribosomes were protected from StxA1 by molar excesses of tRNA and free adenine, indicating that RIPs have the capacity to enter the acceptor site groove prior to binding and depurinating the GAGA tetra-loop.

Adenine↗
PubMed1995

Direct detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction.

We recently reported the development and assessment of a technique for the detection of Shiga-like toxin-producing Escherichia coli (SLTEC) using the polymerase chain reaction (PCR) and a digoxigenin-11-dUTP-labelled DNA probe. This technique has now been adapted for the direct identification of SLTEC in ground beef. Ground beef homogenates were diluted 1000-fold to reduce the concentration of components which inhibit the thermostable polymerase. Assessment of four different ground beef samples using the PCR detection technique revealed that fat content was a major inhibitory component. As few as 30 SLTEC ml-1 of a ground beef homogenate were detected using the PCR technique, although it was necessary to enrich six of the samples for positive detection. These findings indicate that the PCR detection technique is suitable for the identification of SLTEC directly from contaminated ground beef without isolation of the bacterium or purification of its DNA.

Animals↗
PubMed1995

Purification and characterization of a Shiga toxin A subunit-CD4 fusion protein cytotoxic to human immunodeficiency virus-infected cells.

In a previous paper, we reported that a chimeric toxin composed of the enzymatic domain of the Shiga toxin A polypeptide (StxA1) genetically fused to the human CD4 (hCD4) molecule selectively kills cells infected with human immunodeficiency virus type 1 (HIV-1). Although other hCD4-containing chimeras cytotoxic to HIV-infected cells have been developed, there is limited information regarding their receptor binding and internalization. Therefore, the goals of this study were to purify the StxA1-hCD4 fusion protein, identify the receptor(s), and investigate the cytosolic trafficking route used by the chimeric toxin. Sufficient quantities of the StxA1-hCD4 hybrid were isolated for this investigation by using the pET expression and purification system. Cos-1 cells were rendered sensitive to the StxA1-hCD4 chimera by transfection with the env gene, which encodes HIV-1 envelope glycoproteins. The entry and translocation pathway used by the StxA1-hCD4 hybrid toxin was investigated by assessing the protective capacities of chemical reagents which interfere with microfilament movement, acidification of endosomes, and the integrity of the Golgi apparatus. Our findings indicated that the chimera uses HIV-1 glycoprotein gp120, and perhaps gp41, as a receptor which directs its entry through receptor cycling. Uptake is pH independent, and the StxA1-hCD4 hybrid is apparently translocated to the Golgi complex as with other bipartite toxins.

Ammonium Chloride↗
PubMed1995

Analysis of Shiga toxin subunit association by using hybrid A polypeptides and site-specific mutagenesis.

Shiga toxin (STX), a bacterial toxin produced by Shigella dysenteriae type 1, is a hexamer composed of five receptor-binding B subunits which encircle an alpha-helix at the carboxyl terminus of the enzymatic A polypeptide. Hybrid toxins constructed by fusing the A polypeptide sequences of STX and Shiga-like toxin type II were used to confirm that the carboxyl terminus of the A subunits governs association with the B pentamers. The alpha-helix of the 293-amino-acid STX A subunit contains nine residues (serine 279 to methionine 287) which penetrate the nonpolar pore of the B-subunit pentamer. Site-directed mutagenesis was used to establish the involvement of two residues bordering this alpha-helix, aspartic acid 278 and arginine 288, in coupling the C terminus of StxA to the B pentamer. Amino acid substitutions at StxB residues arginine 33 and tryptophan 34, which are on the membrane-contacting surface of the pentamer, reduced cytotoxicity without affecting holotoxin formation. Although these B-subunit mutations did not involve receptor-binding residues, they may have induced an electrostatic repulsion between the holotoxin and the mammalian cell membrane or disrupted cytoplasmic translocation.

Amino Acid Sequence↗
PubMed1994

Cytotoxicity of a shiga toxin A subunit-CD4 fusion protein to human immunodeficiency virus-infected cells.

Shiga toxin (STX) is a ribosome-inactivating cytotoxin produced by Shigella dysenteriae serotype 1. The enzymatic domain of the STX A polypeptide has been defined by introducing amino- and carboxy-terminal deletions in the polypeptide and assessing activity in a cell-free translation system. Three recombinant forms of StxA which possess enzymatic activity were genetically fused to a 165-amino-acid polypeptide derived from CD4, the cellular receptor for human immunodeficiency virus type 1 (HIV-1). This strategy eliminated the STX receptor-binding subunit and directed the hybrid toxins to cells expressing the HIV-1 surface glycoprotein gp120. A bacterial lysate containing these toxin chimeras killed the HIV-1-infected T-cell line 8E5 but was not cytotoxic toward the uninfected parental cell line A3.01. This cytotoxic activity was specifically inhibited by monoclonal antibodies which block the interaction between CD4 and gp120. These StxA-CD4 hybrids add to the repertoire of recombinant fusion proteins which possess the capacity to selectively kill HIV-1-infected T cells.

Bacterial Toxins↗
PubMed1993

Minimum domain of the Shiga toxin A subunit required for enzymatic activity.

The minimum sequence of the enzymatic (A) subunit of Shiga toxin (STX) required for activity was investigated by introducing N-terminal and C-terminal deletions in the molecule. Enzymatic activity was assessed by using an in vitro translation system. A 253-amino-acid STX A polypeptide, which is recognized as the enzymatically active portion of the 293-amino-acid A subunit, expressed less than wild-type levels of activity. In addition, alteration of the proposed nicking site between Ala-253 and Ser-254 by site-directed mutagenesis apparently prevented proteolytic processing but had no effect on the enzymatic activity of the molecule. Therefore, deletion analysis was used to identify amino acid residue 271 as the C terminus of the enzymatically active portion of the STX A subunit. STX A polypeptides with N-terminal and C-terminal deletions were released into the periplasmic space of Escherichia coli by fusion to the signal peptide and the first 22 amino acids of Shiga-like toxin type II, a member of the STX family. Although these fusion proteins expressed less than wild-type levels of enzymatic activity, they confirmed the previous finding that Tyr-77 is an active-site residue. Therefore, the minimum domain of the A polypeptide which was required for the expression of enzymatic activity was defined as StxA residues 75 to 268.

Bacterial Toxins↗
PubMed1993

Identification of the Shiga toxin A-subunit residues required for holotoxin assembly.

Recent X-ray crystallographic analyses have demonstrated that the receptor-binding (B) subunits of Shiga toxin (STX) are arranged as a doughnut-shaped pentamer. The C terminus of the enzymatic (A) subunit presumably penetrates the nonpolar pore of the STX B pentamer, and the holotoxin is stabilized by noncovalent interactions between the polypeptides. We identified a stretch of nine nonpolar amino acids near the C terminus of StxA which were required for subunit association by using site-directed mutagenesis to introduce progressive C-terminal deletions in the polypeptide and assessing holotoxin formation by a receptor analog enzyme-linked immunosorbent assay, immunoprecipitation, and a cytotoxicity assay. Tryptophan and aspartic acid residues which form the N-terminal boundary, as well as two arginine residues which form the C-terminal boundary of the nine-amino-acid sequence, were implicated as the stabilizers of subunit association. Our model proposes that residues 279 to 287 of the 293-amino-acid STX A subunit penetrate the pore while the tryptophan, aspartic acid, and 2 arginine residues interact with other charged or aromatic amino acids outside the pore on the planar surfaces of the STX B pentamer.

Amino Acid Sequence↗
PubMed1993

Roles of a ribosome-binding site and mRNA secondary structure in differential expression of Shiga toxin genes.

The Shiga toxin operon (stx) is composed of two genes for the A and B subunits, which are transcribed from a promoter 5' to the stxA gene. The 1A:5B subunit stoichiometry of the holotoxin suggests that the stxA and stxB genes are differentially regulated. In a previous study, we demonstrated the existence of a second promoter which independently transcribes the stxB gene. However, transcription fusion analysis revealed that the independent stxB gene promoter is not solely responsible for a fivefold increase in B polypeptide production. In this study, we have investigated the role of an independent stxB gene ribosome-binding site (RBS) in the overexpression of STX B subunits. Site-directed mutagenesis was used to eliminate this RBS and establish its role in StxB production. Examination of the nucleotide sequences surrounding the stxB gene RBS revealed a potential for the formation of a stem-loop structure with a calculated delta G of -7.563 kcal/mol (ca. -31.64 kJ/mol). Sequences surrounding the stxA gene RBS were found not to possess a similar potential for secondary-structure formation. Disruption of the stem-loop surrounding the stxB gene RBS by 2- and 4-nucleotide substitutions caused a significant reduction in B polypeptide and holotoxin production, establishing the role of this secondary structure in the enhancement of translation of the stxB gene.

Bacterial Toxins↗
PubMed1993

Evaluation of a technique for identification of Shiga-like toxin-producing Escherichia coli by using polymerase chain reaction and digoxigenin-labeled probes.

A polymerase chain reaction (PCR) technique for the identification of Shiga-like toxin (SLT)-producing Escherichia coli was assessed by using 95 strains of SLT-producing E. coli and 5 Shigella dysenteriae type 1 strains. PCR was used for the amplification of slt gene sequences from whole bacterial colonies. A digoxigenin-labeled DNA probe was used for identification of the PCR products in a spot blot hybridization assay. Modifications were made to adapt this technique for the proper identification of 10 SLT-producing isolates which were refractory to the heat lysis step that was used to liberate whole-cell DNA for PCR and 6 isolates which gave nonspecific amplification products. The sensitivity and specificity of this assay were each 99% when compared with toxin neutralization results by using SLT-specific monoclonal antibodies. These values indicate that this detection technique could be suitable for use in a clinical laboratory.

Antibodies, Monoclonal↗
PubMed1992

Identification of Shiga-like toxin type II producing Escherichia coli using the polymerase chain reaction and a digoxigenin labelled DNA probe.

Epidemiological studies have demonstrated that enterohaemorrhagic strains of Escherichia coli which cause the haemolytic uremic syndrome in humans and the oedema disease in pigs more frequently produce Shiga-like toxin type II (SLT-II) than any other member of the Shiga-like toxin family. A technique has been developed for the identification of SLT-II producing E. coli using the polymerase chain reaction (PCR) and a digoxigenin (DIG)-labelled DNA probe to facilitate the early detection and epidemiological analysis of these pathogens. Whole cell DNA liberated from isolated colonies during the denaturation step of PCR was amplified using a primer pair which is homologous to the slt-II gene sequences. The amplification products were transferred directly to a nitrocellulose membrane or following agarose gel electrophoresis and DNA denaturation. A chemically labelled DNA probe, prepared using PCR with the incorporation of DIG, was used to identify the PCR products of strains which produced SLT-II or a variant of SLT-II.

Bacterial Toxins↗
PubMed1992

Homelessness as a determinant of health.

The homeless population in the United States is variously estimated at between 350,000 and 2.5 million. Families and children are among the fastest-growing segments of this population. A combination of social and economic changes occurring nationally have contributed to this phenomenon. Conditions associated with homelessness have a profound effect on the individual's ability to maintain health, to have access to treatment, and to recuperate. Several health problems are specific to the homeless, and have implications for public health practitioners.

Health Services Needs and Demand↗
PubMed1992

Identification of a B subunit gene promoter in the Shiga toxin operon of Shigella dysenteriae 1.

The Shiga toxin operon (stx) is composed of A and B subunit genes which are transcribed as a bicistronic mRNA from a promoter which lies 5' to the stxA gene. Northern (RNA) blot and primer extension analyses revealed the existence of a second stxB gene transcript. Recombinant plasmids which carried the stxB gene without the stx operon promoter and with the influence of a vector promoter abrogated produced STX B polypeptides, suggesting that the stxB gene mRNA was transcribed from an independent promoter and was not produced by endoribonucleotic processing of the bicistronic mRNA. Examination of the DNA sequences 5' to the stxB gene transcription initiation site which were carried by the recombinant plasmids revealed a region with high homology to the consensus for Escherichia coli promoters. Deletion and mutation of this region affected StxB and holotoxin production, establishing its role in the regulation of the stxB gene. Comparison of the promoters by using a transcription analysis vector revealed that the stxB gene promoter differed from the stx operon promoter in that was approximately sixfold less efficient and was not repressed by iron. Identification of a second promoter in the stx operon indicates that independent transcription of the stxB gene may regulate overproduction of the STX B polypeptides and may contribute to the 1A:5B subunit stoichiometry of the holotoxin.

Amino Acid Sequence↗
PubMed1991

Detection of Shiga toxin-producing Shigella dysenteriae type 1 and Escherichia coli by using polymerase chain reaction with incorporation of digoxigenin-11-dUTP.

A technique has been developed for the detection of Shiga toxin- and Shiga-like toxin type I (ShT/SLT-I)-producing Shigella dysenteriae type 1 and Escherichia coli by using the polymerase chain reaction with the incorporation of digoxigenin-11-dUTP. Target DNA liberated from whole cells was amplified, using primer pairs homologous to the A-subunit genes of ShT/SLT-I. The TTP analog digoxigenin-11-dUTP was incorporated into the reaction mixture, permitting nonradioactive labeling of the amplified DNA. The labeled polymerase chain reaction products were hybridized to specific gene sequences immobilized on a nitrocellulose membrane and detected by using an alkaline phosphatase-conjugated antibody to digoxigenin and the enzyme substrates. Toxin-producing strains of E. coli and S. dysenteriae type 1 were identified as colored spots on the membrane. Because this technique does not require DNA purification, gel electrophoresis, or radioactive DNA probes, it is suitable for the clinical detection of ShT/SLT-I-producing strains of S. dysenteriae type 1 and E. coli.

Bacterial Toxins↗