Tissue factor regulation and gene organization.
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Amino Acid Sequence↗
Biomedical subjects
N Mackman
Publications and source records attributed to N Mackman.
The hemolysin of Escherichia coli.
Many strains of E. coli elaborate a hemolysin which is responsible for the zone of beta-hemolysis surrounding bacterial colonies on blood agar. The significance of this cytolysin as a determinant of bacterial pathogenicity has been established in animal models with the use of genetically engineered, isogenic bacterial strains. An analogous role in human infections has been inferred from the high association of hemolysin production with disease. Studies at a molecular genetical level have defined 4 genes that are required for the synthesis, post-translational modification and secretion of the hemolysin. The structural gene hlyA encodes for a 107-110,000 polypeptide, which must be modified in an unknown manner to its active form by the product of the neighboring hlyC gene. Genes hlyB and hlyD encode for proteins that export the molecule to the extracellular medium. The signal for secretion is contained in the C-terminal portion of the toxin molecule. The secreted hemolysin attacks plasma membranes of target mammalian cells by inserting as a monomer into the bilayer and generating hydrophilic transmembrane pores of approximately 2 nm effective diameter. The pores display a marked selectivity for cations over anions and pore-opening is dependent on the presence of a correct transmembrane potential. Binding to a membrane target does not require the presence of a specific receptor, and pores may be generated in planar lipid membranes consisting solely of phosphatidylcholine. Pore formation in nucleated cells can trigger secondary reactions such as stimulation of arachidonate metabolism with release of lipid mediators, probably initiated by passive influx of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
[Mechanism of cell damage by E. coli hemolysin].
Many strains of E. coli elaborate a hemolysin which is responsible for the zone of beta-hemolysis surrounding bacterial colonies on blood agar. The significance of this cytolysin as a determinant of bacterial pathogenicity has been established in animal models with the use of genetically engineered, isogenic bacterial strains. An analogous role in human infections has been inferred from the high association of hemolysin production with disease. Studies at a molecular genetical level have defined 4 genes that are required for the synthesis, post-translational modification and secretion of the hemolysin. The structural gene hlyA encodes for a 107-110,000 polypeptide which must be modified in an unknown manner to its active form by the product of the neighboring hlyC gene. Genes hlyB and hlyD encode for proteins that export the molecule to the extracellular medium. The signal for secretion is contained in the C-terminal portion of the toxin molecule. The secreted hemolysin attacks plasma membranes of target mammalian cells by inserting as a monomer into the bilayer and generating a hydrophilic transmembrane pore of approximately 2 nm effective diameter. The pore displays a marked selectivity for cations over anions and pore-opening is dependent on the presence of a correct transmembrane potential. Binding to a membrane target does not require the presence of a specific receptor, and pores may be generated in planar lipid membranes consisting solely of phosphatidylcholine. Pore formation in nucleated cells can trigger secondary reactions such as stimulation of arachidonate metabolism with release of lipid mediators, probably initiated by passive influx of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Role of SecA and SecY in protein export as revealed by studies of TonA assembly into the outer membrane of Escherichia coli.
The growth of secAts or secYts mutants at the restrictive temperature has been shown to inhibit the export of many outer membrane proteins. We report here that in two secAts strains the rate of incorporation of newly synthesized protein into both inner and outer membrane fractions decreased by about 70% at the restrictive temperature. The export of the outer membrane protein TonA was used as a model system in which to study the effects of SecA or SecY inactivation. pre-TonA that accumulated at the restrictive temperature was found to co-sediment with the outer membrane fraction. However, the precursor was sensitive to protease and did not float up a sucrose gradient with the membrane fractions. It was therefore concluded that pre-TonA was not integrated into the outer membrane fraction but probably accumulated in the cytoplasm. Studies on the rate of processing of pre-TonA, pulse-labelled at the restrictive temperature then chased at the permissive temperature, revealed differences between secA and secY mutants. In the secAts mutant the great majority of cytoplasmic pre-TonA was not apparently processed to the mature form, whereas in the secYts mutant significant amounts of precursors were rapidly chased into mature TonA, which appeared in the outer membrane. These results suggest that SecA and SecY may act sequentially in the export of proteins to the outer membrane. In particular these data indicate that SecA is required to maintain pre-TonA in a translocationally competent form prior to interaction with the SecY export site.
Bacterial Outer Membrane Proteins↗
Escherichia coli haemolysin forms voltage-dependent ion channels in lipid membranes.
The action of the 107 kDa hemolysin from Escherichia coli on planar lipid membranes was investigated. We report that a single toxin molecule can form a cation-selective, ion-permeable channel of large conductance in a planar phospholipid bilayer membrane. The conductance of the pore is proportional to that of the bulk solution, indicating that the channel is filled with water. A pore diameter of about 2 nm can be evaluated. The pore formation mechanism is voltage-dependent and essentially resembles that of pore-forming colicins; this implies that opening of the channel is dependent on transfer of an electrical charge through the membrane. We propose that the physiological effects of E. coli hemolysin result from its ability to form ion channels in the membrane of attacked cells, and show that there is quantitative agreement between the effects of this toxin on model membranes and its hemolytic properties.
Calcium↗
Release of a chimeric protein into the medium from Escherichia coli using the C-terminal secretion signal of haemolysin.
Recently, we have identified a novel topogenic sequence at the C terminus of Escherichia coli haemolysin (HlyA) which is essential for its efficient secretion into the medium. This discovery has introduced the possibility of using this secretion system for the release of chimeric proteins from E. coli directly into the medium. We have now successfully fused this C-terminal signal to a hybrid protein containing a few residues of beta-galactosidase and the majority of the E. coli outer membrane porin OmpF lacking its own N-terminal signal sequence. We find that this chimeric protein is specifically translocated across the inner and outer membranes and is released into the medium. In addition, we have further localized the HlyA secretion signal to the final 113 amino acids of the C terminus. In fact, a specific secretion signal appears to reside at least in part within the last 27 amino acids of HlyA.
Amino Acid Sequence↗
Genetics and biochemistry of the assembly of proteins into the outer membrane of E. coli.
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Bacterial Outer Membrane Proteins↗
Identification with monoclonal antibodies of hemolysin produced by clinical isolates of Escherichia coli.
Murine monoclonal antibodies were generated against the 107,000-dalton hemolysin encoded by the hemolytic determinant from Escherichia coli LE 2001, and colony blotting was used to assay for production of the hemolysin by 35 hemolytic strains of E. coli and other hemolytic members of the family Enterobacteriaceae of clinical origin. All hemolytic E. coli strains gave positive reactions with two monoclonal antibodies. In contrast, none of the hemolytic, non-E. coli isolates yielded positive colony blots. In addition, Western blotting showed that the hemolysins produced by all clinical E. coli isolates had a similar molecular weight of about 107,000. Discrete antigenic variation may occur in the molecule, since a third monoclonal antibody did not react with the hemolysin from a number of wild-type E. coli strains. Western blot analysis was used to assess the presence of immunoglobulin G (IgG), IgA, and IgM antibodies to E. coli hemolysin in human sera. All 20 of the tested sera from healthy adults contained antibodies to the toxin, with various constellations among the antibody classes. In contrast, sera from five of eight infants aged 8 to 36 months contained no antihemolysin antibodies. We conclude that the 107,000-dalton hemolysin of E. coli is a widespread immunogen that is produced by most or all hemolytic E. coli strains in the human host.
Animals↗
The C-terminal, 23 kDa peptide of E. coli haemolysin 2001 contains all the information necessary for its secretion by the haemolysin (Hly) export machinery.
In this paper we show the construction of a plasmid pLG609 which carries the 3'-end of the haemolysin structural gene, hlyA under tac promoter control. Expression of pLG609 in an E. coli strain carrying the haemolysin export genes hlyB and hlyD led to the efficient secretion of the C-terminal, 23 kDa peptide of haemolysin. The discovery of a C-terminal topogenic sequence, which appears to be all that is required for secretion of the whole toxin, is so far quite unique in protein export.
Biological Transport↗
Secretion of haemolysin by Escherichia coli.
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DNA Restriction Enzymes↗
The carboxy-terminal region of haemolysin 2001 is required for secretion of the toxin from Escherichia coli.
As a first step in the detailed analysis of the mechanism of secretion of haemolysin, we sought to identify sequences or domains within haemolysin A (HlyA) that are essential for its secretion. For this purpose we examined the properties of a deletion and Tn5 insertions into the region of the HlyA gene encoding the C-terminal part of the protein, since both of these are relatively simple to generate. We showed that removal of 27 amino acids from the C-terminus of HlyA is sufficient to inhibit secretion drastically, although the residual polypeptide is still haemolytically active. Cellular fractionation studies showed that haemolytic activity does not accumulate in large amounts within the periplasmic space during normal secretion. More significantly, activity does not appear to accumulate within this compartment when the export functions hlyB and hlyD are removed. These results are consistent with a mechanism in which interaction of the C-terminus of HlyA with the secretion machinery, located in the inner membrane, is followed by direct transfer of haemolysin to the medium.
Amino Acid Sequence↗
Escherichia coli hemolysin may damage target cell membranes by generating transmembrane pores.
Escherichia coli hemolysin is secreted as a water-soluble polypeptide of Mr 107,000. After binding to target erythrocytes, the membrane-bound toxin resembled an integral membrane protein in that it was refractory towards extraction with salt solutions of low ionic strength. Toxin-induced hemolysis could be totally inhibited by addition of 30 mM dextran 4 (mean Mr, 4,000; molecular diameter approximately 3 nm) to the extracellular medium. Uncharged molecules of smaller size (e.g., sucrose, with a molecular diameter of 0.9 nm, or raffinose, with a molecular diameter of 1.2 to 1.3 nm) did not afford such protection. Treatment of erythrocytes suspended in dextran-containing buffer with the toxin induced rapid efflux of cellular K+ and influx of 45Ca2+, as well as influx of [14C]mannitol and [3H]sucrose. [3H]inulin only slowly permeated into toxin-treated cells, and [3H]dextran uptake was virtually nil. Membranes lysed with high doses of E. coli hemolysin exhibited no recognizable ultrastructural lesions when examined by negative-staining electron microscopy. Sucrose density gradient centrifugation of deoxycholate-solubilized target membranes led to recovery of the toxin exclusively in monomer form. Incubation of toxin-treated cells with trypsin caused limited proteolysis with the generation of membrane-bound, toxin-derived polypeptides of Mr approximately 80,000 without destroying the functional pore. We suggest that E. coli hemolysin may damage cell membranes by partial insertion into the lipid bilayer and generation of a discrete, hydrophilic transmembrane pore with an effective diameter of approximately 3 nm. In contrast to the structured pores generated by cytolysins of gram-positive bacteria such as staphylococcal alpha-toxin and streptolysin O, pore formation by E. coli hemolysin may be caused by the insertion of toxin monomers into the target lipid bilayers.
Cell Membrane Permeability↗
Characterisation of HlyC and mechanism of activation and secretion of haemolysin from E. coli 2001.
In this paper the DNA sequence of the cloned hlyC gene from E. coli 2001 is presented. The gene encodes a protein of 20 kDa which is able to activate the 107 kDa polypeptide encoded by hlyA. This gives rise to a haemolytically active protein which differs from the inactive form in stability and by its migration when analysed by polyacrylamide gel electrophoresis under non-denaturing conditions. We also show that the inactive form is secreted in the presence of the transport functions hlyB and hlyD. This result rules out any role for the hlyC gene product in the transport of HlyA across the inner membrane.
Base Sequence↗
Regulation of haemolysin synthesis in E. coli determined by HLY genes of human origin.
We have previously reported the secretion of a 107K polypeptide into the medium from a haemolytic E. coli K12 strain (Mackman and Holland 1984a). In addition, we demonstrated that haemolysin production was correlated with the presence of this polypeptide in the growth medium in a large number of E. coli isolates of human and animal origin (Mackman and Holland 1984b). In this paper we confirm that the 107K polypeptide is indeed haemolysin: both haemolytic activity and the 107K polypeptide show a similar pattern of accumulation during the growth cycle; identical levels are produced in three different growth media; they have the same half-life in minimal medium. The results also show that the expression of haemolysin is not influenced by the growth medium or subject to catabolite repression. However, expression is apparently switched off as cells enter the late exponential phase of growth. Finally, we present data indicating that the previously reported variation in haemolysin production in different media is entirely due to the instability of the haemolysin itself. Degradation of the 107K polypeptide in the medium was accompanied by the accumulation of a major breakdown product of 60K.
DNA, Recombinant↗
Identification of polypeptides required for the export of haemolysin 2001 from E. coli.
We have identified the polypeptides encoded by the haemolysin export genes from a haemolytic determinant 2001 carried by pLG570. This was previously cloned from an E. coli strain, serotype 04 isolated from a human urinary tract infection. Subclones from the recombinant plasmid pLG570 carrying hlyD analysed in vitro and in minicells showed that this gene is transcribed from an independent promoter and encodes a 53 Kd polypeptide. In contrast, detectable levels of the gene products encoded by hlyB were only observed when transcription presumably emanated from a vector promoter. This gene was found to encode at least two polypeptides apparently expressed from alternative translational start sites within a single reading frame. In minicells the major product was a 66 Kd polypeptide whilst after expression in vitro the major product was a 46 Kd polypeptide. Transposon mutagenesis leading to the synthesis of the expected truncated polypeptides was used to confirm the identity of the hlyD and the two hlyB products. Preliminary results suggest that the majority of the 53 Kd polypeptide is located in the inner membrane when cell envelopes from minicells and maxicells were fractionated using sarkosyl, although residual amounts of the 53 Kd polypeptide were also found in the outer membrane.
Bacterial Proteins↗
Genetical and functional organisation of the Escherichia coli haemolysin determinant 2001.
We have identified gene products corresponding to hlyC, hlyA and hlyD encoded by the Escherichia coli haemolytic determinant 2001 of human origin cloned into the recombinant plasmid pLG570. The product of hlyC is required for the "activation" of the inactive 107K polypeptide encoded by the hlyA gene. The activated 107K protein constitutes the active haemolysin secreted into the medium. hlyB and hlyD are separate regions defined by complementation studies and encode functions essential for the export of haemolysin with hlyD encoding a 53K protein. Complementation studies using subclones and Tn5 insertions into pLG570 have revealed the presence of two major promoters upstream of hlyC and hlyD which transcribe the four hly genes in the same direction. Finally, we were able to reconstitute the complete haemolysin system from three different plasmids encoding hlyC, hlyA and hlyB + hlyD, respectively.
Cloning, Molecular↗
Secretion of a 107 K dalton polypeptide into the medium from a haemolytic E. coli K12 strain.
Certain E. coli K12 strains are able to secrete a plasmid encoded 107 K protein into the culture medium. During exponential growth of the cells this protein represents approximately 1% of total cell protein. The presence of the 107 K polypeptide was demonstrated through the fortuitous use of strain MC4100. This gave a largely protein-free culture supernatant, presumably due to minimal lysis of whole cells. Pulse-labelling experiments showed that the secretion of the 107 K polypeptide reached a maximum during the stationary phase of growth, where it represented substantially more than 1% of total cell protein. The 107 K polypeptide is coded by the haemolytic plasmid pHly167, and appears to be related to a previously reported intracellular "precursor" form of the alpha-haemolysin (Goebel and Hedgpeth 1982). However, additional extracellular factors appear to be required for alpha-haemolysin activity since several nonhaemolytic mutants still secrete this protein.
Bacterial Proteins↗
Functional characterization of a cloned haemolysin determinant from E. coli of human origin, encoding information for the secretion of a 107K polypeptide.
We have recently reported the secretion of a 107K polypeptide by an E. coli strain containing the haemolytic plasmid pHly167 (Mackman and Holland 1984). In this paper we show that a large number of haemolytic E. coli strains, apparently including both plasmid and chromosomally located haemolysin genes, secrete similar large molecular weight proteins. Partial purification of one haemolysin suggests that activity co-purifies with a 107K polypeptide. These results were confirmed by cloning the corresponding haemolysin determinant in the form of a recombinant plasmid pLG570, containing chromosomal DNA prepared from a human isolate of E. coli, LE2001. Tn5 was used as a mutagen to localize the haemolysin genes to a 7-kilobase region of pLG570. Structural and export functions were identified by assaying cell sonicates of non-haemolytic mutants. At least one structural gene was identified which coded for a 107K polypeptide. Insertions into this gene completely eliminated haemolysin activity and resulted in truncation of the 107K protein whereas insertions into the adjacent 4-kb region resulted in intracellular haemolytic activity. This internal haemolysin appeared to accumulate in the periplasm which suggests that factors encoded by the 4-kb region are involved in exporting the 107K polypeptide across the outer membrane.
Chromosome Mapping↗