Nigel Mackman

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.

Secretion of Haemolysin by Escherichia coli

Escherichia coli secretes very few proteins into the culture medium and it is presumed that the outer membrane constitutes the major barrier to true secretion. The E. coli envelope, whose structure has been extensively reviewed (Nikaido and Nakae 1979; Osborn and Wu 1980; Hall and Silhavy 1981; Lugtenberg and Van Alphen 1983), is shown in Fig. 1. It is composed of an inner and outer membrane which encloses the peptidoglycan or rigid cell wall. The periplasmic space is also located between the membrane layers. This compartment may contain at least 4% of total cell protein (Nossal and Heppel 1966) and may have a quite viscous or gel-like structure (Hobot et al. 1984). The periplasm contains up to 50 distinct polypeptide species (Copeland et al. 1982), the majority of which are concerned with import mechanisms connecting outer membrane pores (porins) with specific inner membrane permeases. It is difficult to estimate the precise volume of the periplasmic space under normal growth conditions since it is not structurally defined.

Genetical and functional organisation of the Escherichia coli haemolysin determinant 2001

SummaryWe 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.

The carboxy-terminal region of haemolysin 2001 is required for secretion of the toxin from Escherichia coli

SummaryAs 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 theHlyA 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 functionshlyB andhlyD 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.

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

Functional characterization of a cloned haemolysin determinant from E. coli of human origin, encoding information for the secretion of a 107K polypeptide

SummaryWe 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.

Identification of polypeptides required for the export of haemolysin 2001 from E. coli.

SummaryWe 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 nitro 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.

The hemolysin of Escherichia coli.

Many strains of E. coli elaborate a hemolysin which is responsible for the zone of β-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+. Perfusion of subcytolytic doses through isolated and ventilated rabbit lungs induces lung edema that is at least partially due to such secondary events. The capacity of E. coli hemolysin to damage human tissues via primary and secondary processes is consistent with the concept of its pathogenic role in human infections.[/p]

Regulation of haemolysin synthesis in E. coli determined by HLY genes of human origin

SummaryWe 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 haemoolysin itself. Degradation of the 107K polypeptide in the medium was accompanied by the accumulation of a major breakdown product of 60K.

Secretion of a 107 K dalton polypeptide into the medium from a haemolytic E. coli K12 strain.

SummaryCertain 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 α-haemolysin (Goebel and Hedgpeth 1982). However, additional extracellular factors appear to be required for α-haemolysin activity since several nonhaemolytic mutants still secrete this protein.