Timothy John Mitchell

A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae

We report the discovery of a group of highly conserved DNA sequences located, in those cases studied, within intergenic regions of the chromosome of the Gram positive Streptococcus pneumoniae. The S. pneumoniae genome contains about 25 of these elements called BOX. From 5 to 3, BOX elements are composed of three subunits (boxA, boxB, and boxC) which are 59, 45 and 50 nucleotides long, respectively. BOX elements containing one, two and four copies of boxB have been observed; boxB alone was also detected in one instance. These elements are unrelated to the two most thoroughly documented families of repetitive DNA sequences present in the genomes of enterobacteria. BOX sequences have the potential to form stable stem-loop structures and one of these, at least, is transcribed. Most of these elements are located in the immediate vicinity of genes whose product has been implicated at some stage in the process of genetic transformation or in virulence of S. pneumoniae. This location raises the intriguing possibility that BOX sequences are regulatory elements shared by several coordinately controlled genes, including competence-specific and virulence-related genes.

Complement activation and antibody binding by pneumolysin via a region of the toxin homologous to a human acute‐phase protein

Pneumolysin, a membrane‐damaging toxin, is known to activate the classical complement pathway. We have shown that 1 μg ml−1 of pneumolysin can activate complement, which is a much lower level than observed previously. We have identified two distinct regions of pneumolysin which show homology with a contiguous sequence within acute‐phase proteins, including human C‐reactive protein (CRP). Site‐directed mutagenesis of the pneumolysin gene was used to change residues common to pneumolysin and CRP. Some of the modified toxins had a reduced ability both to activate complement and bind antibody. We suggest that the ability of pneumolysin to activate complement is related to its ability to bind the Fc portion of immunoglobulin G.

The effect of Streptococcus pneumoniae pneumolysin on human respiratory epithelium in vitro

Streptococcus pneumoniae culture filtrates and pneumolysin both slow human ciliary beating and damage respiratory epithelium in vitro. A polyclonal pneumolysin antibody bound to sepharose beads removed pneumolysin from culture filtrates and showed that pneumolysin alone was responsible for the effects on epithelium. In a 48-h organ culture pneumolysin caused ciliary slowing and epithelial disruption in a dose-dependent manner down to 5 ng/ml. Comparison of the ciliary slowing activity and pneumolysin concentration in filtrates in a continuous broth culture showed a maximal effect at 16 h (pneumolysin 7.5 micrograms/ml). Later the activity decreased while the pneumolysin concentration increased (8.8 micrograms/ml). This loss of activity was prevented by neutralisation of the acid pH of the culture medium. Eight different culture filtrates produced significant (P less than 0.05) ciliary slowing which correlated (r = 0.95) with simultaneously measured haemolytic (pneumolysin) activity. Substitution of tryptophan (position 433) by phenylalanine reduced the haemolytic and ciliary slowing activity of pneumolysin, but did not affect its ability to activate complement. There was no correlation between the ciliary slowing produced by the culture filtrate and that produced by the autolysate of a particular strain, nor between ciliary slowing and the extent of autolysis or the serotype of the strain.

Expression of the pneumolysin gene in Escherichia coli: rapid purification and biological properties

The gene for pneumolysin, the thiol-activated toxin from Streptococcus pneumoniae, has been expressed in Escherichia coli. The recombinant protein has been purified using a rapid, high yield, purification procedure and has been shown to be identical with respect to N-terminal amino-acid sequence, specific activity, effect on human polymorphonuclear phagocytes and effect on human complement to the native toxin purified from the pneumococcus. This provides a large enough source of material to begin investigation of pneumolysin as a candidate for inclusion in a pneumococcal vaccine.

Subunit organisation and symmetry of pore-forming, oligomeric pneumolysin

We present a detailed analysis of the oligomeric subunit organisation of pneumolysin by the use of negative stain electron microscopy and image processing to produce a projection density map. Analysis of the rotational symmetry has revealed a large and variable subunit number, between 40–50. The projected subunit density by rotational averaging shows at least two distinct subunit domains at different radial positions. Side views of the rings reveal further details concerning the dimensions of the oligomer in the membrane. On the basis of these observations and our previous knowledge of the monomer domain structure we propose that the 4‐domain subunits are packed in a square planar arrangement to form the pneumolysin oligomer.

Growth and virulence of a complement-activation-negative mutant of Streptococcus pneumoniae in the rabbit cornea

Our previous work has demonstrated the importance of pneumolysin in the virulence of S. pneumoniae in a rabbit intracorneal model. This was accomplished by showing that deletion of the gene encoding pneumolysin resulted in reduced virulence, whereas restoration of the wild-type gene resulted in restoration of the virulent phenotype. To assess the importance of a particular domain in the pneumolysin molecule, we have now constructed a strain which produces a pneumolysin molecule which is hemolytic but which bears a site-specific mutation in the domain known to be associated with the complement-activating properties of this molecule. Comparison of the virulence of this strain with that of a strain bearing the wild-type gene showed statistically significantly lower total slit lamp examination (SLE) scores at 12, 18, 24, and 36 h (particularly with respect to fibrin formation), but no difference at 48 h. Determination of colony forming units (CFU) in eyes infected with the two strains showed approximately 10(6) bacteria per cornea until 36 h. Between 36 and 48 h, the bacteria were almost completely cleared with very few bacteria recoverable at the later time point. The loss of virulence observed with this mutation in the complement-activation domain of pneumolysin, though less than that observed with the gene deletion mutant, suggests that complement activation by pneumolysin has a significant role in the pathology observed in this model of corneal infection.

Structural and functional characterisation of two proteolytic fragments of the bacterial protein toxin, pneumolysin

Proteolytic cleavage of the bacterial protein toxin pneumolysin with protease K creates two fragments of 37 and 15 kDa. This paper describes the purification of these two fragments and their subsequent physical and biological characterisation. The larger fragment is directly involved in the cytolytic mechanism of this pore‐forming protein, via membrane binding and self‐association. The smaller fragment lacks ordered structure or discernible activity.

Structure, Function and Role in Disease of Pneumolysin, The Thiol-Activated Toxin of Streptococcus Pneumoniae

Pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae is one of a family of toxins produced by four different genera of Gram positive bacteria (1). This family of toxins share a variety of physical and biological properties and exert their effects via damage to eukaryotic membranes (2). A striking feature of this family is their pronounced immunological cross-reactivity such that sera raised against one member of this family generally reacts with and often neutralizes and precipitates heterologous toxin (1). They are termed thiol-activated since they are inactivated upon oxidation and treatment with reducing agents restores full activity (1). This was thought to reflect the formation and breakage of intra-molecular disulfide bridges, a process which induces conformational changes in the protein which are reflected in their ability to interact with membranes (3). As well as mediating such changes, a single sulphydryl was postulated to be essential for activity (4). However, the role this essential cysteine plays in toxin activity is unclear. The thiol-activated toxins are thought to utilize cholesterol as receptor since their cytolytic activity is only manifest on cells which have cholesterol as part of their membranes and since free cholesterol is a potent inhibitor of cytolytic activity (1, 2). It has been postulated that this essential sulphydryl group may mediate (or is involved in) the interaction of the toxin and cholesterol (6). It should be noted that cholesterol has not been shown conclusively to act as the receptor for these toxins.