Graham J. Boulnois

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.

Structure and function of pneumolysin, the multifunctional, thiol-activated toxin of Streptococcus pneumoniae.

Pneumolysin is a thiol‐activated membrane‐damaging, multifunctional toxin and a known virulence factor of Streptococcus penumoniae. The toxin can interfere with the functioning of both cellular and soluble components of the human immune system which protects against penumococcal infection. Different amino acids within the toxin which are important in promoting oligomerization of the toxin in membranes and for the generation of functional lesions have been identified by site‐directed mutagenesis. Pneumolysin can also activate the classical pathway of complement, and this appears to involve antibody binding (via Fc) by a region of the toxin homologous to C‐reactive protein, a human acute‐phase protein also capable of classical pathway activation and implicated in host defence against pneumococcal infection.

Pneumococcal proteins and the pathogenesis of disease caused by Streptococcus pneumoniae.

Streptococcus pneumoniae is an important agent of disease in man at the extremes of age and in individuals with underlying debilitating conditions. It is responsible for the majority of cases of community-acquired pneumonia, an important cause of septicaemia, one of the three most common pathogens in bacterial meningitis and the most prevalent agent in otitis media (Roberts, 1985). The precise incidence of each of these infections is not known because of problems in diagnosis (particularly with respect to pneumonia and otitis media) and inadequate reporting of cases. However, Austrian (19826), having reviewed a number of studies, concluded that in the USA there are 2-5 cases of pneumonia per 1000 population per annum, equating to between 440000 and 1.1 x lo6 cases each year. Despite antibiotic therapy the case fatality rate is about 5-7%, making pneumonia one of the major causes of morbidity and mortality in the developed world. In the same study, Austrian estimated that the attack rate for septicaemia was between 25 and 50 per 100000 population per annum, with a case fatality rate of between 17 and 24%. Most, but not all, of these cases were associated with lower respiratory tract infections. In an attempt to focus attention on the desirability of developing an effective vaccine to protect against pneumococcal infection, this situation was likened, in terms of incidence and mortality, to that of poliomyelitis prior to the introduction of the vaccine. The incidence of pneumococcal meningitis is estimated to be about 1.5 per 100000 population per annum (Austrian, 1982b). It seems likely that similar attack and fatality rates are

Molecular analysis of the Escherichia coli K5 kps locus: identification and characterization of an inner-membrane capsular polysaccharide transport system

The complete nucleotide sequence has been determined of a region of the Escherichia coli K5 antigen gene cluster postulated to encode functions for the translocation of capsular polysaccharide across the inner membrane. This revealed two genes, designated kpsM and kpsT, organized in a single transcriptional unit. Analysis of the predicted amino acid sequence of the KpsM and KpsT proteins indicates that they may function as dual components in a polysaccharide export system analogous to the periplasmic binding protein‐dependent transport systems of Gram‐negative bacteria. We propose that the KpsT protein acts as an energizer, coupling ATP hydrolysis to the transport process mediated by the KpsM protein. Extensive sequence homology between the KpsM and KpsT proteins and the products of the bexB and bexA genes present in the capsulation (cap) locus of Haemophilus influenzae, indicates that a common mechanism for the export of polysaccharide across the inner membrane may exist in these two microorganisms.

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.

Genetics of Capsular Polysaccharide Production in Bacteria

A feature of many bacteria of diverse genera is the production of extracellular acidic polysaccharides. These polysaccharides may be organised into distinct structures termed capsules, or may be excreted as an extracellular slime. However, this distinction is arbitrary and in practice may be of no functional significance.

Region 2 of the Escherichia coli K5 capsule gene cluster encoding proteins for the biosynthesis of the K5 polysaccharide

The nucleotide sequence of region 2 of the Escherichia coli K5 capsule gene cluster has been determined. This region, essential for the biosynthesis of the K5 poly‐saccharide, contained four genes, termed kfiA‐D. The G+C ratio was 33.4%, which was lower than the typical G+C ratio for E. coli and that of the flanking regions 1 and 3 in the K5 capsule gene cluster. Three major RNA transcripts were detected within region 2 by Northern blotting and three promoters located by transcript mapping. Promoter activity was confirmed by promoter‐probe analysis. The predicted amino acid sequence of KfiC had homology to a number of glycosyl transferase enzymes and overexpression of the kfiC gene resulted in increased K5 transferase activity. The predicted amino acid sequence of KfiD had homology to a number of NAD‐dependent dehydrogenase enzymes and was demonstrated to be a UDP‐glucose dehydrogenase that catalyses the formation of UDP‐glucuronic acid from UDP‐glucose.

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.

Surface Components of Escherichia coli That Mediate Resistance to the Bactericidal Activities of Serum and Phagocytes

Microorganisms that enter the animal body, either actively or passively, encounter a battery of nonspecific and specific chemical and cellular defenses whose role is to inactivate and eliminate foreign materials. Successful pathogens have the ability to avoid, resist, or inactivate these defenses for at least the period necessary for initiation of the disease process.

Analysis of the K1 capsule biosynthesis genes of Escherichia coli: Definition of three functional regions for capsule production

SummaryTransposon and deletion analysis of the cloned K1 capsule biosynthesis genes of Escherichia coli revealed that approximately 17 kb of DNA, split into three functional regions, is required for capsule production. One block (region 1) is required for translocation of polysaccharide to the cell surface and mutations in this region result in the intracellular appearance of polymer indistinguishable on immunoelectrophoresis to that found on the surface of K1 encapsulated bacteria. This material was released from the cell by osmotic shock indicating that the polysaccharide was probably present in the periplasmic space. Insertions in a second block (region 2) completely abolished polymer production and this second region is believed to encode the enzymes for the biosynthesis and polymerisation of the K1 antigen. Addition of exogenous N-acetylneuraminic acid to one insertion mutant in this region restored its ability to express surface polymer as judged by K1 phage sensitivity. This insertion probably defines genes involved in biosynthesis of N-acetylneuraminic acid. Insertions in a third block (region 3) result in the intracellular appearance of polysaccharide with a very low electrophoretic mobility. The presence of the cloned K1 capsule biosynthesis genes on a multicopy plasmid in an E. coli K-12 strain did not increase the yields of capsular polysaccharide produced compared to the K1+ isolate from which the genes were cloned.