Escherichia coli is a common cause of meningitis and sepsis in the newborn infant, and the large majority of isolates from these infections produce a polysialic acid (PSA) capsular polysaccharide, the K1 antigen, that protects the bacterial cell from immune attack. We determined whether a capsule-depolymerizing enzyme, by removing this protective barrier, could alter the outcome of systemic infection in an animal model. Bacteriophage-derived endosialidase E (endoE) selectively degrades the PSA capsule on the surface of E. coli K1 strains. Intraperitoneal administration of small quantities of recombinant endoE (20 g) to 3-day-old rats, colonized with a virulent strain of K1, prevented bacteremia and death from systemic infection. The enzyme had no effect on the viability of E. coli strains but sensitized strains expressing PSA to killing by the complement system. This study demonstrates the potential therapeutic efficacy of agents that cure infections by modification of the bacterial phenotype rather than by killing or inhibition of growth of the pathogen.

A small single dose of capsule-depolymerizing enzyme has therapeutic utility in lethal systemic infection in a non-invasive model that has characteristics of the infectious process in humans. We propose that the enzyme reduces the virulence of E. coli K1 by rapid removal of the protective capsular polysaccharide, sensitizing the pathogen to host defences such as phagocytosis by macrophages. Thus, whilst endoE-mediated therapy may not be a viable approach to the treatment of systemic infection in humans, it does support the concept that alteration of the cell wall phenotype is a valid therapeutic strategy.

African trypanosomes are extracellular pathogens of mammals and are exposed to the adaptive and innate immune systems. Trypanosomes evade the adaptive immune response through antigenic variation, but little is known about how they interact with components of the innate immune response, including complement. Here we demonstrate that an invariant surface glycoprotein, ISG65, is a receptor for complement component 3 (C3). We show how ISG65 binds to the thioester domain of C3b. We also show that C3 contributes to control of trypanosomes during early infection in a mouse model and provide evidence that ISG65 is involved in reducing trypanosome susceptibility to C3-mediated clearance. Deposition of C3b on pathogen surfaces, such as trypanosomes, is a central point in activation of the complement system. In ISG65, trypanosomes have evolved a C3 receptor which diminishes the downstream effects of C3 deposition on the control of infection.

The two epidermolytic toxins were shown to have intrinsic N-t-butyloxycarbonyl-L-glutamic acid alpha-phenyl esterase activity. The activity was dependent on free toxin pKa values of 6.6 and 6.8 for ETA and ETB respectively. ETB incorporated 0.97 mol of radiolabelled di-isopropyl phosphorofluoridate/mol of protein with loss of esterolytic and epidermolytic activities. The correspondence of epidermolytic and esterolytic activities in ETA and ETB during thermal inactivation and reaction with di-isopropyl phosphorofluoridate, together with the inactivity of the mutant protein ETA S195G, demonstrates that the two activities are dependent on a single active serine residue in each protein.

Two epidermolytic toxins, produced by different strains of Staphylococcus aureus, split human skin at a site in the upper epidermis. Clinical effects are most common in infants, but adults are susceptible. Epidermolysis may also be observed in the mouse, in vivo and in vitro, and in a few other mammals. Recent in vitro experiments have demonstrated an inhibition by chelators and point to metal-ion, possibly Ca2+, involvement. The epidermolysis effect is insensitive to a wide range of other metabolic inhibitors. The toxin amino acid sequences are similar to that of staphylococcal proteinase, and new experiments by chemical modification and site-directed mutagenesis have shown that toxicity depends on 'active serine' residues of a catalytic triad similar to that found in serine proteases. Furthermore the toxins possess esterolytic activity, also dependent on the 'active serine' sites. However, the toxins have low or undetectable activity towards a range of peptide or protein substrates. In histological and related studies, the toxins bound selectively to an intracellular skin protein, profilaggrin, but there was no evidence that the toxin can enter intact epidermal cells. Therefore, although the circumstantial evidence that the toxins act by proteolysis is convincing, a specific skin proteolytic substrate for the toxin has not been identified.

The sequences of the epidermolytic toxins and V8 serine proteinase share about 25% identity, including the catalytic triad at the proteinase active centre. Here we have altered the putative ETA active‐site serine‐195 to glycine by site‐directed mutagenesis. No epidermolytic activity was detected when up to 100‐fold greater amounts of the homogeneous mutant ETA were injected subcutaneously into neonatal mice showing that serine‐195 is required for toxicogenesis.

Catalytically active, recombinant fusion proteins of bacteriophage E endosialidase were expressed and purified from Escherichia coli. Constructs with different fusion partners added to the amino terminus of the endosialidase were enzymatically active. A post-translational proteolytic cleavage was shown to occur between serine 706 and aspartate 707 to generate the 76 kDa mature enzyme from the 90 kDa translation product. Endosialidase truncated at the C-terminus from aspartate 707 was observed to have the same 76 kDa molecular weight as wild-type enzyme using denaturing SDS-PAGE but, under native PAGE conditions, was not observed to form the approximately 250 kDa trimeric wild-type enzyme, implying that the C-terminus of the enzyme may be required for correct assembly of active trimer, rather than as part of the active site as has been previously suggested. Mutagenesis of aspartate 138 to alanine greatly reduced enzyme activity whereas conversion of other selected aspartate residues to alanine had less effect, consistent with similarities between the structure and cata-lytic mechanism of bacteriophage E endosialidase and those of exosialidases.

Resistance to antibiotics has made the treatment of human infectious diseases increasingly more difficult, with many pathogenic bacteria now completely resistant to all useful antibiotics. Actinomycetes are a group of Grampositive, filamentous bacteria well known as a source of antibiotics. Isolation of actinomycetes representing rare