Pneumolysin as a vaccine and study of structure and function
thesisposted on 2017-02-27, 11:30 authored by Muhammad Adnan
Streptococcus pneumoniae is a Gram-positive bacterium capable of causing the life threatening invasive diseases pneumonia, septicemia and meningitis. It can also cause the highly prevalent, but less serious diseases, acute otisis media (AOM) and sinusitis. Pneumolysin, a 52Kda protein toxin, is not only an important virulence factor of Streptococcus pneumoniae but toxoid versions can be protective immunogens, although they suffer from residual activity. The toxin is produced by all serotypes and has very limited structural variation. Structurally pneumolysin consists of four domains. The mechanism of action of pneumolysin has been proposed to involve binding to the cell surface via its domain 4, then the toxin monomers oligomerize on the cell surface and finally structural changes in domain 3 results alpha helices being converted into beta hairpins that insert into the membrane to form a pore. To understand how the structure explains this function of pneumolysin, two sets of mutants were constructed to investigate the role of individual amino acids in these events. In one set, cystine bridges were introduced to lock different domains; C262+C278 was found to have locked TMH2. In the second set, mutants were made to test hypotheses on which amino acids took part in oligomerization, a double mutant 18A: 84A was found to be inactive. Currently the available pneumococcal vaccines are polysacchride based (Pneumovax) and a protein conjugate vaccine (Prevenar). The problem with Pneumovax is that it does not elicit protection in people at high risk and it covers only 23 serotypes. Prevenar is expensive and has even more limited serotype coverage. An ideal vaccine solution would be a molecule with little or no variation among different serotypes and capable of inducing a T-cell dependent response. The functionally important C-terminal domain of pneumolysin (D4) was investigated for its immunogenicity as a protective antigen and as an adjuvant. Domain 4 (D4) of pneumolysin elicits antibody response and boost it as an adjuvant but didn’t provide protection on its own. Domain 4 mixed in equimolar amounts with domains 1-3 was non-toxic and it provided protection against a lethal dose of S. pneumoniae.