The work described in this thesis deals with two pore forming toxins, namely Arcanobacterium pyogenes pyolysin and Streptococcus agalactiae CAMP factor. Pyolysin (PLO) belongs to the homologous group of cholesterol-dependent cytolysins. In chapter 2, it is shown that PLO can form small oligomers in solution, without the requirement for any membranes or membrane lipids. These small oligomers may aggregate into larger ones on membranes; however, in solution, they apparently do not grow by addition of further monomers, as their size is virtually unaffected by variations of incubation time or toxin concentration. The small, solution-derived oligomers retain hemolytic activity. The membrane lesions observed by electron microscopy are similar to those that are formed by monomeric PLO, except that they are mostly incomplete and arc-shaped, as opposed to the predominantly ring-shaped ones formed by monomeric PLO when directly incubated with membranes. This structural difference corresponds to a detectable difference in functional pore size, as determined by marker release experiments. Thus, arc-shaped PLO oligomers may form functional pores of reduced size. In chapter 3, we show that liposomes that contain phosphatidylcholine and ceramide but no cholesterol or other sterol are susceptible to the cholesterol-dependent cytolysin pyolysin. Pyolysin, at a low rate, forms small oligomers in solution. The solution-derived oligomers are more effective on ceramide-containing liposomes than the monomeric toxin. In contrast, they have much lower activity on liposome membranes that contain cholesterol but no ceramide. Our findings therefore show that at least one member of the ‘cholesterol-dependent cytolysins’ is in fact not strictly dependent on the sterol. In addition, in conjunction with previous data, they suggest that the requirement for cholesterol involves early or intermediate stages of oligomer formation, rather than the final event of membrane insertion. Chapter 4 of this thesis concerns Streptococcus agalactiae CAMP factor. CAMP factor has previously been reported to bind the Fc fragments of immunoglobulin G (IgG) and has therefore also been called ‘protein B’, in analogy to protein A of Staphylococcus aureus. We attempted to characterize the interaction of protein B with IgG in more detail. In contrast to protein A, CAMP factor does not inhibit the activation of complement by hemolysin antibodies bound to sheep red cell surfaces. IgG also failed to inhibit the cohemolytic activity of CAMP factor, which it displays on sphingomyelinase-treated sheep red cells; this is in disagreement with previous findings. After co-incubation, CAMP factor and IgG were cleanly separated by gel filtration. Therefore, CAMP factor does not detectably bind to IgG.

The work described in this thesis deals with two pore forming toxins, namely Arcanobacterium pyogenes pyolysin and Streptococcus agalactiae CAMP factor. Pyolysin (PLO) belongs to the homologous group of cholesterol-dependent cytolysins. In chapter 2, it is shown that PLO can form small oligomers in solution, without the requirement for any membranes or membrane lipids. These small oligomers may aggregate into larger ones on membranes; however, in solution, they apparently do not grow by addition of further monomers, as their size is virtually unaffected by variations of incubation time or toxin concentration. The small, solution-derived oligomers retain hemolytic activity. The membrane lesions observed by electron microscopy are similar to those that are formed by monomeric PLO, except that they are mostly incomplete and arc-shaped, as opposed to the predominantly ring-shaped ones formed by monomeric PLO when directly incubated with membranes. This structural difference corresponds to a detectable difference in functional pore size, as determined by marker release experiments. Thus, arc-shaped PLO oligomers may form functional pores of reduced size. In chapter 3, we show that liposomes that contain phosphatidylcholine and ceramide but no cholesterol or other sterol are susceptible to the cholesterol-dependent cytolysin pyolysin. Pyolysin, at a low rate, forms small oligomers in solution. The solution-derived oligomers are more effective on ceramide-containing liposomes than the monomeric toxin. In contrast, they have much lower activity on liposome membranes that contain cholesterol but no ceramide. Our findings therefore show that at least one member of the ‘cholesterol-dependent cytolysins’ is in fact not strictly dependent on the sterol. In addition, in conjunction with previous data, they suggest that the requirement for cholesterol involves early or intermediate stages of oligomer formation, rather than the final event of membrane insertion. Chapter 4 of this thesis concerns Streptococcus agalactiae CAMP factor. CAMP factor has previously been reported to bind the Fc fragments of immunoglobulin G (IgG) and has therefore also been called ‘protein B’, in analogy to protein A of Staphylococcus aureus. We attempted to characterize the interaction of protein B with IgG in more detail. In contrast to protein A, CAMP factor does not inhibit the activation of complement by hemolysin antibodies bound to sheep red cell surfaces. IgG also failed to inhibit the cohemolytic activity of CAMP factor, which it displays on sphingomyelinase-treated sheep red cells; this is in disagreement with previous findings. After co-incubation, CAMP factor and IgG were cleanly separated by gel filtration. Therefore, CAMP factor does not detectably bind to IgG.

Streptococcus agalactiae CAMP factor is a protein exotoxin that contains 226 amino acid residues and forms oligomeric pores on susceptible cell membranes and liposomes. In this study, fragments of CAMP factor were created and recombinantly expressed to identify functional domains that are involved in membrane binding, oligomerization, and membrane insertion. Altogether, six truncated forms of CAMP factor were created and assayed. CAMP1-113, CAMP1-170, CAMP57-226, and CAMP171-226 showed different levels of hemolytic activity. CAMP1-56 and CAMP114-226 did not show hemolytic activity or oligomerization ability, but showed binding ability. CAMP114-226 inhibited the hemolytic activity of wild-type CAMP factor, most likely through ‘one-sided’ oligomerization. From the comparison of these fragments, it emerges that the region between residues 57 and 113 plays a crucial role in oligomerization and membrane insertion. The high binding efficiency of CAMP114-226 suggests this region has great responsibility on membrane binding. The hemolytically inactive fragments showed higher binding efficiency than some of the active fragments. For the hemolytic fragments, higher binding efficiency gave stronger hemolysis. These findings support that CAMP factor has different functional regions for pore-formation.

ABSTRACT CAMP factor is a 25kDa extracellular protein from Streptococcus agalactiae (Group B streptococci) that contains 226 amino acid residues. CAMP factor has been characterized as a pore-forming toxin (PFT). The typical mechanism of pore formation of PFTs involves three main stages, namely binding of toxin monomers to the membrane surface, oligomerization of the monomers on the cell membrane, and finally the insertion of oligomers into the membrane. This study focused on second stage, and investigates the oligomerisation of CAMP factor on sheep red blood cell membranes. It is known that the hemolytic activity of CAMP factor is greatly enhanced by interaction with sphingomyelinase from Staphylococcus aureus. We here focused on understanding the role of sphingomyelinase in the oligomerisation step. Experimental data were obtained using Förster resonance energy transfer (FRET) studies. The fluorescence dyes IAEDANS and Fluorescein-5-maleimide were used as donor/acceptor fluorophores and attached to mutant single cysteine residues in CAMP factor. Samples of donor- and acceptor-labelled protein were mixed and incubated with red cell membranes that had or had not been pre-treated with sphingomyelinase. Energy transfer was monitored with time-resolved and steady-state fluorescence measurements. In the time-resolved experiments, the fluorescence lifetime of the donor was measured in the presence and the absence of the acceptor, on membrane samples that were or were not treated with sphingomyelinase. We observed a decrease in the fluorescence lifetime of the donor with the presence of the acceptor. The decrease in lifetime due to acceptor interaction signifies the occurrence of energy transfer between the donor and acceptor fluorophores, which indicates proximity due to oligomerisation of the CAMP factor protein on the cell membrane. This was only observed when the membranes had been treated with sphingomyelinase. When membranes were used that had not been treated with sphingomyelinase, the donor lifetimes are very low, suggesting the inability of the CAMP factor to undergo membrane insertion and oligomerisation.

Streptococcus agalactiae CAMP factor is a protein exotoxin that contains 226 amino acid residues and forms oligomeric pores on susceptible cell membranes and liposomes. In this study, fragments of CAMP factor were created and recombinantly expressed to identify functional domains that are involved in membrane binding, oligomerization, and membrane insertion. Altogether, six truncated forms of CAMP factor were created and assayed. CAMP1-113, CAMP1-170, CAMP57-226, and CAMP171-226 showed different levels of hemolytic activity. CAMP1-56 and CAMP114-226 did not show hemolytic activity or oligomerization ability, but showed binding ability. CAMP114-226 inhibited the hemolytic activity of wild-type CAMP factor, most likely through ‘one-sided’ oligomerization. From the comparison of these fragments, it emerges that the region between residues 57 and 113 plays a crucial role in oligomerization and membrane insertion. The high binding efficiency of CAMP114-226 suggests this region has great responsibility on membrane binding. The hemolytically inactive fragments showed higher binding efficiency than some of the active fragments. For the hemolytic fragments, higher binding efficiency gave stronger hemolysis. These findings support that CAMP factor has different functional regions for pore-formation.

ABSTRACT CAMP factor is a 25kDa extracellular protein from Streptococcus agalactiae (Group B streptococci) that contains 226 amino acid residues. CAMP factor has been characterized as a pore-forming toxin (PFT). The typical mechanism of pore formation of PFTs involves three main stages, namely binding of toxin monomers to the membrane surface, oligomerization of the monomers on the cell membrane, and finally the insertion of oligomers into the membrane. This study focused on second stage, and investigates the oligomerisation of CAMP factor on sheep red blood cell membranes. It is known that the hemolytic activity of CAMP factor is greatly enhanced by interaction with sphingomyelinase from Staphylococcus aureus. We here focused on understanding the role of sphingomyelinase in the oligomerisation step. Experimental data were obtained using Förster resonance energy transfer (FRET) studies. The fluorescence dyes IAEDANS and Fluorescein-5-maleimide were used as donor/acceptor fluorophores and attached to mutant single cysteine residues in CAMP factor. Samples of donor- and acceptor-labelled protein were mixed and incubated with red cell membranes that had or had not been pre-treated with sphingomyelinase. Energy transfer was monitored with time-resolved and steady-state fluorescence measurements. In the time-resolved experiments, the fluorescence lifetime of the donor was measured in the presence and the absence of the acceptor, on membrane samples that were or were not treated with sphingomyelinase. We observed a decrease in the fluorescence lifetime of the donor with the presence of the acceptor. The decrease in lifetime due to acceptor interaction signifies the occurrence of energy transfer between the donor and acceptor fluorophores, which indicates proximity due to oligomerisation of the CAMP factor protein on the cell membrane. This was only observed when the membranes had been treated with sphingomyelinase. When membranes were used that had not been treated with sphingomyelinase, the donor lifetimes are very low, suggesting the inability of the CAMP factor to undergo membrane insertion and oligomerisation.