Chemical andImmunological Analysis oftheComplexStructure of Escherichia coli Alpha-Hemolysin (1985)

Escherichia coli alpha-hemolysin (AH)purified fromculture supernatants bygelfiltration andion-exchange chromatography was heterogeneous in charge andsize. A 107,000-dalton protein wasidentified astheproduct ofthehlyAgenebyitsreactivity withanti-AH monoclonal antibodies. Proteolysis oftheproduct ofthehIyA geneoccurred butwas notrequired fortransport oftheprotein through thecell wall. Active AH hadalarger size andlowerplthananalysis ofthehlyAgenesequencewouldpredict, thussuggesting thatthehlyAprotein iscomplexed withotherbacterial products. Lipopolysaccharide was detected inpurified hemolysin complex preparations andmay bea majorcomponentofthecomplexes. Thesefindings suggest several possible mechanisms forrelease ofAH fromthebacterial cell including release byoutermembranefragmentation. The existence ofAH complexed withlipopolysaccharide may haveimportant implications inunderstanding its toxicity. Escherichia colialpha-hemolysin (AH)isa protein that lyses erythrocytes fromseveral species ofanimals invitro. AH isofinterest forseveral reasons.First, E.coli isolates fromextraintestinal infections produce AH more commonly thanintestinal strains do(19), andthehemolysin isbelieved tobea virulence factor. Further evidence forthis belief is that partially purified AH preparations possessseveral toxic biological properties (3-5). Second, AH isone ofveryfew proteins produced bymembersofthefamily Enterobacteriaceae thatisreleased extracellularly (23). Finally, thegenetic control ofAH production, transport, andrelease from thecelliscomplex. Atleast fourgenes located on the bacterial chromosomne oron large transmissible plasmids are required toelicit a cell-free hemolytic phenotype (8,25). Thehemolysin structural gene(hlyA) codes fora 106,000to110,000-dalton protein that can bedetected inthecytoplasmofE.coli cells (8,10). Initially Noegel etal.(20) and others (12) reported thatproteolytic cleavage oftheproduct ofthehlyAgeneinto a 58,000-dalton protein occursinthe cytoplasm as a prerequisite foractivation, transport, and release fromthecell. According tothis controversial model the107,000-dalton protein represents a hemolytically inactive precursorprotein thatisnotfoundoutside thecell. In contrast, MackmanandHolland(18)demonstrated a 107,000-dalton protein asamajor componentofsupernatants fromcultures ofhemolytic E.coli cultures. Felmlee etal.(7) demonstrated a 110,000-dalton protein inE.coliculture supernatants thatcontains theN-terminal aminoacids predicted bytheDNA sequenceofthehlyA gene.Theselatter findings contradict themodelproposed byNoegel etal.(20) andsuggest thatcleavage ofthehlyAprotein isnotrequired foran active extracellular hemolysin. Neither the58,000-dalton nor the106,000- to110,000dalton putative hemolysin protein hasbeenpurified tohomogeneity ina hemolytically active form. Denaturing conditions required fordemonstration ofextracellular E.coli proteins bygelelectrophoresis inactivate thehemolysin. Measurements havebeenmadeofthesize ofnondenatured active AH bygelfiltration chromatography (13), electron microscopy (22), diffusion coefficient determination (22), and