Active and Inactive Forms of Hemolysin (HlyA) fromEscherichia coli

Abstract

The HlyA protein (Mr 110 kDa) which is the gene product of the hlyA gene encoded by the hemolysin determinant of Escherichia coli (Goebel, W. & Hedgpeth, J. (1982) J. Bacteriol. 151, 1290-1298) was observed to accumulate in the culture supernatant (in the presence of the three other Hly proteins HlyC, B and D) throughout the active growth cycle. However, the amount of extracellular HlyA protein did not correlate with the external hemolytic activity, which declined when the cells entered the stationary phase. External hemolytic activity was highly sensitive to phospholipase C and to ultrasonication. The size of the HlyA protein on SDS-PAGE was not changed by these treatments although the hemolytic activity was entirely abolished. On a polyacrylamide gel containing 2M urea but only 0.1% SDS hemolytically active HlyA migrated slightly ahead of the inactive HlyA suggesting that HlyA is more negatively charged than HlyA. Active hemolysin from unconcentrated hemolytic supernatants migrated on Sephacryl S-400 and on glycerol gradients as large complexes. Analysis of the hemolytically active fractions on SDS-PAGE yielded in both cases only HlyA (110 kDA) as major protein. An internal hemolytic activity appeared in most Escherichia coli K-12 strains in the stationary phase which was independent of the presence of HlyA or any other Hly gene product. This hemolytic activity which reached in some strains about 10% of the level determined by the hly genes was sensitive to proteinase K and disappeared upon shift of the cells to the logarithmic phase.

α‐Haemolysin from E. coli purification and self‐aggregation properties

“…2. Only Wagner et al [20] have published cotnparable gels. This demonstrates the important point that a single 110 kDa polypeptide makes up for the protein moiety of haemolysin, as suggested by previous studies [6,21] but not by the data of Bohach and Snyder [19].…”

“…However, this paper describes for the first time the inhibitory action of trypsin on an c<-haemolysin preparation whose protein component is only the 110 kDa polypeptide, thus relating directl~ this particular polypeptide to c~-haemolysin activity. The role of non-protein contponents in haemolytic activity, suggested by several workers [19,20] is not sufficiently documented as yet.…”

Separable domains define target cell specificities of an RTX hemolysin from Actinobacillus pleuropneumoniae

“…3; Table 1). Because of the higher background activity associated with the hemolysin assay, it was not possible to determine whether LktC-activated AppA displayed a similar, low hemolytic activity (nonspecific lysis of erythrocytes by moderate amounts of cleared E. coli lysates has been previously reported [37]). Lysates from E. coli expressing lktA in the absence of RTX C gene function were found to exhibit leukotoxin activity which was low (1% of that observed with LktC activation) but reproducibly above background levels ( Fig.…”

Synthesis, inactivation, and localization of extracellular and intracellular Escherichia coli hemolysins

“…HlyA is a protein of 110 kilodaltons which is hemolytically inactive in the absence of HlyC. The mechanism by which HlyC converts HlyA to a pore-forming cytolysin (10) is still unknown, but is has been shown that hemolytically active and inactive extracellular HlyA proteins differ in various properties (18). The specific hemolysin transport system consisting of at least the two membrane proteins HlyB and HlyD (19) allows the translocation of HlyA in the presence or absence of HlyC (11,12), indicating that HlyC is not essential for hemolysin transport.…”