Escherichia coli hemolysin mutants with altered target cell specificity.
In order to understand the functional significance of HlyC-dependent acylation of the Escherichia coli hemolysin structural protein (HlyA), random as well as site-directed substitutions at the known regions of modification, i.e., those at lysine residues at amino acid positions 563 and 689 (HlyAK563 and HlyAK689, respectively), were isolated. Sixteen random hlyA mutations were identified on the basis of a screen for loss of immunoreactivity to the hemolysin-neutralizing D12 monoclonal antibody that reacts to only HlyC-activated HlyA. These substitutions occurred at the region from HlyAE684 to HlyAY696. A recombinant glutathione S-transferase-hemolysin gene fusion encoding glutathione S-transferase-HlyAS608-T725 residues reacts with monoclonal antibody when HlyC is coexpressed with the fusion protein. Therefore, at most only 12% of the total HlyA primary sequence is needed for HlyC-facilitated acylation at the HlyAK689 position, and this modification can occur in the absence of the proximal HlyAK563 acylation site. The cytolytic activities of these HlyA mutants against sheep erythrocytes and bovine and human lymphocyte cell lines (BL-3 and Raji cells, respectively) were analyzed. HlyAK563 and HlyAK689 substitutions displayed various degrees of loss of cytotoxicity that depended on the particular amino acid replacement. An HlyAK563C variant retained greater than 59 and 21% of its BL-3-lytic and erythrolytic activities, respectively, but was nearly inactive against Raji cells. An HlyA mutant with a K-to-E substitution at amino acid 689 (HlyAK689E) was essentially inactive against all three cell types, whereas an HlyAK689R substitution had a pattern of activity similar to that of the HlyAK563C mutant. Preceding the two in vitro acylated HlyA lysines are glycines that appear to be the only amino acids conserved in alignments of these regions among the RTX toxins. Remarkably, considering the retention of cytotoxic activity by some HlYAK689 mutants, each of three different substitutions at the HlyAG688 position was relatively inactive against all three cell types tested. This suggests that HlyAG688 plays a significant structural role in cytotoxic activity apart from its possible participation in an HlyC activation process which presumably requires recognition of pro-HlyA structures. The related RTX toxin, the Pasteurella haemolytica leukotoxin structural protein (LktA), can be activated in an E. coli recombinant background by HlyC. In amino acid sequence alignments, LktAK554 is equivalent to the HlyAK563 position but it has an asparagine (LktAN684) at the homologous HlyAK689 site. An LktAN684K substitution possesses wild-type leukotoxin activity against BL-3 cells and does not acquire hemolytic or Raji cell cytotoxic activity. Surprisingly, both LktAK554C and LktAK554T substitutions retain considerable BL-3 cytotoxicity (45 and 49%, respectively), indicating that there may be additional lysines within LktA that the HlyC activation mechanism is capable of acylating. Based on these results and a comparison of amino acid sequence alignments of 12 RTX toxins, a putative consensus structure of the RTX residues necessary for HlyC activation is hypothesized.
Pellett
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Department of Medical Microbiology and Immunology, University of Wisconsin--Madison 53706, USA.
Welch
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RA
D000215:Acylation; D000217:Acyltransferases; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins / Q000235:genetics / Q000633:toxicity*; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D005098:Exotoxins; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D006461:Hemolysis; D006801:Humans; D016978:Mannheimia haemolytica / Q000235:genetics / Q000472:pathogenicity; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000633:toxicity; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013329:Structure-Activity Relationship; D018675:Toxicity Tests
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D005098:Exotoxins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D011993:Recombinant Fusion Proteins; C018579:leukotoxin; D000217:Acyltransferases; C092237:HlyC protein, E coli