Targeted mutations that ablate either the adenylate cyclase or hemolysin function of the bifunctional cyaA toxin of Bordetella pertussis abolish virulence.
Bordetella pertussis, the causative agent of whooping cough, secretes several toxins implicated in this disease. One of these putative virulence factors is the adenylate cyclase (AC) toxin that elevates intracellular cAMP in eukaryotic cells to cytotoxic levels. This toxin is a bifunctional protein comprising both AC and hemolysin (HLY) enzymatic domains. The gene encoding the AC toxin (cyaA) is expressed as part of an operon that includes genes required for secretion or activation of the toxin. Because of this genetic organization, it is difficult to create B. pertussis mutants of cyaA that are ablations of a single enzyme function by conventional means, such as transposon mutagenesis. Therefore, to clarify the role of individual toxin functions in the virulence of B. pertussis, we have used site-directed or deletion mutagenesis and genetic recombination to specifically target the cyaA gene of B. pertussis to produce mutants that lack only the AC or HLY activity of this toxin. A point mutant of B. pertussis with abolished AC catalytic activity was greater than 1000 times less pathogenic to newborn mice than wild-type bacteria, directly demonstrating the importance of the AC toxin in pertussis virulence. Similarly, an in-frame deletion mutant of B. pertussis that lacks HLY is equally avirulent, supporting observations that the HLY domain plays a critical role in AC toxin entry into cells. Furthermore, the genetically inactivated AC toxin produced by the point mutant is antigenically similar to the native toxin, suggesting that this strain may be useful in the development of pertussis component vaccines.
Gross
M K
MK
Department of Pharmacology, University of Washington School of Medicine, Seattle 98195.
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Smith
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Storm
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D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics; D000818:Animals; D001886:Bordetella pertussis / Q000235:genetics / Q000472:pathogenicity*; D004252:DNA Mutational Analysis; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D015183:Restriction Mapping; D013329:Structure-Activity Relationship; D010566:Virulence Factors, Bordetella / Q000737:chemistry / Q000633:toxicity*
D037361:Adenylate Cyclase Toxin; D006460:Hemolysin Proteins; D010566:Virulence Factors, Bordetella; D000262:Adenylyl Cyclases