Identification of a common domain in calmodulin-activated eukaryotic and bacterial adenylate cyclases

Abstract

Bordetella pertussis and Bacillus anthracis, two taxonomically distinct bacteria, secrete adenylate cyclase toxins that are activated by the eukaryotic protein calmodulin. The two enzymes contain a well-conserved stretch of 24 amino acid residues [Escuyer et al. (1988) Gene 71, 293-298]. Antibodies have been obtained against two synthetic heptadecapeptides, covering part of the conserved sequences. The anti-peptide antibodies specifically reacted in Western blots with the rat brain adenylate cyclase as well as with the two bacterial enzymes. Anti-rat brain adenylate cyclase serum contained antibodies that were retained by the immobilized peptides, and the affinity-purified antibodies yielded the same recognition pattern of the eukaryotic enzyme as did the unfractionated serum. These results indicate that the eukaryotic adenylate cyclase contains an epitope closely related to that specified by the conserved bacterial sequence. The synthetic peptides and the bacterial adenylate cyclases appeared to compete for ATP (KD of the ATP-peptide complex ca. 0.2 mM), suggesting that the conserved sequence may be part of the substrate binding site in these two enzymes.

The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies

“…7B). This weak response may reflect structural similarities between CAT and eukaryotic adenylate cyclases, resulting in immunological tolerance or an evolutionary mechanism to protect key toxin components from neutralizing antibodies (60,61). Only one of the four mice immunized with the hydrophobic domain reacted with ACT, supporting the SEC data that this construct is poorly folded (Fig.…”

Analysis of epitope information related toBacillus anthracisandClostridium botulinum

“…Currently, the IEDB contains 15 references [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] dealing with B-cell epitopes and four references [40][41][42][43] with data on T-cell epitopes derived from B. anthracis. The IEDB contains ten references for C. botulinum [23,[44][45][46][47][48][49][50][51][52] dealing solely with B-cell epitopes and three references [53][54][55] containing both T-and B-cell determinants.…”

“…Edema factor 342 GVATKGLNVHGKSSDWG 358 B cell [26] Lethal factor 380 DSLSEEEKELLNRIQVDS 397 B cell [30] Protective antigen 340 (A)SFFD 344 B cell [27,39] Protective antigen 442 SKNLAPI 448 B cell [62] Protective antigen 610 IKLNAKMNILIRDKRFHYDRN 630 B cell [31,34,61] Protective antigen 679 EGLKEVINDRYDMLN 693 B cell [25] Protective antigen 700 DGKTFIDFKKYNDKLPLYISNPNYKVNVYA 729 B cell [32] Protective antigen 715 PLYISNPNY 723 B cell [29] Protective antigen N682, K684, L685, P686, L687, Y688 B cell [35] Protective antigen 93 IWSGFIKVKKSDEY 106 T cell [41] Protective antigen 141 RLYQIKIQYQRENPTE 156 T cell [40] Protective antigen 183 PELKQKSSNSRKKR 196 T cell [41] Protective antigen 411 SLVLGKNQTLAT 422 T cell [40] Protective antigen 573 GKDITEFDFNFDQQTSQNIK 592 T cell [43] Protective antigen 576 ITEFDFNFDQQTSQ 589 T cell [41] Protective antigen 688 RYDMLNISSLRQDG 701 T cell [41] Protective antigen 746 STNGIKKILIFSKK 759 T cell [41] Clostriduim botulinum [46,47,50] Key issues…”

Synthesis and secretion of Bordetella pertussis adenylate cyclase as a 200-kilodalton protein

“…The sera were depleted of contamining antibodies by incubating them with crude bacterial extract of an avirulent strain of Bordetella bronchiseptica, which is a common pathogen of rabbits. The immunochemical detection was performed with 251I-labeled protein A (18) or with alkaline phosphatase-labeled antirabbit immunoglobulins (9).…”