Bordetella Adenylate Cyclase Toxin Induces a Cascade of Morphological Changes of Sheep Erythrocytes and Localizes Into Clusters in Erythrocyte Membranes JANA VOJTOVA ´ , OLGA KOFRON ˇ OVA ´ , PETER S ˇ EBO, AND OLDR ˇ ICH BENADA* Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20, Prague 4, Czech Republic KEY WORDS CyaA; scanning electron microscopy; transmission electron microscopy; immunolabeling; membrane projection; membrane protrusions ABSTRACT Adenylate cyclase toxin (CyaA) of Bordetella pertussis penetrates the membrane of eukaryotic cells, producing high levels of intracellular cAMP, as well as hemolysis that results from the formation of cation-selective toxin channels in the membrane. Using several microscopical approaches we studied the effects of CyaA action on the morphology of sheep erythrocytes during early phases preceding lysis and examined localization of CyaA molecules within the erythrocyte membrane. CyaA induced a cascade of morphological changes of erythrocytes, such as shrinkage, formation of membrane projections, and blebs and swelling. The use of an enzymatically inactive CyaA-AC  toxoid that is unable to produce cAMP and of a CyaA-E581K mutant exhibiting higher hemolytic activity than with CyaA showed that the hemolytic activity is responsible for the induc- tion of morphological changes of erythrocytes. Further, immunolabeling of inserted CyaA-232/ FLAG molecules with specific anti-FLAG antibodies and IgG-gold particles indicated a clustered distribution of CyaA molecules in erythrocyte membrane. This was confirmed by immunofluores- cence and confocal microscopy, which revealed uniform stoichiometry of CyaA clusters, suggesting CyaA binding into specific domains in erythrocyte membrane. Indeed, a decrease of CyaA binding after cholesterol depletion of erythrocytes suggests toxin targeting and binding to membrane microdomains (rafts). Microsc. Res. Tech. 69:119–129, 2006. V V C 2006 Wiley-Liss, Inc. INTRODUCTION The adenylate cyclase toxin (CyaA, referred to also as ACT, AC-toxin, or AC-Hly) is produced by Bordetella pertussis, the etiological agent of whooping cough. CyaA plays an important role in the early stages of bacterial infection, since it paralyzes the host immune defense by inhibiting bactericidal functions of phagocytes and allows Bordetella to colonize the respiratory tract epi- thelium (Confer and Eaton, 1982; Goodwin and Weiss, 1990; Harvill et al., 1999; Khelef et al., 1992). CyaA is a 1706 residue-long bifunctional protein exhibiting a cell-invasive enzymatic adenylate cyclase activity and a hemolytic activity (Bellalou et al., 1990; Confer and Eaton, 1982; Glaser et al., 1988). It is syn- thesized as an inactive precursor, which is converted to the active toxin by palmitoylation of the Lys 983 (Hack- ett et al., 1994) and Lys 860 residues (Havlicek et al., 2001). CyaA consists of an amino-terminal adenylate cyclase (AC) domain, comprising about 400 N-terminal residues, and of a hemolysin moiety of about 1306 resi- dues (Glaser et al., 1988). The toxin can translocate its AC domain into the cytosol of target cell (a cell-invasive activity) by a poorly understood mechanism that re- quires the intact hemolysin moiety. In the cytosol the AC domain binds calmodulin and catalyzes the conver- sion of ATP to cAMP, thereby subverting cellular sig- naling (Confer and Eaton, 1982; Guiso et al., 1991). The hemolysin moiety displays structural characteris- tics that link CyaA to the family of bacterial pore-form- ing toxins known as the RTX ( Repeat in To Xin) family, the prototype of which is HlyA, the Escherichia coli a- hemolysin (Welch, 1991). Upon insertion of CyaA into the target membrane, the hemolysin moiety can form small cation-selective channels of 0.6–0.8 nm in dia- meter (Benz et al., 1994; Szabo et al., 1994), which can cause colloid-osmotic lysis of erythrocytes (Bellalou et al., 1990; Glaser et al., 1988). This results from the formation of pores that allow free movement of solutes across cell membrane and influx of water, inducing cell swelling and lysis (Chou and Fitch, 1981; Harris et al., 1991). Both the translocation of the AC domain into the cytosol (cell-invasive activity) and formation of hemolytic channels (hemolytic or channel-forming ac- tivity) absolutely require binding of calcium ions into the RTX-hemolysin moiety and the subsequent confor- mational changes that promote the penetration of CyaA into cells (Hewlett et al., 1991; Rose et al., 1995). The toxin binds to target cells primarily via the CD11b/CD18 (a M b 2 ) integrin receptor (Guermonprez et al., 2001), which is expressed by myeloid phagocytic *Correspondence to: Oldr ˇich Benada, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vı ´den ˇ ska ´ 1083, CZ-142 20 Prague 4, Czech Republic. E-mail: benada@biomed.cas.cz Received 1 September 2005; accepted in revised form 5 December 2005 Contract grant sponsor: EU; Contract grant numbers: LSHB-CT-2003-503582, AVZ50200510; Contract grant sponsor: Academy of Sciences; Contract grant number: A5020406 of the Academy of Sciences; Contract grant sponsor: Howard Hughes Medical Institute; Contract grant number: 55000334. DOI 10.1002/jemt.20277 Published online in Wiley InterScience (www.interscience.wiley.com). V V C 2006 WILEY-LISS, INC. MICROSCOPY RESEARCH AND TECHNIQUE 69:119–129 (2006)