Retargeting from the CR3 to the LFA-1 receptor uncovers the adenylyl cyclase enzyme–translocating segment of Bordetella adenylate cyclase toxin

Abstract

<jats:p>The <jats:italic>Bordetella</jats:italic> adenylate cyclase toxin-hemolysin (CyaA) and the α-hemolysin (HlyA) of <jats:italic>Escherichia coli</jats:italic> belong to the family of cytolytic pore-forming Repeats in ToXin (RTX) cytotoxins. HlyA preferentially binds the α<jats:sub>L</jats:sub>β<jats:sub>2</jats:sub> integrin LFA-1 (CD11a/CD18) of leukocytes and can promiscuously bind and also permeabilize many other cells. CyaA bears an N-terminal adenylyl cyclase (AC) domain linked to a pore-forming RTX cytolysin (Hly) moiety, binds the complement receptor 3 (CR3, α<jats:sub>M</jats:sub>β<jats:sub>2</jats:sub>, CD11b/CD18, or Mac-1) of myeloid phagocytes, penetrates their plasma membrane, and delivers the AC enzyme into the cytosol. We constructed a set of CyaA/HlyA chimeras and show that the CyaC-acylated segment and the CR3-binding RTX domain of CyaA can be functionally replaced by the HlyC-acylated segment and the much shorter RTX domain of HlyA. Instead of binding CR3, a CyaA<jats:sub>1-710</jats:sub>/HlyA<jats:sub>411-1024</jats:sub> chimera bound the LFA-1 receptor and effectively delivered AC into Jurkat T cells. At high chimera concentrations (25 n<jats:sc>m</jats:sc>), the interaction with LFA-1 was not required for CyaA<jats:sub>1-710</jats:sub>/HlyA<jats:sub>411-1024</jats:sub> binding to CHO cells. However, interaction with the LFA-1 receptor strongly enhanced the specific capacity of the bound CyaA<jats:sub>1-710</jats:sub>/HlyA<jats:sub>411-1024</jats:sub> chimera to penetrate cells and deliver the AC enzyme into their cytosol. Hence, interaction of the acylated segment and/or the RTX domain of HlyA with LFA-1 promoted a productive membrane interaction of the chimera. These results help delimit residues 400–710 of CyaA as an “AC translocon” sufficient for translocation of the AC polypeptide across the plasma membrane of target cells.</jats:p>

Almost half of the RTX domain is dispensable for complement receptor 3 binding and cell-invasive activity of the Bordetella adenylate cyclase toxin

“…However, the functional defect of CyaA Δ1295-1561 was rather mild, showing that the deleted segment does not play any important role in toxin binding and translocation across the plasma membrane of target cells. This conclusion goes well with our recent results showing that the entire RTX domain of CyaA can be functionally replaced by the much shorter RTX domain of the E. coli HlyA ( 21 ). Such chimeric CyaA/HlyA molecule was retargeted for binding to the LFA-1 integrin (CD11a/CD18) of leukocytes and was still capable to deliver the AC domain into cytosol of erythrocytes as well as of LFA-1-expressing cells.…”

“…The N-terminal part of the Hly moiety harbors the AC-to-Hly linking segment linked to the pore-forming domain followed by the acylated domains [18][19][20]. These domains jointly form the membrane translocon assembly that mediates the translocation of the AC domain across the lipid bilayer of target cell membrane [21].…”

“…The N-terminal part of the Hly moiety harbors the AC-to-Hly linking segment linked to the pore-forming domain followed by the acylated domains ( 18 , 19 , 20 ). These domains jointly form the membrane translocon assembly that mediates the translocation of the AC domain across the lipid bilayer of target cell membrane ( 21 ). The second half of the Hly moiety is formed by a large RTX domain that consists of five blocks (I–V) of tandem arrays of characteristic glycine and aspartate-rich nonapeptide (RTX) repeats ( 22 , 23 ).…”

“…Previous work indicated that CR3 recognition by CyaA involves a negatively charged surface (residues 1166–1275) presented at the interface of the folded RTX blocks II and III ( 30 , 31 ). Deletion analysis then indicated that the structural integrity of the entire RTX domain was required for cell binding and cytotoxic activity of CyaA ( 21 , 32 , 33 ). In particular, the capacity of the RTX domain to undergo the calcium-dependent folding appeared to be critical for the capacity of CyaA to engage the CR3 receptor of phagocytes, penetrate the cellular membrane, and deliver the cytotoxic AC domain into target cell membrane ( 25 ).…”

Bordetella Adenylate Cyclase Toxin Elicits Airway Mucin Secretion through Activation of the cAMP Response Element Binding Protein

“…The Hly moiety binds with high affinity the CD11b subunit of the CD11b/CD18 integrin that serves as complement receptor 3 (CR3, α M β 2 , or Mac-1) of myeloid phagocytic cells [13][14][15][16][17]. Upon CD11b/CD18 binding, the Hly moiety inserts into the cellular membrane and mediates rapid translocation of the AC enzyme domain into cell cytosol directly across the plasma membrane of cells without the need for receptor-mediated endocytosis [18,19]. Inside cells, the AC enzyme is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP to cAMP, a key second messenger [20].…”