Acyltransferase-mediated selection of the length of the fatty acyl chain and of the acylation site governs activation of bacterial RTX toxins

Abstract

In a wide range of organisms, from bacteria to humans, numerous proteins have to be posttranslationally acylated to become biologically active. Bacterial repeats in toxin (RTX) cytolysins form a prominent group of proteins that are synthesized as inactive protoxins and undergo posttranslational acylation on ε-amino groups of two internal conserved lysine residues by co-expressed toxin-activating acyltransferases. Here, we investigated how the chemical nature, position, and number of bound acyl chains govern the activities of Bordetella pertussis adenylate cyclase toxin (CyaA), Escherichia coli α-hemolysin (HlyA), and Kingella kingae cytotoxin (RtxA). We found that the three protoxins are acylated in the same E. coli cell background by each of the CyaC, HlyC, and RtxC acyltransferases. We also noted that the acyltransferase selects from the bacterial pool of acyl–acyl carrier proteins (ACPs) an acyl chain of a specific length for covalent linkage to the protoxin. The acyltransferase also selects whether both or only one of two conserved lysine residues of the protoxin will be posttranslationally acylated. Functional assays revealed that RtxA has to be modified by 14-carbon fatty acyl chains to be biologically active, that HlyA remains active also when modified by 16-carbon acyl chains, and that CyaA is activated exclusively by 16-carbon acyl chains. These results suggest that the RTX toxin molecules are structurally adapted to the length of the acyl chains used for modification of their acylated lysine residue in the second, more conserved acylation site. In a wide range of organisms, from bacteria to humans, numerous proteins have to be posttranslationally acylated to become biologically active. Bacterial repeats in toxin (RTX) cytolysins form a prominent group of proteins that are synthesized as inactive protoxins and undergo posttranslational acylation on ε-amino groups of two internal conserved lysine residues by co-expressed toxin-activating acyltransferases. Here, we investigated how the chemical nature, position, and number of bound acyl chains govern the activities of Bordetella pertussis adenylate cyclase toxin (CyaA), Escherichia coli α-hemolysin (HlyA), and Kingella kingae cytotoxin (RtxA). We found that the three protoxins are acylated in the same E. coli cell background by each of the CyaC, HlyC, and RtxC acyltransferases. We also noted that the acyltransferase selects from the bacterial pool of acyl–acyl carrier proteins (ACPs) an acyl chain of a specific length for covalent linkage to the protoxin. The acyltransferase also selects whether both or only one of two conserved lysine residues of the protoxin will be posttranslationally acylated. Functional assays revealed that RtxA has to be modified by 14-carbon fatty acyl chains to be biologically active, that HlyA remains active also when modified by 16-carbon acyl chains, and that CyaA is activated exclusively by 16-carbon acyl chains. These results suggest that the RTX toxin molecules are structurally adapted to the length of the acyl chains used for modification of their acylated lysine residue in the second, more conserved acylation site. The cytolytic (pore-forming) RTX toxins are important virulence factors of many Gram-negative bacterial pathogens (1Linhartová I. Bumba L. Mašín J. Basler M. Osička R. Kamanová J. Procházková K. Adkins I. Hejnová-Holubová J. Sadílková L. Morová J. Sebo P. RTX proteins: a highly diverse family secreted by a common mechanism.FEMS Microbiol. Rev. 2010; 34 (20528947): 1076-111210.1111/j.1574-6976.2010.00231.xCrossref PubMed Scopus (348) Google Scholar). The RTX toxins permeabilize host cell membranes and share several characteristic features. These comprise (i) a C-terminal unprocessed secretion signal, recognized by the type I secretion system and mediating translocation of toxins directly from the bacterial cytosol into the extracellular milieu; (ii) characteristic C-terminal nonapeptide glycine- and aspartate-rich RTX repeats that upon binding of numerous calcium ions fold into a β-barrel structure; (iii) posttranslational modification of internal lysine residues within conserved acylated sites by covalent attachment of fatty acyl residues, and (iv) a hydrophobic pore-forming domain, respectively (1Linhartová I. Bumba L. Mašín J. Basler M. Osička R. Kamanová J. Procházková K. Adkins I. Hejnová-Holubová J. Sadílková L. Morová J. Sebo P. RTX proteins: a highly diverse family secreted by a common mechanism.FEMS Microbiol. Rev. 2010; 34 (20528947): 1076-111210.1111/j.1574-6976.2010.00231.xCrossref PubMed Scopus (348) Google Scholar). RTX cytolysins (RTXA) are synthesized as inactive protoxins (proRTXA) and undergo a posttranslational acylation by toxin-activating acyltransferases (RTXC) co-expressed with the protoxins (2Barry E.M. Weiss A.A. Ehrmann I.E. Gray M.C. Hewlett E.L. Goodwin M.S. Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation.J. Bacteriol. 1991; 173 (1987161): 720-72610.1128/jb.173.2.720-726.1991Crossref PubMed Google Scholar, 3Goebel W. Hedgpeth J. Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli.J. Bacteriol. 1982; 151 (7050085): 1290-129810.1128/JB.151.3.1290-1298.1982Crossref PubMed Google Scholar, 4Mackman N. Nicaud J.M. Gray L. Holland I.B. Genetical and functional organisation of the Escherichia coli haemolysin determinant PubMed Scopus Google Scholar, N. J. M. J. E. R. of Kingella kingae RtxA toxin on acylation of lysine residues and PubMed Scopus Google Scholar, P. P. of active adenylate cyclase toxin of Bordetella pertussis in a Escherichia coli 1991; PubMed Scopus Google Scholar). for the Escherichia coli α-hemolysin (HlyA), acyltransferase as acyl chain and only fatty acyl residues by acyl carrier of an acylation of the ε-amino groups of the and residues of HlyA of Escherichia coli to the toxin by acyl carrier fatty 1991; PubMed Scopus Google Scholar, P. acylation of two internal lysine residues for the of Escherichia coli PubMed Scopus Google Scholar). The two lysine residues of HlyA found to be acylated in E. coli by chains and the acyl chains as the and fatty acyl groups L. J. Hewlett E.L. W. M. Escherichia coli α-hemolysin is acylated in with and fatty PubMed Scopus Google Scholar). The chain also found to be the modification of the and residues of the RtxA cytotoxin of Kingella the toxin molecules modified by and chains N. J. M. J. E. R. of Kingella kingae RtxA toxin on acylation of lysine residues and PubMed Scopus Google Scholar). In the Bordetella pertussis RTX adenylate cyclase toxin found to be acylated by on the residue M. L. J. Hewlett E.L. lysine in adenylate cyclase toxin from Bordetella PubMed Scopus Google and when in also on residue L. W. P. Sebo P. M. of adenylate cyclase toxin from Bordetella pertussis PubMed Scopus Google Scholar). for a number of the CyaA M. M. J. N. of and of the hemolysin by Bordetella pertussis Bordetella Scopus Google Scholar). CyaA in E. coli with acyltransferase CyaC, a acylation by and chains with a of M. Sebo P. of lysine is for toxin of Bordetella pertussis adenylate of acylation of the toxin acyltransferase PubMed Scopus Google Scholar, P. M. Sebo P. The conserved lysine in the of Bordetella pertussis adenylate cyclase is for toxin of acylation PubMed Scopus Google Scholar, M. L. Gray M.C. J. Hewlett E.L. Sebo P. of adenylate cyclase toxin is by in Escherichia coli.J. PubMed Scopus Google Scholar, J. J. P. Bumba L. R. Sebo P. R. The conserved residue a in of Bordetella adenylate cyclase PubMed Scopus Google Scholar). of the RTX toxins to be for of their activities (2Barry E.M. Weiss A.A. Ehrmann I.E. Gray M.C. Hewlett E.L. Goodwin M.S. Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation.J. Bacteriol. 1991; 173 (1987161): 720-72610.1128/jb.173.2.720-726.1991Crossref PubMed Google Scholar, N. J. M. J. E. R. of Kingella kingae RtxA toxin on acylation of lysine residues and PubMed Scopus Google Scholar, P. P. of active adenylate cyclase toxin of Bordetella pertussis in a Escherichia coli 1991; PubMed Scopus Google Scholar, P. acylation of two internal lysine residues for the of Escherichia coli PubMed Scopus Google Scholar, J. Basler M. M. E. I. R. Sebo P. of lysine binding and of Bordetella adenylate cyclase on PubMed Scopus Google Scholar, is for and toxin and host PubMed Scopus Google Scholar). the by the acyl chains to and of by the toxins remains The of the acyl chains to a in the of CyaA into a biologically active and of Bordetella pertussis adenylate cyclase CyaA toxin into a and PubMed Scopus Google Scholar, M. M. acylation the and of the CyaA RTX J. PubMed Scopus Google and in a and of CyaA with the also as the or J. Basler M. M. E. I. R. Sebo P. of lysine binding and of Bordetella adenylate cyclase on PubMed Scopus Google Scholar, M. J. R. Sebo P. of Bordetella pertussis adenylate cyclase with of toxin acylation and of the PubMed Scopus Google Scholar). also to be for the of HlyA to L. chains are for the in the Escherichia coli α-hemolysin binding to PubMed Scopus Google and for in HlyA within the L. of fatty bound to Escherichia coli α-hemolysin and in the PubMed Scopus Google Scholar). The toxin-activating acyltransferase are highly conserved the of bacterial and of acyltransferases to also the hemolysin as as the HlyA in E. a hemolytic on of a bacterial toxin-activating PubMed Scopus Google Scholar, J. J. M. of the haemolysin and the and secretion of the by the HlyC, and proteins of Escherichia Microbiol. PubMed Scopus Google Scholar). the or the same and cell as the activated by the as the HlyA and CyaA in E. coli hemolytic of the of the Escherichia coli hemolysin and PubMed Google Scholar, K. N. R. J. of the adenylate cyclase toxin of Bordetella pertussis and the of by and of Bacteriol. PubMed Google Scholar). has RTX protoxins are by acyltransferases and are Here, we the of the and RtxA each acylated by one of the three CyaC, HlyC, or RtxC acyltransferases and in the same E. coli cell as to the of in pool of the The results that is the acyltransferase that selects the type of the acyl chain of adapted length that is to the and the acyltransferase also selects whether a lysine residue or both modification sites of will be posttranslationally the on the RTX the acyl residue and acylation of the three RTX we the of the three protoxins and with the three acyltransferase CyaC, HlyC, and in the same E. coli cell the of the for of CyaA toxin R. P. M. Sebo P. of into I by Bordetella pertussis adenylate of cell and PubMed Google The in from to by the of the or and the by or respectively In the and in the to a a The of used to the toxins in E. coli These to from by on or and RtxA the acylation of the of the proteins by to in the toxin activated by acyltransferase a acylation with and chains the and of the molecules also modified by and chains to the and residues of the residues by CyaC, the residue of acylated In when in the of or only in or in acylation of the residue in the acylation of CyaA the acyltransferases recognized the acylation of CyaA with In the second acylation of CyaA by and RtxC acyltransferases that acylated the and RtxC exclusively the and chains in and in for modification of the residue of CyaA The and chains only a of the acyl chains to the residue in and in of the toxins modified by the in the of the acyltransferases in E. coli to and by of fatty acyl chains to the ε-amino groups of the lysine residues from the of ions in are from with two toxin the acyl chain The in the of the acyltransferases in E. coli to and by of fatty acyl chains to the ε-amino groups of the lysine residues from the of ions in are from with two toxin the acyl chain in a in the toxin modified by acyltransferase with the and acyl chains the and a of the molecules acylated with the and chains and or the residue The and chains also the acyl groups to the and residues, when HlyA modified by the acyltransferase RtxC RtxC modified the acylation of HlyA with HlyC, and of the residues In the acyltransferase acylated the residues of the HlyA molecules exclusively with the and acyl chains and with the residues modified with the and acyl groups or the RtxA cytotoxin modified by acyltransferase RtxC and also by the acyltransferase the residue with the and acyl chains in and in In the acyltransferase acylated the residue of RtxA with the and acyl groups the the residue of the RtxA only modified with the and chains in and in or of results that the acyltransferases a for of from the E. coli pool exclusively the and acyl chains for acylation of the or of the and In the and RtxC acyltransferases exclusively the and chains for acylation of three that is the acyltransferase that selects the length of the acyl chain that to the the that the and and the second, more conserved acylation sites of the and acylation of the acylation sites in or The of toxins with and of acyl chains to how the or acylation and the length of the acyl chains the activities of the three acylated CyaA we their to and as the CyaA in with acylation of the by the chains, of the residue by the or chains the of and to The modification by the more the of both toxin the of that the and proteins to of by the same of within of The and of the and proteins when their that of to binding of of the three CyaA to and The or and toxins bound with a and of also to that the CyaA the binding of and to as be by the that the of P. N. E. P. P. N. The adenylate cyclase toxin of Bordetella pertussis to the PubMed Scopus Google Scholar, R. Bumba L. J. Sebo P. Bordetella adenylate cyclase toxin is a of the PubMed Scopus Google with the the and proteins in their to the into the cytosol of to the functional on to the of acylation of the residue of the and as a acylated by only on the residue J. Basler M. M. E. I. R. Sebo P. of lysine binding and of Bordetella adenylate cyclase on PubMed Scopus Google to and into as the acylated the of the and proteins to the modification of the residue by the acyl chains, a specific on CyaA the modification of the residue by the acyl chains. In with the cytolytic on the and also a on of as from the of by and with that of the of by and with that of be that the in the number two acylated lysine and the length and chemical of acyl chains the of the of the by the acylated CyaA toxin for the HlyA toxin modified by the CyaC, HlyC, or RtxC acyltransferases. in the acylated exclusively on the residue by the and chains a to as the toxin acylated on both and residues by the and acyl chains. the and toxin as hemolytic as of the length or number or of acyl residues, three acylated HlyA toxin to as by their to of of to the to In with the cytolytic on three HlyA activities on from the with the of that to that of by the and proteins The and toxin also with to of the by the and of results that the acyl chains to the residue on the toxin a pore-forming and as the acylation by acyl chains to both and residues in or the acylation of and acylation of by acyl chains in the that in to of the residue by or acyl chains for of acylation of the residue of RtxA by or acyl chains for cytolytic of the and acylated by RtxC or HlyC, respectively in to the RtxA toxin activated by modification of the residue by the or acyl chains. the of RtxA only upon modification by and acyl chains. In with the and toxins activities on the a the a of and respectively the of of to of and of These results that the in to into the in to form to the modification by the acyl RtxA has with and as and activities of the RtxA on membranes in the of The the the and the of membranes in the of RtxA to in of from on several membranes with or the same as in The the of by the RtxA for on several membranes with or the same as in and the of with a The of by The in each the We that of the three the CyaA toxin is only activated by modification of residue by fatty acyl chains and the RtxA only by the modification of residue by acyl chains. the HlyA toxin be activated by modification with or acyl chains to These results that is the acyltransferase the RTX protoxin that selects the acyl chain of the adapted length from the pool of the the acyltransferase also whether both or only one of the two conserved acylation sites in the RTX protoxin will be recognized and modified by the acyl chains. In the the acylation of the toxin from the pertussis found to be modified by a on the ε-amino group of M. L. J. Hewlett E.L. lysine in adenylate cyclase toxin from Bordetella PubMed Scopus Google Scholar). CyaA with in the pertussis found to be on both the and residues L. W. P. Sebo P. M. of adenylate cyclase toxin from Bordetella pertussis PubMed Scopus Google and the acylation for CyaA from of pertussis M. M. J. N. of and of the hemolysin by Bordetella pertussis Bordetella Scopus Google Scholar). of the in pertussis in of the to and also on the residue of the CyaA M. M. J. N. of and of the hemolysin by Bordetella pertussis Bordetella Scopus Google Scholar). of CyaA with in the E. coli revealed that the toxin acylated by chains the residue and and chains the residue M. L. Gray M.C. J. Hewlett E.L. Sebo P. of adenylate cyclase toxin is by in Escherichia coli.J. PubMed Scopus Google Scholar). that the residue of the CyaA from E. coli only also a of P. M. Sebo P. The conserved lysine in the of Bordetella pertussis adenylate cyclase is for toxin of acylation PubMed Scopus Google Scholar). The and chains also as the fatty acyl of the CyaA from the E. coli J. J. P. Bumba L. R. Sebo P. R. The conserved residue a in of Bordetella adenylate cyclase PubMed Scopus Google Scholar). In with we found that and are the two posttranslational of the and residues also when the CyaA is in the E. coli In the or CyaA on the same background and exclusively on the residue by and acyl chains in and The and activities and to the acylation the residue of and we have that acylation of the residue of and for activities of the toxin on both M. Sebo P. of lysine is for toxin of Bordetella pertussis adenylate of acylation of the toxin acyltransferase PubMed Scopus Google as as on and J. Basler M. M. E. I. R. Sebo P. of lysine binding and of Bordetella adenylate cyclase on PubMed Scopus Google Scholar). is to that the 14-carbon and chains are to the 16-carbon and chains the residue of only the of acylation also the length of the acyl chains to a in of CyaA and in of activities on The specific binding of the and and to only with the and in the to the cell into the cytosol and to by the acyl chains to the and translocation of the CyaA the and a also on that a acyl chain has to be to the ε-amino group of the chain of the residue of CyaA to the on the toxin and to with the of the cell or of the the toxin has into the a the modification of CyaA with the acyl chains the and of by the and the same as by the that in the number and of the acyl chains to the CyaA only the of the toxin to into the and form and on the of the We that CyaA acylated on the of toxin molecules the residue In only or acylation on when CyaA modified with or that have a for the acylation of CyaA the and RtxC acyltransferases. of results that the acyltransferase in pertussis with CyaA to by acyl chains and be by toxin-activating acyltransferases of Gram-negative bacteria that for modification of fatty acyl modification of HlyA activated in in two of E. coli a and an and the and residues of HlyA found to be acylated by a chain The as the and fatty acyl chains L. J. Hewlett E.L. W. M. Escherichia coli α-hemolysin is acylated in with and fatty PubMed Scopus Google Scholar). Here, we that the HlyA toxin in the E. coli acylated by the and chains both the and residue and by the and chains. In to the results of L. J. Hewlett E.L. W. M. Escherichia coli α-hemolysin is acylated in with and fatty PubMed Scopus Google we to and acyl groups to the HlyA the and of the that E. coli have an pool from that of the E. coli used the of HlyA a as to the length and of the acyl chains. the residue of HlyA acylated with the and acyl chains by CyaC, the a to of or to and on as upon modification on both and in to the HlyA toxin be modified by acyl chains of length and HlyA protoxin and as in an in acylation P. K. of the acyltransferase with two of the Escherichia coli toxin Microbiol. PubMed Scopus Google that an for the the residue of HlyA for that the in acylation of residues and only of of the and residues revealed that both sites for hemolytic of HlyA P. acylation of two internal lysine residues for the of Escherichia coli PubMed Scopus Google Scholar). results that the of the and RtxC acyltransferases in to the the as acylated by both in only or of HlyA molecules modified by or RtxC on the a of acylation of the the acylation of the residue on the and proteins a to the and proteins also an on as the acylated acylation of the of HlyA upon of of a of the residue in toxin a upon of the residue of the acylation of is by for CyaA on both as as on M. Sebo P. of lysine is for toxin of Bordetella pertussis adenylate of acylation of the toxin acyltransferase PubMed Scopus Google Scholar, J. Basler M. M. E. I. R. Sebo P. of lysine binding and of Bordetella adenylate cyclase on PubMed Scopus Google Scholar). the is in specific on the M. Sebo P. of lysine is for toxin of Bordetella pertussis adenylate of acylation of the toxin acyltransferase PubMed Scopus Google Scholar, J. Basler M. M. E. I. R. Sebo P. of lysine binding and of Bordetella adenylate cyclase on PubMed Scopus Google Scholar). we that the RtxA with RtxC in the E. coli modified by and acyl chains on the residue N. J. M. J. E. R. of Kingella kingae RtxA toxin on acylation of lysine residues and PubMed Scopus Google Scholar). a of the RtxA molecules found to be acylated on by chains N. J. M. J. E. R. of Kingella kingae RtxA toxin on acylation of lysine residues and PubMed Scopus Google Scholar). of the RtxA molecules also found to be modified by and acyl chains on the residues N. J. M. J. E. R. of Kingella kingae RtxA toxin on acylation of lysine residues and PubMed Scopus Google Scholar). Here, we the type and of acylation of RtxA toxin on the a of modification of the residue that the of acylation as a of the of the as for the acylation of the residue of CyaA M. Sebo P. of lysine is for toxin of Bordetella pertussis adenylate of acylation of the toxin acyltransferase PubMed Scopus Google Scholar). in used for of the have for the in the of the acylated N. J. M. J. E. R. of Kingella kingae RtxA toxin on acylation of lysine residues and PubMed Scopus Google Scholar). of to by to on the of used for of and respectively in to by PubMed Scopus Google Scholar). as the RtxA acylated by and acyl chains on the and only modification by and chains on the The and with a acylation to and on the biologically active the modified by the residue by the and chains to and only a on in to the in the to form as into the and to of In the to the and highly into the with to of the acylated toxins N. J. M. J. E. R. of Kingella kingae RtxA toxin on acylation of lysine residues and PubMed Scopus Google Scholar, J. Basler M. M. E. I. R. Sebo P. of lysine binding and of Bordetella adenylate cyclase on PubMed Scopus Google Scholar, R. J. W. of the in of hemolysin from Escherichia coli.J. Bacteriol. PubMed Google Scholar, R. E. Sebo P. cyclase toxin of Bordetella for the of and with HlyA of Escherichia coli.J. PubMed Google Scholar). In we that RtxA has to be modified by 14-carbon fatty acyl chains to be biologically active, HlyA remains active also when modified by 16-carbon acyl chains and CyaA is only activated by 16-carbon acyl chains. These results the of acyl chains of by the acyltransferase and suggest a of the toxin molecules to the length of the acyl chains used for modification of their acylated residue in the second, more conserved acylation site. The E. coli used for and in The E. coli the and M. and characterization of a for on Escherichia coli Microbiol. Google used for of the RTX number in a in with and and of and to J. Scholar). used as a in The R. P. M. Sebo P. of into I by Bordetella pertussis adenylate of cell and PubMed Google the and of the used to for the of the and RtxA toxins activated by the acyltransferase CyaC, HlyC, or the in from to by of or and the by or respectively The and from the J. R. J. Sebo P. RTX PubMed Scopus Google and the and from the N. J. M. J. E. R. of Kingella kingae RtxA toxin on acylation of lysine residues and PubMed Scopus Google Scholar). In the and in the to a a M. with binding for and on PubMed Scopus Google Scholar). The toxins in E. coli with the with in and with for an the by with and by and the for The in the with and with and the for The the HlyA and RtxA on an with and The with and the HlyA and RtxA with The of HlyA and RtxA in and on a with the same The with and the HlyA and RtxA with and The the CyaA in and and on a with the same The with and the CyaA with The proteins in to of and with modified a of for The second of to a of of and the for the the of the by to and of the into the The a a of of and a a of of directly to a The on a with a and a of the in the within an range of The with a and one of of The in and results in by the of the a of from The as range of of of and The the of a toxin the The for with a of three and modification for with lysine acylation from to and The to and used for The acylation of lysine residues by of the acylated ions and their lysine residues modified specific to the of the proteins posttranslational with used within the are in the on R. K. W. P. of by the of Escherichia PubMed Scopus Google in by a with a the The RTX proteins in and and into the with a The by the in and and the The by with by and and by of a and the with the Rev. Scopus Google with a The with a The by the activities of the CyaA in the of as of Bordetella pertussis adenylate cyclase with of two PubMed Google Scholar). of to of in and as the of the by upon into as J. R. P. Bumba L. I. R. Sebo P. residues of the the and the of adenylate cyclase PubMed Scopus Google Scholar). binding of the CyaA in as J. R. P. Bumba L. I. R. Sebo P. residues of the the and the of adenylate cyclase PubMed Scopus Google Scholar). of CyaA as in with with acylated and RtxA in of and hemolytic in by of the to with and the to in for in a M. J. R. Sebo P. and activities of Bordetella pertussis adenylate cyclase toxin in of PubMed Scopus Google Scholar). in with of the CyaA for to of toxin by three in the to the the with for of with the CyaA for in the by the of in for and by the of and by a J. R. P. Bumba L. I. R. Sebo P. residues of the the and the of adenylate cyclase PubMed Scopus Google Scholar). of CyaA as to the toxin as the of to the to the to the of the The have to the the with the J. M. J. M. E. J. J. and and in for PubMed Scopus Google Scholar). and are for We by with with with repeats in toxin acyl carrier modified

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