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.

Department of Biochemistry Faculty of Science Charles University Prague Prague Czech Republic

Department of Genetics and Microbiology Faculty of Science Charles University Prague Prague Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Prague Czech Republic

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Linhartová I., Bumba L., Mašín J., Basler M., Osička R., Kamanová J., Procházková K., Adkins I., Hejnová-Holubová J., Sadílková L., Morová J., and Sebo P. (2010) RTX proteins: a highly diverse family secreted by a common mechanism. FEMS Microbiol. Rev. 34, 1076–1112 10.1111/j.1574-6976.2010.00231.x PubMed DOI PMC

Barry E. M., Weiss A. A., Ehrmann I. E., Gray M. C., Hewlett E. L., and Goodwin M. S. (1991) PubMed DOI PMC

Goebel W., and Hedgpeth J. (1982) Cloning and functional characterization of the plasmid-encoded hemolysin determinant of PubMed DOI PMC

Mackman N., Nicaud J. M., Gray L., and Holland I. B. (1985) Genetical and functional organisation of the PubMed DOI

Osickova A., Balashova N., Masin J., Sulc M., Roderova J., Wald T., Brown A. C., Koufos E., Chang E. H., Giannakakis A., Lally E. T., and Osicka R. (2018) Cytotoxic activity of PubMed DOI PMC

Sebo P., Glaser P., Sakamoto H., and Ullmann A. (1991) High-level synthesis of active adenylate cyclase toxin of PubMed DOI

Issartel J. P., Koronakis V., and Hughes C. (1991) Activation of PubMed DOI

Stanley P., Packman L. C., Koronakis V., and Hughes C. (1994) Fatty acylation of two internal lysine residues required for the toxic activity of PubMed DOI

Lim K. B., Walker C. R., Guo L., Pellett S., Shabanowitz J., Hunt D. F., Hewlett E. L., Ludwig A., Goebel W., Welch R. A., and Hackett M. (2000) PubMed DOI

Hackett M., Guo L., Shabanowitz J., Hunt D. F., and Hewlett E. L. (1994) Internal lysine palmitoylation in adenylate cyclase toxin from PubMed DOI

Havlícek V., Higgins L., Chen W., Halada P., Sebo P., Sakamoto H., and Hackett M. (2001) Mass spectrometric analysis of recombinant adenylate cyclase toxin from PubMed DOI

Bouchez V., Douche T., Dazas M., Delaplane S., Matondo M., Chamot-Rooke J., and Guiso N. (2017) Characterization of post-translational modifications and cytotoxic properties of the adenylate-cyclase hemolysin produced by various PubMed DOI PMC

Basar T., Havlícek V., Bezouskova S., Hackett M., and Sebo P. (2001) Acylation of lysine 983 is sufficient for toxin activity of PubMed DOI

Basar T., Havlícek V., Bezousková S., Halada P., Hackett M., and Sebo P. (1999) The conserved lysine 860 in the additional fatty-acylation site of PubMed DOI

Hackett M., Walker C. B., Guo L., Gray M. C., Van Cuyk S., Ullmann A., Shabanowitz J., Hunt D. F., Hewlett E. L., and Sebo P. (1995) Hemolytic, but not cell-invasive activity, of adenylate cyclase toxin is selectively affected by differential fatty-acylation in PubMed DOI

Masin J., Roderova J., Osickova A., Novak P., Bumba L., Fiser R., Sebo P., and Osicka R. (2017) The conserved tyrosine residue 940 plays a key structural role in membrane interaction of PubMed DOI PMC

Masin J., Basler M., Knapp O., El-Azami-El-Idrissi M., Maier E., Konopasek I., Benz R., Leclerc C., and Sebo P. (2005) Acylation of lysine 860 allows tight binding and cytotoxicity of PubMed DOI

Balashova N. V., Shah C., Patel J. K., Megalla S., and Kachlany S. C. (2009) PubMed DOI

Karst J. C., Ntsogo Enguéné V. Y., Cannella S. E., Subrini O., Hessel A., Debard S., Ladant D., and Chenal A. (2014) Calcium, acylation, and molecular confinement favor folding of PubMed DOI PMC

O'Brien D. P., Cannella S. E., Voegele A., Raoux-Barbot D., Davi M., Douché T., Matondo M., Brier S., Ladant D., and Chenal A. (2019) Post-translational acylation controls the folding and functions of the CyaA RTX toxin. FASEB J. 33, 10065–10076 10.1096/fj.201802442RR PubMed DOI

El-Azami-El-Idrissi M., Bauche C., Loucka J., Osicka R., Sebo P., Ladant D., and Leclerc C. (2003) Interaction of PubMed DOI

Herlax V., and Bakás L. (2003) Acyl chains are responsible for the irreversibility in the PubMed DOI

Herlax V., Maté S., Rimoldi O., and Bakás L. (2009) Relevance of fatty acid covalently bound to PubMed DOI PMC

Greene N. P., Crow A., Hughes C., and Koronakis V. (2015) Structure of a bacterial toxin-activating acyltransferase. Proc. Natl. Acad. Sci. U.S.A. 112, E3058–E3066 10.1073/pnas.1503832112 PubMed DOI PMC

Gygi D., Nicolet J., Frey J., Cross M., Koronakis V., and Hughes C. (1990) Isolation of the PubMed DOI

Forestier C., and Welch R. A. (1990) Nonreciprocal complementation of the PubMed DOI PMC

Westrop G., Hormozi K., da Costa N., Parton R., and Coote J. (1997) Structure-function studies of the adenylate cyclase toxin of PubMed DOI PMC

Osicka R., Osicková A., Basar T., Guermonprez P., Rojas M., Leclerc C., and Sebo P. (2000) Delivery of CD8 PubMed DOI PMC

Guermonprez P., Khelef N., Blouin E., Rieu P., Ricciardi-Castagnoli P., Guiso N., Ladant D., and Leclerc C. (2001) The adenylate cyclase toxin of PubMed DOI PMC

Osicka R., Osickova A., Hasan S., Bumba L., Cerny J., and Sebo P. (2015) PubMed DOI PMC

Stanley P., Koronakis V., Hardie K., and Hughes C. (1996) Independent interaction of the acyltransferase HlyC with two maturation domains of the PubMed DOI

Grant R. P., and Hoofnagle A. N. (2014) From lost in translation to paradise found: enabling protein biomarker method transfer by mass spectrometry. Clin. Chem. 60, 941–944 10.1373/clinchem.2014.224840 PubMed DOI PMC

Ludwig A., Garcia F., Bauer S., Jarchau T., Benz R., Hoppe J., and Goebel W. (1996) Analysis of the PubMed DOI PMC

Benz R., Maier E., Ladant D., Ullmann A., and Sebo P. (1994) Adenylate cyclase toxin (CyaA) of PubMed

Laviña M., Pugsley A. P., and Moreno F. (1986) Identification, mapping, cloning and characterization of a gene ( PubMed DOI

Sambrook J., Fritsch E. F., and Maniatis T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Morova J., Osicka R., Masin J., and Sebo P. (2008) RTX cytotoxins recognize β2 integrin receptors through PubMed DOI PMC

Khan F., He M., and Taussig M. J. (2006) Double-hexahistidine tag with high-affinity binding for protein immobilization, purification, and detection on Ni-nitrilotriacetic acid surfaces. Anal. Chem. 78, 3072–3079 10.1021/ac060184l PubMed DOI

Benz R., Janko K., Boos W., and Läuger P. (1978) Formation of large, ion-permeable membrane channels by the matrix protein (porin) of PubMed DOI

Nicolai C., and Sachs F. (2013) Solving ion channel kinetics with the QuB software. Biophys. Rev. Lett. 08, 191–211 10.1142/S1793048013300053 DOI

Ladant D. (1988) Interaction of PubMed

Masin J., Osickova A., Sukova A., Fiser R., Halada P., Bumba L., Linhartova I., Osicka R., and Sebo P. (2016) Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin. Sci. Rep. 6, 29137 10.1038/srep29137 PubMed DOI PMC

Basler M., Masin J., Osicka R., and Sebo P. (2006) Pore-forming and enzymatic activities of PubMed DOI PMC

Perez-Riverol Y., Csordas A., Bai J., Bernal-Llinares M., Hewapathirana S., Kundu D. J., Inuganti A., Griss J., Mayer G., Eisenacher M., Perez E., Uszkoreit J., Pfeuffer J., Sachsenberg T., Yilmaz S., et al. (2019) The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47, D442–D450 10.1093/nar/gky1106 PubMed DOI PMC

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