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

Abstract

<jats:p>In a wide range of organisms, from bacteria to humans, numerous proteins have to be posttranslationally acylated to become biologically active. Bacterial <jats:underline>r</jats:underline>epeats in <jats:underline>t</jats:underline>o<jats:underline>x</jats:underline>in (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 <jats:italic>Bordetella pertussis</jats:italic> adenylate cyclase toxin (CyaA), <jats:italic>Escherichia coli</jats:italic> α-hemolysin (HlyA), and <jats:italic>Kingella kingae</jats:italic> cytotoxin (RtxA). We found that the three protoxins are acylated in the same <jats:italic>E. coli</jats:italic> 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.</jats:p>

Binding of Kingella kingae RtxA Toxin Depends on Cell Surface Oligosaccharides, but Not on β2 Integrins

“…RtxA belongs to the RTX (Repeats in ToXin) family of pore-forming cytotoxins that are produced by many Gram-negative bacterial pathogens, including the genera Actinobacillus, Aggregatibacter, Bordetella, Escherichia, Mannheimia, Moraxella, Morganella, Pasteurella, Proteus, and Vibrio [12]. Sequence homology with the RTX toxins revealed four functional segments in the 956 residues-long RtxA: (i) a pore-forming domain encompassing residues~140 to 410, harboring four putative transmembrane α-helices; (ii) an acylated segment, where the RtxA protoxin (proRtxA) is posttranslationally activated; recently, we experimentally demonstrated that the acyltransferase RtxC activates proRtxA by fatty acyl modification on lysine residues 558 and 689, primarily with myristoyl and hydroxymyristoyl chains [9,13]; (iii) a typical calcium-binding RTX domain between residues~730 to 810, harboring the conserved repetitions of a nonapeptide motif X-(L/I/F)-X-G-G-X-G-(N/D)-D (where X is any amino acid residue), which form calcium-binding sites and (iv) a C-terminal secretion signal. Upon binding to target cells that is facilitated by membrane cholesterol, RtxA inserts itself into the cell membrane and forms cation-selective membrane pores, which induce cation flux leading to cell death [9,14].…”

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

“…The acylation status of the proteins was then characterized by mass spectrometry, which revealed that both Lys860 and Lys983 residues of RTX770 and the Lys983 of the RTX908 were predominantly modified by a mixture of palmitoyl (C16:0) and palmitoleyl (C16:1) chains, with a small proportion of myristoyl (C14:0) and octadecenoyl (C18:1) groups (data not shown). Hence, the overall acylation status of the acylated proteins resembled that of intact recombinant CyaA ( 6 ).…”

“…The acylation status of the proteins was determined as described previously ( 6 ). Briefly, the proteins were digested in 25 mM ammonium bicarbonate (pH 8.2) and 4 M urea at a trypsin/protein ratio of 1:50 for 6 h at 30 °C followed by addition of the second portion of trypsin to a final ratio of 1:25 and incubation for another 6 h at 30 °C.…”

“…This 1706-residue-long (177-kDa) bifunctional protein comprises a cell-invasive N-terminal adenylyl cyclase (AC) enzyme domain (∼400 residues) that is fused to a pore-forming Repeat-in-ToXin (RTX) hemolysin (Hly) moiety of ∼1300 residues in length ( 3 ). The Hly moiety itself consists of several functional domains and undergoes posttranslational activation by a coexpressed CyaC acyltransferase that links fatty-acyl residues to the ε-amino groups of the internal lysine residues K860 and K983 ( 4 , 5 , 6 ). The activated Hly moiety binds and penetrates the plasma membrane of various host cells and delivers into their cytosol the N-terminal AC enzyme domain ( 7 , 8 ).…”

Lysine Fatty Acylation: Regulatory Enzymes, Research Tools, and Biological Function

“…Once the proper cofactors and buffer conditions are determined for an enzyme of interest, measuring its activity in regulating KFA can be straightforward. Assays for RtxC enzymes indirectly measure activity by taking advantage of the hemolytic action of their cognate RtxA proteins (Bellalou et al, 1990;Linhartova et al, 2010;Osickova et al, 2020). This is not a broadly applicable technique for KFA enzymes and as such will not be further elaborated on.…”