A C-terminal translocation signal is necessary, but not sufficient for type IV secretion of the Helicobacter pylori CagA protein.
Type IV secretion systems are increasingly recognized as important virulence determinants of Gram-negative bacterial pathogens. While the examination of several type IV-secreted proteins suggested that their secretion depends on C-terminal signals, the nature of these signals and their conservation among different systems remain unclear. Here, we have characterized the secretion signal of the Helicobacter pylori CagA protein, which is translocated by the Cag type IV secretion apparatus into eucaryotic cells. The production of fusion proteins of CagA and green fluorescent protein (GFP) did not result in translocation of GFP to epithelial cells, but a fusion of GFP with the CagA C-terminus exerted a dominant-negative effect upon wild-type CagA translocation. We show that CagA translocation depends on the presence of its 20 C-terminal amino acids, containing an array of positively charged residues. Interestingly, these positive charges are neither necessary nor sufficient for CagA translocation, but replacing the C-terminal region of CagA with that of other type IV-secreted proteins reconstitutes CagA translocation competence. Using a novel type IV translocation assay with a phosphorylatable peptide tag, we show that removal of the N-terminal part of the CagA protein renders the protein translocation-incompetent as well. Thus, the Cag type IV secretion system seems to diverge from other systems not only with respect to its composition and architecture, but also in terms of substrate recognition and transport.
Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, Pettenkoferstr. 9a, D-80336 Munich, Germany.
Sabine
S
Hohlfeld
Isabelle
I
Pattis
Jürgen
J
Püls
Gregory V
GV
Plano
Rainer
R
Haas
Wolfgang
W
Fischer
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016480:Helicobacter pylori / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C080866:cagA protein, Helicobacter pylori; D049452:Green Fluorescent Proteins