title: Quantitative analysis of CagA type IV secretion by Helicobacter pylori reveals substrate recognition and translocation requirements.
abstract: Bacterial type IV secretion systems are protein transporters with a remarkable diversity of substrates and substrate recognition mechanisms. Type IV-secreted proteins often contain C-terminal secretion signals, but may also require other regions for recognition as secretory substrates, or for full secretion efficiency. For example, type IV secretion of CagA, a major pathogenicity factor of the human gastric pathogen Helicobacter pylori, depends on a C-terminal signal and on N-terminal protein regions. To examine the involvement of individual CagA regions for type IV secretion efficiency, we have established and evaluated a β-lactamase-dependent reporter system which allows quantitative determination of translocation into host cells. For validation, we used this reporter system to obtain quantitative data for type IV secretion of CagA variants with sequential C-terminal truncations. Alanine-scanning mutagenesis of the CagA C-terminus revealed that none of the characteristic charged residues in this region is necessary for type IV secretion. Translocation rates measured for CagA variants with N-terminal deletions show that CagA does not have an N-terminal signal sequence, but requires its N-terminal domain for efficient secretion. Finally, we provide evidence that only newly synthesized CagA protein is translocated, supporting a model in which type IV secretion is coupled to protein biosynthesis.
authors.affiliation: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany.|German Center for Infection Research (Deutsches Zentrum für Infektionsforschung; DZIF), Ludwig-Maximilians-Universität, München, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Schindele
authors.affiliation: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Weiss
authors.affiliation: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany.|German Center for Infection Research (Deutsches Zentrum für Infektionsforschung; DZIF), Ludwig-Maximilians-Universität, München, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Haas
authors.affiliation: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Fischer
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport; D004789:Enzyme Activation; D005753:Gastric Mucosa / Q000378:metabolism / Q000382:microbiology; D016480:Helicobacter pylori / Q000502:physiology*; D014176:Protein Biosynthesis; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D017384:Sequence Deletion; D000069319:Type IV Secretion Systems; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D000069319:Type IV Secretion Systems; C080866:cagA protein, Helicobacter pylori; D001618:beta-Lactamases