title: Molecular and functional characterization of type I signal peptidase from Legionella pneumophila.
abstract: Legionella pneumophila is a facultative intracellular Gram-negative rod-shaped bacterium that has become an important cause of both community-acquired and nosocomial pneumonia. Numerous studies concerning the unravelling of the virulence mechanism of this important pathogen have been initiated. As evidence is now accumulating for the involvement of protein secretion systems in bacterial virulence in general, the type I signal peptidase (LepB) of L. pneumophila was of particular interest. This endopeptidase plays an essential role in the processing of preproteins carrying a typical amino-terminal signal peptide, upon translocation across the cytoplasmic membrane. This paper reports the cloning and the transcriptional analysis of the L. pneumophila lepB gene encoding the type I signal peptidase (SPase). Reverse transcription PCR experiments showed clear lepB expression when L. pneumophila was grown both in culture medium, and also intracellularly in Acanthamoeba castellanii, a natural eukaryotic host of L. pneumophila. In addition, LepB was shown to be encoded by a polycistronic mRNA transcript together with two other proteins, i.e. a LepA homologue and a ribonuclease III homologue. SPase activity of the LepB protein was demonstrated by in vivo complementation analysis in a temperature-sensitive Escherichia coli lepB mutant. Protein sequence and predicted membrane topology were compared to those of leader peptidases of other Gram-negative human pathogens. Most strikingly, a strictly conserved methionine residue in the substrate binding pocket was replaced by a leucine residue, which might influence substrate recognition. Finally it was shown by in vivo experiments that L. pneumophila LepB is a target for (5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate, a specific inhibitor of type I SPases.
authors.affiliation: Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
authors.forename: Elke
authors.initials: E
authors.lastname: Lammertyn
authors.affiliation: Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
authors.forename: Lieve
authors.initials: L
authors.lastname: Van Mellaert
authors.affiliation: Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
authors.forename: Eef
authors.initials: E
authors.lastname: Meyen
authors.affiliation: Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
authors.forename: Ilya
authors.initials: I
authors.lastname: Lebeau
authors.affiliation: Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
authors.forename: Emmy
authors.initials: E
authors.lastname: De Buck
authors.affiliation: Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
authors.forename: Jozef
authors.initials: J
authors.lastname: Anné
authors.affiliation: Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
authors.forename: Nick
authors.initials: N
authors.lastname: Geukens
mesh_terms: D000048:Acanthamoeba / Q000382:microbiology; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006801:Humans; D016952:Legionella pneumophila / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; C482639:LepB protein, Legionella pneumophila; D008565:Membrane Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase