title: Significant role for ladC in initiation of Legionella pneumophila infection.
abstract: Previously, we identified ladC in a cohort of genes that were present in Legionella pneumophila but absent in other Legionella species. Here we constructed a ladC mutant of L. pneumophila and assessed its ability to replicate in mammalian cell lines and Acanthamoeba castellanii. The ladC mutant was recovered in significantly lower numbers than wild-type L. pneumophila at early time points, which was reversed upon transcomplementation with ladC but not ladC(N430A/R434A), encoding a putative catalytically inactive derivative of the protein. In fact, complementation of ladC::Km with ladC(N430A/R434A) resulted in a severe replication defect within human and amoeba cell models of infection, which did not follow a typical dominant negative phenotype. Using differential immunofluorescence staining to distinguish adherent from intracellular bacteria, we found that the ladC mutant exhibited a 10-fold reduction in adherence to THP-1 macrophages but no difference in uptake by THP-1 cells. When tested in vivo in A/J mice, the competitive index of the ladC mutant dropped fivefold over 72 h, indicating a significant attenuation compared to wild-type L. pneumophila. Although localization of LadC to the bacterial inner membrane suggested that the protein may be involved in signaling pathways that regulate virulence gene expression, microarray analysis indicated that ladC does not influence the transcriptional profile of L. pneumophila in vitro or during A. castellanii infection. Although the mechanism by which LadC modulates the initial interaction between the bacterium and host cell remains unclear, we have established that LadC plays an important role in L. pneumophila infection.
authors.affiliation: Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Victoria 3800, Australia.
authors.forename: Hayley J
authors.initials: HJ
authors.lastname: Newton
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Sansom
authors.forename: Jenny
authors.initials: J
authors.lastname: Dao
authors.forename: Christel
authors.initials: C
authors.lastname: Cazalet
authors.forename: Holger
authors.initials: H
authors.lastname: Bruggemann
authors.forename: Christiane
authors.initials: C
authors.lastname: Albert-Weissenberger
authors.forename: Carmen
authors.initials: C
authors.lastname: Buchrieser
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Cianciotto
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Hartland
mesh_terms: D048248:Acanthamoeba castellanii / Q000254:growth & development / Q000382:microbiology*; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004847:Epithelial Cells / Q000382:microbiology*; D005260:Female; D006801:Humans; D016952:Legionella pneumophila / Q000254:growth & development / Q000472:pathogenicity*; D007877:Legionnaires' Disease / Q000382:microbiology*; D051379:Mice; D009000:Monocytes / Q000382:microbiology*; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D000262:Adenylyl Cyclases