Flagellum of Legionella pneumophila Positively Affects the Early Phase of Infection of Eukaryotic Host Cells

Abstract

Legionella pneumophila, the etiologic agent of Legionnaires' disease, contains a single, monopolar flagellum which is composed of one major subunit, the FlaA protein. To evaluate the role of the flagellum in the pathogenesis and ecology of Legionella, the flaA gene of L. pneumophila Corby was mutagenized by introduction of a kanamycin resistance cassette. Immunoblots with antiflagellin-specific polyclonal antiserum, electron microscopy, and motility assays confirmed that the specific flagellar mutant L. pneumophila Corby KH3 was nonflagellated. The redelivery of the intact flaA gene into the chromosome (L. pneumophila Corby CD10) completely restored flagellation and motility. Coculture studies showed that the invasion efficiency of the flaA mutant was moderately reduced in amoebae and severely reduced in HL-60 cells. In contrast, adhesion and the intracellular rate of replication remained unaffected. Taking these results together, we have demonstrated that the flagellum of L. pneumophila positively affects the establishment of infection by facilitating the encounter of the host cell as well as by enhancing the invasion capacity.Legionella pneumophila, the etiologic agent of Legionnaires' disease, is a ubiquitous microorganism inhabiting natural and man-made freshwater biotopes (5). In these environments, the gram-negative, rod-shaped bacteria survive as intracellular pathogens of protozoan organisms such as Acanthamoeba castellanii, Hartmannella vermiformis, and Naegleria spp. (15). Upon transmission to individuals via L. pneumophila-containing aerosols generated by showerheads and air-conditioning systems, the bacteria invade and multiply within alveolar macrophages (1, 2, 7) and nonphagocytic cells (17). The infection which mainly affects immunocompromised patients results in a life-threatening atypical pneumonia (7).Detailed ultrastructural and molecular studies of the intracellular fate of the bacterium revealed that human macrophages and protozoan cells infected with L. pneumophila exhibit remarkable similarities concerning the establishment of a replicative phagosome (3,16,22,42,45). However, significant differences were observed during early stages of infection (21). Uptake by Hartmannella is accomplished by a microfilamentindependent mechanism that is sensitive to methylamine, which is an inhibitor of receptor-mediated endocytosis (28). So far, one receptor of Hartmanella vermiformis, a Gal/GalNAc lectin, could be identified (46). Attachment of L. pneumophila to this lectin results in tyrosine dephosphorylation of multiple host cell proteins. However, depending on the type of amoeba, different receptors might be involved (22). In contrast, the uptake by human macrophages occurs following binding of complement receptors CR1 and CR3 via microfilament-dependent phagocytosis (26). In addition to this cytochalasin D-sensitive mechanism, complement-independent mechanisms for uptake by nonphagocytic cells have been described (39).The influence of bacterial motility on infection processes or on survival of legio...

FliA expression analysis and influence of the regulatory proteins RpoN, FleQ and FliA on virulence and in vivo fitness in Legionella pneumophila

“…Screening for Gm r mutants obtained by homologous recombination was performed by PCR with primers FliD_Mut_Test 1U, 2R, 3U and 4R. The construct pKH3 for the flaA mutant has been described before (Dietrich et al 2001). A new flagellin-deficient mutant strain was created via natural transformation instead of electrotransformation.…”

“…FliA is required for the in vivo fitness (Albert-Weissenberger et al 2010;Dietrich et al 2001;Hammer et al 2002;Heuner et al 2002;Jacobi et al 2004;Molofsky et al 2005). Furthermore it is thought that motility but not flagellin promotes contact with host cells and increases infectivity (Molofsky et al 2005).…”

Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila , plaB , Exhibiting Hemolytic Activity

“…L. pneumophila plaB knockout mutants were generated by electroporation of pKH195 into L. pneumophila Corby and allelic exchange. Screening for mutants obtained by double crossover was performed as described recently (17). Integration of the Km r cassette into the chromosomal plaB gene was verified by Southern blot analysis (data not shown).…”

Contribution of FliC to Epithelial Cell Invasion by Enterohemorrhagic Escherichia coli O113:H21

“…Flagellin has been implicated in the invasion of host cells by several pathogens, including L. monocytogenes, Legionella pneumophila, and Burkholderia cepacia (8,9,33). In L. monocytogenes, which also invades human colonic cells, deletion of the gene encoding flagellin, flaA, resulted in an ϳ4-fold reduction in invasion of Caco-2 cells (9).…”