Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells

Abstract

Legionella pneumophila expresses pili of variable lengths, either long (0.8 to 1.5 microm) or short (0.1 to 0.6 microm), that can be observed by transmission electron microscopy. We have identified a gene in L. pneumophila with homology to the type IV pilin genes (pilEL). An insertion mutation was constructed in pilEL and introduced into the L. pneumophila wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The L. pneumophila pilEL mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and Acanthamoeba polyphaga but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that the pilEL locus is present in L. pneumophila serogroups 1 through 13 but is variable in 16 other Legionella species. The presence of a type IV pilin gene and its expression by L. pneumophila may provide an advantage for colonization of lung tissues during Legionnaires' disease and invasion of amoebas in the environment.

FIG. 1

Electron microscopic examination of expression of pili by L. pneumophila AA100. L. pneumophila wild-type strain AA100 expresses short (0.1 to 0.6 μm) (A) and long (0.8 to 1.5 μm) (B) pili. Pili are indicated by small arrowheads. Flagella are noticeably thicker than the pili observed and are indicated by large arrowheads. Bars, 0.5 μm.

FIG. 2

Nucleotide and deduced amino acid sequences of L. pneumophila pilE_L. Potential ribosome binding sites are underlined; potential sites for the start of translation are in boldface. The internal EcoRI site, which is the site of the kanamycin gene insertion in BS100, is indicated by the diamond. The position of cleavage of the leader peptide in type IV pilin genes is indicated by the arrowhead.

FIG. 3

Alignment of the deduced PilE_L sequence with those of type IV pilin proteins. The arrowhead indicates the site of cleavage of the leader peptide between the conserved glycine and phenylalanine in type IV pilin genes. PilA is the type IV pilin gene in P. aeruginosa, PilE is in N. gonorrhoeae, and TfpQ is in M. bovis. Amino acids homologous among all four proteins are in boldface.

FIG. 4

Comparison of pili expressed in L. pneumophila AA100 and in the pilE_L mutant. L. pneumophila wild-type strain AA100 expresses short (A) and long (B) pili. The pilE_L mutant strain BS100 expresses only short pili (C). Cosmid pCC3, which contains the pilE_L locus, is able to reintroduce expression of long pili to BS100 (D). Pili are indicated by small arrowheads; flagella are indicated by large arrowheads. Bars, 0.5 μm.

FIG. 5

Presence of pilE_L in Legionella species. Chromosomal DNA from different Legionella species was subjected to Southern analysis under low-stringency conditions and probed with the pilE_L locus. L. pneumophila AA100 chromosomal DNA hybridizes with a single 2-kb fragment (arrowhead) under high-stringency conditions. Lanes 1 to 17, L. pneumophila, L. dumoffi, L. longbeachae, L. gormanii, L. micdadei, L. wadsworthii, L. oakridgensis, L. feeleii, L. sainthelensis, L. spiritensis, L. jamestowniensis, L. santicrucis, L. cherrii, L. parisiensis, L. erythra, L. hackeliae, and L. israelensis, respectively.