Identification of Legionella pneumophila rcp, a pagP-like gene that confers resistance to cationic antimicrobial peptides and promotes intracellular infection

Abstract

In the course of characterizing a locus involved in heme utilization, we identified a Legionella pneumophila gene predicted to encode a protein with homology to the product of the Salmonella enterica serovar Typhimurium pagP gene. In Salmonella, pagP increases resistance to the bactericidal effects of cationic antimicrobial peptides (CAMPs). Mutants with insertions in the L. pneumophila pagP-like gene were generated and showed decreased resistance to different structural classes of CAMPs compared to the wild type; hence, this gene was designated rcp for resistance to cationic antimicrobial peptides. Furthermore, Legionella CAMP resistance was induced by growth in low-magnesium medium. To determine whether rcp had any role in intracellular survival, mutants were tested in the two most relevant host cells for Legionnaires' disease, i.e., amoebae and macrophages. These mutants exhibited a 1,000-fold-decreased recovery during a Hartmannella vermiformis coculture. Complementation of the infectivity defect could be achieved by introduction of a plasmid containing the intact rcp gene. Mutations in rcp consistently reduced both the numbers of bacteria recovered during intracellular infection and their cytopathic capacity for U937 macrophages. The rcp mutant was also more defective for lung colonization of A/J mice. Growth of rcp mutants in buffered yeast extract broth was identical to that of the wild type, indicating that the observed differences in numbers of bacteria recovered from host cells were not due to a generalized growth defect. However, in low-Mg(2+) medium, the rcp mutant was impaired in stationary-phase survival. This is the first demonstration of a pagP-like gene, involved in resistance to CAMPs, being required for intracellular infection and virulence.

FIG. 1

Sequence analysis of the L. pneumophila pagP-like gene and PagP-like protein. (A) Sequence organization and restriction analysis of pEH12, pBOC16, and pTA2. For all plasmids, only the L. pneumophila strain 130b DNA fragment is depicted. Restriction sites are as follows: A, AflII; B, BamHI; E, EcoRI; N, NdeI; and S, SacI. (B) Alignment (boxed regions) of PagP-like proteins in L. pneumophila (Lpn), Salmonella serovar Typhimurium (Sty) (GenBank accession number AF057021), and E. coli (Eco) (GenBank accession number AE000167). The Clustal method was used to generate alignment data (45).

FIG. 2

Extracellular growth kinetics of L. pneumophila strains. Wild-type strain 130b (●) and mutant NU260 (□) were compared for growth in BYE (A), CDM (0.7 mM Mg²⁺) (B), and CDM with 0.05 mM (C), or 0.005 mM (D) Mg²⁺ cations. Growth was assessed by measuring the OD₆₆₀ of triplicate cultures at various times of incubation. Asterisks indicate significant differences (P < 0.05; Student's t test) between the 130b and NU260 cultures. Standard deviations are shown, but the error bars are too small to be observed in panels A to C. This experiment was performed in triplicate with comparable results, and representative data are presented here.

FIG. 3

Intracellular infection of human macrophages with L. pneumophila strains. U937 cell monolayers (n = 3) were infected at an MOI of 1 with strain 130b (●) and NU260 (□). Asterisks indicate significant differences (P < 0.01; Student's t test) in numbers of recovered bacteria between 130b and NU260. Comparable results were obtained in triplicate experiments, and the data presented here are from one representative experiment. Error bars indicate standard deviations.

FIG. 4

Cytopathicity assay to assess the viability of L. pneumophila-infected human macrophages. U937 cell monolayers (n = 6) were infected at MOIs of 1 (A) and 10 (B) with wild-type strain 130b (●) and mutant NU260 (□). Macrophage viability is expressed as the percentage of alamarBlue dye reduction in infected compared to uninfected monolayers. Asterisks indicate significant differences in cytopathicity (P < 0.0005; Student's t test) between 130b and NU260. Comparable results were obtained from an additional experiment (data not shown). Error bars indicate standard deviations.

FIG. 5

Coculture of L. pneumophila strains with H. vermiformis. (A) Monolayers of amoebae (n = 3) were infected with wild-type strain 130b (●) and mutant NU260 (□) at an MOI of 0.01. Asterisks indicate significant differences (P < 0.05 at 48 h or P < 0.005 at 72 h; Student's t test) in numbers of recovered bacteria between 130b and NU260. The experiment was performed five times with comparable results, and representative data are presented here. (B) Genetic complementation of the rcp mutant's infectivity defect. Monolayers of amoebae were cocultured with either 130b containing the CHL resistance vector pSU2719 (bars 1), NU260 containing the vector (bars 2), or NU260 containing rcp cloned into pSU2719 (bars 3), and the numbers of recovered bacteria were determined at 0 h (solid bars) and 72h (hatched bars) p.i. In experiment 1 (black bars), the complementing plasmid was pS14. In experiment 2 (grey bars), which is representative of a replicate experiment, the complementing plasmid was pS25. Asterisks indicate significant differences (P < 0.005; Student's t test) in numbers of recovered bacteria between NU260 containing the vector and NU260 containing rcp. Error bars indicate standard deviations.

FIG. 6

In vivo competition assays with L. pneumophila 130b and NU260. The wild type (WT) and mutant were inoculated intratracheally into A/J mice (n = 4 to 6). The ratio of wild type to mutant in infected lungs was assessed by dividing the CFU per lung for the wild type by the CFU per lung for NU260. The values expressed are normalized to the deviation observed in the ratio of 130b to NU260 in the initial inoculum (i.e., 0.61).