Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway

Abstract

Legionella pneumophila replicates within a specialized phagosome in cultured cells, a function necessary for its pathogenicity. The replicative phagosome lacks membrane marker proteins, such as the glycoprotein LAMP-1, that are indicators of the normal endocytic pathway. We describe the isolation of several Legionella genes essential for intracellular growth and evasion of the endocytic pathway, using a genetic and cell biological approach. We screened 4,960 ethyl methanesulfonate-mutagenized colonies for defects in intracellular growth and trafficking to the replicative phagosome. Six mutant strains of L. pneumophila that had severe intracellular growth defects in mouse bone marrow-derived macrophages were identified. All six mutants were found in phagosomes that colocalized with LAMP-1, indicating defects in intracellular trafficking. The growth defects of two of these strains were complemented by molecular clones from a bank constructed from a wild-type L. pneumophila strain. The inserts from these clones are located in a region of the chromosome contiguous with several other genes essential for intracellular growth. Three mutants could be complemented by single open reading frames placed in trans, one mutant by a gene termed dotH and two additional mutants by a gene termed dotO. A deletion mutation was created in a third gene, dotI, which is located directly upstream of dotH. The delta dotI strain was also defective for intracellular growth in macrophages, and this defect was complemented by a single open reading frame in trans. Based on sequence analysis and structural predictions, possible roles of dotH, dotI, and dotO in intracellular growth are discussed.

FIG. 1

Colocalization of intracellular growth mutants with late endosomal, lysosomal marker LAMP-1 in mouse bone marrow-derived macrophages by immunofluorescence microscopy. Macrophages were infected with wild-type or mutant strains of L. pneumophila for 2 h, fixed, and stained for LAMP-1 colocalization (A, C, E, G, and I) and intracellular versus extracellular bacteria (B, D, F, H, and J). Neighboring panels show LAMP-1 staining (left) and corresponding intracellular bacteria (right). (A and B) Lp01 (dot⁺); (C and D) formalin-killed Lp01 (dot⁺); (E and F) HL1400 (dotO); (G and H) HL1700 (dotH); (I and J) HL056 (dotI).

FIG. 2

Intracellular growth mutants of L. pneumophila colocalize with LAMP-1. For each sample, mouse bone marrow macrophages were incubated with mutant or wild-type strains for 2 h, fixed, stained for intracellular versus extracellular bacteria and LAMP-1 colocalization, and examined. Data were collected from 100 intracellular bacteria in total. Percent LAMP-1 positive was calculated by dividing the number of intracellular rod-shaped bacteria colocalizing with LAMP-1 by the total number of intracellular rod-shaped bacteria scored. Values shown are averages of duplicate samples from two identical experiments (four samples in total) and their standard deviations.

FIG. 3

Complementation of intracellular growth defects of L. pneumophila mutants HL1700 and HL1400. Growth was monitored for 72 h to measure the ability of p1713 and p1415 to allow mutants HL1700 (A) and HL1400 (B) to grow intracellularly in phorbol ester-treated U937 cells. Data points and error bars represent the mean CFU of triplicate samples from a typical experiment (performed at least twice) and their standard deviations. (A) Lp01 (dot⁺; ▵), HL019 (dot⁺, pMS8; ▴), HL1700 (dotH; □), HL1705 (dotH, pMS8; ┘) and HL1719 (dotH, p1713; ▪). (B) Lp01 (dot⁺; ▵), HL019 (dot⁺, pMS8; ▴), HL1400 (dotO; □), HL1405 (dotO, pMS8; ┘), and HL1419 (dotO, p1415; ▪).

FIG. 4

Physical map of new loci in L. pneumophila essential for intracellular growth. Arrows illustrate the direction of transcription and do not imply operon structure. Inserts of plasmids isolated in the complementation test from L. pneumophila genomic library, as well as inserts from plasmids containing open reading frames generated by PCR or subcloning, are shown.

FIG. 5

dotH complements the intracellular growth defect of mutant HL1700 in U937 cells. L. pneumophila strains harboring plasmids were tested for growth in phorbol ester-treated U937 cells. Mean CFU were calculated as the average of duplicate samples from two identical experiments. Values shown represent fold growth, which was determined by dividing mean CFU at 72 h by the mean CFU at t₀. Error bars represent the total error, which is equal to the total growth multiplied by the combination of the fractional errors for each time point. Strains: Lp01 (dot⁺); HL019 (dot⁺, pMS8); HL1700 (dotH); HL1705 (dotH, pMS8); HL1719 (dotH, p1713); HL1725 (dotH, pH3).

FIG. 6

Mutants HL1400 and HL1000 are complemented for intracellular growth by the dotO gene in trans. Growth was measured for 72 h to examine the ability of p2A (dotO⁺) to allow HL1400 (A) and HL1000 (B) to grow intracellularly. Data points and error bars represent the mean CFU of triplicate samples from a typical experiment (performed at least twice) and their standard deviations. (A) Lp01 (dot⁺; ▵), HL019 (dot⁺, pMS8; ▴), HL1400 (dotO1; □), HL1405 (dotO1, pMS8; ┘), and HL1422 (dotO1, p2A; ▪). (B) Lp01 (dot⁺; ▵), HL019 (dot⁺, pMS8; ▴), HL1000 (dotO2; □), HL1005 (dotO2, pMS8; ┘), and HL1012 (dotO2, p2A; ▪).

FIG. 7

(A) ΔdotI mutation causes defective intracellular growth in phorbol ester-treated U937 cells over 72 h, and this defect is complemented by dotI in trans. Growth of L. pneumophila was monitored for 72 h. Values and error bars represent the average of triplicate samples from a typical experiment (performed at least twice) and their standard deviations. Strains: Lp01 (dot⁺; ▵), HL019 (dot⁺, pMS8; ▴), HL056 (dotI; □), HL057 (dotI, pMS8; ┘), and HL059 (dotI, pI1; ▪). (B) ΔdotI mutants colocalize with LAMP-1. Mouse bone marrow-derived macrophages were incubated with mutant or virulent strains for 2 h, fixed, and stained for intracellular versus extracellular bacteria and LAMP-1 colocalization. Data were collected from 100 intracellular bacteria. Percent LAMP-1 positive was calculated by dividing the number of intracellular rod-shaped bacteria colocalizing with LAMP-1 by the total number of intracellular rod-shaped bacteria scored. Values shown are the averages of duplicate samples from two identical experiments (four samples in total) and their standard deviations.

FIG. 8

Hydrophilicity analyses and model of predicted amphiphilic β-sheet of proteins shown to be essential for intracellular growth of L. pneumophila. (A) Hydrophilicity analysis of DotH predicts an N-terminal secretion signal sequence. (B) Hydrophilicity analysis of DotI predicts a membrane-spanning domain between amino acid residues 20 and 50. (C) Model of amphipathic β-sheets in DotI between amino acids 57 and 64 and amino acids 152 and 164. Hydrophobic faces of the predicted β-sheets are shown in a shaded box, while the polar faces are shown in an unshaded box.