Characterization of a macrophage-specific infectivity locus (milA) of Legionella pneumophila - PubMed (original) (raw)

Characterization of a macrophage-specific infectivity locus (milA) of Legionella pneumophila

O S Harb et al. Infect Immun. 2000 Jan.

Abstract

Legionella pneumophila has been shown to possess multiple genetic loci that play roles in its ability to survive within host cells. The mil (macrophage-specific infectivity loci) mutants of L. pneumophila exhibit a spectrum of defects in intracellular survival in and cytopathogenicity to macrophages and alveolar epithelial cells. This study characterizes one of the mil mutants (GB111). Intracellular growth of GB111 in macrophages was approximately 100- to 1,000-fold less than that of AA100, the parental strain, at 24 and 48 h postinfection. This defect in turn corresponded to a defect in cytopathogenicity. Sequence analysis of the affected GB111 open reading frame (ORF) revealed it to encode a putative transport protein, and the ORF was designated milA. The phenotypic defect of the milA mutant was complemented with a PCR fragment containing only milA, indicating that the defect in GB111 was due to the disruption of milA. Intracellular trafficking of the mutant was examined by laser scanning confocal microscopy. The data showed that 50% of the GB111 phagosomes colocalized with the late endosomal/lysosomal marker LAMP-2 (2 and 4 h postinfection), while less than 10% of the AA100 phagosomes colocalized with this marker. On the other hand, over 80% of the GB111 phagosomes were similar to the AA100 phagosome in that they were devoid of LAMP-1 and cathepsin D, and they were colocalized with the endoplasmic reticulum (ER) marker BiP. However, the number of GB111 phagosomes that colocalized with BiP decreased to 50% 6 h postinfection compared to that of AA100, which remained constant (80% colocalization). Thus, compared to AA100, the milA mutation caused a defect in intracellular replication, which was associated with colocalization of the phagosome with LAMP-2 and BiP, while colocalization with LAMP-1 and cathepsin D was not affected.

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Figures

FIG. 1

Intracellular growth kinetics of L. pneumophila GB111 and AA100 (wild type) in U937 macrophage-like cells (A) and WI-26 alveolar epithelial cells (B) as determined by a gentamicin protection assay. Strain B6 is a cosmid-complemented clone of GB111. ∗, significantly fewer intracellular bacteria than in the wild-type (AA100)-infected cells based on Student's t test (P < 0.05).

FIG. 2

Cytopathogenicity of L. pneumophila GB111 and AA100 (wild type) to U937 macrophage-like cells (A) and WI-26 alveolar epithelial cells (B) as determined by using Alamar Blue dye. Strain B6 is a cosmid-complemented clone of GB111. Percent killing of cells was normalized to that for uninfected cells, which were considered 100% viable. ∗, significantly less survival than for uninfected control cells based on Student's t test (P < 0.05).

FIG. 3

Analysis of the GB111 sequence. (A) Alignment of the predicted amino acid sequence of the GB111 ORF to a predicted permease of B. subtilis, using the Blast2 sequence analysis program. (B) Hydropathy profile of the predicted GB111 amino acid sequence based on the Kyte-Doolittle algorithm with a window size of seven amino acids. Negative scores indicate relative hydrophobicity.

FIG. 4

Colocalization of phagosomes with LAMP-2. (A) Wild-type L. pneumophila (AA100); (B) GB111; (C) paraformaldehyde-killed AA100. LAMP-2 is visualized by using secondary antibodies conjugated to Oregon Green (green pseudo-color). Extracellular bacteria were visualized with secondary antibody conjugated to Alexa Red (red pseudocolor). Nucleic acids were stained with Toto-3 (blue pseudocolor), which labels both intracellular and extracellular bacteria and the cell nucleus. Intensities of fluorescence across the indicated phagosomes (arrows) are shown at the right. Darkened arrows in panel B point to a GB111 phagosome that does not colocalize with LAMP-2, and fluorescence intensity across this phagosome is shown on the far right.

FIG. 5

Colocalization of BiP (ER) with phagosomes of wild-type L. pneumophila (AA100) (A), GB111 (B), and paraformaldehyde-killed AA100 (C). BiP is visualized by using secondary antibodies conjugated to Oregon Green (green pseudocolor). Extracellular bacteria were visualized with secondary antibody conjugated to Alexa Red (red pseudocolor). Nucleic acids were stained with Toto-3 (blue pseudocolor), which labels both intracellular and extracellular bacteria and the cell nucleus. Intensities of fluorescence across the indicated (arrows) phagosomes are shown at the right.

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Characterization of a macrophage-specific infectivity locus (milA) of Legionella pneumophila - PubMed (original) (raw)