Characterization of a macrophage-specific infectivity locus (milA) of Legionella pneumophila.
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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Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington, Kentucky 40536-0084, USA.
Abu Kwaik
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D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D002455:Cell Division; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006461:Hemolysis; D006801:Humans; D020104:Immunophilins / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D016952:Legionella pneumophila / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity*; D008264:Macrophages / Q000382:microbiology*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D019696:Peptidylprolyl Isomerase; D010588:Phagosomes / Q000382:microbiology; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D020298:U937 Cells; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; D008565:Membrane Proteins; D020104:Immunophilins; C512922:Mip protein, Legionella pneumophila; D019696:Peptidylprolyl Isomerase