Affiliation:
1. Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington, Kentucky 40536-0084
Abstract
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.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Immunology,Microbiology,Parasitology
Cited by
21 articles.
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