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Infection and Immunity, January 2000, p. 368-376, Vol. 68, No. 1
Department of Microbiology and Immunology,
University of Kentucky Chandler Medical Center, Lexington, Kentucky
40536-0084
Received 21 June 1999/Returned for modification 10 August
1999/Accepted 4 October 1999
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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Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of a Macrophage-Specific
Infectivity Locus (milA) of Legionella
pneumophila
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington, KY 40536-0084. Phone: (606) 323-3873. Fax: (606)
257-8994. E-mail: yabukw{at}pop.uky.edu.
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