LEGIONELLA-PNEUMOPHILA DOTA PROTEIN IS REQUIRED FOR EARLY PHAGOSOME TRAFFICKING DECISIONS THAT OCCUR WITHIN MINUTES OF BACTERIAL UPTAKE

Numerous intracellular bacterial pathogens modulate the nature of the membrane-bound compartment in which they reside, although little is kn own about the molecular basis for this control. Legionella pneumophila is a bacterial pathogen able to grow within human alveolar macrophage s and residing in a phagosome that does not fuse with lysosomes. This study demonstrates that the dotA product is required to regulate traff icking of the L. pneumophila phagosome. Phagosomes containing L. pneum ophila dotA(+) bacteria exhibited differential trafficking profiles wh en compared with isogenic dotA mutants. Phagosomes containing dotA mut ants showed rapid accumulation of the lysosomal glycoprotein LAMP-1 as early as 5 min after uptake, whereas the majority of wild-type L. pne umophila phagosomes did not acquire LAMP-1. The association of LAMP-1 with phagosomes containing dotA mutant bacteria was concomitant with t he appearance of the small GTP-binding protein Rab7 on the vacuolar me mbrane. These data demonstrate that phagosomes containing replication- competent L. pneumophila evade early endocytic fusion events. In contr ast, the kinetics of LAMP-1 and Rab7 association indicate that the dot A mutants are routed along a well-characterized endocytic pathway lead ing to fusion with lysosomes. Genetic studies show that L. pneumophila requires DotA expression before macrophage uptake in order to establi sh an intracellular site for replication. However, the bacteria do not appear to require continuous expression of the DotA protein to mainta in a replicative phagosome. These data indicate that DotA is one facto r that plays a fundamental role in regulating initial phagosome traffi cking decisions either upon or immediately after macrophage uptake.