Characterization of inhibition of phagosome -lysosome fusion by <italic>Legionella pneumophila</italic>.

Abstract

<italic>Legionella pneumophila</italic>, the causative agent of Legionnaires' disease, survives within macrophages by evading phagosome-lysosome fusion. To understand the mechanism of phagosome-lysosome inhibition by <italic>Legionella pneumophila</italic>, I took two complementary approaches. First, I undertook a phenotypic analysis comparing expression of seven virulence traits by one well characterized <italic>L. pneumophila</italic> strain to four clinical isolates of <italic>L. micdadei</italic>, a species known to form phagolysosomes and to grow poorly in human monocytes. By nearly every measure, <italic>L. micdadei</italic> appeared less virulent than <italic>L. pneumophila</italic>. To identify virulence factors genetically, an <italic>L. pneumophila</italic> genomic library was screened for clones which conferred to <italic>L. micdadei </italic> robust intracellular growth. No such loci were isolated, consistent with the multiple phenotypic differences observed for the two species. The second approach used quantitative fluorescence microscopy to determine whether vacuoles; containing <italic>L. pneumophila</italic> are either completely separate from, or merely stalled along, the endocytic pathway. I analyzed vacuoles as they aged from 2.5 min to 4 h and, as expected, found that the majority of phagosomes containing non-pathogens like exponential phase (E) <italic>L. pneumophila</italic> and <italic>E. coli</italic>, fused sequentially with early endosomes, late endosomes and lysosomes, as judged by their sequential acquisition of transferrin receptor, LAMP-1 and cathepsin D. In striking contrast, phagosomes containing post exponential phase (PE) <italic> L. pneumophila</italic> did not acquire any of these markers, indicating establishing a vacuole which is completely separate from the endocytic pathway. Surprisingly, although phagosomes containing either Dot/Icm secretion system mutants<italic> dotA</italic> and <italic>dotB</italic> or formalin-killed PE <italic>L. pneumophila </italic> rapidly acquired LAMP-1, they rarely acquired lysosomal markers. Nor did these vacuoles accumulate endosomal tracers, TRov, CM-DiI, or Alexa Fluor-streptavidin, which readily labeled phagosomes containing polystyrene beads, formalin-killed <italic>E. coli</italic> or E <italic>L. pneumophila </italic>. Thus virulent PE, <italic>L. pneumophila</italic> require a Dot-dependent factor for inhibiting fusion with LAMP-1 containing vacuoles and a Dot-independent, formalin-resistant factor to evade lysosomes. Taken together our data indicate that <italic>L. pneumophila</italic> has evolved to possess multiple factors which isolate its phagosome from the degradative vacuoles. Consequently, <italic> Legionella</italic> can thrive in macrophages. The results of my studies on <italic> L. pneumophila</italic> can be used as a model to understand pathogenesis of a class of parasites which cause disease by virtue of their replication in macrophages.