Disruption of the Phagosomal Membrane and Egress ofLegionella pneumophilainto the Cytoplasm during the Last Stages of Intracellular Infection of Macrophages andAcanthamoeba polyphaga

Abstract

Although the early stages of intracellular infection by Legionella pneumophila are well established at the ultrastructural level, a detailed ultrastructural analysis of late stages of intracellular replication has never been done. Here we show that the membrane of the L. pneumophila-containing phagosome (LCP) is intact for up to 8 h postinfection of macrophages and Acanthamoeba polyphaga. At 12 h, 71 and 74% of the LCPs are disrupted within macrophages and A. polyphaga, respectively, while the plasma membrane remains intact. At 18 and 24 h postinfection, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and amorphous material are dispersed among the bacteria and these bacteria are considered cytoplasmic. At 18 h, 77% of infected macrophages and 32% of infected A. polyphaga amoebae harbor cytoplasmic bacteria. At 24 h, 99 and 78% of infected macrophages and amoebae, respectively, contain cytoplasmic bacteria. On the basis of lysosomal acid phosphatase staining of infected macrophages and A. polyphaga, the lysosomal enzyme is present among the bacteria when host vesicles are dispersed among bacteria. Our data indicate that bacterial replication proceeds despite physical disruption of the phagosomal membrane. We also show that an lspG mutant that is defective in the type II secretion system and therefore does not secrete the hydrolytic enzymes metalloprotease, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A is as efficient as the wild-type strain in disruption of the LCP. Therefore, L. pneumophila disrupts the phagosomal membrane and becomes cytoplasmic at the last stages of infection in both macrophages and A. polyphaga. Lysosomal elements, mitochondria, cytoplasmic vesicles, and amorphous material are all dispersed among the bacteria, after phagosomal disruption, within both human macrophages and A. polyphaga. The disruption of the LCP is independent of the hydrolytic enzymes exported by the type II secretion system.

Identification of Legionella pneumophila -Specific Genes by Genomic Subtractive Hybridization with Legionella micdadei and Identification of lpnE , a Gene Required for Efficient Host Cell Entry

“…The Legionella vacuole recruits the host GTPase, Rab1, and the v-SNARE, Sec22b, to establish a replicative vacuole surrounded by rough endoplasmic reticulum (RER) (30). The recruitment of RER correlates strongly with the virulence of L. pneumophila, and the manipulation of host cell trafficking pathways allows the bacteria to replicate to high numbers inside cells before eventual bacterial egress, host cell death, and the infection of new host cells (2,39).…”

Legionella pneumophila glucosyltransferase inhibits host elongation factor 1A

“…These organellae are associated with ribosome-studded membranes and support multiplication of the bacteria (6,7). Eventually, host cells die because of induction of apoptosis or necrosis (8)(9)(10), and the pathogens are released to invade new pools of phagocytes of the host (10,11). During recent years several genetic loci and bacterial factors have been identified that are suggested to be implicated in Legionella-host cell interactions (12).…”

Maintenance of vacuole integrity by bacterial pathogens

“…Recent work has shown that the bulk of Salmonella replication observed during growth in epithelial cells occurs in the cytosol; the significance of this during infection is unknown although it may play a role in bacterial dissemination [39,40]. Escape from the vacuole has also been observed for Mycobacterial species and L. pneumophila suggesting that an extra-vacuolar stage occurs in other bacterial infections [41][42][43]. The frequency and outcome of vacuole disruption during disease for both of these organisms is unknown.…”