Mechanisms of invasion of macrophages and lung epithelial cells by filamentous Legionella pneumophila

Legionella species are causative agents of legionellosis, a severe form of pneumonia. These bacteria are pleomorphic and different morphologies have varying virulence potential. Although filamentous forms of Legionella have been reported in patient samples since the first description of legionellosis, their role in disease remains unknown. This work examined the virulence potential of filamentous Legionella pneumophila (FLp) and provides the first description of the invasion mechanisms used by FLp to invade human lung epithelial cells (LECs) and macrophages, and the intracellular differentiation and replication of FLp to produce infectious progeny. FLp were successfully internalized by macrophages and engulfment occurred along the long axis of the filament. However, this uptake process deviated from the canonical phagocytic pathway. Filamentous morphology resulted in the formation of a long-lasting tubular phagocytic cup (T-PC) stage that modulated the outcome of phagocytosis, allowing FLp to escape phagolysosomal killing in a length-dependent manner. Investigation of the mechanism responsible for this revealed that despite being unsealed compartments, the T-PCs fused with endosomes and lysosomes, events linked to the maturation of phagosomes. Nevertheless, these T-PCs were `leaky' and failed to become hydrolytic prior to sealing, impeding their microbicidal capacity. This allowed FLp to secrete effectors while in a non-hydrolytic T-PC and modify their intracellular compartments. FLp attachment to LECs was mediated by E-cadherin and β1 integrin receptors and induced the formation of actin-enriched membrane surface structures that we designated `hooks' and `membrane wraps'. These structures entrapped the filament at the cell surface, and through a variation of the zipper mechanism of invasion, facilitated the actomyosin-dependent internalization of FLp. A supply of E-cadherins from intracellular recycling pathway and β1 integrins released by focal adhesion turnover sustained this internalization process. Collectively, these results demonstrate that FLp can invade both LECs and macrophages, and could play an important role in pathogenesis.Ph.D.2017-06-30 00:00:0