Abstract
Pseudomonas aeruginosa is known to invade epithelial cells during infection and in vitro. However, little is known of bacterial or epithelial factors modulating P. aeruginosa intracellular survival or replication after invasion, except that it requires a complete lipopolysaccharide core. In this study, real-time video microscopy revealed that invasive P. aeruginosa isolates induced the formation of membrane blebs in multiple epithelial cell types and that these were then exploited for intracellular replication and rapid real-time motility. Further studies revealed that the type three secretion system (T3SS) of P. aeruginosa was required for blebbing. Mutants lacking either the entire T3SS or specific T3SS components were instead localized to intracellular perinuclear vacuoles. Most T3SS mutants that trafficked to perinuclear vacuoles gradually lost intracellular viability, and vacuoles containing those bacteria were labeled by the late endosomal marker lysosome-associated marker protein 3 (LAMP-3). Interestingly, mutants deficient only in the T3SS translocon structure survived and replicated within the vacuoles that did not label with LAMP-3. Taken together, these data suggest two novel roles of the P. aeruginosa T3SS in enabling bacterial intracellular survival: translocon-dependent formation of membrane blebs, which form a host cell niche for bacterial growth and motility, and effector-dependent bacterial survival and replication within intracellular perinuclear vacuoles.
Highlights
Pseudomonas aeruginosa infection is a significant cause of human morbidity and mortality [8, 23, 27, 62, 63]
Real-time video microscopy revealed that invasive P. aeruginosa isolates induced the formation of membrane blebs in multiple epithelial cell types and that these were exploited for intracellular replication and rapid real-time motility
It has been shown that invasion requires bacterial interaction with host cell targets such as lipid rafts [39, 66, 67], which are themselves associated with specific targets for P. aeruginosa invasion, for example, the cystic fibrosis transmembrane conductance regulator [38, 49] and asialo-GM1 [10]
Summary
Pseudomonas aeruginosa infection is a significant cause of human morbidity and mortality [8, 23, 27, 62, 63]. The type three secretion system (T3SS) is important for P. aeruginosa virulence in corneal, skin, airway, and systemic infections [53, 61]. This system delivers effector proteins into host cells upon contact by the combination of a needle apparatus (allows effector secretion directly from the bacterial cell) followed by a translocon pore (inserted into host membranes for the intracellular delivery of effectors). Molecular and cellular relationships between in vitrodefined activities and the in vivo roles of the T3SS are yet to be established, in particular as they relate to the sequence of events enabling bacterial survival and to the numerous cell types and conditions under which bacteria interact with the infected host.
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