Legionella pneumophila, the bacterial agent of legionnaires' disease, replicates intracellularly within a specialized vacuole of mammalian and protozoan host cells. Little is known about the specialized vacuole except that the Icm/Dot type IV secretion system is essential for its formation and maintenance. The Legionella genome database contains two open reading frames encoding polypeptides (LepA and LepB) with predicted coiled-coil regions and weak homology to SNAREs; these are delivered to host cells by an Icm/Dot-dependent mechanism. Analysis of mutant strains suggests that the Lep proteins may enable the Legionella to commandeer a protozoan exocytic pathway for dissemination of the pathogen.

Abstract. Amoebae of the eukaryotic microorganism Dictyostelium discoideum were found to contain an interconnected array of tubules and cisternae whose membranes were studded with 15-nm-diameter "pegs" Comparison of the ultrastructure and freeze-fracture behavior of these pegs with similar structures found in other cells and tissues indicated that they were the head domains of vacuolar-type proton pumps. Supporting this identification, the pegs were observed to decorate and clump when broken amoebae were exposed to an antiserum against the B subunit of mammalian vacuolar H+-ATPase. The appearance of the peg-rich cisternae in quick-frozen amoebae depended on their osmotic environment: under hyperosmotic conditions, the cisternae were fiat with many narrow tubular extensions, while under hypo-osmotic conditions the cisternae ranged from bulbous to spherical. In all cases, however, their contents deep etched like pure water. These properties indicated that the interconnected tubules and cisternae comprise the contractile vacuole system of Dictyostelium. Earlier studies had demonstrated that contractile vacuole membranes in Dictyostelium are extremely rich in calmodulin (Zhu, Q., and M. Clarke. 1992. J. Cell Biol. 118: 347-358). Light microscopic immunofluorescence confirmed that antibodies against the vacuolar proton pump colocalized with anti-calmodulin antibodies on these organelles. Time-lapse video recording of living amoebae imaged by interference-reflection microscopy, or by fluorescence microscopy after staining contractile vacuole membranes with potential-sensitive styryl dyes, revealed the extent and dynamic interrelationship of the cisternal and tubular elements in Dictyostelium's contractile vacuole system. The high density of proton pumps throughout its membranes suggests that the generation of a proton gradient is likely to be an important factor in the mechanism of fluid accumulation by contractile vacuoles.