icmTIs Essential for Pore Formation-Mediated Egress ofLegionella pneumophilafrom Mammalian and Protozoan Cells

Abstract

The final step of the intracellular life cycle of Legionella pneumophila and other intracellular pathogens is their egress from the host cell after termination of intracellular replication. We have previously isolated five spontaneous mutants of L. pneumophila that replicate intracellularly similar to the wild-type strain but are defective in pore formation-mediated cytolysis and egress from mammalian and protozoan cells, and the mutants have been designated rib (release of intracellular bacteria). Here, we show that the rib mutants are not defective in the activity of enzymes secreted through the type II secretion system, including phospholipase A, lysophospholipase A, and monoacylglycerol lipase, although they are potential candidates for factors that lyse host cell membranes. In addition, the pilD and lspG mutants, which are defective in the type II secretion system, are not defective in the pore-forming toxin. We show that all five rib mutants have an identical point mutation (deletion) following a stretch of poly(T) in the icmT gene. Spontaneous revertants of the rib mutants, due to an insertion of a nucleotide following the poly(T) stretch in icmT, have been isolated and shown to have regained the wild-type phenotype. We constructed an icmT insertion mutant (AA100kmT) in the chromosome of the wild-type strain by allelic exchange. The AA100kmT mutant was as defective as the rib mutant in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. Both the rib mutant and the AA100kmT mutant were complemented by the icmT gene for their phenotypic defect. rtxA, a gene that is thought to have a minor role in pore formation, was not involved in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. We conclude that the icmT gene is essential for pore formationmediated lysis of mammalian and protozoan cells and the subsequent bacterial egress.

Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila , plaB , Exhibiting Hemolytic Activity

“…Other bacterial factors involved in the destruction of eukaryotic cells, such as the RtxA toxin (12) or the icm/dot gene products (1,36,40), have already been characterized. However, especially in the case of the icm/dot genes, which code for a type IV protein secretion system (52,58), it is possible that these determinants do not represent the structural genes actually accounting for the cell-lysing agent.…”

“…Lipolytic activities of an L. pneumophila plaB mutant. L. pneumophila has been shown to secrete several lipolytic activities into the culture supernatant as well as to possess lipolytic activities associated with the bacterial cell (3,4,22,23,25,40). To assess the plaB mutant with respect to lipid hydrolysis, we tested both culture supernatants and bacterial-cell lysates for their abilities to release FFA from DPPG, DPPC, MPLPG, MPLPC, and 1-MPG.…”

“…Pore formation is furthermore an indispensable step in bacterial egress after bacterial intracellular multiplication in both amoebae and macrophages (1, 40). Pore formation in L. pneumophila depends on the integrity of several genes of the icm/dot locus (e.g., dotA, icmQ, icmR, and icmT), responsible for the expression of a type IV protein secretion machinery involved in pathogenesis (14,36,40).…”

“…Pore formation is furthermore an indispensable step in bacterial egress after bacterial intracellular multiplication in both amoebae and macrophages (1, 40). Pore formation in L. pneumophila depends on the integrity of several genes of the icm/dot locus (e.g., dotA, icmQ, icmR, and icmT), responsible for the expression of a type IV protein secretion machinery involved in pathogenesis (14,36,40).In addition to proteases and pore-forming toxins, bacterial lipolytic enzymes are known to confer hemolytic and cytotoxic activities by the destruction of cell membrane lipids and/or generation of membrane-perturbing reaction products, as shown for Clostridium perfringens alpha-toxin, a phospholipase C, or Campylobacter coli PldA, a phospholipase A (27,54,59). L. pneumophila also possesses a variety of lipolytic activities, including lipase, phospholipase A, and lysophospholipase A activities, secreted into the culture medium as well as associated with the bacterium (3,4,22,23,25).…”

Isolation of Legionella pneumophila from surface and ground waters in Osogbo, Nigeria

“…At least 13 species of amoebae and 2 species of ciliated protozoa support the intracellular growth of L. pneumophila (Fields, 1996). The dot/icm loci have been shown to play important roles in the pathogenicity of Legionellae (Berger and Isberg, 1993;Marra et al, 1992;Molmeret et al, 2002a;2002b). In fact, the loci have been demonstrated to help in the intracellular multiplication in both protozoan and macrophages (Brand et al, 1994;Segal and Shuman, 1999).…”