. In contrast to this single mode of killing of protozoa, we have recently proposed a biphasic model by which L. pneumophila kills macrophages, in which the first phase is manifested through the induction of apoptosis during early stages of the infection, followed by an independent and temporal induction of necrosis during late stages of intracellular replication. Here we show that, similar to the protozoan host, the induction of necrosis and cytolysis of macrophages by L. pneumophila is mediated by the pore-forming toxin or activity. This activity is temporally and maximally expressed only upon termination of bacterial replication and correlates with cytolysis of macrophages and alveolar epithelial cells in vitro. We have identified five L. pneumophila mutants defective in the pore-forming activity. The phagosomes harboring the mutants do not colocalize with the late endosomal or lysosomal marker Lamp-1, and the mutants replicate intracellularly similar to the parental strain. Interestingly, despite their prolific intracellular replication, the mutants are defective in cytotoxicity and are "trapped" within and fail to lyse and egress from macrophages and alveolar epithelial cells upon termination of intracellular replication. However, the mutants are subsequently released from the host cell, most likely due to apoptotic death of the host cell. Data derived from cytotoxicity assays, confocal laser scanning microscopy, and electron microscopy confirm the defect in the mutants to induce necrosis of macrophages and the failure to egress from the host cell. Importantly, the mutants are completely defective in acute lethality (24 to 48 h) to intratracheally inoculated A/J mice. We conclude that the poreforming activity of L. pneumophila is not required for phagosomal trafficking or for intracellular replication. This activity is expressed upon termination of bacterial replication and is essential to induce cytolysis of infected macrophages to allow egress of intracellular bacteria. In addition, this activity plays a major role in pulmonary immunopathology in vivo.

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.

The C-terminus of IcmT is essential for pore formation and for intracellular trafficking of Legionella pneumophila within Acanthamoeba polyphaga respectively, in intracellular growth in A. polyphaga, and the respective defects correlated with fusion of the bacterial phagosomes to lysosomes. Taken together, the data showed that the C-terminus domain of IcmT is essential for the pore-forming activity and is required for intracellular trafficking and replication within A. polyphaga, but not within mammalian cells. IntroductionLegionella pneumophila, the bacterium responsible for the acute pneumonia designated Legionnaire's disease, is able to grow within cells in the alveolar spaces . In the environment, this bacterium replicates within protozoa (Rowbotham, 1980;1986). Intracellular replication is culminated in pore formationmediated cytolysis of the protozoan host and subsequent bacterial egress (Gao and Abu Kwaik, 2000a). At least 15 species of protozoa support the intracellular growth of L. pneumophila, and bacterial replication within amoebae plays a major role in bacterial ecology and pathogenesis .Infection of both mammalian and protozoan cells by L. pneumophila shares similar mechanisms (Gao et al., 1997;. After entry into both host cells, L pneumophila modulates the biogenesis of the vacuole into a replicative niche, which does not fuse to lysosomal compartments and is subsequently surrounded by mitochondria and the rough endoplasmic reticulum Abu Kwaik et al., 1998a). The type IV Dot/Icm secretion system is essential for evasion of lysosomal fusion Vogel et al., 1998). After termination of intracellular replication, L. pneumophila induces expression of the pore-forming toxin, which is essential for cytolysis of the host cell and subsequent bacterial egress (Byrne and Swanson, 1998;Alli et al., 2000). Egress from host cells is a fundamental step in the life cycle of intracellular pathogens that allows the organism to spread to a new susceptible host cell and to search for a new replication niche.Mutants defective in egress from the host cell upon termination of intracellular replication have been isolated in our laboratory and designated rib (release of intracellular bacteria) (Alli et al., 2000;Gao and Abu Kwaik, 2000a SummaryWe have shown previously that the five rib (release of intracellular bacteria) mutants of Legionella pneumophila are competent for intracellular replication but defective in pore formation-mediated cytolysis and egress from protozoan and mammalian cells. The rib phenotype results from a point mutation (deletion) DG 544 in icmT that is predicted to result in the expression of a protein truncated by 32 amino acids from the C-terminus. In contrast to the rib mutants that are capable of intracellular replication, an icmT null mutant was completely defective in intracellular replication within mammalian and protozoan cells, in addition to its defect in pore formation-mediated cytolysis. The icmT wild-type allele complemented the icmT null mutant for both defects of intracellular replicati...

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This study compares the performance of clinical diagnosis and three laboratory diagnostic methods (thick film microscopy (TFM), rapid diagnostic test (RDT), and polymerase chain reaction (PCR)) for the diagnosis of Plasmodium falciparum in Nigeria. Using clinical criteria, 217 children were recruited into the study out of which 106 (48.8%) were positive by TFM, 84 (38.7%) by RDT, and 125 (57.6%) by PCR. Using a composite reference method generated from the three diagnostic methods, 71 (32.7%) patients were found to be truly infected and 90 (41.5%) truly uninfected, while 56 (25.8%) were misidentified as infected or noninfected. When each of the 3 diagnostic methods was compared with the composite reference, PCR had sensitivity of 97.3%, specificity of 62.5%, positive predictive value (PPV) of 56.8%, and negative predictive value (NPV) of 97.8%; microscopy had sensitivity of 77.2%, specificity of 72%, PPV of 66.9%, and NPV of 81.1%, while RDT had sensitivity of 62.3%, specificity of 87.4%, PPV of 67.7%, and NPV of 84.5%. PCR test performed best among the three methods followed by TFM and RDT in that order. The result of this study shows that clinical diagnosis cannot be relied upon for accurate diagnosis of P. falciparum in endemic areas.