Lian-Yong Gao

Temporal Pore Formation-Mediated Egress from Macrophages and Alveolar Epithelial Cells by Legionella pneumophila

ABSTRACT Legionella pneumophila does not induce apoptosis in the protozoan host, but induces pore formation-mediated cytolysis after termination of intracellular replication (L.-Y. Gao and Y. Abu Kwaik, Environ. Microbiol. 2:79–90, 2000). 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 pore-forming 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.

Hijacking of apoptotic pathwaysby bacterial pathogens.

Increasing evidence indicates that apoptosis of the host cell may constitute a defense mechanism to confine the infection by bacterial pathogens. Certain pathogens have developed elegant mechanisms to modulate the fate of the host cell, which include induction or blockage of apoptosis. These studies will promote our understanding of the pathogenesis of infectious diseases and aid the development of means for therapeutic intervention.

Transcriptional regulation of the macrophage-induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant

Expression of the global stress protein gene (gspA) is induced during the intracellular infection of macrophages and upon exposure of Legionella pneumophila to in vitro stress stimuli. Transcription of gspA is regulated by two promoters, one of which is regulated by the σ32 heat‐shock transcription factor. We utilized a gspA promoter fusion to a promoterless lacZ to probe the phagososmal ‘microenvironment’ for the kinetics of exposure of intracellular L. pneumophila to stress stimuli. Expression through the gspA promoter was constitutively induced by approx. 16‐fold throughout the intracellular infection, and occurred predominantly through the σ32‐regulated promoter. Expression of the gspA promoter was induced approx. 4.5‐fold, 5‐, 11‐ and 9‐fold upon exposure of L. pneumophila to heat shock, oxidative stress, acid shock, and osmotic shock, respectively. An isogenic insertion mutant of L. pneumophila in gspA (strain AA224) was constructed by allelic exchange in the wild‐type strain AA200. Compared to in vitro‐grown wild‐type strain AA200, AA224 was more susceptible to all four in vitro stress stimuli. The wild‐type phenotypes were restored to strain AA224 by complementation with a plasmid containing wild‐type gspA. There was no difference between the wild‐type strain and the gspA mutant in cytopathogenicity to U937 cells or in their kinetics of intracellular replication within macrophages and amoebae. However, compared to in vitro‐grown bacteria, macrophage‐grown and amoebae‐grown AA200 and AA224 showed an equal and dramatic increase in resistance to in vitro stress stimuli. Our data showed that regardless of the capacity of L. pneumophila to subvert the microbicidal mechanisms of the macrophage, intracellular L. pneumophila is exposed to a high level of stress stimuli throughout the intracellular infection. Although the GspA protein is required for protection of the bacteria against in vitro stress stimuli, and is induced during intracellular multiplication, the loss of its function is probably compensated for by other macrophage‐induced and stress‐induced proteins within the intracellular environment.

Invasion of mammalian and protozoan cells by Legionella pneumophila

The ability of Legionella pneumophila to cause disease is dependent on its intracellular replication within alveolar cells. In the environment, the bacterium is a parasite of protozoa, which are central to the ecology and pathogenesis of L. pneumophila . This review examines invasion of mammalian and protozoan cells by this bacterium.