HtrA homologue of Legionella pneumophila: an indispensable element for intracellular infection of mammalian but not protozoan cells.

Legionella pneumophila replicates within alveolar macrophages, and possibly, alveolar epithelial cells and also within protozoa in the aquatic environment. Here we characterize an L. pneumophila mutant defective in the HtrA/DegP stress-induced protease/chaperone homologue and show that HtrA is indispensable for intracellular replication within mammalian macrophages and alveolar epithelial cells and for intrapulmonary replication in A/J mice. Importantly, amino acid substitutions of two conserved residues in the catalytic domain of (H103mapstoR and S212mapstoA) and in-frame deletions of either or both of the two conserved PDZ domains of HtrA abolish its function. Interestingly, the htrA mutant exhibits a parental-type phenotype in intracellular replication within the protozoan host Acanthamoeba polyphaga. We used a promoterless lacZ fusion to the htrA promoter to probe the phagosomal microenvironment harboring L. pneumophila within macrophages and within A. polyphaga for the exposure to stress stimuli. The data show that expression through the htrA promoter is induced by 12,000- to 20,000-fold throughout the intracellular infection of macrophages but its induction is by 120- to 500-fold within protozoa compared to in vitro expression. Data derived from confocal laser scanning microscopy reveal that in contrast to the parental strain, phagosomes harboring the htrA mutant within U937 macrophages colocalize with the late endosomal-lysosomal marker LAMP-2, similar to killed L. pneumophila. Coinfection experiments examined by confocal laser scanning microscopy show that in communal phagosomes harboring both the parental strain and the htrA mutant, replication of the mutant is not rescued, while replication of a dotA mutant control, which is normally trafficked into a phagolysosome, is rescued by the parental strain. Our data show, for the first time, that the stress response by L. pneumophila (mediated, at least in part, by HtrA) is indispensable for intracellular replication within mammalian but not protozoan cells.

Temporal pore formation-mediated egress from macrophages and alveolar epithelial cells by Legionella pneumophila.

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.

Abnormal DNA damage-inducible protein in cells from Sjögren's syndrome patients.

Antinuclear antibodies are commonly found in patients with Sjögren's syndrome. It has been suggested that the development of antinuclear antibodies depends on the activation of the spliceosome and other transcription-related subcellular particles, some of which have recently been shown also to function in DNA-modifying processes, such as DNA repair and V(D)J recombination. These observations add weight to a previously proposed model for the aetiology of Sjögren's syndrome. This includes the abnormal processing of the T-cell receptor and immunoglobulin genes. To test this hypothesis further, the present study on DNA-modifying proteins in Sjögren's syndrome was initiated. Gel-shift experiments using protein extracted from UV-treated Sjögren cells provided evidence of high molecular weight DNA-binding protein in six out of 12 Sjögren patients studied (but not among seven healthy controls). Some Sjögren sera displayed antibodies to protein extracts from cells treated with psoralen plus UVA radiation. These results indicate an abnormal DNA damage-inducible response in Sjögren's syndrome. It may therefore be concluded that alterations in nuclear protein may play a role in the aetiology of Sjögren's syndrome.