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 environ ment. Here we characterize an L. pneumophila mutant defective in the HtrA/D egP stress-induced protease/chaperone homologue and show that HtrA is indis pensable for intracellular replication within mammalian macrophages and alv eolar epithelial cells and for intrapulmonary replication in A/J mice. Impo rtantly, amino acid substitutions of two conserved residues in the catalyti c domain of (H-103-->R and S-212-->A) and in-frame deletions of either or b oth of the two conserved PDZ domains of HtrA abolish its function. Interest ingly, the htrA mutant exhibits a parental-type phenotype in intracellular replication within the protozoan host Acanthamoeba polyphaga. We used a pro moterless lacZ fusion to the htrA promoter to probe the phagosomal microenv ironment harboring L. pneumophila within macrophages and within A. polyphag a 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 intr acellular infection of macrophages but its induction is by 120- to 500 fold within protozoa compared to in vitro expression. Data derived from confoca l laser scanning microscopy reveal that in contrast to the parental strain, phagosomes harboring the hh A mutant within U937 macrophages colocalize wi th the late endosomal-lysosomal marker LAMP-2, similar to killed L. pneumop hila. Coinfection experiments examined by confocal laser scanning microscop y show that in communal phagosomes harboring both the parental strain and t he htrA mutant, replication of the mutant is not rescued, while replication of a dotA mutant control, which is normally trafficked into a phagolysosom e, is rescued by the parental strain. Our data show, for the first time, th at the stress response by L. pneumophila (mediated, at least in part, by Ht rA) is indispensable for intracellular replication within mammalian but not protozoan cells.