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Infection and Immunity, April 2001, p. 2569-2579, Vol. 69, No. 4
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.4.2569-2579.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

HtrA Homologue of Legionella pneumophila: an Indispensable Element for Intracellular Infection of Mammalian but Not Protozoan Cells

Lisa L. Pedersen,1 Marina Radulic,2 Miljenko Doric,2 and Yousef Abu Kwaik1,*

Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington, Kentucky 40536-0084,1 and Department of Microbiology and Parasitology, University of Rijeka, Rijeka, Croatia2

Received 29 August 2000/Returned for modification 15 December 2000/Accepted 10 January 2001

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 (H103right-arrow R and S212right-arrow A) 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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington, KY 40536-0084. Phone: (859) 323-3873. Fax: (859) 257-8994. E-mail: yabukw{at}pop.uky.edu.

Infection and Immunity, April 2001, p. 2569-2579, Vol. 69, No. 4
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.4.2569-2579.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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