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

Abstract

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 (H 103 ¦R and S 212 ¦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.

Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection

“…Expression of htrA is upregulated in S. Typhimurium with macrophages (Eriksson et al, 2003;Everest et al, 1995) and other pathogens that reside within a phagosome such as Yersinia enterocolitica (Yamamoto et al, 1997) and Legionella pneumophila (Pedersen et al, 2001). However, there is almost no similarity between the htrA promoter regions of these bacteria and we were unable to locate counterparts of the S. Typhimurium htrA promoters in these promoter regions.…”

“…This is not the case in Salmonella, but S. Typhimurium htrA mutants exhibit increased sensitivity to killing by reactive oxygen species and reduced survival and/or replication within macrophages (Baumler et al, 1994;Humphreys et al, 1999;Johnson et al, 1991), and are highly attenuated in the murine model of systemic Salmonella infection (Chatfield et al, 1992;Humphreys et al, 1999). Since the seminal studies in Salmonella, HtrA has been found to play a role in the virulence of a wide range of Gram-negative and Grampositive bacterial pathogens (Bringer et al, 2005;Cortes et al, 2002;Humphreys et al, 1999;Ibrahim et al, 2004;Johnson et al, 1991;Jones et al, 2001;Pallen & Wren, 1997;Pedersen et al, 2001;Rigoulay et al, 2005;Williams et al, 2000).…”

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors

“…Furthermore, another thiol-disulfide oxidoreductase (ResA) involved in cytochrome C synthesis (85-88), several predicted metabolic or substratebinding proteins, such as amino acid-binding protein Lpg0498, polysaccharide deacetylase Lpg0633, amine oxidase Lpg1153, hydroxymethylglutaryl CoA-reductase Lpg2052, G3P ABC transporter substrate-binding protein UgpB, were detected as omap E-on. Among the remaining 16 E-or PE-up proteins, five belonged to the functional categories "protein fate" (E-up: DsbA, HtrA, LolA, PE-up: metallopeptidase PepO, carboxyl terminal protease Lpg0499) (79,83,89,90), two to "pathogenesis" (E-up: Lpg1585, PE-up: EnhA/ Lpg2641) (91), two to "transport and binding" (PE-up: arginine ABC transporter substrate-binding protein ArtJ (92), E-up: sugar ABC transporter substrate-binding protein Lpg0184), and one to "cellular processes" (PE-up: carbonic anhydrase Lpg2500) (Fig. 4, supplemental Fig.…”

Identification of host factors required for Yersinia pestis macrophage intracellular survival and their impact on vacuole maturation, acidification and trafficking.

“…Bacteria were added to macrophages and the infection was synchronized by centrifugation. At 20 minutes and 80 minutes post-infection cell monolayers were washed three times with PBS and fixed as described below [225,226]. Statistics.…”

“…E. coli was cultivated at 37°C in Luria-Bertani (LB) broth (Difco) supplemented with 50µg/mL carbenicillin. L. pneumophila AA100, a clinical isolate containing pMIP-GFP, was grown on BCYE agar plates for 3 days at 37°C prior to macrophage infection [224][225][226]. The pGEN222::mCherry plasmid was generated by replacing the EGFP gene from pGEN222 with the mCherry gene using…”