Comparative Analysis of Legionella pneumophila and Legionella micdadei Virulence Traits

While the majority of Legionnaire's disease has been attributed to Legionella pneumophila, Legionella micdadei can cause a similar infection in immunocompromised people. Consistent with its epidemiological profile, the growth of L. micdadei in cultured macrophages is less robust than that of L. pneumophila. To identify those features of the Legionella spp. which are correlated to efficient growth in macrophages, two approaches were taken. First, a phenotypic analysis compared four clinical isolates of L. micdadei to one well-characterized strain of L. pneumophila. Seven traits previously correlated with the virulence of L. pneumophila were evaluated: infection and replication in cultured macrophages, evasion of phagosome-lysosome fusion, contact-dependent cytotoxicity, sodium sensitivity, osmotic resistance, and conjugal DNA transfer. By nearly every measure, L. micdadei appeared less virulent than L. pneumophila. The surprising exception was L. micdadei 31B, which evaded lysosomes and replicated in macrophages as efficiently as L. pneumophila, despite lacking both contact-dependent cytopathicity and regulated sodium sensitivity. Second, in an attempt to identify virulence factors genetically, an L. pneumophila genomic library was screened for clones which conferred robust intracellular growth on L. micdadei. No such loci were isolated, consistent with the multiple phenotypic differences observed for the two species. Apparently, L. pneumophila and L. micdadei use distinct strategies to colonize alveolar macrophages, causing Legionnaire's disease.     

Rapid and complete fusion of macrophage lysosomes with phagosomes containing Salmonella typhimurium.

The virulence of Salmonella typhimurium for mice results, in part, from its ability to survive after phagocytosis by macrophages. Although it is generally agreed that intracellular bacteria persist in membrane-bound phagosomes, there remains some question as to whether these phagosomes fuse with macrophage lysosomes. This report describes the maturation of phagosomes containing S. typhimurium inside mouse bone marrow-derived macrophages. Macrophages were infected briefly and incubated for various intervals; then they were examined by fluorescence microscopy for colocalization of bacteria with lysosomal markers. These markers included LAMP-1, cathepsin L, and fluorescent proteins or dextrans preloaded into lysosomes by endocytosis. By all measures, phagosomes containing S. typhimurium merged completely with the lysosomal compartment within 20 min of phagocytosis. The rate of phagosome-lysosome fusion was similar to the rate for phagocytosed latex beads. Phagolysosomes remained accessible to fluid-phase probes and contained lysosomal markers for many hours. Moreover, a large percentage of the wild-type bacteria that were viable 20 min after infection survived longer incubations inside macrophages, indicating that the survivors were not a minor subpopulation that avoided phagosome-lysosome fusion. Therefore, we conclude that S. typhimurium survives within the lysosomal compartments of macrophages.     

Inhibition of macrophage phagosome-lysosome fusion by Salmonella typhimurium.

Salmonella typhimurium-infected macrophages were examined by electron microscopy to determine whether intracellular survival of S. typhimurium is associated with failure of bacteria containing phagosomes to fuse with secondary lysosomes. S. typhimurium 14028 actively inhibited phagosome-lysosome fusion and appeared to preferentially divide within unfused phagocytic vesicles. In comparison with Escherichia coli, S. typhimurium inhibited phagosome-lysosome fusion in peritoneal macrophages, J774 macrophages, and bone marrow-derived macrophages from both BALB/c (itys) and SWR/J (ityr) mice. The mechanism responsible for Salmonella inhibition of phagosome-lysosome fusion is unknown but requires viable salmonellae, is not blocked by opsonization with fresh normal mouse serum, and is not due to lipopolysaccharide. Inhibition of phagosome-lysosome fusion may play a critical role in survival of salmonellae within macrophages and in virulence.     

Lack of lysosomal fusion with phagosomes containing Ehrlichia risticii in P388D1 cells: abrogation of inhibition with oxytetracycline.

Fusion of lysosomes with phagosomes containing Ehrlichia risticii, an obligate intracellular parasite, was evaluated in P388D1 murine macrophagelike cells. Lysosomes in cells ranging in infectivity from 30 to 70% were labeled cytochemically with acid phosphatase or via endocytosis of thorium dioxide or cationized ferritin to document phagosome-lysosome (P-L) fusion in untreated cells and cells treated with oxytetracycline. Regardless of the marker used, P-L fusion was generally not observed in E. risticii-containing vacuoles in untreated cells, while significantly greater P-L fusion with ehrlichia-containing vacuoles was observed after oxytetracycline treatment. When latex beads were introduced into uninfected cell cultures, P-L fusion was observed with vacuoles containing latex. Fusion of lysosomes with latex-containing vacuoles in cells was significantly greater than fusion of lysosomes with ehrlichia-containing vacuoles in the same infected cells. These findings indicate that E. risticii is able to inhibit P-L fusion, whereas oxytetracycline deprives organisms of this ability.     

Intracellular pathogens and antigen presentation-new challenges with Legionella pneumophila

In this issue of Immunity, examine the intracellular life of Legionella pneumophila in dendritic cells (DC) and macrophages, as well as the presentation of its antigens to CD4 T cells. Legionella is a particularly interesting bacterium because of the peculiarities inherent in its intracellular sojourn in phagocytes: it resides in an unusual vesicle characterized by ribosomes studded along its walls. In this compartment, Legionella proteins encoded by the dot gene inhibit phagosome-lysosome fusion and endosomal acidification, yielding a vesicular structure conducive to the multiplication of Legionella, poor in lysosomal contents, and in MHC molecules.     

Potential Virulence Role of the Legionella pneumophila ptsP Ortholog

We previously identified the Legionella pneumophila ptsP (phosphoenolpyruvate phosphotransferase) ortholog gene as a putative virulence factor in a study of signature-tagged mutagenesis using a guinea pig pneumonia model. In this study, we further defined the phenotypic properties of L. pneumophila ptsP and its complete sequence. The L. pneumophila ptsP was 2,295 bases in length. Its deduced amino acid sequence had high similarity with ptsP orthologs of Pseudomonas aeruginosa, Azotobacter vinelandii, and Escherichia coli, with nearly identical lengths. Here we show that while the mutant grew well in laboratory media, it was defective in both lung and spleen multiplication in guinea pigs. It grew slowly in guinea pig alveolar macrophages despite good uptake into the cells. Furthermore, there was minimal growth in a human alveolar epithelial cell line (A549). Transcomplementation of the L. pneumophila ptsP mutant almost completely rescued its growth in alveolar macrophages, in A549 cells, and in guinea pig lung and spleen. The L. pneumophila ptsP mutant was capable of evasion of phagosome-lysosome fusion and resided in ribosome-studded phagosomes. Pore formation activity of the mutant was normal. The L. pneumophila ptsP mutant expressed DotA and IcmX in apparently normal amounts, suggesting that the ptsP mutation did not affect dotA and icmX regulation. In addition, the mutant was resistant to serum and neutrophil killing. Taken together, these findings show that L. pneumophila ptsP is required for full in vivo virulence of L. pneumophila, most probably by affecting intracellular growth.     

Identification and subcellular localization of the Legionella pneumophila IcmX protein: a factor essential for establishment of a replicative organelle in eukaryotic host cells

The gram-negative respiratory pathogen Legionella pneumophila infects and grows within mammalian macrophages and protozoan host cells. Upon uptake into macrophages, L. pneumophila establishes a replicative organelle that avoids fusion with endocytic vesicles. There are 24 dot/icm genes on the L. pneumophila chromosome required for biogenesis of this vacuole. Many of the Dot/Icm proteins are predicted to be components of a membrane-bound secretion apparatus similar to type IV conjugal transfer systems. We have been investigating the function of L. pneumophila dot/icm gene products that do not have obvious orthologs in other type IV transfer systems, since these determinants could govern processes unique to phagosome biogenesis. The icmX gene product falls into this category. To understand the role of the IcmX protein in pathogenesis, we have detailed interactions between an L. pneumophila icmX deletion mutant and murine bone marrow-derived macrophages. These data demonstrate that icmX is required for biogenesis of the L. pneumophila replicative organelle. Immunoblot analysis indicates that the icmX gene product is a polypeptide with an estimated molecular mass of 50 kDa. The IcmX protein was localized to the bacterial periplasm, and periplasmic translocation was mediated by an N-terminal sec-dependent leader peptide. A truncated IcmX product was secreted into culture supernatants by wild-type L. pneumophila growing extracellularly in liquid media; however, transport of the IcmX protein into eukaryotic host cells was not detected. Proteins similar in molecular weight to IcmX were identified in other Legionella species by immunoblot analysis using a monoclonal antibody specific for L. pneumophila IcmX protein. From these data, we conclude that the IcmX protein is an essential component of the dot/icm secretion apparatus, and that a conserved mechanism of host cell parasitism exists for members of the Legionellaceae family.     

Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization

Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L.?pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L.?pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L.?pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.     

Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion.

Legionella pneumophila is a facultative intracellular parasite able to survive within both human monocytes and amoebae. We have demonstrated that processing of L. pneumophila by the free-living amoeba Acanthamoeba castellanii shows many similarities to the processing of L. pneumophila by monocytes. These similarities include uptake of L. pneumophila by coiling phagocytosis and the subsequent confinement of L. pneumophila in a ribosome-studded phagosome. In addition, as in monocytes, inhibition of lysosomal fusion with phagosomes containing L. pneumophila was detected in amoebae. With all clinical isolates, inhibition of phagosomes-lysosome fusion correlated with virulence. However, with one of the environmental isolates tested, no significant difference in phagosome-lysosome fusion was observed between the virulent and avirulent forms. These results indicate that the avirulent form of this isolate differed from the virulent form in some other respect critical to intracellular survival. Therefore, intracellular multiplication of L. pneumophila within A. castellanii may not be solely dependent upon the inhibition of lysosomal fusion.     

Complement (C3)-Receptor-Mediated Phagocytosis of Agarose Beads by Mouse Macrophages

The phagocytosis by macrophages of C3bi-coated agarose heads reached a plateau after 15 min, compared with 30 min for C3b-coated beads. By using ¹²⁵I-Iabelled C3bi or C3b coupled to the agarose beads, we found that 70% and 95% of total radioactivity were removed from the heads after 12 h and 36 h of intracellular digestion, respectively. Intracellular degradation of C3bi linked to agarose beads was also demonstrated by testing binding of monoclonal antibodies against human C3c, C3g and C3d to beads extracted from the cells after phagocytosis. Such extracted beads also showed reduced attachment to new macrophages compared with non-ingested beads. Treatment of the cells with leupeptin, an inhibitor of the lysosomal enzyme cathepsin B, or with dextran sulphate to inhibit phagosome-lysosome fusion greatly reduced the release of labelled protein from the agarose during the first 12 h. These findings show that C3bi and C3b on agarose is destroyed intracellularly by lysosomal enzymes.     

Inhibition of phagosome-lysosome fusion in ovine polymorphonuclear leucocytes by Ehrlichia (Cytoecetes) phagocytophila

Ehrlichia (Cytoecetes) phagocytophila, the causative agent of tick-borne fever, is an intracellular bacterium that survives and multiplies within granulocytes and monocytes. In the present study, the possible fusion of lysosomes with phagosomes containing E. phagocytophila was investigated in poly-morphonuclear (PMN) cells of sheep infected with the agent, acid phosphatase cytochemistry and cationized ferritin being used as markers of primary and secondary lysosomal enzymes. Latex beads or Candida albicans were incubated with infected and uninfected PMN cells and labelled with the same lysosomal markers. Lysosomal enzymes labelled with the markers were commonly found in phagosomes containing latex beads or C. albicans, but there was no evidence of phagosome-lysosome (P-L) fusion in phagosomes containing E. phagocytophila. It was significant that in cells that contained E. phagocytophila, latex beads and C. albicans, P-L fusion occurred only in phagosomes containing latex beads or C. albicans. However, evidence of P-L fusion with phagosomes containing E. phagocytophila was obtained when PMN cells were incubated with oxytetracycline, which is known to inhibit synthesis of bacterial proteins. These findings indicate that E. phagocytophila is capable of inhibiting P-L fusion and that oxytetracycline depresses this capability.     

Disruption of the phagosomal membrane and egress of Legionella pneumophila into the cytoplasm during the last stages of intracellular infection of macrophages and Acanthamoeba polyphaga

Although the early stages of intracellular infection by Legionella pneumophila are well established at the ultrastructural level, a detailed ultrastructural analysis of late stages of intracellular replication has never been done. Here we show that the membrane of the L. pneumophila-containing phagosome (LCP) is intact for up to 8 h postinfection of macrophages and Acanthamoeba polyphaga. At 12 h, 71 and 74% of the LCPs are disrupted within macrophages and A. polyphaga, respectively, while the plasma membrane remains intact. At 18 and 24 h postinfection, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and amorphous material are dispersed among the bacteria and these bacteria are considered cytoplasmic. At 18 h, 77% of infected macrophages and 32% of infected A. polyphaga amoebae harbor cytoplasmic bacteria. At 24 h, 99 and 78% of infected macrophages and amoebae, respectively, contain cytoplasmic bacteria. On the basis of lysosomal acid phosphatase staining of infected macrophages and A. polyphaga, the lysosomal enzyme is present among the bacteria when host vesicles are dispersed among bacteria. Our data indicate that bacterial replication proceeds despite physical disruption of the phagosomal membrane. We also show that an lspG mutant that is defective in the type II secretion system and therefore does not secrete the hydrolytic enzymes metalloprotease, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A is as efficient as the wild-type strain in disruption of the LCP. Therefore, L. pneumophila disrupts the phagosomal membrane and becomes cytoplasmic at the last stages of infection in both macrophages and A. polyphaga. Lysosomal elements, mitochondria, cytoplasmic vesicles, and amorphous material are all dispersed among the bacteria, after phagosomal disruption, within both human macrophages and A. polyphaga. The disruption of the LCP is independent of the hydrolytic enzymes exported by the type II secretion system.     

Polyanionic agents do not inhibit phagosome-lysosome fusion in cultured macrophages

The survival of some intracellular pathogens within macrophages may be aided by an ability of the organism to antagonize, from within the entrapping phagosome, its fusion with lysosomes. On the other hand, certain polyanionic agents have been implicated in imposing a similar block to fusion from the lysosomal domain--because the transfer of various foreign markers from lysosomes to newly formed phagosomes is remarkably inhibited in these polyanion-containing cells. Based on an analysis of various observations and our own recent data, we propose that the polyanionics do not, in fact, prevent phagosome-lysosome fusion but, instead, physically entrap the usual markers in a gelatinous matrix within the lysosomes. This view accounts for many paradoxical consequences of polyanionic accumulation and for the curiously normal behavior of macrophages that are presumed to be suffering from such a crucial intracellular dysfunction.     

Components of the Legionella pneumophila flagellar regulon contribute to multiple virulence traits, including lysosome avoidance and macrophage death

Legionella pneumophila is a motile intracellular pathogen of macrophages and amoebae. When nutrients become scarce, the bacterium induces expression of transmission traits, some of which are dependent on the flagellar sigma factor FliA (sigma(28)). To test how particular components of the L. pneumophila flagellar regulon contribute to virulence, we compared a fliA mutant with strains whose flagellar construction is disrupted at various stages. We find that L. pneumophila requires FliA to avoid lysosomal degradation in murine bone marrow-derived macrophages (BMM), to regulate production of a melanin-like pigment, and to regulate binding to the dye crystal violet, whereas motility, flagellar secretion, and external flagella or flagellin are dispensable for these activities. Thus, in addition to flagellar genes, the FliA sigma factor regulates an effector(s) or regulator(s) that contributes to other transmissive traits, notably inhibition of phagosome maturation. Whether or not the microbes produced flagellin, all nonmotile L. pneumophila mutants bound BMM less efficiently than the wild type, resulting in poor infectivity and a loss of contact-dependent death of BMM. Therefore, bacterial motility increases contact with host cells during infection, but flagellin is not an adhesin. When BMM contact by each nonmotile strain was promoted by centrifugation, all the mutants bound BMM similarly, but only those microbes that synthesized flagellin induced BMM death. Thus, the flagellar regulon equips the aquatic pathogen L. pneumophila to coordinate motility with multiple traits vital to virulence.     

Legionella dumoffii DjlA, a member of the DnaJ family, is required for intracellular growth

Legionella dumoffii is one of the common causes of Legionnaires' disease and is capable of replicating in macrophages. To understand the mechanism of survival within macrophages, transposon mutagenesis was employed to isolate the genes necessary for intracellular growth. We identified four defective mutants after screening 790 transposon insertion mutants. Two transposon insertions were in genes homologous to icmB or dotC, within dot/icm loci, required for intracellular multiplication of L. pneumophila. The third was in a gene whose product is homologous to the 17-kDa antigen forming part of the VirB/VirD4 type IV secretion system of Bartonella henselae. The fourth was in the djlA (for "dnaj-like A") gene. DjlA is a member of the DnaJ/Hsp40 family. Transcomplementation of the djlA mutant restored the parental phenotype in J774 macrophages, A549 human alveolar epithelial cells, and the amoeba Acanthamoeba culbertsoni. Using confocal laser-scanning microscopy and transmission electron microscopy, we revealed that in contrast to the wild-type strain, L. dumoffii djlA mutant-containing phagosomes were unable to inhibit phagosome-lysosome fusion. Transmission electron microscopy also showed that in contrast to the virulent parental strain, the djlA mutant was not able to recruit host cell rough endoplasmic reticulum. Furthermore, the stationary-phase L. dumoffii djlA mutants were more susceptible to H2O2, high osmolarity, high temperature, and low pH than was their parental strain. These results indicate that DjlA is required for intracellular growth and organelle trafficking, as well as bacterial resistance to environmental stress. This is the first report demonstrating that a single DjlA-deficient mutant exhibits a distinct phenotype.     

Interaction of Nocardia asteroides with Rabbit Alveolar Macrophages: Association of Virulence, Viability, Ultrastructural Damage, and Phagosome-Lysosome Fusion

In vitro-maintained rabbit alveolar macrophages were infected with three strains of Nocardia asteroides. It was found that N. asteroides GUH-2 was resistant to macrophage killing, while N. asteroides 14759 was intermediate in resistance, and N. asteroides 10905 had little resistance to killing by macrophages. These observations correlated well with the data on relative virulence previously determined in mice. To establish the intracellular events leading to these differences, we determined the occurrence of phagosome-lysosome fusion in infected macrophages by both electron and fluorescent microscopic methods. It was found that the virulent strain GUH-2 inhibited phagosome-lysosome fusion; the intermediately virulent strain, 14759, partially inhibited fusion; and the less-virulent strain, 10905, was unable to inhibit fusion. In addition, electron microscopy of infected macrophages demonstrated that cells of the virulent strain, GUH-2, were not damaged, and only some of the cells of the intermediately virulent strain, 14759, were damaged, while most of the cells of the less virulent strain, 10905, exhibited considerable cellular destruction. These data indicated a direct correlation between the virulence of these organisms and their resistance to killing by alveolar macrophages, their lack of macrophage-induced ultrastructural damage, and their ability to inhibit phagosome-lysosome fusion. Thus, it appears that inhibition of phagosome-lysosome fusion in alveolar macrophages may be one of the mechanisms of pathogenicity of virulent strains of N. asteroides.     

Phagocytosis of Wild-Type Legionella pneumophila Occurs through a Wortmannin-Insensitive Pathway

Wild-type Legionella pneumophila grows in human macrophages within a replicative phagosome, avoiding lysosomal fusion, while nonreplicative mutants are killed in lysosomes. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocks phagocytosis of an avirulent mutant, but not of wild-type L. pneumophila, without affecting membrane ruffling and actin polymerization. These results show that wild-type and mutant Legionella strains use different entry pathways. They suggest that PI3Ks are involved in phagocytosis of an avirulent L. pneumophila mutant and regulate the ability of microorganisms to generate a replicative phagosome.     

Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate

The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosis phagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development of L. pneumophila and M. tuberculosis phagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-type L. pneumophila never acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis phagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads, M. tuberculosis, and avirulent L. pneumophila but not wild-type L. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-type L. pneumophila phagosomes, functional Rab5 persists on M. tuberculosis phagosomes. The absence of Rab5 on the L. pneumophila phagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis phagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.     

Infection of macrophages with Legionella pneumophila induces phosphorylation of a 76-kilodalton protein.

Infection of peritoneal macrophages from susceptible A/J mice with Legionella pneumophila induced phosphorylation of a 76-kDa protein. The phosphorylation occurred when macrophages were infected with a virulent strain of L. pneumophila but did not occur when they were infected with an avirulent strain or with other bacteria such as either Pseudomonas aeruginosa or Salmonella typhimurium. Also, no phosphorylation of this protein was observed when macrophages were stimulated with either lipopolysaccharide or phorbol myristate acetate. However, phosphorylation did occur in macrophages infected with a virulent strain of L. pneumophila and treated with either erythromycin to inhibit growth or with cytochalasin D to inhibit uptake of L. pneumophila by macrophages. These results support the view that phosphorylation of this protein occurs during the early phases of interaction between L. pneumophila and macrophages. The role of this specific protein in the recognition, intracellular uptake, and growth of L. pneumophila in permissive macrophages remains to be clarified.     

The Legionella pneumophila effector SidJ is required for efficient recruitment of endoplasmic reticulum proteins to the bacterial phagosome

The virulence of Legionella pneumophila is dependent on the Dot/Icm type IV protein secretion system, which translocates effectors into infected cells. A large number of such translocated proteins have been identified, but few of these proteins are necessary for intracellular replication of the pathogen, making it difficult to correlate these genes with specific cell-biological events associated with L. pneumophila infection. We report here the identification and characterization of a family of two substrates, SidJ and SdjA, with distinctive phenotypes. In contrast to many Dot/Icm substrates, whose expression levels are elevated when bacteria are grown to postexponential phase, SidJ is produced at a constant rate during the entire bacterial growth cycle. Mutation in sidJ causes a significant growth defect in both macrophage and amoeba hosts, but an sdjA mutant is detectably defective only in protozoan hosts. However, in the amoeba host a mutant lacking both sidJ and sdjA does not display a more severe growth defect than the sidJ mutant. Despite its significant intracellular growth defect, the sidJ mutant is still able to effectively evade fusion with lysosomes. Importantly, recruitment of endoplasmic reticulum (ER) proteins by vacuoles containing the sidJ mutant was considerably delayed in both mammalian and amoeba cells. Our results suggest that SidJ modulates host cellular pathways, contributing to the trafficking or retention of ER-derived vesicles to L. pneumophila vacuoles.