Legionella pneumophila DotU and IcmF are required for stability of the Dot/Icm complex

Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a DeltadotU DeltaicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the DeltadotU DeltaicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.     

Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System

Legionella pneumophila uses the Icm/Dot type 4B secretion system (T4BSS) to deliver translocated protein substrates to the host cell, promoting replication vacuole formation. The conformational state of the translocated substrates within the bacterial cell is unknown, so we sought to determine if folded substrates could be translocated via this system. Fusions of L. pneumophila Icm/Dot-translocated substrates (IDTS) to dihydrofolate reductase (DHFR) or ubiquitin (Ub), small proteins known to fold rapidly, resulted in proteins with low translocation efficiencies. The folded moieties did not cause increased aggregation of the IDTS and did not impede interaction with the adaptor protein complex IcmS/IcmW, which is thought to form a soluble complex that promotes translocation. The translocation defect was alleviated with a Ub moiety harboring mutations known to destabilize its structure, indicating that unfolded proteins are preferred substrates. Real-time analysis of translocation, following movement during the first 30 min after bacterial contact with host cells, revealed that the folded moiety caused a kinetic defect in IDTS translocation. Expression of an IDTS fused to a folded moiety interfered with the translocation of other IDTS, consistent with it causing a blockage of the translocation channel. Furthermore, the folded protein fusions also interfered with intracellular growth, consistent with inefficient or impaired translocation of proteins critical for L. pneumophila intracellular growth. These studies indicate that substrates of the Icm/Dot T4SS are translocated to the host cytosol in an unfolded conformation and that folded proteins are stalled within the translocation channel, impairing the function of the secretion system.     

Legionella pneumophila entry gene rtxA is involved in virulence

Successful parasitism of host cells by intracellular pathogens involves adherence, entry, survival, intracellular replication, and cell-to-cell spread. Our laboratory has been examining the role of early events, adherence and entry, in the pathogenesis of the facultative intracellular pathogen Legionella pneumophila. Currently, the mechanisms used by L. pneumophila to gain access to the intracellular environment are not well understood. We have recently isolated three loci, designated enh1, enh2, and enh3, that are involved in the ability of L. pneumophila to enter host cells. One of the genes present in the enh1 locus, rtxA, is homologous to repeats in structural toxin genes (RTX) found in many bacterial pathogens. RTX proteins from other bacterial species are commonly cytotoxic, and some of them have been shown to bind to beta(2) integrin receptors. In the current study, we demonstrate that the L. pneumophila rtxA gene is involved in adherence, cytotoxicity, and pore formation in addition to its role in entry. Furthermore, an rtxA mutant does not replicate as well as wild-type L. pneumophila in monocytes and is less virulent in mice. Thus, we conclude that the entry gene rtxA is an important virulence determinant in L. pneumophila and is likely to be critical for the production of Legionnaires' disease in humans.     

The Dot/Icm Effector SdhA Is Necessary for Virulence of Legionella pneumophila in Galleria mellonella and A/J Mice

Legionella pneumophila is an intracellular bacterium that resides within amoebae and macrophages in a specialized compartment termed the Legionella-containing vacuole (LCV). As well as providing an intracellular niche for replication, the LCV helps to prevent the release of bacterial components into the cytoplasm. Recognition of these components as danger signals by the host activates immune responses leading to clearance of the bacterium. Here, we examined the role of two important virulence factors of L. pneumophila, the potent danger signal flagellin and the translocated Dot/Icm type IVB secretion system effector SdhA, which is crucial to maintain LCV integrity, in the Galleria mellonella infection model. We demonstrate that flagellin expression does not contribute to virulence, replication, or induction of clearance mechanisms. Conversely, SdhA expression is important for virulence. We found that in the absence of SdhA, the LCV in hemocytes showed signs of instability and leakage. Furthermore, in contrast to wild-type L. pneumophila, a ΔsdhA mutant caused a transient depletion of hemocytes and reduced mortality. Analysis of the ΔsdhA mutant in the A/J mouse model also showed a significant replication defect. Together, our data underline the crucial importance of SdhA in infection across different model organisms.     

Dot/Icm Effector Translocation by Legionella longbeachae Creates a Replicative Vacuole Similar to That of Legionella pneumophila despite Translocation of Distinct Effector Repertoires

Legionella organisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires'' disease. These bacteria replicate within a pathogen-derived vacuole termed the Legionella-containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis of Legionella pneumophila. Here, we have characterized the Legionella longbeachae replicative vacuole (longbeachae-LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV of L. pneumophila (pneumophila-LCV). We constructed a Dot/Icm-deficient strain by deleting dotB and demonstrated the inability of this mutant to replicate inside THP-1 cells. L. longbeachae does not enter THP-1 cells as efficiently as L. pneumophila, and this is reflected in the observation that translocation of BlaM-RalF_LLO (where RalF_LLO is the L. longbeachae homologue of RalF) into THP-1 cells by the L. longbeachae Dot/Icm system is less efficient than that by L. pneumophila. This difference is negated in A549 cells where L. longbeachae and L. pneumophila infect with similar entry dynamics. A β-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, the Rab-like effector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on the longbeachae-LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to the longbeachae-LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of the L. longbeachae replicative vacuole, highlighting phenotypic similarities to the vacuole of L. pneumophila as well as unique aspects of LCV biology.     

Association of Legionella pneumophila with the macrophage endoplasmic reticulum.

Legionella pneumophila replicates within a membrane-bounded compartment that is studded with ribosomes. In this study we investigated whether these ribosomes originate from the cytoplasmic pool or are associated with host endoplasmic reticulum (ER). Immunofluorescence and electron microscopic localization studies of ER proteins in macrophages infected with L. pneumophila indicated that the bacteria reside in a compartment surrounded by ER. An L. pneumophila mutant that grows slowly in macrophages was slow to associate with host ER, providing genetic evidence in support of the hypothesis that this specialized vacuole is required for intracellular bacterial growth. Ultrastructural studies, in which the ER luminal protein BiP was labeled by immunoperoxidase cytochemistry, revealed that L. pneumophila replication vacuoles resemble nascent autophagosomes. Furthermore, short-term amino acid starvation of macrophages, which stimulated host autophagosomes. Furthermore, short-term amino acid starvation of macrophages, which stimulated host autophagy, increased association of the bacteria with the ER and enhanced bacterial growth. These results are compatible with the hypothesis that L. pneumophila exploits the autophagy machinery of macrophages to establish an intracellular niche favorable for replication.     

The Dot/Icm type IV secretion system of Legionella pneumophila is essential for the induction of apoptosis in human macrophages

We have previously shown that Legionella pneumophila induces caspase 3-dependent apoptosis in mammalian cells during early stages of infection. In this report, we show that nine L. pneumophila strains with mutations in the dotA, dotDCB, icmT, icmGCD, and icmJB loci are completely defective in the induction of apoptosis, in addition to their severe defects in intracellular replication and pore formation-mediated cytotoxicity. Importantly, all nine dot/icm mutants were complemented for all their defective phenotypes with the respective wild-type loci. We show that the role of the Dot/Icm type IV secretion system in the induction of apoptosis is independent of the RtxA toxin, the dot/icm-regulated pore-forming toxin, and the type II secretion system. However, the pore-forming toxin, which is triggered upon entry into the postexponential growth phase, enhances the ability of L. pneumophila to induce apoptosis. Our data provide the first example of the role of a type IV secretion system of a bacterial pathogen in the induction of apoptosis in the host cell.     

Incomplete activation of macrophage apoptosis during intracellular replication of Legionella pneumophila

The ability of the intracellular bacterium Legionella pneumophila to cause disease is totally dependent on its ability to modulate the biogenesis of its phagosome and to replicate within alveolar cells. Upon invasion, L. pneumophila activates caspase-3 in macrophages, monocytes, and alveolar epithelial cells in a Dot/Icm-dependent manner that is independent of the extrinsic or intrinsic pathway of apoptosis, suggesting a novel mechanism of caspase-3 activation by this intracellular pathogen. We have shown that the inhibition of caspase-3 prior to infection results in altered biogenesis of the L. pneumophila-containing phagosome and in an inhibition of intracellular replication. In this report, we show that the preactivation of caspase-3 prior to infection does not rescue the intracellular replication of L. pneumophila icmS, icmR, and icmQ mutant strains. Interestingly, preactivation of caspase-3 through the intrinsic and extrinsic pathways of apoptosis in both human and mouse macrophages inhibits intracellular replication of the parental stain of L. pneumophila. Using single-cell analysis, we show that intracellular L. pneumophila induces a robust activation of caspase-3 during exponential replication. Surprisingly, despite this robust activation of caspase-3 in the infected cell, the host cell does not undergo apoptosis until late stages of infection. In sharp contrast, the activation of caspase-3 by apoptosis-inducing agents occurs concomitantly with the apoptotic death of all cells that exhibit caspase-3 activation. It is only at a later stage of infection, and concomitant with the termination of intracellular replication, that the L. pneumophila-infected cells undergo apoptotic death. We conclude that although a robust activation of caspase-3 is exhibited throughout the exponential intracellular replication of L. pneumophila, apoptotic cell death is not executed until late stages of the infection, concomitant with the termination of intracellular replication.     

Identification of a hypervariable region containing new Legionella pneumophila Icm/Dot translocated substrates by using the conserved icmQ regulatory signature

Legionella pneumophila is an intracellular pathogen that has been shown to utilize the Icm/Dot type IV secretion system for pathogenesis. This system was shown to be composed of Icm/Dot complex components, accessory proteins, and a large number of translocated substrates. In this study, comparison of the icmQ regulatory regions from many Legionella species revealed a conserved regulatory sequence that includes the icmQ −10 promoter element. Mutagenesis of this conserved regulatory element indicated that each of the nucleotides in it affects the level of expression of the icmQ gene but not in a uniform fashion. A genomic analysis discovered that four additional genes in L. pneumophila contain this conserved regulatory sequence, which was found to function similarly in these genes as well. Examination of these four genes indicated that they are dispensable for intracellular growth, but two of them were found to encode new Icm/Dot translocated substrates (IDTS). Comparison of the genomic regions encoding these two IDTS among the four available L. pneumophila genomic sequences indicated that one of these genes is located in a hypervariable genomic region, which was shown before to contain an IDTS-encoding gene. Translocation analysis that was performed for nine proteins encoded from this hypervariable genomic region indicated that six of them are new IDTS which are translocated into host cells in an Icm/Dot-dependent manner. Furthermore, a bioinformatic analysis indicated that additional L. pneumophila genomic regions that contain several neighboring IDTS-encoding genes are hypervariable in gene content.     

Three Antagonistic Cyclic di-GMP-Catabolizing Enzymes Promote Differential Dot/Icm Effector Delivery and Intracellular Survival at the Early Steps of Legionella pneumophila Infection

Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole.     

Signal Transduction during Legionella pneumophila Entry into Human Monocytes

Legionella pneumophila causes Legionnaires’ disease by replication in alveolar macrophages and monocytes. The bacteria are internalized most efficiently by opsonin-dependent, CR3-mediated phagocytosis. This investigation focused on determining the role of actin polymerization and phosphorylation signals in this uptake mechanism. Uptake inhibition assays and confocal microscopic analysis indicated that entry of L. pneumophila activated tyrosine kinase (TK) and protein kinase C (PKC) and induced actin polymerization at the site of bacterial entry. Upon L. pneumophila entry, six major cellular proteins (75, 71, 59, 56, 53, and 52 kDa) were TK phosphorylated in soluble fractions of monocytes, and three of these proteins (52, 53, and 56 kDa) were consistently found in insoluble (i.e., cytoskeletal) fractions of monocytes as well. Tyrosine phosphorylation was suppressed when cells were pretreated with the kinase inhibitor genistein, tyrphostin, or staurosporine. A similar tyrosine-phosphorylated protein pattern was observed with CR3-mediated entry of avirulent L. pneumophila, Escherichia coli, or zymosan into monocytes. This study has shown that PKC and TK signals which activate actin polymerization during the process of phagocytosis are induced upon L. pneumophila entry. In addition, CR3 receptor-mediated phagocytosis into monocytes may involve tyrosine phosphorylation of similar proteins, regardless of the particle being phagocytosed. Therefore, the tyrosine-induced phosphorylation observed during opsonized L. pneumophila entry is not a virulence-associated event.     

Pleomorphism of Legionella pneumophila

Legionella pneumophila (Lp), serogroups 1-6, was grown in vitro on a variety of media, in embryonated hens' eggs, and in guinea pigs. The morphology of the microbe was examined by light, immunofluorescent, and electron microscopy (transmission, scanning, negative staining). The configuration of all serogroups examined differed somewhat on agar media, in liquid media, and in vivo. Each serogroup of Lp showed pleomorphic features indistinguishable from the others. Except for filamentous forms, pleomorphism was least conspicuous on agar. By contrast, pleomorphism was most apparent in yeast extract broth, and it was detected by all of the morphologic techniques employed. Bacilli were seen most commonly, but the spectrum of forms was as follows: cocci, coccobacilli (short bacilli), medium bacilli, bacilli with terminal cocci, filamentous forms, and branches. Diplococci, branches, and stalks were only rarely seen, and the latter form was never visualized by immunofluorescence. In tissue samples from infected guinea pigs and embryonated hens' eggs, Lp was typically a short bacillus, but coccoid and coccobacillary forms were seen. Lp is clearly a pleomorphic bacterium, particularly when grown in yeast extract broth. The variety of forms described herein might provide clues to taxonomy, ecologic niche, and physiology of Lp.     

Differential growth of Legionella pneumophila in guinea pig versus mouse macrophage cultures.

Legionella pneumophila is a facultative intracellular bacterium which replicated well in inbred guinea pig strain 2 peritoneal macrophages at a low infectivity ratio. In contrast, the growth of this organism was markedly restricted in mouse (BDF1) peritoneal macrophages, even at a relatively high infectivity ratio. The initial uptake of L. pneumophila organisms by macrophages was similar in both animal species, and both groups of macrophages supported the growth of Listeria monocytogenes. Treatment of L. pneumophila with immune guinea pig serum did not result in restriction of bacterial growth in macrophages, but guinea pig macrophages were readily induced to suppress the growth of L. pneumophila by preincubation with supernatants obtained from mitogen-activated normal guinea pig splenocyte cultures. Thus, lymphokines generated from mitogen-stimulated guinea pig lymphocytes induced a restriction of growth of these organisms similar to that observed naturally with macrophages from mice, which are considered highly resistant to these bacteria. Although guinea pigs are considered highly susceptible to L. pneumophila infections and mice are considered relatively resistant, the mechanism of this difference is not clear. The results of the present study suggest that the restriction of L. pneumophila growth by macrophages relates to host susceptibility to infection and that cell populations permissive for L. pneumophila can be transformed to nonpermissive by products from stimulated lymphocytes but not by opsonization with immune serum.     

Cyclic AMP inhibition of lipopolysaccharide-induced restriction of Legionella pneumophila growth in macrophage cultures.

The mechanism of the effects of lipopolysaccharide (LPS) on macrophages in terms of replication of intracellular facultative bacteria is unclear. It was found in the present study that the anti-Legionella pneumophila activity induced by LPS in macrophages from susceptible A/J mice was reversed in vitro by dibutyryl cyclic AMP (DcAMP). A 24-h pretreatment of murine thioglycolate-elicited macrophages with LPS resulted in an enhanced ability of these cells to inhibit the intracellular growth of L. pneumophila. This anti-L. pneumophila activity of macrophages induced by LPS was inhibited when DcAMP (10(-3) to 10(-5) M) was present during preincubation with LPS. The addition of DcAMP to the cultures was more effective before LPS treatment than after treatment. The effect of DcAMP was dose dependent. The secretion and production of acid phosphatase by LPS-activated macrophages were also inhibited by the addition of DcAMP before LPS treatment. Furthermore, the anti-L. pneumophila activity of macrophages induced by LPS could also be reversed in vitro by treatment with prostaglandin E2, colchicine, isoproterenol, theophylline, or hydrocortisone, all of which are known to increase the intracellular levels of cyclic AMP in various tissues. These observations indicate that the anti-L. pneumophila activity induced by LPS treatment can be modified by mechanisms involving cyclic nucleotide metabolism.     

Specificity of Legionella pneumophila and Coxiella burnetii vacuoles and versatility of Legionella pneumophila revealed by coinfection

Legionella pneumophila and Coxiella burnetii are phylogenetically related intracellular bacteria that cause aerosol-transmitted lung infections. In host cells both pathogens proliferate in vacuoles whose biogenesis displays some common features. To test the functional similarity of their respective intracellular niches, African green monkey kidney epithelial (Vero) cells, A/J mouse bone marrow-derived macrophages, human macrophages, and human dendritic cells (DC) containing mature C. burnetii replication vacuoles were superinfected with L. pneumophila, and then the acidity, lysosome-associated membrane protein (LAMP) content, and cohabitation of mature replication vacuoles was assessed. In all cell types, wild-type L. pneumophila occupied distinct vacuoles in close association with acidic, LAMP-positive C. burnetii replication vacuoles. In murine macrophages, but not primate macrophages, DC, or epithelial cells, L. pneumophila replication vacuoles were acidic and LAMP positive. Unlike wild-type L. pneumophila, type IV secretion-deficient dotA mutants trafficked to lysosome-like C. burnetii vacuoles in Vero cells where they survived but failed to replicate. In primate macrophages, DC, or epithelial cells, growth of L. pneumophila was as robust in superinfected cell cultures as in those singly infected. Thus, despite their noted similarities, L. pneumophila and C. burnetii are exquisitely adapted for replication in unique replication vacuoles, and factors that maintain the C. burnetii replication vacuole do not alter biogenesis of an adjacent L. pneumophila replication vacuole. Moreover, L. pneumophila can replicate efficiently in either lysosomal vacuoles of A/J mouse cells or in nonlysosomal vacuoles of primate cells.