Identification of non-dot/icm suppressors of the Legionella pneumophila DeltadotL lethality phenotype

Legionella pneumophila, a causative agent of bacterial pneumonia, survives inside phagocytic cells by avoiding rapid targeting to the lysosome. This bacterium utilizes a type IVB secretion system, encoded by the dot/icm genes, to replicate inside host cells. DotL, a critical component of the Dot/Icm secretion apparatus, functions as the type IV coupling protein. In contrast to most dot/icm genes, which are dispensable for growth on bacteriological media, dotL is required for the viability of wild-type L. pneumophila. Previously we reported that DeltadotL lethality could be suppressed by inactivation of the Dot/Icm complex via mutations in other dot/icm genes. Here we report the isolation of non-dot/icm suppressors of this phenotype. These DeltadotL suppressors include insertions that disrupt the function of the L. pneumophila homologs of cpxR, djlA, lysS, and two novel open reading frames, lpg0742 and lpg1594, that we have named ldsA and ldsB for lethality of DeltadotL suppressor. In addition to suppressing DeltadotL lethality, inactivation of these genes in a wild-type strain background causes a range of defects in L. pneumophila virulence traits, including intracellular growth, implicating these factors in the proper function of the Dot/Icm complex. Consistent with previous data showing a role for the cpx system in regulating expression of several dot/icm genes, the cpxR insertion mutant produced decreased levels of three Dot/Icm proteins, DotA, IcmV, and IcmW. The remaining four suppressors did not affect the steady-state levels of any Dot/Icm protein and are likely to represent the first identified factors necessary for assembly and/or activation of the Dot/Icm secretion complex.     

Implication of proteins containing tetratricopeptide repeats in conditional virulence phenotypes of Legionella pneumophila

Legionella pneumophila, the causative agent of Legionnaires' disease, is a ubiquitous freshwater bacterium whose virulence phenotypes require a type IV secretion system (T4SS). L. pneumophila strain JR32 contains two virulence-associated T4SSs, the Dot/Icm and Lvh T4SSs. Defective entry and phagosome acidification phenotypes of dot/icm mutants are conditional and reversed by incubating broth-grown stationary-phase cultures in water (WS treatment) prior to infection, as a mimic of the aquatic environment of Legionella. Reversal of dot/icm virulence defects requires the Lvh T4SS and is associated with a >10-fold induction of LpnE, a tetratricopeptide repeat (TPR)-containing protein. In the current study, we demonstrated that defective entry and phagosome acidification phenotypes of mutants with changes in LpnE and EnhC, another TPR-containing protein, were similarly reversed by WS treatment. In contrast to dot/icm mutants for which the Lvh T4SS was required, reversal for the ΔlpnE or the ΔenhC mutant required that the other TPR-containing protein be present. The single and double ΔlpnE and ΔenhC mutants showed a hypersensitivity to sodium ion, a phenotype associated with dysfunction of the Dot/Icm T4SS. The ΔlpnE single and the ΔlpnE ΔenhC double mutant showed 3- to 9-fold increases in translocation of Dot/Icm T4SS substrates, LegS2/SplY and LepB. Taken together, these data identify TPR-containing proteins in a second mechanism by which the WS mimic of a Legionella environmental niche can reverse virulence defects of broth-grown cultures and implicate LpnE and EnhC directly or indirectly in translocation of Dot/Icm T4SS protein substrates.     

Identification of the DotL coupling protein subcomplex of the Legionella Dot/Icm type IV secretion system

Legionella pneumophila, the causative agent of Legionnaires' disease, survives in macrophages by altering the endocytic pathway of its host cell. To accomplish this, the bacterium utilizes a type IVB secretion system to deliver effector molecules into the host cell cytoplasm. In a previous report, we performed an extensive characterization of the L. pneumophila type IVB secretion system that resulted in the identification of a critical five-protein subcomplex that forms the core of the secretion apparatus. Here we describe a second Dot/Icm protein subassembly composed of the type IV coupling protein DotL, the apparatus proteins DotM and DotN, and the secretion adaptor proteins IcmS and IcmW. In the absence of IcmS or IcmW, DotL becomes destabilized at the transition from the exponential to stationary phases of growth, concurrent with the expression of many secreted substrates. Loss of DotL is dependent on ClpA, a regulator of the cytoplasmic protease ClpP. The resulting decreased levels of DotL in the icmS and icmW mutants exacerbates the intracellular defects of these strains and can be partially suppressed by overproduction of DotL. Thus, in addition to their role as chaperones for Legionella type IV secretion system substrates, IcmS and IcmW perform a second function as part of the Dot/Icm type IV coupling protein subcomplex.     

A Dot/Icm-translocated ankyrin protein of Legionella pneumophila is required for intracellular proliferation within human macrophages and protozoa

The Dot/Icm type IV secretion system of Legionella pneumophila translocates numerous bacterial effectors into the host cell and is essential for bacterial proliferation within macrophages and protozoa. We have recently shown that L. pneumophila strain AA100/130b harbours 11 genes encoding eukaryotic-like ankyrin (Ank) proteins, a family of proteins involved in various essential eukaryotic cellular processes. In contrast to most Dot/Icm-exported substrates, which have little or no detectable role in intracellular proliferation, a mutation in ankB results in a severe growth defect in intracellular replication within human monocyte-derived macrophages (hMDMs), U937 macrophages and Acanthamoeba polyphaga. Single cell analyses of coinfections of hMDMs have shown that the intracellular growth defect of the ankB mutant is totally rescued in cis within communal phagosomes harbouring the wild type strain. Interestingly, distinct from dot/icm structural mutants, the ankB mutant is also rescued in trans within cells harbouring the wild type strain in a different phagosome, indicating that AnkB is a trans-acting secreted effector. Using adenylate cyclase fusions to AnkB, we show that AnkB is translocated into the host cell via the Dot/Icm secretion system in an IcmSW-dependent manner and that the last three C-terminal amino acid residues are essential for translocation. Distinct from the dot/icm structural mutants, the ankB mutant-containing phagosomes exclude late endosomal and lysosomal markers and their phagosomes are remodelled by the rough endoplasmic reticulum. We show that at the postexponential phase of growth, the LetA/S and PmrA/B Two Component Systems confer a positive regulation on expression of the ankB gene, whereas RpoS, LetE and RelA suppress its expression. Our data show that the eukaryotic-like AnkB protein is a Dot/Icm-exported effector that plays a major role in intracellular replication of L. pneumophila within macrophages and protozoa, and its expression is temporally controlled by regulators of the postexponential phase of growth.     

Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth

Legionella pneumophila is a bacterial pathogen that can enter the human lung and grow inside alveolar macrophages. To grow within phagocytic host cells, the bacteria must create a specialized organelle that restricts fusion with lysosomes. Biogenesis of this replicative organelle is controlled by 24 dot and icm genes, which encode a type IV-related transport apparatus. To understand how this transporter functions, isogenic L. pneumophila dot and icm mutants were characterized, and three distinct phenotypic categories were identified. Our data show that, in addition to genes that encode the core Dot/Icm transport apparatus, subsets of genes are required for pore formation and modulation of phagosome trafficking. To understand activities required for virulence at a molecular level, we investigated protein-protein interactions. Specific interactions between different Icm proteins were detected by yeast two-hybrid and gel overlay analysis. These data support a model in which the IcmQ-IcmR complex regulates the formation of a translocation channel that delivers proteins into host cells, and the IcmS-IcmW complex is required for export of virulence determinants that modulate phagosome trafficking.     

Coxiella burnetii express type IV secretion system proteins that function similarly to components of the Legionella pneumophila Dot/Icm system

Coxiella burnetii is an obligate intracellular pathogen that replicates in large endocytic vacuoles. Genomic sequence data indicate that 21 genes encoding products that are similar to components of the Legionella pneumophila Dot/Icm type IV secretion system are located on a contiguous 35 kb region of the Coxiella chromosome. It was found that several dot/icm genes were expressed by Coxiella during host cell infection and that dot/icm gene expression preceded the formation of large replicative vacuoles. To determine whether these genes encode a functional type IV secretion system, we have amplified the Coxiella dotB, icmQ, icmS and icmW genes and produced the encoded proteins in Legionella mutants in which the native copy of each gene had been deleted. The Coxiella dotB, icmS and icmW products restored dot/icm-dependent growth of Legionella mutants in eukaryotic host cells. The Coxiella IcmQ protein and the Legionella IcmR protein did not interact, which could explain why the Coxiella icmQ gene was unable to restore growth to a Legionella icmQ mutant. Thus, Coxiella encodes functional components of a type IV secretion system expressed in vivo that is mechanistically related to the Legionella Dot/Icm apparatus. These studies suggest that a dot/icm-related secretion system could play an important role in creating the specialized vacuole that supports Coxiella replication.     

The Legionella pneumophila IcmS-LvgA protein complex is important for Dot/Icm-dependent intracellular growth

Many bacterial pathogens require a functional type IV secretion system (T4SS) for virulence. Legionella pneumophila, the causative agent of Legionnaires' disease, employs the Dot/Icm T4SS to inject a large number of protein substrates into its host, thereby altering phagosome trafficking. The L. pneumophila T4SS substrate SdeA has been shown to require the accessory factor IcmS for its export. IcmS, defined as a type IV adaptor, exists as a heterodimer with IcmW and this complex functions in a manner similar to a type III secretion chaperone. Here we report an interaction between IcmS and the previously identified virulence factor LvgA. Similar to the icmS mutant, the lvgA mutant appears to assemble a fully functional Dot/Icm complex. Both LvgA and IcmS are small, acidic proteins localized to the cytoplasm and are not exported by the Dot/Icm system, suggesting they form a novel type IV adaptor complex. Inactivation of lvgA causes a minimal defect in growth in the human monocytic cell line U937 and the environmental host Acanthamoeba castellanii. However, the lvgA mutant was severely attenuated for intracellular growth of L. pneumophila in mouse macrophages, suggesting LvgA may be a critical factor that confers host specificity.     

The DotA protein from Legionella pneumophila is secreted by a novel process that requires the Dot/Icm transporter.

Legionella pneumophila requires the dot/icm genes to create an organelle inside eukaryotic host cells that will support bacterial replication. The dot/icm genes are predicted to encode a type IV-related secretion apparatus. However, no proteins have been identified that require the dot/icm genes for secretion. In this study we show that the DotA protein, which was previously found to be a polytopic membrane protein, is secreted by the Dot/Icm transporter into culture supernatants. Secreted DotA protein was purified and N-terminal sequencing of the purified protein revealed that a 19 amino acid leader peptide is removed from DotA prior to secretion. Extracellular DotA protein did not fractionate in membrane vesicles. Structures containing secreted DotA protein were visualized by electron microscopy and were shaped like hollow rings. These data indicate that the large poly topic membrane protein DotA is secreted from L.pneumophila by a unique process. This represents the first target secreted by the dot/icm-encoded apparatus and demonstrates that this transporter is competent for protein secretion.     

The Icm/Dot type-IV secretion systems of Legionella pneumophila and Coxiella burnetii

Type-IV secretion systems are devices present in a wide range of bacteria (including bacterial pathogens) that deliver macromolecules (proteins and single-strand-DNA) across kingdom barriers (as well as between bacteria and into the surroundings). The type-IV secretion systems were divided into two subgroups and Legionella pneumophila and Coxiella burnetii are the only two bacteria known today to utilize a type-IVB secretion system for pathogenesis. In this review we summarized the available information concerning the icm/dot type-IVB secretion systems by comparing the two bacteria that possess this system, the proteins components of their systems as well as the homology of proteins from type-IVB secretion systems to proteins from type-IVA secretion systems. In addition, the phenotypes associated with mutants in the L. pneumophila icm/dot genes, their relations to properties of specific Icm/Dot proteins as well as the protein substrates delivered by this system are described.     

The Coxiella burnetii Dot/Icm system delivers a unique repertoire of type IV effectors into host cells and is required for intracellular replication

Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in an acidified vacuole derived from the host lysosomal network. This pathogen encodes a Dot/Icm type IV secretion system that delivers bacterial proteins called effectors to the host cytosol. To identify new effector proteins, the functionally analogous Legionella pneumophila Dot/Icm system was used in a genetic screen to identify fragments of C. burnetii genomic DNA that when fused to an adenylate cyclase reporter were capable of directing Dot/Icm-dependent translocation of the fusion protein into mammalian host cells. This screen identified Dot/Icm effectors that were proteins unique to C. burnetii, having no overall sequence homology with L. pneumophila Dot/Icm effectors. A comparison of C. burnetii genome sequences from different isolates revealed diversity in the size and distribution of the genes encoding many of these effectors. Studies examining the localization and function of effectors in eukaryotic cells provided evidence that several of these proteins have an affinity for specific host organelles and can disrupt cellular functions. The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors. Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication. Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication.     

The response regulator PmrA is a major regulator of the icm/dot type IV secretion system in Legionella pneumophila and Coxiella burnetii

Legionella pneumophila and Coxiella burnetii have been shown to utilize the icm/dot type IV secretion system for pathogenesis and recently a large number of icm/dot-translocated substrates were identified in L. pneumophila. Bioinformatic analysis has revealed that 13 of the genes encoding for L. pneumophila-translocated substrates and five of the C. burnetii icm/dot genes, contain a conserved regulatory element that resembles the target sequence of the PmrA response regulator. Experimental analysis which included the construction of a L. pneumophila pmrA deletion mutant, intracellular growth analysis, comparison of gene expression between L. pneumophila wild type and the pmrA mutant, construction of mutations in the PmrA conserved regulatory element, controlled expression studies as well as mobility shift assays, demonstrated the direct relation between the PmrA regulator and the expression of L. pneumophila icm/dot-translocated substrates and several C. burnetii icm/dot genes. Furthermore, genomic analysis identified 35 L. pneumophila and 68 C. burnetii unique genes that contain the PmrA regulatory element and few of these genes from L. pneumophila were found to be new icm/dot-translocated substrates. Our results establish the PmrA regulator as a fundamental regulator of the icm/dot type IV secretion system in these two bacteria.     

Host cell-dependent secretion and translocation of the LepA and LepB effectors of Legionella pneumophila

Legionella pneumophila is the Gram-negative bacterial agent of Legionnaires' disease, an acute, often fatal pneumonia. L. pneumophila infects alveolar macrophages, evading the antimicrobial defences of the phagocyte by preventing fusion of the phagosome with lysosomes and avoiding phagosome acidification. The bacteria then modulate the composition of the vacuole so that it takes on the characteristics of the endoplasmic reticulum. Similar events occur when the bacteria infect unicellular protozoa. It is thought that replication in fresh water protozoa provides an environmental reservoir for the organism. Several effector proteins are delivered to the host by the Icm/Dot type IV secretion system (TFSS). Some of these have been shown to participate in the trafficking of the Legionella phagosome. Here we describe the ability of the Icm/Dot TFSS to translocate two effectors, LepA and LepB, that play a role in the non-lytic release of Legionella from protozoa. We report that translocation of the Lep proteins is inhibited by agents that depolymerize actin filaments and that effectors may be secreted into the extracellular medium upon cell contact. Depletion of the Lep proteins by deletion of their genes results in increased ability to lyse red blood cells. In contrast, overexpression of Lep-containing hybrid proteins appears to specifically inhibit the activity of the Icm/Dot TFSS and may prevent the delivery of other effectors that are critical for intracellular multiplication.     

Genetic analysis of the Legionella pneumophila DotB ATPase reveals a role in type IV secretion system protein export

The pulmonary pathogen Legionella pneumophila uses the Dot/Icm type IV secretion system (T4SS) to replicate inside host cells. This apparatus translocates proteins into macrophages to alter their endocytic pathway and enable bacterial growth. Although the secretion ATPase DotB is critical for T4SS function, its specific role in type IV secretion remains undefined. Due to similarity to the VirB11 and PilT ATPases, DotB has been proposed to play a role in assembly of the T4SS, retraction of pili and/or export of substrates. With the goal of understanding the protein's function(s), we isolated and characterized 30 dotB alleles using a variety of phenotypic and biochemical assays. Twenty-four of these alleles possess several dot/icm mutant phenotypes, including a complete lack of intracellular replication, plasmid mobilization and contact-dependent cytotoxicity. These 24 non-functional alleles fall into three classes: those with a known biochemical defect, those with a predicted enzymatic defect and those with an unknown defect. Six other alleles display partial activity in dot/icm phenotypic assays, thus constituting a fourth class. Two mutants in this class are unable to export a subset of T4SS substrates, providing the first evidence for a DotB function in substrate export and suggesting a possible role in substrate selection.     

Relationships between a new type IV secretion system and the icm/dot virulence system of Legionella pneumophila

We describe here a Legionella pneumophila type IV secretion system that is distinct from the previously described icm/dot system. This type IV secretion system contains 11 genes (lvh ) homologous to genes of other type IV secretion systems, arranged in a similar manner. The lvh genes were found to be located on a DNA island with a GC content higher than the L. pneumophila chromosome. In contrast to the icm/dot system that was shown to be required for intracellular growth in HL-60-derived human macrophages and Acanthamoeba castellanii, the lvh system was found to be dispensable for intracellular growth in these two hosts. The lvh system was found to be partially required for RSF1010 conjugation, a process that was previously shown to be completely dependent on several icm/dot genes. However, results obtained from analysis of double mutants in the icm/dot genes and the lvh genes revealed that lvh genes can substitute for some components of the icm/dot system for RSF1010 conjugation, but not for intracellular growth. These results indicate that components of the icm/dot system and components of the lvh type IV secretion system are able to interact with one another.     

The Legionella IcmSW Complex Directly Interacts with DotL to Mediate Translocation of Adaptor-Dependent Substrates

Legionella pneumophila is a Gram-negative bacterium that replicates within human alveolar macrophages by evasion of the host endocytic pathway through the formation of a replicative vacuole. Generation of this vacuole is dependent upon the secretion of over 275 effector proteins into the host cell via the Dot/Icm type IVB secretion system (T4SS). The type IV coupling protein (T4CP) subcomplex, consisting of DotL, DotM, DotN, IcmS and IcmW, was recently defined. DotL is proposed to be the T4CP of the L. pneumophila T4SS based on its homology to known T4CPs, which function as inner-membrane receptors for substrates. As a result, DotL is hypothesized to play an integral role(s) in the L. pneumophila T4SS for the engagement and translocation of substrates. To elucidate this role, a genetic approach was taken to screen for dotL mutants that were unable to survive inside host cells. One mutant, dotLY725Stop, did not interact with the type IV adaptor proteins IcmS/IcmW (IcmSW) leading to the identification of an IcmSW-binding domain on DotL. Interestingly, the dotLY725Stop mutant was competent for export of one class of secreted effectors, the IcmSW-independent substrates, but exhibited a specific defect in secretion of IcmSW-dependent substrates. This differential secretion illustrates that DotL requires a direct interaction with the type IV adaptor proteins for the secretion of a major class of substrates. Thus, by identifying a new target for IcmSW, we have discovered that the type IV adaptors perform an additional role in the export of substrates by the L. pneumophila Dot/Icm T4SS.     

Cell biology of Legionella pneumophila

Legionella pneumophila is the causative agent of a potentially fatal form of pneumonia named Legionnaires' disease. L. pneumophila survives and replicates inside macrophages by preventing phagosome-lysosome fusion. A large number of L. pneumophila genes, called dot or icm, have been identified that are required for intracellular growth. It has recently been shown that the dot/icm genes code for a putative large membrane complex that forms a type IV secretion system used to alter the endocytic pathway.     

Modulation of ubiquitin dynamics and suppression of DALIS formation by the Legionella pneumophila Dot/Icm system

Legionella pneumophila is an intracellular pathogen that uses effector proteins translocated by the Dot/Icm type IV secretion system to modulate host cellular processes. Here we investigate the dynamics of subcellular structures containing ubiquitin during L. pneumophila infection of phagocytic host cells. The Dot/Icm system mediated the formation of K48 and K63 poly-ubiquitin conjugates to proteins associated with L. pneumophila -containing vacuoles in macrophages and dendritic cells, suggesting that regulatory events and degradative events involving ubiquitin are regulated by bacterial effectors during infection. Stimulation of TLR2 on the surface of macrophages and dendritic cells by L. pneumophila- derived molecules resulted in the production of ubiquitin-rich dendritic cell aggresome-like structures (DALIS). Cells infected by L. pneumophila with a functional Dot/Icm system, however, failed to produce DALIS. Suppression of DALIS formation did not affect the accumulation of ubiquitinated proteins on vacuoles containing L. pneumophila. Examining other species of Legionella revealed that Legionella jordanis was unable to suppress DALIS formation after creating a ubiquitin-decorated vacuole. Thus, the L. pneumophila Dot/Icm system has the ability to modulate host processes to promote K48 and K63 ubiquitin conjugates on proteins at the vacuole membrane, and independently suppress cellular events required for the formation of DALIS.     

Manipulation of host vesicular trafficking and innate immune defence by Legionella Dot/Icm effectors

Legionella pneumophila, the causative agent of Legionnaires' disease, infects and replicates in macrophages and amoebas. Following internalization, L. pneumophila resides in a vacuole structure called Legionella-containing vacuole (LCV). The LCV escapes from the endocytic maturation process and avoids fusion with the lysosome, a hallmark of Legionella pathogenesis. Interference with the secretory vesicle transport and avoiding lysosomal targeting render the LCV permissive for L. pneumophila intracellular replication. Central to L. pneumophila pathogenesis is a defect in the organelle trafficking/intracellular multiplication (Dot/Icm) type IV secretion system that translocates a large number of effector proteins into host cells. Many of the Dot/Icm effectors employ diverse and sophisticated biochemical strategies to manipulate the host vesicular transport system, playing an important role in LCV biogenesis and trafficking. Similar to other bacterial pathogens, L. pneumophila also delivers effector proteins to modulate or counteract host innate immune defence pathways such as the NF-κB and apoptotic signalling. This review summarizes the known functions and mechanisms of Dot/Icm effectors that target host membrane trafficking and innate immune defence pathways.     

Legionella secreted effectors and innate immune responses

Legionella pneumophila is a facultative intracellular pathogen capable of replicating in a wide spectrum of cells. Successful infection by Legionella requires the Dot/Icm type IV secretion system, which translocates a large number of effector proteins into infected cells. By co-opting numerous host cellular processes, these proteins function to establish a specialized organelle that allows bacterial survival and proliferation. Even within the vacuole, L. pneumophila triggers robust immune responses. Recent studies reveal that a subset of Legionella effectors directly target some basic components of the host innate immunity systems such as phagosome maturation. Others play essential roles in engaging the host innate immune surveillance system. This review will highlight recent progress in our understanding of these interactions and discuss implications for the study of the immune detection mechanisms.     

Activation of caspase-3 by the Dot/Icm virulence system is essential for arrested biogenesis of the Legionella-containing phagosome

The Dot/Icm type IV secretion system of Legionella pneumophila is essential for evasion of endocytic fusion and for activation of caspase-3 during early stages of infection of macrophages, but the mechanisms of manipulating these host cell processes are not known. Here, we show that caspase-3 activation by L. pneumophila is independent of all the known apoptotic pathways that converge on the activation of caspase-3. The cytoplasmic proteins IcmS, IcmR and IcmQ, which are involved in secretion of Dot/Icm effectors, are required for caspase-3 activation. Pretreatment of U937 macrophages and human peripheral blood monocytes (hPBM) with the capase-3 inhibitor (DEVD-fmk) or the paninhibitor of caspases (Z-VAD-fmk) before infection blocks intracellular replication of L. pneumophila in a dose-dependent manner. Inhibition of caspase-3 results in co-localization of the L. pneumophila-containing phagosome (LCP) with the late endosomal/lysosomal marker Lamp-2, and the LCP contains lysosomal enzymes, similar to the dotA mutant, which is defective in caspase-3 activation. However, activation of caspase-3 before infection does not rescue the replication defect of the dotA mutant. Interestingly, inhibition of caspase-3 after a 15 or 30 min infection period by the parental strain has no detectable effect on the formation of a replicative niche. The Dot/Icm-mediated activation of caspase-3 by L. pneumophila specifically cleaves, in a dose- and time-dependent manner, the Rab5 effector Rabaptin-5, which maintains Rab5-GTP on the endosomal membrane. In addition, PI3 kinase, which is a crucial effector of Rab5 downstream of Rababptin-5, is not required for intracellular replication. Using single-cell analysis, we show that apoptosis is not evident in the infected cell until bacterial replication results in > 20 bacteria per cell. We conclude that activation of caspase-3 by the Dot/Icm virulence system of L. pneumophila is essential for halting biogenesis of the LCP through the endosomal/lysosomal pathway, and that this is associated with the cleavage of Rabpatin-5.