Reassessing the Role of DotF in the Legionella pneumophila Type IV Secretion System

Legionella pneumophila, the causative agent of a severe pneumonia termed Legionnaires’ Disease, survives and replicates within both protozoan hosts and human alveolar macrophages. Intracellular survival is dependent upon secretion of a plethora of protein effectors that function to form a replicative vacuole, evade the endocytic pathway and subvert host immune defenses. Export of these factors requires a type IV secretion system (T4SS) called Dot/Icm that is composed of twenty-seven proteins. This report focuses on the DotF protein, which was previously postulated to have several different functions, one of which centered on binding Dot/Icm substrates. In this report, we examined if DotF functions as the T4SS inner membrane receptor for Dot/Icm substrates. Although we were able to recapitulate the previously published bacterial two-hybrid interaction between DotF and several substrates, the interaction was not dependent on the Dot/Icm substrates’ signal sequences as predicted for a substrate:receptor interaction. In addition, binding did not require the cytoplasmic domain of DotF, which was anticipated to be involved in recognizing substrates in the cytoplasm. Finally, inactivation of dotF did not abolish intracellular growth of L. pneumophila or translocation of substrates, two phenotypes dependent on the T4SS receptor. These data strongly suggest that DotF does not act as the major receptor for Dot/Icm substrates and therefore likely performs an accessory function within the core-transmembrane subcomplex of the L. pneumophila Dot/Icm type IV secretion system.     

The Machinery at Endoplasmic Reticulum-Plasma Membrane Contact Sites Contributes to Spatial Regulation of Multiple Legionella Effector Proteins

The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.     

Intragenic Recombination Has a Critical Role on the Evolution of Legionella pneumophila Virulence-Related Effector sidJ

SidJ is a Dot/Icm effector involved in the trafficking or retention of ER-derived vesicles to Legionella pneumophila vacuoles whose mutation causes an observable growth defect, both in macrophage and amoeba hosts. Given the crucial role of this effector in L. pneumophila virulence we investigated the mechanisms shaping its molecular evolution. The alignment of SidJ sequences revealed several alleles with amino acid variations that may influence the protein properties. The identification of HGT events and the detection of balancing selection operating on sidJ evolution emerge as a clear result. Evidence suggests that intragenic recombination is an important strategy in the evolutionary adaptive process playing an active role on sidJ genetic plasticity. This pattern of evolution is in accordance with the life style of L. pneumophila as a broad host-range pathogen by preventing host-specialization and contributing to the resilience of the species.     

Viewing Legionella pneumophila Pathogenesis through an Immunological Lens

Legionella pneumophila is the causative agent of the severe pneumonia Legionnaires' disease. L. pneumophila is ubiquitously found in freshwater environments, where it replicates within free-living protozoa. Aerosolization of contaminated water supplies allows the bacteria to be inhaled into the human lung, where L. pneumophila can be phagocytosed by alveolar macrophages and replicate intracellularly. The Dot/Icm type IV secretion system (T4SS) is one of the key virulence factors required for intracellular bacterial replication and subsequent disease. The Dot/Icm apparatus translocates more than 300 effector proteins into the host cell cytosol. These effectors interfere with a variety of cellular processes, thus enabling the bacterium to evade phagosome–lysosome fusion and establish an endoplasmic reticulum-derived Legionella-containing vacuole, which facilitates bacterial replication. In turn, the immune system has evolved numerous strategies to recognize intracellular bacteria such as L. pneumophila, leading to potent inflammatory responses that aid in eliminating infection. This review aims to provide an overview of L. pneumophila pathogenesis in the context of the host immune response.     

Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization,Pathogen Vacuole Motility and Infection

The causative agent of Legionnaires'' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct “Legionella-containing vacuole” (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF) domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP) in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED) fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila.     

Refining the Plasmid-Encoded Type IV Secretion System Substrate Repertoire of Coxiella burnetii

The intracellular bacterial agent of Q fever, Coxiella burnetii, translocates effector proteins into its host cell cytosol via a Dot/Icm type IV secretion system (T4SS). The T4SS is essential for parasitophorous vacuole formation, intracellular replication, and inhibition of host cell death, but the effectors mediating these events remain largely undefined. Six Dot/Icm substrate-encoding genes were recently discovered on the C. burnetii cryptic QpH1 plasmid, three of which are conserved among all C. burnetii isolates, suggesting that they are critical for conserved pathogen functions. However, the remaining hypothetical proteins encoded by plasmid genes have not been assessed for their potential as T4SS substrates. In the current study, we further defined the T4SS effector repertoire encoded by the C. burnetii QpH1, QpRS, and QpDG plasmids that were originally isolated from acute-disease, chronic-disease, and severely attenuated isolates, respectively. Hypothetical proteins, including those specific to QpRS or QpDG, were screened for translocation using the well-established Legionella pneumophila T4SS secretion model. In total, six novel plasmid-encoded proteins were translocated into macrophage-like cells by the Dot/Icm T4SS. Four newly identified effectors are encoded by genes present only on the QpDG plasmid from severely attenuated Dugway isolates, suggesting that the presence of specific effectors correlates with decreased virulence. These results further support the idea of a critical role for extrachromosomal elements in C. burnetii pathogenesis.     

Two Fis Regulators Directly Repress the Expression of Numerous Effector-Encoding Genes in Legionella pneumophila

Legionella pneumophila is an intracellular human pathogen that utilizes the Icm/Dot type IVB secretion system to translocate a large repertoire of effectors into host cells. For most of these effectors, there is no information regarding their regulation. Therefore, the aim of this study was to examine the involvement of the three L. pneumophila Fis homologs in the regulation of effector-encoding genes. Deletion mutants constructed in the genes encoding the three Fis regulators revealed that Fis1 (lpg0542 gene) and Fis3 (lpg1743) but not Fis2 (lpg1370) are partially required for intracellular growth of L. pneumophila in Acanthamoeba castellanii. To identify pathogenesis-related genes directly regulated by Fis, we established a novel in vivo system which resulted in the discovery of numerous effector-encoding genes directly regulated by Fis. Further examination of these genes revealed that Fis1 and Fis3 repress the level of expression of effector-encoding genes during exponential phase. Three groups of effector-encoding genes were identified: (i) effectors regulated mainly by Fis1, (ii) effectors regulated mainly by Fis3, and (iii) effectors regulated by both Fis1 and Fis3. Examination of the upstream regulatory region of all of these effector-encoding genes revealed multiple putative Fis regulatory elements, and site-directed mutagenesis confirmed that a few of these sites constitute part of a repressor binding element. Furthermore, gel mobility shift assays demonstrated the direct relation between the Fis1 and Fis3 regulators and these regulatory elements. Collectively, our results demonstrate for the first time that two of the three L. pneumophila Fis regulators directly repress the expression of Icm/Dot effector-encoding genes.     

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.     

Deciphering Legionella effector delivery by Icm/Dot secretion system reveals a new role for c-di-GMP signaling

Secretion of bacterial effector proteins into host cells plays a key role in bacterial virulence. Yet, the dynamics of the secretion systems activity remains poorly understood, especially when machineries deal with the export of numerous effectors. We address the question of multi-effector secretion by focusing on the Legionella pneumophila Icm/Dot T4SS that translocates a record number of 300 effectors. We set up a kinetic translocation assay, based on the β-lactamase translocation reporter system combined with the effect of the protonophore CCCP. When used for translocation analysis of Icm/Dot substrates constitutively produced by L. pneumophila, this assay allows a fine monitoring of the secretion activity of the T4SS, independently of the expression control of the effectors. We observed that effectors are translocated with a specific timing, suggesting a control of their docking/translocation by the T4SS. Their delivery is accurately organized to allow effective manipulation of the host cell, as exemplified by the sequential translocation of effectors targeting Rab1, namely SidM/DrrA, LidA, LepB. Remarkably, the timed delivery of effectors does not depend only on their interaction with chaperone proteins but implies cyclic-di-GMP signaling, as the diguanylate cyclase Lpl0780/Lpp0809, contributes to the timing of translocation.     

Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host–pathogen interactions.     

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen,Legionella pneumophila

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila‐translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.     

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.     

A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization

Successful infection by the opportunistic pathogen Legionella pneumophila requires the collective activity of hundreds of virulence proteins delivered into the host cell by the Dot/Icm type IV secretion system. These virulence proteins, also called effectors modulate distinct host cellular processes to create a membrane-bound niche called the Legionella containing vacuole (LCV) supportive of bacterial growth. We found that Ceg14 (Lpg0437), a Dot/Icm substrate is toxic to yeast and such toxicity can be alleviated by overexpression of profilin, a protein involved in cytoskeletal structure in eukaryotes. We further showed that mutations in profilin affect actin binding but not other functions such as interactions with poly-l-proline or phosphatidylinositol, abolish its suppressor activity. Consistent with the fact the profilin suppresses its toxicity, expression of Ceg14 but not its non-toxic mutants in yeast affects actin distribution and budding of daughter cells. Although Ceg14 does not detectably interact with profilin, it co-sediments with filamentous actin and inhibits actin polymerization, causing the accumulation of short actin filaments. Together with earlier studies, these results reveal that multiple L. pneumophila effectors target components of the host cytoskeleton.     

Targeting eEF1A by a Legionella pneumophila effector leads to inhibition of protein synthesis and induction of host stress response

The Legionella pneumophila Dot/Icm type IV secretion system is essential for the biogenesis of a phagosome that supports bacterial multiplication, most likely via the functions of its protein substrates. Recent studies indicate that fundamental cellular processes, such as vesicle trafficking, stress response, autophagy and cell death, are modulated by these effectors. However, how each translocated protein contributes to the modulation of these pathways is largely unknown. In a screen to search substrates of the Dot/Icm transporter that can cause host cell death, we identified a gene whose product is lethal to yeast and mammalian cells. We demonstrate that this protein, called SidI, is a substrate of the Dot/Icm type IV protein transporter that targets the host protein translation process. Our results indicate that SidI specifically interacts with eEF1A and eEF1Bγ, two components of the eukaryotic protein translation elongation machinery and such interactions leads to inhibition of host protein synthesis. Furthermore, we have isolated two SidI substitution mutants that retain the target binding activity but have lost toxicity to eukaryotic cells, suggesting potential biochemical effect of SidI on eEF1A and eEF1Bγ. We also show that infection by L. pneumophila leads to eEF1A‐mediated activation of the heat shock regulatory protein HSF1 in a virulence‐dependent manner and deletion of sidI affects such activation. Moreover, similar response occurred in cells transiently transfected to express SidI. Thus, inhibition of host protein synthesis by specific effectors contributes to the induction of stress response in L. pneumophila‐infected cells.     

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.     

Legionella pneumophila Strain 130b Possesses a Unique Combination of Type IV Secretion Systems and Novel Dot/Icm Secretion System Effector Proteins

Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.Many bacterial pathogens use specialized protein secretion systems to deliver into host cells virulence effector proteins that interfere with the antimicrobial responses of the host and facilitate the survival of the pathogen (5, 10, 22, 76). The components of these secretion systems are highly conserved. Comparative bioinformatic analysis of pathogen genomes revealed an ever-increasing number of proteins that are likely to be translocated virulence effectors. Only a few effectors have been characterized, and their biochemical functions are unknown, yet the identification of translocated effector proteins and their mechanism of action is fundamental to understanding the pathogenesis of many bacterial infections.Legionella pneumophila is the etiological agent of Legionnaires’ disease, which is an acute form of pneumonia (34, 66). L. pneumophila serogroup 1 accounts for more than 90% of all cases worldwide. Although L. pneumophila is an environmental organism, its ability to survive and replicate in amoebae, such as Acanthamoeba castellanii, has equipped the organism with the capacity to replicate in human cells (45, 58, 68, 80). Following the inhalation of aerosols containing L. pneumophila into the human lung, the bacteria promote their uptake by alveolar macrophages and epithelial cells (21, 44, 71), where they replicate within an intracellular vacuole that avoids fusion with the endocytic pathway (46, 47). L. pneumophila evades endosome fusion by establishing a replicative vacuole that shares many characteristics with the endoplasmic reticulum (ER) (48, 53, 89). The formation of the unique Legionella-containing vacuole (LCV) requires the Dot (defective in organelle trafficking)/Icm (intracellular multiplication) type IV secretion system (T4SS) (85, 91).Type IV secretion systems are versatile multiprotein complexes that can transport DNA and proteins to recipient bacteria or host cells (19, 36). Based on structural and organizational similarity, three main T4SS classes have been distinguished: T4SSA, T4SSB, and genomic island-associated T4SS (GI-T4SS) (3, 51). The genetic organization and components of T4SSA have high similarity to the classical VirB4/VirD4 transfer DNA (T-DNA) transfer system of Agrobacterium tumefaciens (3). In the sequenced L. pneumophila strains, three distinct T4SSAs with different prevalences among strains have been described: Lvh, Trb-1, and Trb-2 (37, 83, 86). The Lvh (Legionella vir homologues) T4SSA is not required for intracellular bacterial replication in macrophages and amoebae but seems to contribute to infection at lower temperatures and inclusion in Acanthamoeba castellanii cysts (6, 78, 86).The Dot/Icm T4SSB secretes and translocates multiple bacterial effector proteins into the vacuolar membrane and cytosol of the host cell (31, 70). The functions of the great majority of these proteins are unknown. Several effectors have similarity to eukaryotic proteins or carry eukaryotic motifs (7, 16, 25). They are predicted to allow L. pneumophila to manipulate host cell processes by functional mimicry (31, 70). Many of the effectors have paralogues or belong to related protein families that are likely to have overlapping functions.Comparative analysis of the recent L. pneumophila genome sequences has revealed their diversity and plasticity (16, 18, 88). This plasticity enables the bacterium to acquire new genetic factors, including new effector proteins that enhance bacterial replication and survival in eukaryotic cells. This has resulted in a diverse species in which 7 to 11% of the genes in each L. pneumophila isolate are strain specific (38). Some of the diversity occurs among genes encoding Dot/Icm effectors, including those within the same family. For example some ankyrin repeat and F-box effector genes are highly conserved, while others vary considerably between L. pneumophila isolates (16, 41, 62, 73, 75). Even though it is not experimentally proven, the subsequent selection of Dot/Icm effectors among different L. pneumophila isolates might reflect their usefulness in host-pathogen interactions, whereby different effector repertoires are maintained during adaptation to different environmental niches or hosts. This may then translate into differences in virulence during opportunistic infection.In this study, we sequenced the genome of L. pneumophila serogroup 1 strain 130b (ATCC BAA-74, also known as Wadsworth or AA100) (29, 30) and analyzed the sequence for T4SSs and novel Dot/Icm effectors. This analysis established that the strain encodes a unique combination of T4SSs and a set of Dot/Icm effectors that had not been described previously but that are present in a range of clinical and environmental L. pneumophila isolates. The new effectors represent the latest members of an ever-growing list of T4SS substrates and presumably reflect the great capacity of L. pneumophila for adaptation to a variety of hosts.     

The Legionella pneumophila IcmSW complex interacts with multiple Dot/Icm effectors to facilitate type IV translocation

Many gram-negative pathogens use a type IV secretion system (T4SS) to deliver effector proteins into eukaryotic host cells. The fidelity of protein translocation depends on the efficient recognition of effector proteins by the T4SS. Legionella pneumophila delivers a large number of effector proteins into eukaryotic cells using the Dot/Icm T4SS. How the Dot/Icm system is able to recognize and control the delivery of effectors is poorly understood. Recent studies suggest that the IcmS and IcmW proteins interact to form a stable complex that facilitates translocation of effector proteins by the Dot/Icm system by an unknown mechanism. Here we demonstrate that the IcmSW complex is necessary for the productive translocation of multiple Dot/Icm effector proteins. Effector proteins that were able to bind IcmSW in vitro required icmS and icmW for efficient translocation into eukaryotic cells during L. pneumophila infection. We identified regions in the effector protein SidG involved in icmSW-dependent translocation. Although the full-length SidG protein was translocated by an icmSW-dependent mechanism, deletion of amino terminal regions in the SidG protein resulted in icmSW-independent translocation, indicating that the IcmSW complex is not contributing directly to recognition of effector proteins by the Dot/Icm system. Biochemical and genetic studies showed that the IcmSW complex interacts with a central region of the SidG protein. The IcmSW interaction resulted in a conformational change in the SidG protein as determined by differences in protease sensitivity in vitro. These data suggest that IcmSW binding to effectors could enhance effector protein delivery by mediating a conformational change that facilitates T4SS recognition of a translocation domain located in the carboxyl region of the effector protein.     

Crystal structure of Legionella DotD: insights into the relationship between type IVB and type II/III secretion systems

The Dot/Icm type IVB secretion system (T4BSS) is a pivotal determinant of Legionella pneumophila pathogenesis. L. pneumophila translocate more than 100 effector proteins into host cytoplasm using Dot/Icm T4BSS, modulating host cellular functions to establish a replicative niche within host cells. The T4BSS core complex spanning the inner and outer membranes is thought to be made up of at least five proteins: DotC, DotD, DotF, DotG and DotH. DotH is the outer membrane protein; its targeting depends on lipoproteins DotC and DotD. However, the core complex structure and assembly mechanism are still unknown. Here, we report the crystal structure of DotD at 2.0 Å resolution. The structure of DotD is distinct from that of VirB7, the outer membrane lipoprotein of the type IVA secretion system. In contrast, the C-terminal domain of DotD is remarkably similar to the N-terminal subdomain of secretins, the integral outer membrane proteins that form substrate conduits for the type II and the type III secretion systems (T2SS and T3SS). A short β-segment in the otherwise disordered N-terminal region, located on the hydrophobic cleft of the C-terminal domain, is essential for outer membrane targeting of DotH and Dot/Icm T4BSS core complex formation. These findings uncover an intriguing link between T4BSS and T2SS/T3SS.