TraK and TraB Are Conserved Outer Membrane Proteins of the Neisseria gonorrhoeae Type IV Secretion System and Are Expressed at Low Levels in Wild-Type Cells

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the medium, and this DNA is effective in transforming other gonococci via natural transformation. In addition, the T4SS is important in the initial stages of biofilm development and mediates intracellular iron uptake in the absence of TonB. To better understand the mechanism of type IV secretion in N. gonorrhoeae, we examined the expression levels and localization of two predicted T4SS outer membrane proteins, TraK and TraB, in the wild-type strain as well as in overexpression strains and in a strain lacking all of the T4SS proteins. Despite very low sequence similarity to known homologues, TraB (VirB10 homolog) and TraK (VirB9 homolog) localized similarly to related proteins in other systems. Additionally, we found that TraV (a VirB7 homolog) interacts with TraK, as in other T4SSs. However, unlike in other systems, neither TraK nor TraB required the presence of other T4SS components for proper localization. Unlike other gonococcal T4SS proteins we have investigated, protein levels of the outer membrane proteins TraK and TraB were extremely low in wild-type cells and were undetectable by Western blotting unless overexpressed or tagged with a FLAG3 triple-epitope tag. Localization of TraK-FLAG3 in otherwise wild-type cells using immunogold electron microscopy of thin sections revealed a single gold particle on some cells. These results suggest that the gonococcal T4SS may be present in single copy per cell and that small amounts of T4SS proteins TraK and TraB are sufficient for DNA secretion.     

A Translocation Motif in Relaxase TrwC Specifically Affects Recruitment by Its Conjugative Type IV Secretion System

Type IV secretion system (T4SS) substrates are recruited through a translocation signal that is poorly defined for conjugative relaxases. The relaxase TrwC of plasmid R388 is translocated by its cognate conjugative T4SS, and it can also be translocated by the VirB/D4 T4SS of Bartonella henselae, causing DNA transfer to human cells. In this work, we constructed a series of TrwC variants and assayed them for DNA transfer to bacteria and human cells to compare recruitment requirements by both T4SSs. Comparison with other reported relaxase translocation signals allowed us to determine two putative translocation sequence (TS) motifs, TS1 and TS2. Mutations affecting TS1 drastically affected conjugation frequencies, while mutations affecting either motif had only a mild effect on DNA transfer rates through the VirB/D4 T4SS of B. henselae. These results indicate that a single substrate can be recruited by two different T4SSs through different signals. The C terminus affected DNA transfer rates through both T4SSs tested, but no specific sequence requirement was detected. The addition of a Bartonella intracellular delivery (BID) domain, the translocation signal for the Bartonella VirB/D4 T4SS, increased DNA transfer up to 4% of infected human cells, providing an excellent tool for DNA delivery to specific cell types. We show that the R388 coupling protein TrwB is also required for this high-efficiency TrwC-BID translocation. Other elements apart from the coupling protein may also be involved in substrate recognition by T4SSs.     

Biological Diversity of Prokaryotic Type IV Secretion Systems

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.     

DNA Substrate-Induced Activation of the Agrobacterium VirB/VirD4 Type IV Secretion System

The bitopic membrane protein VirB10 of the Agrobacterium VirB/VirD4 type IV secretion system (T4SS) undergoes a structural transition in response to sensing of ATP binding or hydrolysis by the channel ATPases VirD4 and VirB11. This transition, detectable as a change in protease susceptibility, is required for DNA substrate passage through the translocation channel. Here, we present evidence that DNA substrate engagement with VirD4 and VirB11 also is required for activation of VirB10. Several DNA substrates (oncogenic T-DNA and plasmids RSF1010 and pCloDF13) induced the VirB10 conformational change, each by mechanisms requiring relaxase processing at cognate oriT sequences. VirD2 relaxase deleted of its translocation signal or any of the characterized relaxases produced in the absence of cognate DNA substrates did not induce the structural transition. Translocated effector proteins, e.g., VirE2, VirE3, and VirF, also did not induce the transition. By mutational analyses, we supplied evidence that the N-terminal periplasmic loop of VirD4, in addition to its catalytic site, is essential for early-stage DNA substrate transfer and the VirB10 conformational change. Further studies of VirB11 mutants established that three T4SS-mediated processes, DNA transfer, protein transfer, and pilus production, can be uncoupled and that the latter two processes proceed independently of the VirB10 conformational change. Our findings support a general model whereby DNA ligand binding with VirD4 and VirB11 stimulates ATP binding/hydrolysis, which in turn activates VirB10 through a structural transition. This transition confers an open-channel configuration enabling passage of the DNA substrate to the cell surface.     

Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis.     

Functional and topological characterization of novel components of the comB DNA transformation competence system in Helicobacter pylori

Helicobacter pylori is one of the most diverse bacterial species known. A rational basis for this genetic variation may be provided by its natural competence for genetic transformation and high-frequency recombination. Many bacterial competence systems have homology with proteins that are involved in the assembly of type IV pili and type II secretion systems. In H. pylori, DNA uptake relies on a transport system related to type IV secretion systems (T4SS) designated the comB system. The prototype of a T4SS in Agrobacterium tumefaciens consists of 11 VirB proteins and VirD4, which form the core unit necessary for the delivery of single proteins or large nucleoprotein complexes into target cells. In the past we identified proteins ComB4 and ComB7 through ComB10 as being involved in the process of DNA uptake in H. pylori. In this study we identified and functionally characterized further (T4SS-homologous) components of the comB transformation competence system. By combining computer prediction modeling, experimental topology determination, generation of knockout strains, and genetic complementation studies we identified ComB2, ComB3, and ComB6 as essential components of the transformation apparatus, structurally and functionally homologous to VirB2, VirB3, and VirB6, respectively. comB2, comB3, and comB4 are organized as a separate operon. Thus, for the H. pylori comB system, all T4SS core components have been identified except for homologues to VirB1, VirD4, VirB5, and VirB11.     

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants

The Type IV Secretion System (T4SS) is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP) is a key cytoplasmic protein that recruits the VirD2–T-DNA complex to the VirD4-coupling protein (VirD4 CP) of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of α-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems.     

Mechanism and structure of the bacterial type IV secretion systems

The bacterial type IV secretion systems (T4SSs) translocate DNA and protein substrates to bacterial or eukaryotic target cells generally by a mechanism dependent on direct cell-to-cell contact. The T4SSs encompass two large subfamilies, the conjugation systems and the effector translocators. The conjugation systems mediate interbacterial DNA transfer and are responsible for the rapid dissemination of antibiotic resistance genes and virulence determinants in clinical settings. The effector translocators are used by many Gram-negative bacterial pathogens for delivery of potentially hundreds of virulence proteins to eukaryotic cells for modulation of different physiological processes during infection. Recently, there has been considerable progress in defining the structures of T4SS machine subunits and large machine subassemblies. Additionally, the nature of substrate translocation sequences and the contributions of accessory proteins to substrate docking with the translocation channel have been elucidated. A DNA translocation route through the Agrobacterium tumefaciens VirB/VirD4 system was defined, and both intracellular (DNA ligand, ATP energy) and extracellular (phage binding) signals were shown to activate type IV-dependent translocation. Finally, phylogenetic studies have shed light on the evolution and distribution of T4SSs, and complementary structure-function studies of diverse systems have identified adaptations tailored for novel functions in pathogenic settings. This review summarizes the recent progress in our understanding of the architecture and mechanism of action of these fascinating machines, with emphasis on the ‘archetypal’ A. tumefaciens VirB/VirD4 T4SS and related conjugation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Structural and dynamic properties of bacterial Type IV secretion systems (Review)

The type IV secretion systems (T4SS) are widely distributed among the Gram-negative and –positive bacteria. These systems mediate the transfer of DNA and protein substrates across the cell envelope to bacterial or eukaryotic cells generally through a process requiring direct cell-to-cell contact. Bacteria have evolved T4SS for survival during establishment of pathogenic or symbiotic relationships with eukaryotic hosts. The Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation machines serve as models for detailed mechanistic studies aimed at elucidating the nature of translocation signals, machine assembly pathways and architectures, and the dynamics of substrate translocation. The A. tumefaciens VirB/D4 T4SS are polar-localized organelles composed of a secretion channel and an extracellular T pilus. These T4SS are assembled from 11 or more subunits. whose membrane topologies, intersubunit contacts and, in some cases, 3-dimensional structures are known. Recently, powerful in vivo assays have identified C-terminal translocation signals, defined for the first time the translocation route for a DNA substrate through a type IV secretion channel, and supplied evidence that ATP energy consumption contributes to a late stage of machine morphogenesis. Together, these recent findings describe the mechanics of type IV secretion in unprecedented detail.     

Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system

Type IV secretion systems (T4SS) are utilized by a wide range of Gram negative bacteria to deliver protein and DNA substrates to recipient cells. The best characterized T4SS are the type IVA systems, which exhibit extensive similarity to the Agrobacterium VirB T4SS. In contrast, type IVB secretion systems share almost no sequence homology to the type IVA systems, are composed of approximately twice as many proteins, and remain largely uncharacterized. Type IVB systems include the Dot/Icm systems found in the pathogens Legionella and Coxiella and the conjugative apparatus of IncI plasmids. Here we report the first extensive characterization of a type IVB system, the Legionella Dot/Icm secretion apparatus. Based on biochemical and genetic analysis, we discerned the existence of a critical five-protein subassembly that spans both bacterial membranes and comprises the core of the secretion complex. This transmembrane connection is mediated by protein dimer pairs consisting of two inner membrane proteins, DotF and DotG, which are able to independently associate with DotH/DotC/DotD in the outer membrane. The Legionella core subcomplex appears to be functionally analogous to the Agrobacterium VirB7-10 subcomplex, suggesting a remarkable conservation of the core subassembly in these evolutionarily distant type IV secretion machines.     

Type VI Secretion System Toxins Horizontally Shared between Marine Bacteria

The type VI secretion system (T6SS) is a widespread protein secretion apparatus used by Gram-negative bacteria to deliver toxic effector proteins into adjacent bacterial or host cells. Here, we uncovered a role in interbacterial competition for the two T6SSs encoded by the marine pathogen Vibrio alginolyticus. Using comparative proteomics and genetics, we identified their effector repertoires. In addition to the previously described effector V12G01_02265, we identified three new effectors secreted by T6SS1, indicating that the T6SS1 secretes at least four antibacterial effectors, of which three are members of the MIX-effector class. We also showed that the T6SS2 secretes at least three antibacterial effectors. Our findings revealed that many MIX-effectors belonging to clan V are “orphan” effectors that neighbor mobile elements and are shared between marine bacteria via horizontal gene transfer. We demonstrated that a MIX V-effector from V. alginolyticus is a functional T6SS effector when ectopically expressed in another Vibrio species. We propose that mobile MIX V-effectors serve as an environmental reservoir of T6SS effectors that are shared and used to diversify antibacterial toxin repertoires in marine bacteria, resulting in enhanced competitive fitness.     

Structural and dynamic properties of bacterial type IV secretion systems (review)

The type IV secretion systems (T4SS) are widely distributed among the gram-negative and -positive bacteria. These systems mediate the transfer of DNA and protein substrates across the cell envelope to bacterial or eukaryotic cells generally through a process requiring direct cell-to-cell contact. Bacteria have evolved T4SS for survival during establishment of pathogenic or symbiotic relationships with eukaryotic hosts. The Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation machines serve as models for detailed mechanistic studies aimed at elucidating the nature of translocation signals, machine assembly pathways and architectures, and the dynamics of substrate translocation. The A. tumefaciens VirB/D4 T4SS are polar-localized organelles composed of a secretion channel and an extracellular T pilus. These T4SS are assembled from 11 or more subunits. whose membrane topologies, intersubunit contacts and, in some cases, 3-dimensional structures are known. Recently, powerful in vivo assays have identified C-terminal translocation signals, defined for the first time the translocation route for a DNA substrate through a type IV secretion channel, and supplied evidence that ATP energy consumption contributes to a late stage of machine morphogenesis. Together, these recent findings describe the mechanics of type IV secretion in unprecedented detail.     

The Small Heat-shock Protein HspL Is a VirB8 Chaperone Promoting Type IV Secretion-mediated DNA Transfer

Agrobacterium tumefaciens is a plant pathogen that utilizes a type IV secretion system (T4SS) to transfer DNA and effector proteins into host cells. In this study we discovered that an α-crystallin type small heat-shock protein (α-Hsp), HspL, is a molecular chaperone for VirB8, a T4SS assembly factor. HspL is a typical α-Hsp capable of protecting the heat-labile model substrate citrate synthase from thermal aggregation. It forms oligomers in a concentration-dependent manner in vitro. Biochemical fractionation revealed that HspL is mainly localized in the inner membrane and formed large complexes with certain VirB protein subassemblies. Protein-protein interaction studies indicated that HspL interacts with VirB8, a bitopic integral inner membrane protein that is essential for T4SS assembly. Most importantly, HspL is able to prevent the aggregation of VirB8 fused with glutathione S-transferase in vitro, suggesting that it plays a role as VirB8 chaperone. The chaperone activity of two HspL variants with amino acid substitutions (F98A and G118A) for both citrate synthase and glutathione S-transferase-VirB8 was reduced and correlated with HspL functions in T4SS-mediated DNA transfer and virulence. This study directly links in vitro and in vivo functions of an α-Hsp and reveals a novel α-Hsp function in T4SS stability and bacterial virulence.     

Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion

Bacteria use type IV secretion systems (T4SS) to translocate DNA (T-DNA) and protein substrates across the cell envelope. By transfer DNA immunoprecipitation (TrIP), we recently showed that T-DNA translocates through the Agrobacterium tumefaciens VirB/D4 T4SS by forming close contacts sequentially with the VirD4 receptor, VirB11 ATPase, the inner membrane subunits VirB6 and VirB8 and, finally, VirB2 pilin and VirB9. Here, by TrIP, we show that nucleoside triphosphate binding site (Walker A motif) mutations do not disrupt VirD4 substrate binding or transfer to VirB11, suggesting that these early reactions proceed independently of ATP binding or hydrolysis. In contrast, VirD4, VirB11 and VirB4 Walker A mutations each arrest substrate transfer to VirB6 and VirB8, suggesting that these subunits energize this transfer reaction by an ATP-dependent mechanism. By co-immunoprecipitation, we supply evidence for VirD4 interactions with VirB4 and VirB11 independently of other T4SS subunits or intact Walker A motifs, and with the bitopic inner membrane subunit VirB10. We reconstituted substrate transfer from VirD4 to VirB11 and to VirB6 and VirB8 by co-synthesis of previously identified 'core' components of the VirB/D4 T4SS. Our findings define genetic requirements for DNA substrate binding and the early transfer reactions of a bacterial type IV translocation pathway.     

Acinetobacter baumannii Utilizes a Type VI Secretion System for Bacterial Competition

Type VI secretion systems (T6SS) are a class of macromolecular secretion machines that are utilized by a number of bacteria for inter-bacterial competition or to elicit responses in eukaryotic cells. Acinetobacter baumannii is an opportunistic pathogen that causes severe infections in humans. These infections, including pneumonia and bacteremia, are important, as they are often associated with hospitals and medical-settings where they disproportionally affect critically ill patients like those residing in intensive care units. While it is known that A. baumannii genomes carry genes whose predicted products have homology with T6SS-associated gene products from other bacteria, and secretion of a major T6SS structural protein Hcp has been demonstrated, no additional work on an A. baumannii T6SS has been reported. Herein, we demonstrated that A. baumannii strain M2 secretes Hcp and this secretion was dependent upon TssB, an ortholog of a bacteriophage contractile sheath protein, confirming that strain M2 produces a functional T6SS. Additionally, we demonstrated that the ability of strain M2 to out-compete Escherichia coli was reliant upon the products of tssB and hcp. Collectively, our data have provided the first evidence demonstrating function in inter-bacterial competition, for a T6SS produced by A. baumannii.     

Impact of Plasmids,Including Those EncodingVirB4/D4 Type IV Secretion Systems,on Salmonella enterica serovar Heidelberg Virulence in Macrophages and Epithelial Cells

Salmonella enterica serovar Heidelberg (S. Heidelberg) can cause foodborne illness in humans following the consumption of contaminated meat and poultry products. Recent studies from our laboratory have demonstrated that certain S. Heidelberg isolated from food-animal sources harbor multiple transmissible plasmids with genes that encode antimicrobial resistance, virulence and a VirB4/D4 type-IV secretion system. This study examines the potential role of these transmissible plasmids in bacterial uptake and survival in intestinal epithelial cells and macrophages, and the molecular basis of host immune system modulation that may be associated with disease progression. A series of transconjugant and transformant strains were developed with different combinations of the plasmids to determine the roles of the individual and combinations of plasmids on virulence. Overall the Salmonella strains containing the VirB/D4 T4SS plasmids entered and survived in epithelial cells and macrophages to a greater degree than those without the plasmid, even though they carried other plasmid types. During entry in macrophages, the VirB/D4 T4SS encoding genes are up-regulated in a time-dependent fashion. When the potential mechanisms for increased virulence were examined using an antibacterial Response PCR Array, the strain containing the T4SS down regulated several host innate immune response genes which likely contributed to the increased uptake and survival within macrophages and epithelial cells.     

A component of the Xanthomonadaceae type IV secretion system combines a VirB7 motif with a N0 domain found in outer membrane transport proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 Å X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7_XAC2622) and its interaction with VirB9. NMR solution studies show that residues 27–41 of the disordered flexible N-terminal region of VirB7_XAC2622 interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7_XAC2622 has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7_XAC2622 oligomerizes through interactions involving conserved residues in the N0 domain and residues 42–49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB7_XAC2622 oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.     

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.