An IcmF Family Protein,ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL,and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens

An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpL_M, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpL_M and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL_M is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impL_M mutants with substitutions or deletions in the Walker A motif failed to complement the impL_M deletion mutant for Hcp secretion, which provided evidence that ImpL_M may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL_M and another essential T6SS component, ImpK_L. Topology and biochemical fractionation analyses suggested that ImpK_L is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL_M-ImpK_L interaction domains suggested that ImpL_M interacts with ImpK_L via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL_M interacts with ImpK_L, and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.Many pathogenic gram-negative bacteria employ protein secretion systems formed by macromolecular complexes to deliver proteins or protein-DNA complexes across the bacterial membrane. In addition to the general secretory (Sec) pathway (18, 52) and twin-arginine translocation (Tat) pathway (7, 34), which transport proteins across the inner membrane into the periplasm, at least six distinct protein secretion systems occur in gram-negative bacteria (28, 46, 66). These systems are able to secrete proteins from the cytoplasm or periplasm to the external environment or the host cell and include the well-documented type I to type V secretion systems (T1SS to T5SS) (10, 15, 23, 26, 30) and a recently discovered type VI secretion system (T6SS) (4, 8, 22, 41, 48, 49). These systems use ATPase or a proton motive force to energize assembly of the protein secretion machinery and/or substrate translocation (2, 6, 41, 44, 60).Agrobacterium tumefaciens is a soilborne pathogenic gram-negative bacterium that causes crown gall disease in a wide range of plants. Using an archetypal T4SS (9), A. tumefaciens translocates oncogenic transferred DNA and effector proteins to the host and ultimately integrates transferred DNA into the host genome. Because of its unique interkingdom DNA transfer, this bacterium has been extensively studied and used to transform foreign DNA into plants and fungi (11, 24, 40, 67). In addition to the T4SS, A. tumefaciens encodes several other secretion systems, including the Sec pathway, the Tat pathway, T1SS, T5SS, and the recently identified T6SS (72). T6SS is highly conserved and widely distributed in animal- and plant-associated Proteobacteria and plays an important role in the virulence of several human and animal pathogens (14, 19, 41, 48, 56, 63, 74). However, T6SS seems to play only a minor role or even a negative role in infection or virulence of the plant-associated pathogens or symbionts studied to date (5, 37-39, 72).T6SS was initially designated IAHP (IcmF-associated homologous protein) clusters (13). Before T6SS was documented by Pukatzki et al. in Vibrio cholerae (48), mutations in this gene cluster in the plant symbiont Rhizobium leguminosarum (5) and the fish pathogen Edwardsiella tarda (51) caused defects in protein secretion. In V. cholerae, T6SS was responsible for the loss of cytotoxicity for amoebae and for secretion of two proteins lacking a signal peptide, hemolysin-coregulated protein (Hcp) and valine-glycine repeat protein (VgrG). Secretion of Hcp is the hallmark of T6SS. Interestingly, mutation of hcp blocks the secretion of VgrG proteins (VgrG-1, VgrG-2, and VgrG-3), and, conversely, vgrG-1 and vgrG-2 are both required for secretion of the Hcp and VgrG proteins from V. cholerae (47, 48). Similarly, a requirement of Hcp for VgrG secretion and a requirement of VgrG for Hcp secretion have also been shown for E. tarda (74). Because Hcp forms a hexameric ring (41) stacked in a tube-like structure in vitro (3, 35) and VgrG has a predicted trimeric phage tail spike-like structure similar to that of the T4 phage gp5-gp27 complex (47), Hcp and VgrG have been postulated to form an extracellular translocon. This model is further supported by two recent crystallography studies showing that Hcp, VgrG, and a T4 phage gp25-like protein resembled membrane penetration tails of bacteriophages (35, 45).Little is known about the topology and structure of T6SS machinery subunits and the distinction between genes encoding machinery subunits and genes encoding regulatory proteins. Posttranslational regulation via the phosphorylation of Fha1 by a serine-threonine kinase (PpkA) is required for Hcp secretion from Pseudomonas aeruginosa (42). Genetic evidence for P. aeruginosa suggested that the T6SS may utilize a ClpV-like AAA⁺ ATPase to provide the energy for machinery assembly or substrate translocation (41). A recent study of V. cholerae suggested that ClpV ATPase activity is responsible for remodeling the VipA/VipB tubules which are crucial for type VI substrate secretion (6). An outer membrane lipoprotein, SciN, is an essential T6SS component for mediating Hcp secretion from enteroaggregative Escherichia coli (1). A systematic study of the T6SS machinery in E. tarda revealed that 13 of 16 genes in the evp gene cluster are essential for secretion of T6S substrates (74), which suggests the core components of the T6SS. Interestingly, most of the core components conserved in T6SS are predicted soluble proteins without recognizable signal peptide and transmembrane (TM) domains.The intracellular multiplication F (IcmF) and H (IcmH) proteins are among the few core components with obvious TM domains (8). In Legionella pneumophila Dot/Icm T4SSb, IcmF and IcmH are both membrane localized and partially required for L. pneumophila replication in macrophages (58, 70, 75). IcmF and IcmH are thought to interact with each other in stabilizing the T4SS complex in L. pneumophila (58). In T6SS, IcmF is one of the essential components required for secretion of Hcp from several animal pathogens, including V. cholerae (48), Aeromonas hydrophila (63), E. tarda (74), and P. aeruginosa (41), as well as the plant pathogens A. tumefaciens (72) and Pectobacterium atrosepticum (39). In E. tarda, IcmF (EvpO) interacted with IcmH (EvpN), EvpL, and EvpA in a yeast two-hybrid assay, and its putative nucleotide-binding site (Walker A motif) was not essential for secretion of T6SS substrates (74).In this study, we characterized the topology and interactions of the IcmF and IcmH family proteins ImpL_M and ImpK_L, which are two essential components of the T6SS of A. tumefaciens. We adapted the nomenclature proposed by Cascales (8), using the annotated gene designation followed by the letter indicated by Shalom et al. (59). Our data indicate that ImpL_M and ImpK_L are both integral inner membrane proteins and interact with each other via their N-terminal domains residing in the cytoplasm. We also provide genetic evidence showing that ImpL_M may function as a nucleoside triphosphate (NTP)-binding protein or nucleoside triphosphatase to mediate T6S machinery assembly and/or substrate secretion.     

Vp1659 Is a Vibrio parahaemolyticus Type III Secretion System 1 Protein That Contributes to Translocation of Effector Proteins Needed To Induce Cytolysis,Autophagy, and Disruption of Actin Structure in HeLa Cells

Vibrio parahaemolyticus harbors two type III secretion systems (T3SSs; T3SS1 and T3SS2), of which T3SS1 is involved in host cell cytotoxicity. T3SS1 expression is positively regulated by ExsA, and it is negatively regulated by ExsD. We compared the secretion profiles of a wild-type strain (NY-4) of V. parahaemolyticus with those of an ExsD deletion mutant (ΔexsD) and with a strain of NY-4 that overexpresses T3SS1 (NY-4:pexsA). From this comparison, we detected a previously uncharacterized protein, Vp1659, which shares some sequence homology with LcrV from Yersinia. We show that vp1659 expression is positively regulated by ExsA and is negatively regulated by ExsD. Vp1659 is specifically secreted by T3SS1 of V. parahaemolyticus, and Vp1659 is not required for the successful extracellular secretion of another T3SS1 protein, Vp1656. Mechanical fractionation showed that Vp1659 is translocated into HeLa cells in a T3SS1-dependent manner and that deletion of Vp1659 does not prevent VopS from being translocated into HeLa cells during infection. Deletion of vp1659 significantly reduces cytotoxicity when HeLa cells are infected by V. parahaemolyticus, while complementation of the Δvp1659 strain restores cytotoxicity. Differential staining showed that Vp1659 is required to induce membrane permeability in HeLa cells. We also show evidence that Vp1659 is required for actin rearrangement and the induction of autophagy. On the basis of these data, we conclude that Vp1659 is a T3SS1-associated protein that is a component of the secretion apparatus and that it is necessary for the efficient translocation of effector proteins into epithelial cells.As a marine pathogen, Vibrio parahaemolyticus is frequently isolated from seafood products such as oysters and shrimp (19, 45). The main symptoms of V. parahaemolyticus infection in humans include diarrhea, nausea, and vomiting. In addition to the gastrointestinal infection, necrotizing fasciitis and septic shock are reportedly associated with V. parahaemolyticus infection (37). V. parahaemolyticus can also cause wound infections after contact with contaminated water (6, 7, 16, 37).V. parahaemolyticus is able to adhere to and invade epithelial cells (1, 38, 43). Pili are involved in the adherence to the intestinal epithelium (32), but it is not clear what factors are required for V. parahaemolyticus to invade epithelial cells. Hemolysins are considered primary factors involved in the pathogenesis of V. parahaemolyticus. For example, a thermostable direct hemolysin (tdh) mutant strain loses the ability to cause fluid accumulation in the intestinal lumen (33), while deletion of a tdh-related gene (trh) results in the complete loss of hemolysis and the partial loss of fluid accumulation in a rabbit intestinal ligation model (42). Recent studies show that the disruption of epithelial tight junctions, which is a hallmark of bacterial dissemination into the circulatory system and subsequent septicemia, is independent of the thermostable direct hemolysin, suggesting that additional factors are required for the pathogenesis of V. parahaemolyticus (27).A broad range of Gram-negative bacteria employ type III secretion systems (T3SSs) to export virulence-related proteins into the extracellular milieu and/or to deliver these proteins directly into host cells (5, 12, 13). T3SSs are composed of three parts: a secretion apparatus, translocators, and effectors (17, 18). The secretion apparatus and translocators are encoded by ca. 25 genes that are conserved and usually located in a genomic island. Genes that encode effectors are less conserved and can be found distal from the T3SS islands. The secretion apparatus serves to secrete both effectors and translocators from bacterial cells, and translocators help the effectors cross into the eukaryotic cells, where they can disrupt normal host cell signal functions.Two distinct T3SSs (T3SS1 and T3SS2) were identified in the genome of V. parahaemolyticus (28). On the basis of the sequence similarity and gene organization, T3SS1 was classified as a member of the Ysc family of secretion systems, while T3SS2 was classified as a member of the Inv-Mxi-Spa family (40). Functional analysis shows that deletion of T3SS1 decreases cytotoxicity against HeLa cells, while deletion of T3SS2 diminishes intestinal fluid accumulation (35). Interestingly, in some strains, T3SS2 can be involved in the cytotoxic effect specifically against Caco-2 and HCT-8 cells (23). One study showed that T3SS1 of V. parahaemolyticus induces autophagy, but blocking autophagy does not completely mitigate cytotoxicity, indicating that other T3SS1-induced mechanisms contribute to cell death (3, 4). Recent work from our laboratory showed that V. parahaemolyticus induces cell rounding, pore formation, and membrane damage, consistent with the induction of an oncosis pathway (46). Importantly, treatment of infected cells with an osmoprotectant (polyethylene glycol 3350) significantly reduced cytotoxicity, indicating that oncosis is the primary mechanism by which T3SS1 of V. parahaemolyticus causes cell death for in vitro cultures (46). Nevertheless, it is unknown which effector protein(s) is involved in cell cytotoxicity. By comparing the secretion protein profiles of wild-type and T3SS1 mutant strains, four T3SS1 proteins have been identified (34). Among these, Vp1680 is translocated into host cells and is required for the induction of autophagy during infection of HeLa cells (3, 34). Recent studies showed that VopS is able to prevent the interaction of Rho GTPase with its downstream factors by a new modification mechanism, called AMPylation (44), and this prevents the assembly of actin fibers. Two proteins (VopT and VopL) have been identified as T3SS2 substrates (23, 26). VopT is a member of ADP-ribosyltransferase and is partially responsible for the cytotoxic effect specific to Caco-2 and HCT-8 cells (23). VopL induces the assembly of actin stress fibers (26) and is potentially responsible for the internalization of V. parahaemolyticus into Caco-2 cells (1). Many other potential effector proteins are encoded proximal to T3SS1 and T3SS2 apparatus genes, but these have not been functionally characterized. The function of structural genes has not been extensively studied for either T3SS1 or T3SS2 in V. parahaemolyticus.T3SSs are expressed after contact with host cells or when cells are grown under inducing conditions (17). Expression of T3SS1 in V. parahaemolyticus is induced when bacteria are grown in tissue culture medium (Dulbecco''s minimal essential medium [DMEM]), although the secretion of one substrate (Vp1656) was not detected under this condition, probably due to the low detection sensitivity (47). T3SS1 genes are not expressed when bacteria are grown in LB medium supplemented with 2.5% NaCl (LB-S). Disruption of the exsD gene or overexpression of exsA results in the constitutive expression of T3SS1 genes and the secretion of Vp1656 even when bacteria are grown in LB-S (47). For the present study, we took advantage of these regulatory mechanisms and compared the proteins secreted by the NY-4 (wild type), ΔexsD, ΔexsD::pexsD (exsD complement), and NY-4:pexsA strains. We identified two proteins (VopS and Vp1659) that are present in the supernatants of the ΔexsD and NY-4:pexsA strains but that are absent in the supernatants of the NY-4 and ΔexsD::pexsD strains. Herein we demonstrate that Vp1659 is secreted into the extracellular milieu and is translocated into HeLa cells by T3SS1. Functional analysis is consistent with the hypothesis that Vp1659 plays a role in actin rearrangement and induction of cytotoxicity and autophagy.     

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.     

Functional Dissection of the Conjugative Coupling Protein TrwB

The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwBΔN70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.Bacterial conjugation can be viewed mechanistically as a rolling-circle replication system linked to a type IV secretion process. The two processes come into contact through the activity of a protein that couples the plasmid replication machinery to the export system in the membrane, allowing horizontal dissemination of the replicating DNA molecule (35). This key protein is called “coupling protein” (here “T4CP” for “type IV CP”). It is present in all conjugative systems as well as in many type IV secretion systems (T4SS) involved in bacterial virulence (16). The secreted substrate in bacterial conjugation is the relaxase or pilot protein, attached to the DNA strand. The shoot-and-pump model for bacterial conjugation proposes that, after secretion of the protein through the T4SS, the T4CP works as a motor for export of the rest of the DNA molecule (36). In addition to its presumed role as a DNA transporter, TrwB is also required for transport of relaxase TrwC in the absence of DNA transfer (15).In accordance with its proposed coupling activity, early genetic experiments made patent that the function of conjugative T4CPs depended on interactions with both the cytoplasmic substrate complex (the relaxosome) and the T4SS (6, 7). Thus, T4CP interactions with other conjugation proteins are a key aspect of their function. There have been several reports of interactions between T4CPs from conjugative plasmids and either relaxosomal components—such as F-TraD with TraM (14, 38), RP4-TraG with TraI (49), and pCF10-PcfC with PcfF and PcfG (11)—or T4SS components such as R27-TraG with TrhB (17). T4CP-T4SS interactions have also been reported for the VirB/D4 T4SS involved in DNA transfer from Agrobacterium tumefaciens to plant cells (1, 9). Both sets of interactions have only been concurrently shown for TrwB, the T4CP of plasmid R388. TrwB interacts with proteins TrwA and TrwC, which form the R388 relaxosome, and with the R388 T4SS component TrwE (37). While the interaction with the relaxosome is highly specific for its cognate system (24, 37, 48), the interaction between the T4CP and the T4SS is less specific: a single T4CP can interact functionally with several conjugative T4SS. Interestingly, a correlation was observed between the strength of the T4CP-TrwE-like interaction and the efficiency of DNA transfer (37). T4CPs also interact with TrwE-like components of T4SS involved in virulence (13). In the case of the highly related Trw T4SS systems of plasmid R388 and the human pathogen Bartonella, it was further demonstrated that R388 TrwE could be functionally replaced by the Bartonella tribocorum TrwE homolog, TrwE_Bt (13).T4CPs are integral membrane proteins anchored to the inner membrane by an N-terminal transmembrane domain (TMD). The soluble cytoplasmic domain of TrwB (TrwBΔN70), lacking this TMD, has been biochemically and structurally analyzed in detail. It retains the ability to bind NTPs and to unspecifically bind DNA (42). The characterization of its DNA-dependent ATPase activity (53) strengthened the possibility that T4CPs work as DNA motors. This activity is also stimulated by the oriT-binding protein TrwA (52).The determination of the three-dimensional (3D) structure of TrwBΔN70 indicated a quaternary structure consisting of hexamers that form an almost spherical, orange-shaped structure with a 20-Å inner channel (ICH) (18, 19). Each monomer is composed of two main structural domains: the nucleotide-binding domain (NBD) and the all-alpha domain (AAD). The NBD has α/β topology and is reminiscent of RecA and DNA ring helicases. The AAD is facing the cytoplasmic side and bears significant structural similarity to the N-terminal domain of site-specific recombinase XerD and also to a 40-residue segment of the DNA binding domain of protein TraM, the component of the relaxosome of F-like plasmids that interacts with its cognate T4CP, TraD. The structure of the hexamer as a whole resembles that of the F₁-ATPase, raising interesting perspectives into the possible way of action of coupling proteins as molecular motors in conjugation (5).There have been several attempts to functionally dissect T4CPs. In F-TraD, it was determined that its C terminus is essential for relaxosomal specificity, probably through an interaction with TraM (4, 39, 48). The cytoplasmic domain of the related TraD protein of plasmid R1 stimulates both transesterase and helicase activities of its cognate relaxase, TraI (41, 51). A series of random mutations were shown to affect TraD oligomerization (23). In VirD4, the T4CP of the VirB T4SS of A. tumefaciens, both the periplasmic domain plus key residues of the NBD are required for its location at the cell poles (31); its interaction with the T4SS protein substrate VirE2 does not require the N-terminal TMD (2). Mutational analysis of R27 TraG showed that the periplasmic residues are essential for interaction with the T4SS (22). An N-terminal deletion variant of PcfC, the T4CP of the Enterococcus plasmid pCF10, loses its membrane localization but retains its ability to bind relaxosomal components (11). Biochemical analysis of full-length R388 TrwB showed that the N-terminal TMD stabilizes the protein, aids oligomerization, and affects nucleotide selection (25-27). This region is essential for T4SS interaction, but TrwBΔN70 retains the ability to interact with the relaxosomal components TrwA and TrwC (37). Taken together, these analyses suggested that the N-terminal TMD of the T4CPs is necessary for T4SS interaction, oligomerization, and cellular location and that the C-terminal cytoplasmic domain is necessary for relaxosomal interactions and ATPase activity associated with DNA transport.In this study, we set up different assays to search for mutants affecting TrwB function in DNA and protein transfer. We constructed a series of TrwB point mutants based on the 3D structure of TrwBΔN70. Most selected residues were essential for TrwB function in conjugation, especially under conditions where TrwB was in limiting quantities. We analyzed the in vivo properties of selected mutants with a battery of in vivo assays to map functional domains. Also, random mutants in the TMD were screened for improved interactions with the T4SS, which allowed mapping of the TrwB-TrwE interaction domain.     

Thioredoxin 1 Participates in the Activity of the Salmonella enterica Serovar Typhimurium Pathogenicity Island 2 Type III Secretion System

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium relies on its Salmonella pathogenicity island 2 (SPI2) type III secretion system (T3SS) for intracellular replication and virulence. We report that the oxidoreductase thioredoxin 1 (TrxA) and SPI2 are coinduced for expression under in vitro conditions that mimic an intravacuolar environment, that TrxA is needed for proper SPI2 activity under these conditions, and that TrxA is indispensable for SPI2 activity in both phagocytic and epithelial cells. Infection experiments in mice demonstrated that SPI2 strongly contributed to virulence in a TrxA-proficient background whereas SPI2 did not affect virulence in a trxA mutant. Complementation analyses using wild-type trxA or a genetically engineered trxA coding for noncatalytic TrxA showed that the catalytic activity of TrxA is essential for SPI2 activity in phagocytic cells whereas a noncatalytic variant of TrxA partially sustained SPI2 activity in epithelial cells and virulence in mice. These results show that TrxA is needed for the intracellular induction of SPI2 and provide new insights into the functional integration between catalytic and noncatalytic activities of TrxA and a bacterial T3SS in different settings of intracellular infections.In Escherichia coli, thioredoxin 1 (TrxA, encoded by trxA) is an evolutionary conserved 11-kDa cytosolic highly potent reductase that supports the activities of various oxidoreductases and ribonucleotide reductases (1, 29) and interacts with a number of additional cytoplasmic proteins through the formation of temporary covalent intermolecular disulphide bonds (32). Consequently, as trxA mutants of E. coli (51), Helicobacter pylori (13), and Rhodobacter sphaeroides (34) show increased sensitivity to hydrogen peroxide, TrxA has been defined as a significant oxidoprotectant. In addition, TrxA possess a protein chaperone function that is disconnected from cysteine interactions (30, 32).Salmonella enterica serovar Typhimurium is closely related to E. coli. During divergent evolution, the Salmonella genome acquired a number of virulence-associated genes (20). Many of these genes are clustered on genetic regions termed Salmonella pathogenicity islands (or SPIs). Of these, SPI1 and SPI2 code for separate type III secretion systems (T3SSs). T3SSs are supramolecular virulence-associated machineries that, in several pathogenic gram-negative bacterial species, enable injection of effector proteins from the bacteria into host cells (22, 57). The effector proteins, in turn, manipulate intrinsic host cell functions to facilitate the infection.The SPI1 T3SS of S. serovar Typhimurium is activated for expression in the intestine in response to increased osmolarity and decreased oxygen tension (22, 57). SPI1 effector proteins are primarily secreted into cells that constitute the epithelial layer and interfere with host cell Cdc42 and Rac-1 signaling and actin polymerization. This enables the bacteria to orchestrate their own actin-dependent uptake into nonphagocytic cells (57). SPI1 effector proteins also induce inflammatory signaling and release of interleukin-1β from infected cells (25, 26).Subsequent systemic progression of S. serovar Typhimurium from the intestinal tissue relies heavily on an ability to survive and replicate in phagocytic cells (18, 46, 53, 54). S. serovar Typhimurium uses an additional set of effector proteins secreted by the SPI2 T3SS for replication inside host cells and for coping with phagocyte innate responses to the infection (10, 11, 54). The functions of SPI2 effectors include diversion of vesicular trafficking, induction of apoptotic responses, and manipulation of ubiquitination of host proteins (28, 40, 45, 53). Hence, SPI2 effector proteins create a vacuolar environment that sustains intracellular replication of S. serovar Typhimurium (28).In addition to pathogenicity islands, the in vivo fitness of Salmonella spp. relies on selected functions shared with other enterobacteria. Thus, many virulence genes are integrated into “housekeeping” gene regulatory networks, coded for by a core genome, which steer bacterial stress responses (12, 17, 27, 55). Selected anabolic pathways also contribute to virulence of S. serovar Typhimurium (18, 27), evidently by providing biochemical building blocks for bacterial replication (36).In S. serovar Typhimurium, TrxA is a housekeeping protein that strongly contributes to virulence in cell culture and mouse infection models (8). However, the mechanism by which TrxA activity adds to virulence has not been defined. Here we show that the contribution of TrxA to virulence of S. serovar Typhimurium associates with its functional integration with the SPI2 T3SS under conditions that prevail in the intracellular vacuolar compartment of the host cell. These findings ascribe a novel role to TrxA in bridging environmental adaptations with virulence gene expression and illuminate a new aspect of the interaction between evolutionary conserved and horizontally acquired gene functions in bacteria.     

Inhibition of Selenium Metabolism in the Oral Pathogen Treponema denticola

In this report we provide evidence that the antimicrobial action of stannous salts and a gold drug, auranofin, against Treponema denticola is mediated through inhibition of the metabolism of selenium for synthesis of selenoproteins.The biological use of selenium as a catalyst, incorporated into proteins as selenocysteine, is broad. It plays an essential role in energy metabolism, redox balance, and reproduction in a variety of organisms, from bacterial pathogens to eukaryotic parasites to humans. The results of several epidemiological studies indicate that higher levels of selenium in the mammalian diet can have a negative effect on dental health (2, 17-19, 39). Although the impact of selenium is attributed to its influence on the physical properties of the enamel surface (10), the role of selenium in supporting the oral microbial community has not been studied.The oral cavity is a highly complex microbiome, with a large proportion of its residents uncharacterized due to their fastidious nature and resistance to traditional culture methods (11). Analysis of whole saliva indicates that bacterial metabolism influences the amino acid composition and indicates a role for amino acid fermentation (38). Curtis et al. demonstrated the occurrence of Stickland reactions in dental plaque (9). These reactions were first described in clostridia (35-37). They involve the coupled fermentation of amino acids in which one amino acid is oxidized (Stickland donor) and another (Stickland acceptor) is reduced (29). Treponema denticola, an established resident of the oral cavity, performs Stickland reactions via the selenoprotein glycine reductase (32). Glycine reductase is composed of a multiprotein complex that contains two separate selenoproteins, termed selenoprotein A and selenoprotein B (1, 7, 8, 15, 16). This complex of proteins converts glycine to acetyl phosphate by using inorganic phosphate and the reducing potential from thioredoxin. For the organisms that use this complex, this is a vital source of ATP. Thus far, the requirement for selenocysteine at the active site of this enzyme complex is universally conserved, even though all other selenoproteins that have been identified using computational techniques have a putative cysteine homologue (24).Treponema denticola is considered one of the primary pathogens responsible for periodontitis, a chronic inflammatory disease that is the major cause of adult tooth loss (11, 27, 33). It is the best-studied oral spirochete, commonly found with other spirochetes within the periodontal pocket. It expresses a variety of virulence factors and is capable of adhering to and penetrating endothelial cell monolayers (31). Its health impact may reach beyond the oral cavity. A recent study linked periodontitis with peripheral arterial disease and detected T. denticola, along with other periodontal pathogens, in atherosclerotic plaque (3). Sequence analysis indicates the presence of several selenoproteins in addition to glycine reductase within the genome of T. denticola (24). This organism exhibits a strict growth requirement for selenium (32).A significant literature exists that clearly demonstrates the antimicrobial activity of fluoride compounds against microorganisms associated with dental decay and periodontitis. Both sodium fluoride and stannous fluoride, as well as stannous ions alone, inhibit the growth of T. denticola (21). The inhibitory effect of stannous salts on T. denticola''s growth is unexplained. It should be noted that toothpastes containing stannous fluoride are more effective in reducing gingivitis and plaque (28, 30).Tin, as well as several other trace elements, modulates the effects of acute selenium toxicity (20). Conversely, selenium affects the activity of tin in animal models (4-6). In this study, we examine the possibility that stannous ions interfere with selenium metabolism in T. denticola.     

Activation of ExoU Phospholipase Activity Requires Specific C-Terminal Regions

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that utilizes a type III secretion system to subvert host innate immunity. Of the 4 known effector proteins injected into eukaryotic cells, ExoS and ExoU are cytotoxic. The cytotoxic phenotype of ExoU depends on the enzymatic activity of the patatin-like phospholipase A₂ domain localized to the N-terminal half of the protein. Amino acid residues located within the C-terminal region of ExoU are postulated to be required for trafficking or localization to the plasma membrane of eukaryotic cells. This report describes the characterization of a transposon-based linker insertion library in ExoU. Utilizing an unbiased screening approach and sensitive methods for measuring enzymatic activity, we identified regions of ExoU that are critical for activation of the phospholipase activity by the only known cofactor, SOD1. Insertions at D572 and L618 reduced the rate of substrate cleavage. Enzymatic activity could be restored to almost parental levels when SOD1 concentrations were increased, suggesting that the linker insertion disrupted the interaction between ExoU and SOD1. An enzyme-linked immunosorbent assay (ELISA)-based binding test was developed to measure ExoU-SOD1 binding. These experiments suggest that ExoU activation by SOD1 is hampered by linker insertion. ExoU derivatives harboring minimal phospholipase activity retained biological activity in tissue culture assays. These proteins affected primarily cellular architecture in a manner similar to that of ExoT. Our studies suggest that conformational changes in ExoU are facilitated by SOD1. Importantly, the level of phospholipase activity influences the biological outcome of ExoU intoxication.Pseudomonas aeruginosa is a Gram-negative bacterium responsible for severe and potentially fatal opportunistic infections. As a contributor to nosocomial infections, P. aeruginosa is a leading cause of hospital-acquired and ventilator-associated pneumonias (40). Furthermore, P. aeruginosa is responsible for ulcerative keratitis and ocular disease found in conjunction with the use of soft contact lenses (2, 10, 54). Infections with this pathogen are of critical concern for individuals admitted with severe burns, due to the bacterium''s ability to colonize and persist in damaged tissues (35). Patients suffering from cystic fibrosis often succumb to severe lung infections and inflammation due to colonization with antibi otic-resistant, mucoid strains of P. aeruginosa (3). The expression of multiple efflux pumps and the ability to inactivate and modify antibiotics make P. aeruginosa dangerous and difficult to treat (27). Several investigators are exploring ways, as adjuncts or alternatives to antibiotic treatment, to neutralize virulence factors that contribute to the ability of P. aeruginosa to suppress host innate and adaptive immune responses (17, 21, 22, 52).Many Gram-negative bacteria, including P. aeruginosa, encode one or more type III secretion systems (T3SS), which are thought to aid in pathogenesis and increase disease severity (19, 32, 39). Four effectors are translocated by the T3SS of P. aeruginosa and include ExoS, ExoT, ExoU, and ExoY (8, 23, 56, 57). The activity of each effector is dependent upon interaction with a cofactor present in eukaryotic but not prokaryotic cells. ExoS and ExoT are bifunctional enzymes that possess both Rho GTPase-activating protein and ADP-ribosyltransferase activities (23, 25, 51). The ADP ribosylation of eukaryotic proteins by ExoS and ExoT requires activation by members of the 14-3-3 family of scaffolding proteins (13). ExoY is an adenylyl cyclase that causes the accumulation of cyclic AMP (cAMP) in intoxicated cells. The eukaryotic cofactor required for ExoY activity has not been identified (57). ExoU, a potent A₂ phospholipase responsible for membrane disruption and cellular lysis, requires superoxide dismutase 1 (SOD1) for the detection of enzymatic activity (43, 46).ExoU is an important virulence factor of P. aeruginosa, as it causes rapid cell death during in vitro infections and is associated with poor clinical outcomes (19, 39, 44). Several studies have used truncation analyses, linker mutagenesis, and site-specific amino acid substitutions to define regions of ExoU important for various functions (7, 36). ExoU is a 74-kDa, hydrophilic, and slightly acidic protein with a pI of 5.9 (8). The first 52 amino acids are required for interaction with the chaperone SpcU and may be important for translocation through the T3SS (7, 9). Enzymatic activity is attributed to the patatin-like phospholipase domain located between residues 107 and 357 (34, 46). Two catalytic residues, S142 and D344, and a sequence encoding an oxyanion hole (₁₁₂GGAK₁₁₅) are located within this domain (34, 46). The oxyanion hole is thought to stabilize the negative charge of the intermediate structure during substrate cleavage (5). C-terminal residues of ExoU, specifically the last 137 amino acids, have been implicated in membrane localization after translocation into mammalian cells (37). The domain or region(s) required for the activation of ExoU by SOD1 have not been identified.In this study, linker-scanning mutagenesis (the insertion of 15 nucleotides randomly throughout the coding sequence) was used to identify regions of exoU that impair activation of phospholipase activity by SOD1. Our data support the model that SOD1 may be facilitating the activation of ExoU by altering the conformational properties of the enzyme. Understanding the molecular mechanisms mediating SOD1 and ExoU interaction may contribute to the design of therapeutics for the treatment of acute P. aeruginosa infections.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.