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.     

Agrobacterium tumefaciens Type IV Secretion Protein VirB3 Is an Inner Membrane Protein and Requires VirB4, VirB7, and VirB8 for Stabilization

Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures.Bacteria use type IV secretion (T4S) to deliver macromolecules to prokaryotes and eukaryotes (12). Animal and human pathogens deliver proteins to their eukaryotic hosts to affect cellular processes causing disease. The plant-pathogenic bacterium Agrobacterium tumefaciens delivers both proteins and DNA to plants and other eukaryotes. DNA delivered by Agrobacterium directs constitutive synthesis of phytohormones in a transformed plant cell, promoting cancerous growth (56). The Ptl toxin of Bordetella pertussis modifies G proteins by ADP-ribosylation, affecting intracellular cell signaling, and CagA of Helicobacter pylori disrupts epithelial cell polarity by inhibiting PAR1 kinase activity (37, 44, 47). T4S is ancestrally related to bacterial conjugation, a mechanism used by bacteria for interbacterial plasmid transfer, enabling them to acquire novel genes for antibiotic resistance, degradation of organic molecules, toxin production, and other virulence traits (29).The VirD4/VirB family of proteins, found conserved in many alphaproteobacteria, mediates T4S (12). The Ti plasmid-encoded Agrobacterium T4S system requires VirD4 and 11 VirB proteins, VirB1 to VirB11, for efficient DNA transfer (7, 54). The membrane and membrane-associated VirB proteins assemble a macromolecular structure at the cell membrane to promote substrate transfer (12). The octopine Ti plasmid pTiA6NC-encoded VirB6 to VirB11 proteins assemble the T4S apparatus at a cell pole (34, 35, 39). The VirD4 coupling protein targets the VirE2 substrate protein to the cell pole (4). A recent study found that the nopaline Ti plasmid pTiC58 T4S system (T4SS) and its substrates form a helical array around the cell circumference (1). Structural studies using Escherichia coli conjugative plasmid pKM101-encoded VirB homologues showed that TraN (VirB7), TraO (VirB9), and TraF (VirB10) form the core complex and that TraF forms a channel at the outer membrane (11, 23). The Agrobacterium VirB proteins assemble a T-pilus, an appendage composed primarily of VirB2, with VirB5 and VirB7 as its minor constituents (38, 40, 41, 48, 50, 55). VirB3, a homolog of the pilin-like TraL protein encoded in E. coli plasmids, is postulated to function in T-pilus assembly (52). Three ATP-utilizing proteins, VirB4, VirB11, and VirD4, supply energy for substrate translocation (3, 9, 34).The membrane topology of all the VirB proteins, except for VirB3, was determined by analyses of random phoA insertion mutants, targeted phoA fusions, and targeted bla fusions (6, 14, 15, 21, 22, 31, 35, 53). phoA and bla, which encode alkaline phosphatase and β-lactamase, respectively, serve as excellent markers for periplasmic proteins, as they are enzymatically active only when targeted to the cell periplasm (8, 30). Green fluorescent protein (GFP) is an ideal cytoplasmic marker because it fluoresces only when located in the cytoplasm (19, 20). When GFP is targeted to the periplasm through fusion with a membrane-spanning domain (MSD), it fails to fold properly and does not fluoresce.The prevailing view, based on in silico analysis, is that VirB3 is a bitopic membrane protein with a periplasmic C terminus. No phoA-positive insertions in virB3, however, were identified in two random mutagenesis studies of the virB operon (6, 15). The small size of VirB3, a polypeptide of 108 amino acids (aa), could be a contributing factor to the negative findings. Yet several PhoA-positive insertions in two smaller VirB proteins, VirB2 (74-aa mature peptide) and VirB7 (41-aa mature peptide), were successfully obtained in both studies. Therefore, the negative findings may also be indicative of the presence of a small periplasmic domain in VirB3. Biochemical studies showed that the nopaline Ti plasmid pTiC58-encoded VirB3 protein (pTiC58 VirB3) associates with the bacterial outer membrane, while VirB2 associates with both the inner and outer membranes (52). The pTiC58 VirB4 protein is required for localization of VirB3 to the outer membrane (33). VirB4 is also required for VirB3 stability (33, 55). A low level of VirB3 accumulated in a nonpolar pTiC58 virB6 deletion mutant; however, addition of virB6 in trans did not restore the level of the protein, even though it restored tumorigenicity (27). VirB3 participates in the formation of protein complexes with the T-pilus proteins VirB2 and VirB5 (55).Homologues of VirB3 are found in many alphaproteobacteria with a T4SS. While most VirB3 homologues are small proteins, several recently identified homologues are fusions of VirB3 and the immediate downstream protein VirB4 (5, 10, 24). These fusion homologs, which include Actinobacillus MagB03 (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAG24434","term_id":"10880920","term_text":"AAG24434"}}AAG24434), Campylobacter CmgB3/4 ({"type":"entrez-protein","attrs":{"text":"EAQ71805","term_id":"87248841","term_text":"EAQ71805"}}EAQ71805), Yersinia pseudotuberculosis TriC ({"type":"entrez-protein","attrs":{"text":"CAF25448","term_id":"51591645","term_text":"CAF25448"}}CAF25448), Citrobacter koseri PilX3-4 ({"type":"entrez-protein","attrs":{"text":"ABV12046","term_id":"157082368","term_text":"ABV12046"}}ABV12046), and Klebsiella pneumoniae PilX3-4 ({"type":"entrez-protein","attrs":{"text":"BAF49490","term_id":"134048868","term_text":"BAF49490"}}BAF49490), have VirB3 at the N terminus and VirB4 at the C terminus. Agrobacterium VirB4 is an integral membrane protein with a cytoplasmic N terminus (14). Its homologues are expected to have a similar topology. The prevailing view that pTi VirB3 has a periplasmic C terminus is inconsistent with the cytoplasmic location of the N terminus of VirB4 in the VirB3-VirB4 fusion protein homologues.In this study, we report the membrane topology of Agrobacterium VirB3 and demonstrate that the C terminus of the protein resides in the cytoplasm. We also demonstrate that VirB3 is an inner membrane protein, not an outer membrane protein as previously reported (52). The octopine Ti plasmid pTiA6NC VirB4 protein does not affect membrane localization of VirB3 but does stabilize VirB3. VirB4, however, is not sufficient for pTiA6NC VirB3 stabilization. Two additional proteins, VirB7 and VirB8, are required for the stabilization of pTiA6NC VirB3.     

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.     

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDΔN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo (ΔL_k = −4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDΔN130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.Type IV secretion systems (T4SS) in gram-negative bacteria mediate translocation of macromolecules out of the bacterial cell (14). The transmission of effector proteins and DNA into plant cells or other bacteria via cell-cell contact is one example of their function, and conjugation systems as well as the transferred DNA (T-DNA) delivery system of the phytopathogen Agrobacterium tumefaciens are prototypical of the T4SS family. Macromolecular translocation is achieved by a membrane-spanning protein machinery comprised of 12 gene products, VirB1 to VirB11 and an associated factor known as the coupling protein (VirD4) (66). The T4SS-associated coupling protein (T4CP) performs a crucial function in recognition of appropriate secretion substrates and governing entry of those molecules to the translocation pathway (7, 8, 10, 30, 41). In conjugation systems substrate recognition is applied to the relaxosome, a nucleoprotein complex of DNA transfer initiator proteins assembled specifically at the plasmid origin of transfer (oriT). In current models, initiation of the reactions that provide the single strand of plasmid (T-strand) DNA for secretion to recipient bacteria is expected to resemble the initiation of chromosomal replication (for reviews, see references 18, 54, and 81). Controlled opening of the DNA duplex is required to permit entry of the DNA processing machinery. The task of remodeling the conjugative oriT is generally ascribed to two or three relaxosome auxiliary factors, of host and plasmid origin, which occupy specific DNA binding sites at this locus. Intrinsic to the relaxosome is also a site- and strand-specific DNA transesterase activity that breaks the phosphodiester backbone at nic (5). Upon cleavage, the transesterase enzyme (also called relaxase) forms a reversible phosphotyrosyl linkage to the 5′ end of the DNA. Duplex unwinding initiating from this site produces the single-stranded T strand to be exported. A wealth of information is available supporting the importance of DNA sequence recognition and binding by relaxosome components at oriT to the transesterase reaction in vitro and for effective conjugative transfer (for reviews, see references 18, 54, and 81). On the other hand, the mechanisms controlling release of the 3′-OH generated at nic and the subsequent DNA unwinding stage remain obscure.Equally little is known about the process of nucleoprotein uptake by the transport channel. DNA-independent translocation of the relaxases TrwC (R388), MobA (RSF1010), and VirD2 (Ti plasmid) has been demonstrated; thus, current models propose that the relaxase component of the protein-DNA adduct is the substrate actively secreted by the transport system after interaction with the T4CP (42, 66). Cotransport of the covalently linked single-stranded T strand occurs concurrently (42). The mechanisms underlying relaxosome recognition by T4CPs are not understood. Direct interactions have been observed biochemically between the RP4 TraG protein and relaxase proteins of the cognate plasmid (65) and heterologous relaxosomes that it mobilizes (73, 76). TrwB of R388 interacts in vitro with relaxase TrwC and an auxiliary component, TrwA (44). TraD proteins of plasmid R1 and F are known to interact with the auxiliary relaxosome protein TraM (20) via a cluster of C-terminal amino acids (3, 62). Extensive mutagenic analyses (45) plus recent three-dimensional structural data for a complex of the TraM tetramerization domain and the C-terminal tail of TraD (46) have provided more detailed models for the intermolecular contacts involved in recognition.Application of the Cre recombinase assay for translocation of conjugative relaxases as well as effector proteins to eukaryotic cells is currently the most promising approach to elucidate protein motifs recognized by T4CPs (56, 68, 78, 79). Despite that progress, the nature of the interactions between a T4CP and its target protein that initiate secretion and the mechanisms controlling this step remain obscure. In contrast to systems dedicated specifically to effector protein translocation, conjugation systems mobilize nucleoprotein complexes that additionally exhibit catalytic activities, which can be readily monitored. These models are therefore particularly well suited to investigate aspects of regulation occurring at the physical interface of a T4CP and its secretion substrate. For this purpose the MOB_F family of DNA-mobilizing systems is additionally advantageous, since DNA processing within this family features the fusion of a dedicated conjugative helicase to the DNA transesterase enzyme within a single bifunctional protein. The TraI protein of F-like plasmids, originally described as Escherichia coli DNA helicase I (1, 2, 23), and the related TrwC protein of plasmid R388 (25) are well characterized (reviewed in reference 18). Early work by Llosa et al. revealed a complex domain arrangement for TrwC (43). Similar analyses with TraI identified nonoverlapping transesterase and helicase domains (6, 77), while the remaining intermediate and C-terminal regions of the protein additionally provide functions essential to effective conjugative transfer (49, 71). The ability to physically separate the catalytic domains of TraI and TrwC has facilitated a detailed biochemical characterization of their DNA transesterase, ATPase, and DNA-unwinding reactions. Nonetheless, failure of the physically disjointed polypeptides to complement efficient conjugative transfer when coexpressed indicates a role(s) for these proteins in the strand transfer process that goes beyond the need for their dual catalytic activities (43, 50). The assignment of additional functional properties to regions within TraI is a focus of current investigation (16, 29, 49).In all systems studied thus far, conditions used to reconstitute relaxosomes on a supercoiled oriT plasmid have not supported the initiation steps necessary to enable duplex unwinding by a conjugative helicase. The question remains open whether additional protein components are required and/or whether the pathway of initiation is subject to specific repression. In the present study, we applied the IncFII plasmid R1 paradigm to investigate the potential for interaction between purified components of the relaxosome and its cognate T4CP, TraD, to exert regulatory effects on relaxosome activities in vitro. In this and in the accompanying report (72), we present evidence for wide-ranging stimulatory effects of the cytoplasmic domain of TraD protein and its interaction partner TraM on multiple aspects of relaxosome function.     

Autoproteolysis of YscU of Yersinia pseudotuberculosis Is Important for Regulation of Expression and Secretion of Yop Proteins

YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU_CC. Autoproteolysis occurs at the conserved N↓PTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca²⁺ conditions) and calcium-depleted medium (−Ca²⁺ conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca²⁺-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca²⁺ conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under −Ca²⁺ conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.The type III secretion system (T3SS) occurs in many gram-negative pathogenic or symbiotic bacteria (6, 16, 19). The T3SS is evolutionarily related to the bacterial flagellum (19, 24), but while the flagellar apparatus is dedicated to bacterial motion, the T3SS specifically allows bacterial targeting of effector proteins across eukaryotic cell membranes into the lumen of the target cell (19). The main function of the effectors is to reprogram the cell to the benefit of the bacterium (28). The two organelles are superficially similar in form and can be divided into two physical substructures; a basal body is connected to a multimeric filamentous protein structure protruding from the bacterial surface. The basal body is embedded in the cell wall and spans from the cytosol to the surface of the bacterium with a cytosolic extension called the C-ring. The proximal center of the basal body is likely involved in the actual export of nonfolded substrates, which are thought to pass through the cell wall through this hollow structure (6, 16, 41). Early and elegant work by Macnab''s group showed that morphogenesis of the flagella is ordered such that first the cell-proximal hook structure is polymerized and then the flagellar filament is assembled on top of the hook structure (43). Thus, there is ordered switching from secretion of hook proteins to flagellin, which was called substrate specificity switching by Macnab et al. (15, 27). Mutants expressing extraordinarily long hooks have been isolated and connected to regulation and determination of hook buildup and subsequent substrate specificity switching (18, 29, 43). A central factor in this process is the integral 42-kDa cytoplasmic membrane protein FlhB, which has four putative transmembrane helices in its N-terminal domain, which is designated FlhB_TM. The hydrophilic C-terminal domain (FlhB_C) is predicted to protrude into the cytosol. In addition, FlhB_C can be further divided into two subdomains, FlhB_CN (amino acids 211 to 269) and FlhB_CC (amino acis 270 to 383), that are connected via a proposed flexible hinge region (27). The hinge region contains a highly conserved NPTH motif, which is found in all T3SSs. Interestingly, FlhB_C is specifically cleaved within this NPTH sequence (N269↓P270) (27). Site-specific mutagenesis of the NPTH site has a significant effect on the substrate switching, and the ability of flhB(N269A) and flhB(P270A) mutants to cleave FlhB is impaired, indicating that autoproteolysis is important (13, 15). Interestingly, the proteolysis is most likely the outcome of an autochemical process rather than an effect of external proteolytic enzymes (13). The FlhB homolog in the Yersinia pseudotuberculosis plasmid-encoded T3SS is the YscU protein, which has been shown to be essential for proper function of the T3SS since a yscU-null mutant is unable to secrete Yop proteins (Yops) into the culture supernatant (1, 21). YscU has been coupled to needle and Yop secretion regulation, as second-site suppressor mutations introduced into YscU_CC restore the yscP-null mutant phenotype. A yscP mutant is unable to exhibit substrate specificity switching and carries excess amounts of the needle protein YscF on the bacterial surface compared to the wild type. (11) Furthermore, YscP has been implicated in regulation of the T3SS needle length as a molecular ruler, where the size and helical content of YscP determine the length of the needle (20, 42). Together, these findings suggest that YscP and YscU interact and that this interaction is important for regulation of needle length, as well as for Yop secretion. As in FlhB, four predicted transmembrane helices followed by a cytoplasmic tail can be identified in YscU (1). In addition, the cytoplasmic part (YscU_C) can be divided into the YscU_CN and YscU_CC subdomains (Fig. ​(Fig.1A).1A). Variants of YscU with a single substitution in the conserved NPTH sequence (N263A) have been found to be unable to generate YscU_CC, suggesting that YscU of Yersinia also is autoproteolysed (21, 33, 38). The T3SS of Y. pseudotuberculosis secretes about 11 proteins, which collectively are called Yops (Yersinia outer proteins). These Yops have different functions during infection. Some are directly involved as effector proteins, attacking host cells to prevent phagocytosis and inflammation, while others have regulatory functions. Although the pathogen is extracellularly located, the Yop effectors are found solely in the cytosol of the target cell, and secretion of Yops occurs only at the zone of contact between the pathogen and the eukaryotic target cell (7, 36). Close contact between the pathogen and the eukaryotic cell also results in elevated expression and secretion of Yops (12, 30). Hence, cell contact induces the substrate switching; therefore, here we studied the connection between YscU autoproteolysis and expression, as well as secretion and translocation of Yops. Previous studies of YscU function were conducted mainly with in trans constructs instead of introduced YscU mutations in cis. Such studies reported loss of T3SS regulation (21). To avoid potential in trans problems, we introduced all mutations in cis with the aim of elucidating the function of YscU in type III secretion (T3S). Our results suggest that YscU autoproteolysis is not an absolute requirement either for Yop/LcrV secretion or for Yop translocation but is important for accurate regulation of Yop expression and secretion.Open in a separate windowFIG. 1.Autoproteolysis of YscU. (A) Schematic diagram of YscU in the bacterial inner membrane. The diagram shows the NPTH motif and the different parts of YscU after autoproteolysis and is the result of a prediction of transmembrane helices in proteins performed at the site http://www.cbs.dtu.dk/services/TMHMM. IM, inner membrane. (B) E. coli expressing C-terminally His-tagged YscU_C was induced with IPTG, which was followed by sonication and solubilization and denaturation of the protein in binding buffer (8 M urea and 10 mM imidazole). The lysate (lane L) was flushed over the Ni column, and the flowthrough (lane FT) was collected. The column was washed five times with binding buffer, and the wash fractions (lanes W1 to W5) were collected. Elution buffer (8 M urea and 300 mM imidazole) was flushed over the column to release proteins bound to the column, resulting in the eluate (lane E). The eluate was diluted 1:30 in 10 mM Tris (pH 7.4) to obtain a urea concentration of 0.2 M and incubated at 21°C overnight. The resulting overnight eluate fraction (lane E/ON) was TCA precipitated and taken up in binding buffer. Samples were analyzed by 15% Tris-Tricine SDS-PAGE. The cleavage of YscU_C-His₆ to YscU_CC-His₆ and YscU_CN was verified by N-terminal sequencing. All fractions were volume corrected. Lane ST contained a protein standard.     

Surface Translocation by Legionella pneumophila: a Form of Sliding Motility That Is Dependent upon Type II Protein Secretion

Legionella pneumophila exhibits surface translocation when it is grown on a buffered charcoal yeast extract (BCYE) containing 0.5 to 1.0% agar. After 7 to 22 days of incubation, spreading legionellae appear in an amorphous, lobed pattern that is most manifest at 25 to 30°C. All nine L. pneumophila strains examined displayed the phenotype. Surface translocation was also exhibited by some, but not all, other Legionella species examined. L. pneumophila mutants that were lacking flagella and/or type IV pili behaved as the wild type did when plated on low-percentage agar, indicating that the surface translocation is not swarming or twitching motility. A translucent film was visible atop the BCYE agar, advancing ahead of the spreading legionellae. Based on its abilities to disperse water droplets and to promote the spreading of heterologous bacteria, the film appeared to manipulate surface tension and, as such, acted like a surfactant. Indeed, a sample obtained from the film rapidly dispersed when it was spotted onto a plastic surface. L. pneumophila type II secretion (Lsp) mutants, but not their complemented derivatives, were defective for both surface translocation and film production. In contrast, mutants defective for type IV secretion exhibited normal surface translocation. When lsp mutants were spotted onto film produced by the wild type, they were able to spread, suggesting that type II secretion promotes the elaboration of the Legionella surfactant. Together, these data indicate that L. pneumophila exhibits a form of surface translocation that is most akin to “sliding motility” and uniquely dependent upon type II secretion.The genus Legionella was established in 1977, following the isolation of Legionella pneumophila from patients with a form of pneumonia now known as Legionnaires'' disease (33). Today, L. pneumophila is recognized as a common cause of both community-acquired and nosocomial pneumonia (84). Legionellosis occurs sporadically and in large outbreaks, with the largest outbreak occurring as recently as 2003 and encompassing 800 suspected and 449 confirmed cases (43). L. pneumophila is especially pathogenic for the elderly and the immunocompromised, large and growing segments of the population (39, 84), and recent studies have been highlighting the growing significance of travel-associated Legionnaires'' disease (107). L. pneumophila is a gram-negative, gammaproteobacterium that is widespread in natural and manufactured water systems (22, 39, 93). Infection occurs after the inhalation of Legionella-contaminated water droplets originating from a wide variety of aerosol-generating devices (39). Alarmingly, outbreaks can occur following the airborne spread of L. pneumophila over distances of >10 km from cooling towers or scrubbers (86). Within its aquatic habitats, L. pneumophila survives over a wide temperature range and grows on surfaces, in biofilms, and as an intracellular parasite of protozoa (9, 39, 110). Within the mammalian lung, the organism has the ability to attach to and invade macrophages and epithelia (27, 106, 113). Among the processes that promote L. pneumophila growth in both the environment and the mammalian lung are Lsp type II protein secretion, Dot/Icm type IVB protein secretion, and Lvh type IVA protein secretion (5, 25, 31, 106). Other key surface features of L. pneumophila are polar flagella that promote swimming motility and type IV pili that help mediate adherence (53, 103, 113). In addition to exporting proteins onto its surface into the extracellular milieu, and/or into host cells, L. pneumophila also secretes a siderophore and pyomelanin pigment that help mediate iron assimilation (23). We now report that L. pneumophila has the ability to translocate or spread across an agar surface. This new form of Legionella “motility” did not require the action of flagella, pili, or type IV secretion but was associated with the export of a surfactantlike material and an intact type II secretion system.     

Evidence for VirB4-Mediated Dislocation of Membrane-Integrated VirB2 Pilin during Biogenesis of the Agrobacterium VirB/VirD4 Type IV Secretion System

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.The Agrobacterium tumefaciens VirB/VirD4 type IV secretion system (T4SS) delivers effector proteins and DNA to plant cells during infection (1, 14). The 11 VirB proteins and VirD4 substrate receptor mediate assembly of the envelope-spanning translocation channel, whereas the VirB proteins independently of VirD4 are required for polymerization of the extracellular T pilus (6, 32, 46). These T4SS subunits include the three ATPases VirD4, VirB4, and VirB11; a trans-envelope core complex comprised of VirB7, VirB9, and VirB10; subunits involved in assembly or spatial positioning of the core complex (VirB1, VirB6, and VirB8); and other structural components (VirB2 pilin, VirB3, and pilus-associated VirB5) (1, 14, 43, 48, 55, 70). The VirB/VirD4 subunits are conserved among many Gram-negative bacterial T4SSs, and recent structures of homologs of VirD4, VirB5, VirB8, VirB10, and VirB11 and a VirB7/VirB9/VirB10 machine subassembly are supplying exciting new information about T4SS machine architectures (11, 28, 29).The pilin subunit VirB2 is a component of both the secretion channel and T pilus (39, 47, 48). Its role in substrate transfer was established with a modified chromatin immunoprecipitation (ChIP) assay termed transfer DNA (T-DNA) immunoprecipitation (TrIP), wherein the pilin (but not the T pilus) was shown to form formaldehyde-cross-linkable contacts with the translocating T-DNA substrate (10). TrIP studies with virB mutant strains also supplied evidence that VirB2 occupies a distal portion of the translocation channel near or at the outer membrane (OM) (10). Complementary genetic studies identified mutations in several VirB subunits, including VirB6, VirB9, VirB10, and VirB11, that selectively block T pilus production without affecting substrate transfer (39, 40, 41, 62). These Tra⁺ Pil⁻ “uncoupling” mutations do not bypass the requirement for VirB2 production for substrate transfer, as the further deletion of virB2 from the Tra⁺ Pil⁻ mutant strains renders these strains transfer defective (39, 41, 62). Therefore, VirB2 pilin, but not an intact T pilus, is required for passage of substrates to target cells.The pathways culminating in the integration of VirB2 into the two terminal organelles, the secretion channel and T pilus, are fundamentally poorly understood. The early VirB protein-independent reactions involve insertion of the 12.3-kDa propilin into the inner membrane (IM); cleavage of a long, 47-residue signal sequence, presumably by LepB signal peptidase; and covalent joining of the N-terminal Gln48 and C-terminal Ser121 to form the mature, cyclic pilin (24). This unusual head-to-tail cyclization reaction was also shown for the VirB2 homolog, TrbC (24/51% sequence identity/similarity) of plasmid RP4 (24, 34, 44). Other VirB2 homologs, such as F plasmid TraA (19/47% identity/similarity) (67), remain linear although their N termini are modified by N acetylation (54).Prevailing models suggest that mature forms of conjugative pilins accumulate in the IM as pools for use in assembly of the channel/pilus upon receipt of an unknown morphogenetic signal(s). The IM-integrated VirB2, TraA_F, and TrbC_RP4 pilins likely adopt similar topologies, as deduced from similar predicted secondary structures and results of reporter fusion studies with periplasmically active alkaline phosphatase (PhoA) (5, 22, 56). Two hydrophobic domains are thought to orient across the IM so that a small, intervening hydrophilic loop is cytoplasmic and the hydrophilic N and C termini are periplasmic. Detailed studies confirming this overall topology are lacking, and limited information exists regarding the nature of pilin interactions with other T4SS subunits (36, 51). Furthermore, little is known about the mechanism or energetic requirements for dislocation of membrane-integrated forms of conjugative pilins during machine morphogenesis.In A. tumefaciens, mutations in the Walker A nucleoside triphosphate (NTP) binding site motifs of the VirB4 and VirB11 ATPases render cells defective for substrate transfer and pilus production, indicating that NTP energy consumption by both ATPases is essential for assembly of the two terminal organelles (6, 7, 58, 62, 68). VirB4-like subunits are signatures of all T4SSs described to date, whereas VirB11-like proteins are common but not ubiquitous among the T4SSs (1). Some T4SSs, such as the conjugation machines encoded by Escherichia coli F-like plasmids, lack VirB11 homologs, and yet their conjugative pili extend and retract dynamically by a mechanism(s) dependent on VirB4 homologs (18, 65). On the basis of these observations, it is reasonable to propose that the VirB4-like subunits catalyze early reactions associated with assembly of conjugative pili.Here, we used the scanning cysteine accessibility method (SCAM) (9) to define the IM topology of cyclized VirB2. We then assayed for contributions of VirB subunits to the pilin structural organization. We present biochemical evidence for VirB4-mediated dislocation of VirB2 pilin from the membrane and also for a contribution by VirB11 in modulating pilin tertiary or quaternary structure. We discuss our findings in the context of recent advances in our understanding of T4SS machine assembly and architecture.     

Pseudomonas syringae HrpP Is a Type III Secretion Substrate Specificity Switch Domain Protein That Is Translocated into Plant Cells but Functions Atypically for a Substrate-Switching Protein

Pseudomonas syringae delivers virulence effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). P. syringae pv. tomato DC3000 HrpP has a C-terminal, putative T3SS substrate specificity switch domain, like Yersinia YscP. A ΔhrpP DC3000 mutant could not cause disease in tomato or elicit a hypersensitive response (HR) in tobacco, but the HR could be restored by expression of HrpP in trans. Though HrpP is a relatively divergent protein in the T3SS of different P. syringae pathovars, hrpP from P. syringae pv. syringae 61 and P. syringae pv. phaseolicola 1448A restored HR elicitation and pathogenicity to DC3000 ΔhrpP. HrpP was translocated into Nicotiana benthamiana cells via the DC3000 T3SS when expressed from its native promoter, but it was not secreted in culture. N- and C-terminal truncations of HrpP were tested for their ability to be translocated and to restore HR elicitation activity to the ΔhrpP mutant. No N-terminal truncation completely abolished translocation, implying that HrpP has an atypical T3SS translocation signal. Deleting more than 20 amino acids from the C terminus abolished the ability to restore HR elicitation. HrpP fused to green fluorescent protein was no longer translocated but could restore HR elicitation activity to the ΔhrpP mutant, suggesting that translocation is not essential for the function of HrpP. No T3SS substrates were detectably secreted by DC3000 ΔhrpP except the pilin subunit HrpA, which unexpectedly was secreted poorly. HrpP may function somewhat differently than YscP because the P. syringae T3SS pilus likely varies in length due to differing plant cell walls.Many proteobacterial pathogens use a type III secretion system (T3SS) as their primary mechanism to overcome and infect eukaryotic hosts. T3SSs are complex macromolecular machines that span both the bacterial cell envelope and host cell barriers to deliver proteins, commonly termed effectors, from the bacterial cytoplasm into the host cytoplasm (13, 19). After delivery into the host, effector proteins manipulate host cell function and suppress host defenses, allowing bacterial proliferation and disease development (6, 20). Bacteria that rely on T3SS to cause disease include plant pathogens such as Pseudomonas syringae, Ralstonia solanacearum, Erwinia and Xanthomonas species and animal pathogens in the genera Yersinia, Salmonella, Shigella, Escherichia, and Pseudomonas. While the repertoire of effectors delivered by a given T3SS is unique, the T3SS machinery is more universal (13). T3SS includes a core set of eight conserved proteins. These proteins, which are also conserved in bacterial flagellar biogenesis machines, make up the multiringed base structure, or basal body, that spans the bacterial membranes and cell wall. T3SS machines are also comprised of less-conserved and unique proteins that vary between systems. These include regulatory proteins that orchestrate construction of the machine and the extracellular components that function to translocate effectors across host barriers.The extracellular portion of the T3SS is comprised of the pilus or needle appendage (in plant or animal pathogens, respectively), which acts as a conduit for effector delivery, and the translocon complex, which creates the pore in the host cell membrane. These substructures vary between different T3SSs; presumably these external structures have adapted to allow different bacteria to infect different types of host cells. For Yersinia enterocolitica to infect macrophage cells, the T3SS needle must be a particular length (∼58 nm) to bridge the lipopolysaccharides extending from the bacterial outer membrane and reach the host cell membrane (35). Several other animal pathogens have T3SS needles of a defined length (48). Enteropathogenic Escherichia coli also has an additional extension beyond the needle called the EspA filament that functions to span the mucous layer found outside enterocyte cells (13). In plant pathogens, however, the extracellular gap between a bacterium and a plant cell includes a thick plant cell wall that is variable in width between plant species. Consequently, plant pathogenic Pseudomonas syringae has a pilus that can measure over 1 μm in vitro (25).Another major difference between the T3SS machineries of animal and plant pathogens is their translocon complexes. In animal pathogens, these are typically comprised of three essential proteins, but there is growing evidence that plant pathogen translocons employ diverse, functionally redundant components (28). There is growing interest in understanding the regulatory players that orchestrate the construction of diverse machinery. It is hypothesized that the assembly of the T3SS must involve several tightly regulated steps that allow secretion of the required components, followed by that of effectors upon completion. Of particular interest here is the control of pilus/needle subunit secretion, which is necessary when the pilus/needle is being constructed but would presumably compete with translocon and effector secretion after the T3SS is complete.We study the model plant pathogen P. syringae pv. tomato (Pto) DC3000, the causal agent of bacterial speck of tomato and Arabidopsis thaliana (8). DC3000 has a T3SS that delivers ca. 28 effectors and is essential for pathogenesis (11, 12, 30, 43). The P. syringae T3SS is encoded by hrp and hrc genes (hypersensitive response and pathogenicity/conserved), which are located in a pathogenicity island on the chromosome (4). hrc genes encode the conserved core components present in every T3SS. hrp genes encode T3SS components that are divergent or unique to P. syringae and enterobacterial plant pathogens, which also possess Hrp1 class T3SS (13). In contrast, plant pathogenic Ralstonia and Xanthomonas spp. have Hrp2 class T3SS, as indicated by several different Hrp proteins and distinct regulatory systems.To better understand the T3SS machinery, we previously conducted a survey of the hrp genes of P. syringae pv. syringae (Psy) 61 to complete the inventory of all those encoding proteins capable of traveling the T3SS into plant cells when expressed from a constitutive promoter (39). We hypothesized that these proteins might aid in pilus or translocon construction or regulate the construction process. HrpP was one protein found to be a T3SS substrate and important for secretion and translocation of the model effector AvrPto. Importantly, HrpP is related to a well-studied protein from Yersinia enterocolitica, YscP, which is a T3SS-secreted protein and a regulator responsible for switching the T3SS from secreting needle subunits to secreting effector proteins (15, 38, 47). It has also been shown that secretion of YscP into the culture medium is not essential for the switch function and that there may be two type III secretion signals embedded in YscP (2).The phenotype of a yscP mutant is unregulated secretion of the needle subunit, no secretion of effectors, and production of needles of indeterminate length. The switching phenotype requires a domain at the C terminus of YscP called the type III secretion substrate specificity switch (T3S4) domain, which is a conserved feature unifying its homologs (1). YscP has been proposed to act as a molecular ruler because the length of the YscP protein is directly correlated with the length of the Ysc needle (26). According to this model, when the needle has reached its proper length, YscP signals to the T3SS machinery to stop secreting needle subunits and begin secreting effector proteins. However, other functional models have been hypothesized for homologs of YscP. A recent study of the Salmonella enterica serovar Typhimurium YscP homolog InvJ showed that an invJ mutant lacked an inner rod. When the inner rod protein PrgJ was overexpressed, the length of the needle decreased relative to that of the wild type, leading the researchers to conclude that InvJ controls the inner rod, which in turn controls needle length (33). Recent evidence in Yersinia has lent more support to this model. YscP was found to negatively control secretion of YscI, the inner rod protein (51). Also, certain YscI mutations affected needle assembly but not effector secretion, implying that YscI may be a key player in substrate switching. Little is known about HrpB, the inner rod homolog in P. syringae (22), other than that the protein can be translocated into plant cells and is essential for T3SS function (39).Other models for length control/substrate switching have been proposed, such as the “C-ring cup model” in flagella, which was based on the observation that certain mutations in proteins that make up the inner membrane C ring of the basal body lead to shorter hooks (the flagellar equivalent of the needle), thus suggesting that C-ring capacity controls hook length (32). A more recent, flagellar “molecular-clock” model suggests that because overexpression of hook subunits leads to longer hooks and hook polymerization-defective mutants make shorter hooks, hook polymerization initiates a countdown, and the timing, in cooperation with the YscP homolog FliK, determines final hook length (34).HrpP is considered a member of the YscP/FliK family due mostly to the presence of a T3S4 domain at its C terminus. HrpP is also proline rich (10.6%), which is considered a characteristic of the family. The most striking feature of HrpP is its small size; the protein is 189 amino acids, compared with YscP from Y. enterocolitica, which is 453 amino acids and 8.4% proline. We were intrigued by how HrpP functions in P. syringae to regulate a pilus that can measure several hundred nanometers in length. Also, unlike animal pathogen needles and flagellar hooks, the pilus of P. syringae is predicted to be indeterminate in length, based on the fact that plant cell walls vary in width between species (40).We hypothesized that HrpP would be a main player in regulating pilus construction in P. syringae by allowing the system to make the transition between secretion of pilus subunits and secretion of translocon or effector proteins, though perhaps by a novel mechanism. In this study, we more precisely define the role of HrpP in the P. syringae T3SS. We show that HrpP is a T3SS substrate in DC3000, is translocated into plant cells at levels equivalent to those of effectors, and is essential for the function of the T3SS. Though it is highly translocated and variable, we found that HrpP from different P. syringae pathovars could complement the DC3000 hrpP mutant. Analysis of truncations of HrpP and an impassible HrpP-green fluorescent protein (GFP) fusion suggests that it has structural similarities to YscP, but surprisingly, HrpP was found to be required for full secretion of the pilus subunit HrpA as well as for translocation of HrpB.     

A Single Tyrosine in the Severe Acute Respiratory Syndrome Coronavirus Membrane Protein Cytoplasmic Tail Is Important for Efficient Interaction with Spike Protein

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 3 major envelope proteins: spike (S), membrane (M), and envelope (E). Previous work identified a dibasic endoplasmic reticulum retrieval signal in the cytoplasmic tail of SARS-CoV S that promotes efficient interaction with SARS-CoV M. The dibasic signal was shown to be important for concentrating S near the virus assembly site rather than for direct interaction with M. Here, we investigated the sequence requirements of the SARS-CoV M protein that are necessary for interaction with SARS-CoV S. The SARS-CoV M tail was shown to be necessary for S localization in the Golgi region when the proteins were exogenously coexpressed in cells. This was specific, since SARS-CoV M did not retain an unrelated glycoprotein in the Golgi. Importantly, we found that an essential tyrosine residue in the SARS-CoV M cytoplasmic tail, Y₁₉₅, was important for S-M interaction. When Y₁₉₅ was mutated to alanine, M_Y195A no longer retained S intracellularly at the Golgi. Unlike wild-type M, M_Y195A did not reduce the amount of SARS-CoV S carbohydrate processing or surface levels when the two proteins were coexpressed. Mutating Y₁₉₅ also disrupted SARS-CoV S-M interaction in vitro. These results suggest that Y₁₉₅ is necessary for efficient SARS-CoV S-M interaction and, thus, has a significant involvement in assembly of infectious virus.Coronaviruses are enveloped positive-strand RNA viruses that infect a wide variety of mammalian and avian species. These viruses generally cause mild disease in humans and are one major cause of the common cold (34). However, severe acute respiratory syndrome coronavirus (SARS-CoV), a novel human coronavirus which emerged in the Guangdong province in China in 2002 (30, 48), caused a widespread pandemic. SARS-CoV caused severe disease with a mortality rate of approximately 10%, the highest for any human coronavirus to date (62). The phylogeny and group classification of SARS-CoV remain controversial (17), but it is widely accepted to be a distant member of group 2. While SARS-CoV is no longer a major health threat, understanding the basic biology of this human pathogen remains important.Coronaviruses encode three major envelope proteins in addition to various nonstructural and accessory proteins. The envelope protein (E) is the least abundant structural protein in the virion envelope, although it is expressed at robust levels during infection (21). E plays an essential role in assembly for some but not all coronaviruses (31-33, 45) and may also be a viroporin (reviewed in reference 21). The spike glycoprotein (S) is the second most abundant protein in the envelope. S determines host cell tropism, binds the host receptor, and is responsible for virus-cell, as well as cell-cell, fusion (15). The S protein is a type I membrane protein with a large, heavily glycosylated luminal domain and a short cytoplasmic tail that has been shown to be palmitoylated in some coronaviruses (47, 58). The membrane protein (M) is the most abundant protein in the virion envelope and acts as a scaffold for virus assembly. M has three transmembrane domains, a long cytoplasmic tail, and a short glycosylated luminal domain (reviewed in reference 21). Unlike many enveloped viruses, coronaviruses assemble at and bud into the lumen of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and exit the infected cell by exocytosis (29). In order to accomplish this, the envelope proteins must be targeted to the budding compartment for assembly.For most coronaviruses, the E and M proteins localize in the Golgi region near the budding site independently of other viral structural proteins. We have previously shown for infectious bronchitis virus (IBV) E protein that the cytoplasmic tail contains Golgi targeting information (9). IBV M contains Golgi targeting information in its first transmembrane domain (57), while the transmembrane domains and cytoplasmic tail of mouse hepatitis virus (MHV) M appear to play a role in Golgi targeting (1, 36). Some coronavirus S proteins contain targeting information in their cytoplasmic tails; however, some do not (38, 39, 52, 63). Since S proteins can escape to the cell surface when highly expressed, S may rely on lateral interactions with other viral envelope proteins to localize to the budding site and be incorporated into newly assembling virions.In line with its role in virus assembly, M is necessary for virus-like particle (VLP) formation (3, 10, 26, 40, 55, 59). M has been shown to interact with itself to form homo-oligomers (12). In addition, M interacts with E, S, and the viral nucleocapsid and is essential for virion assembly (reviewed in reference 21). Lateral interactions between the coronavirus envelope proteins are critical for efficient virus assembly. The interaction of S and M has been studied for MHV, and the cytoplasmic tail of each protein is important for interaction (16, 44). Specifically, deletion of an amphipathic region in the MHV M cytoplasmic tail abrogates efficient interaction with MHV S (11). The S and M proteins of IBV, bovine coronavirus, feline infectious peritonitis virus, and SARS-CoV have been shown to interact; however, information about the specific regions that are important for interaction remains elusive (16, 22, 26, 42, 64). Due to the presence of several accessory proteins in the virion envelope (23-25, 28, 51, 53), it is possible that the requirements for SARS-CoV S and M interaction could be different from those of previously studied coronaviruses.In earlier work, we reported that SARS-CoV M retains SARS-CoV S intracellularly at the Golgi region when both proteins are expressed exogenously (39). We also demonstrated that the SARS-CoV S cytoplasmic tail interacts with in vitro-transcribed and -translated SARS-CoV M (39). Here, we show that the SARS-CoV M cytoplasmic tail is necessary for specific retention of SARS-CoV S at the Golgi region. We found a critical tyrosine residue at position 195 to be important for retaining SARS-CoV S Golgi membranes when coexpressed with M. When Y₁₉₅ was mutated to alanine, the mutant protein, M_Y195A, did not reduce the amount of SARS-CoV S at the plasma membrane or reduce the extent of S carbohydrate processing as well as wild-type SARS-CoV M does. Additionally, mutation of Y₁₉₅ in SARS-CoV M disrupted the S-M interaction in vitro. Thus, Y₁₉₅ is likely to play a critical role in the assembly of infectious SARS-CoV.