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

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

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

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

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

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

The plant sHSP superfamily: five new members in Arabidopsis thaliana with unexpected properties

The small heat shock proteins (sHsps), which are ubiquitous stress proteins proposed to act as chaperones, are encoded by an unusually complex gene family in plants. Plant sHsps are classified into different subfamilies according to amino acid sequence similarity and localization to distinct subcellular compartments. In the whole Arabidopsis thaliana genome, 19 genes were annotated to encode sHsps, of which 14 belong to previously defined plant sHsp families. In this paper, we report studies of the five additional sHsp genes in A. thaliana, which can now be shown to represent evolutionarily distinct sHsp subfamilies also found in other plant species. While two of these five sHsps show expression patterns typical of the other 14 genes, three have unusual tissue specific and developmental profiles and do not respond to heat induction. Analysis of intracellular targeting indicates that one sHsp represents a new class of mitochondrion-targeted sHsps, while the others are cytosolic/nuclear, some of which may cooperate with other sHsps in formation of heat stress granules. Three of the five new proteins were purified and tested for chaperone activity in vitro. Altogether, these studies complete our basic understanding of the sHsp chaperone family in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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

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

Agrobacterium tumefaciens VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion System

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) translocates DNA and protein substrates across the bacterial cell envelope. Six presumptive channel subunits of this T4SS (VirD4, VirBll, VirB6, VirB8, VirB2, and VirB9) form close contacts with the VirD2-T-strand transfer intermediate during export, as shown recently by a novel transfer DNA immunoprecipitation (TrIP) assay. Here, we characterize the contribution of the hydrophobic channel component VirB6 to substrate translocation. Results of reporter protein fusion and cysteine accessibility studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, five transmembrane segments, and a cytoplasmic C terminus. TrIP studies aimed at characterizing the effects of VirB6 insertion and deletion mutations on substrate translocation identified several VirB6 functional domains: (i) a central region composed of a large periplasmic loop (P2) (residues 84 to 165) mediates the interaction of VirB6 with the exiting T-strand; (ii) a multi-membrane-spanning region carboxyl-terminal to loop P2 (residues 165 to 245) is required for substrate transfer from VirB6 to the bitopic membrane subunit VirB8; and (iii) the two terminal regions (residues 1 to 64 and 245 to 290) are required for substrate transfer to the periplasmic and outer membrane-associated VirB2 and VirB9 subunits. Our findings support a model whereby the periplasmic loop P2 comprises a portion of the secretion channel and distinct domains of VirB6 participate in channel subunit interactions required for substrate passage to the cell exterior.     

An essential virulence protein of Agrobacterium tumefaciens,VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA

The 11 gene products of the Agrobacterium tumefaciens virB operon, together with the VirD4 protein, are proposed to form a membrane complex which mediates the transfer of T-DNA to plant cells. This study examined one putative component of that complex, VirB4. A deletion of the virB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the virB4 gene with the kanamycin resistance-conferring nptII gene. The virB4 gene was found to be necessary for virulence on plants and for the transfer of IncQ plasmids to recipient cells of A. tumefaciens. Genetic complementation of the deletion strain by the virB4 gene under control of the virB promoter confirmed that the deletion was nonpolar on downstream virB genes. Genetic complementation was also achieved with the virB4 gene placed under control of the lac promoter, even though synthesis of the VirB4 protein from this promoter is far below wild-type levels. Having shown a role for the VirB4 protein in DNA transfer, lysine-439, found within the conserved mononucleotide binding domain of VirB4, was changed to a glutamic acid, methionine, or arginine by oligonucleotide-directed mutagenesis. virB4 genes bearing these mutations were unable to complement the virB4 deletion for either virulence or for IncQ transfer, showing that an intact mononucleotide binding site is necessary for the function of VirB4 in DNA transfer. The necessity of the VirB4 protein with an intact mononucleotide binding site for extracellular complementation of virE2 mutants was also shown. In merodiploid studies, lysine-439 mutations present in trans decreased IncQ plasmid transfer frequencies, suggesting that VirB4 functions within a complex to facilitate DNA transfer.     

Agrobacterium tumefaciens VirB9, an outer-membrane-associated component of a type IV secretion system, regulates substrate selection and T-pilus biogenesis

Agrobacterium tumefaciens translocates DNA and protein substrates between cells via a type IV secretion system (T4SS) whose channel subunits include the VirD4 coupling protein, VirB11 ATPase, VirB6, VirB8, VirB2, and VirB9. In this study, we used linker insertion mutagenesis to characterize the contribution of the outer-membrane-associated VirB9 to assembly and function of the VirB/D4 T4SS. Twenty-five dipeptide insertion mutations were classified as permissive for intercellular substrate transfer (Tra+), completely transfer defective (Tra-), or substrate discriminating, e.g., selectively permissive for transfer only of the oncogenic transfer DNA and the VirE2 protein substrates or of a mobilizable IncQ plasmid substrate. Mutations inhibiting transfer of DNA substrates did not affect formation of close contacts of the substrate with inner membrane channel subunits but blocked formation of contacts with the VirB2 and VirB9 channel subunits, which is indicative of a defect in assembly or function of the distal portion of the secretion channel. Several mutations in the N- and C-terminal regions disrupted VirB9 complex formation with the outer-membrane-associated lipoprotein VirB7 or the inner membrane energy sensor VirB10. Several VirB9.i2-producing Tra+ strains failed to elaborate T pilus at detectable levels (Pil-), and three such Tra+ Pil- mutant strains were rendered Tra- upon deletion of virB2, indicating that the cellular form of pilin protein is essential for substrate translocation. Our findings, together with computer-based analyses, support a model in which distinct domains of VirB9 contribute to substrate selection and translocation, establishment of channel subunit contacts, and T-pilus biogenesis.     

Interactions between Brucella suis VirB8 and Its Homolog TraJ from the Plasmid pSB102 Underline the Dynamic Nature of Type IV Secretion Systems

The proteinVirB8 plays a critical role in the assembly and function of the Agrobacterium tumefaciens virB type IV secretion system (T4SS). The structure of the periplasmic domain of both A. tumefaciens and Brucella suis VirB8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of VirB8 and interactions with VirB4 and VirB10. We have shown previously that TraJ, the VirB8 homologue from pSB102, and the chimeric protein TraJB8, encompassing the cytoplasmic and transmembrane (TM) domains of TraJ and the periplasmic domain of VirB8, were unable to complement a B. suis mutant containing an in-frame deletion of the virB8 gene. This suggested that the presence of the TraJ cytoplasmic and TM domains could block VirB8 dimerization or assembly in the inner membrane. By bacterial two-hybrid analysis, we found that VirB8, TraJ, and the chimeras can all interact to form both homo- and heterodimers. However, the presence of the TM domain of TraJ resulted in much stronger interactions in both the homo- and heterodimers. We expressed the wild-type and chimeric proteins in wild-type B. suis. The presence of proteins carrying the TM domain of TraJ had a dominant negative effect, leading to complete loss of virulence. This suggests that the T4SS is a dynamic structure and that strong interactions block the spatial flexibility required for correct assembly and function.Brucellosis is a major worldwide zoonosis primarily affecting developing countries and causing them severe economic losses (7). Bacteria of the genus Brucella, the causative agent, are gram-negative facultative intracellular pathogens of various wild and domestic mammals, as well as humans, where it causes a very debilitating disease known as Malta fever (48). In addition, these bacteria are also a focus of concern as possible biological warfare agents (23).The key aspect of Brucella virulence is its ability to survive and proliferate within professional and nonprofessional phagocytes (7). Once phagocytosed, this bacteria subverts the vesicular traffic in the host cell to establish a niche in a compartment derived from the endoplasmic reticulum, where it multiplies (3, 34, 39, 40, 41). Several factors have been reported to be essential for the virulence of this bacterium (16, 17, 18, 19, 21, 22, 29, 31). Strikingly, we (36) and others (44) have demonstrated the presence in Brucella of a type IV secretion system (T4SS) that is encoded by the virB operon and whose integrity is required for virulence (8, 14, 19). Several other species of gram-negative bacteria have been found to rely on the presence of a T4SS for full virulence (10, 13). Both extracellular (Helicobacter) and intracellular (Legionella, Bartonella) pathogens use their T4SSs to inject effector proteins directly into the target cell, where they affect the biology of the cell. Bordetella pertussis uses its T4SS to secrete the pertussis toxin into the extracellular medium, where it is taken up by cells. The T4SS of Agrobacterium tumefaciens translocates both effector proteins and a nucleoprotein complex into target plant cells through a mechanism reminiscent of bacterial conjugation through T4SS.The A. tumefaciens VirB T4SS, which is considered the T4SS paradigm, is composed of 11 different proteins named VirB1 to VirB11 plus VirD4. These proteins can be functionally subdivided in three different groups. The proteins VirB4, VirB11, and VirD4 are inner membrane ATPases with a large cytoplasmic domain and are believed to provide the energy required for T4SS assembly and for the translocation of effectors. VirB2 and VirB5 form an extracellular bacterial appendage believed to anchor the bacteria to the host cell (4, 25). Finally, VirB3 and VirB6 to VirB10 are believed to form a channel-like structure spanning both the inner and outer membranes of the bacteria.Among these structural proteins, VirB8 has been shown to play a key role in the assembly of the T4SS. Recent studies with A. tumefaciens demonstrated that VirB8 acts as a nucleation center required to recruit VirB9 and VirB10 into clusters in the outer membrane (15, 30) and to localize VirB proteins at the cell pole (26). VirB8 is a protein spanning the bacterial inner membrane, with the first 67 amino acids forming a short cytoplasmic tail, followed by a single hydrophobic transmembrane (TM) domain. The carboxy-terminal moiety of the protein, of 172 amino acids, is believed to be entirely periplasmic. Recently, the three-dimensional structures of the periplasmic domains of VirB8 from Brucella suis (46) and A. tumefaciens (5) have been determined. Using these structural data, site-directed mutagenesis has been performed on the periplasmic part of B. suis VirB8, showing that changes in amino acids that inhibit the dimerization of VirB8 or its interactions with VirB4 or VirB10 also affect T4SS assembly and B. suis virulence (37). Among all of the homologs of B. suis VirB8, the closest are the proteins TraJ, encoded in the tra operons of broad-host-range plasmids pSB102 and pIPO2 (43, 45). The TraJ protein from pSB102 shares more than 50% identity with B. suis VirB8 at the amino acid level, and this percentage increases to more than 60% when only the periplasmic domain is considered. In a previous study, we have taken advantage of this close similarity between VirB8 and TraJ to examine the possibility of a functional heterologous complementation of VirB8 by TraJ in BS1008, a B. suis mutant carrying an in-frame deletion of the virB8 gene (38). From our results, it appeared that the protein TraJ was unable to complement BS1008. As the major similarities between VirB8 and TraJ were found in their respective periplasmic domains, we constructed chimeric genes encoding proteins in which the major part of the periplasmic domain of one protein (amino acids 77 to 241 of TraJ and amino acids 76 to 239 of VirB8) was replaced with the corresponding part of the other protein (these proteins are described in Fig. ​Fig.1)1) and studied the ability of these chimeric proteins to restore the virulence of BS1008. The TraJB8 chimera, where the periplasmic part of VirB8 replaces the corresponding part of TraJ, was also unable to complement BS1008. In contrast, B8TraJ, the reverse chimera in which the periplasmic part of TraJ replaces the corresponding part of VirB8, partially restored the virulence of BS1008. These results show that, when fused to the cytoplasmic and TM parts of VirB8, the periplasmic part of TraJ can functionally replace the corresponding part of VirB8 in T4SS assembly. In contrast, the cytoplasmic and TM parts of TraJ cannot replace the corresponding part of VirB8, whether fused to the periplasmic part of TraJ or VirB8. To further elucidate these points, we undertook a more detailed study of the interactions of these proteins by bacterial two-hybrid (BACTH) analysis. Here we show that the proteins VirB8, B8TraJ, TraJB8, and TraJ display strikingly different abilities to interact with themselves, as well as with VirB8 itself. Further, the TM domain of these proteins plays a crucial role in determining the strength of these interactions. Finally we show that, when overexpressed in a wild-type B. suis strain, these various proteins are able to modulate its virulence, even leading to complete loss of virulence. These data give interesting clues concerning the mechanisms of type IV secretion.Open in a separate windowFIG. 1.Schematic representation of the proteins used in this study. Light and dark gray parts represent protein domains from VirB8 and TraJ, respectively. All proteins were synthesized as fusion proteins with the T18 or T25 subunit of the B. pertussis adenylate cyclase domain fused to the amino-terminal end of the VirB8- and/or TraJ-containing part. In the case of the periplasmic domains of VirB8 (VirB8p) and TraJ (TraJp), synthesis of these proteins as fusions with the subunits of B. pertussis adenylate cyclase likely targets them to the bacterial cytoplasm, as suggested by fractionation studies. The rightmost column indicates whether the corresponding proteins have (+) or have not (−) been detected in Western blotting experiments.     

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

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

A novel small heat shock protein of <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>: characterization,structure modeling,and expression profiles under environmental stresses

Small heat shock proteins (sHsps) are a class of chaperones with low molecular weight, feathered by a C-terminal α-crystallin domain (ACD). They participate in reestablishing the stability of partially denatured proteins and therefore contribute to cellular homeostasis. In this work, we identified a sHsp homolog (designated as sHsp19) from Haliotis discus hannai, an economically important farmed mollusk in East Asia. sHsp19 possesses a sHsp hallmark domain, which exhibits the typical fold of ACD as revealed by a three-dimensional model constructed through an iterative threading assembly refinement method. The amino acid sequence sHsp19 shares low identities with any other known sHsps, with percentages below 35 %. Besides, sHsp19 shows relatively distant phylogenetic relationships with sHsps of various mollusks, including two other identified sHsps of abalone subspecies. qRT-PCR analysis indicated that the expression of sHsp19 occurred in multiple tissues. Upon exposure to thermal, oxidative, and multiple toxic metal stresses, the level of sHsp19 mRNA was rapidly elevated in a persistent fashion, with the maximum increase up to 170.58-, 405.84-, and 361.96-fold, respectively. These results indicate sHsp is a novel sHsp that possesses the distinguishing structural feature of sHsps but has remote homologies with known sHsps. It is likely to be important in stress adaptation of abalone and may be applied as a bioindicator for monitoring pollution or detrimental changes of environment in abalone culture.     

Type-IVC Secretion System: A Novel Subclass of Type IV Secretion System (T4SS) Common Existing in Gram-Positive Genus Streptococcus

A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. Type IV secretion system (T4SS) is one of versatile secretion systems essential for the virulence and even survival of some bacteria species, and they enable the secretion of protein and DNA substrates across the cell envelope. T4SS was once believed to be present only in Gram-negative bacteria. In this study, we present evidence of a new subclass of T4SS, Type-IVC secretion system and indicate its common existence in the Gram-positive bacterial genus Streptococcus. We further identified that VirB1, VirB4, VirB6 and VirD4 are the minimal key components of this system. Using genome comparisons and evolutionary relationship analysis, we proposed that Type-IVC secretion system is movable via transposon factors and mediates the conjugative transfer of DNA, enhances bacterial pathogenicity, and could cause large-scale outbreaks of infections in humans.     

A single amino acid change in the transmembrane domain of the VirB8 protein affects dimerization,interaction with VirB10 and Brucella suis virulence

VirB8 is a critical component of the Brucella suis type IV secretion system (T4SS). We previously showed that the transmembrane (TM) domain plays an essential role in interactions of this protein with itself and the other proteins of the T4SS. We report that a point mutation in this TM domain stabilizes homodimers of VirB8 and heterodimers with VirB10. A similar variant of Agrobacterium tumefaciens VirB8 showed the same phenotype. The B. suis VirB8 variant was unable to complement a virB8 mutant and displayed a dominant negative phenotype when expressed in wild type B. suis. We suggest that interaction of VirB8 with VirB10 could play a major role in the correct function of the B. suis VirB T4SS.Structured summary of protein interactionsAtVirB8 physically interacts with AtVirB10 by two hybrid (View interaction)TraJ physically interacts with TraJ by two hybrid (View Interaction 1, 2)AtVirB8 physically interacts with AtVirB8 by two hybrid (View interaction)VirB10 physically interacts with VirB10 by two hybrid (View interaction)VirB8 physically interacts with VirB8 by two hybrid (View Interaction 1, 2)VirB10 physically interacts with VirB8 by two hybrid (View interaction)AtVirB10 physically interacts with AtVirB10 by two hybrid (View interaction)VirB8 physically interacts with VirB10 by two hybrid (View interaction)AtVirB10 physically interacts with AtVirB8 by two hybrid (View interaction)     

The 2.5 ? Structure of the Enterococcus Conjugation Protein TraM resembles VirB8 Type IV Secretion Proteins

Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gram-negative (G−) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G+), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G+ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 Å resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G−) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G+). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G+ and G− bacteria, we propose a new classification scheme of VirB8-like proteins.     

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.     

Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species.     

VirB10 vaccination for protection against <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis>

Background

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the etiologic agent Anaplasma phagocytophilum. HGA was designated a nationally notifiable disease in the United States in 1998. Currently there are no vaccines available against HGA. Conserved membrane proteins that are subdominant in Anaplasma species, such as VirB9 and VirB10, may represent better vaccine targets than the variable immunodominant surface proteins. VirB9 and VirB10 are constituents of the Type 4 secretion system (T4SS) that is conserved amongst many intracellular bacteria and performs essential functions for invasion and survival in host cells.

Results

Immunogenicity and contribution to protection, provided after intramuscular vaccination of plasmid DNA encoding VirB9-1, VirB9-2, and VirB10 followed by inoculation of homologous recombinant proteins, in a prime-boost immunization strategy was evaluated in a murine model of HGA. Recombinant VirB9-1-, VirB9-2-, and VirB10-vaccinated mice developed antibody responses that specifically reacted with A. phagocytophilum organisms. However, only the mice vaccinated with VirB10 developed a significant increase in IFN-γ CD4⁺ T cells and partial protection against challenge with A. phagocytophilum.

Conclusions

This work provides evidence that A. phagocytophilum T4SS VirB10 is partially protective in a murine model against infection in an IFN-γ-dependent fashion and suggests that this protein may be a potential vaccine candidate against this and possibly other pathogenic bacteria with a T4SS.