Recruitment of heterologous substrates by bacterial secretion systems for transkingdom translocation.

Bacterial secretion systems mediate the selective exchange of macromolecules between bacteria and their environment, playing a pivotal role in processes such as horizontal gene transfer or virulence. Among the different families of secretion systems, Type III, IV and VI (T3SS, T4SS and T6SS) share the ability to inject their substrates into human cells, opening up the possibility of using them as customized injectors. For this to happen, it is necessary to understand how substrates are recruited and to be able to engineer secretion signals, so that the transmembrane machineries can recognize and translocate the desired substrates in place of their own. Other factors, such as recruiting proteins, chaperones, and the degree of unfolding required to cross through the secretion channel, may also affect transport. Advances in the knowledge of the secretion mechanism have allowed heterologous substrate engineering to accomplish translocation by T3SS, and to a lesser extent, T4SS and T6SS into human cells. In the case of T4SS, transport of nucleoprotein complexes adds a bonus to its biotechnological potential. Here, we review the current knowledge on substrate recognition by these secretion systems, the many examples of heterologous substrate translocation by engineering of secretion signals, and the current and future biotechnological and biomedical applications derived from this approach.

Conjugative DNA Transfer From E. coli to Transformation-Resistant Lactobacilli.

Lactic acid bacteria (LAB) belonging to the genus classically known as Lactobacillus, recently split into 25 different genera, include many relevant species for the food industry. The well-known properties of lactobacilli as probiotics make them an attractive model also for vaccines and therapeutic proteins delivery in humans. However, scarce tools are available to accomplish genetic modification of these organisms, and most are only suitable for laboratory strains. Here, we test bacterial conjugation as a new tool to introduce genetic modifications into many biotechnologically relevant laboratory and wild type lactobacilli. Using mobilizable shuttle plasmids from a donor Escherichia coli carrying either RP4 or R388 conjugative systems, we were able to get transconjugants to all tested Lactocaseibacillus casei strains, including many natural isolates, and to several other genera, including Lentilactobacillus parabuchneri, for which no transformation protocol has been reported. Transconjugants were confirmed by the presence of the oriT and 16S rRNA gene sequencing. Serendipitously, we also found transconjugants into researcher-contaminant Staphylococcus epidermidis. Conjugative DNA transfer from E. coli to S. aureus was previously described, but at very low frequencies. We have purified this recipient strain and used it in standard conjugation assays, confirming that both R388 and RP4 conjugative systems mediate mobilization of plasmids into S. epidermidis. This protocol could be assayed to introduce DNA into other Gram-positive microorganisms which are resistant to transformation.

Bacterial injection machines: Evolutionary diverse but functionally convergent.

Many human pathogens use Type III, Type IV, and Type VI secretion systems to deliver effectors into their target cells. The contribution of these secretion systems to microbial virulence was the main focus of a workshop organised by the International University of Andalusia in Spain. The meeting addressed structure-function, substrate recruitment, and translocation processes, which differ widely on the different secretion machineries, as well as the nature of the translocated effectors and their roles in subverting the host cell. An excellent panel of worldwide speakers presented the state of the art of the field, highlighting the involvement of bacterial secretion in human disease and discussing mechanistic aspects of bacterial pathogenicity, which can provide the bases for the development of novel antivirulence strategies.

The secret life of conjugative relaxases.

Conjugative relaxases are well-characterized proteins responsible for the site- and strand-specific endonucleolytic cleavage and strand transfer reactions taking place at the start and end of the conjugative DNA transfer process. Most of the relaxases characterized biochemically and structurally belong to the HUH family of endonucleases. However, an increasing number of new families of relaxases are revealing a variety of protein folds and catalytic alternatives to accomplish conjugative DNA processing. Relaxases show high specificity for their cognate target DNA sequences, but several recent reports underscore the importance of their activity on secondary targets, leading to widespread mobilization of plasmids containing an oriT-like sequence. Some relaxases perform other functions associated with their nicking and strand transfer ability, such as catalyzing site-specific recombination or initiation of plasmid replication. They perform these roles in the absence of conjugation, and the validation of these functions in several systems strongly suggest that they are not mere artifactual laboratory observations. Other unexpected roles recently assigned to relaxases include controlling plasmid copy number and promoting retrotransposition. Their capacity to mediate promiscuous mobilization and genetic reorganizations can be exploited for a number of imaginative biotechnological applications. Overall, there is increasing evidence that conjugative relaxases are not only key enzymes for horizontal gene transfer, but may have been adapted to perform other roles which contribute to prokaryotic genetic plasticity. Relaxed target specificity may be key to this versatility.

Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus.

Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.

DNA Delivery and Genomic Integration into Mammalian Target Cells through Type IV A and B Secretion Systems of Human Pathogens.

We explore the potential of bacterial secretion systems as tools for genomic modification of human cells. We previously showed that foreign DNA can be introduced into human cells through the Type IV A secretion system of the human pathogen Bartonella henselae. Moreover, the DNA is delivered covalently attached to the conjugative relaxase TrwC, which promotes its integration into the recipient genome. In this work, we report that this tool can be adapted to other target cells by using different relaxases and secretion systems. The promiscuous relaxase MobA from plasmid RSF1010 can be used to deliver DNA into human cells with higher efficiency than TrwC. MobA also promotes DNA integration, albeit at lower rates than TrwC. Notably, we report that DNA transfer to human cells can also take place through the Type IV secretion system of two intracellular human pathogens, Legionella pneumophila and Coxiella burnetii, which code for a distantly related Dot/Icm Type IV B secretion system. This suggests that DNA transfer could be an intrinsic ability of this family of secretion systems, expanding the range of target human cells. Further analysis of the DNA transfer process showed that recruitment of MobA by Dot/Icm was dependent on the IcmSW chaperone, which may explain the higher DNA transfer rates obtained. Finally, we observed that the presence of MobA negatively affected the intracellular replication of C. burnetii, suggesting an interference with Dot/Icm translocation of virulence factors.

Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB.

Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening-closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA.

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome.

Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery.IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used in vivo to transfer the DNA of interest into the appropriate cell and promote its integration. If used in combination with a site-specific nuclease, it could lead to site-specific integration of the incoming DNA by homologous recombination.

Coupling Proteins in Type IV Secretion.

Type IV coupling proteins (T4CPs) are essential constituents of most type IV secretion systems (T4SSs), and probably the most intriguing component in terms of their evolutionary origin and functional role. Coupling proteins have coevolved with their cognate secretion system and translocated substrates. They are present in all conjugative systems, leading to the suggestion that they play a specific role in DNA transfer. However, they are also part of many T4SSs involved in bacterial virulence, where they are required for protein translocation, with no apparent involvement in DNA secretion. Their name reflects genetic and biochemical evidence of a connecting role between the substrate and the T4SS, thus probably playing a major role in substrate recruitment. Increasing evidence supports also a role in signal transmission leading to activation of secretion. Most studies have addressed conjugative coupling proteins of the VirD4-like protein family. Their conserved features include a nucleotide-binding domain, essential for substrate translocation, a C-terminal domain involved in substrate interactions, and a transmembrane domain anchoring them to the inner membrane, which is an important regulator of protein function. Purified soluble deletion mutants display ATP hydrolysis activity and unspecific DNA binding. Elucidation of the 3D structure of the soluble deletion mutant of the conjugative coupling protein TrwB, TrwBΔN70, provided the basis for further mutagenesis studies rendering interesting insights into the structure-function of these proteins. Their key role as couplers between substrate and transporter provides biotechnological potential as targets for anti-virulence strategies, as well as for customization of substrate delivery through heterologous secretion systems.

A Functional oriT in the Ptw Plasmid of Burkholderia cenocepacia Can Be Recognized by the R388 Relaxase TrwC.

Burkholderia cenocepacia is both a plant pathogen and the cause of serious opportunistic infections, particularly in cystic fibrosis patients. B. cenocepacia K56-2 harbors a native plasmid named Ptw for its involvement in the Plant Tissue Watersoaking phenotype. Ptw has also been reported to be important for survival in human cells. Interestingly, the presence of PtwC, a homolog of the conjugative relaxase TrwC of plasmid R388, suggests a possible function for Ptw in conjugative DNA transfer. The ptw region includes Type IV Secretion System genes related to those of the F plasmid. However, genes in the adjacent region shared stronger homology with the R388 genes involved in conjugative DNA metabolism. This region included the putative relaxase ptwC, a putative coupling protein and accessory nicking protein, and a DNA segment with high number of inverted repeats and elevated AT content, suggesting a possible oriT. Although we were unable to detect conjugative transfer of the Ptw resident plasmid, we detected conjugal mobilization of a co-resident plasmid containing the ptw region homologous to R388, demonstrating the cloned ptw region contains an oriT. A similar plasmid lacking ptwC could not be mobilized, suggesting that the putative relaxase PtwC must act in cis on its oriT. Remarkably, we also detected mobilization of a plasmid containing the Ptw oriT by the R388 relaxase TrwC, yet we could not detect PtwC-mediated mobilization of an R388 oriT-containing plasmid. Our data unambiguously show that the Ptw plasmid harbors DNA transfer functions, and suggests the Ptw plasmid may play a dual role in horizontal DNA transfer and eukaryotic infection.

Chloramphenicol Selection of IS10 Transposition in the cat Promoter Region of Widely Used Cloning Vectors.

The widely used pSU8 family of cloning vectors is based on a p15A replicon and a chloramphenicol acetyltransferase (cat) gene conferring chloramphenicol resistance. We frequently observed an increase in the size of plasmids derived from these vectors. Analysis of the bigger molecular species shows that they have an IS10 copy inserted at a specific site between the promoter and the cat open reading frame. Promoter activity from both ends of IS10 has been reported, suggesting that the insertion events could lead to higher CAT production. Insertions were observed in certain constructions containing inserts that could lead to plasmid instability. To test the possibility that IS10 insertions were selected as a response to chloramphenicol selection, we have grown these constructs in the presence of different amounts of antibiotic and we observed that insertions arise promptly under higher chloramphenicol selective pressure. IS10 is present in many E. coli laboratory strains, so the possibility of insertion in constructions involving cat-containing vectors should be taken into account. Using lower chloramphenicol concentrations could solve this problem.

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.

Structural independence of conjugative coupling protein TrwB from its Type IV secretion machinery.

The stability of components of multiprotein complexes often relies on the presence of the functional complex. To assess structural dependence among the components of the R388 Type IV secretion system (T4SS), the steady-state level of several Trw proteins was determined in the absence of other Trw components. While several Trw proteins were affected by the lack of others, we found that the coupling protein TrwB is not affected by the absence of other T4SS components, nor did its absence alter significantly the levels of integral components of the complex, underscoring the independent role of the coupling protein on the T4SS architecture. The cytoplasmic ATPases TrwK (VirB4) and TrwD (VirB11) were affected by the absence of several core complex components, while the pilus component TrwJ (VirB5) required the presence of all other Trw proteins (except for TrwB) to be detectable. Overall, the results delineate a possible assembly pathway for the T4SS of R388. We have also tested structural complementation of TrwD (VirB11) and TrwJ (VirB5) by their homologues in the highly related Trw system of Bartonella tribocorum (Bt). The results reveal a correlation with the functional complementation data previously reported.

HUH site-specific recombinases for targeted modification of the human genome.

Site-specific recombinases (SSRs) have been crucial in the development of mammalian transgenesis. For gene therapy purposes, this approach remains challenging, because, for example, SSR delivery is largely unresolved and SSR DNA substrates must pre-exist in target cells. In this review, we discuss the potential of His-hydrophobic-His (HUH) recombinases to overcome some of the limitations of conventional SSRs. Members of the HUH protein family cleave single-stranded (ss)DNA, but can mediate site-specific integration with the aid of the host replication machinery. Adeno-associated virus (AAV) Rep remains the only known example to support site-specific integration in human cells, and AAV is an excellent gene delivery vector that can be targeted to specific cells and organelles. Bacterial protein TrwC catalyzes integration into human sequences and can be delivered to human cells covalently linked to DNA, offering attractive new features for targeted genome modification.

New perspectives into bacterial DNA transfer to human cells.

The type IV secretion system (T4SS) VirB/D4 of the facultative intracellular pathogen Bartonella henselae is known to translocate bacterial effector proteins into human cells. Two recent reports on DNA transfer into human cells have demonstrated the versatility of this bacterial secretion system for macromolecular substrate transfer. Moreover, these findings have opened the possibility for developing new tools for DNA delivery into specific human cell types. DNA can be introduced into these cells covalently attached to a site-specific integrase with potential target sequences in the human genome. This novel DNA delivery system is discussed in the context of existing methods for genetic modification of human cells.

Site-specific integration of foreign DNA into minimal bacterial and human target sequences mediated by a conjugative relaxase.

BACKGROUND: Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. CONCLUSIONS/SIGNIFICANCE: The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.

Transfer of R388 derivatives by a pathogenesis-associated type IV secretion system into both bacteria and human cells.

Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies. B. henselae codes for two T4SSs: VirB/VirD4 and Trw. A ΔvirB mutant strain was transfer deficient, while a ΔtrwE mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels. B. henselae bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient Escherichia coli under certain conditions, underscoring the versatility of T4SSs.

Nuclear targeting of a bacterial integrase that mediates site-specific recombination between bacterial and human target sequences.

TrwC is a bacterial protein involved in conjugative transfer of plasmid R388. It is transferred together with the DNA strand into the recipient bacterial cell, where it can integrate the conjugatively transferred DNA strand into its target sequence present in the recipient cell. Considering that bacterial conjugation can occur between bacteria and eukaryotic cells, this protein has great biotechnological potential as a site-specific integrase. We have searched for possible TrwC target sequences in the human genome. Recombination assays showed that TrwC efficiently catalyzes recombination between its natural target sequence and a discrete number of sequences, located in noncoding sites of the human genome, which resemble this target. We have determined the cellular localization of TrwC and derivatives in human cells by immunofluorescence and also by an indirect yeast-based assay to detect both nuclear import and export signals. The results indicate that the recombinase domain of TrwC (N600) has nuclear localization, but full-length TrwC locates in the cytoplasm, apparently due to the presence of a nuclear export signal in its C-terminal domain. The recombinase domain of TrwC can be transported to recipient cells by conjugation in the presence of the helicase domain of TrwC, but with very low efficiency. We mutagenized the trwC gene and selected for mutants with nuclear localization. We obtained one such mutant with a point A904T mutation and an extra peptide at its C terminus, which maintained its functionality in conjugation and recombination. This TrwC mutant could be useful for future TrwC-mediated site-specific integration assays in mammalian cells.

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 TrwB Delta N70, 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.