Anaplasma phagocytophilum effector EgeA facilitates infection by hijacking TANGO1 and SCFD1 from ER-Golgi exit sites to pathogen-occupied inclusions.

The obligatory intracellular bacterium Anaplasma phagocytophilum causes human granulocytic anaplasmosis, an emerging zoonosis. Anaplasma has limited biosynthetic and metabolic capacities, yet it effectively replicates inside of inclusions/vacuoles of eukaryotic host cells. Here, we describe a unique Type IV secretion system (T4SS) effector, ER-Golgi exit site protein of Anaplasma (EgeA). In cells infected by Anaplasma, secreted native EgeA, EgeA-GFP, and the C-terminal half of EgeA (EgeA-C)-GFP localized to Anaplasma-containing inclusions. In uninfected cells, EgeA-C-GFP localized to cis-Golgi, whereas the N-terminal half of EgeA-GFP localized to the ER. Pull-down assays identified EgeA-GFP binding to a transmembrane protein in the ER, Transport and Golgi organization protein 1 (TANGO1). By yeast two-hybrid analysis, EgeA-C directly bound Sec1 family domain-containing protein 1 (SCFD1), a host protein of the cis-Golgi network that binds TANGO1 at ER-Golgi exit sites (ERES). Both TANGO1 and SCFD1 localized to the Anaplasma inclusion surface. Furthermore, knockdown of Anaplasma EgeA or either host TANGO1 or SCFD1 significantly reduced Anaplasma infection. TANGO1 and SCFD1 prevent ER congestion and stress by facilitating transport of bulky or unfolded proteins at ERES. A bulky cargo collagen and the ER-resident chaperon BiP were transported into Anaplasma inclusions, and several ER stress marker genes were not up-regulated in Anaplasma-infected cells. Furthermore, EgeA transfection reduced collagen overexpression-induced BiP upregulation. These results suggest that by binding to the two ERES proteins, EgeA redirects the cargo-adapted ERES to pathogen-occupied inclusions and reduces ERES congestion, which facilitates Anaplasma nutrient acquisition and reduces ER stress for Anaplasma survival and proliferation.

Type IV secretion system effector sabotages multiple defense systems in a competing bacterium.

Effector proteins secreted by bacteria that infect mammalian and plant cells often subdue eukaryotic host cell defenses by simultaneously affecting multiple targets. However, instances when a bacterial effector injected in the competing bacteria sabotage more than a single target have not been reported. Here, we demonstrate that the effector protein, LtaE, translocated by the type IV secretion system from the soil bacterium Lysobacter enzymogenes into the competing bacterium, Pseudomonas protegens, affects several targets, thus disabling the antibacterial defenses of the competitor. One LtaE target is the transcription factor, LuxR1, that regulates biosynthesis of the antimicrobial compound, orfamide A. Another target is the sigma factor, PvdS, required for biosynthesis of another antimicrobial compound, pyoverdine. Deletion of the genes involved in orfamide A and pyoverdine biosynthesis disabled the antibacterial activity of P. protegens, whereas expression of LtaE in P. protegens resulted in the near-complete loss of the antibacterial activity against L. enzymogenes. Mechanistically, LtaE inhibits the assembly of the RNA polymerase complexes with each of these proteins. The ability of LtaE to bind to LuxR1 and PvdS homologs from several Pseudomonas species suggests that it can sabotage defenses of various competitors present in the soil or on plant matter. Our study thus reveals that the multi-target effectors have evolved to subdue cell defenses not only in eukaryotic hosts but also in bacterial competitors.

Streptococcus suis Serotype 2 Type IV Secretion Effector SspA-1 Induces Proinflammatory Cytokine Production via TLR2 Endosomal and Type I Interferon Signaling.

The subtilisin-like protease-1 (SspA-1) plays an important role in the pathogenesis of a highly virulent strain of Streptococcus suis 2. However, the mechanism of SspA-1-triggered excessive inflammatory response is still unknown. In this study, we demonstrated that activation of type I IFN signaling is required for SspA-1-induced excessive proinflammatory cytokine production. Further experiments showed that the TLR2 endosomal pathway mediates SspA-1-induced type I IFN signaling and the inflammatory response. Finally, we mapped the major signaling components of the related pathway and found that the TIR adaptor proteins Mal, TRAM, and MyD88 and the downstream activation of IRF1 and IRF7 were involved in this pathway. These results explain the molecular mechanism by which SspA-1 triggers an excessive inflammatory response and reveal a novel effect of type I IFN in S. suis 2 infection, possibly providing further insights into the pathogenesis of this highly virulent S. suis 2 strain.

VirBR, a transcription regulator, promotes IncX3 plasmid transmission, and persistence of blaNDM-5 in zoonotic bacteria.

IncX3 plasmids carrying the New Delhi metallo-ß-lactamase-encoding gene, blaNDM-5, are rapidly spreading globally in both humans and animals. Given that carbapenems are listed on the WHO AWaRe watch group and are prohibited for use in animals, the drivers for the successful dissemination of Carbapenem-Resistant Enterobacterales (CRE) carrying blaNDM-5-IncX3 plasmids still remain unknown. We observe that E. coli carrying blaNDM-5-IncX3 can persist in chicken intestines either under the administration of amoxicillin, one of the largest veterinary ß-lactams used in livestock, or without any antibiotic pressure. We therefore characterise the blaNDM-5-IncX3 plasmid and identify a transcription regulator, VirBR, that binds to the promoter of the regulator gene actX enhancing the transcription of Type IV secretion systems (T4SS); thereby, promoting conjugation of IncX3 plasmids, increasing pili adhesion capacity and enhancing the colonisation of blaNDM-5-IncX3 transconjugants in animal digestive tracts. Our mechanistic and in-vivo studies identify VirBR as a major factor in the successful spread of blaNDM-5-IncX3 across one-health AMR sectors. Furthermore, VirBR enhances the plasmid conjugation and T4SS expression by the presence of copper and zinc ions, thereby having profound ramifications on the use of universal animal feeds.

Brucella NpeA is a secreted Type IV effector containing an N-WASP-binding short linear motif that promotes niche formation.

The modulation of actin polymerization is a common theme among microbial pathogens. Even though microorganisms show a wide repertoire of strategies to subvert the activity of actin, most of them converge in the ones that activate nucleating factors, such as the Arp2/3 complex. Brucella spp. are intracellular pathogens capable of establishing chronic infections in their hosts. The ability to subvert the host cell response is dependent on the capacity of the bacterium to attach, invade, avoid degradation in the phagocytic compartment, replicate in an endoplasmic reticulum-derived compartment and egress. Even though a significant number of mechanisms deployed by Brucella in these different phases have been identified and characterized, none of them have been described to target actin as a cellular component. In this manuscript, we describe the identification of a novel virulence factor (NpeA) that promotes niche formation. NpeA harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP and stabilizes the autoinhibited state. Our results show that NpeA is secreted in a Type IV secretion system-dependent manner and that deletion of the gene diminishes the intracellular replication capacity of the bacterium. In vitro and ex vivo experiments demonstrate that NpeA binds N-WASP and that the short linear motif is required for the biological activity of the protein.IMPORTANCEThe modulation of actin-binding effectors that regulate the activity of this fundamental cellular protein is a common theme among bacterial pathogens. The neural Wiskott-Aldrich syndrome protein (N-WASP) is a protein that several pathogens target to hijack actin dynamics. The highly adapted intracellular bacterium Brucella has evolved a wide repertoire of virulence factors that modulate many activities of the host cell to establish successful intracellular replication niches, but, to date, no effector proteins have been implicated in the modulation of actin dynamics. We present here the identification of a virulence factor that harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP stabilizing its autoinhibited state. We demonstrate that this protein is a Type IV secretion effector that targets N-WASP-promoting intracellular survival and niche formation.

The F plasmid conjutome: the repertoire of E. coli proteins translocated through an F-encoded type IV secretion system.

Bacterial conjugation systems pose a major threat to human health through their widespread dissemination of mobile genetic elements (MGEs) carrying cargoes of antibiotic resistance genes. Using the Cre Recombinase Assay for Translocation (CRAfT), we recently reported that the IncFV pED208 conjugation system also translocates at least 16 plasmid-encoded proteins to recipient bacteria. Here, we deployed a high-throughput CRAfT screen to identify the repertoire of chromosomally encoded protein substrates of the pED208 system. We identified 32 substrates encoded by the Escherichia coli W3110 genome with functions associated with (i) DNA/nucleotide metabolism, (ii) stress tolerance/physiology, (iii) transcriptional regulation, or (iv) toxin inhibition. The respective gene deletions did not impact pED208 transfer proficiencies, nor did Group 1 (DNA/nucleotide metabolism) mutations detectably alter the SOS response elicited in new transconjugants upon acquisition of pED208. However, MC4100(pED208) donor cells intrinsically exhibit significantly higher SOS activation than plasmid-free MC4100 cells, and this plasmid carriage-induced stress response is further elevated in donor cells deleted of several Group 1 genes. Among 10 characterized substrates, we gained evidence of C-terminal or internal translocation signals that could function independently or synergistically for optimal protein transfer. Remarkably, nearly all tested proteins were also translocated through the IncN pKM101 and IncP RP4 conjugation systems. This repertoire of E. coli protein substrates, here termed the F plasmid "conjutome," is thus characterized by functions of potential benefit to new transconjugants, diverse TSs, and the capacity for promiscuous transfer through heterologous conjugation systems. IMPORTANCE: Conjugation systems comprise a major subfamily of the type IV secretion systems (T4SSs) and are the progenitors of a second large T4SS subfamily dedicated to translocation of protein effectors. This study examined the capacity of conjugation machines to function as protein translocators. Using a high-throughput reporter screen, we determined that 32 chromosomally encoded proteins are delivered through an F plasmid conjugation system. The translocated proteins potentially enhance the establishment of the co-transferred F plasmid or mitigate mating-induced stresses. Translocation signals located C-terminally or internally conferred substrate recognition by the F system and, remarkably, many substrates also were translocated through heterologous conjugation systems. Our findings highlight the plasticity of conjugation systems in their capacities to co-translocate DNA and many protein substrates.

Bactericidal effectors of the Stenotrophomonas maltophilia type IV secretion system: functional definition of the nuclease TfdA and structural determination of TfcB.

Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli, indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase.IMPORTANCEStenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa. We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).

Cryo-EM structure of a conjugative type IV secretion system suggests a molecular switch regulating pilus biogenesis.

Conjugative type IV secretion systems (T4SS) mediate bacterial conjugation, a process that enables the unidirectional exchange of genetic materials between a donor and a recipient bacterial cell. Bacterial conjugation is the primary means by which antibiotic resistance genes spread among bacterial populations (Barlow 2009; Virolle et al, 2020). Conjugative T4SSs form pili: long extracellular filaments that connect with recipient cells. Previously, we solved the cryo-electron microscopy (cryo-EM) structure of a conjugative T4SS. In this article, based on additional data, we present a more complete T4SS cryo-EM structure than that published earlier. Novel structural features include details of the mismatch symmetry within the OMCC, the presence of a fourth VirB8 subunit in the asymmetric unit of both the arches and the inner membrane complex (IMC), and a hydrophobic VirB5 tip in the distal end of the stalk. Additionally, we provide previously undescribed structural insights into the protein VirB10 and identify a novel regulation mechanism of T4SS-mediated pilus biogenesis by this protein, that we believe is a key checkpoint for this process.

PrgE: an OB-fold protein from plasmid pCF10 with striking differences to prototypical bacterial SSBs.

A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded type IV secretion systems (T4SSs). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many, their function remains unclear. Here, we characterize PrgE, a proposed SSB from the Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very unusual DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string supported by its N-terminal tail. In vitro studies highlight the plasticity of PrgE oligomerization and confirm the importance of the N-terminus. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from eukaryotic SSBs.

Translocation of YopJ family effector proteins through the VirB/VirD4 T4SS of Bartonella.

The evolutionary conserved YopJ family comprises numerous type-III-secretion system (T3SS) effectors of diverse mammalian and plant pathogens that acetylate host proteins to dampen immune responses. Acetylation is mediated by a central acetyltransferase domain that is flanked by conserved regulatory sequences, while a nonconserved N-terminal extension encodes the T3SS-specific translocation signal. Bartonella spp. are facultative-intracellular pathogens causing intraerythrocytic bacteremia in their mammalian reservoirs and diverse disease manifestations in incidentally infected humans. Bartonellae do not encode a T3SS, but most species possess a type-IV-secretion system (T4SS) to translocate Bartonella effector proteins (Beps) into host cells. Here we report that the YopJ homologs present in Bartonellae species represent genuine T4SS effectors. Like YopJ family T3SS effectors of mammalian pathogens, the "Bartonella YopJ-like effector A" (ByeA) of Bartonella taylorii also targets MAP kinase signaling to dampen proinflammatory responses, however, translocation depends on a functional T4SS. A split NanoLuc luciferase-based translocation assay identified sequences required for T4SS-dependent translocation in conserved regulatory regions at the C-terminus and proximal to the N-terminus of ByeA. The T3SS effectors YopP from Yersinia enterocolitica and AvrA from Salmonella Typhimurium were also translocated via the Bartonella T4SS, while ByeA was not translocated via the Yersinia T3SS. Our data suggest that YopJ family T3SS effectors may have evolved from an ancestral T4SS effector, such as ByeA of Bartonella. In this evolutionary scenario, the signal for T4SS-dependent translocation encoded by N- and C-terminal sequences remained functional in the derived T3SS effectors due to the essential role these sequences coincidentally play in regulating acetyltransferase activity.

Conjugative type IV secretion systems enable bacterial antagonism that operates independently of plasmid transfer.

Bacterial cooperation and antagonism mediated by secretion systems are among the ways in which bacteria interact with one another. Here we report the discovery of an antagonistic property of a type IV secretion system (T4SS) sourced from a conjugative plasmid, RP4, using engineering approaches. We scrutinized the genetic determinants and suggested that this antagonistic activity is independent of molecular cargos, while we also elucidated the resistance genes. We further showed that a range of Gram-negative bacteria and a mixed bacterial population can be eliminated by this T4SS-dependent antagonism. Finally, we showed that such an antagonistic property is not limited to T4SS sourced from RP4, rather it can also be observed in a T4SS originated from another conjugative plasmid, namely R388. Our results are the first demonstration of conjugative T4SS-dependent antagonism between Gram-negative bacteria on the genetic level and provide the foundation for future mechanistic studies.

Metagenome diversity illuminates the origins of pathogen effectors.

Recent metagenome-assembled genome (MAG) analyses have profoundly impacted Rickettsiology systematics. The discovery of basal lineages (novel families Mitibacteraceae and Athabascaceae) with predicted extracellular lifestyles exposed an evolutionary timepoint for the transition to host dependency, which seemingly occurred independent of mitochondrial evolution. Notably, these basal rickettsiae carry the Rickettsiales vir homolog (rvh) type IV secretion system and purportedly use rvh to kill congener microbes rather than parasitize host cells as described for later-evolving rickettsial pathogens. MAG analysis also substantially increased diversity for the genus Rickettsia and delineated a sister lineage (the novel genus Tisiphia) that stands to inform on the emergence of human pathogens from protist and invertebrate endosymbionts. Herein, we probed Rickettsiales MAG and genomic diversity for the distribution of Rickettsia rvh effectors to ascertain their origins. A sparse distribution of most Rickettsia rvh effectors outside of Rickettsiaceae lineages illuminates unique rvh evolution from basal extracellular species and other rickettsial families. Remarkably, nearly every effector was found in multiple divergent forms with variable architectures, indicating profound roles for gene duplication and recombination in shaping effector repertoires in Rickettsia pathogens. Lateral gene transfer plays a prominent role in shaping the rvh effector landscape, as evinced by the discovery of many effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchange between Rickettsia and Legionella species. Our study exemplifies how MAGs can yield insight into pathogen effector origins, particularly how effector architectures might become tailored to the discrete host cell functions of different eukaryotic hosts.IMPORTANCEWhile rickettsioses are deadly vector-borne human diseases, factors distinguishing Rickettsia pathogens from the innumerable bevy of environmental rickettsial endosymbionts remain lacking. Recent metagenome-assembled genome (MAG) studies revealed evolutionary timepoints for rickettsial transitions to host dependency. The rvh type IV secretion system was likely repurposed from congener killing in basal extracellular species to parasitizing host cells in later-evolving pathogens. Our analysis of MAG diversity for over two dozen rvh effectors unearthed their presence in some non-pathogens. However, most effectors were found in multiple divergent forms with variable architectures, indicating gene duplication and recombination-fashioned effector repertoires of Rickettsia pathogens. Lateral gene transfer substantially shaped pathogen effector arsenals, evinced by the discovery of effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchanges between Rickettsia and Legionella species. Our study exemplifies how MAGs yield insight into pathogen effector origins and evolutionary processes tailoring effectors to eukaryotic host cell biology.

Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines.

Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate-TraD and TraD-T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.

Inhibition of the type IV secretion system from antibiotic-resistant Helicobacter pylori clinical isolates supports the potential of Cagα as an anti-virulence target.

Helicobacter pylori resistance to antibiotics is a growing problem and it increasingly leads to treatment failure. While the bacterium is present worldwide, the severity of clinical outcomes is highly dependent on the geographical origin and genetic characteristics of the strains. One of the major virulence factors identified in H. pylori is the cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) used to translocate effectors into human cells. Here, we investigated the genetic variability of the cagPAI among 13 antibiotic-resistant H. pylori strains that were isolated from patient biopsies in Québec. Seven of the clinical strains carried the cagPAI, but only four could be readily cultivated under laboratory conditions. We observed variability of the sequences of CagA and CagL proteins that are encoded by the cagPAI. All clinical isolates induce interleukin-8 secretion and morphological changes upon co-incubation with gastric cancer cells and two of them produce extracellular T4SS pili. Finally, we demonstrate that molecule 1G2, a small molecule inhibitor of the Cagα protein from the model strain H. pylori 26695, reduces interleukin-8 secretion in one of the clinical isolates. Co-incubation with 1G2 also inhibits the assembly of T4SS pili, suggesting a mechanism for its action on T4SS function.

Structural and functional diversity of type IV secretion systems.

Considerable progress has been made in recent years in the structural and molecular biology of type IV secretion systems in Gram-negative bacteria. The latest advances have substantially improved our understanding of the mechanisms underlying the recruitment and delivery of DNA and protein substrates to the extracellular environment or target cells. In this Review, we aim to summarize these exciting structural and molecular biology findings and to discuss their functional implications for substrate recognition, recruitment and translocation, as well as the biogenesis of extracellular pili. We also describe adaptations necessary for deploying a breadth of processes, such as bacterial survival, host-pathogen interactions and biotic and abiotic adhesion. We highlight the functional and structural diversity that allows this extremely versatile secretion superfamily to function under different environmental conditions and in different bacterial species. Additionally, we emphasize the importance of further understanding the mechanism of type IV secretion, which will support us in combating antimicrobial resistance and treating type IV secretion system-related infections.

Role of the CagY antenna projection in Helicobacter pylori Cag type IV secretion system activity.

Helicobacter pylori strains containing the cag pathogenicity island (PAI) are associated with the development of gastric adenocarcinoma and peptic ulcer disease. The cag PAI encodes a secreted effector protein (CagA) and a type IV secretion system (Cag T4SS). Cag T4SS activity is required for the delivery of CagA and non-protein substrates into host cells. The Cag T4SS outer membrane core complex (OMCC) contains a channel-like domain formed by helix-loop-helix elements (antenna projections, AP) from 14 copies of the CagY protein (a VirB10 ortholog). Similar VirB10 antenna regions are present in T4SS OMCCs from multiple bacterial species and are predicted to span the outer membrane. In this study, we investigated the role of the CagY antenna region in Cag T4SS OMCC assembly and Cag T4SS function. An H. pylori mutant strain with deletion of the entire CagY AP (∆AP) retained the capacity to produce CagY and assemble an OMCC, but it lacked T4SS activity (CagA translocation and IL-8 induction in AGS gastric epithelial cells). In contrast, a mutant strain with Gly-Ser substitutions in the unstructured CagY AP loop retained Cag T4SS activity. Mutants containing CagY AP loops with shortened lengths were defective in CagA translocation and exhibited reduced IL-8-inducing activity compared to control strains. These data indicate that the CagY AP region is required for Cag T4SS activity and that Cag T4SS activity can be modulated by altering the length of the CagY AP unstructured loop.

Functional characterization of VirB/VirD4 and Icm/Dot type IV secretion systems from the plant-pathogenic bacterium Xanthomonas euvesicatoria.

Introduction: Many Gram-negative plant- and animal-pathogenic bacteria employ type IV secretion (T4S) systems to transport proteins or DNA/protein complexes into eukaryotic or bacterial target cells. T4S systems have been divided into minimized and expanded T4S systems and resemble the VirB/VirD4 T4S system from the plant pathogen Agrobacterium tumefaciens and the Icm/Dot T4S system from the human pathogen Legionella pneumophila, respectively. The only known plant pathogen with both types of T4S systems is Xanthomonas euvesicatoria which is the causal agent of bacterial spot disease on pepper and tomato plants. Results and discussion: In the present study, we show that virB/virD4 and icm/dot T4S genes are expressed and encode components of oligomeric complexes corresponding to known assemblies of VirB/VirD4 and Icm/Dot proteins. Both T4S systems are dispensable for the interaction of X. euvesicatoria with its host plants and do not seem to confer contact-dependent lysis of other bacteria, which was previously shown for the chromosomally encoded VirB/VirD4 T4S system from Xanthomonas axonopodis pv. citri. The corresponding chromosomal T4S gene cluster from X. euvesicatoria is incomplete, however, the second plasmid-localized vir gene cluster encodes a functional VirB/VirD4 T4S system which contributes to plasmid transfer. In agreement with this finding, we identified the predicted relaxase TraI as substrate of the T4S systems from X. euvesicatoria. TraI and additional candidate T4S substrates with homology to T4S effectors from X. axonopodis pv. citri interact with the T4S coupling protein VirD4. Interestingly, however, the predicted C-terminal VirD4-binding sites are not sufficient for T4S, suggesting the contribution of additional yet unknown mechanisms to the targeting of T4S substrates from X. euvesicatoria to both VirB/VirD4 and Icm/Dot T4S systems.

Comparative Analysis of T4SS Molecular Architectures.

The recently published high-resolution R388 T4SS structure provides exciting new details about the complete complex of T4SS, including the components making up the stalk and arches, numerous symmetry mismatches between regions of the complex, and an intriguing interpretation of the closed stalk and radial symmetry of the inner membrane complex, which is related to pilus biogenesis assembly. However, there are a few unidentified densities in the electron microscopy map and portions of the identified component sequences for which the structure is not yet known. It is also unclear how well this minimized DNA-transporting T4SS predicts the structure of other T4SSs, such as expanded systems and those that transport proteins rather than DNA. In this review, we evaluate what can be inferred from the recent high-resolution structure of the R388 T4SS with respect to the Cag and Dot/Icm systems. These systems were selected because, given what is currently known about these systems, we expect them to present most structural differences compared to the R388 T4SS structure. Furthermore, we discuss bacterial physiology and diversity, the T4SS structures and their variations between different bacterial species. These insights may prove beneficial for researchers who elucidate the structure and functions of T4SS in different bacterial species.

Genetic diversity of Helicobacter pylori type IV secretion system cagI and cagN genes and their association with clinical diseases.

A number of cagPAI genes in the Helicobacter pylori genome are considered the most evolved genes under a diversifying selection and evolutionary pressure. Among them, cagI and cagN are described as a part of the two different-operon of cagPAI that are involved in the T4SS machinery, but the definite association of these factors with clinical manifestations is still unclear. A total of 70 H. pylori isolates were obtained from different gastroduodenal patients. All isolates were examined for the presence of primary H. pylori virulence genes by PCR analysis. Direct DNA sequence analysis was performed for the cagI and cagN genes. The results were compared with the reference strain. The cagI, cagN, cagA, cagL, vacA s1m1, vacA s1m2, vacA s2m2, babA2, sabA, and dupA genotypes were detected in 80, 91.4, 84, 91.4, 32.8, 42.8, 24.4, 97.1, 84.3, and 84.3% of the total isolates, respectively. The most variable codon usage in cagI was observed at residues 20-25, 55-60, 94, 181-199, 213-221, 241-268, and 319-320, while the most variable codon usage in CagN hypervariable motif (CagNHM) was observed at residues 53 to 63. Sequencing data analysis of cagN revealed a hypothetical hexapeptide motif (EAKDEN/K) in residues of 278-283 among six H. pylori isolates, which needs further studies to evaluate its putative function. The present study demonstrated a high prevalence of cagI and cagN genes among Iranian H. pylori isolates with gastroduodenal diseases. Furthermore, no significant correlation between cagI and cagN variants and clinical diseases was observed in the present study. However, all patients had a high prevalence of cagPAI genes including cagI, cagN, cagA, and cagL, which indicates more potential role of these genes in disease outcome.

DNA Transport through the Dynamic Type IV Secretion System.

The versatile type IV secretion system (T4SS) nanomachine plays a pivotal role in bacterial pathogenesis and the propagation of antibiotic resistance determinants throughout microbial populations. In addition to paradigmatic DNA conjugation machineries, diverse T4SSs enable the delivery of multifarious effector proteins to target prokaryotic and eukaryotic cells, mediate DNA export and uptake from the extracellular milieu, and in rare examples, facilitate transkingdom DNA translocation. Recent advances have identified new mechanisms underlying unilateral nucleic acid transport through the T4SS apparatus, highlighting both functional plasticity and evolutionary adaptations that enable novel capabilities. In this review, we describe the molecular mechanisms underscoring DNA translocation through diverse T4SS machineries, emphasizing the architectural features that implement DNA exchange across the bacterial membrane and license transverse DNA release across kingdom boundaries. We further detail how recent studies have addressed outstanding questions surrounding the mechanisms by which nanomachine architectures and substrate recruitment strategies contribute to T4SS functional diversity.