Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells

Several bacterial pathogens utilize conjugation machines to export effector molecules during infection. Such systems are members of the type IV or 'adapted conjugation' secretion family. The prototypical type IV system is the Agrobacterium tumefaciens T-DNA transfer machine, which delivers oncogenic nucleoprotein particles to plant cells. Other pathogens, including Bordetella pertussis, Legionella pneumophila, Brucellaspp. and Helicobacter pylori, use type IV machines to export effector proteins to the extracellular milieu or the mammalian cell cytosol.     

VirB8: a conserved type IV secretion system assembly factor and drug target.

Type IV secretion systems are used by many gram-negative bacteria for the translocation of macromolecules (proteins, DNA, or DNA-protein complexes) across the cell envelope. Among them are many pathogens for which type IV secretion systems are essential virulence factors. Type IV secretion systems comprise 8-12 conserved proteins, which assemble into a complex spanning the inner and the outer membrane, and many assemble extracellular appendages, such as pili, which initiate contact with host and recipient cells followed by substrate translocation. VirB8 is an essential assembly factor for all type IV secretion systems. Biochemical, cell biological, genetic, and yeast two-hybrid analyses showed that VirB8 undergoes multiple interactions with other type IV secretion system components and that it directs polar assembly of the membrane-spanning complex in the model organism Agrobacterium tumefaciens. The availability of the VirB8 X-ray structure has enabled a detailed structure-function analysis, which identified sites for the binding of VirB4 and VirB10 and for self-interaction. Due to its multiple interactions, VirB8 is an excellent model for the analysis of assembly factors of multiprotein complexes. In addition, VirB8 is a possible target for drugs that target its protein-protein interactions, which would disarm bacteria by depriving them of their essential virulence functions.     

Assembly and mechanisms of bacterial type IV secretion machines

Type IV secretion occurs across a wide range of prokaryotic cell envelopes: Gram-negative, Gram-positive, cell wall-less bacteria and some archaea. This diversity is reflected in the heterogeneity of components that constitute the secretion machines. Macromolecules are secreted in an ATP-dependent process using an envelope-spanning multi-protein channel. Similar to the type III systems, this apparatus extends beyond the cell surface as a pilus structure important for direct contact and penetration of the recipient cell surface. Type IV systems are remarkably versatile in that they mobilize a broad range of substrates, including single proteins, protein complexes, DNA and nucleoprotein complexes, across the cell envelope. These machines have broad clinical significance not only for delivering bacterial toxins or effector proteins directly into targeted host cells, but also for direct involvement in phenomena such as biofilm formation and the rapid horizontal spread of antibiotic resistance genes among the microbial community.     

Type IV secretion systems and their effectors in bacterial pathogenesis

Type IV secretion systems (T4SSs) are membrane-associated transporter complexes used by various bacteria to deliver substrate molecules to a wide range of target cells. T4SSs are involved in horizontal DNA transfer to other bacteria and eukaryotic cells, in DNA uptake from or release into the extracellular milieu, in toxin secretion and in the injection of virulence factors into eukaryotic host target cells by several mammalian pathogens. Rapid progress has been made towards defining the structures and functions of T4SSs, identifying the translocated effector molecules and elucidating the mechanisms by which the effectors subvert eukaryotic cellular processes during infection. These findings have had an important impact on our understanding of how these pathogens manipulate host cell functions to trigger bacterial uptake, facilitate intracellular growth and suppress defence mechanisms, thus facilitating bacterial colonization and disease development.     

Interbacterial macromolecular transfer by the Campylobacter fetus subsp. venerealis type IV secretion system

We report here the first demonstration of intra- and interspecies conjugative plasmid DNA transfer for Campylobacter fetus. Gene regions carried by a Campylobacter coli plasmid were identified that are sufficient for conjugative mobilization to Escherichia coli and C. fetus recipients. A broader functional range is predicted. Efficient DNA transfer involves the virB9 and virD4 genes of the type IV bacterial secretion system encoded by a pathogenicity island of C. fetus subsp. venerealis. Complementation of these phenotypes from expression constructions based on the promoter of the C. fetus surface antigen protein (sap) locus was temperature dependent, and a temperature regulation of the sap promoter was subsequently confirmed under laboratory conditions. Gene transfer was sensitive to surface or entry exclusion functions in potential recipient cells carrying IncPα plasmid RP4 implying functional relatedness to C. fetus proteins. The virB/virD4 locus is also known to be involved in bacterial invasion and killing of cultured human cells in vitro. Whether specifically secreted effector proteins contribute to host colonization and infection activities is currently unknown. Two putative effector proteins carrying an FIC domain conserved in a few bacterial type III and type IV secreted proteins of pathogens were analyzed for secretion by the C. fetus or heterologous conjugative systems. No evidence for interbacterial translocation of the Fic proteins was found.     

Type III effector proteins from the plant pathogen Xanthomonas and their role in the interaction with the host plant

Pathogenicity of Xanthomonas campestris pathovar (pv.) vesicatoria and most other Gram-negative bacterial plant pathogens largely depends on a type III secretion (TTS) system which is encoded by hypersensitive response and pathogenicity (hrp) genes. These genes are induced in the plant and are essential for the bacterium to be virulent in susceptible hosts and for the induction of the hypersensitive response (HR) in resistant host and non-host plants. The TTS machinery secretes proteins into the extracellular milieu and effector proteins into the plant cell cytosol. In the plant, the effectors presumably interfere with cellular processes to the benefit of the pathogen or have an avirulence activity that betrays the bacterium to the plant surveillance system. Type III effectors were identified by their avirulence activity, co-regulation with the TTS system and homology to known effectors. A number of effector proteins are members of families, e.g., the AvrBs3 family in Xanthomonas. AvrBs3 localizes to the nucleus of the plant cell where it modulates plant gene expression. Another family that is also present in Xanthomonas is the YopJ/AvrRxv family. The latter proteins appear to act as SUMO cysteine proteases in the host. Here, we will present an overview about the regulation of the TTS system and its substrates and discuss the function of the AvrRxv and AvrBs3 family members in more detail.     

Profiling the secretomes of plant pathogenic Proteobacteria

Secreted proteins are central to the success of plant pathogenic bacteria. They are used by plant pathogens to adhere to and degrade plant cell walls, to suppress plant defence responses, and to deliver bacterial DNA and proteins into the cytoplasm of plant cells. However, experimental investigations into the identity and role of secreted proteins in plant pathogenesis have been hindered by the fact that many of these proteins are only expressed or secreted in planta, that knockout mutations of individual proteins frequently have little or no obvious phenotype, and that some obligate and fastidious plant pathogens remain recalcitrant to genetic manipulation. The availability of genome sequence data for a large number of agriculturally and scientifically important plant pathogens enables us to predict and compare the complete secretomes of these bacteria. In this paper we outline strategies that are currently being used to identify secretion systems and secreted proteins in Proteobacterial plant pathogens and discuss the implications of these analyses for future investigations into the molecular mechanisms of plant pathogenesis.     

Visualization of secreted Hrp and Avr proteins along the Hrp pilus during type III secretion in Erwinia amylovora and Pseudomonas syringae

Pili are required for protein and/or DNA transfer from bacteria to recipient plant or bacterial cells, based on genetic evidence. However, it has never been shown directly that the effector proteins or DNA are localized along or inside the pili in situ. Failure to visualize an association of effector proteins/DNA with pili is the central issue in the debate regarding the exact function of pili in protein and DNA transfer. In this study, a newly developed in situ immunogold labelling procedure enabled visualization of the specific localization of type III effector proteins of Erwinia amylovora and Pseudomonas syringae pv. tomato along the Hrp pilus, but not along the flagellum or randomly in the intercellular space. In contrast, PelE, a pectate lyase secreted via the type II protein secretion system, was not associated with the Hrp pilus. These results provide direct evidence that type III secretion occurs only at the site of Hrp pilus assembly and that the Hrp pilus guides the transfer of effector proteins outside the bacterial cell, favouring the 'conduit/guiding filament' model.     

Rotting softly and stealthily

The soft rot erwiniae, which are plant pathogens on potato and other crops world-wide, synthesize and secrete large quantities of plant cell wall degrading enzymes that are responsible for the soft rot phenotype, earning them the epithet 'brute force' pathogens. They have been distinguished from classic 'stealth' pathogens, such as Pseudomonas syringae, which possesses an extensive battery of Type III secreted effector proteins and phytotoxins to manipulate and suppress host defences. However, recent studies, including whole-genome sequencing, are revealing many components of stealth pathogenesis within the soft rot erwiniae (SRE), suggesting that 'stealth' and 'brute force' should not be regarded as mutually exclusive modes of pathogenesis.     

IpgD, a protein secreted by the type III secretion machinery of Shigella flexneri, is chaperoned by IpgE and implicated in entry focus formation

Invasion of epithelial cells by Shigella flexneri involves entry and intercellular dissemination. Entry of bacteria into non-phagocytic cells requires the IpaA-D proteins that are secreted by the Mxi-Spa type III secretion machinery. Type III secretion systems are found in several Gram-negative pathogens and serve to inject bacterial effector proteins directly into the cytoplasm of host cells. In this study, we have analysed the IpgD protein of S. flexneri, the gene of which is located on the virulence plasmid at the 5' end of the mxi-spa locus. We have shown that IpgD (i) is stored in the bacterial cytoplasm in association with a specific chaperone, IpgE; (ii) is secreted by the Mxi-Spa type III secretion system in amounts similar to those of the IpaA-D proteins; (iii) is associated with IpaA in the extracellular medium; and (iv) is involved in the modulation of the host cell response after contact of the bacterium with epithelial cells. This suggests that IpgD is an effector that might be injected into host cells to manipulate cellular processes during infection.     

Bacterial Type IV secretion systems: versatile virulence machines

Many bacterial pathogens employ multicomponent protein complexes to deliver macromolecules directly into their eukaryotic host cell to promote infection. Some Gram-negative pathogens use a versatile Type IV secretion system (T4SS) that can translocate DNA or proteins into host cells. T4SSs represent major bacterial virulence determinants and have recently been the focus of intense research efforts designed to better understand and combat infectious diseases. Interestingly, although the two major classes of T4SSs function in a similar manner to secrete proteins, the translocated 'effectors' vary substantially from one organism to another. In fact, differing effector repertoires likely contribute to organism-specific host cell interactions and disease outcomes. In this review, we discuss the current state of T4SS research, with an emphasis on intracellular bacterial pathogens of humans and the diverse array of translocated effectors used to manipulate host cells.     

Functional analysis of HrpF, a putative type III translocon protein from Xanthomonas campestris pv. vesicatoria

Type III secretion systems (TTSSs) are specialized protein transport systems in gram-negative bacteria which target effector proteins into the host cell. The TTSS of the plant pathogen Xanthomonas campestris pv. vesicatoria, encoded by the hrp (hypersensitive reaction and pathogenicity) gene cluster, is essential for the interaction with the plant. One of the secreted proteins is HrpF, which is required for pathogenicity but dispensable for type III secretion of effector proteins in vitro, suggesting a role in translocation. In this study, complementation analyses of an hrpF null mutant strain using various deletion derivatives revealed the functional importance of the C-terminal hydrophobic protein region. Deletion of the N terminus abolished type III secretion of HrpF. Employing the type III effector AvrBs3 as a reporter, we show that the N terminus of HrpF contains a signal for secretion but not a functional translocation signal. Experiments with lipid bilayers revealed a lipid-binding activity of HrpF as well as HrpF-dependent pore formation. These data indicate that HrpF presumably plays a role at the bacterial-plant interface as part of a bacterial translocon which mediates effector protein delivery across the host cell membrane.     

Show me the substrates: modulation of host cell function by type IV secretion systems

Evidence for the involvement of type IV protein secretion systems in bacterial virulence is accumulating. Many of the substrate proteins secreted by type IV systems either hijack or interfere with specific host cell pathways. These substrates can be injected directly into host cells via the type IV apparatus or are secreted by the type IV machinery in a state that allows them to gain access to cellular targets without the further assistance of the type IV system. Arguably, the protein substrates of most type IV secretion systems remain undiscovered. Here, we review the activities of known type IV substrates and discuss the putative roles of unidentified substrates.     

The type III needle and the damage done

Many Gram-negative pathogens translocate virulence proteins directly into host cells using a type III secretion system. This complex secretion machinery is composed of approximately 25 different proteins that assemble to span both bacterial membranes, and contact the host cell to form a direct channel between the bacterial and host cell cytoplasms. Assembly of the system and efficient secretion of virulence proteins through this apparatus require specific chaperones. Although the machinery is morphologically conserved among all bacteria, the secreted proteins vary widely and are responsible for the range of diseases caused by bacterial pathogens. Recent structures have given insights into important chaperone and effector proteins, as well as revealing the first atomic structures of portions of the secretion machinery itself.     

Plant-bacterial pathogen interactions mediated by type III effectors

Effectors secreted by the bacterial type III system play a central role in the interaction between Gram-negative bacterial pathogens and their host plants. Recent advances in the effector studies have helped cementing several key concepts concerning bacterial pathogenesis, plant immunity, and plant-pathogen co-evolution. Type III effectors use a variety of biochemical mechanisms to target specific host proteins or DNA for pathogenesis. The identifications of their host targets led to the identification of novel components of plant innate immune system. Key modules of plant immune signaling pathways such as immune receptor complexes and MAPK cascades have emerged as a major battle ground for host-pathogen adaptation. These modules are attacked by multiple type III effectors, and some components of these modules have evolved to actively sense the effectors and trigger immunity.     

Regulatory Networks of the T4SS Control: From Host Cell Sensing to the Biogenesis and the Activity during the Infection

Delivery of effectors, DNA or proteins, that hijack host cell processes to the benefit of bacteria is a mechanism widely used by bacterial pathogens. It is achieved by complex effector injection devices, the secretion systems, among which Type 4 Secretion Systems (T4SSs) play a key role in bacterial virulence of numerous animal and plant pathogens. Considerable progress has recently been made in the structure–function analyses of T4SSs. Nevertheless, the signals and processes that trigger machine assembly and activity during infection, as well as those involved in substrate recognition and transfer, are complex and still poorly understood. In this review, we aim at summarizing the last updates of the knowledge on signaling pathways that regulate the biogenesis and the activity of T4SSs in important bacterial pathogens.     

Bacterial type IV secretion systems in human disease

Type IV secretion (T4S) systems are versatile machines involved in many processes relevant to bacterial virulence, such as horizontal DNA transfer and effector translocation into human cells. A recent workshop organized by the International University of Andalousia in Baeza, Spain, covered most aspects of bacterial T4S relevant to human disease, ranging from the structural and mechanistic analysis of the T4S systems to the physiological roles of the translocated effector proteins in subverting cellular functions in infected humans. This review reports the highlights from this workshop, which include the first visualization of a T4S system core complex spanning both membranes of Gram-negative bacteria, the identification of the first host receptors for T4S systems, the identification and characterization of novel T4S effector proteins, the analysis of the molecular function of effector proteins in subverting human cellular functions and an analysis of the role of T4S systems in the evolution of pathogenic bacteria. Our increasing knowledge of the biology of T4S systems improves our ability to exploit them as biotechnological tools or to use them as novel targets for a new generation of antimicrobials.     

Biological Diversity of Prokaryotic Type IV Secretion Systems

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.