Type III secretion: a secretory pathway serving both motility and virulence (Review)

‘Type III secretion’ (T3S) refers to a secretion pathway that is common to the flagellae of eubacteria and the injectisomes of some Gram-negative bacteria. Flagellae are rotary nanomachines allowing motility but they contain a built-in secretion apparatus that exports their own distal components to the distal end of the growing structure where they polymerize. In some cases they have been shown to export non-flagellar proteins. Injectisomes are transkingdom communication apparatuses allowing bacteria docked at the surface of a eukaryotic cell membrane to inject effector proteins across the two bacterial membranes and the eukaryotic cell membrane. Both nanomachines share a similar basal body embedded in the two bacterial membranes, topped either by a hook and a filament or by a stiff short needle. Both appear to be assembled in the same fashion. They recognize their substrate by a loose N-terminal peptide signal and the help of individual chaperones of a new type.     

Type III secretion: a bacterial device for close combat with cells of their eukaryotic host

Salmonella, Shigella, Yersinia, Pseudomonas aeruginosa, enteropathogenic Escherichia coli and several plant-pathogenic Gram-negative bacteria use a new type of systems called 'type III secretion' to attack their host. These systems are activated by contact with a eukaryotic cell membrane and they allow bacteria to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane to reach a given compartment and destroy or subvert the target cell. These systems consist of a secretion apparatus made up of about 25 individual proteins and a set of proteins released by this apparatus. Some of these released proteins are 'effectors' that are delivered by extracellular bacteria into the cytosol of the target cell while the others are 'translocators' that help the 'effectors' to cross the membrane of the eukaryotic cell. Most of the 'effectors' act on the cytoskeleton or on intracellular signalling cascades. One of the proteins injected by the enteropathogenic E. coli serves as a membrane receptor for the docking of the bacterium itself at the surface of the cell.     

Type III secretion system translocator has a molten globule conformation both in its free and chaperone-bound forms

Type III secretion systems of Gram-negative pathogenic bacteria allow the injection of effector proteins into the cytosol of host eukaryotic cells. Crossing of the eukaryotic plasma membrane is facilitated by a translocon, an oligomeric structure made up of two bacterial proteins inserted into the host membrane during infection. In Pseudomonas aeruginosa, a major human opportunistic pathogen, these proteins are PopB and PopD. Their interactions with their common chaperone PcrH in the cytosol of the bacteria are essential for the proper function of the injection system. The interaction region between PopD and PcrH was identified using limited proteolysis, revealing that the putative PopD transmembrane fragment is buried within the PopD/PcrH complex. In addition, structural features of PopD and PcrH, either individually or within the binary complex, were characterized using spectroscopic methods and 1D NMR. Whereas PcrH possesses the characteristics of a folded protein, PopD is in a molten globule state either alone or in the PopD/PcrH complex. The molten globule state is known to enable the membrane insertion of translocation/pore-forming domains of bacterial toxins. Therefore, within the bacterial cytoplasm, PopD is preserved in a state that is favorable to secretion and insertion into cell membranes.     

Translocators YopB and YopD from Yersinia enterocolitica form a multimeric integral membrane complex in eukaryotic cell membranes

The type III secretion systems are contact-activated secretion systems that allow bacteria to inject effector proteins across eukaryotic cell membranes. The secretion apparatus, called injectisome or needle complex, includes a needle that terminates with a tip structure. The injectisome exports its own distal components, like the needle subunit and the needle tip. Upon contact, it exports two hydrophobic proteins called translocators (YopB and YopD in Yersinia enterocolitica) and the effectors. The translocators, assisted by the needle tip, form a pore in the target cell membrane, but the structure of this pore remains elusive. Here, we purified the membranes from infected sheep erythrocytes, and we show that they contain integrated and not simply adherent YopB and YopD. In blue native PAGE, these proteins appeared as a multimeric 500- to 700-kDa complex. This heteropolymeric YopBD complex could be copurified after solubilization in 0.5% dodecyl maltoside but not visualized in the electron microscope. We speculate that this complex may not be stable and rigid but only transient.     

The blueprint of the type-3 injectisome

Type-3 secretion systems are sophisticated syringe-like nanomachines present in many animal and plant Gram-negative pathogens. They are capable of translocating an arsenal of specific bacterial toxins (effector proteins) from the prokaryotic cytoplasm across the three biological membranes directly into the eukaryotic cytosol, some of which modulate host cell mechanisms for the benefit of the pathogen. They populate a particular biological niche, which is maintained by specific, pathogen-dependent effectors. In contrast, the needle complex, which is the central component of this specialized protein delivery machine, is structurally well-conserved. It is a large supramolecular cylindrical structure composed of multiple copies of a relatively small subset of proteins, is embedded in the bacterial membranes and protrudes from the pathogen's surface with a needle filament. A central channel traverses the entire needle complex, and serves as a hollow conduit for proteins destined to travel this secretion pathway. In the past few years, there has been a tremendous increase in an understanding on both the structural and the mechanistic level. This review will thus focus on new insights of this remarkable molecular machine.     

Subversion of membrane transport pathways by vacuolar pathogens

Mammalian phagocytes control bacterial infections effectively through phagocytosis, the process by which particles engulfed at the cell surface are transported to lysosomes for destruction. However, intracellular pathogens have evolved mechanisms to avoid this fate. Many bacterial pathogens use specialized secretion systems to deliver proteins into host cells that subvert signaling pathways controlling membrane transport. These bacterial effectors modulate the function of proteins that regulate membrane transport and alter the phospholipid content of membranes. Elucidating the biochemical function of these effectors has provided a greater understanding of how bacteria control membrane transport to create a replicative niche within the host and provided insight into the regulation of membrane transport in eukaryotic cells.     

Bacterial nanomachines: the flagellum and type III injectisome

The bacterial flagellum and the virulence-associated injectisome are complex, structurally related nanomachines that bacteria use for locomotion or the translocation of virulence factors into eukaryotic host cells. The assembly of both structures and the transfer of extracellular proteins is mediated by a unique, multicomponent transport apparatus, the type III secretion system. Here, we discuss the significant progress that has been made in recent years in the visualization and functional characterization of many components of the type III secretion system, the structure of the bacterial flagellum, and the injectisome complex.     

Anchorless adhesins and invasins of Gram-positive bacteria: a new class of virulence factors

Bacterial adherence to and invasion of eukaryotic cells are important mechanisms of pathogenicity. Most Gram-positive bacteria interact with the components of the host extracellular matrix (ECM) to adhere to, colonize and invade cells and tissues. The bacterial proteins that bind to components of the ECM harbour signal sequences for their secretion and mechanisms of anchoring to the host cell surface. However, in recent years, some cell-surface adhesins and invasins of Gram-positive bacteria have been described that do not possess a signal sequence or a membrane anchor. These proteins are secreted by an as-yet-unknown mechanism and are probably localized on the bacterial surface by reassociation. These anchorless but surface-located adhesins and invasins represent a new class of virulence factors.     

Imaging the assembly, structure and activity of type III secretion systems

The type III secretion system (T3SS) is a sophisticated molecular machinery of Gram-negative bacteria used to 'inject' (translocate) bacterial proteins (effectors) into eukaryotic cells. For this, the T3SS has to assemble into a multiprotein complex, which is constituted of distinct parts; a basal body spanning the two bacterial membranes connected with a cytoplasmic bulb, an attached needle structure resembling a molecular syringe, and a distal needle tip structure that re-organizes into a 'translocon', which is a protein complex that inserts into the host cellular membrane. Upon engaging with eukaryotic cells, the T3SSs perform 'single-step' translocation of bacterial effector proteins across three membranes (two bacterial and one eukaryotic). Since the formulation of the major concepts of the T3SS about 15 years ago, imaging has been a major ingredient for elucidating the T3SS structure and function. Direct observation of molecular events in the context of cells will remain a key feature for better understanding of T3SS structure, regulation and function. In this review we describe how light and electron microscopy have been used to shed light on the processes of maturation and activity of the T3SS. Furthermore, we highlight recent imaging innovations with the potential to provide better insight into the T3SS structure and function.     

The type III secretion system tip complex and translocon

The type III secretion machinery of Gram-negative bacteria, also known as the injectisome or needle complex, is composed of a basal body spanning both bacterial membranes and the periplasm, and an external needle protruding from the bacterial surface. A set of three proteins, two hydrophobic and one hydrophilic, are required to allow translocation of proteins from the bacterium to the host cell cytoplasm. These proteins are involved in the formation of a translocation pore, the translocon, in the host cell membrane. Exciting progress has recently been made on the interaction between the translocators and the injectisome needle and the assembly of the translocon in the host cell membrane. As expected, the two hydrophobic translocators insert into the target cell membrane. Unexpectedly, the third, hydrophilic translocator, forms a complex on the distal end of the injectisome needle, the tip complex, and serves as an assembly platform for the two hydrophobic translocators.     

The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus

Gram-negative bacteria commonly interact with eukaryotic host cells by using type III secretion systems (TTSSs or secretons). TTSSs serve to transfer bacterial proteins into host cells. Two translocators, IpaB and IpaC, are first inserted with the aid of IpaD by Shigella into the host cell membrane. Then at least two supplementary effectors of cell invasion, IpaA and IpgD, are transferred into the host cytoplasm. How TTSSs are induced to secrete is unknown, but their activation appears to require direct contact of the external distal tip of the apparatus with the host cell. The extracellular domain of the TTSS is a hollow needle protruding 60 nm beyond the bacterial surface. The monomeric unit of the Shigella flexneri needle, MxiH, forms a superhelical assembly. To probe the role of the needle in the activation of the TTSS for secretion, we examined the structure-function relationship of MxiH by mutagenesis. Most point mutations led to normal needle assembly, but some led to polymerization or possible length control defects. In other mutants, secretion was constitutively turned "on." In a further set, it was "constitutively on" but experimentally "uninducible." Finally, upon induction of secretion, some mutants released only the translocators and not the effectors. Most types of mutants were defective in interactions with host cells. Together, these data indicate that the needle directly controls the activity of the TTSS and suggest that it may be used to "sense" host cells.     

Port of entry--the type III secretion translocon

Many Gram-negative plant and animal pathogenic bacteria use a specialized type III secretion system (TTSS) as a molecular syringe to inject effector proteins directly into the host cell. Protein translocation across the eukaryotic host cell membrane is presumably mediated by a bacterial translocon. The structure of this predicted transmembrane complex and the mechanism of transport are far from being understood. In bacterial pathogens of animals, several putative type III secretion translocon proteins (TTPs) have been identified. Interestingly, TTP sequences are not conserved among different bacterial species, however, there are structural similarities such as transmembrane segments and coiled-coil regions. Accumulating evidence suggests that TTPs are components of oligomeric protein channels that are inserted into the host cell membrane by the TTSS.     

The cytoplasmic portion of the T3SS inner membrane ring components sort into distinct families based on biophysical properties

Type III secretion systems are used by many Gram-negative bacteria to inject effector proteins into eukaryotic cells to subvert their normal activities. Structurally conserved portions of the type III secretion apparatus include a: basal body located within the bacterial envelope; an exposed needle with tip complex that delivers effectors across the target cell membrane; and cytoplasmic sorting platform that selects cargo and powers secretion. While structurally conserved, the individual proteins that make up this nanomachine are typically not interchangeable though they do tend to fall into families. Here we selected a single domain from the inner membrane ring of the basal body from six different type III secretion systems (called SctD using a proposed unifying nomenclature). The selected domain creates an integral interface between the basal body and the sorting platform. Therefore, it represents a pivotal point between two distinct assemblies. All six protein domains possess a structural motif called a forkhead-associated-like (FHA-like) domain but differ greatly in their sequences and solution behaviors. These differences are used here to define family boundaries for these FHA-like domains. The data parallel, though not precisely, family boundaries defined by other proteins within the apparatus and by phylogenetic analysis. Ultimately, differences in the families are likely to reflect differences in the activities of these type III secretion systems or the host niches in which these pathogens are found.     

Type III secretion: the bacteria-eukaryotic cell express

Type III secretion (T3S) is an export pathway used by Gram-negative pathogenic bacteria to inject bacterial proteins into the cytosol of eukaryotic host cells. This pathway is characterized by (i) a secretion nanomachine related to the bacterial flagellum, but usually topped by a stiff needle-like structure; (ii) the assembly in the eukaryotic cell membrane of a translocation pore formed by T3S substrates; (iii) a non-cleavable N-terminal secretion signal; (iv) T3S chaperones, assisting the secretion of some substrates; (v) a control mechanism ensuring protein delivery at the right place and time. Here, we review these different aspects focusing in open questions that promise exciting findings in the near future.     

Omp85 in eukaryotic systems: one protein family with distinct functions

Omp85-like proteins are evolutionary ancient components of bacterial outer membranes and their evolutionary offspring. As a consequence, proteins of this family can be found in the outer membrane systems of Gram-negative bacteria and endosymbiotically derived organelles. In the different membranes, they perform distinct functions such as catalyzing protein insertion into or protein transport across the bilayer. Here, the knowledge on the Omp85-like proteins in the eukaryotic system with regard to structural properties and physiological behavior is summarized.     

Genomic analysis of secretion systems

Secretion of proteins into the extracellular environment is important to almost all bacteria, and in particular mediates interactions between pathogenic or symbiotic bacteria with their eukaryotic hosts. The accumulation of bacterial genome sequence data in the past few years has provided great insights into the distribution and function of these secretion systems. Three systems are responsible for secretion of proteins across the bacterial cytoplasmic membrane: Sec, SRP and Tat. Many novel examples of systems for transport across the Gram-negative bacterial cell envelope have been discovered through genome sequencing and surveys, including many novel type III secretion systems and autotransporters. Similarly, genomic data mining has revealed many new potential secretion substrates and identified unsuspected domains in secretion-associated proteins. Interestingly, genomic analyses have also hinted at the existence of a dedicated protein secretion system in Gram-positive bacteria, targeting members of the WXG100/ESAT-6 family of proteins, and have revealed an unexpectedly wide distribution of sortase-driven protein-targeting systems.     

Type III machines of pathogenic yersiniae secrete virulence factors into the extracellular milieu

Gram-negative bacteria use type III machines to inject toxic proteins into the cytosol of eukaryotic cells. Pathogenic Yersinia species export 14 Yop proteins by the type III pathway and some of these, named effector Yops, are targeted into macrophages, thereby preventing phagocytosis and allowing bacterial replication within lymphoid tissues. Hitherto, YopB/YopD were thought to insert into the plasma membrane of macrophages and to promote the import of effector Yops into the eukaryotic cytosol. We show here that the type III machines of yersiniae secrete three proteins into the extracellular milieu (YopB, YopD and YopR). Although intrabacterial YopD is required for the injection of toxins into eukaryotic cells, secreted YopB, YopD and YopR are dispensable for this process. Nevertheless, YopB, YopD and YopR are essential for the establishment of Yersinia infections in a mouse model system, suggesting that type III secretion machines function to deliver virulence factors into the extracellular milieu also.     

Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA

The cytokinetic Z ring is required for bacterial cell division. It consists of polymers of FtsZ, the bacterial ancestor of eukaryotic tubulin, linked to the cytoplasmic membrane. Formation of a Z ring in Escherichia coli occurs as long as one of two proteins, ZipA or FtsA, is present. Both of these proteins bind FtsZ suggesting that they might function to tether FtsZ filaments to the membrane. Although ZipA has a transmembrane domain and therefore can function as a membrane anchor, interaction of FtsA with the membrane has not been explored. In this study we demonstrate that FtsA, which is structurally related to eukaryotic actin, has a conserved C-terminal amphipathic helix that is essential for FtsA function. It is required to target FtsA to the membrane and subsequently to the Z ring. As FtsA is much more widely conserved in bacteria than ZipA, it is likely that FtsA serves as the principal membrane anchor for the Z ring.