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Secretion in Gram negative bacteria

Secretion in Gram negative bacteria


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Among the 6 secretion systems in bacteria, can these 6 ways be sorted out in the order of how harmful it is to the human host? Like say type 3 is highly virulent so that comes first, but I don't know about the rest of the systems. Being a computer science student, I have failed to understand the Wikipedia article to derive this order. Does a discrete order even exist? Can anyone answer this question?


Theoretically there are two very basic things to consider in judging virulence for the secretion systems: a) Can it secrete into any human cell? (If not, chances of being virulent are smaller, but not zero as whatever is secreted in the environment of a cell could also be harmful for the cell.) b) Does it secrete some agent (protein, RNA, molecule), which is potentially harmful for a human cell?

a) alone is not sufficient for it to be virulent if b) is not also given. (As it could inject something in a cell that the cell can handle without a problem.) b) alone is not sufficient, as it has to get inside the cell somehow. Although, b) alone can work, if the agent has another way to get inside a cell or to harm the cell from outside.

So, generally, without knowing ALL the bacterial secretion systems and secreted agents, it is impossible to decide if one secretion system (alone) is always, sometimes or never harmful.


I think you cannot find a better answer then the following review:

http://www.nature.com/nrmicro/journal/v13/n6/full/nrmicro3456.html

It was published in 2015, and it gives a good overview about the secretion systems. Anyway, T6SS can also be virulent. Vibrio cholerae can use an effector of this SS to cross-link actin in the host cell and modify its morphology and its cytoskeleton behaviour. In general I think that there is not a general order of virulence, also because the T3SS was discovered long ago. Whereas, the T6SS just recently, so the amount of information about its function is still somehow limited.

Hope this helps.


Two-partner Secretion of Gram-negative Bacteria

The mechanisms of protein secretion by pathogenic bacteria remain poorly understood. In Gram-negative bacteria, the two-partner secretion pathway exports large, mostly virulence-related “TpsA” proteins across the outer membrane via their dedicated “TpsB” transporters. TpsB transporters belong to the ubiquitous Omp85 superfamily, whose members are involved in protein translocation across, or integration into, cellular membranes. The filamentous hemagglutinin/FhaC pair of Bordetella pertussis is a model two-partner secretion system. We have reconstituted the TpsB transporter FhaC into proteoliposomes and demonstrate that FhaC is the sole outer membrane protein required for translocation of its cognate TpsA protein. This is the first in vitro system for analyzing protein secretion across the outer membrane of Gram-negative bacteria. Our data also provide clear evidence for the protein translocation function of Omp85 transporters.


Structural Biology of Bacterial Secretion Systems in Gram-Negative Pathogens- Potential for New Drug Targets

Author(s): E. Durand, D. Verger, A. Toste Rego, V. Chandran, G. Meng, R. Fronzes, G. Waksman Institute of Structural and Molecular Biology, Birkbeck and University College London, Malet Street, London, WC1E 7HX.

Affiliation:

Journal Name: Infectious Disorders - Drug Targets
Formerly Current Drug Targets - Infectious Disorders

Volume 9 , Issue 5 , 2009




Abstract:

Gram-negative bacteria have evolved diverse secretion systems/machineries to translocate substrates across the cell envelope. These various machineries fulfil a wide variety of functions but are also essential for pathogenic bacteria to infect human or plant cells. Secretion systems, of which there are seven, utilize one of two secretion mechanisms: (i) the one-step mechanism, whereby substrates are translocated directly from the bacterial-cytoplasm to the extracellular medium or into the eukaryotic-target cell (ii) the two-step mechanism, whereby substrates are first translocated across the bacterial-inner membrane once in the periplasm, substrates are targeted to one of the secretion systems that mediate the transport across the outer membrane and the release outside the bacterial cell. This review describes in details the main structural features of these secretion systems. Structural biology offers the possibility to understand the molecular mechanisms at play in the various secretion systems. It also helps to design specifically drugs that can block these machineries and thus attenuate the virulence of pathogenic bacteria.

Infectious Disorders - Drug Targets

Title: Structural Biology of Bacterial Secretion Systems in Gram-Negative Pathogens- Potential for New Drug Targets

VOLUME: 9 ISSUE: 5

Author(s):E. Durand, D. Verger, A. Toste Rego, V. Chandran, G. Meng, R. Fronzes and G. Waksman

Affiliation:Institute of Structural and Molecular Biology, Birkbeck and University College London, Malet Street, London, WC1E 7HX.

Abstract: Gram-negative bacteria have evolved diverse secretion systems/machineries to translocate substrates across the cell envelope. These various machineries fulfil a wide variety of functions but are also essential for pathogenic bacteria to infect human or plant cells. Secretion systems, of which there are seven, utilize one of two secretion mechanisms: (i) the one-step mechanism, whereby substrates are translocated directly from the bacterial-cytoplasm to the extracellular medium or into the eukaryotic-target cell (ii) the two-step mechanism, whereby substrates are first translocated across the bacterial-inner membrane once in the periplasm, substrates are targeted to one of the secretion systems that mediate the transport across the outer membrane and the release outside the bacterial cell. This review describes in details the main structural features of these secretion systems. Structural biology offers the possibility to understand the molecular mechanisms at play in the various secretion systems. It also helps to design specifically drugs that can block these machineries and thus attenuate the virulence of pathogenic bacteria.


Protein Secretion Pathways

Bacteria and eukaryotic cells have specialized systems for exporting certain proteins (secretory proteins) to the external environment. Generally, secretory proteins are required for acquiring nutrients, cell-to-cell communication, protection, and structures that reside on the outer surface of the cell membrane. The primary impediment to the release of a secretory protein is a membrane. The processes that facilitate secretion of proteins through such a formidable barrier are similar among all organisms, although there are significant differences between organisms. For example, gram-negative and gram-positive bacteria do not have the same secretory pathways.

Schematic representation of secretion in gram-positive bacteria. A signal recognition particle (SRP) binds to the signal peptide of a secretory protein,
and this complex binds to a membrane protein that directs the secretory protein (1) to the Sec complex. There is also an SRP-independent pathway (2), where a signal peptide alone makes contact with the Sec complex. The secretory protein is translocated through a channel within the Sec complex (3), and the signal peptide is removed by a signal peptidase(s). Proper folding of the secretory protein occurs as it passes through the cell wall (4).

A secreted protein in gram-negative bacteria must pass through an inner membrane, a periplasmic space, and an outer membrane to exit the cell, whereas in gram-positive bacteria, secretory proteins are transported only across a single cytoplasmic membrane. In contrast, the secretory system in higher organisms is more complex. Unlike prokaryotic proteins, many eukaryotic proteins require a number of highly specific modifications, such as glycosylation, acetylation, sulfation, and phosphorylation, to produce functional secretory proteins. Some of these protein modifications and various processing steps are carried out in the endoplasmic reticulum, and others take place in the Golgi apparatus, where proteins are also sorted according to their final cellular destinations, including those that exit through the cell membrane. The property that distinguishes a protein that remains in the cytoplasm from one that is secreted is often an amino acid sequence (a signal peptide, signal sequence, leader sequence, or leader peptide) at its N terminus. In gram-positive bacteria, the signal peptide of some secretory proteins makes direct contact with a membrane-bound assembly of proteins (a secretion complex, or Sec complex) that facilitates the passage of these proteins through the membrane and their release to the external environment.

Schematic representation of a type II secretion pathway in gram-negative bacteria. The SecB protein binds to a secretory protein in the cytoplasm (1),
SecB attaches to the SecA protein that is part of the Sec complex of the inner membrane (2), and the secretory protein is translocated through the inner membrane (3). A signal peptidase removes the signal peptide, and the secretory protein is properly folded in the periplasm (4) the secretory protein combines with the Gsp complex (5) and it is translocated to the external environment (6).

Alternatively, for other secretory proteins, a group of proteins called a signal recognition complex binds to a signal peptide, and this combination attaches to a membrane-bound signal recognition complex receptor before making contact with the Sec complex. In both cases, the secretory protein is translocated through a channel formed by the Sec complex, and its release depends on removal of the signal peptide by a membrane-bound enzyme called a signal peptidase. Subsequently, proteins that have crossed the cytoplasmic membrane readily pass through the porous cell wall, where they encounter metal ions and other components that promote proper folding and molecular stabilization. Gram-negative bacteria have multiple pathways for the secretion of various proteins. Some of these systems (Sec-dependent pathways) use the same membrane-bound Sec complex for transmitting a secretory protein through the inner membrane into the periplasm. Collectively, the Sec dependent pathways are designated the general secretion pathway.

In these instances, a cytoplasmic protein (SecB) binds an amino acid sequence (domain) of a secretory protein that has a signal peptide. In turn, the SecB protein combines with a protein (SecA) of the membrane bound Sec complex. The secretory protein is translocated into the periplasm, and the signal peptide is removed. At this point, the secretory protein encounters various periplasmic proteins that ensure proper folding. Thereafter, Secdependent secretory proteins exit through the outer membrane by different routes. A region of some proteins is capable of forming a channel in the outer membrane that allows part of the remaining protein to be selectively extruded (autotransporter pathway). In these cases, proteolytic cleavage releases the functional portion of the protein to the external environment. Other proteins are able to pass through an outer membrane channel that is formed by a separate protein (single accessory pathway).

The type III secretion system is made up of about 20 different proteins
that form a continuous channel through the inner and outer membranes of gram negative bacteria. The type III secretion system is used by bacterial pathogens to secrete toxins and other proteins into plant and animal host cells. A hollow needle like protein structure extends from the bacterial surface into the host cell.

Another pathway (chaperone/usher pathway) is used by specific proteins that form fimbriae on the surface of the bacterial cell. A fourth general secretion pathway branch called the type II secretion pathway consists of a protein complex (the Gsp complex) that spans the periplasmic space and forms a channel through the outer membrane. Most secreted proteins pass through the type II pathway. In these cases, secretory proteins earmarked for the type II pathway are first transported to the periplasmic space via the Secdependent pathway, where they bind to the Gsp complex and are shunted through the outer membrane. Other Sec-dependent pathways have been found in various gram-negative bacteria. In contrast to the type II pathway, the type I and type III secretion pathways are Sec independent, and each has its own protein complex that extends from the inner to the outer membrane, forming a continuous channel from the bacterial cytoplasm to the external environment.

For example, bacterial flagellar proteins reach the outer surface of the cell by means of a type III secretion pathway. Type III secretion pathways are often used by bacterial pathogens to secrete bacterial proteins into the cytoplasm of eukaryotic host cells. Signal peptides are recognized by the Sec-independent systems but are not necessarily cleaved during the secretion process.

Schematic representation of the secretion pathway in eukaryotes. (A) A signal recognition particle (SRP) binds to the signal sequence of a secretory protein. (B) The SRP attaches to an SRP receptor on the endoplasmic reticulum (ER) membrane. (C) The secretory protein is translocated into the lumen of the ER, and a signal peptidase removes the signal sequence. (D) The secretory protein is folded, partially modified, and packaged in a transport vesicle intended for the Golgi network. (E) The ER-released vesicle carrying the secretory protein enters the Golgi network at the cis face and passes through the Golgi stack, where it is further modified after it is sorted, a plasma membrane-specific vesicle is formed at the trans face of the Golgi network. The secretory transport vesicle fuses with the plasma membrane and releases the secretory protein to the extracellular environment.

Protein secretion is basically the same in all eukaryotic organisms from yeast to plant and animal cells. Briefly, the signal sequence of a secretory protein is bound by a signal recognition particle during protein synthesis the signal recognition particle attaches to a receptor on the membrane ofthe endoplasmic reticulum, and the secretory protein passes through a channel in the membrane as translation proceeds a signal peptidase removes the signal sequence and the secretory protein is released into the lumen of the endoplasmic reticulum, where it is folded and, if required, glycosylated. A vesicle containing a processed secretory protein buds off from the endoplasmic reticulum and is transported to and fuses with the cis face of the Golgi apparatus.

Additional processing, glycosylations, and posttranslation modifications take place in the Golgi stack. The secretory protein then emerges from the trans face of the Golgi apparatus enclosed in a vesicle that is transported to and fuses with the plasma membrane, where the contents are released to the external environment. In eukaryotic organisms, some proteins are secreted continuously (constitutive secretion). Others remain in vesicles (mature secretory granules near the plasma membrane and are released only after a hormone ormembrane depolarization signal is received.


PROTEIN SECRETION BY GRAM-NEGATIVE BACTERIA

A number of Gram-negative bacteria rely on dedicated secretion systems to transport virulence proteins outside of the cell and, in some cases, directly into the cytoplasm of a eukaryotic or prokaryotic target cell. Extracellular protein secretion can be a challenge for Gram-negative bacteria, because these secreted proteins must cross two (and, in some cases, three) phospholipid membranes in order to reach their final destination ( Fig 3 ). Some secreted proteins in Gram-negative bacteria traverse these membranes in two separate steps, where they are first delivered to the periplasm through the Sec or Tat secretion systems, as discussed in the preceding section, and are then transferred across the outer membrane by a second transport system. This process is known as Sec- or Tat-dependent protein secretion. Additionally, many other proteins are secreted through channels that span both the inner and outer bacterial membranes through a process known as Sec- or Tat-independent protein secretion. The dedicated secretion systems in Gram-negative bacteria are numbered Type I through Type VI, with each system transporting a specific subset of proteins. These systems all rely on β-barrel channels that form a ring in the outer membrane of the bacterial cell, but otherwise exhibit a fair amount of diversity in their structures and mechanistic functions, as will be outlined below.

Gram-negative bacteria utilize a number of dedicated protein secretion systems to transport proteins across 1, 2, or 3 phospholipid membranes. Some proteins are secreted in a two-step, Sec- or Tat-dependent mechanism. These proteins cross the inner membrane with the help of either the Sec or Tat secretion pathways and are then transported across the outer membrane using a second secretion system. The T2SSs and T5SSs secrete proteins in this manner. Because it secretes folded substrates, the T2SS translocates proteins initially transported by either the Tat or Sec pathway (where Sec substrates are folded in the periplasm). In contrast, autotransporters of the T5SS must be unfolded prior to outer membrane transport and thus must be secreted across the inner membrane by the Sec pathway. Additionally, several Gram-negative protein secretion systems transport their substrates across both bacterial membranes in a onestep, Sec- or Tat-independent process. These include the T1SSs, T3SSs, T4SSs, and T6SSs. All of these pathways contain periplasm-spanning channels and secrete proteins from the cytoplasm outside the cell, however, their mechanisms of protein secretion are quite different. Three of these secretion systems, the T3SS, T4SS, and T6SS can also transport proteins across an additional host cell membrane, delivering secreted proteins directly to the cytosol of a target cell.

The Type I Secretion System

Type I secretion systems (T1SSs) have been found in a large number of Gram-negative bacteria, including pathogens of plants and animals, where they transport their substrates in a one-step process (as demonstrated in Fig. 3 ) across both the inner and outer bacterial membranes (recently reviewed in (25)). Unlike other protein transport systems found in Gram-negative bacteria, T1SSs closely resemble a large family of ATP-binding cassette (ABC) transporters, which export small molecules such as antibiotics and toxins out of the cell (26). Some bacteria may have several T1SSs, each of which is dedicated to transporting one or a few unfolded substrates (27). These substrates range in function and include digestive enzymes, such as proteases and lipases, as well as adhesins, heme-binding proteins, and proteins with repeats-in-toxins (RTX) motifs. T1SS substrates are generally Sec-independent and typically, but don’t always, contain a C-terminal signal sequence that is recognized by the T1SS and remains uncleaved (see below).

T1SSs have three essential structural components: an ABC transporter protein in the inner membrane, a membrane fusion protein (MFP) that crosses the inner membrane and bridges it to the outer membrane factor (OMF) in the outer membrane (25). The ABC transporter component associated with the T1SS has several critical functions – it catalyzes ATP to provide the energy to transport the substrate, interacts with the MFP, and participates in substrate recognition (28). The MFP associates with the ABC transporter in the inner membrane and spans the periplasm to associate with the OMF (29�). In addition, the cytoplasmically located N-terminus of the MFP is believed to play a role in substrate selection (30, 32). The OMF generates a pore in the outer membrane, through which the substrate passes in an unfolded state. Interestingly, T1SSs often use the multi-purpose protein TolC as their OMF (27). This pore-forming protein is also used to export molecules and other compounds, and is recruited to the MFP after the ABC transporter and MFP have contacted a substrate(32).

The T1SS ABC transporters have been further divided into three groups based on their N-terminal sequences (reviewed in (28)). One class of ABC transporters contains a C39 peptidase domain, which belongs to the papain superfamily structural motif. The C39-peptidase-containing ABC-transporters are critical for recognizing and cleaving the N-termini of substrates. An example of a T1SS substrate with a C39 peptidase domain is Colicin V of E. coli (33). A second class of ABC transporters contains a C39-like peptidase domain (CLD) that lacks proteolytic activity and, therefore, does not cleave its designated substrates (34). Substrates of CLD-containing ABC transporters generally contain RTX motifs and are much larger than those secreted by a C39-containing peptidase ABC transporter. Interestingly, RTX motifs bind to calcium at extracellular, but not intracellular levels. Because calcium binding promotes the folding of these proteins, these large substrates are able to remain unfolded inside the cell (35). Finally, a third class of T1SS ABC transporters lacks any additional sequences in the N-terminal domain. Their substrates may or may not contain RTX motifs but are smaller in size than substrates transported by CLD-containing ABC transporters and contain secretion signals at their C-termini (27).

T1SS substrates contribute to virulence in a variety of bacterial pathogens, including V. cholerae, which uses its T1SS to secrete the MARTX toxin (36), and Serratia marcescens, which secretes the hemophore HasA via the T1SS pathway (29). One of the best-studied T1SS substrates is the HlyA hemolysin protein of uropathogenic E. coli (37�). This RTX-family toxin inserts into the membranes of both erythrocytes and nucleated eukaryotic cells, causing them to rupture (37). Rupture of host cells by HlyA can help the bacteria to cross mucosal barriers, and additionally, can damage effector immune cells, which prevents clearance of the infection.

The Type II Secretion System

Type II secretion systems (T2SSs) are conserved in most Gram-negative bacteria, where they transport folded proteins from the periplasm into the extracellular environment. Because the T2SS channel is only found in the outer membrane ( Fig. 3 ), proteins secreted through this apparatus must first be delivered to the periplasm via the Sec or Tat secretion pathways, which transfer protein substrates across the inner membrane, as described earlier in this chapter. This secretion system was originally called the main terminal branch of the Sec secretion pathway due to its ability to export proteins transported across the inner membrane by the Sec secretion system (3). However, this nomenclature has since been updated to the T2SS to reflect the ability of these secretion systems to transport Tat-secreted proteins as well (40). Because proteins destined for secretion by the T2SS apparatus must first pass through the Sec or Tat inner membrane transporters, T2SS substrates must have a Sec- or Tat-type cleavable signal sequence at their N termini (3). Additionally, because the T2SS secretes folded substrates, proteins transported across the cytoplasmic membrane by the Sec pathway must be folded in the periplasm prior to export through the T2SS.

T2SSs have a broad specificity and are capable of secreting a diverse array of substrates outside of the bacterial cell, some of which contribute to the virulence of bacterial pathogens (3). In some bacterial species, the T2SS is required for the secretion of multiple substrates, while in others, it is only used to transport a single protein (41). These secreted proteins have a range of biological functions, but are generally enzymes, such as proteases, lipases, and phosphatases, as well as several proteins that process carbohydrates (3).

T2SSs are complex and consist of as many as 15 different proteins, which can be broken into four subassemblies: the outer-membrane complex, the inner-membrane platform, the secretion ATPase, and the pseudopilus (3). As its name suggests, the outer-membrane complex resides in the outer membrane, where it serves as the channel through which folded periplasmic T2SS substrates are translocated (42). This channel is composed of a multimeric protein called the secretin. The secretin has a long N terminus, which is believed to extend all the way to the periplasm to make contact with other T2SS proteins in the inner membrane (42). The inner membrane platform, which is composed of multiple copies of at least 4 proteins, is embedded in the inner membrane and extends into the periplasm, contacting the secretin. This platform plays a crucial role in the secretion process, by communicating with the secretin, pseudopilus, and the ATPase to coordinate export of substrates (3). The ATPase is located in the cytoplasm and provides the energy to power the system. As its name implies, the T2SS pseudopilus is evolutionarily related and structurally similar to proteins that comprise type IV pili on bacterial cell surfaces, as well as some bacterial competence systems (43). Therefore, one model for secretion through the T2SS channel proposes that these pseudopili retract in order to push the folded T2SS substrate through the outer membrane channel. In this “piston” model, “secretion-competent” proteins in the periplasm contact the periplasmic domain of the secretin. This interaction is believed to stimulate the cytoplasmic ATPase to drive retraction of the T2SS pseudopili, which push proteins through the secretin channel (3, 44, 45).

A number of bacterial pathogens employ T2SSs to transport virulence factors outside of the cell. Examples of T2SS substrates that are important for virulence in a mammalian host include the cholera toxin of V. cholerae (46), which causes the watery diarrhea associated with the disease cholera, and exotoxin A of P. aeruginosa (47), which blocks protein synthesis in host cells, leading to lethal infection by this bacterium. Still, other pathogens use their T2SSs to secrete enzymes that help them adapt to their environment, which can include plant and animal hosts. These pathogens include Legionella pneumophila (48), enterotoxigenic and enterohemorrhagic E. coli (ETEC and EHEC) (49�), K. pneumoniae (52), Aeromonas hydrophila (53), and Dickeya dadantii (54).

The Type III Secretion System

Type III secretions systems (T3SSs) are found in a large number of Gram-negative bacterial pathogens and symbionts (reviewed in (55)). T3SSs have been described as “injectisomes” and “needle and syringe”-like apparatuses because of their structure (see Fig. 3 ). They secrete a wide variety of proteinaceous substrates across both the inner and outer bacterial membranes. In addition, most T3SSs also transport substrates into a target eukaryotic cell membrane in the same step and, therefore, actually transport proteins across three membranes. Secretion of T3SS substrates is generally thought to be a one-step process, although recently this notion has been challenged in Yersinia (discussed below). T3SS substrates are generically called effector proteins. Pathogens may secrete only a few effectors proteins, as in the cases of Pseudomonas and Yersinia, or several dozen, as in the cases of Shigella and EHEC. Secretion signals are embedded within the N-termini of T3SS substrates and are not cleaved. Many, but not all T3SS effectors have chaperones that guide them to the T3SS base, where they are secreted in an ATP-dependent, unfolded state.

The T3SS has a core of 9 proteins that are highly conserved among all known systems (reviewed in (56, 57)). They share 8 of these proteins with the flagellar apparatus found in many bacteria and are evolutionarily related to flagellin (58). In addition to these 9 core proteins, T3SSs have an additional 10 to 20 proteins that play either essential or important roles in their function. The structural components of T3SSs are typically encoded in a few operons, which can be found either in pathogenicity islands in the bacterial chromosome or on plasmids. Because T3SSs are typically horizontally acquired, bacteria that are evolutionary distinct may have closely related systems and vice versa (58). For example, the genomes of Shigella and E. coli are highly homologous, yet the Shigella T3SS is more similar to the Salmonella T3SS than it is to systems found in the E. coli pathogens EHEC and EPEC. Seven families of T3SSs have been proposed primarily based on the homology of their extracellularly elaborated needles, tips, and translocons (58).

The T3SS can be broken down into three main components: a base complex or basal body, the needle component, and the translocon (56). The base complex contains cytoplasmic components and spans the inner and outer membrane, forming a socket-like structure consisting of several rings with a center rod (59). In most systems it is comprised of at least 15 proteins (56, 57). Encased by and emanating from this socket and rod-like structure is a filament called the needle, which extends through the secretin and into the extracellular space (59). The T3SS needle has an inner hollow core that is wide enough to permit an unfolded effector to traverse (60, 61). Excitingly, recent work has visualized a ‘trapped’ effector protein by cryo-EM and single particle analysis, supporting the model that substrates can traverse through the needle (62, 63).

The T3SS tip complex, which resides on the outer end of the needle, is critical for sensing contact with host cells and regulating secretion of effectors (64, 65). It is also necessary for insertion of the translocon into host cell membranes (65, 66). The T3SS translocon is essential for passage of effectors through host cell membranes, but not for secretion of effectors outside of the bacterium (67, 68). Translocons are assembled upon contact with host cells and form a pore that is essential for effector delivery (66). Recently, however, an alternative two-step model of translocation of Type 3 effectors has been proposed, where effectors and translocon components are secreted prior to host cell contact and remain associated with the bacteria, perhaps in lipid vesicles (69, 70). After contact with host cells, perhaps sensed through the needle, the translocon and tip proteins form a pore through which the effectors pass. Additional experiments are needed to determine the mechanism by which translocation occurs.

Translocation of T3SS effectors into host cells is essential for the virulence of many pathogens, including pathogenic species of Yersinia, Salmonella, and Shigella (55). Over the last 25 years, much work has focused on understanding the functions of T3SS effector proteins. Their functions vary widely among different pathogens and how they jointly orchestrate their effects on host cells is still being elucidated (71�). Many of these effectors remodel normal cellular functions to enable the pathogen to establish an infectious niche either within the host cell or in mammalian tissue sites. Impressively, the study of these effectors has provided fundamental insights into a number of different facets of eukaryotic cell biology.

The Type IV Secretion System

Type IV secretion systems (T4SSs) are ancestrally related to bacterial DNA conjugation systems and can secrete a variety of substrates, including single proteins and protein-protein and DNA-protein complexes (75). T4SSs secrete substrates into a wide range of target cells, including other bacteria (of the same or different species) and eukaryotic cells. These macromolecular complexes are largely found in Gram-negative bacteria, where they transport substrates across both the inner and outer membranes ( Fig. 3 ). Like T3SSs, T4SSs can span an additional, host cell membrane, allowing for direct transfer of substrates into the cytoplasm of the recipient cell. Because T4SSs are capable of transferring both DNA and proteins, they can serve a variety of functions, including conjugative transfer of DNA, DNA uptake and release, and translocation of effector proteins or DNA/protein complexes directly into recipient cells.

Despite the diversity in their substrates and functions, all T4SSs are evolutionarily related, sharing common components and operating in a similar manner (76). For that reason, the remainder of this section will focus on the VirB/D system of Agrobacterium tumeficans as a model of Type IV Secretion. A. tumeficans uses its T4SS to transport oncogenic T-DNA into plant cells, and has served as the paradigm for studying T4SS assembly and function (77). The VirB/D T4SS contains 12 proteins, named VirB1-VirB11 and VirD4 (78). Most of these proteins are membrane associated and multi-copy, interacting with themselves and each other. The VirB6-10 proteins are found in the periplasm, inner and outer membranes, and form the secretion channel as well as its accessory proteins. VirB4, VirB11, and VirD4 localize to the inner membrane and serve as the ATPases that power the system. VirD4 also functions as a coupling protein, binding proteins prior to secretion through the channel. Generally, T4SSs also include an extracellular pilus, composed of a major (VirB2) and minor (VirB5) subunit.

The process of substrate secretion through the T4SS apparatus is still an active area of investigation. However, it is believed that substrate DNA or protein first makes contact with VirD4, which functions as a molecular “gate” at the base of the secretion apparatus (79). VirD4 then transfers the substrate to VirB11, which delivers the substrate to the inner membrane channel complex. Finally, the substrate is transferred across the periplasm to the outer membrane protein complex. It is not currently known what role the T4SS pilus plays in the secretion process. Some believe that the pilus may serve merely as an attachment device, allowing bacteria to come into tight contact with target cells (78). Still, others have predicted that the pilus may actually serve as the conduit for substrate translocation, particularly into target cells (80). Work to determine which of these two models is correct is currently ongoing.

T4SSs play pivotal roles in the pathogenesis of a wide range of bacteria. Notable examples of bacterial pathogens that employ T4SSs for virulence are Neisseria gonorrhoeae, which uses its T4SS to mediate DNA uptake (which promotes virulence gene acquisition)(81), and L. pneumophila, Brucella suis, and Helicobacter pylori, which use their T4SSs to translocate effector proteins into host cells during infection to disrupt their defense strategies (82). These effector proteins have a wide range of functions. For example, the intracellular pathogen L. pneumophila uses its T4SS to translocate more than 200 effector proteins into the host cell, where they play important roles in remodeling the host cell architecture in order to create a vacuole suitable for bacterial replication (83). A major focus in the T4SS field is now on understanding the roles these effector proteins affect host cell functions. In addition to enhancing our understanding of host-pathogen interactions, these studies have also led to novel insights into eukaryotic cellular biology.

The Type V Secretion System

Type V secretion system (T5SS) substrates are unique in that, unlike other secreted substrates, which cross the bacterial membrane with the help of a dedicated secretion apparatus or membrane channel, they secrete themselves. These proteins or groups of proteins carry their own β-barrel domain, which inserts into the outer membrane and forms a channel that either the remainder of the protein or a separate protein is transported through (84, 85). Because protein secretion by T5SSs only occurs in the outer membrane, these proteins must first be translocated across the inner membrane and into the periplasm in an unfolded state by the Sec apparatus ( Fig. 3 ). Therefore, T5SS proteins carry an N-terminal Sec signal sequence that is cleaved off as they pass into the periplasm (86).

Most well known T5SS substrates are virulence proteins, serving as toxins and receptor-binding proteins. Some examples of T5SS substrates that play important roles in pathogenesis include the immunoglobulin A protease of N. gonorrhoeae, which cleaves host antibodies (87), the IcsA protein of Shigella flexneri (88), which promotes actin-based intracellular motility and also serves as an adhesin (89), and YadA of Y. enterocolitica (90), which helps to promote translocation of T3SS substrates into host cells, and assists in mediating resistance to attack by the host complement system (91). T5SSs can be separated into three classes, depending on the number of proteins involved in the secretion process. These classes include autotransporter secretion, two-partner secretion, and chaperone-usher secretion (92).

Autotransporter secretion

The most simplistic form of Type V secretion is known as the autotransporter system. As its name implies, autotransporters contain components that allow them to secrete themselves (92). More specifically, autotransporters contain 3𠄴 domains: a translocator domain at the C-terminus that forms the outer membrane channel, a linker domain, a passenger domain that contains the functional part of the autotransporter protein, and sometimes, a protease domain that cleaves off the passenger domain once it passes through the channel (85).

Following secretion of the unfolded autotransporter protein through the inner membrane, the translocator domain assembles in the outer membrane, forming a 12-stranded β-barrel, usually with the help of a number of accessory factors, including the periplasmic chaperone Skp and the Bam complex (93, 94). The flexible linker domain then leads the passenger domain through the channel to the outside of the cell. Once the transporter domain has reached the outside of the cell, it is either released by its own protease domain or remains attached to the translocator domain and protrudes outside the cell (85).

Two-partner secretion

While the majority of T5SS substrates are secreted via the autotransporter mechanism, a few rely on different polypeptides for transport outside of the cells. In a process called two-partner secretion, a pair of proteins participates in the secretion process, in which one partner carries the β-barrel domain, while the other partner serves as the secreted protein (95). Two-partner secretion has been observed in a large variety of Gram-negative bacteria and is primarily responsible for transporting large virulence proteins, such as the filamentous haemagglutinin of Bordetella pertussis and the high-molecular weight adhesins HWM1 and HWM2 of Haemophilus influenzae (96, 97).

Chaperone-usher secretion

A third subcategory of T5SSs involves proteins secreted with the help of two other proteins: the usher protein, which forms the β-barrel channel in the outer membrane, and the chaperone, a periplasmic protein that facilitates folding of the secreted protein prior to delivery to the channel (98). Chaperone-usher systems are commonly used to assemble pilins on the surface of Gram-negative bacteria, such as the P pilus of uropathogenic E. coli (98).

The Type VI Secretion System

Type VI secretion systems (T6SSs) are the most recent bacterial secretion systems to be discovered (99) and, therefore, there is still much to learn about their structure and functions. T6SSs translocate proteins into a variety of recipient cells, including eukaryotic cell targets and, more commonly, other bacteria (100). These systems are fairly well conserved in a wide-range of Gram-negative bacterial species, with nearly a quarter of sequenced genomes containing genes for T6SS components (101). Unlike many of the other characterized Gram-negative secretion systems, T6SSs are capable of transporting effector proteins from one bacterium to another in a contact-dependent manner, which is believed to play a role in bacterial communication and interactions in the environment (100).

T6SSs are very large, with up to 21 proteins encoded within a contiguous gene cluster (100). Thirteen of these proteins appear to be conserved in all T6SSs and are thought to play a structural role in the secretion apparatus. Intriguingly, T6SSs share structural homology to phage tails, and it has been hypothesized that T6SSs may have arisen from inverted phage tails that eject proteins outside of the bacterial cell rather than injecting them inside the cell ( Fig. 3 ) (102). It has been proposed that some structural components of the T6SS apparatus may also serve as effector proteins, though other T6SS effector proteins have also been identified. These effectors have many forms and functions, with many directed against the bacterial cell wall and membrane, which supports a role for this secretion apparatus in promoting interspecies bacterial competition (100, 101). Lending further credence to this hypothesis, many T6SS effectors are encoded alongside a gene that provides immunity to the effector, thereby preventing self-intoxication (100).

T6SSs are hypothesized to contribute to the virulence of some bacterial pathogens, both through delivery of protein substrates to host cells, and by secreting substrates into neighboring bacteria that may be competing to exploit a specific host niche. While we know that many bacterial pathogens, including P. aeruginosa, V. cholerae, and S. marcescens are able to use their T6SSs under laboratory conditions (101�), the mechanisms of how these T6SSs contribute to survival in the environment (and in mammalian infection) have not been determined.


Two-partner secretion of gram-negative bacteria: a single β-barrel protein enables transport across the outer membrane

The mechanisms of protein secretion by pathogenic bacteria remain poorly understood. In gram-negative bacteria, the two-partner secretion pathway exports large, mostly virulence-related "TpsA" proteins across the outer membrane via their dedicated "TpsB" transporters. TpsB transporters belong to the ubiquitous Omp85 superfamily, whose members are involved in protein translocation across, or integration into, cellular membranes. The filamentous hemagglutinin/FhaC pair of Bordetella pertussis is a model two-partner secretion system. We have reconstituted the TpsB transporter FhaC into proteoliposomes and demonstrate that FhaC is the sole outer membrane protein required for translocation of its cognate TpsA protein. This is the first in vitro system for analyzing protein secretion across the outer membrane of gram-negative bacteria. Our data also provide clear evidence for the protein translocation function of Omp85 transporters.


6 Towards a unifying model for macromolecular traffic across the outer membrane

Even though the GSP machineries from various bacteria share homologous components, they remain highly specific with respect to the range of secreted proteins and to the molecular interactions taking place between the secretion components. As mentioned before, although the GspO peptidase is efficient on heterologous substrates, in most cases exchange of Gsp components, such as XcpYL or XcpZM with PulL or PulM respectively [112], is not functional. In fact, it seems that the closer are the organisms phylogenetically related, the higher are the chances of producing functional hybrid machineries. Indeed, the work of Lindeberg et al. [37] proved that most of the out genes are exchangeable between E. carotovora and E. chrysanthemi, while the strict specificity is given by OutC and OutD.

Analysis of sequence data revealed that a number of Gsp proteins are similar to components involved in type-4 pilus biogenesis in P. aeruginosa [39], N. gonorrhoeae [149], V. cholerae [150] and other bacteria [151], and DNA uptake associated with natural competence of Bacillus subtilis [152]. In addition, these homologous systems often exhibit comparable genetic organization with GSP systems. Related components include putative nucleotide-binding proteins (the GspE homologues), inner membrane proteins (the GspF homologues), type-4 prepilin-like proteins (the GspG, -H, -I, -J and -K homologues), as well as outer membrane secretins (the GspD homologues) in Gram-negative bacteria.

Considering the multiple similarities between components of these transport systems, it has been suggested that they might form membrane-associated complexes that are assembled by similar mechanisms and share a common final organization [ 23, 41]. They might have evolved from a common ancestor to form complexes that have been adapted to function in outer membrane traffic of macromolecules (proteins or DNA), macromolecular structures (filamentous phages) or cell surface organelles (pili). At this stage of our knowledge, it is difficult to predict whether beside a possible common architecture, these systems might share related functional principles. In particular, the presence of subunits with type-4 leader sequences suggests the existence of a rod-like protein structure analogous to that formed by assembled pilins, that could act as a scaffold for a final trans-envelope structure [23]. However, as it has been previously discussed, there is actually no clear evidence for such a pilus-like structure. Nevertheless, it has been proposed that subunit interactions between proteins with type-4 pilin leader sequences could provide the basis for a ratchet-like process ensuring the vectoriality of the transport mechanism in the various systems [153]. An alternative hypothesis may be to consider that a growing trans-periplasmic structure of packed subunits might help punching out the transported substrate, i.e. the exoproteins bound to the GspD pore in the case of protein secretion ( Fig. 5). Such models are also consistent with the involvement of nucleotide-binding proteins in these different transport systems, that might energize in some way the rotational motion, or the assembly of an helical core complex involving multiple subunits with pilin-like leader sequences. Recent works, based on the identification of suppressor mutations, have suggested that XcpRE interacts with the pseudopilin XcpTG [154]. This observation supports the concept of XcpRE moving XcpTG through the cytoplasmic membrane. Following this idea, the presence of multiple GspE homologues involved in pilin biogenesis could be related to the reversibility of pilus assembly, the PilA subunit being extruded and retracted depending on the direction of the ratchet rotation driven by the different nucleotide-binding proteins [ 151, 155]. Implicitly, these fascinating speculations also highlight that investigating the molecular mechanisms of such complex membrane systems will be extremely difficult and much remains to be learned about the physical basis of the unidirectional outer membrane translocation catalyzed by these systems. As similar components are also involved in transport and assembly of various macromolecules through the membranes of Gram-negative bacteria, these studies might end in understanding specific mechanisms required for accommodating particular region of the cell envelope, including the peptidoglycan layer, to such transport processes.

Model for Xcp machinery functioning in P. aeruginosa. A: The exoprotein is translocated from the cytoplasm (Cyto) to the periplasm (Peri) across the inner membrane (IM) by the Sec machinery. The exoprotein is further released after cleavage of the signal peptide (wavy line) by the leader peptidase (chisel). It acquires a certain level of folding (a) which might require disulfide bond formation (b) or chaperone activity (c). The exoprotein is then targeted to the Xcp secretion machinery in which XcpPC and XcpQD might be involved in specific recognition of this substrate. XcpPC could also control the closing and opening of the pore formed by twelve XcpQD monomers in the outer membrane (OM). Among the other Xcp components, the pseudopilins XcpTG, -UH, -VI, -WJ and -XK are processed by the leader peptidase XcpAO which removes the leader peptide. The translocation of the mature pseudopilins and their assembly into a pseudopilus could require the ATPase activity of XcpRE. This latter protein might push the subunits across the IM, as shown by the arrow, and the XcpSF, -YL and -ZM proteins might also contribute to the formation of this specialized translocation apparatus. B: (1) Upon binding of the exoproteins, the XcpPC protein relieves its interaction with XcpQD and allows a conformational change of the XcpQD monomers leading to an enlargement of the central cavity. This step might also require the intervention of the pmf as discussed in the text. The number of interacting XcpPC proteins is purely speculative. Concomitantly to this event, the pseudopilus continues to extend towards the OM, upon incorporation of new pseudopilin subunits. (2) When the pseudopilus reaches the OM, it expels the exoproteins from the XcpQD channel into the medium. At the same time, upon XcpXK pseudopilin incorporation (in black), pseudopilus extension stops. (3) The incorporation of XcpXK in the pseudopilus might also command disassembly of the structure, the subunits being recycled by moving from the bottom of the pseudopilus into the plan of the membrane. The XcpPC protein then reinitiates interaction with XcpQD to restore the closed state of the channel.

Model for Xcp machinery functioning in P. aeruginosa. A: The exoprotein is translocated from the cytoplasm (Cyto) to the periplasm (Peri) across the inner membrane (IM) by the Sec machinery. The exoprotein is further released after cleavage of the signal peptide (wavy line) by the leader peptidase (chisel). It acquires a certain level of folding (a) which might require disulfide bond formation (b) or chaperone activity (c). The exoprotein is then targeted to the Xcp secretion machinery in which XcpPC and XcpQD might be involved in specific recognition of this substrate. XcpPC could also control the closing and opening of the pore formed by twelve XcpQD monomers in the outer membrane (OM). Among the other Xcp components, the pseudopilins XcpTG, -UH, -VI, -WJ and -XK are processed by the leader peptidase XcpAO which removes the leader peptide. The translocation of the mature pseudopilins and their assembly into a pseudopilus could require the ATPase activity of XcpRE. This latter protein might push the subunits across the IM, as shown by the arrow, and the XcpSF, -YL and -ZM proteins might also contribute to the formation of this specialized translocation apparatus. B: (1) Upon binding of the exoproteins, the XcpPC protein relieves its interaction with XcpQD and allows a conformational change of the XcpQD monomers leading to an enlargement of the central cavity. This step might also require the intervention of the pmf as discussed in the text. The number of interacting XcpPC proteins is purely speculative. Concomitantly to this event, the pseudopilus continues to extend towards the OM, upon incorporation of new pseudopilin subunits. (2) When the pseudopilus reaches the OM, it expels the exoproteins from the XcpQD channel into the medium. At the same time, upon XcpXK pseudopilin incorporation (in black), pseudopilus extension stops. (3) The incorporation of XcpXK in the pseudopilus might also command disassembly of the structure, the subunits being recycled by moving from the bottom of the pseudopilus into the plan of the membrane. The XcpPC protein then reinitiates interaction with XcpQD to restore the closed state of the channel.


Type IV secretion in Gram-negative and Gram-positive bacteria

For correspondence. E-mail [email protected] Tel. +493045043942 Fax +493045043983. E-mail [email protected] Tel. +4991318528081 Fax +4991318528082.Search for more papers by this author

Department of Microbiology and Molecular Genetics, The University of Texas Medical School at Houston, 6431 Fannin St, Houston, TX 77030 USA

Institute of Structural and Molecular Biology, University College London and Birkbeck College, London WC1E 7HX UK

Division of Microbiology, Department of Biology, Friedrich Alexander University Erlangen-Nuremberg, D-91058 Erlangen Germany

For correspondence. E-mail [email protected] Tel. +493045043942 Fax +493045043983. E-mail [email protected] Tel. +4991318528081 Fax +4991318528082.Search for more papers by this author

Life Sciences and Technology, Beuth University of Applied Sciences Berlin, D-13347 Berlin Germany

For correspondence. E-mail [email protected] Tel. +493045043942 Fax +493045043983. E-mail [email protected] Tel. +4991318528081 Fax +4991318528082.Search for more papers by this author

Department of Microbiology and Molecular Genetics, The University of Texas Medical School at Houston, 6431 Fannin St, Houston, TX 77030 USA

Institute of Structural and Molecular Biology, University College London and Birkbeck College, London WC1E 7HX UK

Division of Microbiology, Department of Biology, Friedrich Alexander University Erlangen-Nuremberg, D-91058 Erlangen Germany

For correspondence. E-mail [email protected] Tel. +493045043942 Fax +493045043983. E-mail [email protected] Tel. +4991318528081 Fax +4991318528082.Search for more papers by this author

Summary

Type IV secretion systems (T4SSs) are versatile multiprotein nanomachines spanning the entire cell envelope in Gram-negative and Gram-positive bacteria. They play important roles through the contact-dependent secretion of effector molecules into eukaryotic hosts and conjugative transfer of mobile DNA elements as well as contact-independent exchange of DNA with the extracellular milieu. In the last few years, many details on the molecular mechanisms of T4SSs have been elucidated. Exciting structures of T4SS complexes from Escherichia coli plasmids R388 and pKM101, Helicobacter pylori and Legionella pneumophila have been solved. The structure of the F-pilus was also reported and surprisingly revealed a filament composed of pilin subunits in 1:1 stoichiometry with phospholipid molecules. Many new T4SSs have been identified and characterized, underscoring the structural and functional diversity of this secretion superfamily. Complex regulatory circuits also have been shown to control T4SS machine production in response to host cell physiological status or a quorum of bacterial recipient cells in the vicinity. Here, we summarize recent advances in our knowledge of ‘paradigmatic’ and emerging systems, and further explore how new basic insights are aiding in the design of strategies aimed at suppressing T4SS functions in bacterial infections and spread of antimicrobial resistances.


Catarina Felisberto-Rodrigues, Amit Meir, Marie S. Prevost, Adam Redzej and Martina Trokter: These authors contributed equally to this work.

Affiliations

Institute of Structural and Molecular Biology, University College London and Birkbeck, Malet Street, London, WC1E 7HX, UK

Tiago R. D. Costa, Catarina Felisberto-Rodrigues, Amit Meir, Marie S. Prevost, Adam Redzej, Martina Trokter & Gabriel Waksman

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Secretion in prokaryotes

Prokaryotes, such as bacteria, lack membrane-bound organelles such as the ER and Golgi apparatus, but secretion can occur through many other pathways. For example, gram-negative bacteria use six different methods, labelled Types I-VI, all of which use unique molecular structures to move products across the cell membrane. Much of what bacteria actively secrete is harmful to other cells, and ongoing research into these secretory systems is useful for developing antibiotic treatments.

1. What uses secretion?
A. Neurons
B. Bacteria
C. Glands
D. All of the above

2. Which region of a eukaryotic cell is NOT part of the ER-Golgi secretion pathway?
A. The endoplasmic reticulum
B. The Golgi apparatus
C. The cytosol
D. Vesicles

3. Which of the following is true about porosomes?
A. They produce proteins and package them into vesicles.
B. They interact with vesicles to release proteins from the cell.
C. They exist in vesicle walls.
D. None of the above.