Abstract:
Salmonella, among other Gram-negative bacteria, possess a molecular syringe-like apparatus known as a type III secretion system (T3SS). T3SSs are virulence factors utilized by the bacterium to increase pathogenicity and to evade host immune processes that could potentially be harmful to the bacterial cell. The T3SS is expressed once Salmonella comes into contact with host macrophages and is engulfed. The secretion of effector proteins by the T3SS disrupts host cell processes, creates a safe intracellular environment for Salmonella, assists in Salmonella’s motility and position within its host and prevents the pathogen’s digestion by host macrophages. The T3SS expression, assembly, activation and its distinct functions are highly dependent upon genetics. In this essay, I have highlighted the importance of the genes corresponding to the T3SS as well as its’ associated effector proteins and translocator proteins. I have discussed what we can learn about individual effectors, their functions and how this impacts the well-oiled system that is the T3SS by learning what impact certain deletions in the genes encoding these components of the system will have upon the greater model.
Introduction:
Salmonella enterica (S.enterica) is a gram-negative, disease-causing bacterium responsible for a huge proportion of highly virulent diseases worldwide (Chappell et al., 2009). Salmonella enterica serovar typhi is responsible for an estimated 21 million cases of Typhoid fever every year and a subsequent 200,000 deaths globally (Solomon & Milner, 2017). Furthermore, S.enterica serovar typhimurium(S.typhimurium) and other non-typhoidal Salmonella(NTS) including Salmonella enterica serovar entiritidis(S.enteritidis) are among the leading causes of food poisoning resulting in gastroenteritis and occasional death, if the usually self-limiting bacterium causes a disseminated infection in the host (Hong et al., 2016). There are a variety of factors that contribute to the virulence of S.enterica, including areas on the genome containing virulence genes, known as Pathogenicity Islands 1 and 2(SPI-1 and SPI-2 respectively), fimbriae, flagella, a virulence plasmid and lastly, two Type Three Secretion Systems(T3SS1 and T3SS2), with T3SS1 being encoded by SPI-1 and T3SS2 being encoded by SPI-2(12). For the rest of my discussion, when referring to the SPI-2 encoded T3SS2, it will be shortened to simply ‘T3SS’.
SPI-2 and the T3SS
Genes encoded by SPI-1 result in the pathogen’s ability to invade the host epithelial cells of the gastro-intestinal tract, therefore resulting in Salmonella colonisation within the intestine leading to enteritis; whereas the SPI-2 encoded genes are essential for the replication and survival of S.enterica within host macrophages. Thus, SPI-2 genes contribute greatly to the spread of Salmonella, which could potentially result in the systemic(disseminated) infection (Martínez et al., 2011). SPI-2 is a locus on the S.enterica genome containing horizontally-acquired genes which contribute to the bacterium’s virulence. However, some of these SPI-2 associated genes are located away from the SPI-2 locus, yet are expressed together with the genes present in the SPI-2 locus (Fass & Groisman, 2009). The SPI-2 encoded T3SS can be found in all subspecies of S.enterica (Figueira & Holden, 2012). The primary function of this secretion system is believed to be the translocation of effector proteins across the membrane of the Salmonella-containing vacuole (SCV) in infected host macrophages or epithelial cells (Figueira & Holden, 2012). The SCV is a modified phagosome of sorts and is responsible for intracellular survival and replication of S.enterica, allowing the bacterium to evade host anti-microbial activity within the membrane-bound vacuole of the SCV (Steele-Mortimer, 2008). The SPI-2 and T3SS are expressed mainly when S.enterica is in the SCV after having invaded its host, (Ochman et al., 1996, Shea et al., 1996).
The SCV, SCV biogenesis and F-actin.
Following the phagocytic uptake of Salmonella into host immune cells (eg. macrophages) the SCV undergoes maturation by limiting interactions with the endocytic pathway of the host (this prevents the hydrolytic enzymes produced by the host immune system reaching the SCV) and inhibits the fusion of the vacuole with host lysozomes (Coburn et al., 2007a). The T3SS also plays an important role in SCV biosynthesis. In early SCV biogenesis, T3SS secretes an effector protein that prevents this SCV-lysozome membrane fusion, SpicC (Steele-Mortimer, 2008). In the next stage of SCV biosynthesis, protein effectors secreted by T3SS2 such as SifA, Sseg and Ssef maintain the positioning of the SCV over a long period of time. Similarly, in the final stages of SCV biosynthesis, the T3SS2 secretes effectors such as SteC, SseI and SspH2 to maintain SCV integrity and prevent the movement of any harmful molecules into the SCV, or alternatively the movement of essential molecules out of the membrane-enclosed vacuole (Steele-Mortimer, 2008). In addition, T3SS protein effectors are also associated with the formation of an actin coat around intracellular Salmonella, this is believed to promote SCV fusion with actin-containing vesicles, thus enabling SCV transport, and also offers protection to the SCV by preventing the vacuole from fusing with unfavorable membrane bound-structures. The assembly of this F-actin coat is dependent on the T3SS, specifically the secreted effectors SteC, SseB, SseC, and SseD. Therefore, it can be said that T3SS is almost essential for formation and presumably maintenance of the SCV. As it is known that the T3SS is encoded by SPI-2, it can be assumed that these virulence factors are equally as important for the pathogenicity of the Salmonella enterica bacterium, as the SCV is a necessity for survival and replication of Salmonella within its host. This raises the question of whether a SPI-2 T3SS mutant would have the ability to survive and effectively replicate within the SCV without the use of a T3SS and associated effectors. (See paragraph on Salmonella and the host immune system)
Salmonella-induced filaments
Around 4-6 hours post-infection, long tubular structures can be seen extended from the SCV. These structures are known as Salmonella-induced filaments, or Sifs (Abrahams & Hensel, 2006). The SPI-2 encoded effector protein SifA is responsible for the formation of these filaments as well as a variety of other effectors, including SseF, SseG, SopD2 and PipB2 (Zhao et al., 2015, Krieger et al., 2014). SifA is secreted by the T3SS. Sifs contain late endosomal markers, including lysosomal glycoproteins including LAMPs, similar to the SCV (Mota et al., 2009). Mutants lacking the effector SifA are greatly reduced in virulence and they lack the ability to intracellularly replicate and thus their ability to cause systemic disease is greatly reduced. Mutant strains lacking the sifA gene are unable to produce Sifs and thus the pathogen leaks into the host cytoplasm as a result of the SCVs lacking in membrane integrity (Beuzón et al., 2000). These results highlight the necessity of sifA and its corresponding SPI-2 encoded effector protein SifA in Salmonella virulence. Strains lacking in SseF and SseG effector proteins present morphological differences in Sifs produced, such as thinner-appearing filaments, however the greatest impact upon Salmonella virulence with respect to Sifs is following the deletion of sifA (Kuhle & Hensel, 2002). It was then discovered that the explanation behind thinner filaments in sseF and sseG mutants was due to the fact that the formation of the double membrane SIF depends fully on the T3SS effectors SseF and SseG (Krieger et al., 2014). This double-membraned filament proved twice as large in mean diameter when compared with the membrane of Sifs produced by sseF and sseG mutants. The single membraned Sifs produced showed to have lower levels of LAMP-1 and also were more susceptible to fragmentation following chemical fixation with para-formaldehyde (PFA).
The genetics behind the T3SS
T3SSs are complex structures used by S.enterica and a variety of other Gram-negative bacteria such as Shigella spp., Chlamydia spp. etc. as a device to translocate effector proteins from the prokaryote into the eukaryotic cell (Green & Mecsas, 2016) (Zigangirova et al., 2012). The secretion of these effectors often enables the bacteria to survive in subcellular niches and to interfere with the signal-transduction pathways and other common cellular pathways of the host (Cordero-Alba et al., 2012). The SPI-2 genes that encode the T3SS are located in 26 kb of the pathogenicity island, and are categorized as four operons: regulatory, structural I, structural II and effector/chaperone (Hensel et al., 1998). The expression of the T3SS, along with other SPI-2 related genes, is reliant on three two-component systems, EmvZ/OmpR and PhoQ/PhoP (Forest et al., 2010), and finally the SsrA/SsrB system. The SsrA/SsrB system is activated by the action of the EmvZ/OmpR and PhoQ/PhoP systems, resulting in the expression of the ssra gene (Bijlsma & Groisman, 2005, Feng et al., 2004). The SsrA/SsrB two-component system controls its own expression as well as the expression of the secretion apparatus and its subsequent effector proteins (Lee et al., 2000).
The SsrA/SsrB two-component system
OmpR is responsible for activating the expression of SsrA of the SsrA/SsrB complement system shortly after S.enterica’s initial interaction with host macrophages (Lee et al., 2000). SsrA is a sensor kinase which, after being activated by OmpR, autophosphoryates and activates SsrB, the response regulator (Osborne & Coombes, 2011). To regulate the expression of the T3SS, SsrB binds to the promoters of all gene clusters associated with SPI-2, this step is essential for the expression of the T3SS and its effector proteins, even those that are not located within the SPI-2 locus (Fass & Groisman, 2009). Furthermore, SsrB regulates transcription from both the SsrA promoter and its own (Feng et al., 2003). The additional features of this complement system can be seen by examining its relationship with nucleoid-associated proteins(NAPs). NAPs control a large proportion of gene expression in enteric bacteria, for example, H-NS is an NAP that controls the expression of horizontally acquired genes, such as the SPI-2 and its associated genes (Stoebel et al., 2008). In an experiment conducted by X, the deletion of the gene encoding H-NS resulted in the reversal of gene repression in a variety of genes that usually appear silent under laboratory conditions (Stoebel et al., 2008). Similarly, SPI-2 encoded genes were derepressed upon silencing of the hns gene (Walthers et al., 2007). As previously discussed, SsrB is responsible for expression of the SPI-2 by binding to its promoter. However, SsrB serves a second function wherein it appears to counteract H-NS associated gene silencing (Walthers et al., 2007), thus contributing greatly to the expression of the T3SS, not only by regulating its expression but also by preventing its repression by antagonizing the effects of the negative regulator, H-NS (Walthers et al., 2011).
T3SS structure and constituents
The T3SS has a complex modular structure comprising of a basal body which spans the inner and outer membrane of the bacterium, as well as the peri-membrane space. The needle and tip are inserted into the basal body of the T3SS (Diepold et al., 2010, Sadarangani et al., 2013). The TTSS apparatus is made up of between 20-25 proteins. Around half of these T3SS proteins can be found in other Type 3 structures (ie. They are not specific to the Salmonella secretion system). The conserved proteins that make up the T3SS structure are often similar in sequence to the basal body proteins of the flagella (Aizawa, 2001, Ghosh, 2004). This information indicates that there is a common evolutionary past between the two structures (Nguyen et al., 2000). Phylogenetic research has brought about two possibilities; the first being that the T3SS evolved from the flagellum, the second alluding that both structures are as ancient as the other, with a similar ancestry (Gophna et al., 2003). Both hypotheses would explain the similarities between both apparatus’; for example, both the flagella and the T3SS are involved in the secretion of proteins that contribute to the virulence of S.enterica and both obtain energy from ATPase (Sadarangani et al., 2013). The hypotheses would also give rise to functional and biochemical differences, such as role in pathogenicity, proteins secreted etc., (Ghosh, 2004). The proteins associated with the bulk of the T3SS consist of YscC, YscJ, YscN, YscQ, YscR, YscS, YscT, YscU and YscV which make up the body of the structure. The ‘needle’ of the T3SS, known as the ‘injectisome’ is composed of the protein YscF (Sadarangani et al., 2013). The above proteins are highly conserved in Salmonella T3SS structures (as well as the T3SS structure in other gram negative bacteria) (Troisfontaines & Cornelis, 2005).
Figure 1:
Figure 1: Adapted from an image in ‘Bacterial Secretion’, a lecture given in SVIMS, Tirupati in 2012 by Dr.B.V.Ramana,MD.
The above figure shows the basal body structure of the T3SS and its protein components, YscC, YscJ, YscN, YscQ, YscR, YscS, YscT, YscU and YscV. In addition, the figure shows the injectisome and its protein constituent, YscF. The diagram shows the location of the T3SS in the bacterial cell, and exhbits how the basal body of the apparatus is present between the bacterial inner and outer membranes, as well as through the peri-membrane space. The injectisome is extracellular and can be seen secreting an effector protein, for example SteC, into the host.
Effector proteins vs translocator proteins
There are 19 known effectors secreted by the T3SS, however many of these effectors have no known function to date. Known SPI-2 encoded effectors include GogB, PipB, PipB2, SifA, SifB, SopD2, SseF, SlrP, SseG, SseI/SrfH, SseJ, SseK1, SseK2, SseL, SspH1, SspH2, SteA, SteB, and SteC (Ramsden et al., 2007). These effectors are secreted by the T3SS through the injectisome and into the host by passing through pores on the host cell membrane that are formed by translocator proteins, also secreted by the T3SS. The effector-translocator system is a complementary system, conserved to pathogens with a T3SS (Coburn et al., 2007b). However, effectors and translocators differ in their dependence upon the T3SS (Boyd et al., 2000). After effector proteins SopE and SptP were internally deleted under experimental conditions, the proteins were shown to be secreted from flagellar structures instead, highlighting their non-dependence upon the T3SS (Ghosh, 2004). Conversely, the translocation process is entirely dependent upon the T3SS and the translocators will not be secreted from the flagellar apparatus in the case of a deletion (Lee & Galán, 2004).
Salmonella protein translocation and the translocon
In pathogens that infect animals, including S.enterica, it is believed that there are two translocators responsible for the creation of the membrane pore formed for efficient effector secretion into the host cell. There is an apparent third hydrophilic translocator protein involved in this process that is located at the tip of the injectisome and controls secretion of effector proteins (Tomalka et al., 2012). This pore is known as the translocon pore and is composed of around 6-8 proteins (Ide et al., 2001). In Salmonella, SseB, SseC and SseD are the three translocator proteins secreted by the T3SS and encoded by the SPI-2 that form the translocon (Nikolaus et al., 2001). SsaM is a protein encoded in the structural II operon of the SPI-2 (Hensel et al., 1997), its’ absolute function remains unknown due to its interaction with a variety of processes contributing to the virulence of S.enterica. ssaM mutants bear a very similar phenotype to spiC mutants. ssaM mutants fail to produce Sifs. Similarly, SsaM is known to be essential for correct secretion of translocon proteins and effector proteins, however a ssaM- mutant resulted in the over-secretion of at least two effector proteins. In a similar study, spiC- mutants produced similar results. SsaM and SpiC were then shown to interact within the bacterial cell, and so it is believed that the two form a complex that can distinguish between the effectors and translocators and thus orders their secretion from the T3SS (Yu et al., 2004).
Effector proteins – an overview
As previously stated, the T3SS is responsible for the secretion of effector proteins from the pathogen to the host cell via the injectisome (Dean, 2011). These effectors contribute greatly to the pathogenicity of disease-causing micro-organisms as a result of their wide range of biochemical activities (Galán, 2009). Oftentimes, these proteins possessing eukaryotic protein motifs have the ability to mimic the functions the host counterparts and therefore these pathogens are able to modulate a range of host signaling pathways, often enabling them to evade host immune responses (Stavrinides et al., 2008). A large proportion of the effectors (with known functions) are involved in interactions with the host endosomal membrane system, including SseF, SseG, SseJ, SifA, SifB, PipB, PipB2 and SopD2. Following sequence analysis of these effectors, it was found that the ability of these proteins to mediate membrane integration and association could be related to the presence of hydrophobic domains in the protein structure of the effectors (Abrahams & Hensel, 2006). *return*
Table 1:
Effector Protein:
Role/function:
Translocation:
Gene Location:
SopD2
Targeted to Sifs and late endosomes
SPI-2
Pathogenicity islet
SpiC
Interferes with vesicular trafficking (among other known functions including involvement in Sif formation)
SPI-2
SPI-2
SseF
Contributes to Sif formation
SPI-2
SPI-2
SseG
Contributes to Sif formation
SPI-2
SPI-2
SifA
Required for SCV membrane integrity, Sif formation
SPI-2
Pathogenicity islet and Sif formation
SifB
Targeted to Sifs
SPI-2
Pathogenicity islet
SspH1*
Interferes with the host\’s ubiquitination pathway
SPI-1/SPI-2
Gifsy-3 prophage
SspH2
Actin remodelling
SPI-2
Phage
SlrP*
Interacts with mammalian thioredoxin-1
SPI-1/SPI-2
Plasmid/transposase
SseI/SrfH
Actin remodelling
SPI-2
Gifsy-2 prophage
SseJ
Acyl transferase/SCV membrane dynamics
SPI-2
Phage
PipB
Targeted to Sifs
SPI-2
SPI-5
Table 1: Adapted from ’Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system’ (Waterman & Holden, 2003).
Table 1 is a table showing a selection of effector proteins (with known functions) encoded by the SPI-2 locus. All of these proteins are dependent on the SsrA/SsrB two-component system with the exceptions of SspH1 and SlrP. The table states the known functions of the effector proteins, the area of translocation and the location of the genes
*Although the roles for SlrP and SspH1 were unknown to Waterman and Holden in 2003, Bernal-Beyard and Ramos-Morales provided a function for SlrP in 2009 (Bernal-Bayard & Ramos-Morales, 2009) and Haraga and Miller for SspH1 in 2006 (Haraga & Miller, 2006).
SpiC – a dynamic effector protein
SpiC was the first T3SS-secreted effector to be identified (Uchiya et al., 1999). In earlier laboratory-conducted analyses of infected macrophages using electron microscopes, it became known that a large proportion of SCV containing wild type S.Typhimurium did not fuse with host lysozomes and endosomes, prolonging their survival within host macrophages and enabling them to replicate and proliferate (Rathman et al., 1997, Buchmeier & Heffron, 1991). However, similar experiments conducted using S.typhimurium with a mutant spiC gene showed a much larger number of SCV fusion with host compartments (Uchiya et al., 1999). Therefore, it can be concluded that SpiC certainly contributes towards maintenance of the SCV, allowing S.typhimurium to replicate and colonise within the host, eventually causing disease. Other work has shown that SpiC has additional functions for the pathogenic bacteria, funcions which contradict its previous role as an effector (Waterman & Holden, 2003). It has been shown that SpiC is essential for the secretion of SscB, SscC and SscD, which are translocator proteins, and also for the translocation of effector proteins into infected host macrophages (Freeman et al., 2002). SpiC is involved in the translocation of SifA, the protein involved in Sif formation, and therefore, spiC mutants do not form Sifs (Guy et al., 2000). As discussed previously, SpiC has an important role in protein effector and translocator secretion (Yu et al., 2004). For these reasons, SpiC is both the most controversial and best-understood protein effector, as although it appears to have a variety of functions, its’ importance cannot be disputed.
Salmonella T3SS and the host immune system
As discussed previously, the T3SS and its’ secreted protein effectors are directly linked to the virulence of S.enterica, demonstrating the contribution of the structure and effectors to disease. The build-up of fluid in intestinal bovine loops was shown to be caused by effectors SipA, SopA, SopB, SopD, and SopE2. In vitro and in vivo, the effectors were additionally proven to induce extreme diarrhoea in young calves (Boyle et al., 2006, Zhang et al., 2002, Coburn et al., 2007b). Although hosts offer physical protection against the invasion of pathogens in the form of mucosal barriers, intestinal epithelium etc., the host’s immune cells offer the most dynamic and effective protection to the host. Therefore, it is of a pathogen’s best interest to adapt to cheat the immune system in order to aid replication and colonisation. T3SSs have adapted and evolved strategies that enable them to evade host immune responses. Such strategies include the activation of host signalling cascades by pathogens. However, the T3SS is not always of benefit to Salmonella as the presence of a T3SS can trigger extracellular and intracellular pattern recognition receptors (PRRs) (Coburn et al., 2007b). For example, interleukin-8 (IL-8) is produced as a host immune response to Salmonella flagellin (Gewirtz et al., 2001). This occurs as a result of recognition of the flagellin by the host toll-like receptor (TLR), known as TLR5, the flagellin PRR (Zeng et al., 2006). The SPI-2 encoded T3SS does not secrete flagellin, however, a functional T3SS necessary for flagellin delivery to the basolateral membrane where it’s PRR is identified (Lyons et al., 2004). The identification by the host of a foreign cell and potential pathogen triggers an inflammatory response in the host which can result in some symptoms such as acute swelling, heat or pain (Gewirtz et al., 2001).
Moreover, Salmonella’s T3SS can evade the host defence mechanism wherein cells infected by S.enterica are immobilized. Instead, the infected cells enclosing the pathogen become increasingly motile. This promotes the colonization and spread of S.enterica through the body, eventually leading to systemic disease (Worley et al., 2006). The SrfH/SseI effector is secreted by the T3SS and interacts with the protein TRIP6 in the host. TRIP6 accelerates cellular motility (Coburn et al., 2007b). Additionally, Salmonella can evade phagocytosis by host macrophages. Salmonella protects itself from damage as a result of reactive oxygen and nitrogen intermediates. The pathogen prevents delivery of NADPH oxidase and nitric oxide synthase to the SCV. This strategy to preserve the SCV is dependent on the T3SS, thus emphasizing its importance in Salmonella colonization and systemic disease (Coburn et al., 2007b). Due to the host immune system recognising Salmonella as a pathogen, thus triggering immune responses by the host to eliminate the pathogen, recent work has been carried out to develop new vaccines. Vaccinations are being developed both by using secreted effectors as vaccines and also by producing live-attenuated vaccines. Currently, there is an effective vaccine for typhoid fever (caused by S.typhi) (Ivanoff et al., 1994).
A strain with a silent T3SS gene is used, in addition to extra features such as mutations in genes involved in important cellular pathways, such as aroC, the gene involved in aromatic amino acid synthesis (Coburn et al., 2007b).
Conclusion:
By examining the diverse functions of the T3SS, it is evident how essential the complex structure is for Salmonella as a pathogen. Not only does the T3SS increase the virulence of S.enterica but it is entirely essential for the spread of Salmonella around the body, resulting in the systemic disease. By viewing each individual aspect of the T3SS, including the physical structure, the effectors and the translocon-forming proteins etc., it becomes clear that each component is essential for smooth running of the complicated system. By examining research experiments wherein certain genes are silenced, deleted or mutated, the results are resounding in proving not only the impact of the T3SS upon Salmonella replication and colonisation, but also the impact each individual gene has upon the well-oiled machine that is the T3SS.
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