Bacteria are required to adapt to and survive in constantly changing and harsh environments. In order to respond to and alter their environment, secretion systems have evolved in bacteria to export proteins into the surrounding milieu (reviewed in Costa et al., 2015). One secretion system in Gram-negative bacteria that has been the focus of much research in the last three decades is the Type III secretion system (T3SS) (reviewed in Galán et al., 2014). Type III Secretion Systems (T3SSs) have been shown to be important for virulence in many Gram-negative bacterial pathogens of animals and plants; including Pseudomonas syringae, Xanthomonas, Ralstonia solanacearum, Erwinia, pathogenic Escherichia coli, Salmonella, Shigella, Yersinia, and Burkholderia (Gemski et al., 1980; Maurelli et al., 1985; Galán and Curtiss, 1989; Jarvis et al., 1995; Stevens et al., 2002; Büttner and He, 2009). T3SSs span the bacterial inner and outer membranes forming a “molecular syringe” which allows bacteria to export proteins, called effectors, from the bacterial cytoplasm into a target eukaryotic cell (reviewed in Galán et al., 2014).

The focus of this review is T3SSs in the pathogenic bacterium Burkholderia pseudomallei, and to some extent the closely related species B. mallei and B. thailandensis. Originally described in drug addicts in Rangoon in the early twentieth century by Alfred Whitmore (Whitmore, 1913), Burkholderia pseudomallei is a facultative intracellular pathogen (Pruksachartvuthi et al., 1990) that is the causative agent of melioidosis, or Whitmore's disease. Melioidosis is a severe disease of humans and animals, causing an estimated 165,000 cases of human melioidosis per year resulting in a predicted 89,000 deaths (Limmathurotsakul et al., 2016). Infection with B. pseudomallei is usually associated with environmental exposure and can occur through breaks in the skin, inhalation or ingestion (reviewed in Cheng and Currie, 2005). In the majority of cases, the incubation period for melioidosis is between 1 and 21 days following infection (Ngauy et al., 2005). About 50% of melioidosis cases affect people with diabetes and other important risk factors include lung disease, cystic fibrosis and excessive alcohol consumption (Currie et al., 2010). There are varied clinical presentations of B. pseudomallei infection ranging from skin infections to pneumonia and septic shock, which hampers accurate diagnosis in a clinical setting (Currie et al., 2010). B. pseudomallei is reported to be able to reactivate after remaining latent following a primary infection. The longest reported period between infection and reactivation occurred in a World War II veteran who manifested symptoms 62 years after exposure (Ngauy et al., 2005). B. pseudomallei has been classified as a bioterrorism agent by both the UK government and the US Centres for Disease Control and Prevention (reviewed in Rotz et al., 2002; Cheng and Currie, 2005).

B. mallei, the causative agent of glanders in horses and other solipeds, is a zoonotic pathogen with restricted host range (Yabuuchi et al., 1992; Srinivasan et al., 2001). In humans, B. mallei causes a disease similar to melioidosis and has been similarly classified as a potential bioterrorism agent in the UK and US (Rotz et al., 2002; Van Zandt et al., 2013). The soil saprophyte B. thailandensis is non-pathogenic and present in high numbers in the soils and standing waters of endemic areas. This species is commonly used as an alternative model system for B. pseudomallei and B. mallei studies as its genome encodes many homologs of virulence factors from these pathogenic species (Brett et al., 1998; Moore et al., 2004; Yu et al., 2006; Haraga et al., 2008).

The B. pseudomallei genome encodes three T3SSs which are referred to as T3SS-1, T3SS-2, and T3SS-3. The genome of B. pseudomallei consists of two circular chromosomes, with all three T3SSs residing on chromosome 2 (Holden et al., 2004). T3SS-2 and T3SS-3 are present in the genomes of B. mallei and B. thailandensis, whereas T3SS-1 is absent from both (Rainbow et al., 2002). T3SS-1 and T3SS-2 are relatively poorly characterized and share homology with the Hrp2 family of T3SSs found in plant pathogens (Winstanley et al., 1999; Rainbow et al., 2002). The best characterized of the B. pseudomallei T3SSs, T3SS-3, is also known as the Burkholderia secretion apparatus (Bsa) T3SS. It is a member of the Inv-Mxi-Spa family of T3SSs from Salmonella spp. (SPI-1) and Shigella flexneri (Attree and Attree, 2001; Stevens et al., 2002; Egan et al., 2014).

Burkholderia pseudomallei T3SS-1 (BPSS1390-BPSS1410) and T3SS-2 (BPSS1610-BPSS1629) show closest homology to the Hrp2 T3SS of the plant pathogen Ralstonia solanacearum (Angus et al., 2014). B. pseudomallei T3SS-2 expression is activated by the AraC-type regulator HrpB (BPSS1610) (Lipscomb and Schell, 2011). HrpB also regulates the expression of a type IV pilus encoded directly upstream of T3SS-2, but does not appear to regulate the other T3SSs in B. pseudomallei (Lipscomb and Schell, 2011). In order to investigate the role that T3SS-1 and T3SS-2 play in plants, a tomato plant infection model was established for B. pseudomallei and B. thailandensis (Lee et al., 2010). B. pseudomallei KHW T3SS-1 and T3SS-2 mutants were reported to be attenuated in tomato plants (Lee et al., 2010). However, in a more recent study, B. thailandensis did not display phytopathogenic activity in tomato plants which were treated identically to those in Lee et al. (2010), Lipscomb and Schell (2011). The question of whether B. pseudomallei is capable of infecting plants, and what if any role T3SS-1 and -2 have in this process, remain important unanswered questions.

The function of T3SS-1 and -2 in mammalian systems has also been investigated to some extent. T3SSs-1 and -2 do not appear to be required for vacuole escape of the bacterium into the cytoplasm of infected macrophages (Burtnick et al., 2008), and are dispensable in a Syrian hamster model of infection (Warawa and Woods, 2005). However, a B. pseudomallei T3SS-1 mutant displayed increased co-localisation with the autophagy marker LC3 and a reduction in intracellular survival in RAW264.7 cells (D'Cruze et al., 2011). In the same study, T3SS-1 was required for full virulence in a respiratory murine model of melioidosis (D'Cruze et al., 2011). Both earlier studies inactivated T3SS-1 by mutating the structural auto-protease component (BpscU, Table 1) (Warawa and Woods, 2005; Burtnick et al., 2008) while the latter study generated a system knockout by mutation of the ATPase (BpscN, Table 1), which may account for the different phenotypes observed.

The transcriptional regulation of T3SS-3 has been elucidated (Sun et al., 2010). At the top of the regulatory hierarchy is the gene bspR (BPSL1105), which, when disrupted led to a reduced level of expression of genes in the T3SS-3 locus as shown by microarray and real time PCR (Sun et al., 2010). bspR signals through the membrane bound regulator bprP (BPSS1553) which controls expression levels of both structural and secreted components of T3SS-3 (Sun et al., 2010). bprP further signals through bsaN (BPSS1546) and its co-activator, the chaperone bicA (BPSS1533), controlling transcription of the known effectors bopC, bopE and bopA, as well as the chaperone bicP (BPSS1523) and the regulators bprB-D (BPSS1520-22) (Sun et al., 2010; Chen et al., 2014). bsaN also relays the regulation signal to the virulence-associated Type 6 secretion system and virulence factors such as bimA and virAG (Sun et al., 2010; Chen et al., 2014).

The presence of genes allowing for arabinose assimilation was one of the first methods used to differentiate between virulent B. pseudomallei and avirulent B. thailandensis (Smith et al., 1997; Moore et al., 2004). Expressing the B. thailandensis arabinose assimilation operon in B. pseudomallei causes a down-regulation of expression of the T3SS-3 genes, notably the T3SS regulator bsaN, and also results in a reduction in virulence in a Syrian hamster model of infection (Moore et al., 2004). This suggests that loss of the arabinose assimilation operon may account for some of the differential virulence observed between these two species of Burkholderia (Moore et al., 2004).

The structure of the T3SS is well conserved and is similar to that of the bacterial flagella system (Kubori et al., 1998; Young et al., 1999; Gophna et al., 2003). It is thought that the flagella and the T3SS evolved from a similar ancestor, but the T3SS has been the product of a large amount of horizontal gene transfer (Gophna et al., 2003). T3SSs are separated into seven different families named after the archetype system in each family, each with slight differences in structure and host cell target, with multiple types of T3SS present in some bacterial species (e.g., plant or animal) (reviewed by Büttner, 2012). In recent years the structure of the Salmonella T3SS has been solved using cryo-EM (Schraidt and Marlovits, 2011) and cryo-ET (Hu et al., 2017). The structure of the Salmonella T3SS (reviewed by Galán et al., 2014) consists of inner and outer membrane rings connected by a rod, the extracellular needle and a secreted translocation pore which spans the target cell membrane. Assembly of the T3SS appears to be hierarchical and is the subject of multiple recent reviews (Büttner, 2012; Diepold and Wagner, 2014). Based on evidence from Yersinia and Salmonella, it appears construction of the T3SS begins with the formation of the outer-membrane/structural ring (Secretin) and independently the inner membrane export machinery, which are then linked by YscJ (Yersinia) or PrgK (Salmonella), followed by assembly of the ATPase/C-ring and the formation of the mature needle complex (Diepold et al., 2010, 2011; Wagner et al., 2010). The structural proteins in the B. pseudomallei T3SS-3 (Figure 2) that have been studied specifically are described below.

Proteins are secreted in an unfolded state due to the narrow width of the needle channel which is 2–3 nm in S. flexneri (Blocker et al., 2001; Radics et al., 2014). This was proven by cryo-EM imaging of a substrate which was engineered to become trapped in the secretion channel (Radics et al., 2014). The ATPase at the base of the secretion system has been shown to dissociate proteins from their chaperones and is also thought to be involved in unfolding of the protein (Akeda and Galán, 2005). The source of energy for exporting proteins is the subject of some debate. In the flagellar T3SS of Yersinia and Salmonella, secretion can take place in the absence of the ATPase (Wilharm et al., 2004; Erhardt et al., 2014). This evidence, combined with a study which showed flagellar T3S was halted in the absence of a proton gradient, has led to the hypothesis that the ATPase is primarily required for protein unfolding and that proton motive force energizes secretion of the unfolded substrate (Paul et al., 2008).

The B. pseudomallei T3SS-3 ATPase is BsaS (BPSS1541). A B. pseudomallei bsaS mutant was unable to secrete the known effector protein BopE, demonstrated a defect in intracellular survival in RAW264.7 cells and was attenuated in BALB/c mice infected intra-nasally, demonstrating that the ATPase is required for T3S of effector proteins and has a similar phenotype to other T3SS-3 structural mutants (Gong et al., 2015). This agrees with a previous study in which B. pseudomallei and B. thailandensis containing in-frame deletions in bsaS, were impaired in their ability to escape the endosomal compartment and form plaques in host cell monolayers as a result of cell-to-cell spread (French et al., 2011). Salmonella lacking a functional ATPase will still assemble a mature T3SS, and will still secrete effector proteins, though at a reduced level (Erhardt et al., 2014). The effect that a bsaS mutation in B. pseudomallei T3SS-3 may have on either assembly of the T3SS apparatus or the secretion of early/middle substrates, such as the major needle component (BsaL) and the needle tip protein (BipD) remains an interesting open question.

Upon host cell contact, the translocators of the T3SS are inserted into the host cell membrane forming a pore through which effector proteins may pass. Shigella is able to lyse red blood cells by inserting the translocators IpaB and IpaC into the RBC membrane forming a 25 angstrom pore (Blocker et al., 1999). Secretion of the translocator proteins is controlled by the needle tip protein. A Shigella needle tip protein mutant (ipaD), constitutively secreted the translocators IpaB and IpaC (Picking et al., 2005). Immunofluorescence microscopy demonstrated the presence of the Shigella IpaD protein on the surface of the bacteria in the absence of a host cell membrane, a finding that was further confirmed by electron microscopy (Espina et al., 2006). The needle tip protein probably acts to sense host cell contact, inhibiting premature secretion of translocators (Espina et al., 2006). In the same study it was shown that antibodies to the tip protein disrupted the haemolysis of sheep erythrocytes indicating the functional importance of IpaD in the insertion of the translocator complex into eukaryotic cell membranes, as well as regulating effector protein secretion (Espina et al., 2006). Next, the T3SS senses host cell contact when IpaD binds to bile salts causing IpaB to be exposed at the needle tip (Olive et al., 2007; Stensrud et al., 2008).

The final step in secretion of the translocation pore is dependent on Shigella interacting with host cell lipids to induce IpaC secretion (Epler et al., 2009). When cultured in the presence of liposomes, IpaC localizes to the bacterial surface and is secreted (Epler et al., 2009). This agrees with earlier evidence that cholesterol is bound by translocation components (Salmonella SipB and Shigella IpaB), is important for entry into host cells and is required for efficient translocation of effector proteins into host cells (Lafont et al., 2002; Hayward et al., 2005). There is some evidence to suggest that the translocator proteins, along with the needle tip and a functional T3SS, may be sufficient to determine the intracellular niche of Salmonella and Shigella in the absence of any of the effector proteins (Du et al., 2016).

Type III secretion signals are located at the N-terminus of a protein, are not cleaved and do not share primary sequence identity with each other (Michiels and Cornelis, 1991; Schesser et al., 1996). In Yersinia, it was demonstrated that as little as 15 amino acids of the N-terminus of YopE were required for secretion (Sory et al., 1995). Interestingly, substituting the alanines at position 2 and 15 in the YopE secretion signal with glutamic acid, did not affect secretion (Anderson and Schneewind, 1997). Even shifting the reading frame did not prevent the secretion of YopE, which the authors suggested may indicate a secretion signal located in the mRNA encoding the effector protein (Anderson and Schneewind, 1997).

A second important signal in the N-terminal region of T3SS effector proteins is the chaperone binding domain (CBD) which is required for the specific secretion of effector proteins (Abe et al., 1999; Ehrbar et al., 2003, 2006; Lee and Galán, 2003, 2004) as well as for their stability in the bacterial cytoplasm (Frithz-Lindsten et al., 1995; Abe et al., 1999). By determining the crystal structure of an effector protein bound to its chaperone, it is known that T3SS chaperones in Yersinia and Salmonella bind the N-terminal amino acids of their effector protein, just after the T3S signal, but before any functional domains, and maintain the bound effector protein in a partially unfolded state that may be more competent for secretion (Birtalan and Ghosh, 2001; Stebbins and Galán, 2001).

The T3S signal also appears to be promiscuous between systems, allowing secretion of effector proteins from bacteria in an unrelated host bacterium containing a T3SS (Rossier et al., 1999; Hovis et al., 2013). This is even true of less similar T3SSs, as demonstrated by the ability of the Hrp Plant T3SS of Xanthomonas to secrete the mammalian effector protein Yersinia YopE (Rossier et al., 1999). Perhaps because of the similarity between the two, virulence-associated T3SS effectors can be secreted by the bacterial flagellar T3SS system in Yersinia and Salmonella (Young and Young, 2002; Lee and Galán, 2004; Warren and Young, 2005; Ehrbar et al., 2006). It is only through interaction with the appropriate chaperone that specific secretion through a single system is achieved. Inhibition of the ability of Salmonella SopE to bind its chaperone InvB, caused secretion through both the flagellar and SPI-1 virulence associated T3SS (Lee and Galán, 2004; Ehrbar et al., 2006). The genome of B. pseudomallei encodes five putative T3SS-3 chaperones, though little work concerning their function has yet been described.

The role of the T3SS is to deliver an array of effector proteins into the target cell to subvert host cell functions. The function of different effector proteins is extremely varied, ranging from blocking apoptosis (E. coli NleH) (Hemrajani et al., 2010), prevention of phagocytosis (Yersinia YopH) (Persson et al., 1997), cytotoxic activity (Pseudomonas aeruginosa ExoU) (Sato et al., 2003) and disruption of the actin cytoskeleton (Salmonella SopE) (Hardt et al., 1998). B. pseudomallei encodes seven effector proteins known to be secreted by T3SS-3 (CHBP, BopC, BopA, BapA, BprD, BapC, and BopE), as well as one hypothetical T3SS effector BopB (Pumirat et al., 2014; Vander Broek et al., 2015).

While there has been a wealth of new information concerning B. pseudomallei T3S, there are still many significant gaps in knowledge, particularly with regard to the importance of T3SS-1 and T3SS-2 in melioidosis. It is possible that the plant pathogen-like T3SS-1 and T3SS-2 add fitness to B. pseudomallei in the environment, allowing infection/colonization of adjacent plant life. However, it is also possible that these systems are relevant in mammalian hosts, given their conservation amongst the B. pseudomallei strains sequenced to date, and the finding that T3SS-1 is required for full virulence in a murine model of melioidosis (D'Cruze et al., 2011).

It is still unclear whether T3SS-3 plays a significant role in invasion of host cells, particularly non-phagocytic cells. There have been many reports of T3SS-3 or its effector proteins playing a role in invasion (Stevens et al., 2003; Suparak et al., 2005; Muangsombut et al., 2008; Muangman et al., 2011; Kang et al., 2015). Yet in another study, T3SS-3 ATPase (bsaS) mutants did not show a decrease in invasion efficiency in HEK293 or HeLa cells (French et al., 2011). This observation is important because it questions the involvement of T3SS-3 in invasion as well as highlighting a general lack of understanding of the pathways and mechanisms involved in B. pseudomallei entry into host cells.

To date CHBP is the only effector protein secreted by the B. pseudomallei T3SS-3 that is encoded outside of the T3SS-3 locus, and which is not present in the genome of all strains (Pumirat et al., 2014). This raises the question of whether other effector proteins secreted by T3SS-3, but encoded outside of the T3SS-3 locus, may be present in other strains. Full genome comparisons of almost 100 strains of B. pseudomallei identified 86% of genes as being conserved and present in all strains (Sim et al., 2008). The other 14% of genes, considered accessory genes, were disproportionally present in genomic islands and were associated with clinical isolates (Sim et al., 2008). The GIs in B. pseudomallei are highly variable between strains. A study of five clinical B. pseudomallei strains identified a total of 71 GIs distributed between the strains, with at least half being unique to the strain in which they were identified (Tuanyok et al., 2008). The variability in these regions is largely due to horizontal gene transfer and B. pseudomallei has a relatively high rate of lateral gene transfer compared to the mutation rates of other bacterial species (Pearson et al., 2009). As B. pseudomallei has a large amount of genomic diversity in its accessory genome and a high rate of lateral gene transfer, there is a strong possibility that other novel effector proteins are encoded in other strains of B. pseudomallei that have yet to be identified. Study of these effector proteins could provide important insights into strain differences as well as the potential for novel effector biology, but it is a difficult task as the secretome of B. pseudomallei is very complex (Vander Broek et al., 2015). Also, due to the temporal and hierarchical control of the T3SS, there is always the possibility that an effector protein will not be secreted under the conditions used in a given experiment (reviewed in Büttner, 2012). Previous methods of identifying T3SS-3 effector proteins have relied on initial bioinformatics prediction (Stevens et al., 2003; Muangman et al., 2011) or high throughput screens (Vander Broek et al., 2015), both of which may prove to be useful tools for further studies of B. pseudomallei T3S. Indeed it is timely to apply these approaches to the identification and characterization of effector proteins secreted by the lesser studied T3SS-1 and T3SS-2.

Another important outstanding area of research is characterizing the functions of those effector proteins that have already been identified. One common characteristic of many T3SS effector proteins that complicates this task is their functional redundancy (reviewed in Galán, 2009). Commonly, deletion of one effector protein yields little or no phenotype in infection models because other effector proteins target either the same host cell protein or pathway (reviewed in Galán, 2009). In a biological system this would increase the chance of effectors successfully carrying out their intended function and decrease the likelihood of host cell interference. This functional redundancy also highlights the evolutionary importance of dysregulating specific cellular pathways from the standpoint of the bacterium. Even where redundancy does not exist, the function of effector proteins are often subtle when compared to bacterial toxins and may not be easily measurable in in vitro or in vivo models of infection (reviewed in Dean, 2011). This lack of a phenotype to inform focused studies presents a challenge for investigators, creating the need for high throughput “fishing” assays, such as protein immunoprecipitation/pull-downs and yeast two-hybrid assays. These assays have been used successfully to identify host cell binding partners and the subsequent functions of effector proteins from other bacteria (Zhou et al., 2013; Pallett et al., 2014). Understanding the functions of T3SS-3 effector proteins may provide new insights into host-pathogen interactions in B. pseudomallei infection.

Finally, the T3SS-3 has been shown to be one of the most important virulence factors in B. pseudomallei models of infection, raising the question of whether T3SS-3 could be a useful target for protective vaccines or therapeutic intervention in melioidosis patients. Although several attempts have been made to use live-attenuated vaccines based on mutation of key T3SS-3 genes in murine models of melioidosis, none have proven to provide sterilizing immunity. Despite being a potent B- and T-cell antigen, attempts to utilize BipD as a subunit vaccine in murine models of melioidosis have shown little promise (Stevens et al., 2004; Druar et al., 2008). There have also been attempts to use the translocator proteins of T3SS-3 as subunit vaccines. The N-terminal region of BipB was tested as a protective antigen in mice, but showed no protection against subsequent challenge (Druar et al., 2008). The C-terminal and N-terminal regions of BipC have also been separately tested as a subunit vaccine in mice, but neither antigen showed any protection (Druar et al., 2008). Some studies have focused on the use of effector proteins as subunit vaccines. Out of these studies the BopA protein shows most promise, since mice immunized with recombinant BopA were protected against subsequent intranasal challenge with both B. mallei and B. pseudomallei (Whitlock et al., 2010). More recently interest in the use of small molecule inhibitors of the T3SS-3 has arisen (Gong et al., 2015). Treatment of B. pseudomallei infected RAW264.7 cells with a small molecule inhibitor targeting BsaS of the T3SS-3, resulted in a decrease in bacterial intracellular survival (Gong et al., 2015). However, the use of such inhibitors is still very much in its infancy, with important in vivo studies being required to determine whether such small molecules would be effective in murine models of melioidosis.

CV and JS contributed equally to the writing of this review article.