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Regulation of type III secretion systems Matthew S Francis*, Hans Wolf-Watz* and Åke Forsberg*†‡ Type III secretion systems are utilised by numerous Gramnegative bacteria to efficiently interact with a host. Appropriate expression of type III genes is achieved through the integration of several regulatory pathways that ultimately co-ordinate the activity of a central transcriptional activator usually belonging to the AraC family. The complex regulatory cascades allow this virulence strategy to be utilised by different bacteria even if they occupy diverse niches that define a unique set of environmental cues. Simulating the appropriate combination of signals in vitro to allow a meaningful interpretation of the type III assembly and secretion regulatory cascade remains a common goal for researchers. Pieces of the puzzle slowly emerge to provide insightful views into the complex regulatory networks that allow bacteria to assemble and utilise type III secretion to efficiently colonise a host. Addresses *Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden † Department of Medical Countermeasures, Swedish Defence Research Agency, SE-901 82 Umeå, Sweden ‡ e-mail: [email protected] Author for correspondence: Åke Forsberg Current Opinion in Microbiology 2002, 5:166–172 1369-5274/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 28 February 2002 Abbreviations AI-2 autoinducer-2 ECF extracytoplasmic factor EHEC enterohemorrhagic Escherichia coli EPEC enteropathogenic E. coli LEE locus of enterocyte effacement TTSS type III secretion system
Introduction Type III secretion systems (TTSSs) are complex protein secretion and delivery machines utilised by many animaland plant-interacting bacteria that occupy diverse niches. More than 25 genes are required to establish a functional TTSS. In animal pathogens, the principle function of TTSS is to deliver anti-host-virulence-determinants into mammalian cells. This results in a cascade of regulated events to overcome animal defence mechanisms necessary to establish bacterial infection. By analogy, plant pathogens utilise TTSSs to cause disease in ‘susceptible’ plants and trigger the hypersensitive response in ‘resistant’ plants. With this level of complexity, it is not surprising that multifaceted regulatory systems are required to impart spatial and temporal control of type III gene expression. It is apparent that regulation occurs in at least two distinct steps: expression of genes required for assembly of the secretion apparatus followed by expression of genes whose products are substrates for TTSSs. In this review, we illustrate some recent developments in the regulation of TTSSs.
Sensing environmental cues Pathogenic bacteria occupy very different infection foci. The animal pathogens Salmonella, Shigella and Chlamydia all actively invade host cells. Upon invasion, however, Salmonella and Chlamydia persist within host vacuoles and modulate them, whereas Shigella freely replicate within the cytosol and eventually spread from cell to cell. In contrast, Yersinia spp. and Pseudomonas aeruginosa predominantly remain extracellular, and enteropathogenic Escherichia coli (EPEC) colonise the small intestine. Moreover, phytopathogens remain extracellular in susceptible plants, occupying the intercellular spaces of parenchymatus tissue. As each niche exerts an assortment of unique environmental pressures, and TTSSs are essential for virulence of these pathogens, it follows that a vast array of environmental signals influences the expression of TTSS assembly genes and secretion substrate genes (Table 1). Most bacteria employ phosphorelay mechanisms to sense the surrounding environment. In animal pathogens, these signals are most often routed to AraC-like transcriptional activators that establish a cascade of type III gene activation [1–7]. A role for this activator family was first discovered by Cornelis et al. in Yersinia [2]. The involvement of small nucleoid-associated proteins in this process likely reflects the need to fine-tune gene expression with the physiological or nutritional status of the bacteria. The phytopathogens Xanthomonas campestris and Ralstonia solanacearum utilise related AraC-like regulators [8,9]. In addition, Pseudomonas syringae, Erwinia amylovora and R. solanacearum employ alternative sigma factors belonging to the extracytoplasmic factor (ECF) family [10–12]. Expression of the latter requires RpoN [13] and enhancer-binding proteins that function as response regulators [14,15]. In this section, we illustrate how different bacteria utilise diverse environmental cues to regulate TTSS assembly and TTSS-mediated secretion. We consider selected examples that best reflect actual in vivo regulatory events (we apologise to our many colleagues whose inspiring work has not been cited, owing to space restrictions, in our review). Temperature
Most bacterial pathogens normally reside in the surrounding environment. However, on contact with an animal host, these bacteria encounter an increase in their growth temperature to 37°C. Furthermore, temperature increase has long been known to induce TTSS virulence gene expression in Shigella spp. [16] and Yersinia spp. [17,18]. Therefore, how do these pathogens utilise temperature as a stimulus for TTSS gene activation? In Shigella spp., on temperature increase to 37°C, an AraC-like transcriptional activator, VirF, responds by activating a regulatory cascade mediated by VirB [19]. In turn, VirB activates genes required for TTSS assembly and secretion substrates involved in Shigella invasiveness and
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Table 1 A summary of regulatory characteristics of type III secretion systems. Pathogen
Stimuli
Two-component regulators AraC-like Additional regulatory transcriptional elements activators
Animal pathogen Salmonella Mg2, oxygen, osmolarity, pH, inorganic phosphate, PhoP/PhoQ, PhoR/PhoB, HilC, HilD, spp. SPI-1 RNA stability Bar/SirA, OmpR/EnvZ, HilA InvF Salmonella spp. Mg2, phosphate starvation, pH media composition SPI-2 Shigella spp. Cell contact, media composition, serum, Congo Red, temperature, pH, tRNA modification Yersinia spp. Cell contact, Ca2, temperature, amino acids, albumin Pseudomonas Cell contact, serum, Ca2 aeruginosa EPEC Temperature, pH, cell density, media composition, growth phase Chlamydia spp. Cell contact or intracellular signals? Bordetella spp. ? Plant pathogens Pseudomonas Media composition, pH, osmolarity, temperature, syringae Erwinia spp.
plant factor(s) pH, media composition temperature, ammonium, nicotinic acid Media composition, plant factor(s)
Xanthomonas campestris Ralstonia Cell contact, media composition, Congo Red solanacearum Rhizobium spp. Flavanoids ? indicates presently unknown.
intracellular spread [20]. The major nucleiod protein, H-NS, which is encoded by the virR gene [21], negatively controls TTSS gene expression in a temperature-dependent manner. In fact, Falconi et al. [22] elegantly showed that H-NS is only able to bind to and repress the promoter of the primary regulator, VirF, at temperatures below 32°C. This is due to a subtle, temperature-dependent change in the DNA structure of the virF promoter, such that, above 32°C, the promoter is insensitive to the repressive effects of H-NS. Similarly, DNA topology has been implicated in the temperature-dependent expression of TTSS genes expressed from large virulence plasmids encoded by Yersinia spp., EPEC and enterohemorrhagic E. coli (EHEC) [23]. Rohde et al. [23] observed intrinsic DNA bends in the virulence plasmids that were melted at elevated temperature. Therefore, it is likely that these four pathogens all exhibit a comparable physical basis for thermoregulation that probably involve the accessibility of target DNA to H-NS. This is supported by the finding that H-NS represses transcription of TTSS genes in EPEC [24]. In addition to H-NS, other nucleoid-associated proteins are emerging as potential modulators of TTSS gene expression. At least in Shigella, Falconi et al. [25] indicate that factor inversion stimulation (FIS) antagonises the repressive function of H-NS bound to the VirF regulator in a temperature-dependent manner. Thus, they propose that modulation of the DNA architecture has been universally
SsrA/SsrB, PhoP/PhoQ, ? OmpR/EnvZ CpxA/CpxR, OmpR/EnvZ VirF
SicA, H-NS, HupB, Hha, Fis, CsrA, FliZ, FadD, SipB ? VirB, LuxS, IHF, H-NS (VirR), Fis, LuxS, IpaB LcrQ, YopD, LcrH, SycH, YopN, LcrV, LcrG, YmoA PcrV?
?
LcrF
?
ExsA
?
Per, GadX
? BvgAS
? ?
?
?
HrpL, HrpR, HrpS, HrpV, RpoN
HrpX/HrpY
?
HrpL, HrpS
HrpG
HrpX
?
HrpG
HrpB
PrhA, PrhI, PrhJ, PrhR
y4xI
?
NodD1
Ler, H-NS, Fis, Hha, IHF, LuxS ? ?
evolved by pathogens as a strategy to regulate virulence gene expression in response to environmental stimuli. It follows that several signals, such as temperature, osmolarity, pH and oxygen tension, have all been implicated to cause topological changes in local DNA structure. Not surprisingly, these environmental stimuli are all encountered by a pathogen during infection of an animal. Divalent cations
Some pathogens generally remain intracellular throughout the infection. To establish this niche, bacteria need to differentiate between an extracellular and intracellular location. Divalent cations can act as an extracellular signal to regulate virulence gene expression [26]. The facultatively intracellular pathogen Salmonella typhimurium employs one TTSS (encoded by Salmonella pathogenicity island [SPI]-1) to ensure initial invasion of host cells and another TTSS (encoded by SPI-2) for subsequent intracellular survival and multiplication within vacuoles of macrophages. This strategy is essential for S. typhimurium to cause systemic infection in mice. To strictly co-ordinate expression of the two TTSSs, these bacteria utilise Mg2+ concentration to sense a subcellular locality [27,28]. During the invasion process, bacteria are subject to high Mg2+ concentrations outside the cell compared to inside host cell vacuoles [29]. It follows that growth in medium containing a low Mg2+ concentration induced expression of several SPI-2 genes [30], although the mediator(s) of this upregulation is not
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known [31]. In contrast, after bacterial invasion, the PhoP/PhoQ two-component regulatory system responds to Mg2+-limiting conditions [26,28] by downregulating SPI-1 gene expression [32]. Thus, S. typhimurium senses the surrounding Mg2+ concentration to inversely regulate two independent TTSSs: SPI-1, which is important in ensuring initial invasion, then SPI-2, which is important for intracellular survival and proliferation. Acidity
In order to colonise or invade the intestinal epithelium, pathogens must first overcome the hostile acidic environment encountered during passage through the stomach. In fact, there are few environments that compare to the low pH found in this organ. Colonisation of the proximal regions of the small intestine by EPEC and EHEC requires a TTSS encoded on a pathogenicity island termed locus of enterocyte effacement (LEE). The plasmidlocated per locus, which incorporates a gene encoding an AraC-like transcriptional activator [4], activates the global regulator Ler (LEE-encoded regulator) to initiate a cascade of TTSS gene activation from LEE [33]. However, EPEC has also evolved acidic pH neutralisation mechanisms, such as the glutamate decarboxylase system encoded by gadAB to enable survival in the stomach [34]. As such, EPEC provides an interesting example of TTSS regulation in response to external pH. Shin et al. [35••] recently identified the product of the gadX gene — an AraC-like transcriptional regulator that inversely controls expression of the gadAB acid tolerance genes and the per regulon in response to low pH and growth conditions. In particular, as EPEC passes through the acidic environment of the stomach, GadX activates gadAB and represses per transcription. However, for colonisation of the small intestine, EPEC must express the TTSS while adapting to the pH increase and variable nutrient composition caused by actively digested food. In this environment, GadX represses transcription from the gadAB promoter and activates per to promote a cascade of TTSS gene expression. Therefore, GadX activity enables EPEC to sense the pH of the surrounding environment to co-ordinately regulate essential acid tolerance and TTSS virulence genes. Quorum sensing
Bacteria determine cell population density by cell-to-cell communication. This bacterial global regulatory network is termed quorum sensing. Recently, a universal quorum sensing mechanism has been recognised in both Gramnegative and Gram-positive bacteria. The product of the luxS gene, conserved in numerous bacterial species, acts upon a biosynthetic intermediate to generate the autoinducer-2 (AI-2) signalling molecule [36]. Conceivably, numerous bacteria may use AI-2 in intra- and interspecies communication. In both EPEC and EHEC, high concentrations of AI-2 in the culture media directly (and indirectly through Ler) activate TTSS structural and secreted effector genes [37]. This may offer an explanation for the unusually low infectious dose observed for some
EHEC strains. Sperandio et al. [37] suggest that, rather than sensing self-signalling molecules, this pathogen may activate the TTSS by sensing autoinducers synthesised by commensal E. coli resident in the large intestine. In contrast, the AI-2 signalling mechanism is not a prerequisite for full virulence of Shigella spp., although it does influence virB transcription [38]. Though these findings suggest that quorum sensing may only be adapted to regulate TTSS in bacterial pathogens that colonise host tissues occupied by commensal flora [38], they do point to a superbly cunning way for these bacteria to sense their surrounding environment.
Host cell contact Several laboratories have shown that cell contact by most animal pathogens expressing a functional TTSS is a prerequisite for increased production and subsequent delivery of anti-host-effector proteins across host cell plasma membranes (see below). However, recent findings by Aldon et al. [39••] are the first to show that initial TTSS gene transcription and subsequent apparatus assembly are also induced upon cell contact in the plant pathogen R. solanacearum. Intriguingly, this occurs independently of a functional TTSS [39••]. As the signal for cell contact is likely to be a non-diffusible macromolecule, the mechanism by which this signal is recognised and subsequently transmitted through the bacterial envelope is of great significance. In R. solanacearum, transcription of the HrpB transcriptional activator, which controls TTSS gene expression, is significantly induced upon cell contact [40]. Three proteins, PrhA, PrhI and PrhR, whose expression and cellular localisation are TTSS-independent, are required for transmitting the cell contact signal across the bacterial envelope [12]. PrhA probably interacts with a non-diffusible plant cell wall component during contact. The function of PrhR and PrhI has been derived from functions of analogous proteins involved in induction of the the iron transport system in E. coli [41,42]. It is proposed that this signal is first transmitted across the outer membrane by PrhA to the periplasmic carboxy-terminal region of the inner membrane protein PrhR, which subsequently mediates signalling through the inner membrane. In turn, the amino terminus of PrhR, located at the cytoplasmic face of the inner membrane, activates the alternate sigma factor, PrhI (which belongs to the ECF family). Activated PrhI is presumably released from the inner membrane to initiate transcription of regulatory genes necessary for the transcription of additional TTSS-encoding operons [12]. Thus, R. solanacearum has evolved a three-component signalling module that spans the bacterial envelope to activate pathogenicity determinants in response to a nondiffusible plant cell wall signal.
Regulation of TTSS organelle assembly For several animal pathogens, the type III organelle has been isolated and analysed. The basal portion of these structures possesses two sets of rings resembling the flagellar basal body (Figure 1). The type III export machinery apparently associates with the cytoplasmic face of the rings
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Figure 1 Schematic representation of the stepwise assembly of the TTSS apparatus encoded by SPI-1 of S. typhimurium. The grey shading indicates the order of protein assembly: first, the inner rings (PrgH); second, the periplasmic channel (PrgK); third, the outer rings (InvG/InvH in the outer membrane, then InvA, InvC, SpaP, SpaQ, SpaR and SpaS to form the export machinery in the inner membrane); and fourth, the external needle appendage (possibly PrgI and PrgJ). Protein secretion in steps 1–3 is sec-dependent, whereas it is sec-independent in step 4. InvJ (and its homologues) is required for regulating the assembly switch mechanism after completion of needle complex assembly to permit the secretion of translocator and antihost-effector proteins. An InvJ mutant causes extended needles. This illustration is adapted from [48•].
Step 1 Step 2 Step 3 Step 4
Inner rings Periplasmic channel Outer rings Needle appendage Needle appendage Needle complex
Outer membrane Basal portion
Inner membrane Wild-type InvJ mutant (normal needle length) (extended needle length)
Current Opinion in Microbiology
in the inner membrane. A feature common to all type III organelles so far isolated is the needle-like structure that protrudes from the ring located in the outer membrane. This needle is required for secretion, suggesting that combination of the basal portion and needle extension, collectively termed the needle complex, is an intact secretion organelle. In plant pathogens, the needle is replaced by a broader, more extended pilus-like structure. Although secreted proteins are most likely channelled through these structures [43,44••], they are apparently not involved in binding to plant cells [45]. Interestingly, an EspA-containing filamentous structure not required for secretion associates with the needle complex of EPEC [46••,47]. The likely role for these filaments is in channelling anti-host-effector proteins across the host cell plasma membrane.
active secretion of its substrates. This would be analogous to the mechanism involved in switching from hook to filament in flagella biosynthesis (see the review by Aldridge and Hughes). Briefly, fliK mutants are unable to make this switch to filament assembly, and this results in a distinctive poly-hook phenotype. Apparently, FliK senses a completed hook structure and transmits this signal to FlhB, which replaces hook export with flagellin export. In TTSSs, defects in distantly related FliK homologues, such as InvJ (which is encoded by SPI-1), result in longer needle complexes with reduced secretion potential (Figure 1) [49••]. Although the mechanism of substrate switching in TTSSs is not known, the presence of a FlhB homologue in all type III systems indicates that this function is conserved in assembly of both flagella and TTSSs.
Information concerning TTSS organelle assembly is derived from what is known about flagella biosynthesis. Flagella basal body assembly is a highly ordered process that is regulated at the transcriptional level by complex feedback mechanisms (see the review by Aldridge and Hughes in this issue). Although the assembly process for the TTSS needle complex is unclear, details of SPI-1encoded TTSS assembly are now emerging. The structure spanning the bacterial envelope follows a stepwise assembly: first, the inner rings; second, the periplasmic channel; and third, the outer rings (Figure 1) [48•]. The final incorporation of the external needle appendage is not sec-dependent. Instead, it requires an assembled basal structure that also includes the type-III-specific export machinery localised to the cytoplasmic face of the inner ring (Figure 1) [48•]. In principle, the completed needle complex is competent for secretion of translocator and anti-host-effector proteins. Interestingly, however, upon needle completion, a switching mechanism may signal the readiness of the TTSS for
Several TTSSs, such as those of Yersinia spp., P. aeruginosa and Shigella spp., require intimate cell contact to induce secretion of translocater and anti-host-effector proteins. In addition, cell contact also elevates effector gene transcription [50]. Induction requires a functional needle complex, as gene expression in assembly defective mutants is severely repressed. This is consistent with the requirements for successful flagellar assembly, which include secretion of the anti-sigma factor FlgM (see the review by Aldridge and Hughes). By analogy, induction of Yersiniaspecific TTSSs by cell contact requires the rapid secretion of a negative regulatory element, LcrQ [50]. However, the molecular mechanism by which the cell contact signal is sensed and transmitted across the bacterial envelope is unknown. Interestingly, serum and amphipathic agents like Congo Red can also induce TTSSs. In P. aeruginosa and Shigella, these agents are believed to mimic receptor binding and induction normally mediated by cell contact [51,52]. As a completed needle complex is a prerequisite
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for both substrate switching and secretion of translocator and anti-host-effector proteins, it may also be a component of the module involved in sensing and transmitting cell contact.
The regulatory role of TTSS chaperones A unique feature of TTSSs concerns the requirement of a specialised intracellular chaperone for the pre-secretory stabilisation and efficient secretion of a cognate protein predestined for an extracellular function [53]. Thus, bacteria defective in one chaperone cannot efficiently secrete its cognate partner, though secretion of other proteins remains unaffected. Chaperones do not generally share significant homology. However, they are recognised by similar physical characteristics that are also observed in TTSS molecules required for flagellar biosynthesis. Surprisingly, not all secreted proteins appear to require a specialised chaperone. This observation prompted Boyd et al. [54] to suggest that chaperones establish a hierarchy for effector protein delivery into target eukaryotic cells. On the basis of work in Yersinia, these researchers found that, in the presence of the SycE chaperone, the chaperone-binding site located near the amino terminus of the YopE cytotoxin (a GTPaseactivating protein for Rho-GTPases) gave this protein a competitive advantage for secretion and subsequent delivery into target cells. Perhaps the chaperone presents YopE to the secretion/delivery machinery to allow more rapid secretion [55]. This is consistent to in vivo-like conditions, in which the Yersinia type III effector YopH (tyrosine protein phosphatase) is delivered by a chaperonedependent mechanism into macrophages within a minute of infection [56]. Recently, a new role for chaperones in the regulation of TTSS virulence determinants has been established. SicA, a TTSS chaperone specific for SipB and SipC of Salmonella spp., acts together with the DNA-binding protein InvF (a member of the AraC-like family of transcriptional activators) to activate the expression of a subset of genes encoding type-III-secreted products that are essential for Salmonella pathogenicity [57••]. It is proposed that, as SicA co-purifies with InvF and is necessary for InvF-dependent promoter activity, it is likely that SicA functions as an InvF cofactor to establish an open promoter complex to enable transcription initiation [57••]. SicA-dependent transcriptional activation is probably contingent on the secretion of SipB and SipC. This view has been pursued in Yersinia pseudotuberculosis. The complex between the TTSS chaperone LcrH/SycD and one of its cognate partners, YopD, appears to play a very important role in establishing negative feedback regulation of TTSS genes encoding type-III-secreted substrates [58•]. Although YopD is an important negative regulatory element [59], LcrH also plays an active role by interacting with YscY, a member of the Yersinia secretion apparatus [58•]. Therefore, it is interesting to speculate that early secretion of YopD may relieve the transcription block and enable LcrH to activate gene expression analogous to its distant homologue, SicA.
As previously indicated, high concentrations of the intracellular regulatory factor, LcrQ, inhibits TTSS gene expression in Yersinia spp. [50]. Intriguingly, the SycH chaperone is required to overcome the LcrQ-dependent negative feedback by physically interacting with LcrQ to ensure its rapid secretion and probable inactivation of residual intracellular LcrQ [60•]. By analogy to LcrQ function, a controlled release of the anti-FliA sigma factor, FlgM, fine-tunes expression of genes essential for complete flagella assembly (see the review by Aldridge and Hughes). Interestingly, translation of the flgM transcript whose product is predominantly predestined for secretion requires the TTSS chaperone FlgN [61]. Therefore, even though the regulatory pathways involving LcrQ and FlgM are evolutionary unrelated, chaperone involvement at least confirms the additional regulatory role of a subset of these unique molecules. Whether the primary role of TTSS chaperones is to maintain their cognate partner in an unfolded, secretioncompetent state or as a regulator of gene expression is not known. It is worth noting that some TTSS substrates are secreted in the absence of their chaperones, whereas others simply do not require chaperone assistance. Considering the tight coupling between cell contact and virulence gene induction (or secretion), and that this secretion probably occurs in an ordered manner during infection, the regulatory phenotype of a subset of these chaperones may reside at the level of secretion control. This would ensure immediate, ordered substrate delivery into eukaryotic cells in response to physical contact. Thus, loss of regulatory control would ensue if ordered substrate delivery was not maintained.
Conclusions We note that several environmental cues, nucleiod-associated proteins, phosphorelay two-component systems, AraC-like transcriptional activators and global regulators cross-talk to modulate expression of TTSS genes. Realistically, however, the use of in vivo gene expression technologies, summarised by Hautefort and Hinton [62], is required to accurately determine the spatial and temporal control of these impressively complex regulatory cascades. It is worth noting, however, that recent developments indicate that cell contact, assembly switching and specific chaperone molecules play a unique role in regulation of TTSSs. We envisage that future work will address the specific receptor–ligand interactions, nondiffusible signals and modes of transmission that initiate induction of target cell contact. It is also necessary to determine the role, if any, of specific chaperones in type III gene regulation and effector molecule secretion by plant pathogens. Finally, details of the pattern of TTSS assembly are now emerging. As type III structures spanning the bacterial envelope are similar among the bacteria studied, the assembly mechanism probably involves common sec-dependent and sec-independent mechanisms. Therefore, targeting of the assembly pathway may provide a common antibacterial strategy for disease prevention.
Regulation of type III secretion systems Francis, Wolf-Watz and Forsberg
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36. Schauder S, Shokat K, Surette MG, Bassler BL: The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule. Mol Microbiol 2001, 41:463-476. 37.
Sperandio V, Mellies JL, Nguyen W, Shin S, Kaper JB: Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli. Proc Natl Acad Sci USA 1999, 96:15196-15201.
38. Day WA Jr, Maurelli AT: Shigella flexneri LuxS quorum-sensing system modulates virB expression but is not essential for virulence. Infect Immun 2001, 69:15-23. 39. Aldon D, Brito B, Boucher C, Genin S: A bacterial sensor of plant •• cell contact controls the transcriptional induction of Ralstonia solanacearum pathogenicity genes. EMBO J 2000, 19:2304-2314. Expression of TTSS genes in R. solanacearum is induced when bacteria are grown in the presence of plant cells. This work establishes that an outer membrane protein, PrhA, encodes a receptor that interacts with a nondiffusible plant cell component. This ultimately results in transcriptional activation of TTSS-encoding operons. 40. Brito B, Marenda M, Barberis P, Boucher C, Genin S: prhJ and hrpG, two new components of the plant signal-dependent regulatory cascade controlled by PrhA in Ralstonia solanacearum. Mol Microbiol 1999, 31:237-251. 41. Stiefel A, Mahren S, Ochs M, Schindler PT, Enz S, Braun V: Control of the ferric citrate transport system of Escherichia coli: mutations in region 2.1 of the FecI extracytoplasmic-function sigma factor suppress mutations in the FecR transmembrane regulatory protein. J Bacteriol 2001, 183:162-170. 42. Enz S, Mahren S, Stroeher UH, Braun V: Surface signalling in ferric citrate transport gene induction: Interaction of the FecA, FecR, and FecI regulatory proteins. J Bacteriol 2001, 182:637-646. 43. Jin Q, Hu W, Brown I, McGhee G, Hart P, Jones AL, He SY: Visualisation of secreted Hrp and Avr proteins along the Hrp pilus during type III secretion in Erwinia amylovora and Pseudomonas syringae. Mol Microbiol 2001, 40:1129-1139. 44. Jin Q, He SY: Role of the Hrp pilus in type III protein secretion in •• Pseudomonas syringae. Science 2001, 294:2556-2558. In this report, newly secreted effector proteins were visualised by in situ immunogold labelling and shown to associate with the distal part of Hrp pilus of Erwinia amylovora and Pseudomonas syringae. This strongly suggests that the secreted effector proteins are channeled through the Hrp pilus. 45. Van Gijsegem F, Vasse J, Camus J-C, Marenda M, Boucher C: Ralstonia solanacearum produces Hrp-dependent pili that are required for PopA secretion but not for attachment to plant cells. Mol Microbiol 2000, 36:249-260. 46. Sekiya K, Ohishi, M, Ogino T, Tamano K, Sasakawa C, Abe A: •• Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and its direct interaction with the EspA-sheath-like structure. Proc Natl Acad Sci USA 2001, 98:11638-11643. In this study, the needle complex of EPEC was isolated and visualised by electron microscopy. The authors of this paper observed that the needle extensions in EPEC are at least 10 times longer than those in other pathogens, such as Salmonella and Shigella. Moreover, the needle base is composed of the EscF ‘needle homologue’ protein and the long extensions are formed by EspA. The latter is a known requirement for the delivery of EspB and EspD into host cells. 47.
Wilson RK, Shaw RK, Daniell S, Knutton S, Frankel G: Role of EscF, a putative needle complex protein, in the type III protein translocation system of enteropathogenic Escherichia coli. Cell Microbiol 2001, 3:753-762.
48. Sukhan A, Kubori T, Wilson J, Galán J: Genetic analysis of assembly • of the Salmonella enterica serovar Typhimurium secretionassociated needle complex. J Bacteriol 2001, 183:1159-1167. This is the first genetic study to address the assembly process of a type III secretion needle complex. Evidence is provided to show that the basal portion and the export system assemble together in a sec-dependent process, whereas the needle appendage is assembled in a sec-independent but type-IIIexport-dependent process only after the completion of the basal portion.
49. Kubori T, Sukhan A, Aizawa, S-I, Galán J: Molecular characterization •• and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system. Proc Natl Acad Sci USA 2000, 97:10225-10230. In this elegant work, the needle component of the type III secretion organelle was first identified and found to be essential for a functional TTSS. In addition, the needle length was shown to be controlled by InvJ. 50. Pettersson J, Nordfelth R, Dubinina E, Bergman T, Gustafsson M, Magnusson K-E, Wolf-Watz H: Modulation of virulence factor expression by pathogen target cell contact. Science 1996, 273:1231-1233. 51. Vallis AJ, Yahr T L, Barbieri JT, Frank DW: Regulation of ExoS production and secretion by Pseudomonas aeruginosa in response to tissue culture conditions. Infect Immun 1999, 67:914-920. 52. Demers B, Sansonetti PJ, Parsot C: Induction of type III secretion in Shigella flexneri is associated with differential control of transcription of genes encoding secreted proteins. EMBO J 1998, 17:2894-2903. 53. Wattiau P, Woestyn S, Cornelis GR: Customized secretion chaperones in pathogenic bacteria. Mol Microbiol 1996, 20:255-262. 54. Boyd AP, Lambermont I, Cornelis GR: Competition between the Yops of Yersinia enterocolitica for delivery into eukaryotic cells: role of the SycE chaperone binding domain of YopE. J Bacteriol 2000, 182:4811-4821. 55. Lloyd SA, Norman M, Rosqvist R, Wolf-Watz H: Yersinia YopE is targeted for type III secretion by N-terminal, not mRNA, signals. Mol Microbiol 2001, 39:520-531. 56. Andersson K, Magnusson KE, Majeed M, Stendahl O, Fallman M: Yersinia pseudotuberculosis-induced calcium signaling in neutrophils is blocked by the virulence effector YopH. Infect Immun 1999, 67:2567-2574. 57. ••
Darwin KH, Miller VL: Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium. EMBO J 2001, 20:1850-1862. This elegant study described the regulatory function of the TTSS chaperone SicA in the absence of its cognate partners, SipB and SipC. The authors showed that SicA acts as a cofactor of the AraC-like regulator LcrF. SicA did not interact with DNA directly or alter stability of InvF. However, co-purification of SicA with InvF suggested that they interact to activate transcription from TTSS promoters. 58. Francis MS, Lloyd SA, Wolf-Watz H: The type III secretion • chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis. Mol Microbiol 2001, 42:1075-1094. The authors of this paper indicated that the LcrH chaperone acts in complex with its cognate partner YopD as a repressor of protein synthesis. In addition, they were the first to report that chaperone binding to a member of the type III apparatus occurred after secretion of the cognate partner. They suggest that this complex de-represses TTSS gene expression. 59. Williams AW, Straley SC: YopD of Yersinia pestis plays a role in negative regulation of the low-calcium response in addition to its role in translocation of Yops. J Bacteriol 1998, 180:350-358. 60. Cambronne ED, Cheng LW, Schneewind O: LcrQ/YscM1, regulators • of the Yersinia yop virulon, are injected into host cells by a chaperone-dependent mechanism. Mol Microbiol 2000, 37:263-273. Secretion of the negative regulatory element LcrQ by Yersinia is essential for the cell-contact-dependent induction of virulence genes. This study clearly showed that the cognate chaperone of the YopH effector, SycH, also interacts with LcrQ and is required for its secretion. This important finding highlights a unique regulatory role for SycH and establishes a possible mechanism for negative feedback regulation in Yersinia. 61. Karlinsey JE, Lonner J, Brown KL, Hughes KT: Translation/secretion coupling by type III secretion systems. Cell 2000, 102:487-497. 62. Hautefort I, Hinton JC: Measurement of bacterial gene expression in vivo. Philos Trans R Soc Lond B Biol Sci 2000, 355:601-611.
Regulation of type III secretion systems Matthew S Francis*, Hans Wolf-Watz* and Åke Forsberg*†‡ Type III secretion systems are utilised by numerous Gramnegative bacteria to efficiently interact with a host. Appropriate expression of type III genes is achieved through the integration of several regulatory pathways that ultimately co-ordinate the activity of a central transcriptional activator usually belonging to the AraC family. The complex regulatory cascades allow this virulence strategy to be utilised by different bacteria even if they occupy diverse niches that define a unique set of environmental cues. Simulating the appropriate combination of signals in vitro to allow a meaningful interpretation of the type III assembly and secretion regulatory cascade remains a common goal for researchers. Pieces of the puzzle slowly emerge to provide insightful views into the complex regulatory networks that allow bacteria to assemble and utilise type III secretion to efficiently colonise a host. Addresses *Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden † Department of Medical Countermeasures, Swedish Defence Research Agency, SE-901 82 Umeå, Sweden ‡ e-mail: [email protected] Author for correspondence: Åke Forsberg Current Opinion in Microbiology 2002, 5:166–172 1369-5274/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 28 February 2002 Abbreviations AI-2 autoinducer-2 ECF extracytoplasmic factor EHEC enterohemorrhagic Escherichia coli EPEC enteropathogenic E. coli LEE locus of enterocyte effacement TTSS type III secretion system
Introduction Type III secretion systems (TTSSs) are complex protein secretion and delivery machines utilised by many animaland plant-interacting bacteria that occupy diverse niches. More than 25 genes are required to establish a functional TTSS. In animal pathogens, the principle function of TTSS is to deliver anti-host-virulence-determinants into mammalian cells. This results in a cascade of regulated events to overcome animal defence mechanisms necessary to establish bacterial infection. By analogy, plant pathogens utilise TTSSs to cause disease in ‘susceptible’ plants and trigger the hypersensitive response in ‘resistant’ plants. With this level of complexity, it is not surprising that multifaceted regulatory systems are required to impart spatial and temporal control of type III gene expression. It is apparent that regulation occurs in at least two distinct steps: expression of genes required for assembly of the secretion apparatus followed by expression of genes whose products are substrates for TTSSs. In this review, we illustrate some recent developments in the regulation of TTSSs.
Sensing environmental cues Pathogenic bacteria occupy very different infection foci. The animal pathogens Salmonella, Shigella and Chlamydia all actively invade host cells. Upon invasion, however, Salmonella and Chlamydia persist within host vacuoles and modulate them, whereas Shigella freely replicate within the cytosol and eventually spread from cell to cell. In contrast, Yersinia spp. and Pseudomonas aeruginosa predominantly remain extracellular, and enteropathogenic Escherichia coli (EPEC) colonise the small intestine. Moreover, phytopathogens remain extracellular in susceptible plants, occupying the intercellular spaces of parenchymatus tissue. As each niche exerts an assortment of unique environmental pressures, and TTSSs are essential for virulence of these pathogens, it follows that a vast array of environmental signals influences the expression of TTSS assembly genes and secretion substrate genes (Table 1). Most bacteria employ phosphorelay mechanisms to sense the surrounding environment. In animal pathogens, these signals are most often routed to AraC-like transcriptional activators that establish a cascade of type III gene activation [1–7]. A role for this activator family was first discovered by Cornelis et al. in Yersinia [2]. The involvement of small nucleoid-associated proteins in this process likely reflects the need to fine-tune gene expression with the physiological or nutritional status of the bacteria. The phytopathogens Xanthomonas campestris and Ralstonia solanacearum utilise related AraC-like regulators [8,9]. In addition, Pseudomonas syringae, Erwinia amylovora and R. solanacearum employ alternative sigma factors belonging to the extracytoplasmic factor (ECF) family [10–12]. Expression of the latter requires RpoN [13] and enhancer-binding proteins that function as response regulators [14,15]. In this section, we illustrate how different bacteria utilise diverse environmental cues to regulate TTSS assembly and TTSS-mediated secretion. We consider selected examples that best reflect actual in vivo regulatory events (we apologise to our many colleagues whose inspiring work has not been cited, owing to space restrictions, in our review). Temperature
Most bacterial pathogens normally reside in the surrounding environment. However, on contact with an animal host, these bacteria encounter an increase in their growth temperature to 37°C. Furthermore, temperature increase has long been known to induce TTSS virulence gene expression in Shigella spp. [16] and Yersinia spp. [17,18]. Therefore, how do these pathogens utilise temperature as a stimulus for TTSS gene activation? In Shigella spp., on temperature increase to 37°C, an AraC-like transcriptional activator, VirF, responds by activating a regulatory cascade mediated by VirB [19]. In turn, VirB activates genes required for TTSS assembly and secretion substrates involved in Shigella invasiveness and
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Table 1 A summary of regulatory characteristics of type III secretion systems. Pathogen
Stimuli
Two-component regulators AraC-like Additional regulatory transcriptional elements activators
Animal pathogen Salmonella Mg2, oxygen, osmolarity, pH, inorganic phosphate, PhoP/PhoQ, PhoR/PhoB, HilC, HilD, spp. SPI-1 RNA stability Bar/SirA, OmpR/EnvZ, HilA InvF Salmonella spp. Mg2, phosphate starvation, pH media composition SPI-2 Shigella spp. Cell contact, media composition, serum, Congo Red, temperature, pH, tRNA modification Yersinia spp. Cell contact, Ca2, temperature, amino acids, albumin Pseudomonas Cell contact, serum, Ca2 aeruginosa EPEC Temperature, pH, cell density, media composition, growth phase Chlamydia spp. Cell contact or intracellular signals? Bordetella spp. ? Plant pathogens Pseudomonas Media composition, pH, osmolarity, temperature, syringae Erwinia spp.
plant factor(s) pH, media composition temperature, ammonium, nicotinic acid Media composition, plant factor(s)
Xanthomonas campestris Ralstonia Cell contact, media composition, Congo Red solanacearum Rhizobium spp. Flavanoids ? indicates presently unknown.
intracellular spread [20]. The major nucleiod protein, H-NS, which is encoded by the virR gene [21], negatively controls TTSS gene expression in a temperature-dependent manner. In fact, Falconi et al. [22] elegantly showed that H-NS is only able to bind to and repress the promoter of the primary regulator, VirF, at temperatures below 32°C. This is due to a subtle, temperature-dependent change in the DNA structure of the virF promoter, such that, above 32°C, the promoter is insensitive to the repressive effects of H-NS. Similarly, DNA topology has been implicated in the temperature-dependent expression of TTSS genes expressed from large virulence plasmids encoded by Yersinia spp., EPEC and enterohemorrhagic E. coli (EHEC) [23]. Rohde et al. [23] observed intrinsic DNA bends in the virulence plasmids that were melted at elevated temperature. Therefore, it is likely that these four pathogens all exhibit a comparable physical basis for thermoregulation that probably involve the accessibility of target DNA to H-NS. This is supported by the finding that H-NS represses transcription of TTSS genes in EPEC [24]. In addition to H-NS, other nucleoid-associated proteins are emerging as potential modulators of TTSS gene expression. At least in Shigella, Falconi et al. [25] indicate that factor inversion stimulation (FIS) antagonises the repressive function of H-NS bound to the VirF regulator in a temperature-dependent manner. Thus, they propose that modulation of the DNA architecture has been universally
SsrA/SsrB, PhoP/PhoQ, ? OmpR/EnvZ CpxA/CpxR, OmpR/EnvZ VirF
SicA, H-NS, HupB, Hha, Fis, CsrA, FliZ, FadD, SipB ? VirB, LuxS, IHF, H-NS (VirR), Fis, LuxS, IpaB LcrQ, YopD, LcrH, SycH, YopN, LcrV, LcrG, YmoA PcrV?
?
LcrF
?
ExsA
?
Per, GadX
? BvgAS
? ?
?
?
HrpL, HrpR, HrpS, HrpV, RpoN
HrpX/HrpY
?
HrpL, HrpS
HrpG
HrpX
?
HrpG
HrpB
PrhA, PrhI, PrhJ, PrhR
y4xI
?
NodD1
Ler, H-NS, Fis, Hha, IHF, LuxS ? ?
evolved by pathogens as a strategy to regulate virulence gene expression in response to environmental stimuli. It follows that several signals, such as temperature, osmolarity, pH and oxygen tension, have all been implicated to cause topological changes in local DNA structure. Not surprisingly, these environmental stimuli are all encountered by a pathogen during infection of an animal. Divalent cations
Some pathogens generally remain intracellular throughout the infection. To establish this niche, bacteria need to differentiate between an extracellular and intracellular location. Divalent cations can act as an extracellular signal to regulate virulence gene expression [26]. The facultatively intracellular pathogen Salmonella typhimurium employs one TTSS (encoded by Salmonella pathogenicity island [SPI]-1) to ensure initial invasion of host cells and another TTSS (encoded by SPI-2) for subsequent intracellular survival and multiplication within vacuoles of macrophages. This strategy is essential for S. typhimurium to cause systemic infection in mice. To strictly co-ordinate expression of the two TTSSs, these bacteria utilise Mg2+ concentration to sense a subcellular locality [27,28]. During the invasion process, bacteria are subject to high Mg2+ concentrations outside the cell compared to inside host cell vacuoles [29]. It follows that growth in medium containing a low Mg2+ concentration induced expression of several SPI-2 genes [30], although the mediator(s) of this upregulation is not
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known [31]. In contrast, after bacterial invasion, the PhoP/PhoQ two-component regulatory system responds to Mg2+-limiting conditions [26,28] by downregulating SPI-1 gene expression [32]. Thus, S. typhimurium senses the surrounding Mg2+ concentration to inversely regulate two independent TTSSs: SPI-1, which is important in ensuring initial invasion, then SPI-2, which is important for intracellular survival and proliferation. Acidity
In order to colonise or invade the intestinal epithelium, pathogens must first overcome the hostile acidic environment encountered during passage through the stomach. In fact, there are few environments that compare to the low pH found in this organ. Colonisation of the proximal regions of the small intestine by EPEC and EHEC requires a TTSS encoded on a pathogenicity island termed locus of enterocyte effacement (LEE). The plasmidlocated per locus, which incorporates a gene encoding an AraC-like transcriptional activator [4], activates the global regulator Ler (LEE-encoded regulator) to initiate a cascade of TTSS gene activation from LEE [33]. However, EPEC has also evolved acidic pH neutralisation mechanisms, such as the glutamate decarboxylase system encoded by gadAB to enable survival in the stomach [34]. As such, EPEC provides an interesting example of TTSS regulation in response to external pH. Shin et al. [35••] recently identified the product of the gadX gene — an AraC-like transcriptional regulator that inversely controls expression of the gadAB acid tolerance genes and the per regulon in response to low pH and growth conditions. In particular, as EPEC passes through the acidic environment of the stomach, GadX activates gadAB and represses per transcription. However, for colonisation of the small intestine, EPEC must express the TTSS while adapting to the pH increase and variable nutrient composition caused by actively digested food. In this environment, GadX represses transcription from the gadAB promoter and activates per to promote a cascade of TTSS gene expression. Therefore, GadX activity enables EPEC to sense the pH of the surrounding environment to co-ordinately regulate essential acid tolerance and TTSS virulence genes. Quorum sensing
Bacteria determine cell population density by cell-to-cell communication. This bacterial global regulatory network is termed quorum sensing. Recently, a universal quorum sensing mechanism has been recognised in both Gramnegative and Gram-positive bacteria. The product of the luxS gene, conserved in numerous bacterial species, acts upon a biosynthetic intermediate to generate the autoinducer-2 (AI-2) signalling molecule [36]. Conceivably, numerous bacteria may use AI-2 in intra- and interspecies communication. In both EPEC and EHEC, high concentrations of AI-2 in the culture media directly (and indirectly through Ler) activate TTSS structural and secreted effector genes [37]. This may offer an explanation for the unusually low infectious dose observed for some
EHEC strains. Sperandio et al. [37] suggest that, rather than sensing self-signalling molecules, this pathogen may activate the TTSS by sensing autoinducers synthesised by commensal E. coli resident in the large intestine. In contrast, the AI-2 signalling mechanism is not a prerequisite for full virulence of Shigella spp., although it does influence virB transcription [38]. Though these findings suggest that quorum sensing may only be adapted to regulate TTSS in bacterial pathogens that colonise host tissues occupied by commensal flora [38], they do point to a superbly cunning way for these bacteria to sense their surrounding environment.
Host cell contact Several laboratories have shown that cell contact by most animal pathogens expressing a functional TTSS is a prerequisite for increased production and subsequent delivery of anti-host-effector proteins across host cell plasma membranes (see below). However, recent findings by Aldon et al. [39••] are the first to show that initial TTSS gene transcription and subsequent apparatus assembly are also induced upon cell contact in the plant pathogen R. solanacearum. Intriguingly, this occurs independently of a functional TTSS [39••]. As the signal for cell contact is likely to be a non-diffusible macromolecule, the mechanism by which this signal is recognised and subsequently transmitted through the bacterial envelope is of great significance. In R. solanacearum, transcription of the HrpB transcriptional activator, which controls TTSS gene expression, is significantly induced upon cell contact [40]. Three proteins, PrhA, PrhI and PrhR, whose expression and cellular localisation are TTSS-independent, are required for transmitting the cell contact signal across the bacterial envelope [12]. PrhA probably interacts with a non-diffusible plant cell wall component during contact. The function of PrhR and PrhI has been derived from functions of analogous proteins involved in induction of the the iron transport system in E. coli [41,42]. It is proposed that this signal is first transmitted across the outer membrane by PrhA to the periplasmic carboxy-terminal region of the inner membrane protein PrhR, which subsequently mediates signalling through the inner membrane. In turn, the amino terminus of PrhR, located at the cytoplasmic face of the inner membrane, activates the alternate sigma factor, PrhI (which belongs to the ECF family). Activated PrhI is presumably released from the inner membrane to initiate transcription of regulatory genes necessary for the transcription of additional TTSS-encoding operons [12]. Thus, R. solanacearum has evolved a three-component signalling module that spans the bacterial envelope to activate pathogenicity determinants in response to a nondiffusible plant cell wall signal.
Regulation of TTSS organelle assembly For several animal pathogens, the type III organelle has been isolated and analysed. The basal portion of these structures possesses two sets of rings resembling the flagellar basal body (Figure 1). The type III export machinery apparently associates with the cytoplasmic face of the rings
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Figure 1 Schematic representation of the stepwise assembly of the TTSS apparatus encoded by SPI-1 of S. typhimurium. The grey shading indicates the order of protein assembly: first, the inner rings (PrgH); second, the periplasmic channel (PrgK); third, the outer rings (InvG/InvH in the outer membrane, then InvA, InvC, SpaP, SpaQ, SpaR and SpaS to form the export machinery in the inner membrane); and fourth, the external needle appendage (possibly PrgI and PrgJ). Protein secretion in steps 1–3 is sec-dependent, whereas it is sec-independent in step 4. InvJ (and its homologues) is required for regulating the assembly switch mechanism after completion of needle complex assembly to permit the secretion of translocator and antihost-effector proteins. An InvJ mutant causes extended needles. This illustration is adapted from [48•].
Step 1 Step 2 Step 3 Step 4
Inner rings Periplasmic channel Outer rings Needle appendage Needle appendage Needle complex
Outer membrane Basal portion
Inner membrane Wild-type InvJ mutant (normal needle length) (extended needle length)
Current Opinion in Microbiology
in the inner membrane. A feature common to all type III organelles so far isolated is the needle-like structure that protrudes from the ring located in the outer membrane. This needle is required for secretion, suggesting that combination of the basal portion and needle extension, collectively termed the needle complex, is an intact secretion organelle. In plant pathogens, the needle is replaced by a broader, more extended pilus-like structure. Although secreted proteins are most likely channelled through these structures [43,44••], they are apparently not involved in binding to plant cells [45]. Interestingly, an EspA-containing filamentous structure not required for secretion associates with the needle complex of EPEC [46••,47]. The likely role for these filaments is in channelling anti-host-effector proteins across the host cell plasma membrane.
active secretion of its substrates. This would be analogous to the mechanism involved in switching from hook to filament in flagella biosynthesis (see the review by Aldridge and Hughes). Briefly, fliK mutants are unable to make this switch to filament assembly, and this results in a distinctive poly-hook phenotype. Apparently, FliK senses a completed hook structure and transmits this signal to FlhB, which replaces hook export with flagellin export. In TTSSs, defects in distantly related FliK homologues, such as InvJ (which is encoded by SPI-1), result in longer needle complexes with reduced secretion potential (Figure 1) [49••]. Although the mechanism of substrate switching in TTSSs is not known, the presence of a FlhB homologue in all type III systems indicates that this function is conserved in assembly of both flagella and TTSSs.
Information concerning TTSS organelle assembly is derived from what is known about flagella biosynthesis. Flagella basal body assembly is a highly ordered process that is regulated at the transcriptional level by complex feedback mechanisms (see the review by Aldridge and Hughes in this issue). Although the assembly process for the TTSS needle complex is unclear, details of SPI-1encoded TTSS assembly are now emerging. The structure spanning the bacterial envelope follows a stepwise assembly: first, the inner rings; second, the periplasmic channel; and third, the outer rings (Figure 1) [48•]. The final incorporation of the external needle appendage is not sec-dependent. Instead, it requires an assembled basal structure that also includes the type-III-specific export machinery localised to the cytoplasmic face of the inner ring (Figure 1) [48•]. In principle, the completed needle complex is competent for secretion of translocator and anti-host-effector proteins. Interestingly, however, upon needle completion, a switching mechanism may signal the readiness of the TTSS for
Several TTSSs, such as those of Yersinia spp., P. aeruginosa and Shigella spp., require intimate cell contact to induce secretion of translocater and anti-host-effector proteins. In addition, cell contact also elevates effector gene transcription [50]. Induction requires a functional needle complex, as gene expression in assembly defective mutants is severely repressed. This is consistent with the requirements for successful flagellar assembly, which include secretion of the anti-sigma factor FlgM (see the review by Aldridge and Hughes). By analogy, induction of Yersiniaspecific TTSSs by cell contact requires the rapid secretion of a negative regulatory element, LcrQ [50]. However, the molecular mechanism by which the cell contact signal is sensed and transmitted across the bacterial envelope is unknown. Interestingly, serum and amphipathic agents like Congo Red can also induce TTSSs. In P. aeruginosa and Shigella, these agents are believed to mimic receptor binding and induction normally mediated by cell contact [51,52]. As a completed needle complex is a prerequisite
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for both substrate switching and secretion of translocator and anti-host-effector proteins, it may also be a component of the module involved in sensing and transmitting cell contact.
The regulatory role of TTSS chaperones A unique feature of TTSSs concerns the requirement of a specialised intracellular chaperone for the pre-secretory stabilisation and efficient secretion of a cognate protein predestined for an extracellular function [53]. Thus, bacteria defective in one chaperone cannot efficiently secrete its cognate partner, though secretion of other proteins remains unaffected. Chaperones do not generally share significant homology. However, they are recognised by similar physical characteristics that are also observed in TTSS molecules required for flagellar biosynthesis. Surprisingly, not all secreted proteins appear to require a specialised chaperone. This observation prompted Boyd et al. [54] to suggest that chaperones establish a hierarchy for effector protein delivery into target eukaryotic cells. On the basis of work in Yersinia, these researchers found that, in the presence of the SycE chaperone, the chaperone-binding site located near the amino terminus of the YopE cytotoxin (a GTPaseactivating protein for Rho-GTPases) gave this protein a competitive advantage for secretion and subsequent delivery into target cells. Perhaps the chaperone presents YopE to the secretion/delivery machinery to allow more rapid secretion [55]. This is consistent to in vivo-like conditions, in which the Yersinia type III effector YopH (tyrosine protein phosphatase) is delivered by a chaperonedependent mechanism into macrophages within a minute of infection [56]. Recently, a new role for chaperones in the regulation of TTSS virulence determinants has been established. SicA, a TTSS chaperone specific for SipB and SipC of Salmonella spp., acts together with the DNA-binding protein InvF (a member of the AraC-like family of transcriptional activators) to activate the expression of a subset of genes encoding type-III-secreted products that are essential for Salmonella pathogenicity [57••]. It is proposed that, as SicA co-purifies with InvF and is necessary for InvF-dependent promoter activity, it is likely that SicA functions as an InvF cofactor to establish an open promoter complex to enable transcription initiation [57••]. SicA-dependent transcriptional activation is probably contingent on the secretion of SipB and SipC. This view has been pursued in Yersinia pseudotuberculosis. The complex between the TTSS chaperone LcrH/SycD and one of its cognate partners, YopD, appears to play a very important role in establishing negative feedback regulation of TTSS genes encoding type-III-secreted substrates [58•]. Although YopD is an important negative regulatory element [59], LcrH also plays an active role by interacting with YscY, a member of the Yersinia secretion apparatus [58•]. Therefore, it is interesting to speculate that early secretion of YopD may relieve the transcription block and enable LcrH to activate gene expression analogous to its distant homologue, SicA.
As previously indicated, high concentrations of the intracellular regulatory factor, LcrQ, inhibits TTSS gene expression in Yersinia spp. [50]. Intriguingly, the SycH chaperone is required to overcome the LcrQ-dependent negative feedback by physically interacting with LcrQ to ensure its rapid secretion and probable inactivation of residual intracellular LcrQ [60•]. By analogy to LcrQ function, a controlled release of the anti-FliA sigma factor, FlgM, fine-tunes expression of genes essential for complete flagella assembly (see the review by Aldridge and Hughes). Interestingly, translation of the flgM transcript whose product is predominantly predestined for secretion requires the TTSS chaperone FlgN [61]. Therefore, even though the regulatory pathways involving LcrQ and FlgM are evolutionary unrelated, chaperone involvement at least confirms the additional regulatory role of a subset of these unique molecules. Whether the primary role of TTSS chaperones is to maintain their cognate partner in an unfolded, secretioncompetent state or as a regulator of gene expression is not known. It is worth noting that some TTSS substrates are secreted in the absence of their chaperones, whereas others simply do not require chaperone assistance. Considering the tight coupling between cell contact and virulence gene induction (or secretion), and that this secretion probably occurs in an ordered manner during infection, the regulatory phenotype of a subset of these chaperones may reside at the level of secretion control. This would ensure immediate, ordered substrate delivery into eukaryotic cells in response to physical contact. Thus, loss of regulatory control would ensue if ordered substrate delivery was not maintained.
Conclusions We note that several environmental cues, nucleiod-associated proteins, phosphorelay two-component systems, AraC-like transcriptional activators and global regulators cross-talk to modulate expression of TTSS genes. Realistically, however, the use of in vivo gene expression technologies, summarised by Hautefort and Hinton [62], is required to accurately determine the spatial and temporal control of these impressively complex regulatory cascades. It is worth noting, however, that recent developments indicate that cell contact, assembly switching and specific chaperone molecules play a unique role in regulation of TTSSs. We envisage that future work will address the specific receptor–ligand interactions, nondiffusible signals and modes of transmission that initiate induction of target cell contact. It is also necessary to determine the role, if any, of specific chaperones in type III gene regulation and effector molecule secretion by plant pathogens. Finally, details of the pattern of TTSS assembly are now emerging. As type III structures spanning the bacterial envelope are similar among the bacteria studied, the assembly mechanism probably involves common sec-dependent and sec-independent mechanisms. Therefore, targeting of the assembly pathway may provide a common antibacterial strategy for disease prevention.
Regulation of type III secretion systems Francis, Wolf-Watz and Forsberg
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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2.
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Darwin KH, Miller VL: InvF is required for expression of genes encoding proteins secreted by the SPI-1 type III secretion apparatus in Salmonella typhimurium. J Bacteriol 1999, 181:4949-4954. Eichelberg K, Galán JE: Differential regulation of Salmonella typhimurium type III secreted proteins by pathogenicity island 1 (SPI-1)-encoded transcriptional activators InvF and HilA. Infect Immun 1999, 67:4099-4105. Schechter LM, Damrauer SM, Lee CA: Two AraC/XylS family members can independently counteract the effect of repressing sequences upstream of the hilA promoter. Mol Microbiol 1999, 32:629-642.
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Genin S, Gough CL, Zischek C, Boucher CA: Evidence that the hrpB gene encodes a positive regulator of pathogenicity genes from Pseudomonas solanacearum. Mol Microbiol 1992, 6:3065-3076.
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Wengelnik K, Bonas U: HrpXv, an AraC-type regulator, activates expression of five of the six loci in the hrp cluster of Xanthomonas campestris pv. vesicatoria. J Bacteriol 1996, 178:3462-3469.
10. Xiao Y, Heu S, Yi J, Lu Y, Hutcheson SW: Identification of a putative alternate sigma factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonas syringae Pv. syringae Pss61 hrp and hrmA genes. J Bacteriol 1994, 176:1025-1036. 11. Wei ZM, Beer SV: HrpL activates Erwinia amylovora hrp gene transcription and is a member of the ECF subfamily of sigma factors. J Bacteriol 1995, 177:6201-6210. 12. Brito B, Aldon D, Barberis P, Boucher C, Genin S: A signal transfer system through three compartments transduces the plant cell contact-dependent signal controlling Ralstonia solanacearum hrp genes. Mol Plant–Microbe Interact 2001, 15:109-119. 13. Hendrickson EL, Guevera P, Ausubel FM: The alternative sigma factor RpoN is required for hrp activity in Pseudomonas syringae pv. maculicola and acts at the level of hrpL transcription. J Bacteriol 2000, 182:3508-3516. 14. Wei Z, Kim JF, Beer SV: Regulation of hrp genes and type III protein secretion in Erwinia amylovora by HrpX/HrpY, a novel two-component system, and HrpS. Mol Plant–Microbe Interact 2000, 13:1251-1262. 15. Hutcheson SW, Bretz J, Sussan T, Jin S, Pak K: Enhancer-binding proteins HrpR and HrpS interact to regulate hrp-encoded type III protein secretion in Pseudomonas syringae strains. J Bacteriol 2001, 183:5589-5598. 16. Maurelli AT, Blackmon B, Curtiss R III: Temperature-dependent expression of virulence genes in Shigella species. Infect Immun 1984, 43:195-201. 17.
Gemski P, Lazere JR, Casey T: Plasmid associated with pathogenicity and calcium dependency of Yersinia enterocolitica. Infect Immun 1980, 27:682-685.
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18. Portnoy DA, Moseley SL, Falkow S: Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis. Infect Immun 1981, 31:775-782. 19. Adler B, Sasakawa C, Tobe T, Makino S, Komatsu K, Yoshikawa M: A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence-associated antigens of Shigella flexneri. Mol Microbiol 1989, 3:627-635. 20. Tobe T, Nagai S, Okada N, Adler B, Yoshikawa M, Sasakawa C: Temperature-regulated expression of invasion genes in Shigella flexneri is controlled through the transcriptional activation of the virB gene on the large plasmid. Mol Microbiol 1991, 5:887-893. 21. Maurelli AT, Sansonetti PJ: Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence. Proc Natl Acad Sci USA 1988, 85:2820-2824. 22. Falconi M, Colonna B, Prosseda G, Micheli G, Gualerzi CO: Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS. EMBO J 1998, 17:7033-7043. 23. Rohde JR, Luan XS, Rohde H, Fox JM, Minnich SA: The Yersinia enterocolitica pYV virulence plasmid contains multiple intrinsic DNA bends which melt at 37 degrees C. J Bacteriol 1999, 181:4198-4204. 24. Bustamante VH, Santana FJ, Calva E, Puente JL: Transcriptional regulation of type III secretion genes in enteropathogenic Escherichia coli: Ler antagonizes H-NS-dependent repression. Mol Microbiol 2001, 39:664-678. 25. Falconi M, Prosseda G, Giangrossi M, Beghetto E, Colonna B: Involvement of FIS in the H-NS-mediated regulation of virF gene of Shigella and enteroinvasive Escherichia coli. Mol Microbiol 2001, 42:439-452. 26. Garcia Véscovi E, Soncini FC, Groisman EA: Mg2++ as an extracellular signal: environmental regulation of Salmonella virulence. Cell 1996, 84:165-174. 27.
Pegues DA, Hantman MJ, Behlau I, Miller SI: PhoP/PhoQ transcriptional repression of Salmonella typhimurium invasion genes: evidence for a role in protein secretion. Mol Microbiol 1995, 17:169-181.
28. Groisman E: The pleiotropic two-component regulatory system PhoP-PhoQ. J Bacteriol 2000, 182:3508-3516. 29. Groisman E: The inns and out of virulence gene expression: Mg2+ as a regulatory signal. Bioessays 1998, 20:96-101. 30. Deiwick J, Nikolaus T, Erdogan S, Hensel M: Environmental regulation of Salmonella pathogenicity island 2 gene expression. Mol Microbiol 1999, 31:1759-1773. 31. Beuzon CR, Unsworth KE, Holden DW: In vivo genetic analysis indicates that PhoP-PhoQ and the Salmonella pathogenicity island 2 type III secretion system contribute independently to Salmonella enterica serovar Typhimurium virulence. Infect Immun 2001, 69:7254-7272. 32. Bajah V, Lucas RL, Hwang C, Lee CA: Co-ordinate regulation of Salmonella typhimurium invasion genes by environmental and regulatory factors mediated by control of hilA expression. Mol Microbiol 1996, 22:703-714. 33. Mellies JL, Elliott SJ, Sperandio V, Donnenberg MS, Kaper JB: The Per regulon of enteropathogenic Escherichia coli: identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler). Mol Microbiol 1999, 33:296-306. 34. Castanie-Cornet MP, Penfound TA, Smith D, Elliott JF, Foster JW: Control of acid resistance in Escherichia coli. J Bacteriol 1999, 181:3525-3535. 35. Shin S, Castanie-Cornet MP, Foster JW, Crawford JA, Brinkley C, •• Kaper JB: An activator of glutamate decarboxylase genes regulates the expression of enteropathogenic Escherichia coli virulence genes through control of the plasmid-encoded regulator, Per. Mol Microbiol 2001, 41:1133-1150. This detailed study describes the cross-talk between two independent virulence mechanisms required by EPEC to eventually colonise the small intestine. The GadX-encoding gene coordinates appropriate temporal expression of acid tolerance genes (during passage in the stomach) and type III secretion genes (within the small intestine) by recognising the environmental cues of pH and growth media composition.
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Cell regulation
36. Schauder S, Shokat K, Surette MG, Bassler BL: The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule. Mol Microbiol 2001, 41:463-476. 37.
Sperandio V, Mellies JL, Nguyen W, Shin S, Kaper JB: Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli. Proc Natl Acad Sci USA 1999, 96:15196-15201.
38. Day WA Jr, Maurelli AT: Shigella flexneri LuxS quorum-sensing system modulates virB expression but is not essential for virulence. Infect Immun 2001, 69:15-23. 39. Aldon D, Brito B, Boucher C, Genin S: A bacterial sensor of plant •• cell contact controls the transcriptional induction of Ralstonia solanacearum pathogenicity genes. EMBO J 2000, 19:2304-2314. Expression of TTSS genes in R. solanacearum is induced when bacteria are grown in the presence of plant cells. This work establishes that an outer membrane protein, PrhA, encodes a receptor that interacts with a nondiffusible plant cell component. This ultimately results in transcriptional activation of TTSS-encoding operons. 40. Brito B, Marenda M, Barberis P, Boucher C, Genin S: prhJ and hrpG, two new components of the plant signal-dependent regulatory cascade controlled by PrhA in Ralstonia solanacearum. Mol Microbiol 1999, 31:237-251. 41. Stiefel A, Mahren S, Ochs M, Schindler PT, Enz S, Braun V: Control of the ferric citrate transport system of Escherichia coli: mutations in region 2.1 of the FecI extracytoplasmic-function sigma factor suppress mutations in the FecR transmembrane regulatory protein. J Bacteriol 2001, 183:162-170. 42. Enz S, Mahren S, Stroeher UH, Braun V: Surface signalling in ferric citrate transport gene induction: Interaction of the FecA, FecR, and FecI regulatory proteins. J Bacteriol 2001, 182:637-646. 43. Jin Q, Hu W, Brown I, McGhee G, Hart P, Jones AL, He SY: Visualisation of secreted Hrp and Avr proteins along the Hrp pilus during type III secretion in Erwinia amylovora and Pseudomonas syringae. Mol Microbiol 2001, 40:1129-1139. 44. Jin Q, He SY: Role of the Hrp pilus in type III protein secretion in •• Pseudomonas syringae. Science 2001, 294:2556-2558. In this report, newly secreted effector proteins were visualised by in situ immunogold labelling and shown to associate with the distal part of Hrp pilus of Erwinia amylovora and Pseudomonas syringae. This strongly suggests that the secreted effector proteins are channeled through the Hrp pilus. 45. Van Gijsegem F, Vasse J, Camus J-C, Marenda M, Boucher C: Ralstonia solanacearum produces Hrp-dependent pili that are required for PopA secretion but not for attachment to plant cells. Mol Microbiol 2000, 36:249-260. 46. Sekiya K, Ohishi, M, Ogino T, Tamano K, Sasakawa C, Abe A: •• Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and its direct interaction with the EspA-sheath-like structure. Proc Natl Acad Sci USA 2001, 98:11638-11643. In this study, the needle complex of EPEC was isolated and visualised by electron microscopy. The authors of this paper observed that the needle extensions in EPEC are at least 10 times longer than those in other pathogens, such as Salmonella and Shigella. Moreover, the needle base is composed of the EscF ‘needle homologue’ protein and the long extensions are formed by EspA. The latter is a known requirement for the delivery of EspB and EspD into host cells. 47.
Wilson RK, Shaw RK, Daniell S, Knutton S, Frankel G: Role of EscF, a putative needle complex protein, in the type III protein translocation system of enteropathogenic Escherichia coli. Cell Microbiol 2001, 3:753-762.
48. Sukhan A, Kubori T, Wilson J, Galán J: Genetic analysis of assembly • of the Salmonella enterica serovar Typhimurium secretionassociated needle complex. J Bacteriol 2001, 183:1159-1167. This is the first genetic study to address the assembly process of a type III secretion needle complex. Evidence is provided to show that the basal portion and the export system assemble together in a sec-dependent process, whereas the needle appendage is assembled in a sec-independent but type-IIIexport-dependent process only after the completion of the basal portion.
49. Kubori T, Sukhan A, Aizawa, S-I, Galán J: Molecular characterization •• and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system. Proc Natl Acad Sci USA 2000, 97:10225-10230. In this elegant work, the needle component of the type III secretion organelle was first identified and found to be essential for a functional TTSS. In addition, the needle length was shown to be controlled by InvJ. 50. Pettersson J, Nordfelth R, Dubinina E, Bergman T, Gustafsson M, Magnusson K-E, Wolf-Watz H: Modulation of virulence factor expression by pathogen target cell contact. Science 1996, 273:1231-1233. 51. Vallis AJ, Yahr T L, Barbieri JT, Frank DW: Regulation of ExoS production and secretion by Pseudomonas aeruginosa in response to tissue culture conditions. Infect Immun 1999, 67:914-920. 52. Demers B, Sansonetti PJ, Parsot C: Induction of type III secretion in Shigella flexneri is associated with differential control of transcription of genes encoding secreted proteins. EMBO J 1998, 17:2894-2903. 53. Wattiau P, Woestyn S, Cornelis GR: Customized secretion chaperones in pathogenic bacteria. Mol Microbiol 1996, 20:255-262. 54. Boyd AP, Lambermont I, Cornelis GR: Competition between the Yops of Yersinia enterocolitica for delivery into eukaryotic cells: role of the SycE chaperone binding domain of YopE. J Bacteriol 2000, 182:4811-4821. 55. Lloyd SA, Norman M, Rosqvist R, Wolf-Watz H: Yersinia YopE is targeted for type III secretion by N-terminal, not mRNA, signals. Mol Microbiol 2001, 39:520-531. 56. Andersson K, Magnusson KE, Majeed M, Stendahl O, Fallman M: Yersinia pseudotuberculosis-induced calcium signaling in neutrophils is blocked by the virulence effector YopH. Infect Immun 1999, 67:2567-2574. 57. ••
Darwin KH, Miller VL: Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium. EMBO J 2001, 20:1850-1862. This elegant study described the regulatory function of the TTSS chaperone SicA in the absence of its cognate partners, SipB and SipC. The authors showed that SicA acts as a cofactor of the AraC-like regulator LcrF. SicA did not interact with DNA directly or alter stability of InvF. However, co-purification of SicA with InvF suggested that they interact to activate transcription from TTSS promoters. 58. Francis MS, Lloyd SA, Wolf-Watz H: The type III secretion • chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis. Mol Microbiol 2001, 42:1075-1094. The authors of this paper indicated that the LcrH chaperone acts in complex with its cognate partner YopD as a repressor of protein synthesis. In addition, they were the first to report that chaperone binding to a member of the type III apparatus occurred after secretion of the cognate partner. They suggest that this complex de-represses TTSS gene expression. 59. Williams AW, Straley SC: YopD of Yersinia pestis plays a role in negative regulation of the low-calcium response in addition to its role in translocation of Yops. J Bacteriol 1998, 180:350-358. 60. Cambronne ED, Cheng LW, Schneewind O: LcrQ/YscM1, regulators • of the Yersinia yop virulon, are injected into host cells by a chaperone-dependent mechanism. Mol Microbiol 2000, 37:263-273. Secretion of the negative regulatory element LcrQ by Yersinia is essential for the cell-contact-dependent induction of virulence genes. This study clearly showed that the cognate chaperone of the YopH effector, SycH, also interacts with LcrQ and is required for its secretion. This important finding highlights a unique regulatory role for SycH and establishes a possible mechanism for negative feedback regulation in Yersinia. 61. Karlinsey JE, Lonner J, Brown KL, Hughes KT: Translation/secretion coupling by type III secretion systems. Cell 2000, 102:487-497. 62. Hautefort I, Hinton JC: Measurement of bacterial gene expression in vivo. Philos Trans R Soc Lond B Biol Sci 2000, 355:601-611.