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Keywords:

  • Type-III secretion;
  • Chaperones;
  • Evolution;
  • Bacterial virulence;
  • Symbiosis;
  • Bioinformatics;
  • Genomics;
  • Homology

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2The post-genomic landscape
  5. 3Conservation and variation among type-III secretion systems
  6. 4Towards a taxonomy of type-III secretion
  7. 5Conclusions: from deep past to future perspectives
  8. Acknowledgements
  9. References

We review the biology of non-flagellar type-III secretion systems from a Darwinian perspective, highlighting the themes of evolution, conservation, variation and decay. The presence of these systems in environmental organisms such as Myxococcus, Desulfovibrio and Verrucomicrobium hints at roles beyond virulence. We review newly discovered sequence homologies (e.g., YopN/TyeA and SepL). We discuss synapomorphies that might be useful in formulating a taxonomy of type-III secretion. The problem of information overload is likely to be ameliorated by launch of a web site devoted to the comparative biology of type-III secretion (http://3base.bham.ac.uk).


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2The post-genomic landscape
  5. 3Conservation and variation among type-III secretion systems
  6. 4Towards a taxonomy of type-III secretion
  7. 5Conclusions: from deep past to future perspectives
  8. Acknowledgements
  9. References

Nothing in biology makes sense except in the light of evolution ”. Theodosius Dobzhansky [1]

Type-III secretion is one of at least five different types of protein secretion employed by Gram-negative bacteria to transport proteins from the cytoplasm to the external milieu [2,3]. Type-III secretion systems (T3SSs) can be classified into two major groups: those associated with flagellar biosynthesis and those that mediate interactions between bacteria and eukaryotic cells. In this review, for reasons of space, we will focus on the latter (but will draw parallels with the flagellar systems when necessary). Similarly, we will tend to draw on examples from animal-associated systems, rather than plant-associated (for more on protein secretion in phytopathogens see review in this issue by Preston et al. [4]).

Unfortunately, there is no commonly agreed designation for the class of type-III secretion systems that mediate interactions between bacteria and eukaryotic cells. Although they are sometimes termed ‘virulence-associated T3SSs’, this term can no longer be justified, given the recognition of broader roles, for example in symbiosis (see below). One defining characteristic of these systems might be that they possess a translocation apparatus in addition to the secretion apparatus – thus, they could be described as ‘translocation-proficient type-III secretion systems’. However, as there is some, albeit preliminary, evidence that some flagellar systems might also mediate translocation of proteins into eukaryotic cells [5], we prefer the more neutral term ‘non-flagellar type-III secretion system’ (or NF-T3SS), to distinguish these systems from flagellar systems, and we shall adopt this nomenclature in this review.

A type-III secretion system is an exquisitely engineered molecular pump, harnessing hydrolysis of ATP to the export of proteins from the bacterial cytoplasm across both the inner and outer membranes and the periplasm so that proteins are delivered to the exterior without a periplasmic intermediate (see recent reviews: [2,6–8]). An additional key feature of the non-flagellar systems, shared with type-IV secretion, is their ability to deliver so-called “effector” proteins into the cytoplasm of a target eukaryotic cell through a translocation apparatus – a molecular syringe – that connects the bacterial and eukaryotic cells and facilitates passage of proteins through the target cell plasma membrane [9]. Type-III secretion provides a paradigm of how hierarchical gene regulation, complex protein–protein interactions and controlled protein secretion can result in the assembly of a complex multi-protein structure tightly orchestrated in time and space. Type-III secretion is also central to our understanding of bacterial virulence, symbiosis, and ecology, provides an attractive drug and vaccine target [10] and has been exploited in the biotechnology arena as a antigen delivery system [11,12].

Among animal and human pathogens, interest has focussed on the ‘big five’– a small group of intensively studied T3SSs, which comprises the Ysc-Yop system from Yersinia[13–15], the Mxi-Spa system from Shigella[16–19], the system encoded by the locus for enterocyte effacement (LEE) in attaching and effacing strains of Escherichia coli and Citrobacter rodentium[20–23], and two T3SSs in Salmonella enterica[24–26], encoded by the two Salmonella pathogenicity islands SPI1 and SPI2. Studies on these systems and the gene clusters encoding them have clarified many of the mechanistic details of type-III secretion and identified numerous secreted ’effector’ proteins. In one recent study, every gene in the LEE from C. rodentium was subjected to deletion-mutation and the effect on secretion determined [27]. However, given the existence of several recent reviews on these systems [13–16,20–26], we will omit detailed discussion of their contents and effects. Instead, in this review, we will attempt to stand back from detailed consideration of individual systems and focus on broad themes that link all systems.

The last attempt to appraise the whole literature on type-III secretion was Hueck's heroic review in 1998 [2], which has been cited over 750 times in the last seven years. We cannot hope to match the breadth of that review, given the explosion of information just on the five canonical systems. Instead, we will focus on another theme that has emerged dramatically since Hueck's review: the explosion in genome-sequencing efforts, which has delivered a dramatic increase in the number of known type-III secretion systems and associated sequences [7]. Many of these newly discovered systems have yet to be linked to virulence, or even shown to be functional at all. However, their very diversity raises the issue of whether the most intensely studied systems are still representative of T3SSs in general. In addition, scrutiny of the newly discovered systems promises to deliver new insights into the older better-understood systems, particularly through careful bioinformatics analyses. For this reason, we will review evidence for interesting new assignments of homology, even if not dependent on genome sequencing.

We shall also attempt to interpret the post-genomic explosion of knowledge from a Darwinian perspective [28], following Dobzhansky's dictum, that “Nothing in biology makes sense except in the light of evolution[1]. In particular, we will explore and interpret type-III secretion in the light of three Darwinian axioms:

  • 1
    Evolution matters. We should expect to see homology (similarity as a result of common ancestry) between type-III secretion systems underpinning a natural taxonomy, based on lines of phylogenetic descent. We should also anticipate that the discovery of distant, otherwise overlooked homologies between proteins in different systems will inform our understanding of the structure, function and evolution of type-III secretion systems.
  • 2
    Variation matters. We should distrust the typological approach that implies that one type-III secretion system stands for all and that all proteins can be shoe-horned into a ‘periodic table’ of equivalences [29,30]; instead we should anticipate, and even relish, numerous small, but often significant differences between systems. In addition, using molecular phylogenetics as a guide, we should seek out shared derived characters (synapomorphies in the jargon of cladistics) that differentiate the various classes of non-flagellar T3SS.
  • 3
    Expect imperfection. On the view of descent with modification [28], we should expect to find examples of degenerate T3SSs and be aware that not everything glimpsed in genome sequences still has a function.

2The post-genomic landscape

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2The post-genomic landscape
  5. 3Conservation and variation among type-III secretion systems
  6. 4Towards a taxonomy of type-III secretion
  7. 5Conclusions: from deep past to future perspectives
  8. Acknowledgements
  9. References

2.1Beyond virulence

Non-flagellar type-III secretion (NF-T3SS) first came to the attention of the scientific community through the crucial role it plays in the pathogenesis of many human, animal and plant diseases [2,3]. Since Hueck's review, many more NF-T3SSs have been discovered (often more than one per genome) in a highly diverse group of clinically and/or economically important animal and human pathogens (Table 1 and Fig. 1). Many, if not most, of these have been discovered through genome sequencing. Some have already been linked to virulence, for example: in the chlamydias [31–33], Pseudomonas aeruginosa[34–38], Burkholderia pseudomallei and Burkholderia cepacia[39–43], Edwardsiella tarda[44], the bordetellas [45,46], Aeromomas salmonicida[47–49], Vibrio parahaemolyticus[50], and Photorhabdus luminescens[51]. However, for several of them, any role in pathogenesis remains unproven or obscure: examples here include Chromobacterium violaceum[52].

Table 1.  Some organisms recently found to possess type-III secretion genes Reference list is illustrative rather than exhaustive
OrganismReferences
  1. Note the existence of type-III secretion genes in the Leishmania and Anopheles genomes could come from an as yet unknown endosymbiont or could simply represent contamination of the genomic shot gun libraries.

Aeromonas hydrophila[277]
Aeromonas salmonicida subsp. salmonicida[47]
Anopheles gambiae str. PEST (endosymbiont?)Unpublished: see text
Bordetella spp.[278]
Bradyrhizobium japonicum[279]
Burkholderia cepacia[280]
Burkholderia mallei[43]
Burkholderia multivorans[280]
Burkholderia pseudomallei[281]
Burkholderia thailandensis[43]
Chlamydia muridarum[117]
Chlamydia trachomatis[108]
Chlamydophila psittaci[282]
Chlamydophila pneumoniae[117,283]
Chromobacterium violaceum[52,109]
Citrobacter rodentium[284]
Desulfovibrio vulgaris subsp. vulgaris str. Hildenborough megaplasmid[97]
Erwinia chrysanthemi[285]
Escherichia coli (ETT2)[118–120]
Leishmania major (endosymbiont?)Unpublished: see legend & Table 2
Myxococcus xanthusUnpublished: see text
Pantoea stewartii subsp. stewartii[286]
Parachlamydia sp. UWE25[103]
Photorhabdus asymbiotica[287]
Photorhabdus luminescens[51]
Primary endosymbiont of Sitophilus zeamais[76]
Proteus mirabilisUnpublished: see text
Pseudomonas fluorescens[74]
Pseudomonas sp. KD[74]
Ralstonia solanacearum[61]
Sinorhizobium fredii[288]
Sodalis glossinidius[77]
Verrucomicrobium spinsoumUnpublished: see text
Vibrio harveyi[289]
Vibrio parahaemolyticus[50]
Xanthomonas axonopodis pv. glycines[290]
Xanthomonas oryzae pv. oryzae[291]
image

Figure 1. A schematic representation of the type-III secretion system encoded by the locus for enterocyte effeacement in E. coli and C. rodentium. Model based on the type III secretion model from KEGG [http://www.genome.jp/kegg/pathway/map/map03070.html], and additional published data and images [15,172,196,312,313]. IM, inner membrane; PG, peptidoglycan layer; OM, outer membrane; EM, eukaryotic membrane. Note that the EspA pilus is a characteristic of this system (and probably also of close relatives), but is not a general feature of non-flagellar type-III secretion systems.

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Type-III secretion also underlies the ability of most bacterial phytopathogens to multiply in the plant apoplast and to cause disease and/or evoke the hypersensitivity response. The best-studied system is in Pseudomonas syringae[53–56] (a species of huge economic importance with numerous pathovars which infect a huge number of crop plants, such as soya, peas, oats, tomatoes, tobacco, olive trees). Other examples include T3SSs in Erwinia amylovora[57–59] (causes fireblight in apple, pear trees etc), Erwinia carotovora[60] (causes soft rot in a variety of crops), Ralstonia solanacearum[61,62] (causes southern wilt in over 200 species) and several species of Xanthomonas[63,64].

One key point emerges from the discovery of so many new NF-T3SSs: the anthropocentric view that they are only of importance as causes of disease in humans and in animals and plants of agricultural importance can no longer be sustained. Evidence against a universal link between type-III secretion and virulence in plants and animals comes from several quarters.

Firstly, non-flagellar systems occur in a number of opportunistic pathogens that only occasionally infect humans (for example, P. aeruginosa, C. violaceum, Burkholderia spp, the parachlamydias: Refs in Table 1). Thus, even where there is a credible link between virulence in humans/animals and type-III secretion (e.g., in P. aeruginosa[65,66]), the chief target of the T3SS must lie elsewhere. In other words, retention of these systems relies on selective pressures in non-human, often obscure, environments. This applies also to those human pathogens that exist chiefly as commensals in animals, including most serovars of S. enterica– these T3SSs must provide some selective advantage in colonisation of and transmission between their usual hosts [67,68].

Secondly, it is clear that NF-T3SSs can facilitate symbiotic rather than pathogenic interactions between bacteria and eukaryotes. They occur in several agriculturally and ecologically important rhizobial symbionts of plants, including Rhizobium sp. NGR234, Mesorhizobium loti, Bradyrhizobium japonicum and Rhizobium fredii. Importantly, they have been shown to mediate protein secretion and influence symbiosis in the NGR234 and R. fredii strains [69–72]. In addition, a cryptic T3SS is found in the nonpathogenic saprophyte Pseudomonas fluorescens SBW25, a plant-growth promoting bacterium that colonizes both plant leaves and roots [73,74], with recent evidence suggesting that T3SS genes are widespread among biocontrol fluorescent pseudomonads [75].

Three proteobacterial endosymbionts of metazoa have now been shown to possess NF-T3SSs: P. luminescens, an endosymbiont of nematodes pathogenic to insects, which possesses a Ysc-Yop-like system [51]; Sodalis glossinidius, an endosymbiont of the tsetse fly Glossina spp., which uses a SPI1-like system for cell invasion; and the primary endosymbiont of the grain weevil Sitophilus zeamais, which also possesses a SPI1-like system [76,77]. The presence of T3SS-associated sequences in the unfinished Anopheles gambiae str. PEST genome sequence (Pallen, unpublished) suggests that type-III secretion might also play a role in bacterial-mosquito interactions (alternatively, these sequences might simply represent contamination of the mosquito shotgun library).

Thirdly, some pathogens containing T3SSs can mediate pathogenic effects in a wide range of hosts. For example, P. aeruginosa can infect humans [65], mice [78], plants [79], greater wax moth caterpillars [36,80], fruit flies [81], nematodes [82], and slime molds [83]. T3SS-related genes have been shown to contribute to this organism's virulence in several organisms (humans, mice, caterpillars [36,80,83,84]) and type-III secretion has been suggested as an example in which plants can be used to model human infection [85]. Indeed, it is tempting to speculate that T3SSs traditionally associated with plant-bacterium interactions might also play a role in human disease during the rare episodes of clinical infection with plant-associated bacteria, such as P. fluorescens and Pantoea spp [86–89].

In like vein, clearly homologous type-III effectors are common to animal and plant pathogens. For example, homologous effectors from the animal pathogen Yersinia (YopT) and from the plant pathogen P. syringae (AvrPphB) define a new family of cysteine proteases [90]. Similarly, the putative effector SrfC is found in the plant-associated bacteria P. syringae, Erwinia spp. and Mesorhizobium meliloti and in the animal-associated species S. enterica and Yersinia spp, while the effector HopPmaA from the plant pathogen P. syringae has a homologue, Z3920/ECs3487, in the human pathogen E. coli O157 (Pallen, unpublished observations). Furthermore, several studies have shown that the effectors of T3SS-related virulence mediate similar effects in yeast cells as those seen in animal or plant cells [90–95]. These effectors must therefore target aspects of physiology common to plants and animals and, by implication, probably all eukaryotes. In other words, these effectors, like the secretion systems, are general-purpose tools for subverting eukaryotic cell biology.

Finally, the recent discovery of type-III secretion genes in non-pathogenic organisms from soil or water environments is likely – assuming these genes prove to be functional – to sound the death-knell for any universal link between non-flagellar type-III secretion and disease in animals or higher plants (although perhaps disappointingly, the sequencing of environmentally derived DNA from the Sargasso sea revealed no new insights into type-III secretion [96]– merely one Burkholderia-like system; Pallen, unpublished). Examples of T3SS gene clusters from environmental bacteria include:

  • A plasmid-encoded NF-T3SS in Desulfovibrio vulgaris, an anerobic sulfate-reducing bacterium, widely prevalent in the environment and of economic importance because of its involvement in biocorrosion of ferrous metals [97]. There has been a single case report linking D. vulgaris to human disease [98,99].
  • Type-III-secretion genes in the unfinished genome sequence of Myxococcus xanthus, a myxobacterium with a complex lifecycle that displays gliding motility, forms multicellular fruiting bodies and usually lives in the soil (Pallen, unpublished; preliminary sequence data obtained from The Institute for Genomic Research website at http://www.tigr.org). M. xanthus has never been linked to human, animal or plant infection.
  • Type-III-secretion genes in the unfinished genome sequence of Verrucomicrobium spinosum, an obscure heterotrophic, Gram-negative, nonmotile bacterium with wart-like or extended appendages called prosthecae, found in eutrophic ponds and lakes (Pallen, unpublished; preliminary sequence data obtained from The Institute for Genomic Research website at http://www.tigr.org), [100]. Like Myxococcus, it has never been linked to infection.

Thus, although non-flagellar type-III secretion might now confer selective advantages on animal or plant pathogens by enhancing colonization or transmission, it is clear that this type of secretion predates and need not be associated with virulence in these ‘higher’ organisms. One is thus left asking what is the primary or primordial target of most NF-T3SSs? As Gophna et al. [101] have suggested, one obvious answer comes from the emerging evidence for near-ubiquitous interactions between bacteria and amoebae [102] and the presence of these systems in organisms that primarily reside in amoebae (e.g., the parachlamydias [103]). In conclusion, the NF-T3SSs have not evolved merely to create a nuisance for humanity and our agricultural companions: instead, they represent a leitmotif in the billion-year-old interaction between two branches of Darwin's “great tree of life”: the bacteria and the eukarya.

2.2Emerging diversity

Genome-sequencing efforts have revealed a modest increase in the phylogenetic diversity among the bacteria known to carry NF-T3SSs. These systems have now been found in species from all four branches of the proteobacteria (α, β, γ and δ: Table 1). In addition, they have been found in all genome-sequenced representatives of the order Chlamydiales (including Parachlamydia), and from phylogenetic analyses appear to be ancestral to that group [104]. This implies that T3SSs are likely to occur in other unsequenced species from the same taxon (Neochlamydia hartmannellae, Protochlamydia amoephila, Fritschea, Rhabdochlamydia porcellionis, Simkania negevensis, Waddlia chondrophila) and may mediate interactions between these species and eukaryotes (note a role for many of these as emerging human pathogens [105]). However, NF-T3SSs still appear to be restricted to the two bacterial phyla, the proteobacteria and the Chlamydia/Verrucomicrobium phylum. This is in marked contrast to the flagellar T3SSs, which have now been found in six different bacterial phyla (Proteobacteria, Spirochaetes, Aquificae, Thermotogae, Firmicutes, Planctomycetes). Only time will tell whether this limited phylogenetic distribution is a true reflection of the prevalence of non-flagellar type-III secretion in the biosphere, or whether it represents an artefact of sampling (proteobacteria are grossly over-represented among current bacterial genomes sequencing projects).

2.3Genomic context

A few NF-T3SSs are encoded on plasmids, including the well-characterized Yersinia and Shigella systems and those described more recently in A. salmonicida and D. vulgaris (Table 1); the majority, however, are encoded on the chromosome. In most cases, it appears that these chromosomal type-III-secretion gene clusters have been acquired through horizontal gene transfer, as “pathogenicity islands”[106,107]. The most obvious evidence for this is a difference between the G + C% content of the cluster and its resident genome. However, the mechanisms by which such clusters are mobilized between genomes remain obscure, as do the identities of the primordial donors of these islands.

A couple of examples stand against the dogma that all NF-T3SS gene clusters have originated outside their current host genome. In chlamydias, they have a G + C% content identical to the host genome: this has been taken as evidence of a chlamydial origin for the primordial NF-T3SS [104,108]. However, a similar concordance between base composition of NF-T3SS gene cluster and host genome has also been noted for the Cpi-1 island from C. violaceum[109] suggesting that it has been resident in the C. violaceum genome for a long time and may even have originated in this species or in a close relative.

Another interesting feature of the genetics of type-III secretion is the extent to which genes associated with the same system are located close together in the genome. Initially, it appeared from studies on the ‘big five’ that effector genes were restricted to the same gene clusters that encoded the secretion apparatus. However, from studies on several pathogens (P. syringae, S. enterica, C. rodentium, E. coli), it is clear that this need not be the case – instead, it appears that effector genes can be scattered around the bacterial chromosome, commonly occurring within prophage genomes [27,53,110–116]. This realization raises a number of interesting questions: how and where do new effectors evolve, how are new genes recruited into an organism's effector repertoire, where are they acquired from, how do these genes get into phage genomes, and do plasmid-encoded T3SSs such as the Mxi-Spa system from Shigella or the Ysc-Yop system from Yersinia also translocate effectors encoded on the bacterial chromosome?

In the same way, exceptions have now appeared to the rule that the entire secretion and translocation apparatus must be encoded in the same gene cluster. An obvious separation into multiple clusters occurs in the chlamydial genomes [108,117]. However, exceptions have appeared even among the proteobacterial systems. The Cpi-1 gene cluster in C. violaceum resembles the SPI1 island from S. enterica, with one notable exception: in C. violaceum the genes encoding homologues of the needle-complex genes prgHIJK and of the orgABC genes are found in a separate gene cluster located more than 200 kilobases upstream of Cpi-1 [109]. Similarly, in the SPI1-like ETT2 system of enteroaggregative E. coli strain 042, the genes encoding the translocation apparatus are in an entirely separate cluster (the eip cluster) from the secretion apparatus genes [118–120]. Interestingly, this eip cluster also encodes a homologue of intimin/invasin, a HilA-like regulator and a SicA-like chaperone. This is puzzling as the primary ETT2 gene cluster also encodes a HilA-like regulator and a SicA-like chaperone. This curious redundancy, together with phylogenetic data on the chaperones, suggests that the ETT2 and eip clusters were once part of a larger cluster that underwent fission, accompanied by duplication of chaperones and regulators, after its divergence from its common ancestor with SPI1. The functional significance, if any, of this duplication is unclear.

2.4Degenerate type-III secretion systems

On the view of descent with modification, we may conclude that the existence of organs in a rudimentary, imperfect and useless condition, or quite aborted, far from presenting a strange difficulty …might even have been anticipated and can be accounted for by the laws of inheritance ”. Charles Darwin, Origin of Species [28]

Analysis of the complete genome sequences of two strains of EHEC O157:H7 revealed genes potentially encoding a second cryptic SPI1-like T3SS, which was termed ETT2 (for E. coli type-III secretion system 2) [118,119]. Initial studies proceeded on the assumption that the system was functional and linked to virulence [121–123]. However, through bioinformatics and laboratory-based studies, we came to a number of surprising conclusions about the ETT2 genes.

We found that the ETT2 gene cluster is present in whole or part in the majority of E. coli strains, making it the most prevalent NF-T3SS gene cluster in E. coli, far surpassing the LEE in its distribution [120]. However, as ETT2-associated genes are distributed equally among pathogenic and commensal strains (they occur even in the originally commensal but now laboratory-adapted K-12 strains) we rejected the notion that ETT2-associated genes might be a marker of virulence, particularly as apparently complete ETT2 gene sets occur in several commensal strains from the ECOR collection. Instead, ETT2 genes appear to be a marker of phylogenetic origin, in that they are absent in the early-diverging B2 lineage of E. coli, but present in all other major lineages [120].

We also concluded that, in most cases, the ETT2 gene cluster has undergone mutational attrition, so that it can no longer encode a functioning type-III secretion system. In most strains, there are evident multi-gene deletions [120]. However, even in the O157 genomes in which it was first described, the ETT2 gene cluster contains numerous inactivating mutations which, by analogy with SPI1, must abrogate function [120]. Thus, in most strains, the ETT2 cluster represents a ‘rudimentary, atrophied or aborted organ’ of the sort predicted to occur by Darwin as a consequence of the Theory of Evolution [28]. Plausible exceptions, which might possess intact systems, include the enteroaggregative E. coli strain 042 and a handful of ECOR strains [120]– the hunt is on for a functioning ETT2 NF-T3SS among these strains. There are few clues as to what the function of this system might be, particularly as there are no homologues of any known effectors encoded within the ETT2 cluster. However, we have suggested that a gene at one end of the cluster might encode a candidate effector. More particularly, we have identified a glucoamylase-like domain in the associated protein, leading to speculation that, if it is indeed a translocated effector, it might interfere with glycogen metabolism within host cells [124].

The ETT2 cluster simultaneously provides a model of gene flux and mobility on the one hand and a model of genetic stasis and loss on the other. The shuffling of homologues of genes from three distinct Salmonella pathogenicity islands (SPI1, SPI2, Spi-3) into one cluster in E. coli represents “pathogenicity genes in motion”[120]. However, the single insertion of the cluster into an early lineage, followed by mutational attrition in most lines of descent, provides a model for how genes are lost from a genome once they no longer provide any selective advantage [125]. As the island decays away, frameshift mutations are followed by the arrival of insertion sequences, which may then provoke deletions through homologous recombination. With the ETT2 cluster, we see the whole spectrum of reductive evolution – from an apparently intact 27.5-kilobase cluster in E. coli 042 to just two residual gene fragments in Shigella flexneri[120].

A salient lesson from scrutiny of ETT2 is that not every T3SS gene cluster should be expected to encode a functioning secretion system. Indeed, we have recently found a second example of a degenerate T3SS gene cluster, albeit associated with a broken flagellar system rather than NF-T3SS [126]. We have found that around 20% of E. coli strains carry a ?40-gene cluster potentially encoding an entirely novel second flagellar system (Flag-2) within the species. However, in the only genome-sequenced strain bearing this cluster (E. coli 042), the system is inactivated by a frameshift in a crucial gene.

Evidence for the existence of additional degenerate T3SS gene clusters comes from Yersinia pestis, where there is a chromosomal SPI2-like system that lacks genes for the translocation apparatus and also shows strain-specific inactivation of other genes (the homologue of ssaJ is disrupted by a frame shift in the KIM strain but is intact in strain CO92) [127]. In addition, it is clear that T3SS effector genes encoded outside of structural gene clusters can also undergo loss of function: for example, the Salmonella T3SS effector genes sopD2 and sopE2 have become pseudogenes in S. enterica Typhi strain CT18 [128]. Whether genes from degenerating T3SSs are still transcribed remains an interesting question – one that could be addressed through microarray studies.

The ETT2 gene cluster provide a cautionary tale on the perils of automatically dismissing any degenerate T3SS gene clusters as utterly without phenotype. We have recently shown that two regulators, EtrA and EivF, encoded within the ETT2 gene cluster, exert a profound effect on gene transcription in the LEE. Mutational ablation of these regulators produces a marked increase in the secretion of proteins by the LEE-encoded system through an effect on LEE gene expression in the EHEC O157:H7 Sakai strain [129]. In this regard, we propose that a useful metaphor for the ETT2 cluster might be that of the grin of the Cheshire cat in Alice in Wonderland [130]. We speculate that this metaphor – that powerful regulatory effects might outlive structural decay through mutational attrition – might also apply to other decaying prophages and pathogenicity islands.

The role of ETT2 regulators in influencing the LEE observation adds an additional level of complexity to what is already a highly complex regulatory network governing expression of the LEE genes. Furthermore, from our surveys of the ETT2 gene clusters, it is clear that the ETT2 regulator repertoire varies from one attaching and effacing strain lineage to the next [120–122]. Thus, it seems likely that variations in ETT2 regulator repertoire might account for some of the known variation in LEE-mediated protein secretion among attaching and effacing strains [129,131,132].

2.5Multiple systems in the same cell

The co-existence of the ETT2 and LEE gene clusters within the genome of enterohaemorrhagic E. coli provides a telling example of the potential for regulatory cross-talk between two or more T3SSs in the same cell. Many NF-T3SSs co-exist with flagellar systems; one would anticipate careful co-ordinate regulation of deployment of such energy-hungry systems. E. coli again provides many examples of such a phenomenon, in that the BipA GTPase and IHF co-ordinately regulate expression of two T3SSs in the enteropathogenic pathovar – both regulators positively influence LEE gene expression, while repressing flagellar biosynthesis [133–135]. Evidence for similar co-ordinate regulation has been found in Salmonella, where: mutations in SPI2 genes affect transcription of SPI1 genes [136]; nucleoid-associated protein Fis positively influences expression of both flagellar and non-flagellar T3SS genes [137]; and global regulators such as BarA/SirA, RcsB-RcsC and RtsA/RtsB influence both types of system [138–141]. Salmonella also provides an example of co-dependence of two non-flagellar systems on common post-translational influences, as both the SPI1 and SPI2 systems depend on the periplasmic disulfide bond isomerase DsbA [142]. Furthermore, this pathogen also furnishes evidence of the interplay between flagellar and non-flagellar type-III secretion in virulence [143].

If more than one T3SS is active in a cell, this begs the question of how substrates are targeted to one system rather than another. Evidence from Yersinia suggests that substrates can indeed be secreted by more than one system [144]. A recent study on Salmonella identifies one plausible specificity mechanism – substrate selection by chaperones, although it remains to be seen how far this phenomenon can be generalized across all T3SSs [145].

In addition to the ETT2/LEE and SPI1/SPI2 pairings in enterohaemorrhagic E. coli and S. enterica, respectively, double or multiple NF-T3SSs have recently been described in several other species. Interestingly, the prevalence of these systems often varies within a single species. For example the genome-sequenced strain of V. parahaemolyticus possesses two separate NF-T3SSs [50]. One of the systems is encoded by genes within a pathogenicity island on the V. parahaemolyticus chromosome 2 and is limited to strains pathogenic to humans, while the other T3SS is encoded within chromosome 1 and appears to be common to all strains within the species [50]. Recent functional studies suggest that both systems are active, but have distinct functions – the ubiquitous T3SS is implicated in cytotoxicity, whereas the chromosome-2 system mediates enterotoxicity [146].

There is an even greater multiplicity of T3SSs in B. pseudomallei and related species, where two systems, TTS1 and TTS2, most closely resemble T3SSs from plant pathogens, whereas a third system, TTS3 or Bsa, is similar to those from animal pathogens (e.g., SPI1 from S. enterica) [41,43]. TTS2 and TTS3 occur in B. pseudomallei, B. mallei, and some B. cepacia strains, whereas TTS1 appears to be specific to B. pseudomallei strains that are pathogenic to humans (i.e., absent from environmental biotypes of B. pseudomallei, such as B. thailandensis[43]). Initial functional characterisations of Bsa/TTS3 in B. pseudomallei and B. mallei suggest that it plays an essential role in the virulence of both pathogens in animals [41,147,148]. However, a mutation in the TTS2 system of B. cepacia genomovar III was recently reported to be attenuating in a murine model of respiratory infection [39]– providing a key link between Hrp2 systems and virulence in animals (as opposed to plants). Interestingly, a recent report suggests that loss of an arabinose-assimilation operon contributed to the evolution of B. pseudomallei and B. mallei from B. thailandensis by de-repressing expression of the TTS3/Bsa locus – the TTS3/Bsa genes are apparently intact in the avirulent B. thailandensis but are repressed owing to presence of the ara genes [149]. This provides a useful cautionary tale, showing how acquisition or loss of genes apparently unrelated to Type III secretion might shape the evolution of virulence. It remains to be seen how much regulatory and functional cross-talk there is between the various Burkholderia T3SSs.

The situation within the genus Yersinia is also complex, in that while the well-characterized plasmid-encoded Ysc-Yop system is present in all three pathogenic species, the genome sequence of the Y. pestis chromosome revealed an as yet uncharacterised second degenerate SPI2-like T3SS. Furthermore, Biotype IB strains of Y. enterocolitica possess a SPI1-like chromosomally encoded T3SS, that has been termed Ysa (for Yersinia secretion apparatus) [150]. Cross-talk and functional overlaps between the Ysa and the Ysc systems have been demonstrated, and recent studies have illuminated the regulation of the Ysa system [144,151,152].

3Conservation and variation among type-III secretion systems

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2The post-genomic landscape
  5. 3Conservation and variation among type-III secretion systems
  6. 4Towards a taxonomy of type-III secretion
  7. 5Conclusions: from deep past to future perspectives
  8. Acknowledgements
  9. References

Nature is prodigal in variety, but niggard in innovation”

Let two forms have not a single character in common, yet if these extreme forms are connected together by a chain of intermediate groups, we may at once infer their community of descent, and we put them all into the same class ”. Charles Darwin, Origin of Species [28]

Type-III secretion systems consist of four different sorts of proteins. From the cytoplasm outwards, these are: transcriptional regulators [6], which often vary from one system to another; chaperones, which bind to secreted proteins [153]; the components of the secretion apparatus itself, [2]; and the components of an extracellular filamentous organelle (which can range in length from a short needle complex via the EspA and Hrp pili, to flagellar hooks and filaments [154–157]) (Fig. 1). Furthermore, in non-flagellar systems, there are several proteins that make up the transclocation apparatus, aimed at polarized secretion of effector proteins into the host cell cytoplasm [158,159]. And, then, finally, there are the effectors themselves, which can, in some systems, run to several dozen proteins; [16,53,55,160–164].

Type-III secretion systems represent an excellent case study in the potential and perils of sequence analysis. Attempting to shoehorn all T3SS proteins into a ‘periodic table’ of equivalences, at best ignores subtle variations in protein sequence, domain organization and structure, and at worst can lead to erroneous conclusions. At the other extreme, many well-founded sequence (and presumably structural and functional) similarities between components of different systems have been overlooked by experimentalists in the field. As sequence-based observations and hypotheses can help interpret or suggest laboratory-based experiments, there is a pressing need for careful and comprehensive analyses of T3SS sequences, using methods that provide statistical assessments of significance and leave a clear evidence trail (such as PSI-BLAST [165]). A selection of problems drawn from our own experiences will illustrate this point.

3.1The secretion apparatus

The secretion apparatus contains the most conserved components of type-III secretion, that even span the divide between the flagellar and non-flagellar systems [2,30,166]. Unequivocal homologues between core proteins common to all NF-T3SSs suggest that observations on these proteins in any one system can be generalised, provisionally at least, to the any other system and can be used to frame novel hypotheses about their function. However, it is important to remain on guard for minor but, perhaps, important differences between systems. For example, YscC secretins are conserved among all NF-T3SSs. However, the C-terminal ?100-residue domain from YscC, which is thought to house two DsbA-dependent disulfide bridges [167], is missing from EscC and many other homologues suggesting subtle, but potentially important differences in function [168].

Some distant homologies between secretion system components, and even evidence of internal domain structure, have been overlooked, despite statistically valid evidence from methods like PSI-BLAST. One example is the homology between the SPI-1-encoded protein PrgH [169] and the YscD group of proteins (membrane-associated proteins that form the base of the needle complex; these include YscD from Yersinia and HrpQ from E. amylovora). Furthermore, bioinformatic analyses reveal an intriguing domain structure in these proteins: an FHA (forkhead-associated) domain, a trans-membrane domain, and then a series of BON domains [168,170,171]. FHA domains usually bind to phospho-serine or phospho-threonine peptides. Their presence in the YscD-like proteins points to a link between regulation of type-III secretion and reversible Ser/Thr phosphorylation. This connection is strengthened by close proximity of a gene for a chlamydial FHA-domain protein (CT664 from C. trachomatis and its orthologues), other T3SS genes, and a gene encoding a serine-threonine protein kinase (CT673) [170]. Whether the FHA-like domain in non-chlamydial YscD-like proteins also has this function or represents a molecular fossil is unclear. Even if not involved in reversible binding to pSer/or pThr residues, the cytoplasmic FHA-like domains in the YscD group of proteins might still be involved in signal transduction.

BON domains are though to mediate binding to phospholipids in a variety of other proteins [171]. It thus seems likely that the periplasmic BON domains in the YscD-like proteins play a similar role in the needle complex. Given the recent high-resolution structures of the SPI-1 secretion apparatus [172], it should soon be possible to map these domains onto this structure.

In some cases, apparently missing members of a secretion apparatus can be identified through careful homology searches. For example, in a recent bioinformatics survey of the LEE-encoded system, we noted that in the current annotation of the LEE, there are no equivalents of two proteins from the Ysc-Yop system: YscQ (a member of the FliN family of proteins and a component of the basal secretion complex [173]) and YscL (a member of the FliH family of proteins that bind to and regulates the activity of the T3SS ATPase [173,174]). However, a search of the NCBI's CDD database identified a FliN/SpoA domain in the C-terminal half of SepQ, suggesting that it should be re-named EscQ [168]. Furthermore, scrutiny of the recently solved structure of the homologous domain from HrcQB confirms that SepQ possesses most of the conserved motifs common to this domain family [168,175]. Similarly, PSI-BLAST searches confirmed that the protein encoded by Orf 5 of the LEE belongs to the FliH family and should probably be re-named as EscL [168].

The problem of establishing homology between different components of the secretion apparatus gets worse the shorter the sequence of the protein in question. For example, despite its name, EscF from the LEE-encoded system (only 73 residues long) does not show any significant similarity to YscF on a simple BLASTP search (19% identity; e value of only 0.88). However, more sophisticated approaches, such as PSI-BLAST, confirm the homology. It is also clear from published experimental work that YscF and EscF play equivalent roles as needle components [176–178].

In the SPI1 and Mxi-Spa systems, two small proteins are associated with the needle complex – PrgI/PrgJ and MxiH/MxiI, respectively [169,179–181]. There is experimental evidence from both systems to suggest that one of the proteins (PrgI/MxiH) is the major subunit component of the needle. Recently the function of the other protein (PrgJ/MxiI) has been elucidated – it forms the inner rod within the base of the needle [172]. A PrgJ-like component has yet to be described in either the Ysc-Yop or the LEE-encoded systems. However, PSI-BLAST searches indicate that rORF8 (as designated in the original desciption of the LEE sequence [182]) is homologous to EprJ, PrgJ, MxiI and to an uncharacterised protein from the Ysc-Yop system, YscI [168]. This suggests that rORF8 and YscI play similar roles to PrgJ/MxiI as components of the inner rod and that rORF should be re-named EscI. Interestingly, PSI-BLAST searches also suggest that the PrgI-like and PrgJ-like proteins are paralogous [168].

There are structural similarities between the flagellar hook and the NF-T3SS needle that hint at homology, despite the absence of significant sequence similarity between hook-associated proteins and needle components [183]. This raises an interesting question: do these two structures employ similar mechanisms for controlling their length? In the S. enterica flagellum, FliK acts as a bifunctional protein involved in determining hook length as well as in modulating export pathway specificity at the hook-filament checkpoint. FliK homologues can be identified in a wide range of phylogenetically diverse flagellar systems [184]. Needle-length-determining substrate-specificity-switching proteins have been described in three non-flagellar type-III secretion systems: InvJ in the SPI1 system, Spa-32 in the Mxi-Spa system and YscP in the Ysc-Yop system [180,185–189]. However, any attempt at a grand unified theory of hook/needle length-determination is thwarted by several awkward spanners-in-the-works.

The first of these is the mechanism of action. Interestingly, YscP has recently been shown to operate by a molecular-ruler mechanism [187]. A key feature of such mechanisms is that the size of the overall structure is compared with and governed by the size of an individual molecule [190]. We have shown through PSI-BLAST searches that there is sequence homology between the C-termini of FliK and YscP [184] (Fig. 2). On this basis, it seems reasonable to suggest that a YscP-like molecular ruler mechanism probably also modulates hook length in flagellar systems. However, just such a mechanism of hook-length control has – apparently – been ruled out for FliK, on the basis of experimental investigations in Aizawa's laboratory. Instead, Aizawa proposes a model in which hook length is controlled by the amount of subunit protein secreted by the flagellar export apparatus [191]. Only time – and additional experiments – will tell whether one or other model is incorrect, whether both contain elements of the truth or whether the region of the protein showing homology mediates another function (e.g., substrate switching) [189].

image

Figure 2. Multiple sequence alignment of YscP and homologs. Alignment is presented using CHROMA [314] and default settings: Consensus abbreviations (amino acids): a, aromatic (FHWY, blue lettering on a dark yellow background); b, big (EFHIKLMQRWY, blue on light yellow); h, hydrophobic (ACFGHILMTVWY, black on dark yellow); l, aliphatic (ILV, grey on dark yellow); p, polar (CDEHKNQRST, blue on white); s, small (ACDGNPSTV, dark green on white); t, tiny (AGS, light green on white); -, negatively-charged (DE, red on white); and, +, positively-charged (KR, blue on white). Organism names are abbreviated as follows: Bs, Bacillus subtilis; Cj, Campylobacter jejuni; Cv, Chromobacterium violaceum; Ec, Escherichia coli K12; Pa, Pseudomonas aeruginosa PAO1; Pl, Photorhabdus luminescens; Tm, Thermotoga maritima; Tp, Treponema pallidum; Vp, Vibrio parahaemolyticus; Yp, Yersinia pestis (reproduced from Ref. [168], q. v. for accession numbers, procedural details etc.).

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Another problem besets the ‘unified theory’. Although InvJ and Spa-32 show sequence homology with one another (albeit weakly) and can substitute for one another in vivo [186], despite exhaustive efforts, we have never been able to demonstrate any statistically significant sequence homology between InvJ/Spa-32 on the one hand and YscP/FliK on the other. Nor can we find any similarity between any of these proteins and any proteins from the Esc/Ssa, Hrp1 or Hrp2 families of NF-T3SSs. Despite the lack of sequence similarity or of experimental data, some authorities have already asserted that InvJ and FliK are homologues [29,30]. It is feasible that these proteins have diverged so greatly as to lose any detectable sequence similarity. However, in the absence of structural or experimental data, it would appear to be premature to make an assertion of homology on the current evidence, particularly as tentative assignments tend to harden into dogma and then preclude consideration of other possibilities (i.e., that these systems employ different mechanisms for length control). Clearly, additional experimental data is needed to resolve this issue.

3.2The translocation apparatus

Pallen et al. [192] was the first to report the presence of coiled-coil domains in T3SS proteins and, particularly, in the translocation apparatus proteins from animal pathogens, priming a successful programme of experimental work on the coiled-coil domains of EspA and EspD from the LEE-encoded system of enteropathogenic E. coli[154,159,193]. The translocation apparatus in this T3SS is constructed from EspA, EspB and EspD. Attempts to generalise observations on EspB and EspD through homology searches meet with mixed success. PSI-BLAST searches with EspB prove unhelpful, as the compositional bias of the protein, particularly in its coiled-coil domains, attracts numerous simple-sequence proteins, even when a filter and composition-based statistics are used [192,194]. However, a comparison of domain architecture and genetic location with translocation-pore proteins/genes from other systems (e.g., SipC, IpaB, YopD) suggests functional and structural similarities [192,194]. PSI-BLAST searches with EspD and related proteins prove more fruitful, especially when composition-based statistics are employed – they reveal significant similarities to TTSS translocon-pore proteins from all other proteobacterial NF-T3SSs (YopB and relatives in the Ysc group; IpaB and relatives in the Mxi-Spa group; HrpK in P. syringae, HrpF in Xanthomonas, NolX in rhizobia and PopF1/F2 in Ralstonia.) [168].

In 1998, Knutton and colleagues described the EspA pilus and noted a resemblance to the flagellar filament in both structure and sequence that suggested a common ancestry [154]. Flagellin was predicted to contain N- and C-terminal coiled coils, and the prevailing assumption at that time was that these coiled-coil domains mediated inter-subunit interactions, so that adjacent flagellar monomers were slotted together head-to-tail in a ‘molecular daisy chain’, to polymerise into the flagellar filament [195]. EspA was also predicted to contain a coiled-coil domain at its C-terminus [154,192]. We hypothesised, by analogy with flagellin, that this coiled-coil domain might be required for normal polymerisation of EspA into the EspA pilus. This hypothesis was confirmed in a series of mutagenesis experiments, in which we showed that removal of the propensity for coiled-coil interactions from the C-terminal domain of EspA led to loss of the production of a normal or, in some mutants, even any, EspA pilus [159].

The assumption that the flagellum and EspA filament were homologous was strengthened by the determination of the 3D structure of EspA filaments by image processing of electron micrographs [196]. The EspA filament was found to comprise a helical tube with a diameter of 120 Å enclosing a central channel of 25 Å diameter through which effector proteins could be transported. Even though EspA is significantly smaller than flagellin, the flagellum and EspA filament were found to possess very similar helical symmetry parameters: EspA: ?5.6 subunits/turn, 26 Å the pitch of helix, 4.6 Å axial rise; flagellin: ?5.5 subunits/turn, 26 Å the pitch of helix, 4.7 Å axial rise.

Recently, two papers from Namba's laboratory describe an atomic-level-resolution structure of flagellin and of the flagellar filament [197,198]. Contrary to expectations, these structures reveal that coiled-coil interactions between N- and C-terminal domains do not mediate contacts between adjacent subunits. Instead, these domains engage in an intra-subunit coiled-coil interaction that enables the flagellin to fold into a monomer capable of subsequent polymerisation. Assembly of the filament then relies on complex interactions between residues exposed on the surface of the flagellin monomer.

These findings have prompted us to re-consider our model for EspA filament formation and to re-examine the sequence homology data. Our initial assumption that the C-terminal coiled-coil domain of EspA was equivalent to the extreme C-terminal coiled-coil region of flagellin (part of the D0 domain) now appears erroneous, as the homology data suggest that the C-terminal coiled coil of EspA is in fact homologous to the flagellar D1 domain (Fig. 3), and that EspA lacks the D0 and D3 domains of flagellin. In the flagellar filament, D0 forms the inner tube, while D1 forms the outer tube [197] (Fig. 3). Although D0 interactions are important in filament stability, mutant flagellins that lack the D0 domain can still assemble into straight filaments, albeit with a structure, termed Lt, distinct from the native flagellar filaments [199]. It is thus entirely plausible that the EspA pilus assembles without the need for a D0 domain and resembles the Lt filament structure. The lack of an equivalent of the D3 domain in EspA fits in with the observation that D3 is highly variable in size and in sequence among flagellins and is also not essential for flagellar filament formation in Salmonella.

image

Figure 3. Multiple sequence alignment of EspA and homologues. Alignment is presented using CHROMA [314] and default settings (Consensus abbreviations as for Fig. 2). Organism names are abbreviated as for Fig. 2 and: Ap, Aquifex pyrophilus; Bb, Bartonella bacilliformis; Bh, Bacillus halodurans; Bo, Bordetella bronchiseptica; EHEC, E. coli O157:H7; EPEC, E. coli E2348/69; Et, Edwardsiella tarda Hm, Helicobacter mustelae; Le, Legionella micdadei; Lm, Listeria moncytogenes; Pm, Proteus mirabilis; Rm, Rhizobium meliloti; Th, Treponema hydodysenteriae. 1UCU is the PDB code of Salmonella typhimurium FliC structure [198]. (reproduced from Ref. [168], q. v. for accession numbers, procedural details etc.).

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Recent structural information on EspA complexed with its chaperone CesA [200] indicates that EspA contains a short N-terminal coiled-coil domain in addition to the already recognized much longer C-terminal coiled-coil domain. It is thus possible that together these two coiled-coil domains mediate an intra-subunit interation similar to that seen in flagellin. However, this does not fit well wih the apparent discrepancy in length between the two domains. An alternative possibility is that EspA acts as ‘half-a-flagellin’, so that it dimerizes through an inter-subunit coiled-coil interaction, and then the dimer polymerizes in a fashion similar to flagellin. This model opens up the potential for construction of a filament from heterodimers (see below).

3.3Chaperones

Efficient type-III secretion depends on molecular chaperones, which bind specifically to the effectors or translocators within the bacterial cytoplasm [153,201]. Loss of a T3SS chaperone generally results in rapid degradation, aggregation or reduced secretion of its cognate secretion substrate(s). Taking a broader view of type-III chaperone biology, sequence and structural analyses have highlighted the twin themes of conversation and diversity.

Although sequence identity between T3SS chaperones is low or even absent, it has been claimed that they possess common features such as small size (100–150 residues), a C-terminal amphipathic helix and a tendency towards an acidic pI. This has led some to the view that all non-flagellar T3SS chaperones can be forced into a single structural and functional class and that one can safely generalise from one chaperone to any other [201–203]. This view has been reinforced by the clear structural homology between several NF-T3SS chaperones of known structure (SicP, SycE, SigE, CesT, SycH, Spa15) which all bind to T3SS effectors rather than to translocators [204–210].

Despite this assumption of conservation, most authorities, curiously, have overlooked the statistically significant sequence homology between the various chaperones of the effectors. We have found that the above chaperones of known structure can be connected at the sequence level by PSI-BLAST, even though it has been claimed that so-called Type 1a chaperones (which, it is thought, tend to chaperone a single effector) and Type 1b chaperones (which chaperone multiple effectors) cannot be linked by sequence analysis. Such PSI-BLAST searches also identify numerous other uncharacterised T3SS-associated proteins as CesT-like chaperones (Table 2) [168].

Table 2.  Selected chaperones of the effectors identified by PSI-BLAST searches with CesT
OrganismChaperone description/annotationTarget effector(s)ReferencesAccession No.
  1. The list is illustrative rather than exhaustive. Asterixes indicate potential target effectors identified by gene clustering awaiting experimental confirmation.

Aeromonas salmonicidaUnknownAexT* gb?http://AAK83051.1
Acr2 proteinAopN* emb?http://CAD30215.1
     
Bordetella pertussisHypothetical protein BP2265unknown ref?http://NP_880908.1
 Hypothetical protein BP2236unknown ref?http://NP_880880.1
 Hypothetical protein BP0499BP0500?* N-terminal similar to plu4750 plu0822 ref?http://NP_879351.1
 Hypothetical protein BP2258BopN* ref?http://NP_880901.1
     
Chlamydia trachomatisSecretion Chaperone CT088CT089 (YopN homologue)* ref?http://NP_219591.1
 Hypothetical protein CT663unknown ref?http://NP_220182.1
 Hypothetical protein CT043unknown ref?http://NP_219546.1
     
Chromobacterium violaceumHypothetical protein CV0975CV0974 (SptP homologue)* ref?http://NP_900645.1
     
Desulfovibrio vulgarisCesT-like chaperone DVU2392Unknown: chromosomal when T3SS is plasmid-encoded ref?http://YP_011605.1
 Hypothetical protein DVU2312  ref?http://YP_011525.1
     
Erwinia amylovoraPotential ORFB-specific chaperoneORFB, AvrA-like effector* gb?http://AAF63396.1
 Pathogenicity factor DspB/DspFDspA/DspE, AvrE-like effector[292,293]gb?http://AAC04851.1
     
Escherichia coliCesTTir, Map[207,294–296]pdb?1K3E?A
 CesFEspF[297]gb?http://AAC38388.1
     
Leishmania majorPossible hypothetical 85.8 Kd proteinUnknown emb?http://CAC37204.1
 Hypothetical protein L4830.13Unknown emb?http://CAC22615.1
     
Photorhabdus luminescensplu3777 SctB/LscBYopN homologue Plu3766* ref?http://NP_930983.1
 plu3789YopT homologue plu3788* gb?http://AAO18077.1
 plu3764 LssNYopN homologue Plu3766* ref?http://NP_930970.1
 plu4749Unknown ref?http://NP_931911.1
 plu0829Unknown ref?http://NP_928172.1
 plu3753 LscYUnknown ref?http://NP_930959.1
     
Pseudomonas aeruginosa PAO1PscB PA1715PopN* gb?http://AAC44773.1
 ExsC PA1710Unknown; acts as anti-anti-activator gb?AAC46214.1
 Pcr2 PA1700PopN* gb?http://AAC45941.1
 Probable chaperone PA3842ExoS* ref?http://NP_252531.1
     
Pseudomonas syringae pv. tomato DC3000Conserved effector locus protein PSPTO1369HopPtoN* ref?http://NP_791196.1
 Conserved effector locus protein PSPTO1374HopPtoM* ref?http://NP_791201.1
 Type III chaperone protein SchF PSPTO0503HopPtoF[298]ref?http://NP_790352.1
 Hypothetical protein PSPTOA0017HopPtoS1/ HopPtoO* ref?http://NP_808676.1
 Type III chaperone protein SchA(Pto) PSPTO5353HopPsyA(Pto)* ref?http://NP_795083.1
 Avirulence protein AvrF PSPTO1376AvrE*[292]ref?http://NP_791203.1
 Hypothetical protein PSPTO4589Candidate effector protein PSPTO4588* ref?http://NP_794340.1
 Hypothetical protein PSPTO4599HolPtoZ* ref?http://NP_794350.1
 Hypothetical protein PSPTO4721Unknown ref?http://NP_794464.1
     
Salmonella entericaSicP (of known structure)SptP[208,299]pdb?1JYO?A
 SigE (of known structure)SopB[207,300]pdb?1K3S?A
 InvBSopA, SopE, SopE2, SipA[301–304] 
     
Shigella flexneriIpgEIpgD[305]gb?http://AAP78997.1
Spa15 (of known structure)IpA, IpgB1, OspC3[209,306] 
     
Vibrio parahaemolyticusPutative type III secretion protein VP1665YopN homologue VP1667* ref?http://NP_798044.1
Hypothetical protein VP1682VP1680?* ref?http://NP_798060.1
Putative type III chaperone VP1687VP1686?* ref?http://NP_798066.1
Hypothetical protein VP1684VP1683?* ref?http://NP_798063.1
Putative type III export apparatus protein NosA VP1697YopN homologue VP1667* ref?http://NP_798076.1
     
Xanthomonas spp.HpaB proteinAvrBs1, AvrBs3[307]ref?http://NP_640751.1
     
Yersinia enterocoliticaOrf155Unknown (not YopO)[308]ref?http://NP_783720.1
SycNYopN[309]ref?http://NP_052398.1
SycHYopH, YscM1 and YscM2[210,213,310]ref?http://NP_863547.1
SycTYopT[311]ref?http://NP_783658.1
SycE (of known structure)YopE[204,310]pdb?1JYA?A
YscBYopN[309]ref?http://NP_052412.1
YsaKUnknown gb?http://AAB69191.1

The discovery of so many new chaperones raises a number of issues. For example, the identification of ExsC from P. aeruginosa as a member of this chaperone family is interesting, as this protein has recently been reported to act as an anti-anti-activator controlling transcription of T3SS genes in P. aeruginosa[168,211,212]. This link between a chaperone-of-the-effectors and regulation of gene expression mirrors the better-established links between SycH/YscM1 binding and effector expression in Yersinia spp. [213,214] and the links, in several species, between the chaperones-of-the-translocators and gene regulation [6,215–217]– it should thus prompt researchers working on any such chaperones in other systems to investigate their potential roles in gene regulation.

Another striking finding from homology searches is the abundance of these chaperones in many organisms, especially in plant-associated bacteria such as P. syringae, where they have been under-investigated (Table 2). Also, identification of novel effector-chaperones can also allow us, through guilt-by-association, to identify novel candidate effectors. For example, the hypothetical proteins BP0500 from B. pertussis and plu4750 from P. luminescens are both encoded by genes adjacent to chaperone genes and show homology to each other at the N-terminus, consistent with an effector role (Pallen, unpublished). The association with one or even two chaperones-of-the-effectors with YopN homologues in several species is additional evidence that the YopN-like proteins are a class of effectors (see below).

Recently, Pallen et al. [218] used sequence analysis to identify a structural feature – an array of three tandem tetratricopeptide repeats (TPRs) – within the chaperones-of-the-translocators (includes, e.g., CesD from the LEE-encoded T3SS, LcrH from the Ysc system) that sets them apart from the chaperones of known structure. Use of confirmed structures of eukaryotic tetratricopeptide repeats as structural templates led to a construction of a structural model for this class of chaperones. This TPR model implied that these chaperones form a peptide-binding groove out of an all-α-helical array that is utterly distinct from the helix-binding groove in the SycE-like chaperones of known structure [204–208,218]. The model was found to be consistent with a large body of existing mutagenesis data that had been generated for the chaperone from Y. pseudotuberculosis, LcrH [218] (Betts, Francis, Pallen, unpublished). Thus, although no experimental structure yet exists for the TPR class of chaperones, it is clear that they can no longer be lumped together with the CesT-like chaperones. Instead, it is now certain that non-flagellar type-III secretion chaperones fall into at least two structurally and functionally distinct classes.

Oddly, TPR chaperones appear to be absent from the Hrp1 and Hrp2 types of NF-T3SS, even though, as noted, these systems possess homologues of EspD (HrpK in P. syringae, HrpF in Xanthomonas, NolX in rhizobia and PopF1/F2 in Ralstonia[168]). This raises the question of which proteins, if any, act as chaperones-of-the-translocators in plant-associated NF-T3SSs.

Although, as noted, PSI-BLAST searches support the existence of two large classes of NF-T3SS chaperones, several T3SS chaperones have been described which fall into neither class. Indeed, once one moves beyond the two large classes, it becomes hard to sustain any unified view of type-III-secretion chaperone biology, whether taking the broad view – there appear to be no homologies between chaperones from flagellar systems and non-flagellar systems [219]– or a narrower view: for example, the LEE-encoded chaperone CesAB, which binds EspA [220], appears to have no homologues, even in other members of the Ssa-LEE class of T3SSs (Pallen, unpublished). Discovery of additional T3SSs and elucidation of more protein structures are likely to shed light on the evolution of proteins belonging to the two large classes and to those outside these groups. However, given the already known structural and functional diversity, one has to question whether lumping so many diverse proteins under a single heading of ‘chaperones’ is likely to limit rather than advance our understanding of type-III secretion.

3.4Effectors

Much of the earliest work on non-flagellar type-III secretion proceeded on the assumption that any given system was likely to translocate only a handful of effectors, which were likely to be encoded within the same gene cluster as the secretion system itself. As noted above, this complacent view has been shattered by several groups who, with brilliant success, have adopted a genomic mining approach to identify type-III effectors in the plant pathogen, P. syringae[53,55,161,163,221,222]. One inventory from this organism lists 37 experimentally confirmed secreted/effector proteins, 15 strongly predicted but unconfirmed candidates and many more, weaker, candidates that await testing [53]. This is in stark contrast to the much smaller number of type-III effectors that have been characterized in the big five animal/human-associated systems. However, even here, it is clear that the landscape is changing – with a handful of effectors now known to be encoded outside the secretion system gene clusters in S. enterica, C. rodentium and E. coli[27,110–116]. Our own analyses suggest that the current known T3SS repertoire in E. coli is only the tip of the iceberg – for example, we have found over a dozen homologues of just one effector gene, nleG, in the enterohaemorrhagic E. coli genome (Pallen, unpublished). Such profligate redundancy means that traditional mutagenesis approaches to identifying effector function, exploiting molecular Koch's postulates [223], are unlikely to reveal a phenotype. However, the recent development of an efficient β-lactamase fusion assay for identifying translocated proteins [224] should assist in high-throughput confirmation of candidate effectors, particularly if adapted for use in organisms other than E. coli.

Another important emerging research theme is that subtle strain-to-strain variations in effector repertoire and in the sequences of individual effectors are likely to profoundly alter pathogenic mechanisms and disease outcome. To provide just one example: in both EPEC O127 and EHEC O157, the translocated intimin receptor, Tir, is required for actin nucleation, but behaves in subtly different ways in the two strain lineages. Unlike in EPEC, in O157 actin nucleation requires a second type-III secreted molecule (EspFU/TccP) that interacts with Tir [225,226]. How such finely tuned differences evolve remains a mystery. However, it is clear that our view of effector repertoires is likely to change dramatically in coming years.

3.5Regulators

Gene regulation is probably the most variable aspect of type-III secretion and it is hard to draw out any generic conclusions from genomics or bioinformatics [6]. However, our own analyses have illustrated some interesting variations on common themes. For example, we have reported that the SPI-1 master regulator, HilA, contains nine TPR-like repeats occupying the protein C-terminal to the known regulatory domain [218]. As noted above, tandem TPRs tend to form all-α-helical arrays with peptide-binding grooves, strongly suggesting that the HilA TPRs mediate homologous or heterologous protein–protein interactions, and linking studies on HilA to other regulators with TPR-like repeats (e.g., MalT [227]). Database searches show that there are at least three other close relatives of HilA, all associated with other T3SSs (ETT2 from E. coli, CPI-1 from C. violaceum, and the SPI1-like T3SS from Sodalis glossina). However, there are subtle differences between them, in that the Salmonella HilA and its homologue from C. violaceum both contain 9 TPRs, while the homologous protein YgeH from ETT2 contains only 5 TPRs [218] and the S. glossina homologue possesses only one or two, if any.

Interestingly, TPRs also occur in transcriptional regulators of type-III secretion in some plant pathogens – three tandem TPRs occur at the N-terminus of HrpB from R. solanacearum[218]. However, the recruitment of TPR-like repeats into regulators appears to have occurred independently in plant and animal pathogens as the domain organisation of HilA (N-terminal trans_reg_c domain, C-terminal TPR-like repeats) differs dramatically from that of HrpB/HrpX (N-terminal TPR-like repeats, C-terminal HTH_AraC subdomains). These differences in the domain environment of TPRs in T3SS regulators illustrate the importance of meticulous sequence comparisons and provide an intriguing target for laboratory-based studies.

4Towards a taxonomy of type-III secretion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2The post-genomic landscape
  5. 3Conservation and variation among type-III secretion systems
  6. 4Towards a taxonomy of type-III secretion
  7. 5Conclusions: from deep past to future perspectives
  8. Acknowledgements
  9. References

The affinities of all the beings of the same class have sometimes been represented by a great tree. I believe this simile largely speaks the truth ”. Charles Darwin, Origin of Species [28]

In 2002, Foultier et al. [150] concluded from molecular phylogenetic analyses that the proteobacterial NF-T3SSs could be classified into five large classes: the Ysc group, the Esc/Ssa group, the Hrp1 and Hrp2 groups, and the Inv/Mxi/Spa groups. In addition, they found two orphan systems that did not comfortably fit into any of these large classes, the Bordetella T3SS (which in some analyses fell within the Ysc group) and the rhizobial system. Largely similar conclusions have been reached in other such analyses [101,228,8]. All analyses agree that the chlamydial T3SSs represent a highly divergent out-group to the proteobacterial systems.

Each of the five classes of NF-T3SS has expanded in recent years, with the addition of newly discovered systems (Table 1). Alongside the plasmid-encoded system from Yersinia spp. and the chromosomally encoded T3SS from P. aeruginosa, the Ysc-Yop class now includes systems from Aeromonas, Photorhabdus, V. parahaemolyticus and V. harveyi. The Esc/Ssa group has now expanded to include Cpi-2 from C. violaceum, the degenerate chromosomally encoded system from Y. pestis and the E. tarda T3SS. New members of the Inv-Spa group include the Bop system from Burkholderia, two T3SSs from insect endosymbionts, the Cpi-1 system, an uncharacterised system from Proteus mirabilis, and, probably, novel systems from the environmental organisms M. xanthus and D. vulgaris. The Hrp1 group has expanded with the inclusion of systems from additional pathvars of P. syringae and from additional species of Pectobacterium (formerly Erwinia). Novel members of the Hrp2 lineage include NF-T3SSs in Burkholderia spp.

Traditional molecular phylogenetic analyses allow reconstruction of the evolutionary history of NF-T3SSs and provide a framework for a natural taxonomy of type-III secretion. However, we propose that an alternative, cladistic approach, based on the analysis of higher-order features (gene/protein content and arrangement, with consequent effects on T3SS structure and function) and on the search for shared derived features diagnostic of each group (synapomorphies) is likely to provide more fruitful insights into the evolution and function of non-flagellar T3SSs. In the following sections, we shall describe potential synapomorphies for two of the five major classes. As this kind of cladistic analysis of type-III secretion is still in its infancy, what follows should be viewed as a series of illustrative vignettes, rather than an exhaustive enumeration of synapomorphies.

4.1Synapomorphies in the Ysc group: YopN/TyeA, LcrV and LcrG

SepL is a LEE-encoded protein that is required for A/E lesion formation, secretion of translocators and for the translocation of – but not the secretion of – effectors [229,230]. Although SepL has been said to have no homologues [229,230], our PSI-BLAST searches have revealed homology to two proteins from the Ysc-Yop system: most of the SepL sequence (up to residue 267) is homologous to YopN, while the C-terminal 83 amino acids are homologous to TyeA (Fig. 4) [168]. Through careful use of PSI-BLAST, we have established unequivocal homology between YopN-TyeA and SepL-like proteins from each of the well-characterised animal-pathogen-related T3SSs (InvE from the SPI1 system and MxiC from the Shigella system) (Fig. 4) [168].

image

Figure 4. Multiple sequence alignment of SepL and YopN/TyeA homologues. Alignment is presented using CHROMA [314] and default settings (consensus abbreviations as for Fig. 2). Organism names are abbreviated as for Fig. 2, and: Ah, Aeromonas hydrophila; Bp, Bordetella pertussis; Cc, Chlamydophila caviae; Cp, Chlamydophila pneumoniae; Er, Erwinia chrysanthemi; Pt, Pectobacterium atrosepticum; Se, Salmonella enterica Typhi str. CT18; Sf, Shigella flexneri; St, S. typhimurium LT2. (reproduced from Ref. [168], q. v. for accession numbers, procedural details etc.).

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This assertion of homology between SepL and YopN/TyeA is supported by the observations that the yopN and tyeA genes lie adjacent to one another and the YopN and TyeA proteins interact with one another. Furthermore, shortly after we made this discovery in silico, experimental evidence emerged from another group that a chimaeric YopN/TyeA protein is produced by programmed frameshifting in some Yersinia strains, greatly strengthening this argument [231].

On examing the results of PSI-BLAST searches, it becomes clear that the Ysc-Yop system is unusual in separating out the two SepL-related sequences into two proteins (Fig. 4). In fact, this separation appears to be a synapomorphy diagnostic of the Ysc group, occuring only in systems closely related to the Ysc-Yop system (i.e., T3SSs from P. aeruginosa, P. luminescens, A. salmonicida, V. parahaemolyticus). In the other systems, there is a single protein corresponding to a YopN-TyeA chimaera. The discovery that YopN and TyeA correspond to a single protein in many other systems also implies that targeting of the TyeA and YopN moieties in Yersinia to distinct ultimate locations (bacterial cytoplasm and host cell) is a recent peculiarity of the Ysc-Yop-like systems that may lack any general or fundamental importance.

These assertions of homology should help unify the often apparently discordant literature on all of these proteins [27,229,230,232–243]. Furthermore, these assertions are underpinned by one broadly conserved property of the proteins – they all appear to play a role in differential regulation of translocator secretion and/or effector translocation [229,230,234–237,240,241]. However, given the evidence that one of these proteins, YopN [239,244], is translocated into eukaryotic cells, while another, SsaL [233], is involved in transcriptional regulation, related functions for the whole family of proteins should be investigated.

In addition to YopN-TyeA, another pair of proteins also appears to represent a synapomorphy, unique to the Ysc class of T3SSs: LcrV and LcrG. LcrV (‘the V antigen’) is an essential virulence component in the Ysc-Yop system, with multiple functions. In the yersinial cytoplasm, LcrV stimulates type III secretion by binding to LcrG, a negative regulatory protein, and neutralizing its ability to block secretion [245]. LcrV is also exposed at the bacterial cell surface prior to contact with mammalian cells and is essential along with YopB/D for effector translocation and is itself translocated [246–249]. In addition, LcrV mediates a direct immune suppression by interaction with TLR2 to upregulate IL-10 levels by an unknown mechanism [250,251]. With so many functions, the V antigen is an obvious vaccine target, and, in this regard, its homology with proteins in other Ysc-type T3SSs has not gone unnoticed [252–261].

The evolutionary events that led to the fission of the YopN/TyeA ancestor protein and the recruitment of LcrV/LcrG into the primordial Ysc T3SS have yet to be elucidated, as have the full functional consequences of these events. However, the lack of these synapomorphies in the Bordetella systems sets them apart from the Ysc family of T3SSs, even though according to some traditional molecular phylogenetic analyses they should be lumped together (Pallen, unpublished). It is likely that a thorough analysis of the Ysc family will reveal additional synapomorphies (YscW is one plausible candidate).

4.2A shared derived character state of the Esc/Ssa group: the EspA-like proteins

Although the plant-associated T3SSs produce pili that resemble extended needles, the LEE-encoded T3SS is, so far, unique in possessing a filamentous organelle, the EspA pilus that forms a structurally distinct extension to the needle complex, resembling the flagellar filament [154,196]. As noted above, sequence homology can be detected between EspA and flagellin. Around the same time that it was realised that EspA assembled into a pilus, a homologue of EspA, SseB, was discovered in the SPI-2 system of S. enterica[262]. However, to date, no one has shown that SseB can form a filament, although by analogy with the LEE, this seems plausible. Since the LEE and SPI2 systems were discovered, three new members of the Esc/Ssa group have been discovered: the Cpi-2 system from C. violaceum, an uncharacterised degenerate chromosomal T3SS from Y. pestis and a novel functional T3SS from the fish pathogens, Edwardsiella ictaluri and E. tarda[44,52,109,263–265].

Leaving aside the fact that Y. pestis system lacks any genes for a translocation apparatus – presumably a derived degenerate state – one might anticipate that the presence of EspA-like proteins, and even of associated pili, might represent a shared derived characteristic of the Esc/Ssa group. In this regard, the Edwardsiella systems are known to secrete a single EspA homologue, although it is not yet known whether this assembles into a filament [44,265]. However, scrutiny of the Cpi-2 gene cluster springs a surprise – this cluster encodes four espA homologues [109]! This is the first time multiple EspA homologues have been found in the same type-III secretion system, and begs the question of whether and how they form filaments. A hint comes from the arrangement of the genes – there are two pairs of adjacent espA-like genes, separated by a gene encoding a coiled-coil protein (a potential chaperone?). More interesting still is the fact that, in each pair, the first gene encodes a short conventional EspA homologue, while the second gene encodes a long EspA homologue containing a central insertion [109], reminiscent of, but not homologous to, the flagellin D3 domain (Fig. 3). This arrangement of genes, when interpreted in the light of the half-flagellin model of EspA, might suggest that each pair of EspA-like proteins forms a heterodimer, which then polymerises into a filament. Thus, according to this model, C. violaceum is predicted to produce two types of EspA filament, each built from a distinct heterodimer. Clearly, experimental investigation of the contribution of the C. violaceum EspA homologues to filament formation is required.

5Conclusions: from deep past to future perspectives

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2The post-genomic landscape
  5. 3Conservation and variation among type-III secretion systems
  6. 4Towards a taxonomy of type-III secretion
  7. 5Conclusions: from deep past to future perspectives
  8. Acknowledgements
  9. References

To study history one must know in advance that one is attempting something fundamentally impossible, yet necessary and highly important. To study history means submitting to chaos and nevertheless retaining faith in order and meaning. It is a very serious task, young man, and possibly a tragic one ”. Father Jacobus in Magister Ludi, Herman Hess [266]

Never make predictions, especially about the future”. Casey Stengel

What if one wishes to progress beyond the common ancestor of all NF-T3SSs, further back in time into the deep past, in search of the primordial type-III secretion system? All traditional phylogenetic analyses, based on sequence alignments, separate out the flagellar and non-flagellar systems as monophyletic groups, with distinct evolutionary histories [150,101,228,8]. Along with Gophna et al. [101], we interpret this to mean that both types of system evolved from a common ancestor that predated the origin of either group. However, it has been suggested instead that the non-flagellar systems evolved from a flagellar-like system [228,267], largely on the grounds that flagellar systems are more widespread in nature and because motility appears, intuitively, to be an more ancient phenotype than subversion of eukaryotic cells.

To help answer this question, perhaps a cladistic approach independent of the analysis of individual sequences, and instead based on gene/protein repertoires, might be useful? However, in any discussion of bacterial evolution, one encounters the problem of distinguishing vertical lines of descent from the effects of horizontal gene transfer. Horizontal gene transfer is known to be rife among NF-T3SSs, but it has been assumed that flagellar systems evolve only by vertical transmission. However, the Flag-2-like lateral flagellar systems provide a counter-example, in that they appear to be acquired by horizontal gene transfer [126]. Furthermore, although widespread, flagellar motility is far from universal among bacteria – by our reckoning (allowing for the difficulties of proving a negative), it has been found in only eight out of nineteen bacterial phyla (apparently absent from the Bacteroidetes/Chlorobi group, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deinococcus-Thermus, Dictyoglomi, Fibrobacteres/Acidobacteria group, Fusobacteria, Gemmatimonadetes, Nitrospirae, Thermodesulfobacteria; Pallen, unpublished). Thus, it seems equally, or more, likely that bacterial flagellar motility originated after the last common bacterial ancestor and was then disseminated horizontally among a few distantly related bacterial phyla (with, say, loss of the secretin representing an adaptation to life in Gram-positives), than to accept that it was present in the last common ancestor and then lost in the majority of lineages.

Allowing for the caveat of horizontal gene transfer, are there any shared derived character states particular to the flagellar systems? An obvious candidate is the flagellar motor. Additionally, almost all flagellar systems are associated with a chemotaxis system (missing only in Aquifex and lateral flagellar systems [126,268,269]), so it seems likely that this system was acquired very early in flagellar evolution and may even have been present in the last common ancestor of all bacterial flagellar systems. Another synapomorphy associated with the flagellar systems is the sigma (FliA)-anti-sigma (FlgM) combination, where type-III secretion of the anti-sigma factor influences regulation of flagellar gene expression. In a recent survey, we found FliA/FlgM-like proteins even in systems only distantly related to the proteobacterial flagellum (from Spirochaetes, Aquifex, Thermotoga, Gram-positives, Planctomycetes) [184]. Its near-universal distribution (absent only fromCaulobacter crescentus and Borrelia burgdorferi, which is likely to reflect recent losses) suggests that this system was also present in the last common ancestor of all bacterial flagellar systems.

What of the non-flagellar systems? Do they possess any synapomorphies that set them apart from the flagellar systems? A clear contender is the translocation apparatus – in particular, the translocation pore, formed from EspD and its relatives. Our observation that sequence homology can be established between EspD-like proteins in all NF-T3SSs (YopB and relatives in the Ysc group; IpaB and relatives in the Mxi-Spa group; HrpK in P. syringae, HrpF in Xanthomonas, NolX in rhizobia and PopF1/F2 in Ralstonia) suggests this is indeed an ancient feature of these systems, present in their most recent common ancestor [168].

If we accept the above analysis, then parsimony demands that we also accept the notion that the flagellar and non-flagellar systems have evolved from a common ancestor, rather than the idea that non-flagellar systems have evolved from flagellar systems. The reasoning for this assertion is as follows. To get from the common ancestor of all type-III secretion systems via a flagellar system to a non-flagellar system would require seven steps: gain of the motor, acquisition of the chemotaxis apparatus and recruitment of the sigma/anti-sigma mechanism during the evolution of the flagellar system, and then, in the lineage leading to the NF-T3SSs, loss of the motor, uncoupling from the chemotaxis apparatus and demise of the sigma/anti-sigma mechanism, followed by gain of the translocation apparatus. However, if the flagellar and non-flagellar systems diverged from a common ancestor, then we need posit only four steps: gain of the motor, gain of the chemotaxis apparatus and gain of the sigma/anti-sigma mechanism in the flagellar lineage, and gain of the translocation apparatus in the non-flagellar lineage.

Although parsimony would appear to favour divergent evolutionary trajectories for the flagellar and non-flagellar T3SSs, a number of unresolved problems remain in this view of evolutionary history. Are the hook and needle homologous structures? Gross structural similarities would seem to suggest so, even in the absence of sequence homology between the structural components of the hook and needle [183]. However, the recent publication of high-resolution structures of the components of the flagellar hook (FlgE) and filament (FliC) show that it is possible for proteins with completely different structures to form tubular assemblies with essentially similar architectures and helical symmetry [197,198,270]. Thus, in the absence of atomic resolution structures of the needle proteins, it is premature to assume homology with the hook.

Another problem is the two instances of sequence homology between proteins associated with the flagellar tubular structures and proteins associated with similar structures in the NF-T3SSs: the homology between the EspA-like proteins and flagellin and the homology between the C-terminal domains of FliK and the YscP-like proteins. Does the presence of these apparently ‘flagellar’ proteins in NF-T3SSs represent a pair of shared primitive character states (sympleisiomorphies in the jargon of cladistics) among the NF-T3SSs, which have then been lost from all but one class of NF-T3SS (YscP-like proteins lost from all but the Ysc systems, EspA-like proteins lost from all but the Esc/Ssa systems)? If so, this would tend to support a flagellar origin of the non-flagellar systems. However, a more parsimonious explanation is horizontal gene transfer, as this would require only a single evolutionary event in each of two lineages (acquisition of a flagellin-like gene in the ancestor of the Esc/Ssa systems; acquisition of a FliK-like sequence in the ancestor of the Ysc group), rather than several independent losses in multiple lineages. Further light might be shed on this issue by molecular phylogenetic analyses of these genes/proteins.

Another approach to investigating the early evolutionary history of type-III secretion is to search for a ‘missing link’, or more accurately, an early branching lineage, that more closely resembles the common ancestor of extant T3SSs. Molecular phylogenetic analyses suggest that the NF-T3SS from the chlamydias and parachlamydias represents the earliest diverging member of the NF-T3SS lineage, and that this phylum might represent the evolutionary home of non-flagellar type-III secretion [101,108,228]. In support of an ancient origin of non-flagellar type-III secretion in this group is the fact that the T3SS genes are scattered around the genome in several clusters, rather than in a single cluster suitable for/indicative of horizontal gene transfer, plus the fact that these groups of genes have a G + C% in common with the rest of the genome [108]. In addition, recent genomic analysis of the chlamydia-related symbiont of amoebae, Parachlamydia sp. UWE25 suggests that non-flagellar type-III secretion genes were likely to be present in this lineage more than 700 million years ago, around the time that symbiotic and pathogenic chlamydiae diverged [103,108]. Interestingly, there is now increasing evidence that genes/proteins within the chlamydial system are active and expressed and that the system is likely to translocate effector proteins into the host cell [31–33,232,271–274].

In addition to housing the most divergent NF-T3SS, chlamydias also demand our attention in that they encode proteins that either more closely resemble components of flagellar systems or stand equidistant between flagellar and non-flagellar systems [104]. As chlamydias lack any components of the motor, the chemotaxis system or of the flagellar filament, these flagellar-like proteins might provide a glimpse of the last common ancestor of flagellar and non-flagellar systems. Alternatively, as there is no evidence so far that the relevant genes are expressed or functional, they could simply represent degenerate fragments of an ancient flagellar system. Interestingly, there appears to be clustering of a flagellar-like gene (a homologue of flhA) with a gene for a FliA-like sigma factor in the chlamydial genomes, perhaps providing further evidence for an ancient origin of the sigma-anti-sigma system (although a chlamydial FlgM homologue remains elusive) [104]. However, it is worth stressing that these chlamydial ‘missing link’ genes and proteins appear to be absent from the parachlamydias (Pallen, unpublished). Nonetheless, investigation of the chlamydial ‘missing link’ genes and proteins clearly remains a priority for future research into the origins of type-III secretion.

What about evolution of type-III secretion before the last common ancestor of current systems? It is clear that these systems have a modular architecture, sharing proteins and domains with other biological pathways or systems. For example, secretins and membrane-associated ATPases are shared with type-IV secretion systems (which also mediate protein translocation) [9]. At the level of protein domains, BON domains, found within the YscD-like proteins, are shared with OsmY, some secretins (e.g., NolX), mechanosensitive ion channels and with putative haemolysins, while FHA domains are widespread in nature [170,171]. The components of the secretion apparatus associated with the inner membrane assemble first – and can do so in the absence of the distal components [169]. We thus suspect that type-III secretion might illustrate Haekel's otherwise discredited dictum that “ontology recapitulates phylogeny”[275]. In other words, the multiple core components of the inner ring probably evolved into a unified entity, as a way of traversing the inner membrane, before the machinery to traverse the periplasm and bacterial outer membrane were recuited into the primordial secretion system. More detailed genomics, bioinformatics and structural analyses are likely to shed light on the earliest stages in the evolution of type-III secretion.

Looking to the future, it is clear that current sequencing efforts have far from saturated the type-III secretion gene pool. In the years to come, we anticipate the discovery of many more type-III secretion systems and many more of their effectors. Among unfinished and recently finished genome sequences, several newly discovered systems already demand characterization: in the human pathogen, P. mirabilis, in environmental organisms such as Myxococcus and Desulfovibrio and in the opportunistic pathogen C. violaceum (what are those four EspA homologues doing!). New discoveries might help fill gaps in “sequence space”, so that otherwise obscure evolutionary relations between systems and their components are revealed. However, we must be wary of easy conclusions and continually question assumptions.

One final example illustrates the perils of hasty analysis. As noted, NF-T3SSs are common to the symbiotic and parasitic chlamydias. Thus, the discovery of type-III secretion genes in the genome of V. spinosum, which belongs in the same phylum as the chlamydias, might not seem so surprising and might be taken to indicate a common origin of such systems in the progenitor of the entire phylum. This was our initial conclusion. However, additional scrutiny suggests that the V. spinosum NF-T3SS is quite distinct from those in the chlamydias; instead it most closely resembles the Ysc group of systems, leading to an entirely unexpected conclusion: that NF-T3SS genes have moved horizontally between the proteobacterial and Chlamydia/Verrucomicrobium phyla on more than occasion! (Pallen, unpublished).

An obvious problem with future research on type-III secretion is information overload. Holding in one's head the gene/protein names, gene order and organsiation and all the published data on the system's structure, function and regulation represents a considerable challenge even for one system – the task becomes nearly impossible when applied to the whole range of known type-III secretion systems. In addition to this data deluge, the very diverse contexts in which type-III secretion occurs makes it hard for a scientist working on any one T3SS to follow the literature on any other system, as this requires familiarity with alien jargon, concepts and techniques. This problem is compounded by the fact that type-III secretion research is fragmented into numerous organism-specific communities that seldom interact, attend different research meetings and may even publish in different journals. As a solution to these problems and bulding on our success with coliBASE [276], we have set up a web site and database, 3Base (http://3base.bham.ac.uk) dedicated to the comparative biology of type-III secretion. We invite the reader to exploit and contribute to this facility.

In this review, we have attempted to provide “one long argument” for a comparative, evolutionary, Darwinian approach to the study of type-III secretion. We hope that we have convinced the reader of the utility of this approach, and, in particular, of the value added by bioinformatics to our understanding of type-III secretion. With this in mind, it seems appropriate to close with a final quotation from Origin of Species [28], which applies just as well to type-III secretion as to entire organisms:

… “ when we regard every production of nature as one which has had a long history; when we contemplate every complex structure and instinct as the summing up of many contrivances, each useful to the possessor, in the same way as any great mechanical invention is the summing up of the labour, the experience, the reason, and even the blunders of numerous workmen; when we thus view each organic being, how far more interesting – I speak from experience – does the study of natural history become!”

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2The post-genomic landscape
  5. 3Conservation and variation among type-III secretion systems
  6. 4Towards a taxonomy of type-III secretion
  7. 5Conclusions: from deep past to future perspectives
  8. Acknowledgements
  9. References

We thank the MRC for funding SB's Bioinformatics Fellowship; we thank the Division of Infection and Immunity and the Medical School, University of Birmingham for funding CMB and the 3Base project. We thank the UK Biotechnology and Biological Sciences Research Council for funding other experimental and bioinformatics work within the Pallen group on E. coli and type-III secretion (grant references EGA16107, D13414, 02B1D08033, BBC5167011).

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2The post-genomic landscape
  5. 3Conservation and variation among type-III secretion systems
  6. 4Towards a taxonomy of type-III secretion
  7. 5Conclusions: from deep past to future perspectives
  8. Acknowledgements
  9. References
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