Take Five — Type VII Secretion Systems of Mycobacteria

Biochimica et Biophysica Acta xxx (2013) xxx–xxx

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Take five — Type VII secretion systems of Mycobacteria☆

Edith N.G. Houben a,b,⁎, Konstantin V. Korotkov c, Wilbert Bitter a,b

a VU University, Amsterdam, The Netherlandsb VU University Medical Center, Amsterdam, The Netherlandsc University of Kentucky, Lexington, KY, USA

☆ This article is part of a Special Issue entitled: Protein T⁎ Corresponding author at: VUUniversity, SectionMolec

1085, 1081 HV Amsterdam, The Netherlands. Tel.: +5987155.

E-mail address: [email protected] (E.N.G. Houben).

0167-4889/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.bbamcr.2013.11.003

Please cite this article as: E.N.G. Houben, etdx.doi.org/10.1016/j.bbamcr.2013.11.003

a b s t r a c t
a r t i c l e i n f o
Article history:Received 18 September 2013Received in revised form 7 November 2013Accepted 9 November 2013Available online xxxx

Keywords:Protein secretionMycobacteriumSecretion signalChaperoneProtease

Mycobacteria use type VII secretion (T7S) systems to secrete proteins across their complex cell envelope. Patho-genicmycobacteria, such as the notorious pathogenMycobacterium tuberculosis, have up to five of these secretionsystems, named ESX-1 to ESX-5. At least three of these secretion systems are essential formycobacterial virulenceand/or viability. Elucidating T7S is therefore essential to understand the success ofM. tuberculosis and other path-ogenic mycobacteria as pathogens, and could be instrumental to identify novel targets for drug- and vaccine-development. Recently, significant progress has been achieved in the identification of T7S substrates and ageneral secretion motif. In addition, a start has been made with unraveling the mechanism of secretion andthe structural analysis of the different subunits. This review summarizes these recent findings, which are incor-porated in a working model of this complex machinery. This article is part of a Special Issue entitled: ProteinTrafficking & Secretion.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction on mycobacterial cell envelope

Most bacterial infections are caused by “classical” Gram-positive orGram-negative pathogens. Notable exceptions are the pathogenicmycobacteria, such as Mycobacterium tuberculosis and Mycobacteriumleprae. These bacteria belong to the new order of Corynebacteriales [1].Members of this order are generally characterized by the presence ofunusual fatty acids known as mycolic acids in their cell envelope. De-pending on the species, these lipids contain between 30 and 100 carbonatoms [1]. The mycolic acids form the main constituent of a second hy-drophobic layer surrounding the cytoplasmic membrane. Electronmicroscopic analyses have shown that this layer is almost indistinguish-able from the outer membrane of Gram-negative bacteria [2,3].Furthermore, the available biochemical and biological evidence indi-cate that this second membrane indeed forms a functional equiva-lent of the classical Gram-negative outer membrane [4,5]. Becausethe Corynebacteriales are deeply rooted within the group of highGC Gram-positive bacteria and because the composition and biogen-esis of this mycolic acid-containing membrane is completelydifferent from an LPS-containing outer membrane, the special mem-brane of Corynebacteriales must be the result of convergent evolu-tion. The presence of such a protective outer membrane is oneof the main reasons why Corynebacteriales are tough persistentorganisms, protected against harsh conditions such as desiccation,

rafficking & Secretion.ularMicrobiology, deBoelelaan31 20 5983579; fax: +31 20

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al., Take five — Type VII secre

antimicrobial enzymes/chemicals and mechanic stress. However,this protective shield also poses a problem: how to secrete proteinsto the cell surface or in the environment? Recently, it has becomeclear that the type VII secretion (T7S) pathway, which is alsoknown as the ESX pathway in mycobacteria, forms the major secre-tion route of these proteins.

2. ESX gene clusters

The first T7S system to be discoveredwas the ESX-1 system inmem-bers of the M. tuberculosis complex. This system was shown to be re-sponsible for the secretion of two small proteins that were among themajor culture filtrate proteins [6]. These two secreted proteins arenow known as EsxA and EsxB, but are perhaps better known by theirinitially designated names, ESAT-6 and CFP-10, respectively. The ESX-1system quickly became one of the major topics in tuberculosis researchwhen it was discovered that ESX-1 is a major virulence factor and thatthe absence of this system is responsible for the attenuation of the live-vaccine strain Mycobacterium bovis BCG [7,8]. During prolonged in vitrogrowth this attenuated strain incurred the so-called RD1 deletion,which removes a major part of the ESX-1 locus. The role of the ESX-1system in virulence is diverse and complex and has recently beenreviewed elsewhere [9,10]. Therefore, we will not elaborate on thistopic here. With the identification of the ESX-1 locus, it was also clearthat multiple ESX loci are present in mycobacteria. In fact, up to five dif-ferent loci can be present in a single species, designated ESX-1 throughESX-5 [11]. Although the number of genes and the overall size of theESX loci vary significantly, a set of conserved genes that are coding forthe secretion machinery can be identified. A few years ago a general no-menclature for the ESX components was proposed [12]: components

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present in at least four of the five ESX loci were designated Ecc, forESX-conserved component, whereas other components were calledEsp, for ESX-specific protein. For historic reasons there are a few ex-ceptions to this rule: (i) mycosin or MycP, this component has ho-mology to subtilisin-like proteases and is present in all ESX loci,but has kept its previously designated name [13], (ii) the small se-creted Esx proteins, the eponym for the system, and (iii) two othergroups of substrates that are present in most systems, the so-calledPE and PPE proteins (recently reviewed in [14]).

Althoughfive different ESX loci can bepresent inmycobacteria,mostspecies have a lower number of ESX loci. This is partially due to an ex-pansion of ESX clusters in specific Mycobacterium lineages by duplica-tion events. For instance ESX-2 and ESX-5 evolved relatively recentlyand are therefore only present in specific subsets of the Mycobacte-riaceae. Especially the generation of ESX-5 is noteworthy, because itspresence seems to coincide with the differentiation between the fastand slow-growingmycobacteria [11]. The lineage of slow-growing spe-cies evolved from a clade of fast-growingmycobacteria and they do notformvisible colonieswithin 7 days (formanyof themevenmuch longerincubation times are required). Importantly, all major pathogenswithinthe mycobacteria belong to the slow-growing lineage. It is not knownwhether the presence of ESX-5 is functionally coupled to the slow-growing phenotype.

Another mechanism for differentiation in the number of ESX lociis deletion events. Especially ESX-1, despite its importance as a viru-lence factor, is (partially) lost by numerous mycobacteria.The list ofESX-1-deficient mycobacteria also includes several pathogenic species,such as Mycobacterium avium and Mycobacterium ulcerans, whichmeans that the function of the ESX-1 substrates can be compensatedfor. For M. ulcerans the nature of this compensation is in the secretionof a special toxin, mycolactone, which is cytotoxic for macrophages[15], whereas for M. avium produces glycopeptidolipids (GPLs) thatare not present in other pathogenic mycobacteria. Thus far, only ESX-4, the most archaic system, and ESX-3, which is essential in a numberof species, are omnipresent in mycobacterial species thus far analyzed.

ESX systems can also be categorized on the basis of their function;thus far only ESX-1, ESX-3 and ESX-5 have been shown to be involvedin the secretion of proteins [6,16,17], whereas active secretion of sub-strates via ESX-2 or ESX-4 has not been proven.

Fig. 1. Structures of the T7S substrates. Ribbon representations of EsxA–EsxB (PDB1WA8; [20]),red; EsxG has an HxG variant. Secretion signal motifs YxxxD/E [29] are shown in orange; this mregion of EsxB that interacts with EccC [34] is shown in magenta.

Please cite this article as: E.N.G. Houben, et al., Take five — Type VII secredx.doi.org/10.1016/j.bbamcr.2013.11.003

3. Substrates

The ESX loci are named after the first secreted substrate that wasidentified, EsxA [18]. EsxA belongs to a larger family of proteins,known as theWxG100 proteins [19].WxG stands for the two conservedamino acids tryptophan and glycine, separated by a single other aminoacid, and 100 indicates the average size of these proteins. The otherESX-1 substrate, EsxB, also belongs to theWxG100 family and togetherthese two proteins form a heterodimer that is secreted in a folded con-formation [20]. The structure of this heterodimer has been resolved;both proteins have two α-helices, separated by a turn formed by theWxG motif (Fig. 1; [20]). These proteins are oriented antiparallel andhave flexible N and C-terminal tails. The flexible C-terminal tail ofEsxB contains a motif crucial for secretion (see also below).

The esxBA genes form an operon in the ESX-1 locus (Fig. 2). All otherESX loci also have two esx homologues that are juxtaposed to each other.The Esx proteins encoded by the ESX-3 and ESX-5 loci have been shownto be secreted as well [17,21]. Furthermore, it has recently been shownthat EsxGH, encoded by the ESX-3 locus, also forms a heterodimerwith similar structural characteristics as EsxAB (Fig. 1; [22]). In myco-bacterial genomes one can also find esx gene pairs outside the ESX loci[12]. Usually these isolated gene pairs are nearly identical to esx pairswithin one of the ESX loci, indicating that they are the result of recentgene duplication events. The esx genes of the WxG100 family seem toplay a central role in T7S systems, as homologues are present in all T7Ssystems that have been identified in a wide range of bacterial species[19]. Sometimes, esx genes are not part of an operon togetherwith a sec-ond esx gene; this is often the case for distantly related T7S systems inFirmicutes. In accordance with the observed secretion of folded dimersby T7S systems, one of the WxG100 members of Staphylococcus aureus,which is encoded by such a “single” esx gene, forms a homodimer in-stead of a heterodimer [23].

Although the small Esx proteins are clearly the most archaic sub-strates of the T7S systems, other substrates have been identified. Especial-ly for the ESX-1 systema growingnumber of the so-called esp genes seemto encode secreted substrates (EspA,B,C,E,J,K; [24–26]). Apart from espAand espC that are located in a separate operon, all other genes are locatedwith the ESX-1 locus [12]. As the other ESX loci do not have numerous espgenes, this variation in substrates might be unique for ESX-1.

EsxG–EsxH (PDB 2KG7; [22]) and PE25–PPE41 (PDB2G38; [28]).WxGmotifs are shown inotif is also present in the PE25 sequence, but disordered in the structure. The C-terminal


image of Fig.�1



Fig. 2. Comparison of different gene clusters that encode type VII secretion systems. The color coding for the figure is presented in the key. The black arrows indicate region-specific genes.The dotted line represents a considerable distance between the two adjacent genes and is not in scale.

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Another class of substrates that is secreted by different ESX systems isthepreviouslymentionedPE andPPEproteins [16,21,27]. Perhaps not sur-prisingly, these proteins have several characteristics in common with theEsx proteins. The most important similarity is in the structure: the onlystructure available for PE and PPE proteins is that of PE25 and PPE41,which shows that they also formaheterodimer (Fig. 1; [28]). The domainsof the proteins that are responsible for the interaction form a four-helixbundle in an antiparallel fashion, similar to the EsxAB pair. In fact, thePPE proteins usually also have theWxGmotif located in the turn betweenthe twoα-helices [28]. Finally, the C-terminal tail of the PE protein is flex-ible and contains a secretion motif identical to EsxB [29].

Another similarity is the genetic organization. The pe and ppe geneswithin the ESX loci generally form an operon, which is located next tothe esx genes (Fig. 2). Only the archaic ESX-4 system does not have ape/ppe pair. Finally, similar to the esx genes, pe and ppe genes havebeen duplicated and can be found also at other positions in the genome.However, in contrast to the esx genes, the evolution of pe/ppe genes hasresulted in an extensive diversification [30]. In some species, such asM.tuberculosis and Mycobacterium marinum, nearly 10% of the coding ca-pacity is devoted to these two families [30]. This runaway evolution isalso seen in the size and composition of these proteins: the C-terminaldomain of both PE and PPE proteins can be extremely long, resultingin proteins up to 3300 amino acids for M. tuberculosis (i.e. PPE8). Al-though PE and PPE proteins have been shown or predicted to be secret-ed by different ESX systems, the vast majority of these proteins seem tobe secreted by ESX-5 [21], which makes the ESX-5 system the most ac-tive secretion system in mycobacterial species that have many pe/ppegenes. In line with this, proteomic analysis showed that structuralESX-5 proteins ofM. marinum are among the most abundant cell enve-lope proteins [31]. In contrast to the Esx proteins, PE and PPE proteinsare often not present in the culture supernatant, but end up at the bac-terial cell surface. In M. marinum these proteins can be easily removedusing mild detergents, whereas in M. tuberculosis they seem to bemore stably associated with the cell envelope [32].


3.1. Secretion signal and substrate specificity

What do all the ESX/T7S substrates have in common? As mentionedpreviously, the Esx proteins and the PE/PPEproteins have a similar struc-ture, a heterodimer forming a four-helix bundle. Although the homologyin the primary sequence is very low, both PE/PPE and Esx proteins be-long to the same Pfam protein superfamily, designated the EsxAB clan(CL0352; http://pfam.sanger.ac.uk/clan/EsxAB). Interestingly, also Espsubstrates belong to this clan; some of these Esp proteins are small pro-teins predicted to form a helix-turn-helix fold similar to Esx proteins,whereas other Esp proteins, such as EspB, are larger and predicted toform a four-helix bundle by itself. Therefore, this folding pattern seemsto be a conserved and potentially crucial aspect of T7S substrates, espe-cially because these dimers seem to be secreted in a folded conforma-tion. The only exceptions seem to be EspA and EspE, but the genesencoding these substrates are located next to espC and espF, respectively,each of which is coding for a small protein belonging to the EsxAB clan.Therefore, EspA and EspE might be secreted as heterodimers togetherwith EspC and EspF, respectively, similar to Esx andPE/PPEproteins. Fur-thermore, not only does EspA depend on the presence of EspC [25], butthis protein is also predicted to form a helix-turn-helix in its N-terminaldomain. Therefore, a four-helix bundle seems a general characteristic ofall T7S substrates known to date. Interestingly, EspA additionally con-tains a WxG motif, which has recently been shown to be important forsecretion of EspA and EsxAB [33].

Another conserved characteristic is a short secretion motif. Secretionof the EsxAB dimer was shown to depend on the C-terminal flexible tailof EsxB [34]. This tail specifically interacted in yeast-two-hybrid experi-ments with the EccC component of the transport machinery, indicatingthat it could confer specificity to the system. In this first study however,crucial conserved residues could not be identified. In a later study, itwas shown that secretion of the PE/PPE pairs also depended on a flexibleC-terminal tail; in this case it was the tail of the PE protein [29]. Align-ment of these various C-terminal tails again showed no conserved region


http://pfam.sanger.ac.uk/clan/EsxAB

image of Fig.�2




in the extreme C-terminal part. However, near the C-terminus, at thestart of the flexible tail, two highly conserved residues can be found, a ty-rosine and a negative charge (D/E), that are separated by three other res-idues. This motif is not only present in all PE proteins, but also in EsxBhomologues andmany of the Esp substrates. Mutagenic analysis showedthat both of these residues are crucial for T7S [29]. Furthermore, also thespacing of these two residues was shown to be critical. Therefore, T7Ssubstrates can be distinguished by a helix-turn-helix domain followedby the YxxxD/E secretion motif. The secretion motif does not have tobe at the C-terminus, because many PE proteins and also EspB have afunctional secretion motif adjacent to the helix-turn-helix domain,followed by a large C-terminal extension. Even though the characteristicsrequired for recognition by ESX systems are relatively unrestricted, an al-gorithm has been developed and used to identify a number of new (po-tential) substrates [29].

The substrate characteristics described above seem to apply generallyfor all different ESX systems. This raises the question of how systemspec-ificity is generated. Swapping experiments in which the C-terminal tailsof two PE substrates secreted by ESX-1 and ESX-5, respectively, were ex-changed showed that secretion still occurred, but that secretion specific-itywas not altered [29]. This experiment indicates that system specificityis not enforced by the C-terminal tail for the PE substrates.

3.2. Substrate regulation

The production and regulation of ESX substrates are highly variable.The ESX-3 system is regulated by the Zur repressor and is as suchincreased under conditions of low iron and/or low zinc availability[17,35]. Despite this, the Esx proteins of ESX-1, ESX-3 and ESX-5 aregenerally secreted in different media and not strictly regulated. There-fore, secretion via T7S systems does not seem to depend on some hostsignal or contact with host cells, as has been shown for other secretionsystems. This situation could be different for ESX-2 and ESX-4, as theirputative substrates cannot be found in the culture filtrate. The potentialsubstrates esxTU of ESX-4 are under control of the alternative sigma fac-tor SigM and therefore probably only produced under specificconditions [36,37].

The regulation of some ESX substrates however can be far morecomplex, which is exemplified by the espAC operon of M. tuberculosis.Upstream of this operon is usually a large intergenic region of almost1.5 kb. An increasing number of regulators have been shown to bindthe espAC promoter region and influence its expression, includingEspR,MprA, CRP and Lsr2 [38–42]. This accumulation of regulators indi-cates that strict control of this operon is important formycobacteria. Thenumber of pe and ppe genes is very high and so is their gene transcrip-tion profiles [14]. Each condition that has been tested seems to either re-press or induce specific pe/ppe genes, indicating that they have specificand diverse functions.

A particular property of T7S substrates is that these proteins in gen-eral do not accumulate in the bacterial cell when the secretion system isblocked [6,13,16,21,27,43]. This feature is seen for different ESX systemsand different mutations, indicating that it is a conserved property sens-ing cytoplasmic accumulation of substrates. Apparently, there is a specif-ic feedback mechanism for substrate regulation. This mechanism doesnot (solely) work at the level of transcription initiation (Weerdenburgand Bitter, unpublished results) and therefore either RNA stability, pro-tein translation initiation and/or protein stability is involved.

Another particular characteristic of ESX substrates is co-dependentsecretion. Of course it is logical that the secretion of heterodimers,such as EsxA and EsxB, depends on the presence of both these proteins.However, especially for several ESX-1 substrates this dependence seemsto be extended. For instance, secretion of EspA and its partner proteinEspC is dependent on the presence of EsxAB and vice versa [25,44,45].Similarly, secretion of EspB depends on the expression but not on thesecretion of EsxAB [26,46]. Unfortunately, this co-dependence has notbeen studied in great detail, for instance we do not know whether


EspB and EspA are secreted in a co-dependent manner, and the molec-ular mechanism behind this phenomenon is not known. Perhaps thisfeature is specific for the Esx and Esp proteins of the ESX-1 system, asESX-5 substrates do not seem to show interdependence [14].

4. Specific chaperones

Many different bacterial secretion systemsmake use of both generaland more specific chaperones, with the chaperone-usher pathway andtype III secretion as the most prominent examples. The latter systemmakes use of different classes of chaperones (I to V), which are usedboth for secreted substrates and for secretion machinery components[47]. The major task of these specific chaperones is either to shieldaggregation-prone domains of their substrates or to prevent their pre-mature polymerization. Another task of specific chaperones can be thedirection of substrates to the translocation machinery. In analogy tothe type III secretion system, similar specific chaperones might bepresent in T7S. For instance, the subunit of dimeric substrates contain-ing the secretion signal, i.e. the EsxB homologues, the PE proteins andsome Esp proteins, could function as secretion chaperones, escortingtheir partner proteins through the cell envelope. Interestingly, YscE-like type III chaperones are, just like EsxB, small helix-turn-helix pro-teins [48,49]. These chaperones interact (together with an additionalchaperone) with the small subunits that form the needle of the typeIII secretionmachinery. Together, the two chaperones shield the contactsides of the needle subunits that form subunit–subunit interaction inthe assembled needle. For the EsxAB complex, there is evidence thatEsxA needs to dissociate from EsxB to play a direct role in virulence,by rupturing the phagosomal membrane of the host cell [50–53]. Thisscenario would indeed suggest that EsxB is a chaperone-like protein.However, because we do not knowwhether EsxB has a specific intrinsicfunction once secreted, the term chaperone seems premature.

Another protein that could function as a specific chaperone is EspD.A recent study showed that EspD is crucial for both stabilization and se-cretion of EspA and EspC, although this protein itself is not a substrate ofthe ESX-1 system [54]. In line with this function the espD gene is alsopart of the espAC operon. Unfortunately, the exact function of this pro-tein is not known at this point.

The most prominent chaperone candidate is EspG. Although thisprotein was first thought to be limited to only two Esx systems (hencethe name Esp), we now know that functional equivalents (with low ho-mology) are present in all systems, except for ESX-4. The first indicationfor the chaperone function of this protein was that EspG5 is requiredfor the secretion of PE/PPE proteins via the ESX-5 system [21,27].Subsequently, pull-down experiments showed that EspG5 specificallyinteracted with PE/PPE proteins [55]. Detailed analysis showed thatEspG5 interacted with the PE/PPE complex in a 1:1:1 fashion. Althoughthese proteins showed a tight interaction, EspG5 is not identified in theculture supernatant and seems to be strictly located in the cytosol.Therefore, this complex probably dissociates upon contacting the secre-tion machinery.

Another interesting aspect of EspG chaperones is that they showconsiderable specificity. EspG5 binds different PE/PPE proteins, but itdoes not interact with the PE35/PPE68_1 complex that is secreted viaESX-1 [55]. Conversely, the EspG homologue of ESX-1 (EspG1) doesbind this PE/PPE complex, but not those secreted via ESX-5. Thus,whereas the secretion motif does not seem to differentiate betweenPE proteins of different secretion systems, the EspG proteins do. Ofcourse, this points to the EspG proteins as the markers for system spec-ificity. But this hypothesis still needs to be rigorously tested.

EspG proteins are not general T7S chaperones, as they do not inter-act with the Esx proteins [55]. This could also explain the absence ofEspG in ESX-4, since this system is devoid of pe and ppe genes. Thisleaves the obvious question whether Esx proteins have chaperonesand how Esx proteins are selected for by the secretion system.




Fig. 3. Structure of theN-terminal domain of EccA1. (A) The TPR domain ofM. tuberculosis EccA1 (PDB 4F3V; [61]) is colored in rainbow colors fromN-terminus to C-terminus. The β-fingerinsertion is highlighted in gray. (B) A hexamer model of full-length EccA1 [61] based on a hexamer of Rubisco activase [90]. The TPR and AAA+ domains are shown in pink and purple,respectively.

Fig. 4.Model for type VII secretion. Both Esx and PE/PPE proteins are exported as dimersby the T7S secretion machinery. Substrate recognition occurs via a C-terminal secretionmotif on one of the dimer subunits (indicated by a red box). The cytosolic componentEspG specifically recognizes PE/PPE proteins and possibly targets these substrates to theputative membrane channel that consists of EccB, EccC, EccD and EccE. The three nucleo-tide binding domains of EccC are likely involved in energizing translocation of substratesthrough this channel. In this model, T7S is a two-step process, in which the channel inthe outer membrane refers to a hypothetical, so far unidentified pore.


5. AAA+ ATPase

In addition to EspG there is another cytosolic protein involved inmost ESX secretion systems, which is EccA. EccA belongs to the AAA+(ATPases associated with various cellular activities) protein family,members of which are involved in a large number of diverse processes,including protein degradation, signal transduction and assembly anddisassembly of protein complexes. AAA+ proteins usually form ahexameric ring-shaped structure with a central pore. This hexamericcomplex couples ATP hydrolysis to a conformational change of the com-plex that is propagated to the bound substrate(s). The presence of anAAA+ protein is unusual for a bacterial secretion system, since thema-jority of bacterial traffic ATPases belong to the RecA family of ATPases[56]. An exception is the type VI secretion system, which depends onan AAA+ ATPase to disassemble tubules formed by two small proteins[57,58]. This disassembly step is required for type VI secretion, althoughthe details are not fully known.

All EccA homologs have a conserved domain architecture; they con-sist of two domains joined by a linker. The C-terminal domain of EccAhas all typical sequencemotifs characteristic for AAA+proteins, includ-ing a Walker A andWalker B motif, sensor 1 and 2 regions, putative ar-ginine fingers and an oligomerization domain. In line with this, it hasbeen shown that both full-length EccA1 and its C-terminal domainhave ATPase activity in vitro and full-length EccA1 can form oligomers,possibly hexamers, similar tomany othermembers of the AAA+ family[59]. The N-terminal domain of approximately 30 kDa probably func-tions to regulate the ATPase activity of the C-terminal domain. Thestructure of the N-terminal domain ofM. tuberculosis EccA1 has recentlybeen solved and displays 6 terminal tetratricopeptide repeat (TPR)motif, known for mediating protein–protein interactions in other pro-teins [60], arranged in a right-handed superhelix (Fig. 3; [61]). The con-cave groove of the TPR superhelix is occupied by a β finger insertion, afeature that is also observed in some other TPR domain structures [62].Interestingly, at the structural level the closest bacterial homologue ofEccA1 is the PilF protein of Pseudomonas aeruginosa, which is involvedin the assembly of the type IV pili system [61].

EccA seems to be important for T7S, as disruption of eccA genes inESX-1 and ESX-5 clusters results in the loss of secretion [16,63,64] andEccA3 is, similar to other core components of the ESX-3 system, essentialfor viability [65]. Moreover, an ATPase activity-defective mutant ofEccA1 is also inactive in secretion [66]. However, also EccA independentsecretion through ESX-1 and ESX-5 ([67,68], Houben and Bitter, unpub-lished results) has been observed and the precise role of EccA in the se-cretion process is still unclear. Since EccA is not present in the mostarchaic ESX system, i.e. ESX-4, it would be logical to assume that thefunction of this accessory protein is connected to other ESX proteinswith a similar distribution profile. One possibility is the interaction


with specific (larger) substrates (Esp or PPE proteins) and their chaper-ones, which fitswith the observation that EccA1 interacts with EspC andEspF substrates in yeast two-hybrid studies [25]. In analogy with theAAA+ protein in the type VI secretion system, EccA might be involvedin the disassembly of secreted substrate dimers or secreted substratesand their chaperones. A remarkable and unexplained observation isthat an M. marinum strain with ATPase activity-defective mutant ofEccA1 is partially defective in the production of mycolic acids, revealingan unexpected link with lipid biosynthesis pathways [66].

6. Membrane events in type VII secretion

6.1. The T7S membrane complex

Once T7S substrates are recognized in the cytosol, they should betargeted to the inner membrane and subsequently transported overthemycobacterial cell envelope. This transport will be (at least partially)mediated by the conserved T7S membrane components, EccB, EccC,


image of Fig.�3

image of Fig.�4




EccD, EccE andMycP,which for the ESX-1 systemhave all been shown tobe essential for secretion [6,43,67]. While EccB, EccC, EccE and MycP arerelatively hydrophilic, only having one or two predicted transmembranedomains, EccD is highly hydrophobic, usually having 11 predicted trans-membrane domains. Because of its hydrophobicity, EccD has been hy-pothesized to constitute the membrane pore through which substratesare transported [6], but there is no formal evidence for this. Recently, itwas shown that four of these membrane components, EccB, EccC, EccDand EccE of the ESX-5 system of M. marinum andM. bovis BCG togetherformamembrane complexwith a size of ~1.5 MDa [67]. This largemem-brane complex likely forms the translocation channel through whichsubstrates are transported (Fig. 4), although channel activity has notyet been proven. Since substrates are likely transported as dimers andtherefore are already folded in the cytosol, the translocation pore shouldbe relatively large to be able to accommodate folded structures. Conse-quently, opening and closing of the channel should be highly controlledto maintain the proton motif force across the inner membrane.

Based on mass spectrometry analysis and the apparent molecularweight of a purified T7Smembrane complex, itwas estimated that a sin-gle complex consists of six copies of the components EccB, EccC andEccD and three copies of EccE [67]. Because EccE homologues are notpresent in all T7S systems, this protein is probably located at the periph-ery of the complex. In linewith this reasoning, the EccE protein is highlysensitive to proteolytic degradation when the complex is treated withsmall amounts of trypsin [67]. Of these four subunits, only EccC has pre-dicted functional domains; it shows homology to members of the FtsK/SpoIIIE family of ATPases. This large protein family consists of proteinsthat are involved in a wide range of cellular processes, best-known areFtsK and SpoIIIE that actively transport DNA into the daughter cell dur-ing cell division and sporulation, respectively [69]. Interestingly, mem-bers of this protein family usually form ring-like hexamers, suggestingthat EccC is also functional as a hexameric complex. However, whileFtsK/SpoIIIE-like ATPases usually have a single nucleotide-bindingdomain, EccC has three successive FtsK/SpoIIE-like ATP binding do-mains, indicating that EccC is structurally distinct. In addition, it shouldbe noted that the EccC component of the ESX-1 system (EccC1) differsfrom other EccC homologues in that it is encoded by two separategenes ([11]; Fig. 2). The two gene products presumably form a function-al unit together, which is supported by the observation the EccC1 sub-units interact in a yeast two-hybrid system [6].

Another interesting finding is that a member of FtsK/SpoIIE proteinfamily is a component of the type IV secretion system, the type IV cou-pling protein (T4CP). This protein plays a key role in secretion as it is im-portant for substrate recognition and transport [70]. Possibly, EccCperforms similar functions in T7S. Accordingly, EccC of the ESX-1 systemwas shown to interact in vitro with the substrate EsxB, which was de-pendent on the presence of the C-terminal secretion signal [34].

6.2. Mechanism of transport: a one or two-step process

The T7S substrates need to cross two lipid membranes to end up inthe extracellular environment, analogous to proteins that are exportedby the specialized secretion systems found in Gram-negative bacteria.Protein secretion in this latter group of bacteria can occur in a one-step fashion through a transport channel that spans both the innerand outer membrane, e.g. in type I, III IV and VI secretion (see otherchapters in this volume). In type II and V secretion on the other hand,inner and outer membrane transport is uncoupled, where the Sec andTat machinery mediate the transport over the inner membrane, whileouter membrane transport is mediated by a separate machinery. SinceT7S substrates do not contain classical signal sequences and thereforedo not depend on Sec or Tat for secretion, one might therefore assumethat T7S is a one-step process. Indeed, the size of the T7S membranecomplex allows it to span both the inner and theoutermembrane. How-ever, all four subunits of the T7S complex have predicted classical α-helical transmembrane domains, suggesting that they all are inserted


into the inner membrane. We have postulated previously that theEccE component might be the outer membrane pore-forming unit ofthe complex, in a model where it spans both membranes [67]. The rea-soning for this model was that EccE seemed to be absent in T7S systemsfound in suborders of Actinobacteria that do not contain mycolic acids,such as Streptomycineae. However, we could identify eccE homologueswith low homology in Streptomycineae and other Actinobacteria thatdo not have mycolic acids after closer examination of these T7S geneclusters (see below). Therefore, EccE might not be the outer membranechannel and possibly, the identifiedmembrane complex is inserted onlyin the inner membrane. This suggests that outer membrane transport ismediated by other (unidentified) proteins that are perhaps more loose-ly associated with the inner membrane complex. Although hard evi-dence for a one step secretion process is still missing, there iscurrently also no data that the T7S substrates are exposed to the myco-bacterial periplasm at any point during the translocation process.

6.3. Outer membrane transport

Finding the outer membrane proteins that form the pore throughwhich T7S substrates are transported is difficult, mainly because ingeneral our knowledge of mycobacterial outer membrane proteins isvery limited [71]. Mycobacteria do not have any homologues of outermembrane proteins of Gram-negative bacteria, which is not surprisingsince these proteins evolved independently. The only mycobacterialouter membrane protein that is sufficiently characterized is MspA,which forms pores in the outer membrane of the non-pathogenicMycobacterium smegmatis [72]. The crystal structure of MspA showsthat this porin is octameric and forms an atypical β-barrel [4]. Unfortu-nately, neither this porin nor any homologous protein is presentin mycobacterial pathogens such asM. tuberculosis. Based on the struc-ture of MspA it has been suggested that other mycobacterial outermembrane proteins are also β-barrel proteins, but there is no formalproof for this. Alternatively, similar to type IV secretion [73], the T7Schannel in the outer membrane could be formed by a ring of amphi-pathic α-helices that would be difficult to predict.

6.4. Subcellular location

In recent years, efforts have beenmade to localize T7S systems insidethe mycobacterial cell. Visualization of these systems in whole bacteriausing immunofluorescence is difficult because of the highly impermeablecell envelope. Therefore, either immuno-labeling of newly synthesizedESX-1 substrate EspE on the bacterial surface, or immuno-labeling ofthe EccC component of the ESX-1 system in a “leaky” cell envelope bio-synthesis mutant was conducted to show that both proteins localizedto the bacterial pole of M. marinum [24]. Polar localization was alsoshown in a separate study, in which the same ESX-1 proteins werefused to YFP in M. smegmatis, although it is not clear whether theseYFP-fusions were still functional [74]. It is not known whether otherESX systems are polar localized or not.

6.5. Mycosin

The only conserved membrane protein of ESX systems that is notpart of the ESX-5 membrane complex was the mycosin or MycP.Mycosins are subtilisin-like proteases with a classical signal sequence,followed by a (periplasmic) protease domain and a putative C-terminaltransmembrane region [13,75]. The available experimental evidence in-dicates that mycosins indeed associate with the cell envelope. Althoughthese proteases seem to play a crucial role in various ESX systems, theirfunctional role in the secretion process is unclear. The only known sub-strate for any mycosin is EspB, which is an ESX-1 secreted proteincleaved by MycP1 [76]. Although this finding suggests that mycosinsare involved in substrate processing, the classical substrates, i.e. the Esxproteins, are not cleaved upon transport. Furthermore, the proteolytic




Fig. 5. Structures of mycosin proteases.M. smegmatisMycP1 (PDB 4J94; [77]) andMycP3 (PDB 4KG7; [78]) are shown in ribbon representation. Structure of subtilisin (PDB 3UNX; [91]) isshown for comparison. TheN-terminal extension ofmycosins is shown inblue; three extended loops that are not present in the subtilisin structures are shown in purple. The catalytic triadresidues are shown in orange. The conserved disulfidebridges inmycosins are shown in ball and stick representation.Note thatmycosins lackCa2+ ions (green spheres) that are present insubtilisin.


activity of MycP seems to be dispensable for the secretion process:whereas deletion ofmycP1 abolishes secretion, a catalytically inactivatedMycP1 is capable of supporting secretion of the ESX-1 substrates, even athigher levels than thewild-type protein [76]. This surprising observationled to a proposed dual function formycosins,with a role in substrate pro-cessing and in negative regulation of the secretion process [76].

Recently, the structures ofM. smegmatis andMycobacterium thermo-resistibile MycP1 have been solved, which show a typical subtilisin coredomain decoratedwith several extended loops (Fig. 5; [77,78]). The big-gest surprise of the MycP1 structure is the presence of a unique N-terminal extension. Subtilisins are usually produced with an N-terminalpropeptide that prevents substrate access to the active site until degrada-tion of the propeptide occurs [79]. Although the N-terminal extension ofmycosins does not display homology to the subtilisin propeptide se-quences, it was previously suggested to function as such [76]. However,both MycP1 structures show that this N-terminal extension is wrappedaround the protease domain and does not obstruct or block the activesite. In line with this observation, MycP1 with intact N-terminal exten-sion cleaves EspB in vitro, although with low efficiency [77,78]. Interest-ingly, the structure of M. smegmatis MycP3 reveals the similar fold withthe N-terminal extension in the conformation similar to theMycP1 struc-ture ([78], Fig. 5). While the available mycosin structures are similar infold, the surface properties of the active site clefts are quite distinct(Fig. 5), reflecting ~45% sequence identity and implying different sub-strate specificity for MycP1 and MycP3. Undoubtedly, there are multipleunanswered questions regarding mycosins' specificity and role in secre-tion. What are the substrates for other mycosins? How do the mycosinsinteract with the other components of ESXmachinery? Could these pro-teases be targeted for drug design? Addressing these questions will ad-vance our understanding of the T7S mechanism.

7. Function of type VII secretion seemingly not related to secretion

Although protein secretion via three different ESX systems (ESX-1,ESX-3 and ESX-5) has been firmly established by now, ESX systemshave also been implicated in processes that seem more difficult to rec-oncile with this function. For instance, although ESX-1 of pathogenicslow-growing mycobacteria is responsible for the secretion of the cru-cial virulence factors EsxA, EspB and EspA, the orthologous system inM. smegmatis is involved in a special form of conjugation [80,81]. Inthis unconventional conjugation process different large chromosomalDNA fragments can be efficiently exchanged between special donorand recipient strains [82]. ESX-1 plays a major role in this process,since ESX-1 mutations of the recipient strains are unable to receivedonor DNA [80]. Interestingly, M. smegmatis donor strains with an


inactive ESX-1 system are hyperconjugative [81]. Sometimes, this con-jugation process results in transfer of the donor phenotype and by ge-nome sequencing of various transconjugants it has recently beensuggested that the Esp proteins of the ESX-1 system determine donor/recipient specificity [82]. These results suggest that secreted factorsplay a role in establishing functional interactions between two differentcell types. At this point it is not known whether actual DNA transfer isalso mediated through the ESX-1 machinery, as has been shown fortype IV-like conjugation systems [83], but since ESX-1 donor mutantsare hyperconjugative this does not seem to be the case.

Another interesting function of the T7S pathway is the uptake of ironor zinc via the ESX-3 system. Two different groups have shown thatESX-3 is an essential locus in M. tuberculosis and M. bovis BCG, whichcan be rescued by the addition of extra iron or zinc to the growthmedi-um [17,35]. Because slow-growing mycobacteria produce a single classof siderophores for iron uptake, i.e. (carboxy)mycobactin, and becauseESX-3 mutants show no defect in mycobactin production, it has beenproposed that iron uptake throughmycobactin is blocked in ESX-3 mu-tants. However, although this would explain the effect of iron, it doesnot explain the zinc effect. Zinc ions are not efficiently bound tosiderophores and usually special siderophore-independent zinc trans-porters are present in bacteria. Furthermore, whereas ESX-3 genes areabsolutely required for M. tuberculosis growth, not all mycobactin bio-synthesis genes are [65]. Therefore, more research is required to deter-mine what the exact function is of ESX-3 in metal ion uptake. BecauseESX-3 is responsible for the secretion of EsxGH and possibly other sub-strates, one of these substrates should probably be involved in this pro-cess. This function could be a protein involved in uncoupling of thesiderophore-iron complex or a surface-bound protein functioning as asiderophore or metal-ion receptor.

8. Type VII secretion systems in non-mycobacteria

Although T7S systems are mostly studied in mycobacteria, homolo-gous systems can be found throughout the phylum of Actinobacteria.Here, we assessed the presence of the individual T7S components andsubstrates in the various orders and families of the Actinobacteriausing the Pfam database of protein families (http://pfam.sanger.ac.uk/;[84]). Most T7S associated proteins are categorized in a Pfam familyand the presence of members of these families in the various bacterialfamilies can readily be determined. It should be noted that althoughthe EccD component belongs to the Pfam family YukD, named after asmall cytosolic component of the T7S-like systems found in the phylumof Firmicutes, only the soluble N-terminal domain of EccD shows homol-ogy to these proteins. Therefore, we verified individual YukDhits for the


http://pfam.sanger.ac.uk/

image of Fig.�5



Table 1Presence of T7S genes in the phylum Actinobacteria. Only orders that contain T7S genes are shown. The family Dietziaceae of the order Corynebacteriales is omitted from this table as onlyone draft genome sequence is available for this bacterial group. Pseud, Pseudonocardiales; Micro mono, Micromonosporales; Strept myc, Streptomycetales; Caten, Catenulisporales; Franki,Frankiales; Actino, Actinomycetales; Micro coc, Micrococcales; Propio, Propionibacteriales; Glyco, Glycomycetales; Strept spor, Streptosporangiales; Bifido, Bifidobacteriales; Corio,Coriobacteriales; Nocar, Nocardiaceae; Myco, Mycobacteriaceae; Gord, Gordoniaceae; Tsuka, Tsukamurellaceae; Coryn, Corynebacteriaceae; Segnil, Segniliparaceae.

Gene Pfam Order Corynebacteriales Pseud Micromono

Streptmyc

Caten Franki Actino Micrococ

Propio Glyco Streptspor

Bifido Corio

Family Nocar Myco Gord Tsuka Coryn Segnil

esx WXG100 + + + + + + + + + + + + + + + + + +eccC + + + + + + + + + + + + + + + + + +eccB DUF690 + + + + + + + + + + + + + + + + + +d

eccD YukD + + + + + + + + + + + + + + + + +c ?d

mycP + + + + + + + + + + + + + + + + + +d

eccE DUF2984 + + + + − + + + + + − − − + + − − −eccA + + + + − + + + + − + − − + − + − −espG ESX-

1_EspG+ + + + − + + − +a − − − − − − − − −

pe/ppe

PE, PPE + + + + − + + − + − − − +b − − − − −

a One Pfam ESX-1_EspG member identified in draft genome sequence of Streptomyces sp. AA4.b One Pfam PPE member identified in Kocuria rhizophila.c EccD identified by analysis of specific T7S gene clusters.d One eccB and mycP homologue identified by BLAST searching in a draft genome sequence of Atopobium sp. ICM58; homology between eccD genes is too low for determining the

presence of eccD by BLAST search.


presence of multiple transmembrane domains, EccD commonly has 11transmembrane domains, to establish them as genuine EccD proteins.In addition, the components with a predicted function (i.e. the ATPasesEccA and EccC and the serine protease MycP) belong to larger proteinfamilies also containing proteins involved in other processes. We there-fore conducted BLAST searches using mycobacterial sequences to findhomologous genes and their location near other T7S genes finallyestablished them as eccA, eccC or mycP genes. Through this analysis wecould determine a list of T7S genes that are present in every suborderand therefore seem to encode for the core T7S system in Actinobacteria(Table 1). These core proteins are the membrane components EccB,EccC, EccD and MycP, and the secreted Esx proteins. The central role ofEccB, EccC and EccD indicates that they form the core membrane chan-nel through which substrates are transported. The high conservation ofthe Esx proteins might indicate that these are not just substrates buthave a central role in the secretion process itself. The high conservationofMycP ismore puzzling, considering its postulated role in cleaving spe-cific substrates. MycP might therefore have an additional, more generalrole in secretion.

One intriguing finding is that mycP homologues of T7S systemsfound in Bifidobacterium dentium are in fact chimeric genes, with the3′ end coding for the protease and the 5′ end showing high homologyto eccB (Fig. 2). This chimeric gene indicates that mycosins function inclose proximity of the T7S membrane complex, of which EccB is a sub-unit, and could even be part of this complex. The fact that MycP5 isnot co-purified with the ESX-5 membrane complex could be due to amore labile interaction.

The remaining components, the cytosolic ATPase EccA, the cytosolicchaperone EspG and themembrane component EccE, can be consideredas accessory components that are not required for the core system tofunction. As already mentioned above, it is conceivable that the role ofthe two cytosolic components is dedicated to the membrane targetingof specific substrates that are not present in species that lack these com-ponents. Indeed, EspG has been shown to specifically interact with PEand PPE in the cytosol [55], which coincides with the observation thatthe orders that lack PE and PPE members also lack EspG. In addition,espG genes generally cluster together with pe/ppe genes, as can be seenin a T7S gene cluster in Gordonia polyisoprenivorans that contains twoespG genes both adjacent to a ppe gene (Fig. 2). Finally, while EccE iswidely present in Corynebacterineae, the in silico analysis shows thateccE homologs can also be found in some bacterial families that lackmycolic acids. This makes it unlikely that this component of the putativemembrane channel forms the previously postulated outer membrane


pore in mycolic acid containing species [67]. The accessory role of EccEin the functioning of the membrane complex therefore remains to bedetermined.

Besides the phylum of Actinobacteria, also in the closely related phy-lum of Firmicutes T7S-like systems can be found. These related systemsare composed of various components, of which only the EccC compo-nent and the ESX proteins are conserved with the T7S systems foundin Actinobacteria. This secretion pathway has been shown to be func-tional in S. aureus, Listeria monocytogenes, Bacillus anthracis and Bacillussubtilis by secreting Esx proteins [85–89].While EccC plays a central rolealso in these systems the accessory components play essential roles insecretion as well. The mechanism of T7S in Firmicutes is therefore dis-tinctive from the mechanism of secretion in Actinobacteria.

9. Future perspectives

Since the discovery of T7S systems in 2003 [6], a considerable re-search effort has been made on identifying the proteins that are secret-ed via these systems, the genes that are required for secretion and theeffect of T7S systems onmycobacterial virulence. Thiswork has resultedin the identification of specific T7S substrate families, the Esx, Esp andPE/PPE proteins, and a set of core components that are essential fortheir secretion.More recently, themechanismof T7S is starting to be ad-dressed by elucidating the T7S signal [29,34], the composition of the T7Smembrane complex [67] and the role of individual components in se-cretion. In addition, high-resolution structures of both substrates andindividual components are accumulating [20,22,28,61,77,78]. Importanttopics that still need to be addressed are the mechanism of substraterecognition,what determines system specificity and the role of cytosoliccomponents, especially EspG and EccA, in these processes. Anothermajor issue is the mechanism of substrate targeting to and transportover the mycobacterial cell envelope. What is the role of the T7S mem-brane complex and particularly the FtsK/SpoIIE-like ATPase EccC in sub-strate recognition and transport? In addition, the mechanism oftransport over the outer membrane is still a crucial topic that needs tobe addressed. Are known T7S components also involved in this processor are additional proteins responsible? Structural information on theT7S membrane complex will give information on this matter. An addi-tional issue is the central role of the mycosins in secretion. Have theseproteases an additional function besides cleaving specific substratesandwhat is this crucial function? Finally, it is still almost completely un-known what the functions are of the secreted T7S substrates. Althoughthere is evidence that EsxA permeabilizes membranes [51–53], it is not





known whether this is a general characteristic of all Esx proteins. Simi-larly, the role of PE and PPE proteins in virulence is still largely amystery[14]. What should be realized is that some of these substrates might ac-tually have a function in the secretion process itself, e.g. the presence ofEsx proteins in all T7S systems found so far suggests that they are compo-nents of the systems themselves. In addition, as also mentioned aboveone partner of the substrate couples might be dedicated to chaperonethe other partner to the T7S machinery. Addressing all these topics willreveal the inner workings of T7S, which is crucial to understand the viru-lence mechanisms of pathogenic mycobacteria. As these systems are es-sential for mycobacterial virulence and/or viability, the understandingof T7S could direct research to improve the current vaccine or developnovel drugs.

Acknowledgements

Ourwork is supported by a grant (P20GM103486) from the NationalInstitute of General Medical Sciences (to KVK) and by a VIDI grant fromthe Netherlands Organization for Scientific Research (NWO) (to ENGH).

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Take Five — Type VII Secretion Systems of Mycobacteria