Available online at www.sciencedirect.com

an

d

or the twin-arginine translocation (TAT) system. How- Mycobacterium leprae and the fish pathogen Mycobacterium

ever, culture filtrate of M. tuberculosis also contains manysmall, highly immunogenic proteins that lack classicalsignal sequences. The 6 kDa early secreted antigenictarget (ESAT-6) [1] is the prototype of these proteins,which are characterized by the amino acid (aa) motif Trp-Xaa-Gly (WXG), and a size of approximately 100 aa [2].Multi-genome analyses predict the presence ofWXG-100proteins in a wide range of actinobacteria and Gram-

marinum. Whereas work on ESX-1 in M. leprae has beendiscouraged by the non-culturable status of the bacter-ium, the genomic resemblance of ESX-loci inM. marinumand M. tuberculosis [13] inspired numerous studies thatuse M. marinum as a model [14]. The only available dataon ESX cluster 5 (ESX-5) also come from M. marinum,and revealed that ESX-5 is implicated in the secretion ofPE and PPE proteins [15,16].

Current Opinion in Microbiology 2009, 12:410 www.sciencedirect.comCurrent Opinion in Microbiology 2009, 12:410

This review comes from a themed issue on

Host-microbe interactions: Bacteria

Edited by Brendan Kenny and Raphael Valdivia

Available online 18th January 2009

1369-5274/$ see front matter

# 2008 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.mib.2008.11.003

IntroductionSecreted proteins are among the most important mol-ecules involved in hostpathogen interaction of Mycobac-terium tuberculosis, the etiological agent of humantuberculosis (TB). Proteins containing typical signalsequences, such as the Ag85 T cell antigens get secretedby the Sec mediated secretory pathway, and secretion ofother proteins may occur via the alternative Sec2 pathwayESX/type VII secretion systemsinteractionRoxane Simeone1, Daria Bottai1,2 an

The ESX-1 system is responsible for the secretion of the

prototypic ESX proteins, namely the 6 kDa early secreted

antigenic target (ESAT-6) and the 10 kDa culture filtrate protein

(CFP-10). These two proteins, which form a 1:1 heterodimeric

complex, are among the most important proteins of

Mycobacterium tuberculosis involved in hostpathogen

interaction. They induce a strong T cell mediated immune

response, are apparently involved in membrane and/or host

cell lysis and represent key virulence factors. There are four

other paralogous ESX systems in M. tuberculosis, some of

which are essential for in vitro growth. ESX systems also exist in

many other actinobacteria and Gram-positive bacteria, and

have recently been suggested to be named type VII secretion

systems.

Addresses1 Institut Pasteur, UP Pathogenomique Mycobacterienne Integree, 25

Rue du Dr. Roux, 75724 Paris, France2University of Pisa, Dipartimento di Patologia Sperimentale,

Biotecnologie Mediche, Infettivologia ed Epidemiologia, Pisa, Italy

Corresponding author: Brosch, Roland (roland.brosch@pasteur.fr)d their role in hostpathogen

Roland Brosch1

positive bacteria [2], where they are thought to besecreted via novel secretion systems that were namedESAT-6 systems (ESX-1-ESX-5) [3], or more recentlytype VII secretion systems (T7S system) [4]. In myco-bacteria these clusters typically also encode PE and PPEproteins characterized by their N-terminal motifs proline-glutamic acid (PE) and proline-proline-glutamic acid(PPE) [5].

ESX-1 of M. tuberculosis and related tubercle bacilli isthe ESX/T7S system that has probably attracted themost attention in recent years. Located in the region ofdifference 1(RD1), ESX-1 is deleted in the vaccinestrain Mycobacterium bovis BCG and other attenuatedmembers of the M. tuberculosis complex like the volebacillus Mycobacterium microti, suggesting that secretionof ESAT-6 is involved in virulence of tubercle bacilli.First experimental evidence confirming this hypothesiswas obtained by complementing BCG and M. microti,with differently sized portions of the RD1 region fromM. tuberculosis [6,7]. Only strains that were complemen-ted with the entire RD1 locus and its flanking regionsshowed secretion of ESAT-6 whereas strains comple-mented with smaller constructs expressed ESAT-6 butfailed to secrete it (Figure 1). Recombinant BCG::RD1and M. microti::RD1 strains with a fully restored ESX-1secretion system grew more vigorously in severe com-bined immunodeficient mice and also persisted longerin the organs of immunocompetent mice [7]. However,they induced considerably less pathology than M. tuber-culosis, suggesting that the loss of the ESX-1 systemfrom BCG was only one of several factors that con-tributed to the attenuation of the vaccine strain, as wasrecently confirmed by comparative and functionalgenomics [8].

In parallel, studies using transposon mutants and/or RD1knockout strains of M. tuberculosis came to the sameconclusion that RD1 dependent ESAT-6 secretion wasessential for virulence of M. tuberculosis [912].

ESX-1 is conserved in several other mycobacteria, such as

ESX/type VII secretion systems and their role in hostpathogen interaction Simeone, Bottai and Brosch 5Figure 1Surprisingly, an orthologous ESX-1 system is also presentin the avirulent saprophyte Mycobacterium smegmatis,where it is involved in conjugation [17]. Finally it shouldbe mentioned that the more distantly related ESX/T7Ssystems in Gram-positive bacteria, may or may not have arole in pathogenicity. In Listeria monocytogenes, no effecton virulence could be attributed to ESX [18], while inStaphylococcus aureus, the two ESAT-6-like proteins EsxAand EsxB were associated with abscess formation in amurine infection model [19]. Although the structures ofEsxA and EsxB from S. aureus resemble those of ESAT-6(EsxA) and the 10 kDa culture filtrate protein CFP-10(EsxB) of M. tuberculosis that are known to form a 1:1heterodimeric complex [20], the staphylococcal Esxproteins form mainly homodimers [21]. Finally, anESX/T7S system was also identified in Bacillus antrax,where EsxB is required for secretion of EsxW and induc-

Diagram of the M. tuberculosis H37Rv genomic region showing the position

indicates that genes are essential for expression of ESAT-6 and CFP-10, whe

and CFP-10 (after references [6,23]). The figure also shows the RD1mic reginserts delineated by blue or red lines contained in integrating cosmids RD1-

of M. microti via the attB site. The results of western blot analyses using anti-

left portion of the figure and clearly show that complementation with cosmid

while complementation with construct pe-ppe-esxB-esxA (pAP34) leads to e

that brackets showing RD1BCG (Rv3871Rv3879) were omitted for simplicity

www.sciencedirect.comtion of humoral immune responses in infected guinea pigs[22].

Taken together, it is clear that ESX/T7S systems ofactinobacteria andGram-positive bacteria are highly inter-esting scientific topics studied by many research groupsworldwide. In this reviewwewill present some key aspectson the functional characterization of these systems andtheir secreted proteins. Owing to space constraints, how-ever, we will focus on the ESX-1 system ofM. tuberculosis,which may serve as a paradigm for the other systems.

The ESX-1 secretion machineThe contribution of individual ESX-1 proteins tosecretion of ESAT-6/CFP-10 is well documented[6,10,11,23]. Whereas genes rv3867, rv3873, rv3876,rv3878 and rv3879 do not appear to be essential for

of various genes, depicted as differently colored arrows. Blue color

reas green color indicates that genes are needed for secretion of ESAT-6

ion absent from M. microti (black bracket) [6,7] and the M. tuberculosis

2F9 and pe-ppe-esxB-esxA (pAP34) that were used for complementation

ESAT-6 and anti-ESAT-CFP-10 antibodies (Ab) are depicted in the upper

2F9-RD1 leads to secretion of ESAT-6 and CFP-10 into the supernatant,

xpression but not secretion of the two antigens (after reference [6]). Note

.

Current Opinion in Microbiology 2009, 12:410

6 Host-microbe interactions: BacteriaESAT-6 and CFP-10 secretion in tubercle bacilli, theremaining genes encoded in the extended RD1 region areinvolved in this process (Figure 1). Rv3868 resembles anATPase associated with various cellular activities (AAA)that, unlike previous predictions, may not fulfil a generalchaperone-like function [24]. Three other proteinsencoded upstream and downstream of esxA, Rv3869,Rv3870 and Rv3877, exhibit 1, 3 or 11 predicted trans-membrane domains, respectively. Together with Rv3871,a cytosolic component of theESX-1 system, these proteinsare thought to form a membrane-bound ESX-1 secretorycomplex, with protein export driven by ATP hydrolysis[3,4]. Recently, a C-terminal signal sequence has beenidentified that allows theunstructuredC-terminus ofCFP-10 to be recognized byRv3871 that itself interacts with themembrane protein Rv3870. Point mutation in this signalsequence abolished binding of CFP-10 to Rv3871 andprevented secretion of ESAT-6 and CFP-10 [25]. AsRv3870 and Rv3871 both show similarity to proteins ofthe FtsK-SpoIIIE ATPase family, these proteins mightperform an essential part of the work necessary to secreteESX-1 substrates. This mechanism resembles type IVsecretion system inGram-negative bacteria, where amem-brane-bound SpoIIIE/FtsK-like ATPase recognizes anunstructuredC-terminal sequence anddirects the secretedsubstrate to the cytoplasmic membrane [26].

Apart from genes localized in the RD1 region, a secondgene cluster (rv3616c-rv3614c) situated elsewhere in thegenome of M. tuberculosis, was shown to contribute to thesecretion of ESAT-6 and CFP-10 [27,28]. Inactivation ofthese genes in M. tuberculosis abolished the secretion ofESAT-6 and CFP-10, in spite of a complete RD1 locus[27,28].

Regulation of gene expression in ESX-1Until very recently the regulation of theESX-1 systemwasunknown. As ESAT-6 and CFP-10 are found in super-natants of in vitrogrownM. tuberculosis cultures, secretionofthese antigens does not seem to dependent on interactionwith host cells.This feature is different tomechanisms thatgovern regulation of type III and type IV secretion systemsof Gram-negative bacteria, which are switched on uponhostcell contact. These characteristics of ESAT-6secretion may imply that ESX systems, although beingessential for the pathogenicity of certain virulent myco-bacteria, may fulfil more general roles in the physiology ofthe bacteria than strictly hostcell oriented functions.Adaptation towards pathogenesis may have evolved viathe second genomic region involved in ESAT-6 secretion,carrying rv3616c-rv3614c. Orthologues of these genes aremissing fromM. smegmatis and it remains to be determinedwhether some distantly related, structurally similarproteins may intervene in ESX-1 secretion inM. smegmatis

[17]. As the M. tuberculosis rv3616c-rv3614c gene cluster isclearly involved in the regulation of ESAT-6 secretion,such interspecies differencesmaybeof importance.Oneof

Current Opinion in Microbiology 2009, 12:410the identified key players in this process is PhoP, theresponse regulator of the two-component system PhoR/PhoP,which is essential for virulence ofM. tuberculosis [29].The link between a functional PhoP protein and ESAT-6secretion was recently demonstrated by complementationof the attenuatedM. tuberculosis strain H37Ra with a wild-type copy of gene phoP that restoredESAT-6 secretion andpartially increased the virulence of the recombinantH37Ra::phoP strain [30]. M. tuberculosis H37Ra carries apoint mutation in its phoP gene that interferes with theDNA binding capacities of PhoP [30,31,32] and appar-ently abrogates secretion of ESAT-6 via reduction ofrv3616c-rv3614c expression [30]. Previous transcriptomeanalyses that compared wild-type M. tuberculosis H37RvwithM. tuberculosisH37Raor a phoPknockoutmutant ofM.tuberculosis H37Rv, respectively identified at least 30genes, including cluster rv3616c-rv3614c, that were signifi-cantly lower expressed in both PhoP inactivated strains[33,34]. However, PhoP is not the only regulator thatinfluences rv3616c-rv3614c expression and therebyESAT-6 secretion.A transcription factor namedEspR, encodedbygene rv3849, binds to the rv3616c-rv3614c promoter and isitself secreted from M. tuberculosis by the ESX-1 system[35].Whether or notEspR is itself controlledby thePhoR/PhoP remains to be determined. Available transcriptomedata are not conclusive, as in some studies that used strainswith inactivated or mutated PhoP, the expression level ofEspR was low [34,36], while in others such downregula-tion was not observed [33].

Apart from ESX-1, transcriptome data show that expres-sion of proteins belonging to the ESX-3 cluster is con-trolled by the iron-dependent regulator IdeR [37] and thezinc uptake regulator Zur (previously named FurB) [38].This suggests that ESX-3 might be involved in funda-mental biological processes such as metal ion homeostasisof mycobacteria, which is consistent with the essentialityof ESX-3 for in vitro growth ofM. tuberculosis [39] and theconservation of orthologous ESX-3 systems in a widerange of mycobacterial species.

The ESX-1/T7S system effector proteinsinvolved in hostpathogen interactionAlthough several features related to pathogenicity werefound to be associated with ESX-1, the primary biologicalfunction of this system inM. tuberculosis and other ESX-1harboring bacteria still remains unclear. Reported ESX-1-related effects in tubercle bacilli include the suppressionof proinflammatory responses [11], necrosis [40], apopto-sis [41], membrano-lysis [42] and cytolysis [10,12]. Oneof the most intriguing questions in this respect is thenature of the ESX effector molecules. Several lines ofevidence suggest that ESAT-6 and CFP-10 are the keyeffector molecules of the ESX-1 system. However, it has

also been suggested that ESAT-6 and CFP-10 togetherwith Rv3616c (EspA) could serve as building blocks of theESX-1 secretion machinery in the cell envelope [27]. In

www.sciencedirect.com

this case ESAT-6, CFP-10 and EspA may simply besloughed off into the supernatant by mechanical forces.However, the fact that certain single amino acid changes inESAT-6did not prevent secretion of themodifiedESAT-6molecules but caused attenuation of the recombinant M.tuberculosis strains [43] argues in favor of ESAT-6 being asecreted effector molecule (Figure 2). Furthermore, thefinding that the C-terminal signal sequence of CFP-10 issufficient to secrete yeast fusion proteins via the ESXsystem argues for CFP-10 being a secreted protein[25]. Among the different proposed functions forESAT-6 and ESX-1 in the present scientific literature,the most frequently reported observations point towards afunction linked to lysis of cells and/or membranes. Indeed,Guinn et al.have reported thatESAT-6deletionmutants ofM. tuberculosis couldmultiply withinmacrophage-like cellslines but were unable to spread to uninfectedmacrophages[10]. It was also described that ESAT-6 deleted mutants

show reduced tissue invasiveness due to lacking cytolyticactivity [12], and it was shown that ESAT-6 and CFP-10interacted in a pH-dependent fashion with liposomes thatcontained membranes with biologically relevant phospho-lipids [42], (Figure 2). Furthermore, cryoelectron micro-scopy revealed that ESAT-6 destabilized and lysedliposomes, whereas CFP-10 did not [42]. Thus, the bio-logical activity of ESAT-6 might depend on the acidity ofthe environment, which is an important factor insidephagosomes. By contrast, a different study using circulardichroism spectroscopy suggested that complexes formedby ESAT-6 and CFP-10 and/or other ESX heterocomplexcouples were too stable to dissociate at lower pH [44]. Apossible explanation for this discrepancymight be that thefirst study [42] used culture filtrates from tubercle bacilli,which normally contain acetylated ESAT-6, whereas thesecond study [44] used non-acetylated ESAT-6 likeproteins expressed in Escherichia coli. In previous blot

ESX/type VII secretion systems and their role in hostpathogen interaction Simeone, Bottai and Brosch 7

Figure 2

w

ed

tion

te o

f pa

er

urfaPanel (a) shows a model of the ESAT-6-CFP-10 complex, with CFP-10 in

color codes. Red, enhanced virulence abolished; orange, reduced, interm

[43]). Panel (b) Presence of ESAT-6 in the top fraction of a liposome floata

observed when the same amount of ESAT-6 is supplied via culture filtra

complex with CFP-10, hiding its hydrophobic interphase. The right side o

derived culture filtrates in buffers with different pHs, suggesting that at low

binding of ESAT-6 to liposomes [42]. Panel (c) shows the results from s

dissociation of the CFP-10-ESAT-6 complex [42]. Please note that the ESATcarries an N-terminal acetylation [45], which might influence the stability of th

showing that ESAT-6 destabilizes and lyses liposomes, a feature that is not

www.sciencedirect.comhite and ESAT-6 in blue. The mutations and their effect are indicated by

iate virulence; green, ESX-1-enhanced virulence retained (after reference

assay indicates interaction between ESAT-6 and liposomes, which is not

f M. tuberculosis and/or BCG::RD1 wherein ESAT-6 is present as a 1:1

nel (b) shows results from liposome floatation analysis using BCG::RD1-

pH the complex formed by ESAT-6 and CFP-10 may dissociate and allow

ce plasmon resonance that confirm the effect of acidic pH on the-6 used in that experiment was derived from M. tuberculosis that usually

e complex. Panel (d) shows images obtained by cryoelectronmicroscopy

observed for CFP-10 [42].

Current Opinion in Microbiology 2009, 12:410

8 Host-microbe interactions: Bacteriaoverlay assays it was observed that CFP-10 interacted withnon-acetylated ESAT-6, while binding with acetylatedESAT-6 was less pronounced [45].

Together, these features appear important when put incontext with the recently described translocation of M.tuberculosis from the phagosome into the host cell cyto-plasm at later stages of infection [46]. While at days 4 to7 after infection withM. tuberculosismore than one third ofthe non-apoptotic human dendritic cells contained trans-located bacteria, this effect was not observed when BCGorM. tuberculosisESX-1 transposon mutants were used forinfection [46]. As M. tuberculosis is generally consideredto remain in phagosomes that resist maturation and acid-ification [47], these novel data on the translocation of M.tuberculosis are still controversial. However, a recent inde-pendent study using human macrophages, also reportedthat 25% of theM. tuberculosis cells at day 5 post-infectionwere not enclosed by a phagosomal membrane [48]. Assuch, the ESX-1 systemmight provideM. tuberculosiswiththe tools necessary for escaping from the phagosomalcompartment of professional phagocytic cells and/orreleasing ESAT-6 proteins to the cytoplasm, where theygain access to the class I-processing machinery containedin the proteasome. Such events would explain the strik-ingly higher recruitment and activation of CD8+ T cellsfound in the lungs of mice that were aerosol infected withM. tuberculosis or BCG::RD1 than uninfected or BCG-challenged mice [49]. Indeed, there is also more andmoreevidence from immunologic studies that ESX-1 proteinsgain access to the cytoplasm of the host cell [50].

In conclusion, the ESX/T7S systems represent novelsecretion systems, some of which are clearly involvedin pathogenicity and hostcell interaction. Great progresshas been made in the past five years to characterize thesesystems, but there are still many details of their biology tobe elucidated that might have important consequencesfor the design of novel antituberculosis vaccines andtherapeutic agents.

AcknowledgementsWe are grateful to our colleagues Marien de Jonge, Alexander Pym, PriscilleBrodin, Marjan Fretz, Wafa Frigui, Laleh Majlessi, Claude Leclerc, FelixRomain, Caroline Demangel, Patrick England, Gerard Pehau-Arnaudet,Michael Nilges and Stewart Cole who in recent years contributedsubstantially to the studies that are reviewed in this article. This work wassupported by the European Union (contracts LHSP-CT-2005018923,HEALTH-F3-2007-201762) and the Institut Pasteur.

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

of special interest of outstanding interest1. Sorensen AL, Nagai S, Houen G, Andersen P, Andersen AB:Purification and characterization of a low-molecular-massT-cell antigen secreted byMycobacterium tuberculosis. InfectImmun 1995, 63:1710-1717.

Current Opinion in Microbiology 2009, 12:4102. Pallen MJ: The ESAT-6/WXG100 superfamilyand a newGram-positive secretion system? Trends Microbiol 2002,10:209-212.

3. Brodin P, Rosenkrands I, Andersen P, Cole ST, Brosch R: ESAT-6proteins: protective antigens and virulence factors? TrendsMicrobiol 2004, 12:500-508.

4.

Abdallah A, Gey van Pittius N, Champion P, Cox J, Luirink J,Vandenbroucke-Grauls C, Appelmelk B, Bitter W: Type VIIsecretionmycobacteria show the way.Nat Rev Microbiol 2007,5:883-891.

Very complete and comprehensive overview of the ESX-related featuresin a large range of actinobacteria.

5.

Gey van Pittius NC, Sampson SL, Lee H, Kim Y, van Helden PD,Warren RM: Evolution and expansion of the Mycobacteriumtuberculosis PE and PPE multigene families and theirassociation with the duplication of the ESAT-6 (esx) genecluster regions. BMC Evol Biol 2006, 6:95.

This paper nicely shows evidence that PE and PPE proteins have co-evolved and have preceded the PE-PGRS and PPE-MPTR proteins.

6. Pym AS, Brodin P, Majlessi L, Brosch R, Demangel C, Williams A,Griffiths KE, Marchal G, Leclerc C, Cole ST: Recombinant BCGexporting ESAT-6 confers enhanced protection againsttuberculosis. Nat Med 2003, 9:533-539.

7. Pym AS, Brodin P, Brosch R, Huerre M, Cole ST: Loss of RD1contributed to the attenuation of the live tuberculosis vaccinesMycobacterium bovis BCG and Mycobacterium microti. MolMicrobiol 2002, 46:709-717.

8.

Brosch R, Gordon SV, Garnier T, Eiglmeier K, Frigui W, Valenti P,Dos Santos S, Duthoy S, Lacroix C, Garcia-Pelayo C et al.:Genome plasticity of BCG and impact on vaccine efficacy.Proc Natl Acad Sci U S A 2007, 104:5596-5601.

First whole genome analysis of BCG showing the genome sequence andthe in vitro evolution of one of the most often used live vaccines in thehistory of human vaccination; also reveals several molecular reasons whyBCG in immunocompetent individuals is safe.

9. Lewis KN, Liao R, Guinn KM, Hickey MJ, Smith S, Behr MA,Sherman DR: Deletion of RD1 from Mycobacteriumtuberculosis mimics bacille Calmette-Guerin attenuation.J Infect Dis 2003, 187:117-123.

10. Guinn KI, Hickey MJ, Mathur SK, Zakel KL, Grotzke JE,Lewinsohn DM, Smith S, Sherman DR: Individual RD1-regiongenes are required for export of ESAT-6/CFP-10 and forvirulence of Mycobacterium tuberculosis. Mol Microbiol 2004,51:359-370.

11. Stanley SA, Raghavan S, Hwang WW, Cox JS: Acute infectionand macrophage subversion by Mycobacterium tuberculosisrequire a specialized secretion system. Proc Natl Acad Sci U SA 2003, 100:13001-13006.

12. Hsu T, Hingley-Wilson SM, Chen B, Chen M, Dai AZ, Morin PM,Marks CB, Padiyar J, Goulding C, Gingery M et al.: The primarymechanism of attenuation of bacillus Calmette-Guerin is aloss of secreted lytic function required for invasion of lunginterstitial tissue. Proc Natl Acad Sci U S A 2003,100:12420-12425.

13.

Stinear TP, Seemann T, Harrison PF, Jenkin GA, Davies JK,Johnson PD, Abdellah Z, Arrowsmith C, Chillingworth T,Churcher C et al.: Insights from the complete genomesequence of Mycobacterium marinum on the evolution ofMycobacterium tuberculosis. Genome Res 2008, 18:729-741.

The comparative genomics approach in this paper identifies key regionsof M. tuberculosis that were horizontally transferred and contributed topathogenicity of M. tuberculosis.

14. Tobin DM, Ramakrishnan L: Comparative pathogenesis ofMycobacterium marinum and Mycobacterium tuberculosis.Cell Microbiol 2008, 10:1027-1039.

15.

Abdallah AM, Verboom T, Hannes F, Safi M, Strong M,Eisenberg D, Musters RJ, Vandenbroucke-Grauls CM,Appelmelk BJ, Luirink J et al.: A specific secretion system

mediates PPE41 transport in pathogenic mycobacteria. MolMicrobiol 2006, 62:667-679.

First report on the potential function of amycobacterial ESX-5 system andthe link to PPE proteins.

www.sciencedirect.com

ESX/type VII secretion systems and their role in hostpathogen interaction Simeone, Bottai and Brosch 916.

Abdallah AM, Savage ND, van Zon M, Wilson L, Vandenbroucke-Grauls CM, van der Wel NN, Ottenhoff TH, Bitter W: The ESX-5secretion system of Mycobacterium marinum modulates themacrophage response. J Immunol 2008, 181:7166-7175.

Further report on the potential function of a mycobacterial ESX-5 systemand the link to PPE proteins in relation with the immune system.

17. Coros A, Callahan B, Battaglioli E, Derbyshire KM: Thespecialized secretory apparatus ESX-1 is essential for DNAtransfer in Mycobacterium smegmatis. Mol Microbiol 2008,69:794-808.

18. Way SS, Wilson CB: The Mycobacterium tuberculosis ESAT-6homologue in Listeria monocytogenes is dispensable forgrowth in vitro and in vivo. Infect Immun 2005, 73:6151-6153.

19. Burts ML, WilliamsWA, DeBord K, Missiakas DM: EsxA and EsxBare secreted by an ESAT-6-like system that is required for thepathogenesis of Staphylococcus aureus infections. Proc NatlAcad Sci U S A 2005, 102:1169-1174.

20. Renshaw PS, Lightbody KL, Veverka V, Muskett FW, Kelly G,Frenkiel TA, Gordon SV, Hewinson RG, Burke B, Norman J et al.:Structure and function of the complex formed by thetuberculosis virulence factors CFP-10 and ESAT-6. EMBO J2005, 24:2491-2498.

21.

Sundaramoorthy R, Fyfe PK, Hunter WN: Structure ofStaphylococcus aureus EsxA suggests a contribution tovirulence by action as a transport chaperone and/or adaptorprotein. J Mol Biol 2008, 383:603-614.

This paper reveals the structural differences that exist between the ESXsystems of S. aureus and M. tuberculosis, in particular relating to homo-dimerization versus heterodimerization of EsxA/B complexes.

22. Garufi G, Butler E, Missiakas D: ESAT-6-like protein secretion inBacillus anthracis. J Bacteriol 2008, 190:7004-7011.

23.

Brodin P, Majlessi L, Marsollier L, de Jonge MI, Bottai D,Demangel C, Hinds J, Neyrolles O, Butcher PD, Leclerc C et al.:Dissection of ESAT-6 system 1 ofMycobacterium tuberculosisand impact on immunogenicity and virulence. Infect Immun2006, 74:88-98.

Comprehensive and extensive evaluation of involvement of RD1 proteinsin secretion of ESAT-6 and virulence of tubercle bacilli.

24. Luthra A, Mahmood A, Arora A, Ramachandran R:Characterization of Rv3868: An essential hypothetical proteinof the ESX-1 secretion system in M. tuberculosis. J Biol Chem2008, 283:36532-36541.

25.

Champion PA, Stanley SA, Champion MM, Brown EJ, Cox JS: C-terminal signal sequence promotes virulence factor secretionin Mycobacterium tuberculosis. Science 2006, 313:1632-1636.

This paper elucidates the mechanisms by which ESAT-6 and CFP-10 gettransported out of the bacterial cell and provides evidence that the C-terminal signal sequence of CFP-10 is sufficient for secretion of unrelatedyeast proteins via ESX-1.

26. Christie PJ, Atmakuri K, Krishnamoorthy V, Jakubowski S,Cascales E: Biogenesis, architecture, and function of bacterialtype IV secretion systems. Annu Rev Microbiol 2005,59:451-485.

27. Fortune SM, Jaeger A, Sarracino DA, Chase MR, Sassetti CM,Sherman DR, Bloom BR, Rubin EJ: Mutually dependentsecretion of proteins required for mycobacterial virulence.Proc Natl Acad Sci U S A 2005, 102:10676-10681.

28. MacGurn JA, Raghavan S, Stanley SA, Cox JS: A non-RD1 genecluster is required for Snm secretion in Mycobacteriumtuberculosis. Mol Microbiol 2005, 57:1653-1663.

29. Perez E, Samper S, Bordas Y, Guilhot C, Gicquel B, Martin C: Anessential role for phoP in Mycobacterium tuberculosisvirulence. Mol Microbiol 2001, 41:179-187.

30.

Frigui W, Bottai D, Majlessi L, Monot M, Josselin E, Brodin P,Garnier T, Gicquel B, Martin C, Leclerc C et al.: Control of M.tuberculosis ESAT-6 secretion and specific T cell recognitionby PhoP. PLoS Pathog 2008, 4:e33.

This paper provides for the first time evidence that ESAT-6 secretion is

regulated inM. tuberculosis and finds a link between the well-known two-component virulence regulator PhoP and ESAT-6 secretion that involvesthe rv3616c-rv3614c gene cluster. This link opens new perspectives toelucidate ESX and virulence regulation of M. tuberculosis.

www.sciencedirect.com31. Wang S, Engohang-Ndong J, Smith I: Structure of theDNA-binding domain of the response regulator PhoPfrom Mycobacterium tuberculosis. Biochemistry 2007,46:14751-14761.

32. Lee JS, Krause R, Schreiber J, Mollenkopf HJ, Kowall J, Stein R,Jeon BY, Kwak JY, Song MK, Patron JP et al.: Mutation in thetranscriptional regulator PhoP contributes to avirulence ofMycobacterium tuberculosis H37Ra strain. Cell Host Microbe2008, 3:97-103.

33.

Walters SB, Dubnau E, Kolesnikova I, Laval F, Daffe M, Smith I:The Mycobacterium tuberculosis PhoPR two-componentsystem regulates genes essential for virulence andcomplex lipid biosynthesis. Mol Microbiol 2006,60:312-330.

This analysis provides at least 44 candidate genes that are regulated bythe two-component regulator PhoP, some of which have recently beenshown to be involved in ESAT-6 secretion.

34.

Gao Q, Kripke K, Arinc Z, Voskuil M, Small P: Comparativeexpression studies of a complex phenotype: cord formation inMycobacterium tuberculosis. Tuberculosis (Edinb) 2004,84:188-196.

The supplementary data of this paper provide the results of an extra-ordinary large range of transcriptome comparisons between the virulentH37Rv and the attenuated H37Ra strains of M. tuberculosis.

35.

Raghavan S, Manzanillo P, Chan K, Dovey C, Cox J: Secretedtranscription factor controls Mycobacterium tuberculosisvirulence. Nature 2008, 454:717-721.

This paper describes the regulation of ESAT-6 secretion via influencingexpression of gene cluster rv3616crv3614c by the protein EspR(Rv3849), which is itself secreted by ESX-1.

36. Gonzalo-Asensio J, Mostowy S, Harders-Westerveen J,Huygen K, Hernandez-Pando R, Thole J, Behr M, Gicquel B,Martin C: PhoP: a missing piece in the intricate puzzle ofMycobacterium tuberculosis virulence. PLoS ONE 2008,3:e3496.

37. Rodriguez GM, Voskuil MI, Gold B, Schoolnik GK, Smith I: ideR, anessential gene in Mycobacterium tuberculosis: role ofIdeR in iron-dependent gene expression, iron metabolism,and oxidative stress response. Infect Immun 2002,70:3371-3381.

38.

Maciag A, Dainese E, Rodriguez GM, Milano A, Provvedi R,Pasca MR, Smith I, Palu G, Riccardi G, Manganelli R: Globalanalysis of the Mycobacterium tuberculosis Zur (FurB)regulon. J Bacteriol 2007, 189:730-740.

First evidence that the highly conserved ESX-3 system is regulated by theZink-uptake regulator Zur (previously named FurB) andmight therefore beinvolved in metal ion homeostasis.

39. Sassetti CM, Boyd DH, Rubin EJ: Genes required formycobacterial growth defined by high density mutagenesis.Mol Microbiol 2003, 48:77-84.

40. Junqueira-Kipnis AP, Basaraba RJ, Gruppo V, Palanisamy G,Turner OC, Hsu T, Jacobs WR Jr, Fulton SA, Reba SM, BoomWHet al.: Mycobacteria lacking the RD1 region do not inducenecrosis in the lungs of mice lacking interferon-gamma.Immunology 2006, 119:224-231.

41. Derrick SC, Morris SL: The ESAT6 protein of Mycobacteriumtuberculosis induces apoptosis of macrophages byactivating caspase expression. Cell Microbiol 2007,9:1547-1555.

42.

de Jonge MI, Pehau-Arnaudet G, Fretz MM, Romain F, Bottai D,Brodin P, Honore N, Marchal G, Jiskoot W, England P et al.: ESAT-6 from Mycobacterium tuberculosis dissociates from itsputative chaperone CFP-10 under acidic conditions andexhibits membrane-lysing activity. J Bacteriol 2007,189:6028-6034.

This paper provides evidence that suggests that ESAT-6 and CFP-10bind to biological membrane when they are not forming a complex, whichmay dissociate under certain conditions. The results shown in the paperalso suggest that ESAT-6 has membrane-lysing activity.43. Brodin P, de Jonge MI, Majlessi L, Leclerc C, Nilges M, Cole ST,Brosch R: Functional analysis of early secreted antigenictarget-6, the dominant T-cell antigen of Mycobacteriumtuberculosis, reveals key residues involved in secretion,

Current Opinion in Microbiology 2009, 12:410

complex formation, virulence, and immunogenicity. J BiolChem 2005, 280:33953-33959.

44. Lightbody KL, Ilghari D, Waters LC, Carey G, Bailey MA,Williamson RA, Renshaw PS, Carr MD: Molecular featuresgoverning the stability and specificity of functional complexformation by Mycobacterium tuberculosis CFP-10/ESAT-6family proteins. J Biol Chem 2008, 283:17681-17690.

45. Okkels LM, Muller EC, Schmid M, Rosenkrands I, Kaufmann SH,Andersen P, Jungblut PR: CFP10 discriminates betweennonacetylated and acetylated ESAT-6 of Mycobacteriumtuberculosis by differential interaction. Proteomics 2004,4:2954-2960.

46.

van der Wel N, Hava D, Houben D, Fluitsma D, van Zon M,Pierson J, Brenner MJPP: M. tuberculosis and M. lepraetranslocate from the phagolysosome to the cytosol in myeloidcells. Cell 2007, 129:1287-1298.

The work described in this paper shows evidence that at later stages ofinfection one third to one half of the M. tuberculosis cells in professionalphagocytes leave the phagosomes and enter the host cell cytoplasm, afeature that is not observed for ESX-1 lacking BCG and CFP-10/ESAT-6mutants. This paper provides clear-cut cryoelectronmicroscopicalimages of M. tuberculosis cells in the hostcell cytoplasm. The paperis controversial as it tries to change a long lasting dogma that M.tuberculosis resides and multiplies only in the phagosomal compartment.

47. Russell DG: Phagosomes, fatty acids and tuberculosis. Nat CellBiol 2003, 5:776-778.

48.

Lee BY, Clemens DL, Horwitz MA: The metabolic activity ofMycobacterium tuberculosis, assessed by use of a novelinducible GFP expression system, correlates with its capacityto inhibit phagosomal maturation and acidification in humanmacrophages. Mol Microbiol 2008, 68:1047-1060.

Although the central message of this paper is not on phagosomal escapeofM. tuberculosis, the reported observation that after 5 days of infection25% of the infecting M. tuberculosis cells were found without phagoso-mal membrane in the cytoplasm of human macrophages, confirms thedata on phagosomal escape proposed by van der Wel and colleagues.

49. Majlessi L, Brodin P, Brosch R, Rojas MJ, Khun H, Huerre M,Cole ST, Leclerc C: Influence of ESAT-6 Secretion System 1(RD1) of Mycobacterium tuberculosis on the interactionbetween mycobacteria and the host immune system.J Immunol 2005, 174:3570-3579.

50.

Stanley SA, Johndrow JE, Manzanillo P, Cox JS: The Type I IFNresponse to infection with Mycobacterium tuberculosisrequires ESX-1-mediated secretion and contributes topathogenesis. J Immunol 2007, 178:3143-3152.

This paper suggests that the ESX-1 secretion system secretes effectorsinto the cytoplasm of infected macrophages, thereby triggering the type IIFN response for the manipulation of host immunity.

10 Host-microbe interactions: BacteriaCurrent Opinion in Microbiology 2009, 12:410 www.sciencedirect.com

ESX/type VII secretion systems and their role in host-pathogen interactionIntroductionThe ESX-1 secretion machineRegulation of gene expression in ESX-1The ESX-1/T7S system effector proteins involved in host-pathogen interactionAcknowledgementsReferences and recommended reading