Cell Host & Microbe

Article

Strong Immunogenicity and Cross-Reactivity ofMycobacterium tuberculosis ESX-5 Type VII Secretion-Encoded PE-PPE Proteins Predicts Vaccine PotentialFadel Sayes,1,4 Lin Sun,1,4,7 Mariagrazia Di Luca,5 Roxane Simeone,2 Nathalie Degaiffier,1,4 Laurence Fiette,3,6

Semih Esin,5 Roland Brosch,2 Daria Bottai,5 Claude Leclerc,1,4 and Laleh Majlessi1,4,*1Unite de Regulation Immunitaire et Vaccinologie2Unite de Pathogenomique Mycobacterienne Integree3Unite d’Histopathologie Humaine et Modeles Animaux

Institut Pasteur, Paris F-75015, France4INSERM U1041, Paris F-75015, France5Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Universita di Pisa, 56100 Pisa, Italy6Faculte de Medecine, DER Histologie, Universite Versailles-Saint Quentin en Yvelines, 78000 Versailles, France7Present address: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, 225009 Yangzhou, Jiangsu, China

*Correspondence: laleh.majlessi@pasteur.fr

DOI 10.1016/j.chom.2012.03.003

SUMMARY

The genome of Mycobacterium tuberculosis (Mtb)encodes five type VII secretion systems, ESX-1 toESX-5, most of which are associated with genesencoding PE/PPE proteins, named after theirN-terminal Pro-Glu (PE) or Pro-Pro-Glu (PPE) motifs.Here, we describe the strong T cell immunogenicityof the ESX-5-encoded PE/PPE proteins, which sharea large panel of cross-reactive CD4+ epitopes withsubstantial numbers of their ESX-5-nonassociatedPE/PPE homologs. The immunogenicity of thesenumerous PE/PPE proteins is dependent on theirexport by a functional EccD5, the predicted trans-membrane channel of the ESX-5 secretion appa-ratus. The Mtb Dppe25-pe19 mutant deleted for allESX-5-associated pe and ppe genes, although highlyattenuated in immunocompetent mice, remains ableto induce immunity against the ESX-5-associatedPE/PPE virulence factors, via cross-reactivity withtheir numerous homologs, and against the ESX-1virulence factors ESAT-6/CFP-10. The Dppe25-pe19strain is strongly protective against Mtb infectionin mice and represents a potential antituberculosisvaccine candidate.

INTRODUCTION

Mycobacterium tuberculosis (Mtb) remains one of the world’s

most successful pathogens, infecting one-third of humans,

with �8 million new infections/year leading to �2 million

deaths/year. The currently available Mycobacterium bovis

Bacillus Calmette-Guerin (BCG) vaccine is of limited efficacy

against adult pulmonary tuberculosis (http://www.who.int/tb/

publications/global_reports/2009/). Identification of mycobacte-

rial virulence determinants and components that interact with the

352 Cell Host & Microbe 11, 352–363, April 19, 2012 ª2012 Elsevier

host immune system is critical to understand the mechanisms of

pathogenesis and immunogenicity of mycobacteria to develop

new drugs and vaccines. Compared to the virulent strain Mtb

H37Rv, BCG has numerous genetic deletions (Brosch et al.,

2000), including the 9.8 kb region of difference 1 (RD1). RD1

encodes members of a secretion system dedicated to the

export/secretion of the highly immunogenic major virulence

factors 6 kDa early secreted antigenic target (ESAT-6) and

10 kDa culture filtrate protein (CFP-10) (Brodin et al., 2004;

Mahairas et al., 1996; Simeone et al., 2009), as well as the Esp

proteins, which are putative virulence factors (Abdallah et al.,

2007; Bitter et al., 2009b; DiGiuseppe Champion et al., 2009).

ESAT-6 is the canonical member of the ESX protein family and

is secreted by the ESX-1 type VII secretion system (Abdallah

et al., 2007; Bitter et al., 2009b; DiGiuseppe Champion et al.,

2009). The products exported by the ESX-1 system modulate

the interaction of Mtb with the host immune system (Majlessi

et al., 2005; Stanley et al., 2007; van der Wel et al., 2007).

In addition to the esx-1 (RD1) region, the genome of Mtb

contains four other esx clusters, esx-2 to esx-5, which encode

paralogous ESX type VII secretion systems (Brodin et al., 2004;

Gey Van Pittius et al., 2001; Tekaia et al., 1999). These conserved

clusters contain tandem repeats of esx genes flanked by

genes encoding putative members of type VII secretion systems

(Bitter et al., 2009a; Brodin et al., 2004; DiGiuseppe Champion

et al., 2009). All esx clusters except for esx-4 include one

or more pe and ppe genes. The PE/PPE proteins, named for

their characteristic N-terminal Pro-Glu (PE) or Pro-Pro-Glu

(PPE) motifs (Bottai and Brosch, 2009; Brennan and Delogu,

2002; Cole et al., 1998), are two large protein families that are

unique to mycobacteria. The Mtb genome contains �100 pe

and �69 ppe genes (Cole et al., 1998), which may have

evolved from esx-encoded ancestral pe/ppe copies and are

characterized by homologous repetitive sequences (Gey van

Pittius et al., 2006). This close evolutionary link between esx

clusters and pe/ppe genes suggests that PE/PPE proteins

contribute to infection and pathogenesis. PE/PPE proteins

have been associated with virulence, persistence, and patho-

genesis in the host and may play a role in antigenic variation

Inc.

Figure 1. Identification of T Cell Epitopes of PPE25 or PE19

(A) Part of the Mtb esx-5 locus, encompassing the ppe25-pe19 region, ac-

cording to TubercuList.

(B and C) IFN-g responses of C57BL/6 (H-2b) mice infected s.c. with 1 3

106 CFU/mouse of Dppe25-pe19 or WTMtb. At d 21 p.i., pools of splenocytes

were stimulated in vitro with individual 15-mers from PPE25 (B) or PE19 (C).

Error bars represent ± SD. Solid outlines depict the epitopes that are specific

to ESX-5, while dotted outlines indicate epitopes that are shared with ESX-5-

nonassociated PE/PPE proteins. The results are representative of three

independent experiments.

See also Figure S1.

Cell Host & Microbe

ESX-5 System and Host-Mycobacteria Interaction

and immunodominance (Banu et al., 2002; Cole et al., 1998;

Iantomasi et al., 2011).

The esx-5 cluster is the most recently evolved cluster and is

only present in slow-growing pathogenic mycobacterial species

(Gey Van Pittius et al., 2001). Studies inM. marinum, a fish path-

ogen phylogenetically related to Mtb, revealed that in this

species ESX-5 system is involved in cell-to-cell spread and

secretion of several PPE proteins and all PE members of the

PE-polymorphic GC-rich sequences (PE-PGRS) subfamily

(Abdallah et al., 2006; Abdallah et al., 2009).

Here, we demonstrate themarked T cell immunodominance of

the ESX-5-associated PE/PPE proteins and their strong cross-

Cell H

reactivity with their numerous PE/PPE homologs encoded

outside of esx-5. This cross-reactivity is consistent with the

largely redundant nature of these proteins in Mtb. Moreover,

by characterization of an Mtb H37Rv strain knockout for the

esx conserved component D5 (eccD5, rv1795) gene (Bottai

et al., 2012), encoding the predicted transmembrane channel

of the ESX-5 secretion apparatus (Bitter et al., 2009a), we

show that the immunogenicity of these numerous proteins is

highly dependent on their export by a functional EccD5-contain-

ing secretion apparatus.

Furthermore, we provide evidence that an Mtb mutant lacking

the genomic segment containing all the ESX-5-associated

pe/ppe genes (Dppe25-pe19) (Bottai et al., 2012) is not virulent

in immunocompetent mice. The Dppe25-pe19 strain induces

substantial cross-reactive responses to the missing PE/PPE

virulence factors, due to the extensive redundancy of their

T cell epitopes, which are shared with numerous ESX-5-nonas-

sociated PE/PPE homologs. The Dppe25-pe19 strain also

induces potent T cell immunity against themajor ESX-1 virulence

factors ESAT-6 and CFP-10 and protects against Mtb infection

better than BCG.

RESULTS

T Cell Immunogenicity of ESX-5-AssociatedPE/PPE Proteins and Their Strong CD4+ T CellCross-Reactivities with Other PE/PPE ProteinsWe first investigated the T cell immunogenicity of the ESX-5-

associated PE/PPE proteins in Mtb-infected mice. The esx-5-

associated ppe25-pe19 (rv1787-rv1791) region (Figure 1A)

encodes (1) PE18 and PE19, with 89.9% sequence identity (Fig-

ure S1A available online) and (2) PPE25, PPE26 and PPE27, with

44.7% identity between PPE25 and PPE26 (Figure S1B) and

83.9% identity between PPE25 and PPE27 (Figure S1C).

We identified two groups of PPE25-derived T cell epitopes,

based on epitope mapping and IFN-g responses of C57BL/6

mice infected with WT Mtb or an Mtb strain deleted for the

ESX-5-associated ppe25-pe19 region (Dppe25-pe19) (Bottai

et al., 2012). One group of epitopes, consisting of PPE25:236-

255, PPE25:281-295, and PPE25:321-335, was recognized

by splenocytes from mice infected with WT Mtb but not from

mice infected with the Dppe25-pe19 strain (Figure 1B) and

were thereby specific to the ESX-5-associated PPE proteins.

Consistent with these results, a BLAST analysis of these

sequences against the TubercuList database (http://genolist.

pasteur.fr/TubercuList/index.html) detected no homologies

with other proteins. Another group of epitopes, composed of

PPE25:1-20, PPE25:41-65, PPE25:186-200, and PPE25:201-

215 (Figure 1B), was recognized by splenocytes from both

WT Mtb- or Dppe25-pe19-infected mice, indicating that they

are shared with PPE homologs encoded outside of esx-5.

Consistent with these results, a BLAST analysis identified

substantial sequence similarities among these PPE25 epitopes

and numerous ESX-5-nonassociated PPE proteins, including

PPE32, PPE22, PPE45, PPE30, PPE29, PPE60, PPE19, PPE18,

or PPE50, listed in order of decreasing sequence identity with

PPE25 (Table 1).

PE19:1-18 was identified as an ESX-5-specific epitope, based

on its recognition by splenocytes from mice infected with WT

ost & Microbe 11, 352–363, April 19, 2012 ª2012 Elsevier Inc. 353

Table 1. Paralogs of PPE25 or PE19 Proteins Encoded outside ESX-5 as Selected by BLAST Analysis of the Identified Immunogenic

Regions

Location Epitopes Sequence Sequence Identitya Percent Sequence Identity

PPE25

ESX-5 PPE25:1-20 (Rv1787) LDFGALPPEINSGRMYCGPG

ESX-5 PPE27:1-20 (Rv1790) MbDFGALPPEINSGRMYCGPG 19/20 95

Outside ESX-5 PPE32:1-20 (Rv1808) LDFGALPPEINSGRMYAGPG 19/20 95

Outside ESX-5 PPE22:1-20 (Rv1705c) MDFGALPPEVNSGRMYCGPG 18/20 90

Outside ESX-5 PPE45:1-20 (Rv2892c) LDFGVLPPEINSGRMYAGPG 18/20 90

Outside ESX-5 PPE30:1-20 (Rv1802) MDFGVLPPEINSGRMYAGPG 17/20 85

Outside ESX-5 PPE29:1-20 (Rv1801) MDFGLLPPEINSGRMYTGPG 17/20 85

Outside ESX-5 PPE60:2-21 (Rv3478) VDFGALPPEINSARMYAGPG 17/20 85

Outside ESX-5 PPE19:2-21 (Rv1361c) VDFGALPPEINSARMYAGPG 17/20 85

Outside ESX-5 PPE18:2-21 (Rv1196) VDFGALPPEINSARMYAGPG 17/20 85

ESX-5 PPE25:41-65 (Rv1787) AATGYASVIAELTGAPWVGAASLSM

ESX-5 PPE27:41-65 (Rv1790) AATGYASVIAELTGAPWVGAASLSM 25/25 100

Outside ESX-5 PPE50:41-65 (Rv3135) AAENYGSVIARLTGMHWWGPASTSM 16/25 64

ESX-5 PPE25:186-200 (Rv1787) VAAMPGLLQRLSSAA

Outside ESX-5 PPE45:197-211 (Rv2892c) TAAVPQLLQQLSSTS 9/15 60

ESX-5 PPE25:201-215 (Rv1787) SVSWSNPNDWWLVRL

ESX-5 PPE27:201-215 (Rv1790) SVSWPNPNDWWLVRL 14/15 93

Outside ESX-5 (Rv0525):99-113 DGAWRDPRVWWQLRN 5/15 33

PE19

ESX-5 PE19:34-51 PTTGVVPAAADEVSALTA

ESX-5 PE18:34-51 PTTGVVPAAADEVSALTA 18/18 100

Outside ESX-5 PE13:35-52 (Rv1195) PTTGVVPPAADEVSALTA 17/18 94

Outside ESX-5 PE32:35-52 (Rv3622c) PTTGVVPAAADLVSLLTA 16/18 88

Outside ESX-5 PE31:34-51 (Rv3477) PTTGVVPPAADEVSLLLA 15/18 83

Outside ESX-5 PE-PGRS46:35-52 (Rv2634c) PTTGVVAAAADEVSAAVA 15/18 83

ESX-5 PE19:82-99 ASSGSYAATEAANAAAAG

ESX-5 PE18:82-99 MSSGSYAATEAANAAAAG 17/18 94

Outside ESX-5 PE32:82-99 (Rv3622c) ISAGSYAATEAANAATIA 13/18 72

Outside ESX-5 PE13:82-99 (Rv1195) SSASSYAATEVANAAAAS 13/18 72

Outside ESX-5 PE20:82-99 (Rv1806) ASADSYAATEAANIIAVS 12/18 66

Bolded PE/PPE proteins are coded inside the ESX-5 region.aIdentical aa/epitope length.bUnderlined aa show sequence variability compared to PPE25 or PE19.

Cell Host & Microbe

ESX-5 System and Host-Mycobacteria Interaction

Mtb but not frommice infectedwith theDppe25-pe19 strain (Fig-

ure 1C). In contrast, PE19:34-51 and PE19:82-99 contain

epitopes that are shared with PE proteins encoded outside of

esx-5, as shown by the responses of both Mtb WT- or

Dppe25-pe19-infected mice (Figure 1C). A BLAST analysis

detected strong similarities between PE19:34-51 or PE19:82-

99 and several ESX-5-nonassociated PE proteins, including

PE13, PE32, PE31, PE-PGRS46, and PE20 (Table 1).

All of these PPE25 or PE19 regions were CD4+ T cell epitopes,

as they were only recognized by CD4+ T splenocytes sorted from

WTMtb- (Figure 2A) or Dppe25-pe19- (Figure 2B) infected mice.

Therefore, the presence of large numbers of PE/PPE proteins in

mycobacteria with highly conserved sequences leads to strong

redundancy among T cell epitopes and substantial CD4+ T cell

cross-reactivity.

354 Cell Host & Microbe 11, 352–363, April 19, 2012 ª2012 Elsevier

CD8+ T Cell Immunogenicity of ESX-5-AssociatedPPE ProteinsBased on the predicted affinity of 9-mers for major histocompat-

ibility complex-I (MHC-I) molecules (http://www.syfpeithi.de/),

we selected the sequences PPE26:44-52 and PPE25/27:44-52,

which are potentially H-2Kd restricted. Splenocytes from Mtb-

infected BALB/c mice produced IFN-g after in vitro stimulation

with these peptides (Figure S2A). As determined by intracellular

staining and FACS analyses, CD8+ T splenocytes from infected

mice produced IFN-g/TNF-a in response to these peptides

(Figure S2B). Despite their poor stability in culture, CD8+ T cell

hybridomas specific to PPE26:44-52 (Figure S2C) were gener-

ated from the lymphoid organs of BCG-immunized mice. These

hybridomas were able to produce IL-2 when cocultured with

dendritic cells (DCs) loaded with the PPE26:44-52 peptide

Inc.

Figure 2. Determination of the Phenotype of

T Cells Specific to the Identified Epitopes of

PPE25 or PE19

Responses of magnetically sorted CD4+ or CD8+ T

splenocytes from Mtb WT- (A) or Dppe25-pe19-

(B) infected mice to the identified PPE25 or PE19

epitopes that are either highly specific to ESX-5 or

shared with PE/PPE encoded outside of ESX-5.

The CD4+ or CD8+ fractions contained, respec-

tively, at least 99% CD4+ T cells and 97% CD8+

T cells. Error bars represent ± SD. The results with

WT strain are representative of two independent

experiments and one experiment was performed

with Dppe25-pe19 strain. See also Figure S2.

Cell Host & Microbe

ESX-5 System and Host-Mycobacteria Interaction

(Figure S2C) and were all restricted by H-2Kd, as only L fibro-

blasts transfected with H-2Kd were able to present the

PPE26:44-52 peptide to these T cell hybridomas (Figure S2D).

The TCR of these hybridomas interacted specifically with the

PPE26:44-52 peptide bound to H-2Kd Dextramer (Figure S2E).

The latter allowed ex vivo detection of PPE26:44-52-specific

CD8+ T cells in the lymphocytes from lungs and spleen of Mtb-

infected mice, and stimulated in vitro with the PPE26:44-52

peptide (Figure S2F). Based on a BLAST analysis against the

TubercuList database, and in contrast to the numerous MHC-

II-restricted T cell epitopes identified above, these MHC-I-

restricted T cell epitopes from ESX-5-associated PPE proteins

do not exhibit homology with other Mtb proteins.

Therefore, we conclude that ESX-5-associated PPE proteins

gain efficient access to the MHC-I presentation pathway of the

infected host cells and elicit specific CD8+ T cell effectors that

specifically produce key cytokines that are involved in the control

of mycobacterial growth.

EccD5 Controls the Immunogenicity of a Plethoraof PE/PPE Proteins Encoded in or outside of ESX-5Located downstream of the ppe25-pe19 region, the eccD5 gene

(Figure 1A) is a paralog of the eccD1 gene from the ESX-1 cluster.

The eccD1 gene encodes a presumed transmembrane channel

protein that is required for the secretion of ESAT-6 and CFP-10

(Abdallah et al., 2007; Simeone et al., 2009). T cell immunoge-

nicity of ESX-1 antigens is directly dependent on their active

secretion (Pym et al., 2003). To address whether EccD5 is

involved in the immunogenicity of ESX-5-encoded PE/PPE

proteins, we investigated the capacity of an Mtb eccD5KO strain

(Bottai et al., 2012) to induce CD4+ T cell responses to ESX-5-

associated PE/PPE proteins. We observed that the WT Mtb

strain induced T cell responses to PPE25 (Figure 3A) or PE19

(Figure 3B) epitopes, either highly specific to ESX-5 or shared

with their ESX-5-nonassociated homologs. As expected, the

Dppe25-pe19 strain induced responses only to epitopes shared

with PE/PPE proteins encoded outside of esx-5. The eccD5KO

strain induced responses neither to the epitopes specific to

Cell Host & Microbe 11, 352–3

ESX-5, nor to those shared with the

ESX-5-no-encoded PE/PPE homologs.

In the complemented eccD5KO::esx-5

derivative, the capacity to induce

responses to all of these epitopes was

fully restored (Figures 3A and 3B). All of

the strains induced PPD- or Ag85A-specific T cell responses.

This indicates that the ESX-5 system is involved in T cell immu-

nogenicity of ESX-5-associated PE/PPE proteins as well as

numerous PE/PPE proteins encoded outside of ESX-5, likely

due to its role in their export/secretion. Based on T cell cross-

reactivity and the BLAST analyses, cross-reactive PE/PPE

proteins encoded outside of esx-5 include the PPE25 and

PE19 homologs listed in Table 1. In vitro expression studies

demonstrated that all ESX-5-encoded pe and ppe genes were

transcribed in the eccD5KO strain (Figure 3C), further suggesting

that the lack of T cell response against the ESX-5 PE/PPE

proteins observed in the eccD5KO-immunized mice was due to

a defect in transport/secretion of these proteins. Thus, the

ESX-5 system, by controlling the immunogenicity of large

numbers of PE/PPE proteins, appears to impact the profile of

the host anti-mycobacterial adaptive immunity.

Role of the ESX-5-Associated ppe25-pe19 Regionin the Virulence and Pathogenic Potential of Mtbin Immunocompetent MiceTo investigate the virulence of the Dppe25-pe19 strain in immu-

nocompetent mice, C57BL/6 mice were infected i.n. (intranasal

route) with �1 3 104 colony forming units (CFU)/mouse of

Dppe25-pe19, its complemented Dppe25-pe19::esx-5 deriva-

tive or the WT strain (Figure 4A). In contrast to the WT and

complemented strains, on day 14 postinfection (p.i.), the

Dppe25-pe19 strain did not grow, and at days 24 and 28 p.i. it

could not be cultured from the lungs or spleens of the infected

mice (Figure 4A). At day 28 p.i. in the lungs of Dppe25-pe19-in-

fected mice, only minimal infiltration of the peribronchial and

perivascular spaces by macrophages (MF) and lymphocytes/

plasmocytes was noted, and no acid-fast bacilli were observed

on Ziehl-Neelsen-stained sections (Figure 4B). By contrast,

extensive infiltration by MF, epitheloid cells, lymphocytes/plas-

mocytes and neutrophils was noted in the lungs of Dppe25-

pe19::esx-5-infected mice, and numerous acid-fast bacilli were

detected. On day 14 p.i., substantial recruitment of CD11b+ cells

was detectable in the lungs from C57BL/6 mice infected with the

63, April 19, 2012 ª2012 Elsevier Inc. 355

Figure 3. EccD5 Controls the Immunogenicity of ESX-5-Encoded or -Nonencoded PE/PPE Proteins

(A and B) IFN-g responses of C57BL/6mice injected s.c. with 13 106 CFU/mouse of theMtbWT,Dppe25-pe19, eccD5KO, or eccD5KO::esx-5 strain to PPE (A) or

PE (B) protein. At day 21 p.i., pools of splenocytes were stimulated in vitro with PPD, Ag85A:241-260, or control individual immunogenic PPE25 (A) or PE19 (B)

peptides that are either highly specific to ESX-5 or shared with PE/PPE encoded outside of ESX-5. Error bars represent ± SD. Data are representative of two

independent experiments.

(C) Expression of ESX-5-associated pe and ppe genes in WT or eccD5KO mutant strain. As control, the expression of the housekeeping gene sigA was tested.

See also Table S1.

Cell Host & Microbe

ESX-5 System and Host-Mycobacteria Interaction

complemented or WT Mtb strains, as judged by the increase of

percentages (Figure 4C) and absolute numbers (Figure 4D) of

these cells per mouse. On day 28 p.i., lung T cells from WT

Mtb-infected mice displayed modulation of CD44 expression

and increased percentages (Figures 4E and 4F) and absolute

numbers (data not shown) of inflammatory CD27- CD45RB- cells

(Lyadova et al., 2000). These disease-associated cell activation

and recruitment of innate and adaptive immune cells were only

barely detected or not observed in Dppe25-pe19-infected

356 Cell Host & Microbe 11, 352–363, April 19, 2012 ª2012 Elsevier

mice (Figures 4C–4F). Consistently, the Dppe25-pe19 strain dis-

played attenuated growth capacity both in SCID mice and in

C57BL/6 bone-marrow-derived (BM) MF (Bottai et al., 2012).

Role of the ppe25-pe19 Region in the Interactionof Mycobacteria with Effector Cells of the InnateImmune SystemAt day 21 after i.v. (intravenous route) infection of SCIDmice with

13 106 CFU of WT Mtb, we detected substantial increase in the

Inc.

Figure 4. Attenuated Virulence and Pathogenic Potential of the Dppe25-pe19 Strain in Immunocompetent Mice

(A) C57BL/6 mice (n = 3) were infected i.n. with �1 3 104 CFU/mouse of the Dppe25-pe19, Dppe25-pe19::esx-5, or WT strain and mycobacterial loads were

determined at day 1, 7, 14, 24, or 28 p.i. in the lungs or spleen of individual mice. The absence of proliferation of the Dppe25-pe19 strain at day 28 p.i. was

observed in three independent experiments.

(B) Lesions in the lung at day 28 p.i., as shown by hematoxylin and eosin (H&E) staining (upper and middle panels) and Ziehl-Neelsen staining (lower panel). Scale

bars represent 662.5 mm (upper panel), 100 mm (middle panels), and 25 mm (lower panels).

(C and D) Percentages of CD11b+ cells, compared to whole lung low-density cells (C) or absolute numbers (D) of these cells per mouse at day 14 p.i.

In (A) and (D), error bars represent ± SD.

(E and F) Expression of activation markers by lung CD4+ or CD8+ T subsets at day 28 p.i. The results are representative of at least two independent experiments.

See also Figure S3.

Cell Host & Microbe

ESX-5 System and Host-Mycobacteria Interaction

percentages of CD11b+ cells in the lungs. Part of these cells had

upregulated BST-2 (bone marrow stromal cell antigen-2) (Fig-

ure S3A), most likely due to systemic type-I/II IFN production

(Blasius et al., 2006). This population was mainly composed of

activated MF (CD11bhi F4/80bright), neutrophils (CD11b+ Ly6C+

Ly6G+), and monocytes (CD11b+ Ly6C+ Ly6G–) (Figure S3B). In

the Dppe25-pe19-infected mice, fewer of these cells were re-

cruited to the lungs, as judged by evaluation of their percentages

Cell H

and absolute numbers per mouse (Figures S3A and S3B). Lower

level of lung CD11c+ cell maturation occurred, as well (Fig-

ure S3C). At this time point, the mycobacterial loads in the lungs

and spleens of Dppe25-pe19-infected mice were 2 log10 less

compared to WT Mtb-infected controls (data not shown). In

correlation with the lower bacterial burdens, weaker levels of

pro-inflammatory cytokines or chemoattractants were detected

in the organ homogenates of Dppe25-pe19-infected SCID mice,

ost & Microbe 11, 352–363, April 19, 2012 ª2012 Elsevier Inc. 357

Cell Host & Microbe

ESX-5 System and Host-Mycobacteria Interaction

as compared to their WT Mtb-infected counterparts (Figures

S3D and S3E). We wondered whether this weak inflammation

reflects the reduced virulence/persistence of the Dppe25-pe19

strain in a SCID host or its altered capacity to simulate cells of

the innate immune system. In vitro infection with the Dppe25-

pe19, Dppe25-pe19::esx-5 or WT Mtb strains induced compa-

rable phenotypic maturation of DCs (Figure S4A). DCs (Fig-

ure S4B) or MF (Figure S4C) infected with the Dppe25-pe19,

Dppe25-pe19::esx-5 or WT strain also expressed similar levels

of several inflammatory cytokines and chemokines which play

key roles in the innate immunity against mycobacteria. There-

fore, the attenuated Dppe25-pe19 strain remains a potent acti-

vator of the innate immune system.

The Attenuated Virulence of the Dppe25-pe19 Strain IsNot Due to Defective Expression of ESX-1 VirulenceFactorsWe next asked whether the observed role of the ppe25-pe19

region in virulence was due to any interference with the expres-

sion of the ESX-1-associated major virulence factors ESAT-6

and CFP-10. The MHC-II-restricted presentation and immuno-

genicity of ESAT-6 and CFP-10 by antigen-presenting cells are

directly proportional to the functionality of the ESX-1 system

and secretion of these factors (Brodin et al., 2005; Pym et al.,

2003). Here, we show that Dppe25-pe19-immunized mice

mounted robust ESAT-6- or CFP-10-specific T cell responses

that were comparable to WT Mtb-immunized controls (Fig-

ure 5A). Moreover, using an I-Ab-restricted, ESAT-6:1-20-

specific T cell hybridoma (Frigui et al., 2008), we showed that

DC infected with the Dppe25-pe19, Dppe25-pe19::esx-5 or

WT Mtb strain presented comparable amounts of ESAT-6;

presentation of Ag85A to a specific T cell hybridoma (Majlessi

et al., 2006) was used as an internal control (Figure 5B). The

absence of the ppe25-pe19 region also did not alter the secre-

tion or antigenic presentation of the ESX-3-associated putative

virulence factor TB10.4, based on its presentation, which was

proportional to that of Ag85A, by DC infected with the

Dppe25-pe19, Dppe25-pe19::esx-5 or WT Mtb strain to an

I-Ad-restricted TB10.4:74-88-specific T cell hybridoma (Fig-

ure 5C). The eccD5KO strain, which is over-attenuated in viru-

lence (Bottai et al., 2012), also exhibited similar behavior (Fig-

ure 5D, E). Thus, the ppe25-pe19 region and eccD5 gene do

not affect the secretion of ESX-1/3-associated virulence factors.

Protective Capacity of theDppe25-pe19 Strain as a LiveAttenuated VaccineImmune responses to virulence factors are usually associated

with protection. Our results demonstrate that the Dppe25-pe19

strain displays the unique phenotype of being drastically attenu-

ated, yet still inducing Th1 responses against ESAT-6 and CFP-

10. Due to the presence of an intact eccD5 gene, this strain

induces T cell immunity to large numbers of PE/PPE proteins en-

coded by regions outside of ESX-5 (Figures 2 and 3), which is

potentially protective (Sampson, 2011). Moreover, the Dppe25-

pe19 strain induces immunity to virulence factors that it does

not express due to cross-reactivity among ESX-5-encoded PE/

PPE proteins and their homologs. This strain also keeps its

potential to activate the innate immunity (Figure S4). We thus

evaluated the protective potential of the Dppe25-pe19 strain

358 Cell Host & Microbe 11, 352–363, April 19, 2012 ª2012 Elsevier

against Mtb. C57BL/6 mice were immunized s.c. (subcutaneous

route) with BCG or Dppe25-pe19. We also included mice vacci-

nated with BCG complemented with the complete RD1

(BCG::RD1), which has an improved protective effect compared

to BCG, yet also has increased virulence and pathogenic poten-

tial (Majlessi et al., 2005; Pym et al., 2002; Pym et al., 2003). Mice

were challenged one month later via the aerosol route with�100

CFU of Mtb H37Rv. At 1 month p.i., the protection conferred by

Dppe25-pe19 was comparable to that conferred by BCG::RD1,

i.e., significantly improved compared to BCG (Figure 6A). In an

independent experiment, the improved protective effect of

Dppe25-pe19 was also observed in the lungs and spleen (Fig-

ure 6B). Lungs of Dppe25-pe19-immunized mice showed less

extensive lesions compared to BCG-immunized mice; however,

the composition of MF, epitheloid cells, lymphocytes/plasmo-

cytes and neutrophils was qualitatively similar (Figure 6C). In

contrast, the mice immunized with the attenuated eccD5KO

strain (Bottai et al., 2012) were protected less efficiently than their

counterparts immunizedwith theDppe25-pe19strain (Figure6B).

This observation suggests that immunity to large numbers of PE/

PPE proteins is likely to be correlated with protection.

DISCUSSION

In this study, we demonstrate the critical impact of the Mtb ESX-

5 secretion system in antimycobacterial immunity and patho-

genic potential in immunocompetent mice. While the ESX-5

system of the aquatic M. marinum plays a role in its interaction

with its natural fish host (Abdallah et al., 2007; Weerdenburg

et al., 2012), little is known regarding the ESX-5 of the human

pathogen Mtb. We first showed the strong CD4+ T cell immuno-

genicity of ESX-5-associated PE/PPE proteins. Moreover, we

presented evidence that the extensive redundancy of PE/PPE

proteins in mycobacteria results in substantial CD4+ T cell

cross-reactivity, allowing the Mtb Dppe25-pe19 strain to induce

CD4+ T cell effectors against ESX-5-associated PE/PPE proteins

via their homologs encoded by regions outside of ESX-5. Using

PE/PPE-derived CD4+ T cell epitopes specific to ESX-5 or

shared with their homologs encoded by regions outside of

ESX-5, we elucidated the instrumental role of EccD5 in the immu-

nogenicity of large numbers of PE/PPE proteins.

Reminiscent of the secretion of the ESAT-6:CFP-10 hetero-

dimer (Brodin et al., 2004), PE/PPE proteins do not contain

N-terminal secretion signals andmay require other PE/PPE part-

ners to form soluble heterodimers (Strong et al., 2006) to be

successfully secreted. EccD5 is a paralog of EccD1 (encoded

by the esx-1 cluster) which is responsible for the export of

ESAT-6:CFP-10. EccD5 is a putative membrane protein with 11

transmembrane domains predicted to form the core component

of amembrane-associated complex involved in the translocation

of ESX substrates (Bitter et al., 2009a). Here, we demonstrate

that the T cell immunogenicity of numerous PE/PPE proteins,

whether or not they are encoded by ESX-5, is dependent on

the EccD5 transmembrane channel and therefore probably on

the ESX-5 secretion system. Thus, ESX-5 controls the host

adaptive immunity to dozens or hundreds of Mtb immunogens.

This also correlates well with the fact that several PE/PPE

proteins, including PPE41, are secreted via the ESX-5 system

in M. marinum (Abdallah et al., 2006).

Inc.

Figure 5. Absence of Interference of the ppe25-pe19 Region or the eccD5 Gene with the Expression of ESX-1- or ESX-3-Related Virulence

Factors

(A) IFN-g production by splenocytes fromC57BL/6mice that were uninfected or infected s.c. with 13 106CFU/mouse of theDppe25-pe19 orWT strain, at day 18

p.i., subsequent to in vitro stimulation with PPD, rESAT-6, rCFP-10, or the control rMalE protein or with ESAT-6:1-20 or MalE:100-114 synthetic peptides.

(B) In vitro presentation of ESAT-6 or the control Ag85A by BM-DC- (H-2b) infected with various concentrations of different mycobacterial strains, asmeasured by

their capacity to stimulate the I-Ab-restricted T cell hybridomas, specific to ESAT-6:1-20 (NB11) or Ag85A:241-260 (DE10), respectively.

(C) In vitro presentation of the ESX-3-associated TB10.4, shown as a ratio compared to Ag85A presentation, by BM-DC (H-2d) infected with different myco-

bacteria, as indicated by their capacity to stimulate the I-Ad-restricted TB10.4:74-88-specific (1H2) over the capacity to stimulate the I-Ad-restricted Ag85A:101-

120-specific (2A1) T cell hybridomas.

(D) IFN-g production by splenocytes from C57BL/6 mice, injected s.c. with 1 3 106 CFU/mouse of the eccD5KO, eccD5KO::esx-5 or WT strain.

(E) Presentation of TB10.4 by BM-DC (H-2d) infected with the eccD5KO or eccD5KO::esx-5 strain, as measured using the TB10.4-specific (1H2) hybridoma. Error

bars represent ± SD. The results are representative of three independent experiments.

Cell Host & Microbe

ESX-5 System and Host-Mycobacteria Interaction

In the ESX-5 system, EsxN (TB9.9A) is the paralog of ESAT-6

(Bitter et al., 2009a). The T cell immunogenicity of this protein

has been demonstrated in human (Al-Attiyah et al., 2004;

Alderson et al., 2000) and bovine (Jones et al., 2010). Notably,

Esx-N protein was produced by Dppe25-pe19 and eccD5KO

strains but was not detected in the corresponding culture super-

natants, which demonstrates that the export/secretion of EsxN is

also controlled by the ESX-5 secretion system (Bottai et al.,

2012).

By identifying PPE25-26-27-derived MHC-I-restricted T cell

epitopes, we also demonstrated that in Mtb-infected mice,

ESX-5-associated PPE proteins gain efficient access to the

Cell H

MHC-I presentation machinery of the host antigen presenting

cells and elicit, at the site of the infection, specific CD8+ T cell

effectors that produce key cytokines to control mycobacterial

growth. The fact that the Dppe25-pe19 strain, lacking these

PPE25/26/27-derived CD8+ T cell epitopes, still provides very

good protection, questions whether these particular CD8+

T cell responses have a strong impact on the outcome of infec-

tion. However, the identification and characterization of PPE25/

26/27-derived CD8+ T cell epitopes contribute to enlarge the

very limited numbers of MHC-I-restricted mycobacterial epi-

topes so far described for further investigation of anti-mycobac-

terial CD8+ T cell responses.

ost & Microbe 11, 352–363, April 19, 2012 ª2012 Elsevier Inc. 359

Figure 6. Protective Potential of the

Dppe25-pe19 Strain against Mtb Infection

(A and B) C57BL/6 mice were immunized s.c. with

BCG, BCG::RD1, Dppe25-pe19, or eccD5KO

strain and were challenged 1 month later with

�100 CFU/mouse of WT Mtb H37Rv via aerosol

route. Themycobacterial loads in the lungs/spleen

were determined at 1 month p.i. Two independent

experiments are depicted. * and ** indicate

statistical significance, as determined by the

Student’s t and Mann-Whitney tests, p < 0.01 and

p < 0.008, respectively. Horizontal bars represent

the means of six mice.

(C) Histological sections showing lesions of the

lung, H&E staining. Scale bars represent 662.5 mm

(left panel) and 100 mm (right panel).

See also Figure S4.

Cell Host & Microbe

ESX-5 System and Host-Mycobacteria Interaction

Only a few mycobacterial PE/PPE proteins in different myco-

bacterial species are known to be involved in host-pathogen

interactions. PPE46 was the first member of the PPE family to

be implicated in mycobacterial growth, as shown in a mouse

BM-MF model (Camacho et al., 1999). ESX-5-encoded PPE

proteins have been shown to be involved in the virulence of

M. avium (Li et al., 2005), and PE-PGRS30 was proposed to be

required for full virulence of Mtb (Iantomasi et al., 2011). Here,

by investigating mycobacterial loads, tuberculous lung lesions,

and disease-associated inflammation in C57BL/6 mice, we

showed a drastic reduction in the virulence and pathogenic

properties of the Dppe25-pe19 strain.

ESX secretion systems can be transregulated (Frigui et al.,

2008) or exhibit crosstalk among themselves, as is the case

with ESX-1-encoded PE35 (Brodin et al., 2006). Therefore, it is

possible that ppe25-pe19 depletion and the resulting ESX-5

deficiency may impair the expression of the major virulence

factors related to other ESX systems, thereby leading to this

drastic reduction in virulence. We ruled out this possibility, at

least for the known major ESX-associated virulence factors, by

showing that the expression of neither the ESX-1 virulence

factors ESAT-6 and CFP-10, nor the ESX-3-associated putative

virulence factor TB10.4, was affected in Dppe25-pe19 or

eccD5KO strains. Therefore, the ESX-5-associated PE/PPE

proteins and EccD5 play an essential role in Mtb virulence.

However, we cannot exclude the possibility that the virulence

functions of ESAT-6 and CFP-10 may be dependent on ESX-5-

associated PE/PPE proteins. The biochemical mechanism of

virulence mediated by the ESX-5-associated PE/PPE proteins

remains to be elucidated.

It is important to note that other ESX-5-associated KOmutants

affecting genes immediately downstream of the ppe25-pe19

region, i.e., esxNKO or rv1794KO, retained a virulent phenotype

(Bottai et al., 2012). This fact together with our genetic comple-

360 Cell Host & Microbe 11, 352–363, April 19, 2012 ª2012 Elsevier Inc.

mentation strategy confirm that the atten-

uated virulence and other phenotypic

traits of the Dppe25-pe19 strain were

not due to polar effects of the genetic

construct on downstream genes.

One of the most important findings in

this study is the particularly high protec-

tive effect of the Dppe25-pe19 strain over that of BCG against

infection with virulent Mtb. The Dppe25-pe19 strain displays

the unique characteristics of being drastically attenuated in viru-

lence, while, in contrast to BCG, it secretes ESAT-6 and CFP-10

and induces Th1 responses to these antigens, which are known

to be protective immunogens and also seem to intervene in intra-

cellular localization and phagosomal escape of mycobacteria

(Simeone et al., 2012; van der Wel et al., 2007). The better

protective effect of the Dppe25-pe19 strain over that of the

eccD5KO mutant can be explained by the presence of an intact

eccD5 gene in the former and therefore its capacity at inducing

T cell immunity against a plethora of ESX-5-nonassociated

PE/PPE proteins and, due to their strong cross-reactivity,

against the ESX-5-associated PE/PPE virulence factors that

this strain has lost. Moreover, the Dppe25-pe19 strain remains

potent at inducing the activation of the innate system, which is

instrumental in the initiation of anti-mycobacterial adaptive

immunity. Therefore, overall, the Dppe25-pe19 strain meets the

three necessary conditions to represent an attractive live vaccine

candidate: (1) drastically attenuated virulence, (2) capacity to

induce immunity against ESX-1 and ESX-5 virulence factors,

and (3) capacity to initiate appropriate innate immunity. Indeed,

we demonstrated that this attenuated ESX-5 mutant had

a protective effect comparable to BCG::RD1, which is known to

be more protective than BCG. However, unlike BCG::RD1

(Majlessi et al., 2005; Pym et al., 2002), the Dppe25-pe19 strain

is neither virulent nor pathogenic in immunocompetent mice.

During the preparation of this manuscript, the prominent

protective effect of a recombinantM. smegmatis Desx-3mutant

complemented with the orthologous Mtb esx-3 was reported

(Sweeney et al., 2011). Altogether, the strongly improved

protective capacity of BCG::RD1 (esx-1) (Pym et al., 2003),

M. smegmatis Desx-3::Mtb esx-3 (Sweeney et al., 2011), and

the Dppe25-pe19 esx-5 Mtb mutant demonstrate that genetic

Cell Host & Microbe

ESX-5 System and Host-Mycobacteria Interaction

manipulation of the type VII secretion systems constitutes a

powerful strategy to generate live attenuated anti-Mtb vaccines.

One of the most prominent characteristics of the Dppe25-pe19

strain is its capacity to induce T cell responses against ESX-1

virulence factors, and—by cross reactivity—against the ESX-5-

associated PE/PPE virulence factors that it has lost. Therefore,

the Dppe25-pe19 mutant may pave the way to set up a new

generation of safe live attenuated vaccine against infection

with pathogenic Mtb.

EXPERIMENTAL PROCEDURES

Mice and Infections

C57BL/6 (Janvier, Le Genest-Saint-Isle, France) or BALB/c (Charles River,

Arbresle, France) mice were anesthetized by i.p. injection of 100 mg/kg

Ketamine (Imalgene, Lyon, France) and 10 mg/kg Xylazine (Rompun, KVP

Kiel, Germany) and were infected i.n. with 60 ml/mouse of mycobacterial

suspensions at 2.5 3 105 CFU/ml for a total inoculation of �1 3 104 CFU/

mouse. Subcutaneous (s.c.) immunization was performed at the base of the

tail. Six-week-old female SCID mice (Charles River) were infected i.v. via

the tail vein with 1 3 106 CFU/mouse. All animal studies were approved by

the Institut Pasteur Safety Committee, in accordance with French and Euro-

pean guidelines and regulations.

Cytofluorometry

Low-density cells or adaptive immune cells from infected mice were prepared

as previously described (Majlessi et al., 2005). They were first incubated with

an anti-CD16/CD32 monoclonal antibody (mAb), followed by the indicated

mAbs, all of which were purchased from BD PharMingen (Le Pont-de-Claix,

France). Intracellular cytokine staining was performed as described previously

(Mouries et al., 2008). The PPE26:44-52 peptide, bound to polymerized H-2Kd,

immobilized on a Dextramer backbone and conjugated to PE was from

Immundex (Copenhagen, Denmark). The stained cells were fixed with 4%

paraformaldehyde overnight at 4�C and were acquired with a CyAn system

using Summit software (Beckman Coulter, Villepinte, France). Data were

analyzed with FlowJo software (Treestar, OR).

RNA Extraction and RT-PCR Reactions

RNA was extracted from mycobacteria in exponential growth phase, as previ-

ously described (Bottai et al., 2012). RNA (300 ng) was denatured by incuba-

tion at 70�C for 5 min and subjected to RT-PCR reactions with the SS-One

Step RT-PCR kit (Life Technologies). Retrotranscription and amplification

reactions were carried out at 50�C (retrotranscription) and 94�C, 58�C, and72�C (denaturation, annealing and extension in amplification cycles), respec-

tively. Sequences of primers used are listed in Table S1.

Generation of Anti-TB10.4 or PPE-Specific T Cell Hybridomas

T cell hybridomas specific to mycobacterial antigens were generated from

BALB/c (H-2d) mice immunized s.c. with 1 3 107 CFU/mouse of the BCG

Pasteur 1173P2 strain, as previously described (Majlessi et al., 2003). The

BW5147 cell line or BW5147::CD8 cells were used as fusion partners for

MHC-II-restricted hybrids and MHC-I-restricted hybrids, respectively. The

resulting T cell hybridomas were screened for their capacity to recognize

BM-DC loaded with various peptides.

Antigen Presentation Assays

BM-DC were infected at 13 105 cells/well with different mycobacterial strains

at various multiplicities of infection in antibiotic-free complete RPMI, as previ-

ously described (Majlessi et al., 2003). After overnight incubation at 37�C, cellswere washed and incubated overnight with 1 3 105 cells/well of the appro-

priate T cell hybridomas. The amount of IL-2 produced by the specific T cell

hybridomas was measured by ELISA analysis of the coculture supernatants.

T Cell Assays

Pooled splenocytes from groups of infected mice were cultured at 1 3

106 cells/well in HL-1 medium (Biowhittaker, Lonza, France) complemented

Cell H

with 2 mM GlutaMax (Invitrogen, Life Technologies, France), 5 3 10�5 M

b-mercaptoethanol, 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma-

Aldrich, France) in the presence of PPD, rESAT-6 or rCFP-10 proteins or

appropriate peptides. After 72 hr of incubation, IFN-g in the culture superna-

tants was quantified by ELISA.

Pepscan Assay

Mice were immunized s.c. with 1–53 106 CFU/mouse of different mycobacte-

rial strains. Two to 3 weeks p.i., total splenocytes or sorted CD4+ or CD8+ T

splenocytes were stimulated with 10 mg/ml of individual synthetic 15-mers

from PPE25 (71 15-mers, overlapping by 5 aa) or PE19 (29 15-mers, overlap-

ping by 3 aa) (Mimotopes, Melbourne, Australia). After 72 hr of incubation,

IFN-g was quantified by ELISA. CD4+ or CD8+ T splenocytes from H37Rv

WT Mtb-immunized mice were positively sorted with anti-CD4 or anti-CD8

mAbs coupled to magnetic beads (Miltenyi Biotec, Mercoeur, France).

ELISA

ELISA assays of the culture supernatants or organ homogenates were per-

formed as previously described (Majlessi et al., 2005). mAbs specific to IL-2,

IL-6, IL-10, IL-12p40, or IFN-g were from BD PharMingen. mAbs specific to

IL-1b, CCL-2, and CCL-3 were from R&D Systems (Lille, France). Anti-TNF-a

mAbs were from eBioscience (Paris, France).

Protection Assay

Female C57BL/6 mice were immunized s.c. with 1 3 106 CFU/mouse of

different mycobacterial strains. One month later, mice were challenged as

detailed elsewhere (Jaron et al., 2008). The infected mice were placed in

isolator in A3 protection-level facilities. One month p.i., the lungs and

spleen were individually homogenized with a MillMixer organ homogenizer

(QIAGEN, Courtaboeuf, France). Serial 5-fold dilutions were plated on

7H11 Agarmedium supplementedwith ADC (Difco, Becton Dickinson, Sparks,

MD). The CFU were counted after 18 days of incubation at 37�C. For histolog-ical analyses, the left lobe of the lungs was fixed in formalin and embedded

in paraffin. Five micrometer sections were stained with H&E and Ziehl-

Neelsen.

Statistical Analyses

Statistical analyses were performedwith GraphPad Prism software (GraphPad

Software, La Jolla, CA). The Student’s t and Mann-Whitney tests were

employed based on the distribution of the data.

SUPPLEMENTAL INFORMATION

Supplemental Information includes four figures and one table and can be

found with this article online at doi:10.1016/j.chom.2012.03.003.

ACKNOWLEDGMENTS

This work was supported by the European Community’s FP7 program under

grant agreement number 201762. F.S. is a recipient of a PhD fellowship from

the University of Damascus, Syria. The authors gratefully acknowledge

Dr. Francoise Guinet (Institut Pasteur) and Dr. Richard Lo-Man (Institut

Pasteur) for critical reading of the manuscript, Dr. Peter Sebo (Academy of

Sciences, Czech Republic) for providing recombinant ESAT-6 and CFP-10,

and Eddie Maranghi and Karim Sebastien for expert assistance in animal

care in the A3 facilities. F.S., D.B. and L.M. performed the experiments and

analyzed data. L.S. performed several pepscan assays and generated

MHC-I restricted T-cell hybridomas. N.D. generated and characterized the

anti-TB10.4 T-cell hybridoma. M.D.L., D.B., R.S., R.B., and S.E. designed

and constructed mycobacterial mutants. L.F. performed histopathological

analyses. D.B., R.B., C.L., and L.M. designed and interpreted experiments.

D.B., C.L., and L.M. wrote the manuscript.

Received: November 28, 2011

Revised: February 23, 2012

Accepted: March 22, 2012

Published: April 18, 2012

ost & Microbe 11, 352–363, April 19, 2012 ª2012 Elsevier Inc. 361

Cell Host & Microbe

ESX-5 System and Host-Mycobacteria Interaction

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