M Terrae

M Interjectum

M Genavense

M Paratuberculosis

M Szulgaï

Keywords: M. xenopi, M. kansasii, M. peregrinum, M. fortuitum, M

Using an ELISA test we found that that the antibody immune response to P27-PPE36 in the sera of patients was dominated by an IgA antibody response accompanied by the absence of IgG response. The immune response against the P27-PPE36 protein was investigated in mice. It was studied in the context of different pathogen associated molecular patterns (PAMPs). BALB/c mice were immunized either with the P27-PPE36 recombinant protein in Freund's adjuvant or in phosphate saline buffer (PBS), with a pcDNA3 plasmid containing the gene encoding the P27-PPE36 protein, or with the Escherichia coli bacteria expressing the P27-PPE36 protein genetically fused into the flagellin. We found that P27-PPE36 expressed into the flagellin led to the strongest cellular responses, where we obtained the highest production of IFN- and cell proliferation, an indication of specific Th1-like orientation of the immune response. ¶(14pt) 2. Early works on Rv2108 and genetic analysis ¶(14pt)

Mtb PCR-based assay detection test ¶(6pt)

, one pair of primers and two oligonucleotide probes were successfully used to amplify and to detect the DNA of strains belonging to the M. tuberculosis complex. These primers and probes did not hybridize with DNA from any of the 21 other mycobacterial species tested (M. avium, M. intracellulare, M. gordonae, M. chelonae

paratuberculosis [START_REF] Stinear | Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis[END_REF] and those despite some bacteria like M. marinum have an higher number of PPE genes than M. tuberculosis (106 vs. 69) ( [START_REF] Stinear | Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis[END_REF]. However an other analysis found a Rv2108 ortholog in the same strain (Agy99) of Mycobacterium ulcerans [START_REF] Riley | Identifying cognate binding pairs among a large set of paralogs: the case of PE/PPE proteins of Mycobacterium tuberculosis[END_REF]. This work presents also that Rv2108 gene is deleted in the strain C of Mycobacterium tuberculosis while it is present in the stains CDC1551, F11, H37Rv and Harlem as in the two strains of Mycobacterium bovis tested (BCG stain Pasteur 1173 and AF2122/97). According another study [START_REF] Gey Van Pittius | Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions[END_REF], Rv2108 have no orthologues in M. smegmatis, M avium paratuberculosis, M. leprae, M. ulcerans or M. marinum. This results confirm the interest of this gene in terms of diagnostic tool. M. tuberculosis has become highly specialized for intracellular survival in a very restricted range of mammalian hosts, and several recent studies have shown that lateral gene transfer (LGT) has been a major force in the evolution of the M. tuberculosis complex from an environmental Mycobacterium [START_REF] Kinsella | Fatty acid biosynthesis in Mycobacterium tuberculosis: Lateral gene transfer, adaptive evolution, and gene duplication[END_REF][START_REF] Gutierrez | Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis[END_REF][START_REF] Rosas-Magallanes | Horizontal transfer of a virulence operon to the ancestor of Mycobacterium tuberculosis[END_REF][START_REF] Becq | Contribution of horizontally acquired genomic islands to the evolution of the tubercle bacilli[END_REF]. In fact, Rv2108 appears to belong to one of the 80 regions (minimal number identified containing 360 Protein coding sequences (CDS)) that have probably been acquired by LGT in Mtb [START_REF] Stinear | Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis[END_REF]. Whether acquired by LGT or other means, some of these M. tuberculosis-specific regions contain known virulence genes or code for adaptation factors making them pottentially important for bacteria belonging to Mtb-complex. ¶(14pt)

Genomic organizations ¶(6pt)

Analysis of the genomic environment of the Rv2108 gene reveals that it is situated downstream a member of the pe gene family, Rv2107, coding for the PE22 protein (Fig. 1). These ajacent Rv2107 and Rv2108 genes lie in the same orientation. Occasionally, it can be noted that an insertion site IS6110 is localized between this two genes in the strains H37Rv and CDC1551 [START_REF] Beggs | Mapping of IS6110 insertion sites in two epidemic strains of Mycobacterium tuberculosis[END_REF]Sampson et al., 2001). Genome analysis by the operon/gene cluster method [START_REF] Strong | Inference of protein function and protein linkages in Mycobacterium tuberculosis based on prokaryotic genome organization: a combined computational approach[END_REF][START_REF] Bowers | Prolinks: a database of protein functional linkages derived from coevolution[END_REF] suggests that the PE and PPE families are functionally linked [START_REF] Gey Van Pittius | Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions[END_REF][START_REF] Tekaia | Analysis of the proteome of Mycobacterium tuberculosis in silico[END_REF][START_REF] Strong | Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis[END_REF][START_REF] Tundup | Clusters of PE and PPE genes of Mycobacterium tuberculosis are organized in operons: evidence that PE Rv2431c is co-transcribed with PPE Rv2430c and their gene products interact with each other[END_REF]. That is, the two genes tend to be in close chromosomal proximity on the Mtb genome [START_REF] Strong | Inference of protein function and protein linkages in Mycobacterium tuberculosis based on prokaryotic genome organization: a combined computational approach[END_REF][START_REF] Bowers | Prolinks: a database of protein functional linkages derived from coevolution[END_REF]. Based on their short intergenic distance (56 bp) and same transcription direction, Rv2107 and Rv2108 were assumed to belong to the same operon (Fig. 1) and so be co-transcribed. In Mtb genome, these same-operon PE/PPE pairs comprise less than 10% of the total number of PE and PPE genes (14 pairs of PE and PPE genes are found adjacent -same orientation, minimal intergenic distance -in the genome) [START_REF] Riley | Identifying cognate binding pairs among a large set of paralogs: the case of PE/PPE proteins of Mycobacterium tuberculosis[END_REF]. Genes separated by short intergenic sequences tend to have related function and interact physically [START_REF] Jacob | Genetic regulatory mechanisms in the synthesis of proteins[END_REF]. The structure of a complex of one PE/PPE protein pair was recently characterized [START_REF] Strong | Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis[END_REF][START_REF] Tundup | Clusters of PE and PPE genes of Mycobacterium tuberculosis are organized in operons: evidence that PE Rv2431c is co-transcribed with PPE Rv2430c and their gene products interact with each other[END_REF]. These results indicate that there may be many other instances of interactions between PE and PPE proteins. Like the PE and PPE proteins from the gene Rv2431c (PE25) and Rv2430c (PPE41) that interact together in vitro as probably in vivo [START_REF] Strong | Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis[END_REF][START_REF] Tundup | Clusters of PE and PPE genes of Mycobacterium tuberculosis are organized in operons: evidence that PE Rv2431c is co-transcribed with PPE Rv2430c and their gene products interact with each other[END_REF], it is stongly probable that PPE36 and PE22 have the same behavior. In fact, computational methods predict that the PE22/PPE36 interaction probability is almost the strongest of all the PE/PPE possible combinations tested [START_REF] Riley | Identifying cognate binding pairs among a large set of paralogs: the case of PE/PPE proteins of Mycobacterium tuberculosis[END_REF]. Furthermore, according to this analysis, this putative complexe is predicted to interact specifically, that is, PPE36 do not appear to interact with PEs other than its operon partner PE22, and vice versa [START_REF] Riley | Identifying cognate binding pairs among a large set of paralogs: the case of PE/PPE proteins of Mycobacterium tuberculosis[END_REF] but this supposition would need to be experimentaly confirmed. However due to the fact that Rv2108 is absent in M. tuberculosis strain C and Rv2107 is absent in M. tuberculosis strain F11, it is possible that another interacting partner is able to interact with the orphaned gene, possibly restoring the PE/PPE complex's function, or introducing new complexes that help these strains survive in their environmental niches [START_REF] Riley | Identifying cognate binding pairs among a large set of paralogs: the case of PE/PPE proteins of Mycobacterium tuberculosis[END_REF]. A putative interaction PE22/PPE36 is probably under the form of a 1:1 heterodimeric complex [START_REF] Strong | Inference of protein function and protein linkages in Mycobacterium tuberculosis based on prokaryotic genome organization: a combined computational approach[END_REF]. In their study, they found, as us [START_REF] Le Moigne | Expression, immunochemical, characterisation and localisation of the Mycobaterium tuberculosis protein p27[END_REF], that PPE36 is insoluble when expressed alone. The association with the relative PE protein would lead to a soluble complex: their experiments showed that proteins PE Rv2431c and PPE Rv2430 that are insosuble when expressed on their own are soluble when they are expressed together [START_REF] Strong | Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis[END_REF][START_REF] Tundup | Clusters of PE and PPE genes of Mycobacterium tuberculosis are organized in operons: evidence that PE Rv2431c is co-transcribed with PPE Rv2430c and their gene products interact with each other[END_REF]. ¶(9pt) Fig. 1. Genomic environment of Rv2108 gene. ¶(9pt) ¶(14pt)

Regulation expression of Rv2108 ¶(6pt)

A fundamental step in understanding the role of pe and ppe genes is elucidating how their expression is regulated. Although various studies have demonstrated that pe and ppe genes are expressed under a range of in vitro and in vivo conditions, they have not revealed any obvious indication of global pe and ppe gene regulation (Voskuil et al., 2004 (b)). This group of Voskuil and Smith has tested a large variety of diverse conditions to analyse gene expression in Mtb [START_REF] Manganelli | The Mycobacterium tuberculosis ECF sigma factor sigmaE: role in global gene expression and survival in macrophages[END_REF][START_REF] Sherman | Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha -crystallin[END_REF][START_REF] Manganelli | Role of the extracytoplasmic-function sigma factor sigma(H) in Mycobacterium tuberculosis global gene expression[END_REF][START_REF] Rodriguez | IdeR, an essential gene in Mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response[END_REF][START_REF] Schnappinger | Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment[END_REF][START_REF] Voskuil | Inhibition of respiration by nitric oxide includes a Mycobacterium tuberculosis dormancy program[END_REF]Voskuil et al., 2004 (a); Voskuil et al., 2004 (b)). Among them, only two conditions induce a variation in Rv2108 expression (at least two fold): in presence of 0.05% sodium dodecyl sulfate (SDS) 90 min, Rv2108 expression is repressed [START_REF] Manganelli | The Mycobacterium tuberculosis ECF sigma factor sigmaE: role in global gene expression and survival in macrophages[END_REF] as after 14 days of stationnary phase culture (Voskuil et al., 2004 (a)). Other conditions (Macrophage IFN activated, 24 h; diethylenetriamine/nitric oxide adduct (DETA/NO or DNO) 0.5 mM, 40 min; hydrogen peroxide (H2O2) 10 mM, 40 min; hypoxia (oxygen from 20% to 0.20%), 2 h [START_REF] Sherman | Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha -crystallin[END_REF][START_REF] Rustad | The enduring hypoxic response of Mycobacterium tuberculosis[END_REF]; palmitic acid 50 µm, 4h; non-replicating persistence (NRP) dormancy model 20 days; Iron high vs. low; diamide 5 mM, 1 h; potassium cyanide (KCN) 0.5 mM, 1 h; carbonyl-cyanide 3-chlorophenylhydrazone (CCCP) 0.5 mM, 1 h; ethambutol 10 µm, 24 h; nutrient starvation 24 h [START_REF] Betts | Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling[END_REF]; heat shock (45°C), 30 min [START_REF] Stewart | Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays[END_REF]; acid shock (pH 5.5 vs. 6.9) [START_REF] Fisher | Microarray analysis of the Mycobacterium tuberculosis transcriptional response to the acidic conditions found in phagosomes[END_REF]) do not appear to generate variation (more than 2 fold) in Rv2108 expression. The associated pe gene, Rv2107 (pe22), is found to be induced in macrophage culture of Mtb [START_REF] Schnappinger | Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment[END_REF] and in presence of 0.5 mM DETA/NO [START_REF] Voskuil | Inhibition of respiration by nitric oxide includes a Mycobacterium tuberculosis dormancy program[END_REF]. However, inversely, [START_REF] Park | Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis[END_REF] found that Rv2108 gene is induced by hypoxia (even if this needs confirmation since standard error deviation is elevated). However, contrarly to the majority of genes powerfully regulated by hypoxia, its induction does not requires the putative traznscription factor Rv3133/DosR. Like the majority of other PPE gene (54 of 69), Lsr2, a small basic protein highly conserved in mycobacteria that binds DNA and is implicated in gene regulation, is able to bind Rv2108 sequence [START_REF] Gordon | Lsr2 is a nucleoid-associated protein that targets AT-rich sequences and virulence genes in Mycobacterium tuberculosis[END_REF]. The binding of Lsr2 to the majority of pe/ppe genes suggests that this factor may negatively affect the expression of these antigenic proteins to modulate interactions with the host. More generally, Rv2108 has a low expression in the diverse M. tuberculosis strains that have been tested [START_REF] Gao | Gene expression diversity among Mycobacterium tuberculosis clinical isolates[END_REF] and it does not seem that there is a difference of Rv 2108 gene expression between M. bovis and M. tuberculosis in microarray analysis [START_REF] Rehren | Differential gene expression between Mycobacterium bovis and Mycobacterium tuberculosis[END_REF]. Furthermore, a study showed, by hy density mutagenesis experiments, that Rv2108 is not an essential gene for mycobacterial growth [START_REF] Sassetti | Genes required for mycobacterial growth defined by high density mutagenesis[END_REF]. In these experiments, only three of pe and ppe genes met the criteria for defining growth-attenuating mutations (Rv1807, Rv3872, and Rv3873). Although mutations in several other pe and ppe genes appeared to have subtle defects, the fact that such a small fraction are detected in this system suggests either that most of these genes are able to functionally complement each other, or that they are required under conditions that have not been testes. In the same study, the Mycobacterium leprae gene ML0411 is presented as an orthologue of Rv2108. In the Sanger Institute Mycobacterium leprae genome project, ML0411 is in fact described as being similar to Rv2108. ML0411 is coding for a protein 408-amino acid long named as a serinerich antigen (Sra) that have been largelly described (Vega-Lopez et al., 1993;[START_REF] De Wit | A Mycobacterium leprae-specific gene encoding an immunologically recognized 45 kDa protein[END_REF][START_REF] Macfarlane | Presence of human T-cell responses to the Mycobacterium leprae 45-kilodalton antigen reflects infection with or exposure to M. leprae[END_REF][START_REF] Parkash | Evaluation of recombinant serine-rich 45-kDa antigen (ML0411) for detection of antibodies in leprosy patients[END_REF] Rv2108 belongs the the 27% of genes that are not required for in vitro growth having M. leprae orthologues while the majority (78%) of the genes that they predict to be required for the optimal growth of M. tuberculosis have an orthologue in M. leprae genome. Thus, M. leprae appears to have selectively conserved the majority of genes that are necessary for optimal growth [START_REF] Cole | Massive gene decay in the leprosy bacillus[END_REF]. ¶(14pt)

Characterization of P27-PPE36 protein ¶(6pt)

The Rv2108 nucleotide sequence encoded for a 243 amino acid length protein. The P27-PPE36 antigen belongs to the PPE protein family, large family of protein present in Mtb, which represent 3% of the genome of this bacterium [START_REF] Cole | Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence[END_REF]. With the related PE protein family, they account for 10% of the genome. These families appear to have originated in the fast growing mycobacterial species before undergoing extensive expansion and diversification in certain slow growing species, particularly M. ulcerans, M. marinum and members of the M. tuberculosis complex (Gey van [START_REF] Gey Van Pittius | Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions[END_REF]. This asparagine or glycine-rich protein family containing 69 members has been termed PPE after the characteristic Pro-Pro-Glu motifs near the N-termini, in position 8-10. The relatively conserved N-terminal domain is about 180 amino acids lenght while C-terminal segments vary in sequence and length. According this C-terminal region, the PPE proteins are classified into four subfamilies: the first subfamily (24 members) , named PPE-SVP, has the well conserved motif Gly-X-X-Ser-Val-Pro-X-X-Trp located approximately at position 350; the second (23 members) constitutes the major polymorphic tandem repeats (MPTR) subfamily and is characterized by the presence of multiple tandem repeats of the motif Asn-X-Gly-X-Gly-Asn-X-Gly; the third subfamily (10 members), named PPE-PPW, is characterized by a highly conserved region comprising Gly-Phe-X-Gly-Thr and Pro-X-X-Pro-X-X-Trp motifs; and the last PPE subfamily (12 members) includes proteins with a low percentage of homology at the C-terminus that are unrelated other than having the PPE motif [START_REF] Gordon | Genomics of Mycobacterium bovis[END_REF][START_REF] Adindla | Sequence analysis corresponding to the PPE and PE proteins in Mycobacterium tuberculosis and other genomes[END_REF][START_REF] Gey Van Pittius | Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions[END_REF]. P27-PPE36 belongs to this last subfamily. A recent phylogenetic analysis of the 69 ppe genes present in the M. tuberculosis reference strain H37Rv has uncovered their evolutionary relationships and reveals that they can be divided into 5 sublineages which globally match the subfamilies described above (Gey van [START_REF] Gey Van Pittius | Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions[END_REF]. Rv2108 is classified in the sublineage III, having the most similarity with Rv3892c.

The role of the PPE proteins is still unknown. Firstly, they have been thought to be implicated in increasing antigenic variation and immune evasion due to the highly polymorphic nature of their C-terminal domains [START_REF] Cole | Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence[END_REF][START_REF] Cole | Learning from the genome sequence of Mycobacterium tuberculosis H37Rv[END_REF][START_REF] Karboul | Frequent homologous recombination events in Mycobacterium tuberculosis PE/PPE multigene families: potential role in antigenic variability[END_REF]. Concerning this, an interessant study realized by [START_REF] Plotkin | Detecting selection using a single genome sequence of M. tuberculosis and P. falciparum[END_REF] shows that PE/PPE proteins are under strong selection for amino acids substitution. They calculate volatility of codons which is the proportion of their point-mutaions neighbours that encode different amino acids. The volatility of a codon is used to quantify the chance that the most recent nucleotide mutation to that codon caused an amino-acid subtitution. According their calcul, Rv2108 has a volatility value of 0.1029, which place it at the 594 th rank of genes with the higher volatility among the 4099 genes values calculated. Furthermore, in agreement with the theory of an antigenic variation role, it has been observed that many PPE proteins present high levels of polymorphism like for exemple PPE38 (Rv2352c), PPE39 (Rv2353c) and PPE40 (Rv2356c) [START_REF] Mcevoy | Evidence for a rapid rate of molecular evolution at the hypervariable and immunogenic Mycobacterium tuberculosis PPE38 gene region[END_REF], PPE34 (Rv1917c) (Sampson et al., 2001(a)), PPE42 (Rv2608) [START_REF] Chakhaiyar | Regions of high antigenicity within the hypothetical PPE major polymorphic tandem repeat open-reading frame, Rv2608, show a differential humoral response and a low T cell response in various categories of patients with tuberculosis[END_REF], PPE8 (Rv0355c) [START_REF] Srivastava | Identification of a repetitive sequence belonging to a PPE gene of Mycobacterium tuberculosis and its use in diagnosis of tuberculosis[END_REF] or PPE18 (Rv1196) [START_REF] Hebert | DNA polymorphisms in the pepA and PPE18 genes among clinical strains of Mycobacterium tuberculosis: implications for vaccine efficacy[END_REF] and sequence variation has been observed between the orthologues of the PE and PPE protein families in in silico analyses of the sequenced genomes of M. tuberculosis H37Rv, M. tuberculosis CDC1551 and M. bovis [START_REF] Gordon | Genomics of Mycobacterium bovis[END_REF][START_REF] Fleischmann | Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains[END_REF]Garnier et al., 2003). However, this variability can not be extended to all pe/ppe family members since some are in fact conserved across strains and species [START_REF] Cubillos-Ruiz | Analysis of the genetic variation in Mycobacterium tuberculosis strains by multiple genome alignments[END_REF]. It has then been suggested that the PPE proteins may play a role in the virulence of Mtb [START_REF] Rindi | Search for genes potentially involved in Mycobacterium tuberculosis virulence by mRNA differential display[END_REF][START_REF] Li | A Mycobacterium avium PPE gene is associated with the ability of the bacterium to grow in macrophages and virulence in mice[END_REF], in the maintenance of bacterial growth in macrophages [START_REF] Camacho | Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis[END_REF][START_REF] Dubnau | Mycobacterium tuberculosis genes induced during infection of human macrophages[END_REF][START_REF] Hou | Mycobacterium avium genes expressed during growth in human macrophages detected by Selective Capture of Transcribed Sequences (SCOTS)[END_REF][START_REF] Li | A Mycobacterium avium PPE gene is associated with the ability of the bacterium to grow in macrophages and virulence in mice[END_REF][START_REF] Sassetti | Genes required for mycobacterial growth defined by high density mutagenesis[END_REF] and in the regulation of bacterial iron starvation and oxidative stress responses [START_REF] Rodriguez | Identification and characterization of two divergently transcribed iron regulated genes in Mycobacterium tuberculosis[END_REF][START_REF] Rodriguez | IdeR, an essential gene in Mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response[END_REF]. In addition, PPE might be a target for the protective immune response in experimental mouse models [START_REF] Skeiky | T cell expression cloning of a Mycobacterium tuberculosis gene encoding a protective antigen associated with the early control of infection[END_REF]. It has also be emitted the hypothesis that PPE proteins, due to their abundance of asparagine, could have a possible storage function for this amino acid which is one of the preferred nitrogen sources of the tubercle bacilli [START_REF] Tekaia | Analysis of the proteome of Mycobacterium tuberculosis in silico[END_REF]. Some PPE proteins, like PPE31 (Rv1807) could be involved in the protection from antibiotic stress targeting the envelope and help to confer the basal level of Mtb resistance to antibacterial drugs [START_REF] Provvedi | Global transcriptional response to vancomycin in Mycobacterium tuberculosis[END_REF]. Many PPE proteins are also known to induce a strong T cell and B cell responses and associate with the cell wall. Following surface exposure, these PPE proteins could act as agonists to various surface receptors of APCs resulting in modulation of the host immune responses [START_REF] Choudhary | PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response[END_REF][START_REF] Tundup | The co-operonic PE25/PPE41 protein complex of Mycobacterium tuberculosis elicits increased humoral and cell mediated immune response[END_REF][START_REF] Mishra | Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY[END_REF]Chaitra et al., 2008 (a); Chaitra et al., 2008 (b)). Recently, two PPE proteins, PPE18 (Rv1196) and PPE34 (Rv1917c), were found to specifically interact with the innate immune receptor TLR2 [START_REF] Nair | The PPE18 of Mycobacterium tuberculosis interacts with TLR2 and activates IL-10 induction in macrophage[END_REF][START_REF] Bansal | Src homology 3-interacting domain of Rv1917c of Mycobacterium tuberculosis induces selective maturation of human dendritic cells by regulating PI3K-MAPK-NF-kappaB signaling and drives Th2 immune responses[END_REF].

Very little is known about the protein encoded by the Rv2108 gene. Theoretical properties of P27-PPE36 protein are a low pHi (4.59) and representative amino acid composition is 12% for alanine and 9% for glutamic acid. Predictive secondary structure shows that this protein would be mainly constitued of alpha-helix (58,5%) and the absence of -feuillet. The resting amino acids (31%) would be in random coil. ¶(14pt)

Expression and purification of the PPE36 protein ¶(6pt)

The Rv2108 gene was amplified, inserted into bacterial vectors, sequenced, and expressed as a recombinant protein. Either the GST (pGEX-4T-3) in E. coli DH5 or the pET (pET15b) in E. coli BL21 (DE3) plasmid were used. Induction of the PPPE36 protein by these various expression systems lead to the expression of a protein with an apparent molecular mass of 43 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Fig. 2A andB). ¶(9pt)

(A) (B)

PPE36 GST-PPE36

GST ¶(9pt) This value was higher than the theoretical mass predicted by its DNA sequence translation of 27 kDa. Mass spectrometric analysis of the expressed protein in the pET system revealed a molecule at 29 kDa, which corresponds to the P27-PPE36 putative protein estimated mass plus 2 kDa for the polyHistidine fusion Tag [START_REF] Le Moigne | Expression, immunochemical, characterisation and localisation of the Mycobaterium tuberculosis protein p27[END_REF]. This result was confirmed by partial sequencing of the N-terminal region of the recombinant protein. The reason for this difference may be due to the nature of the P27-PPE36 protein, which belongs to a family of intrinsically unstructured proteins (IUP) with an atypical composition of amino acid sequences [START_REF] Tompa | Intrinsically unstructured proteins[END_REF]. It presents notably a high proportion of Proline dimers (3 for 243 amino acids). These proteins bind less to SDS than most other proteins and their apparent molecular mass is often 1.2-1.8 times higher than the real value calculated from sequence data or measured by mass spectrometry [START_REF] Dunker | Intrinsically disordered protein[END_REF]. Such a phenomen of electrophoresis abnormal migration has been observed for another protein belonging to the PE protein family of Mtb: the product of the gene Rv1441c has an apparent molecular weight of about 60 kDa instead of a theorical MW of 40,7 kDa [START_REF] Banu | Are the PE-PGRS proteins of Mycobacterium tuberculosis variable surface antigens?[END_REF].

Generally, PE and PPE proteins did not express well or expressed in insoluble or unfolded forms [START_REF] Strong | Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis[END_REF]. Our attemps to express P27-PPE36 under the form of a recombinant proteins confirm this rules and lead to the obtention of an insoluble protein [START_REF] Le Moigne | Expression, immunochemical, characterisation and localisation of the Mycobaterium tuberculosis protein p27[END_REF], as confirmed later by an other study [START_REF] Strong | Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis[END_REF]. The lack of apparent transmembrane elements is a possible explanation for their failure to express on their own is that they need protein partners to fold [START_REF] Strong | Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis[END_REF] like explained above in the Genomic organization paragraph. ¶(9pt) ¶(14pt)

Physico-chemical caracteristic of the PPE36 protein ¶(6pt)

Based on the DNA and protein sequences, the expected pI value of the P27 protein should be 4.8. To determine the PI value of the expressed p27 protein, a two-dimensional gel was applied to the cell lysates from the BCG strain. After gel transfer to a nitrocellulose membrane and blotting with the P27-PPE36-specific antibodies, only one spot with a pI between 4.5 and 5 at the same molecular mass level observed by SDS-PAGE was recognized on the membrane [START_REF] Le Moigne | Expression, immunochemical, characterisation and localisation of the Mycobaterium tuberculosis protein p27[END_REF]. ¶(14pt)

Anti-P27-PPE36 antibodies production and localization of P27-PPE36 ¶(6pt)

Very little is known about the cellular localization of the PPE protein family, a 143 kDa PPE protein encoded by the Rv1917c gene (PPE34) was found to be a cell-wall associated protein and probably surface exposed (Sampson et al., 2001) as well as the PPE68 protein (Rv3873 gene) located in the cell envelope [START_REF] Pym | Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti[END_REF][START_REF] Okkels | PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis: strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family[END_REF][START_REF] Demangel | Cell envelope protein PPE68 contributes to Mycobacterium tuberculosis RD1 immunogenicity independently of a 10-kilodalton culture filtrate protein and ESAT-6[END_REF]. We have generated specific mouse monoclonal and rabbit polyclonal antibodies to P27-PPE36 and used them for the immunochemical characterization and cellular localization of this protein. Specific immunoblot analysis confirmed the presence of the P27-PPE36 antigen in Mycobacterium bovis BCG strain and in human clinical isolates of M. tuberculosis from infected patients (Fig. 3), but not in other mycobacteria tested which does not belong to the Mtb complex [START_REF] Le Moigne | Expression, immunochemical, characterisation and localisation of the Mycobaterium tuberculosis protein p27[END_REF]. ¶(9pt) Fig. 3. Western blot analysis of bacterial lysate from different mycobacterial species. P27-PPE36 is the recombinant PPE36 protein. TB1, TB2 and TB3 are Mycobacterium tuberculosis clinical strains isolated from infected patients. Mycobacterium aurum is a fastgrowing mycobacteria, and BCG is the Calmette-Guérin bacillus. The first antibody is a mouse monoclonal IgG antibody directed against PPE36. ¶(9pt) Then, after demonstrating that the P27-PPE36 protein was present in the M. bovis BCG strain and in clinical isolates of M. tuberculosis, we attempted to localize this PPE protein in the BCG strain. To achieve this, bacteria were washed, fixed and ultrathin sections were prepared to be analysed by electron microscopy using immunohistochemistry test with specific anti-P27-PPE36 antibodies. Results generated with monoclonal (Fig. 4 A) and polyclonal antibodies (Fig. 4 B) revealed a peripheral localization of this protein on the cell membrane. Similar results were obtained using western-blot analysis (Fig. 4 D) of the Mtb cell fractions with the monoclonal anti-P27-PPE36 antibody indicating that the P27-PPE36 protein is localized in the membrane of the cell [START_REF] Le Moigne | Expression, immunochemical, characterisation and localisation of the Mycobaterium tuberculosis protein p27[END_REF].

This protein was the third member of its family to be localized at the periphery of the cell (Sampson et al., 2001;[START_REF] Pym | Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti[END_REF][START_REF] Okkels | PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis: strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family[END_REF][START_REF] Demangel | Cell envelope protein PPE68 contributes to Mycobacterium tuberculosis RD1 immunogenicity independently of a 10-kilodalton culture filtrate protein and ESAT-6[END_REF] and since, the same localization was assigned to other PPE proteins like for exemple Map3420c and Map1506 in Mycobacterium avium subsp. paratuberculosis [START_REF] Newton | Presence of PPE proteins in Mycobacterium avium subsp. paratuberculosis isolates and their immunogenicity in cattle[END_REF]. In Mycobacterium immunogenum, a PPE protein (accessio no. YP_001288073) have been found to be a cell-membrane-associated antigen [START_REF] Gupta | Immunoproteomic identification of secretory and subcellular protein antigens and functional evaluation of the secretome fraction of Mycobacterium immunogenum a newly recognized species of the Mycobacterium chelonae-Mycobacterium abscessus group[END_REF]. In a recent detailed analysis of the Mycobacterium marinum capsule using cryoelectron microscopy in conjunction with liquid chromatography mass spectrometry (LC-MS) demonstrated that 5 (MM1129, MM1402, MM0186, MM5047 and MM1497) of the 25 major cell surface proteins were members of the PPE familie [START_REF] Sani | Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins[END_REF]. Similarly high-throughput proteomics MALDI-MS and LC-MS approaches have been utilized by [START_REF] Målen | Definition of novel cell envelope associated proteins in Triton X-114 extracts of Mycobacterium tuberculosis H37Rv[END_REF] to identify 8 PPEs in the M. tuberculosis envelope fractions (PPE18, PPE20, PPE26, PPE32, PPE33, PPE51, PPE60 and PPE68). ¶(9pt) Therefore, these results suggest that cell wall/surface localization is a characteristic of several PE/PPE proteins although another PPE protein, PPE41, have been shown to be secreted by pathogenic mycobacteria [START_REF] Abdallah | Specific secretion system mediates PPE41 transport in pathogenic mycobacteria[END_REF]. So, if for the majority of PE and PPE proteins are localize to the cell wall, some of them could be secreted into the extracellular environment.

Like explained above, P27-PPE36 should be, as a disordered protein which need a partner to fold, associated with the PE protein PE22 (Gey van [START_REF] Gey Van Pittius | Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions[END_REF][START_REF] Strong | Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis[END_REF]. Moreover, this putative complex PPE36-PE22 could be associated with a system dedicated to the secretion of members of the potent T-cell antigen 6-kDa Early Secreted Antigenic Target (ESAT-6) family (Gey van [START_REF] Gey Van Pittius | Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions[END_REF]. According this last computational study constructing an evolutionary history of the pe and ppe genes families, Rv2107 and Rv2108 genes are hypothesized to have been duplicated from the ESAT-6 (esx) gene cluster regions, as they are very homologous to their paralogues within the ESAT-6 (esx) gene clusters and have the same paired genomic orientation. These esx clusters encode the socalled Type VII or ESX secretion systems, of which there are 5 in Mtb (Gey van [START_REF] Van Pittius | The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria[END_REF]. Thus, Rv2107 and Rv2108 would derive from ESAT-6 gene cluster Region 2, i.e. from Rv3893c (PE36) and Rv3892c (PPE69). ¶(14pt)

Serological studies ¶(6pt)

Diverse reports point out the potential immunodominant nature of PPE proteins. Presence of antibodies against other PPE proteins have been found in mycobacterium infected human or animals: in human against the PPE17 (Rv1168c) [START_REF] Khan | Association of strong immune responses to PPE protein Rv1168c with active tuberculosis[END_REF], PPE41 (Rv2340c) [START_REF] Choudhary | PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response[END_REF], PPE42 (Rv2608) [START_REF] Chakhaiyar | Regions of high antigenicity within the hypothetical PPE major polymorphic tandem repeat open-reading frame, Rv2608, show a differential humoral response and a low T cell response in various categories of patients with tuberculosis[END_REF], PPE55 (Rv3347c) [START_REF] Singh | Immunogenicity of the Mycobacterium tuberculosis PPE55 (Rv3347c) protein during incipient and clinical tuberculosis[END_REF], PPE57 (Rv3425) [START_REF] Zhang | PPE protein (Rv3425) from DNA segment RD11 of Mycobacterium tuberculosis: a potential B-cell antigen used for serological diagnosis to distinguish vaccinated controls from tuberculosis patients[END_REF], in human and mice against PPE68 (Rv3873) [START_REF] Daugelat | The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization[END_REF], in human [START_REF] Rindi | Variation of the expression of Mycobacterium tuberculosis ppe44 gene among clinical isolates[END_REF] and mice [START_REF] Romano | Immunogenicity and protective efficacy of tuberculosis subunit vaccines expressing PPE44 (Rv2770c)[END_REF][START_REF] Bonanni | Immunogenicity of mycobacterial PPE44 (Rv2770c) in Mycobacterium bovis BCG-infected mice[END_REF] against the PPE44 (Rv2770c), in cattle against PPE68 (Rv3873) [START_REF] Cockle | Identification of novel Mycobacterium tuberculosis antigens with potential as diagnostic reagents or subunit vaccine candidates by comparative genomics[END_REF], and against a PPE protein of Mycobacterium avium subsp paratuberculosis (Newton et al., 2008). Other studies highlight the capacity of PPE proteins to induce high B cell response in TB patients like PPE41 (Rv2340c) [START_REF] Tundup | The co-operonic PE25/PPE41 protein complex of Mycobacterium tuberculosis elicits increased humoral and cell mediated immune response[END_REF]. Inversely, a study shows that patients with tuberculosis do not develop a strong humoral response against the PPE44 protein [START_REF] Zanetti | Patients with pulmonary tuberculosis develop a strong humoral response against methylated heparin-binding hemagglutinin[END_REF]. In cattle, no difference is seen in the humoral response to the PPE44 (Rv2770c) between infected and TB-free animals [START_REF] Molicotti | Heparin binding haemagglutin as potential diagnostic marker of Mycobacterium bovis[END_REF].

The P27-PPE36 expressed protein is immunologically active, and reacts, in western-blot and ELISA, with antibodies from sera of patients infected with Mtb (Le [START_REF] Le Moigne | Expression, immunochemical, characterisation and localisation of the Mycobaterium tuberculosis protein p27[END_REF]. (Fig. 5). ¶(9pt) Fig. 5. Western blot analysis of the presence of anti-recombinant PPE36 antibodies in the sera of TB patients in comparison with serum from healthy donors and recombinant PPE36hyperimmunized rabbit. ¶(9pt) So, we then studied PPE36 specific antibody isotype distribution in sera of pulmonary tuberculosis patients and compared them to those in sera from healthy control by enzymelinked immunosorbent assay (ELISA). Our result showed a significant increase of IgA antibody response in patient's sera, but a less important IgM response accompanied by total absence of IgG2, 3, and 4 responses and a weak IgG1 response in few patients' sera (unpublished results).

The absence of IgG response in the sera of patients allowed verifying for the presence of immune complexes that may inhibit the interaction of antibodies with our antigen on the plate. Using an immunoprecipitation test with goat anti human immunoglobulin antibodies, no immune complex containing P27-PPE36 was present in the patient's sera.

Rec. PPE36

Rabbit anti-PPE36

TB infected patient serum

Healthy patient serum 45 kDa

The significance of IgA and IgM is not clear. The IgA response is the more interesting and intriguing results for this protein, because this is the first study showing the presence of IgA alone and the absence of an IgG response against a peptidic antigen. The IgA response is often considered to be local (mucosa and body surface) and non systemic (sera). It has also been reported as a more specific for the non peptidic antigens comparing to the IgG response, which was more reactive (Julián et al., 2005). The IgM response is in general related to the natural auto antibodies found in the sera of healthy and infected peoples and animals, and we couldn't ascribe it a diagnostic value 6 , though we note its augmentation during infection. These antibodies are in general polyspecific with weak affinity for their Ag.

The occurrence of antibodies against the PPE proteins is highly controversial; different studies highlighted the capacity of PPE proteins to induce high B cell response in TB human patients or infected animals [START_REF] Tundup | The co-operonic PE25/PPE41 protein complex of Mycobacterium tuberculosis elicits increased humoral and cell mediated immune response[END_REF][START_REF] Singh | Immunogenicity of the Mycobacterium tuberculosis PPE55 (Rv3347c) protein during incipient and clinical tuberculosis[END_REF]. Inversely, a study showed that patients with tuberculosis do not develop a strong humoral response against a PPE protein [START_REF] Zanetti | Patients with pulmonary tuberculosis develop a strong humoral response against methylated heparin-binding hemagglutinin[END_REF].

In comparison with other PPE proteins, P27-PPE36 proved to be less useful as a basis for the development of a TB diagnostic test. However, the presence of an IgA response in the absence of an IgG one, could be exploited as an indicator for Mtb diagnosis. A large number of sera should be tested to gather further information on the immune responses to this antigen. ¶(14pt)

Immune response against P27-PPE36 by different immunisation ways ¶(6pt)

We have studied the immune response of mice against the Mtb P27-PPE36 protein.

The peripheral localization of the P27-PPE36 protein led to the belief that they might play an important immunological role either in diagnosis or in protection. So, we examined the immune response against the P27-PPE36 protein using different Pathogen associated molecular patterns (PAMPs) as adjuvants and vectors for immunization. PAMPs are expressed only by micro-organisms and are recognized by the eukaryotic cells through the pattern recognition receptors (PRRs) of the innate immune system such as the Toll-like receptors (TLRs) [START_REF] Medzhitov | Innate immune recognition: mechanisms and pathways[END_REF]. The interaction of PAMPs with their corresponding TLRs helps to identify the nature of the PAMP and to guide the adequate adaptive immune response [START_REF] Medzhitov | Innate immune recognition: mechanisms and pathways[END_REF]. Muramyl dipeptides, a major element of the Freund's complete adjuvant, bacterial DNA, and bacterial flagellin are three PAMPs recognized by TLR2, TLR9, and TLR5, respectively. Different immunization protocols were used to study immunological potential of the P27-PPE36 protein. BALB/c mice were immunized either with the P27-PPE36 recombinant protein in Freund's adjuvant or in phosphate saline buffer (PBS) (classical immunization), with a pcDNA3 plasmid containing the gene encoding the P27-PPE36 protein (DNA immunization), or with the Escherichia coli bacteria expressing the P27-PPE36 protein genetically fused into the flagellin (flagellin immunization) [START_REF] Le Moigne | Flagellin as a good carrier and potent adjuvant for Th1 response: Study of mice immune response to the p27 (Rv2108) Mycobacterium tuberculosis antigen[END_REF]. (A): Proliferation of splenic cells of immunized mice after incubation in vitro with different concentrations (( ) 0.0 µg/ml, ( ) 1.1µg/ml, ( ) 3.3 µg/ml and ( ) 10µg/ml) of purified p27 recombinant protein. The proliferation was monitored by [ 3 H] thymidine uptake at 66 h after stimulation. (B): Cytokine secretion by splenic cells of immunized mice. Splenic cells were stimulated in vitro by the recombinant P27-PPE36 protein and IFN- was quantified in the supernatant after one week of culture. Results are presented as mean cytokine concentrations (±standard errors) compared to a standard curve of purified cytokines. (C): Specific anti-P27-PPE36 antibodies responses. Mice sera diluted at 1/500 were tested in ELISA for the presence of anti-P27-PPE36 antibodies of the different isotypes IgG1, IgG2a, IgG2b, IgG3, IgM and IgA one week after the third immunization. The results are presented as the optical density of the different isotypes. ¶(9pt) We found that P27-PPE36 expressed into the flagellin led to the strongest cellular responses, where we obtained the highest production of IFN-(Fig. 6 B)and cell proliferation (Fig. 6 A), an indication of specific Th1-like orientation of the immune response. DNA immunization was less potent in the induction of such responses. We confirmed the role of flagellin in this response by using different immunization combinations [START_REF] Le Moigne | Flagellin as a good carrier and potent adjuvant for Th1 response: Study of mice immune response to the p27 (Rv2108) Mycobacterium tuberculosis antigen[END_REF]. However, the specific antibody response was weak with either method (Fig. 6 C). On the other hand, classical immunization with the recombinant protein, soluble or incorporated in Freund's adjuvant still yielded the best antibody response (Fig. 6 C). The best cellular and humoral responses were obtained in the group of mice primed with the recombinant protein and boosted by the antigen presented on the modified flagellin [START_REF] Le Moigne | Flagellin as a good carrier and potent adjuvant for Th1 response: Study of mice immune response to the p27 (Rv2108) Mycobacterium tuberculosis antigen[END_REF]. In general, the P27-PPE36 PPE antigen induced a strong proliferative response accompanied by high production of IFN- and low amount of IL-4 (Le Moigne et al., 2008), independently of the PAMP used. The results indicated that this antigen may be involved in the establishment of the host cellular immune responses against the Mtb.

Protective anti-mycobacterial immunity is primarily mediated by cellular immune responses [START_REF] Flynn | Major histocompatibility complex class I restricted T cells are required for resistance to Mycobacterium tuberculosis infection[END_REF][START_REF] Caruso | Mice deficient in CD4 T cells have only transiently diminished levels of IFN-γ, yet succumb to tuberculosis[END_REF]. Mtb is rich in antigens that induce IFN- secretion, and the presence of such antigens has been reported in purified cell walls, the cytosolic fraction, and short-term culture filtrates (ST-CF) [START_REF] Mustafa | Biotechnology in the development of new vaccines and diagnostic reagents against tuberculosis[END_REF]. The importance of antibodies in tuberculosis is much debated, but it has been suggested that certain antibody specificities against bacterial surface epitopes and with the correct isotype may confer protection against intracellular infections [START_REF] Glatman-Freedman | Advances in antibody-mediated immunity against Mycobacterium tuberculosis: implications for a novel vaccine strategy[END_REF][START_REF] Glatman-Freedman | Serum therapy for tuberculosis revisited: reappraisal of the role of antibody-mediated immunity against Mycobacterium tuberculosis[END_REF][START_REF] Casadevall | Antibody immunity and invasive fungal infections[END_REF].

Other PPE proteins have been reported to be strongly immunogenic [START_REF] Choudhary | PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response[END_REF][START_REF] Demangel | Cell envelope protein PPE68 contributes to Mycobacterium tuberculosis RD1 immunogenicity independently of a 10-kilodalton culture filtrate protein and ESAT-6[END_REF][START_REF] Okkels | PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis: strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family[END_REF][START_REF] Dillon | Molecular characterization and human T-cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family[END_REF][START_REF] Skeiky | T cell expression cloning of a Mycobacterium tuberculosis gene encoding a protective antigen associated with the early control of infection[END_REF]. Antibodies against PPE41 (Rv2430c) are present in TB patients and not in healthy individuals [START_REF] Choudhary | PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response[END_REF]; PPE68 (Rv3873) induces IFN- production from splenocytes of M. tuberculosis-infected mice and from peripheral blood mononuclear cells of TB patients and PPD+ healthy individuals [START_REF] Demangel | Cell envelope protein PPE68 contributes to Mycobacterium tuberculosis RD1 immunogenicity independently of a 10-kilodalton culture filtrate protein and ESAT-6[END_REF][START_REF] Okkels | PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis: strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family[END_REF] and from cattle blood cells [START_REF] Cockle | Identification of novel Mycobacterium tuberculosis antigens with potential as diagnostic reagents or subunit vaccine candidates by comparative genomics[END_REF][START_REF] Mustafa | Immunogenicity of Mycobacterium tuberculosis RD1 region gene products in infected cattle[END_REF]. Immune responses elicited by PPE18 (Rv1196) and PPE14 (Rv0915c) have been shown to provide some protection in mice infected with M. tuberculosis [START_REF] Dillon | Molecular characterization and human T-cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family[END_REF][START_REF] Skeiky | T cell expression cloning of a Mycobacterium tuberculosis gene encoding a protective antigen associated with the early control of infection[END_REF]. Together, these studies suggest that several PPE proteins are expressed in vivo. In other mycobacteries, other PPE proteins have been shown to induce immune responses. For exemple in M. avium subs. paratuberculosis, two PPE proteins named Map39 and Map41 significantly elicited IFN- production in peripheral blood mononuclear cells from infected cattle [START_REF] Nagata | Expression cloning of gamma interferon-inducing antigens of Mycobacterium avium subsp[END_REF]. When immunized in mice, PPE57 (Rv3425) and PPE46 (Rv3018c) induce also strong humoral and cellular responses [START_REF] Wang | PPE protein (Rv3425) from DNA segment RD11 of Mycobacterium tuberculosis: a novel immunodominant antigen of Mycobacterium tuberculosis induces humoral and cellular immune responses in mice[END_REF][START_REF] Chaitra | Modulation of immune responses in mice to recombinant antigens from PE and PPE families of proteins of Mycobacterium tuberculosis by the Ribi adjuvant[END_REF] ¶(14pt)

Conclusion ¶(6pt

The P27-PPE36 protein is the third member of its family to be localized at the periphery of the cell (Sampson et al., 2001;[START_REF] Pym | Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti[END_REF][START_REF] Okkels | PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis: strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family[END_REF]. Now others PPE have been found to have a similar localization. This may shed some light on its role in the diagnosis and pathogenesis of Mtb. In conclusion, the P27-PPE36 protein was found to be a specific antigen for the Mtb complex and was recognized by sera of tuberculosis patients and localized in the membrane of the bacterial cell. ¶(14pt)

Acknowledgment ¶(6pt)

We thank Georges Robreau, Jean-Luc Guesdon and Caroline Borot for their help.

(14pt)

Fig. 2 .

 2 Fig. 2. Coomassie blue staining of bacterial lysates and purified recombinant PPE36 protein expressed with His-Tag (A) or with GST (B). (A): Lanes 1 and 2: bacterial extracts of E. coli BL21 (DE3) without or with IPTG induction, respectively. Lane 3: purified PPE36 protein. (B): Lanes 1 and 2: PPE36, GST-PPE36 fusion protein, partially cleaved or not cleaved by thrombin, respectively.

Fig. 4 .

 4 Fig. 4. Localization of P27-PPE36 antigen: Immunogold electron microscopic image (A-C) showing the peripheral localization of the P27-PPE36 protein in cryosectioned M. bovis BCG and western-blot on various M. tuberculosis cell fractions (D). Incubation was realized either with a mouse monoclonal antibody (A), or rabbit polyclonal anti-P27-PPE36 antibodies (B). Negative control was done using normal rabbit serum (C). (A: 49 000, B and C: 23 000). (D): Immunoblot analysis of different cell fractions of M. tuberculosis obtained from the Tuberculosis Research Materials and Vaccine Testing Laboratory, Colorado State University using monoclonal anti-P27-PPE36 antibody. Recombinant P27-PPE36 (rP27), cytosol fraction (CYT), cell membrane fraction (MEM), culture filtrate proteins (CFP), cell wall fraction (CW), and SDS-soluble cell wall proteins (SCWP).¶(9pt) Therefore, these results suggest that cell wall/surface localization is a characteristic of several PE/PPE proteins although another PPE protein, PPE41, have been shown to be secreted by pathogenic mycobacteria[START_REF] Abdallah | Specific secretion system mediates PPE41 transport in pathogenic mycobacteria[END_REF]. So, if for the majority of PE and PPE proteins are localize to the cell wall, some of them could be secreted into the extracellular environment.

Fig. 6 .

 6 Fig. 6. Immune response generated against P27-PPE36 from mice immunized either with flagellin-modified bacteria (B), DNA plasmid containing the Rv2108 gene (D) or with the P27-PPE36 recombinant protein associated (P) or not (sP) with Freund's adjuvant. Control