Abstract. Introduction. Asma Ahmed 1 Arghya Das 1,2 Sangita Mukhopadhyay 1 *

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1 Critical Review Immunoregulatory Functions and Expression Patterns of PE/PPE Family Members: Roles in Pathogenicity and Impact on Anti-tuberculosis Vaccine and Drug Design Asma Ahmed 1 Arghya Das 1,2 Sangita Mukhopadhyay 1 * 1 Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad, Telengana, India 2 Manipal University, Manipal, Karnataka, India Abstract The Mycobacterium tuberculosis genome was sequenced more than 15 years ago. It revealed a lot of interesting information, one of which was that 10% of the total coding capacity of the M. tuberculosis genome is dedicated to the PE/PPE family. There is a gradual expansion of these proteins from nonpathogenic to pathogenic mycobacteria, and there is increasing evidence that PE/PPE proteins play important roles in mycobacterial pathogenesis. In this review, we discuss PE/ PPE proteins, their close functional association with the ESX clusters, their immunomodulatory functions, and their important roles in mycobacterial virulence. In addition, we have attempted to review and compile information available in the literature detailing the expression patterns of PE/PPE family members in different mycobacterial species and also during infection. Our attempt has been to provide a succinct overview of this interesting family. VC 2015 IUBMB Life, 67(6): , 2015 Keywords: mycobacteria; M. tuberculosis; PE/PPE family proteins; ESX system; immunomodulation; virulence factors; antituberculosis vaccines and drugs Introduction Mycobacterium tuberculosis infection accounted for about 1.5 million deaths in 2013 (1). The mortality and spread of tuberculosis (TB) has been further aggravated by the alarming rise in multidrug-resistant (MDR) and totally drug-resistant cases. The PE and PPE families comprise 10% of the coding capacity of the M. tuberculosis genome (2). Members of the PE and PPE VC 2015 International Union of Biochemistry and Molecular Biology Volume 67, Number 6, June 2015, Pages *Address correspondence to: Sangita Mukhopadhyay, Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD), Bldg. 7, Gruhakalpa /B, Nampally, Hyderabad , Telengana, India. Tel: Fax: sangita@cdfd. org.in (and) sangitacdfd@gmail.com Received 7 January 2015; Accepted 29 April 2015 DOI /iub.1387 Published online 24 June 2015 in Wiley Online Library (wileyonlinelibrary.com) families are characterized by the presence of a conserved Pro- Glu (PE) and Pro-Pro-Glu (PPE) motif in their N-terminal region, respectively (3). There is a conserved N-terminal of about 110 amino acids in case of PE and 180 amino acids in case of PPE proteins. The C-terminal, however, remains highly variable. The members of the PE_PGRS and PPE_MPTR subfamilies are characterized by the presence of GC-rich stretches in their C terminals, which are thought to be hotspots for recombination events and other mutations (3). This leads to a great deal of sequence variation and polymorphism in these proteins (4). There is also speculation that this high variability may contribute to antigenic variation that helps the pathogen to evade host-protective immune responses (5). The characteristic PE and PPE domains have recently been found to be very crucial for targeting of PE/PPE family members to the cell wall. Cell wall localization enables the PE/PPE proteins to elicit potent B- and T-cell responses and also to modulate host immune responses. The PGRS domain of PE_PGRS30, a virulence factor, is responsible for targeting the protein to mycobacterial cell poles in M. tuberculosis and M. bovis (6,7). In 414 IUBMB Life

2 FIG 1 Comparison of the genetic composition of ESX-1 and ESX-5 type VII secretion systems. The red dotted region shows the increase of the pe/ppe genes from ESX-1 to ESX-5; the light blue circle indicates the close liaison between pe/ppe and the esx genes in both ESX-1 and ESX-5. esp, ESX-1 secretion-associated protein; ecc, ESX conserved component. case of other proteins like PPE17, it is the PPE domain which is crucial for cell surface localization (8). The PE/PPE family of proteins with its unique sequence, structural, and expression properties has been found to influence macrophage signaling and pathogenesis of mycobacteria (9 11). M. tuberculosis is a slow growing microaerophilic pathogen which resides predominantly inside macrophages where it replicates and establishes a productive infection (12,13). This is achieved by its ability to circumvent destruction by the host cell, which among all depends on a large variety of mycobacterial lipids and proteins (14,15). The lipids/proteins are either displayed on the bacterial cell surface or secreted into the extracellular milieu or directly secreted inside the macrophages after gaining entry inside it (16). The presence of a specialized type VII secretion system (T7SS) known as the ESX [early secretory antigenic target 6 (ESAT-6)] secretion system contributes to virulence of mycobacteria (17,18). In this review, we discuss the close association of PE/PPE proteins with the ESX system along with their expression patterns and immunomodulatory properties. Finally, we discuss the vaccine potential of candidate PE/PPE proteins and the impact of their interactions at the host pathogen interface on anti-tb drug design and therapy. The pe/ppe and the esx Genes and Their Coevolution Like most other bacteria, M. tuberculosis possesses a general secretion system or the Sec pathway for export of unfolded proteins harboring characteristic N-terminal signal sequences. M. tuberculosis also possesses a twin-arginine transporter (Tat) pathway for the export of folded proteins. Proteins exported by the Tat pathway have a signal sequence more or less similar to proteins exported by the Sec pathway except for the presence of a double arginine motif followed by two uncharged residues near the N-terminus region (19). Other than the Sec and Tat systems, secretion of the pathogenic mycobacterial proteins is dominantly regulated by the ESX secretion system, also known as the unique T7SS. The T7SS is involved in regulating pathogenesis and virulence of mycobacteria (17,18). Five such ESX secretion systems (ESX-1 to ESX- 5) are present in M. tuberculosis. At the center of an ESX cluster are two esx genes, the products of which form a 1:1 dimer (classical examples are ESAT-6 and CFP-10). Flanking the esx genes are ecc or ESX core component and esp or ESX secretion-associated genes (reviewed in 18). The esx proteins are the substrates of the ESX secretion systems. The arrangement of genes in the ESX cluster is shown in Fig. 1. The PE/ PPE family proteins seem to have evolved and duplicated in association with the duplication of the esx gene cluster regions (20). The ESX system, with the most recently emerging yet important ESX-5 and the well-established ESX-1, seems to have a significant role in the export of PE/PPE proteins. Interestingly, they are found to be strongly associated with the unique PE/PPE family gene components (21,22). Understanding how the ESX secretion system in association with the PE/PPE family proteins offers survival advantage to M. tuberculosis is important to get insights into the virulence strategies of mycobacteria which in turn can provide insights into possible antimycobacterial targets. Bioinformatic analyses by Gey van Pittius et al. (20) indicate that the PE/PPE families of proteins are in close association with ESX gene clusters (3,5,20). The pe/ ppe genes seem to have evolved from esx-associated ancestral pe/ppe copies and are characterized by homologous repetitive sequences (2,20). This can be understood from the fact that Ahmed et al. 415

3 IUBMB LIFE starting from the most primitive ESX-4 which contains no PE/ PPE protein in its assembly to the most recent ESX-5 which has 2 PE (PE18 and PE19) and 3 PPE (PPE 25, PPE26 and PPE27) proteins (20,23) and ESX-1 which contains only one PE (PE35) and one PPE (PPE68) protein, respectively (Fig. 1). The recently evolved PE_PGRS (the PE domain is followed by a C- terminal domain with a highly variable Gly-Ala rich sequence) and the PPE_MPTR (major polymorphic tandem repeat subfamily of PPE proteins) subfamilies are thought to originate from PE and PPE genes within the ESX-5 cluster. Recent studies have indicated that ESX-5 is responsible for exporting various PE, PE_PGRS, and PPE proteins of M. marinum and M. tuberculosis (24,25), which lack Sec or Tat signal peptides. PE and PPE proteins are probably targeted to ESX-5 in pairs (20,25). The ESX secretion-associated protein G (EspG) molecules play a role in expression, stability, and secretion of the type VII substrates (24 26). There are several EspG proteins associated with ESX clusters. The EspG homolog of each ESX system recognizes its cognate PE/PPE proteins, maintains them in a stable conformation, and helps in the secretion process (27). The recently solved structure of PE25-PPE41-EspG 5 of M. tuberculosis has answered some important questions about secretion of PE/PPE proteins (28). First, the study showed that EspG interacts with the PPE domain and that the EspG-interacting PPE domain remains fairly conserved. Second, EspGs in an ESX cluster bind PE/PPE proteins in that cluster as well as PE/PPE proteins coded by other ESX clusters as well as non-esx-coded PE/PPE proteins. Therefore, a lot of cross-talk takes place between EspGs and different PE/PPE proteins, and apparently EspG 5 of ESX-5 is responsible for the secretion of maximum PE/PPE proteins (28). The fact that expansion of PE/PPE proteins is linked to the evolution of ESX gene clusters clearly suggests coevolution of the PE and PPE family with the ESX system. Thus, probably these proteins are functionally associated with the ESX secretion systems (20) to regulate mycobacterial physiological processes and virulence (Table 1). The orthologs of ESX-associated PE and PPE proteins are also found in the phylogenetically more distant, fast-growing mycobacteria, such as M. smegmatis or M. abscessus, which do not contain the PGRS or MPTR repeat containing proteins (20,29). This suggests that ESXassociated PE and PPE proteins represent the most ancestral forms of PE and PPE proteins. However, there is a gradual expansion of PE, PPE, and ESX proteins from fast-growing nonpathogenic to slow-growing pathogenic mycobacteria, indicating that these clusters are probably associated with intracellular lodging and survival of the bacilli. Looking deeper into the organization of the five esx gene clusters in M. tuberculosis, it is revealed that the gene content and gene organization are highly conserved and typically carry a pair of esx genes in the center of the cluster with PE and PPE genes located upstream of the esx genes, which again emphasizes the interdependencies of the ESX and the PE/PPE gene families (Table 1 and Fig. 1; ref. (30). The esx and pe/ppe gene cluster is surrounded by other genes which encode membrane proteins and other components of an ATP-powered secretion machine which is typical of a highly evolved active secretion system (31). The close relationship between the PE/PPE proteins and ESX members can also be ascertained from the fact that in M. microti (causes TB in wild voles), a deletion causes simultaneous removal of five genes that encode for ESAT-6 and PE/PPE family proteins (32). This probably explains the reason why M. microti is unable to infect humans despite the fact that it is evolutionarily very close to M. tuberculosis. Another feature of members of the PE and PPE families is that they are often found as co-operonic pairs of mostly one PE and one PPE gene whose products interact with each other (33). There is experimental evidence for some interactions like PPE41 or Rv2430c and PE25 or Rv2431c (34) and PE35 or Rv3872 and PPE68 or Rv3873 (35), and many more interactions have been predicted using bioinformatic tools (36). Structural information of PE and PPE proteins is scarce. However, recently, the structure of the PE25/PPE41 pair bound to EspG was solved. The structure of the PE-PPE-EspG complex yielded a lot of vital information regarding the structure of PE and PPE pairs, their association with ESX components, and the mechanism of their secretion by ESX proteins (28). Expression Patterns of PE/PPE Proteins PE and PPE proteins have evolved along with the duplication of the esx cluster genes and expanded from nonpathogenic to pathogenic mycobacteria. The result of this has been that fastgrowing nonpathogenic mycobacteria like M. smegmatis do not have the PGRS or MPTR repeat containing PE/PPE proteins (20,29) and that the family is most well represented in the slow-growing pathogenic mycobacteria like M. marinum, M. ulcerans, and members of the M. tuberculosis complex, which include M. microti, M. bovis, M. tuberculosis, and M. africanum (20). However, even among M. tuberculosis complex members, there are differences in the expression patterns of PE and PPE proteins. A study which compared steady-state gene expression levels between M. tuberculosis H37Rv and M. bovis AF2122 found that Rv3347c/Mb3379c (PPE55), Rv3872/ Mb3902 (PE35), and Rv3873/Mb3903 (PPE68) are expressed about four times more in M. bovis, whereas Rv1809/ Mb1838,1839 (PPE33) and Rv3136/Mb3160 (PPE51) are expressed more in M. tuberculosis H37Rv (37). Another study which compared steady-state gene expression levels of M. tuberculosis H37Rv and M. bovis Ravenel (TMC401) found that RV1135c/Mb1166c (PPE16), Rv2431/Mb2457c (PE25), and MT3248/Mb3184c (PPE70) are expressed more in M. bovis, whereas 18 genes coding for PE/PPE proteins like PPE18, PPE38, PPE33, PPE51, PE_PGRS33, PE_PGRS38, PE13, and PE15 are expressed more in M. tuberculosis (38). Genomic comparison between M. bovis AF2122/97 and M. tuberculosis H37Rv revealed differences in a whopping 60% or 29 PE and PE_PGRS genes resulting from insertions, deletions, and frame shifts (39). Comparative genome analysis revealed differences like frame shift, insertions, and deletions in several PE/PPE 416 Expression And Functions Of Mycobacterial Pe/Ppe Proteins

4 TABLE 1 Comparison of functionally related components of ESX-1 and ESX-5 type VII secretion systems ESX-1 Functions ESX-5 Functions ecca1 AAA 1 ATPase ecca5 Probable ATP binding, ATPase activity, involved in secretion of ESX-N eccb1 Possible membrane protein eccb5 Possible membrane protein eccca1 FtsK eccca5 Possible transmembrane protein ecccb1 SpoIIIE ecccb5 Possible transmembrane protein cyp143 Probable cytochrome P450 Rv1786 Probable ferredoxin pe35 Immune-modulation ppe25 pe19-ppe25 responsible for export of ESX-N, but not responsible for its expression, have role in macrophage infection. ppe68 Immune-modulation pe18 ppe26 ppe27 pe19 esxb esxa CFP-10 (exported protein cotranscribed with ESAT-6) ESAT-6 (exported protein cotranscribed with CFP-10, elicits high level of IFN-g from memory effector cells during the first phase of a protective immune response, inhibits class I antigen presentation) esxm ESAT-6 like protein, involved in secretion of ESX-N esxn Putative ESAT-6-like protein (ESAT-6-like protein 5); secreted by ESX-5 espi Conserved Pro and Ala rich protein Rv1794 No obvious phenotype eccd1 Probable transmembrane protein eccd5 Probable membrane protein, involved in secretion of ESX-N, role in macrophage infection and cell wall integrity of M. tuberculosis espj espk espl Conserved alanine rich protein Alanine and proline rich protein Conserved protein espb Proteolytic substrate of MYCP1, conserved Ala and Gly rich protein ecce1 Probable membrane protein ecce5 Probable membrane protein mycp1 Has proteolytic activity, cleaves espb, expressed during macrophage infection mycp5 Probable proline rich membrane-anchored mycosin MycP5 (serine protease) (subtilisin-like protease) (subtilase-like) (mycosin-5) The genes given in boldface are the pe/ppe genes of either of ESX-1 or ESX-5. The genes mentioned are not necessarily in the same sequence as they are present in the genome of M. tuberculosis and have been grouped together as per their known or probable functions. esp, ESX-1 secretion-associated protein; ecc, ESX conserved component. Ahmed et al. 417

5 IUBMB LIFE proteins between H37Ra and H37Rv strains of M. tuberculosis, which could possibly contribute to virulence attenuation of M. tuberculosis H37Ra (40,41). A good example of these differences is a frame shift mutation in MRA_1205a of H37Ra which causes a premature C-terminal termination. The H37Rv ortholog of MRA_1205a is Rv1196 which codes for PPE18, a characterized virulence factor (40,42 44). PE_PGRS genes have been shown to be differentially expressed between M. tuberculosis and M. canettii (45). Differences in the expression of PE and PPE proteins have been observed among M. tuberculosis clinical isolates as well (45,46). The major reason of virulence attenuation in M. bovis bacille Calmette-Guerin (BCG) is the absence of RD1-associated genes Rv3872 (PE35), Rv3873 (PPE68), Rv3874 (CFP10), Rv3875 (ESAT-6), and Rv3876 (47). Several functions have been attributed to PE and PPE proteins. They are virulence factors (3,48), contribute to cell wall integrity and structure (22,49), and have also been shown to have enzymatic activity (50,51). Differences in the expression patterns of PE and PPE proteins can provide vital clues about host specificity and virulence of mycobacteria, crucial for designing effective anti-tb vaccines and drugs. For example, M. marinum codes for far more PE and PPE proteins when compared with M. tuberculosis, and this might be one of the reasons why M. marinum has a broader host range (52). Apart from differences in steady-state expression between mycobacterial species, in vivo and stress-induced levels of PE and PPE proteins also have variations. Their expression has been shown to be regulated by conditions of hypoxia, iron, nutrient deprivation, phagosomal acidity, and other host environmental conditions (53,54). IdeR, a metal-dependent regulator, transcription factor Rv0485, PhoP, EspR, and SigF are some of the protein factors which have been found to regulate the expression of PE and PPE proteins in mycobacteria (55 59). Microarray analysis of mycobacteria under various host stress conditions which the bacilli encounters such as hypoxia, acidity, nutrient deprivation, and also analysis of bacilli from granulomas and infected tissues has yielded important information regarding gene expression patterns. Granulomas are organized structures comprising bacilli and immune cells that are formed upon persistent infection and are characteristic of TB disease (60). The granuloma is viewed as a host-protective mechanism; however, recently, it is also being viewed as a means of dissemination of infection and may also contribute toward increased disease pathology (61). PE and PPE family proteins have been shown to have granuloma-specific expression (62,63). PE_PGRS proteins are differentially expressed in granulomas and other infected tissues such as spleen in infected mice (63). A study which used aerosol infection of M. tuberculosis in guinea pigs found several PE/PPE proteins consistently upregulated in lungs throughout the 90-day course of study (64). Interestingly, among the upregulated genes are immunomodulators and virulence factors such as PPE38 (65). PE/PPE proteins which are nonessential for mycobacterial growth are often essential virulence factors during infection. Studies which combine the evaluation of expression patterns of PE/PPE proteins and their in vivo functions will yield vital information about interactions and changes at the host pathogen interface in TB. Immunomodulatory Properties of PE/PPE Proteins and Their Roles in Mycobacterial Virulence Mycobacteria are intracellular pathogens armed with many strategies to subvert and evade the host immune response to better their chances of survival. Many PE/PPE proteins have been found to contribute toward this and are described as mycobacterial virulence factors (3,11,42 44,66). The PE/PPE proteins are found to influence manifold cellular processes and innate as well as adaptive immune responses during infection, for example, macrophage immune responses, cell death, and secretion of cytokines. The influence of PE/PPE family members on various immune responses is summarized below. Mycobacterial Survival in Macrophages and Modulation of Macrophage Immune Responses One of the earliest reports which documented the virulence properties of PE/PPE family members was one which showed that two PE_PGRS of M. marinum homologous to M. tuberculosis Rv1651c and Rv3812 proteins are essential for survival in macrophages (J774) and granulomas in a frog model of infection (62). The M. avium gene bearing 52% homology to M. tuberculosis, Rv1787, was found to prevent phagosome lysosome fusion, an important immune evasion mechanism, and to promote intracellular survival of the bacilli. A mutant strain lacking Rv1787 was cleared more effectively when compared with the wild type in a mouse model of infection (66). The phagosomes containing M. avium subspecies hominissuis bacilli deficient in MAV_2928 (homologous to Rv1787) had differences in zinc, manganese, calcium, and potassium when compared with the phagosomes containing wild-type bacilli (67). Infection studies with a transposon mediated mutant library generated in M. bovis BCG-identified PPE10 (Rv0422c) insertion mutants present in acidified phagosomes, suggesting that this gene might be involved in the arresting phagosomal maturation in infected macrophages (68). Other insertion mutants that were compromised in their ability to survive in macrophages were PE_PGRS62 (Rv3812) and PPE13 (Rv0878c) (68). M. tuberculosis PE_PGRS62, which remains remarkably conserved in the M. tuberculosis complex (69), when overexpressed in M. smegmatis, has been shown to cause phagosome maturation arrest by deterring the recruitment of Rab7 and LAMP1 to bacilli-containing phagosomes (70). M. bovis PE_PGRS62 also has the same capability of inhibiting phagosome maturation and IL-1b production, both of which were reversed on priming with IFN-c (70). The M. tuberculosis mutant strain lacking PE_PGRS30 exhibited compromised ability to colonize lung tissue and cause tissue damage. PE_PGRS30 interferes with phagosomal maturation and is a 418 Expression And Functions Of Mycobacterial Pe/Ppe Proteins

6 possible virulence factor (7). The macrophage is known to be a hostile environment for pathogens. Mycobacterial invasion of and survival in host macrophages is key for establishing a successful infection and subsequent dissemination. When a library of mutants of M. marinum was generated and their ability to associate with and grow inside macrophages revealed that PPE24 promotes survival inside macrophages and that PPE53 is required for association as well as survival inside macrophages (71). Another macrophage defense mechanism that has been found to be affected by PE/PPE family members is the generation of bactericidal nitric oxide. The deficiency of inducible nitric oxide synthase (inos) leads to exacerbated bacterial loads and TB-associated immunopathology in mouse infection models (72,73). M. tuberculosis has been found to inhibit the generation of nitric oxide by inos (74). M. smegmatis strains overexpressing PE5 and PE15, two proteins that remain highly conserved across the M. tuberculosis cluster, reduce inos transcript levels in infected J774.1 and THP-1 macrophages. These strains also showed enhanced survival in infected macrophages (75). Interestingly, PE15 is sixfold more expressed in M. tuberculosis when compared with M. bovis (38). Purified recombinant PPE2 has also been found to reduce the levels of inos in activated macrophages (76). M. tuberculosis PE_PGRS62 when overexpressed in M. smegmatis leads to reduced inos expression in infected macrophages (70). Apart from nitric oxide, mycobacteria also face the danger of extermination due to intracellular oxidative stress. PE_PGRS11, a prototype hypothetical, hypoxia-induced gene with a phosphoglycerate mutase activity, was found to impart resistance against oxidative stress by upregulating Bcl2 and COX2 (77). Cell Death The co-operonic protein complex of PE25/PPE41 secreted by the T7SS of M. tuberculosis was reported to induce necrosis but not apoptosis in RAW264.7 macrophages (78). Induction of necrosis is one of the strategies used by M. tuberculosis to enhance its multiplication and dissemination (79,80). Virulent mycobacterial strains inhibit induction of apoptosis in infected macrophages (81,82). This is thought to help the bacteria escape CD4 1 and CD8 1 immune responses that would be generated when apoptotic vesicles containing bacterial antigens are taken up and presented by dendritic cells (DCs; ref. (83)). However, purified PE_PGRS33 and M. smegmatis overexpressing PE_PGRS33 have been shown to induce macrophage apoptosis by a TLR2-dependent release of TNF-a in RAW macrophages (84). Interestingly, deletion of the PGRS domain of PE_PGRS33 resulted in a reduction in its ability to induce apoptosis (84). It should be noted that the PGRS domain of PE_PGRS33 is highly variable in clinical isolates (85). Another study by Dheenadhayalan et al. (86) showed that M. smegmatis overexpressing PE_PGRS33 induced necrosis and had better survival in both cells and a mouse model of infection (86). A recent study showed that PE-PGRS33 localizes to the host cell mitochondria leading to apoptosis (87), a fact that was previously demonstrated in another study using a system of overexpressed PE_PGRS33 in Jurkat T cells (88). However, the wild-type PE_PGRS33 is capable of inducing necrosis, and the PE domain is crucial for this (87). The mycobacterial surfacelocalized PE_PGRS33 (49) is one of the most well-studied PE/ PPE proteins, and it appears that it can influence macrophage cell death in many ways. The exact mechanism it uses for inducing cell death might depend on its sequence and perhaps expression and stage of infection. Modulation of Cytokine Responses Cytokines like IL-10, IL-12, and TNF-a secreted by macrophages not only control macrophage and other innate immune cell functions but also have effects on the type of effector T- cell response that is generated during infection (89). IL-12 (90,91) and TNF-a (92) boost macrophage antibacterial defenses as well as induce a protective T-helper (Th) 1 response (93,94), whereas IL-10 in addition to being immunosuppressive (95) favors the generation of a Th2 response that is nonprotective for the host and promotes mycobacterial survival (96,97). The PPE18 protein that is 14.8-fold more expressed in M. tuberculosis H37Rv when compared with M. bovis (38) was found to dampen the secretion of both IL-12 and TNF-a in activated macrophages by suppressing NF-jB signaling (43). In addition, it promotes IL-10 production in macrophages and skews the PPD specific T-cell responses to Th2 type (42). Consequently, the M. tuberculosis CDC1551 strain lacking PPE18 was found to be attenuated in a mouse model of low-dose aerosol infection (44), suggesting that PPE18 could indeed be a virulence factor. Interestingly, Rv0485, transcriptional regulator of the co-operonic pair of PE13 and PPE18 when disrupted led to attenuation of M. tuberculosis virulence in a mouse model of infection (57). M. tuberculosis proteins PE_PGRS33 and PE_PGRS26 when overexpressed in M. smegmatis were found to cause increased IL- 10 and reduced IL-12 coupled with enhanced macrophage death and reduced NO, all of which perhaps could contribute toward persistence of these strains in infected mice (98). The RD-1 located PE-PPE pair of PE35 and PPE68 was also found to increase IL-10 and decrease IL-12 levels in THP-1 macrophages (32). The pair also increased monocyte chemoattractant protein (MCP)-1 levels in the same cultures (35). MCP-1 knockout mice are shown to have defects in granuloma formation and are resistant to M. tuberculosis infection (99). Another member of the family, PPE38, was found to increase TNF-a and IL-6 in macrophage cultures, and its deletion resulted in the attenuation of M. marinum in a zebrafish model of infection (65). On the other hand, PPE37, a protein that is found to be highly expressed in phagocytosed bacteria and in bacilli isolated from lungs of infected mice, reduced TNF-a and IL-6 in macrophage cultures infected with M. smegmatis overexpressing it (100). TNF-a-deficient mice are undoubtedly unable to contain mycobacterial infection; however, there is also increasing emerging evidence which shows that excessive levels of this pleiotropic cytokine contribute to disease pathology Ahmed et al. 419

7 IUBMB LIFE (101,102). IL-6 too has been shown to have both antiinflammatory and proinflammatory properties (103). Apart from macrophages, DC-derived cytokines too can influence the generation of a CD4 1 Th response (104). M. tuberculosis protein PPE34 was found to induce maturation of human DCs and enhance IL-10 secretion in these cells. PPE34 primed DCs when cocultured with CD4 1 T cells induced them to secrete high levels of Th2 cytokines such as IL-4, IL-5, and IL-10 but not IFN-c (105). In another set of experiments by the same group, it was observed that PE_PGRS11 and PE_PGRS17 of M. tuberculosis can cause maturation of DCs and increase secretion of proinflammatory cytokines IL-6, IL-8, and IL-12p40 by activating ERK1/2, p38mapk, and NF-jB signaling cascades (106). This is another example of PE/PPE family members inducing both proinflammatory and anti-inflammatory immune responses in one single type of immune cell which happens to be DC in this case. A single PE/PPE protein can impact several immune responses. A proteomic study done to understand the global effects of PPE38 using a system of infection of THP-1 macrophages with M. marinum strains carrying either the wild type or a mutant version of PPE38 also revealed elevated levels of IL-6 and TNF-a in addition to several other inflammatory factors downstream of TLR2, which was shown to interact with PPE38 (107). In addition, proteins involved in the class I antigen presentation pathway seemed to be downregulated (107), suggesting that PPE38 might interfere with the generation of a CD8 1 T-cell response, which is important for clearing intracellular pathogens like M. tuberculosis (108). The bioinformatic tools like DAVID and STRING were also used to construct pathways involving transcription factors that were modulated by PPE38 (107). It was found that PPE38 apparently regulates cross-talk between various transcription factors, the end result being that several immune responses are affected (107). Such approaches which attempt to look at global changes answer more questions and shed more light on the exact mechanisms of action of PE/PPE proteins which exhibit enormous diversity in sequence across mycobacterial species and clinical strains and have varied expression patterns during infection (109). In addition, animal infection studies using mycobacterial species capable of infecting humans like M. tuberculosis, M. marinum, M. bovis, M. avium, which are deficient in PE/PPE proteins, will shed more light on the role of PE/PPE proteins in virulence and their actual mechanism(s) of action. Immunomodulatory functions of PE/PPE proteins are summarized in Table 2. As the ESX systems, especially the ESX-5, plays an essential role in transport and secretion of PE/PPE proteins (21,22), it is probable that the ESX by regulating PE/PPE protein export maintains cell wall integrity and contributes to disease pathogenicity. Many ESX components are also directly involved in the modulation of macrophage signaling cascades to support mycobacterial growth. Although the role of ESX-1 in cell wall integrity is reported, this is probably PE/PPE independent. ESX-5 seems to have a more direct contribution in the cell wall composition with reference to the PE/PPE proteins, which can be understood from a recent study carried out by Daleke et al. (25). The group shows that the conserved PE/PPE protein domain targets the LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway (25). PE/PPE Proteins and ANTI-TB Vaccine and Drug Design Mycobacterium bovis BCG is the sole vaccine used for control of TB. BCG vaccine has been found to confer protection against TB in children; however, it is not so efficacious in adults (110). About one-third of the world population is latently infected with M. tuberculosis. These individuals have a 10% lifetime risk of reactivating the infection and succumbing to active TB disease. In addition, there is now increased threat of infection with MDR and XDR M. tuberculosis strains. Therefore, there is an immense effort globally to develop new vaccines and therapeutic agents to counteract the menace of TB. The most recent vaccine that was developed and is undergoing clinical trials is MVA85A, a modified Vaccinia Ankara virus expressing Ag85A which has been found to induce long-lasting Ag85A-specific CD4 1 T-cell response in HIV-negative as well as HIV-positive MVA85A-vaccinated individuals (111). However, the vaccine was found to be ineffective in reducing incidences of M. tuberculosis infection in already BCG-vaccinated infants in a phase 2b clinical trial (111). Another candidate vaccine designated as Mtb72F/AS02A comprising a polyprotein of Mtb32 (PepA) and Mtb39 (PPE18) in an adjuvant system AS02A was found to offer protection against M. tuberculosis infection, disease reversal, and increment in IL-6 and Th1 cytokines IL-12, IFNc, and TNF-a when used along with BCG in cynomolgus monkeys (112). In a clinical trial, Mtb72F was administered to BCG-vaccinated individuals and also to those who had been infected with M. tuberculosis. In both sets of individuals, vaccination was found to induce Mtb72F-specific polyfunctional CD4 1 T cells and humoral responses (113). The vaccine is well tolerated, immunogenic, and does not induce any adverse effects, and therefore, it deserves to be further tested to determine efficacy. A separate polyprotein vaccine ID93 comprising Rv2608 (PPE44), Rv3619, Rv3620, and Rv1813 when administered to mice in an adjuvant preparation of GLA-SE was found to induce generation of polyfunctional T cells and offer protection in virulent and multidrug resistant M. tuberculosisinfected mice and guinea pigs (114). Apart from PPE18 and PPE44, other PE/PPE family members have also shown promise as vaccine candidates. Several PE/PPE proteins on account of being surface exposed and containing repetitive sequences can serve as targets for the humoral immune system. Indeed, several PE/PPE family members have been shown to induce strong antibody responses (3), examples include Rv0160c or PE4 (115); the PE25/PPE41 protein complex (116); Rv1169c, Rv0978c, and Rv1818c (117); Rv2608 (118); PPE44 (119); Rv2430c (120); Rv0256c (121); and Rv1168c or PPE17 (122). The expression of PE/PPE members has often been found to 420 Expression And Functions Of Mycobacterial Pe/Ppe Proteins

8 TABLE 2 Summary of modulation of immune responses by PE/PPE proteins Family Protein Effect on immune response PE PE4 Increases IL-2, TNFa and IL-6 in a mouse model of infection. Improves mycobacterial survival in macrophages. PE5 PE15 Reduces NO Reduces NO PPE PPE2 Reduces NO in macrophages PPE10 PPE13 PPE18 PPE25 PPE34 PPE37 PPE38 Inhibits phagosomal acidification Important for mycobacterial survival in macrophages Increases IL-10, downregulates IL-12 and TNFa in macrophage cultutres Inhibits phagosomal maturation Increases IL-10 in dendritic cells, increases IL-4 and IL-5 in T cells Reduces TNFa and IL-6 in macrophages Increases IL-6 and TNFa in macrophages. Downregulates MHC-I antigen presentation PE_PGRS PE_PGRS11 Imparts resistance against oxidative stress PE_PGRS17 PE_PGRS26 PE_PGRS30 PE_PGRS33 Increases IL-6, IL-8 and IL-12p40 in dendritic cells Reduces generation of NO Inhibits phagosomal maturarion Induces necrosis and apoptosis.inhibits phagocytosis. Increases IL- 10 and decreases IL-12 and TNFa in macrophage cultures PE_PGRS62 PE-PPE pairs PE25-PPE41 Induce necrosis Inhibits phagosomal maturation and generation of NO PE35-PPE68 Increases IL-10 and decreases IL-12 and TNFa in macrophage cultures vary, and seroreactivity against them can help to differentiate between different clinical categories of patients with TB like pulmonary, extrapulmonary, smear-negative, or relapsed (117,121). The role of humoral immunity in TB is highly underappreciated. Recent studies have demonstrated a role for B cells during TB (123). B-cell-deficient mice receiving 300 bacilli via aerosol showed higher bacterial load and more susceptibility to TB (123). Antibody-mediated protection against mycobacteria could possibly contribute to success of new TB vaccines (112,115,116). The PE/PPE family members are also strong inducers of cell-mediated immune responses, which are essential for countering intracellular pathogens like mycobacteria (124). PE/PPE proteins are major targets of the CD4 1 T-cell immune response. A proteome-wide antigen screen showed that 45% of CD4 1 mycobacterial T-cell responses were generated against PE/PPE antigens (125). There is evidence to show that several PE/PPE proteins have T-cell epitopes which can bind MHC molecules with high affinity and elicit a strong T-cell cytokine response, examples of such proteins are Rv3812 or PE_PGRS62, Rv3018c or PPE46 (126), and cell wall-associated Rv3873 (127). Some of these candidate antigens when administered along with an adjuvant elicited a strong B- and T-cell response (128). The product of Rv0915c, MTB41 or PPE14, identified in a screen for T-cell clones from infected mice was shown to induce MTB41-specific CD4 and CD8 T-cell responses and confer protection under M. tuberculosis challenge when administered as a DNA vaccine (129). An extensive study which attempted to identify novel T-cell antigens for TB vaccine development used bioinformatics to initially screen Ahmed et al. 421

9 IUBMB LIFE potential antigens which met certain criteria like expression in stress conditions, essential for growth, and then 94 of these antigens were tested for their ability to elicit IFN-c in healthy PPD2 and PPD 1 individuals. Finally, a set of 49 antigens including six members of the PE/PPE family were administered prophylactically in M. tuberculosis-infected mice along CpG as adjuvant to test their ability to provide protection. It was observed that PE/PPE proteins Rv2608 and Rv3478 along with other membrane-associated and secretory proteins could significantly reduce cfu burden and induce Th1 responses and polyfunctional T cells, which are increasingly being found to be crucial for control of chronic mycobacterial infections (130,131). The same PE/PPE protein can elicit both T- and B- cell responses. A good case in point is a PE_PGRS protein coded by Rv1818 whose Gly-Ala-rich PGRS domain elicits a good antibody response, whereas its PE domain induces a protective T-cell response (11). Another interesting feature is that PE and PPE proteins despite sequence variability are capable of generating crossreactive responses (127), a fact that was brought to light by a study which used a M. tuberculosis strain deficient in all ESX-5- associated PPE25 to PE19 genes (Dppe25-pe19) for mouse infection studies and found that infected mice could generate T-cell immunity against all deleted proteins despite the Dppe25-pe19 being highly attenuated (132). Most immunodominant epitopes also map to the N-terminal PE domain rather than the variable C-terminal domains. An extensive study in which 27 pe_pgrs genes from 94 clinical strains of five phylogenetic lineages of the M. tuberculosis complex were sequenced, it was observed that despite high sequence variability in the pe_pgrs genes when compared with the rest of the genome, most T-cell epitopes mapped to the invariable PE domain. Very few T-cell epitopes mapped to the highly variable PGRS domain (133). Therefore, escape from T-cell recognition is not the positive selection pressure leading to polymorphism or sequence variation especially in the C-terminus in PE/PPE family members (133,134). The reasons for sequence variability might be something not determined yet. Numerous studies have been carried out to evaluate the vaccine potential of PE/PPE family members, and several of the results appear promising. However, there is a downside to selecting PE/PPE as potential vaccine candidates as many of them are involved in immune evasion mechanisms as discussed above and their expression is highly variable in vivo. In addition, they have been found to be polymorphic within clinical isolates (135) and can be degradation resistant and hence avoid presentation by the MHC (136). Both these properties can severely hamper vaccine efficacy. Thorough characterization of the candidate protein or utilization of only immunodominant epitopes of PE/PPE family members can perhaps help to overcome these hurdles in vaccine development. Apart from being vaccine candidates, PE/PPE family proteins on account of their unique structural properties and owing to the fact that they are exclusive to mycobacteria can also serve as novel drug targets (137). Several PE/PPE proteins exert their immunoregulatory effects downstream of TLR2. Such TLR2-binding PE/PPE proteins include PPE18 (41), PPE17 (138), PE35/PPE68 pair (35); PE_PGRS33 (84,139); PPE38 (107), PPE34 (105), and PE_PGRS11 (77). TLR2 does not seem to play an essential role in TB pathogenesis (140). However, polymorphisms in TLR2 in humans have been reported to be associated with susceptibility as well as resistance to TB (141,142). Although TLR2 serves as a receptor for numerous PE/PPE proteins, there might be subtle differences in interaction patterns and domains which might lead to differences in downstream responses. A good example is the case of Rv1168c and Rv1196c, both of which bind to TLR2 (41,138). However, Rv1168c which activates NF-jB-mediated proinflammatory cytokines such as TNF-a binds to TLR2 leucine-rich repeats (LRR) motifs (138), whereas Rv1196c which dampens NF-jB signaling and induces anti-inflammatory cytokines such as IL-10 binds to TLR2 LRR motifs (41). Thus, inhibition of binding of M. tuberculosis PPE/PE proteins to TLR2 LRR domain can specifically block the antiinflammatory responses and activate proinflammatory protective cytokines. Drugs or small molecules that intervene with binding of PE/PPE proteins with TLR2 or TLR2 LRR domain can be developed as therapeutics. Conclusions Mycobacteria have several strategies to survive inside macrophages (their preferred niche) as well as outside, and once they overcome the challenges posed by the host or rather when the time arrives, they induce colossal damage (143). Mycobacterial defenses comprise proteins which have an N- terminal signal sequence as well as nonclassical secretory products such as superoxide dismutase and other enzymes to alleviate the oxidative stress induced by the host (144). M. tuberculosis proteins can also downregulate the expression of proinflammatory effectors known to be cytotoxic to the bacilli (145). In this context, an important role of the PE/PPE family proteins is documented (3,48,146). These proteins are shown to provide significant survival advantage to the bacilli by suppressing various host-protective responses. The aim of the bacilli is to activate the immune system just enough to ensure survival and dissemination and to suppress it to avoid clearance. This balance between inflammation and suppression probably determines the course of TB disease (147,148). The PE/PPE family is exclusive to mycobacteria, is responsible for virulence, has complex expression patterns, exhibits polymorphism, and most importantly, members of the family despite sharing sequence similarities have their unique expression profiles and mechanisms of action. An additional interesting feature of PE/PPE family members expressed in the pathogenic mycobacterial species is their strong association with esx genes (20). A safe inference from the available facts is that, on one hand, the membrane-associated members form a definite part in the secretion of other components either by directly acting in the process of secretion or by regulating the process tightly (3). Mycobacterial-secreted proteins like 422 Expression And Functions Of Mycobacterial Pe/Ppe Proteins

10 ESAT-6/CFP-10 modulate several host immune responses such as class I antigen presentation (149). Several PE/PPE members also act as effector molecules/virulence factors for intruding through the host defenses and reaching locations which are physically not accessible to the pathogen itself (33,48). The close coordination of these novel proteins and the complex secretion systems used by this clever pathogen only goes on to say that this enormous evolutionary experience has ironically made it a global health emergency. More information on how PE/PPE and ESX secretion systems regulate each other as well as host targets, structure, and expression of PE/PPE proteins will better our understanding of mycobacteria and help to unravel reasons which contribute to their success as pathogens and make them so difficult to eradicate. Acknowledgements The laboratory of Sangita Mukhopadhyay is supported by grants from the Department of Biotechnology, Government of India (BT/PR5496/MED/29/512/2012) and the Department of Science and Technology, Government of India (SR/SO/HS/0120/ 2010) and also core grants from the Centre for DNA Fingerprinting and Diagnostics by the Department of Biotechnology. Asma Ahmed was supported by the Research Associate Fellowship from the Department of Biotechnology, Government of India, and Arghya Das was supported by a fellowship from the Council of Scientific and Industrial Research (CSIR), Government of India. References [1] World Health Organisation (2014) World Health Organisation Global Tuberculosis Report: Executive Summary. Available from: Accessed in February [2] Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 93, [3] Sampson, S. L. 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