Phylogeny to function: PE/PPE protein evolution and impact ...umanitoba.ca/faculties/health_sciences/medicine/units/medical...global burden of tuberculosis (TB) remains enormous’

MicroReview

Phylogeny to function: PE/PPE protein evolution and impacton Mycobacterium tuberculosis pathogenicity

S. Fishbein,1,2 N. van Wyk,2 R. M. Warren2 andS. L. Sampson2*1Harvard School of Public Health, Boston, MA, USA2DST/NRF Centre of Excellence for BiomedicalTuberculosis Research, SAMRC Centre for TuberculosisResearch, Division of Molecular Biology and HumanGenetics, Faculty of Medicine and Health Sciences,Stellenbosch University, Tygerberg 7505, South Africa

Summary

The pe/ppe genes represent one of the most intriguingaspects of the Mycobacterium tuberculosis genome.These genes are especially abundant in pathogenicmycobacteria, with more than 160 members in M.tuberculosis. Despite being discovered over 15 yearsago, their function remains unclear, although variouslines of evidence implicate selected family members inmycobacterial virulence. In this review, we use PE/PPEphylogeny as a framework within which we examinethe diversity and putative functions of these proteins.We report on the evolution and diversity of the respec-tive gene families, as well as the implications thereoffor function and host immune recognition. We summa-rize recent findings on pe/ppe gene regulation, alsoplacing this in the context of PE/PPE phylogeny. Wecollate data from several large proteomics datasets,providing an overview of PE/PPE localization, anddiscuss the implications this may have for hostresponses. Assessment of the current knowledge ofPE/PPE diversity suggests that these proteins are notvariable antigens as has been so widely speculated;however, they do clearly play important roles in viru-lence. Viewing the growing body of pe/ppe literaturethrough the lens of phylogeny reveals trends in fea-tures and function that may be associated with theevolution of mycobacterial pathogenicity.

Introduction

In 2012, the World Health Organization reported that ‘theglobal burden of tuberculosis (TB) remains enormous’(Williams, 2012). The disease burden is exacerbated bythe concurrent HIV epidemic (Corbett et al., 2003) and theemergence of multidrug and extensively drug-resistantMycobacterium tuberculosis strains (Dheda et al., 2014),prompting an urgent need for new diagnostics and thera-pies. To produce more effective interventions against M.tuberculosis, it is critical to improve our understanding ofthe intracellular lifestyle of the pathogen. Upon phagocy-tosis by the host macrophage, M. tuberculosis engagesvarious mechanisms to evade macrophage killing andsurvive adverse environmental conditions, including oxi-dative and nitrosative stress, nutrient limitation and acidicpH (Russell, 2011). Many of the mechanisms that aid M.tuberculosis survival within the host are adaptations ofstrategies used by environmental mycobacteria to survivewithin diverse niches (Cosma et al., 2003).

Some clues to mycobacterial survival strategieswere provided by the publication of the M. tuberculosisH37Rv genome sequence in 1998. Remarkably, the newlyidentified pe/ppe gene families were shown to compriseapproximately 10% of the genome (Cole et al., 1998).Reexamination of the updated Tuberculist database (Lewet al., 2011) shows that this value is a slight overestimation,with pe/ppe coding bases accounting for 7% of the totalcoding potential (Sampson et al., unpublished data).Nonetheless, their prominence in the slow-growingmembers of the Mycobacterium genus is striking. Thereare 99 pe genes and 69 ppe genes in M. tuberculosisH37Rv, characterized by conserved N-terminal proline-glutamate (PE) and proline-proline-glutamate (PPE)motifs. The genes encode a relatively conservedN-terminal sequence of ∼110 and ∼180 amino acids in thePE and PPE families respectively (Cole et al., 1998).Despite this grouping, a rudimentary analysis of sequencefeatures immediately reveals a significant amount of diver-sity within both families. Within these groupings, memberscan be subclassified by C-terminal amino acid sequencefeatures. The pe family can be divided into pe_pgrs

Accepted 23 February, 2015. *For correspondence. [email protected]; Tel. +27 21 9389 073; Fax +279389403.

Molecular Microbiology (2015) 96(5), 901–916 ■ doi:10.1111/mmi.12981First published online 30 March 2015

© 2015 John Wiley & Sons Ltd

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(polymorphic GC-rich sequences) and pe (with no distinc-tive features) genes. The ppe family comprises ppe_mptr(major polymorphic tandem repeats), ppe_svp (with a Gxx-SVPxxW motif), ppe_ppw (with a PxxPxxW motif) and ppegenes with no distinctive features. This subclass diversityprovides the first indication that family members could playfunctionally distinct roles.

In this review, we examine the PE/PPE proteins throughthe lens of their phylogeny; using this as a framework withinwhich we examine the diversity and putative functions ofthese proteins to better understand their role in M. tuber-culosis pathogenesis. Within this analysis framework, wehighlight the potentially diverse functional roles of PE/PPEproteins and challenge previous tendencies to describeboth families as functionally homogeneous entities.

Evolution and variation of the PE/PPE families

PE/PPE proteins are present in both pathogenic and sap-rophytic mycobacteria, and are perhaps more central tomycobacterial metabolism than previously thought (Geyvan Pittius et al., 2006; Akhter et al., 2012). Nonpatho-genic mycobacteria tend to have fewer pe/ppe genes, witha general expansion especially within sublineage V of thegene families in pathogenic species (Fig. 1). Mycobacterialspecies within the M. tuberculosis complex (MTBC), Myco-bacterium leprae, Mycobacterium marinum, Mycobacte-rium ulcerans and Mycobacterium avium possess thehighest number of pe/ppe genes (McGuire et al., 2012).Additionally, the polymorphic subfamilies pe_pgrs andppe_mptr are only present in the MTBC; these representthe most recent sublineages of the pe/ppe gene family(Gey van Pittius et al., 2006).

Although the origin of the pe/ppe families is stillunknown, the ancestral pe/ppe genes are associated withthe type VII or ESX (early secretory antigenic target 6system) mycobacterial secretion systems (Abdallah et al.,2007). The M. tuberculosis genome possesses five esxgene clusters (van Pittius et al., 2001), some of which havebeen implicated in pathogenesis (Simeone et al., 2009).pe/ppe gene family expansion is thought to have beeninitiated by a series of esx gene cluster duplications thatbegan with duplication of an ancestral esx region (Gey vanPittius et al., 2006; Akhter et al., 2012). Phylogeneticreconstructions show that the esx gene clusters expandedthrough multiple gene duplications, accompanied by aconcomitant expansion of the pe/ppe gene family. Sublin-eage I constitutes the pe/ppe genes inserted into and/orassociated with the esx-1 gene cluster. A similar relation-ship is hypothesized for esx-3/sublineage II, esx-2/sublineage III and esx-5/sublineage IV. Phylogeneticanalysis suggests a slow initial expansion of pe/ppe genesuntil the emergence of the most recent and expansivesublineage, sublineage V. In this review, the pe/ppe genes

and PE/PPE proteins will be annotated with their respec-tive phylogenetic sublineage numbers (I–V), denoted by asubscript annotation following the gene/protein name.

Several authors have speculated that the PE/PPE pro-teins represent a source of antigenic variation (Akhteret al., 2012), but this has yet to be supported by experi-mental data (Vordermeier et al., 2012). However, selectedmembers of the family do exhibit extensive genetic varia-tion, with multiple mechanisms contributing to altering theircoding sequences. Spontaneous homologous recombina-tion occurs intergenically mainly in the pe_pgrs genes thatshare extensive homologous regions (Karboul et al.,2008), This can result in deletions or duplications. Alterna-tive start codons, in-frame and frameshifting insertions/deletions, single nucleotide polymorphisms (SNPs) andtransposable element insertions also contribute to geneticvariation (Gey van Pittius et al., 2006; McEvoy et al.,2009a; 2012). A high level of sequence polymorphismhas been recorded within orthologous genes of thefamilies when compared among different clinical isolates.Genetic variation is very frequent in sublineage V membersincluding pe_pgrs33V, pe_pgrs16V, pe_pgrs26V andppe_mptr34V, although variation has also been reported inother sublineage members (e.g. ppe38IV) (Sampson et al.,2001; Banu et al., 2002b; Talarico et al., 2005; 2008;McEvoy et al., 2009b; Copin et al., 2014).

Emerging data suggest that pe/ppe polymorphisms donot evolve in response to immune selection, as has beenpreviously suggested, but rather by some other (as yetunknown) mechanism. McEvoy et al. found that while bothpe and ppe genes had approximately threefold highermutational rates than non-pe/ppe genes, no selective con-straints were present, although this study excluded thepe_pgrs subset (McEvoy et al., 2012). In contrast, an insilico examination of all H37Rv genes suggested that onesublineage IV ppe gene and three sublineage V pe_pgrsgenes displayed positive selection (Zhang et al., 2011).Interestingly, the pe_pgrs62V gene shows relatively littlesequence variation, suggestive of purifying selectionacting upon this gene (Namouchi et al., 2013). The reasonfor such discrepant findings is unclear but could be relatedto small sample size and biased gene selection. Thiscontention is perhaps supported by results from a recentextensive and systematic analysis of pe_pgrs sequencediversity, in 27 pe_pgrs genes from 94 clinical isolatesrepresenting five phylogenetic sublineages (Copin et al.,2014). This study demonstrated that while the pe_pgrsgenes as a whole do exhibit significant genetic variation incomparison to the rest of the genome, individual pe_pgrsgenes differed substantially with respect to the nucleotideand insertion/deletion diversity and dN/dS ratios. The lattermeasure indicated the existence of neutral evolution, puri-fying selection and diversifying selection for different familymembers, suggestive of functionally distinct roles.

902 S. Fishbein, N. van Wyk, R. M. Warren and S. L. Sampson ■

© 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96, 901–916

PE3 PE4

PE-PGRS11 PE-PGRS46

PE-PGRS58 PE12

PE-PGRS39 PE21+PE-PGRS36

PE-PGRS7 PE-PGRS59

PE-PGRS29 PE-PGRS55+PE-PGRS56

PE-PGRS57 PE-PGRS54

PE-PGRS40 PE-PGRS28

PE-PGRS27 PE-PGRS18 PE-PGRS17 PE-PGRS45

PE14 PE-PGRS23 PE-PGRS51 PE-PGRS26

PE-PGRS15 PE-PGRS44

PE-PGRS8 PE-PGRS34

PE-PGRS33 PE-PGRS52

PE-PGRS38 PE-PGRS47

PE-PGRS19 PE-PGRS6

PE-PGRS2 PE-PGRS25

PE-PGRS32 PE-PGRS1

PE16 PE23

PE-PGRS30 PE17

PE-PGRS62 PE1

PE-PGRS31 PE-PGRS49+PE-PGRS50

PE-PGRS41 PE9+PE10

PE-PGRS24 PE-PGRS22

PE-PGRS21 PE-PGRS10

PE-PGRS60+PE-PGRS61 wag22

PE-PGRS53 PE-PGRS4

PE-PGRS14 PE-PGRS9

PE33 PE-PGRS5

PE-PGRS53 PE-PGRS63 (lipY)

PE-PGRS12+PE-PGRS13 PE6

PE-PGRS48 PE-PGRS42 PE-PGRS43

PE-PGRS35 PE26

0.1

PE35

PE34 Sublineage I

PE5 PE15

PE36 Sublineage III

Sublineage II

PE25

PE11 (lipX) PE22

PE20 PE18 PE19

PE32 PE13

PE31 PE7

PE8 PE27

PE2

Sublineage V (PGRS subfamily)

PE-PGRS20 PE24

PE-PGRS16

Sublineage IV

10

PPE68

Sublineage I PPE4 PPE11 PPE37

PPE67 PPE2 PPE3

PPE46 PPE48

PPE1 PPE20

Sublineage II (PPW subfamily)

PPE36 PPE69

PPE59

PPE41 PPE57

PPE58

Sublineage III

PPE9 PPE17

PPE29 PPE30

PPE32 PPE33 PPE65

PPE10 PPE12

PPE21 PPE40

PPE54 PPE6

PPE8 PPE13

PPE16 PPE24

PPE34 PPE35 PPE28

PPE29 PPE53 PPE62

PPE52 PPE64

PPE55 PPE56

PPE38 PPE49

(MPTR subfamily) Sublineage V

PPE27

PPE14 PPE50

PPE51 PPE61 PPE44

PPE15 PPE43 PPE18 PPE19

PPE60 PPE22 PPE26

PPE23 PPE45 PPE25

Sublineage IV (SVP subfamily)

PPE31

SigD

A B

Lsr2SigBmprABphoPRDevRSEspRcAMP-dependent

Fig. 1. Regulation of the pe/ppe gene family. Adapted from ‘Evolution and expansion of the Mycobacterium tuberculosis PE and PPEmultigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions’ (Gey van Pittius et al., 2006). Symbolsnext to gene names in the phylogeny identify the regulator(s) controlling expression of the corresponding gene based on published literature(Dahl et al., 2003; Raman et al., 2004; Bai et al., 2005; Calamita et al., 2005; Rickman et al., 2005; He et al., 2006; Walters et al., 2006; Panget al., 2007; Lee et al., 2008; Fontan et al., 2009; Gazdik et al., 2009; Gordon et al., 2010; Blasco et al., 2012; Majumdar et al., 2012; Solanset al., 2014).A. PE family.B. PPE family.

Evolution of PE/PPE-associated virulence 903


It is increasingly apparent that insertion elements play arole in the presence and arrangement of pe/ppe genes inthe mycobacterial genome. In particular, IS6110 elementsdrive genetic variation that could contribute to phenotypicchanges (Sampson et al., 1999; McEvoy et al., 2007),frequently inserting into pe/ppe genes and their flankingregions, causing mutations as described above (Perez-Lago et al., 2011). The polymorphic or hypervariablenature of certain ppe genes has been correlated withIS6110 integrations and IS6110-mediated recombination,in a sampling of M. tuberculosis strains (McEvoy et al.,2009b). Illumina sequencing of IS6110 insertion sites in asampling of 519 clinical isolates demonstrated that IS6110frequently interrupted pe/ppe genes (Reyes et al., 2012).The majority of reported IS6110 insertions were into ppegenes, predominantly into the later sublineages (16/21IS6110 insertions into pe/ppe genes occurred in subline-age IV and sublineage V members). Limitations in thesegenomic studies exist where repetitive sequences ofmembers of the pe/ppe genes prevent accurate align-ments, which could account for the relative underrepresen-tation of pe_pgrs insertions. However, the associationbetween this gene family and transposable elementsremains. In addition to driving genetic variation, the pres-ence of IS6110 elements near and within pe/ppe genesmay influence gene expression levels, as has beenreported for other genes in M. tuberculosis (Safi et al.,2004).

The impact of genetic variation on structure and conse-quently function of PE/PPE proteins remains largelyunknown. The first structure for a PE/PPE protein pair(PE25III/PPE41III) was determined in 2006 (Strong et al.,2006). The crystal structure of a putative enzymaticdomain of PE_PGRS16V was reported in 2013 (Barathyand Suguna, 2013), and two complete PE25III/PPE41III

structures (in complex with EspG) only followed in 2014(Ekiert and Cox, 2014; Korotkova et al., 2014), underlin-ing the inherently challenging nature of these proteins.The limited experimentally confirmed structural dataimpose limitations on the ability to predict the functionalconsequences of PE/PPE variability.

Nonuniform regulation of pe/ppe transcription

Regulation of the pe/ppe gene family is controlled by aheterogeneous group of transcriptional regulators (Fig. 1)(Voskuil et al., 2004). pe/ppe operonic arrangementsand transcriptional regulation are reviewed in detail byMohareer et al. (2011); here we focus on selected pe/pperegulators in the context of pe/ppe phylogenetic groupings.

Mycobacterial sigma factors facilitate physiologicaladaptation to environmental stimuli and virulence in thepathogenic mycobacteria (Sachdeva et al., 2010); multi-ple sigma factors regulate pe/ppe gene expression. SigF

has been shown to regulate genes from all sublineages ofthe pe gene family, as well as genes from the ppe sublin-eages I–IV. SigF is present in both saprophytic and patho-genic mycobacteria, and is consistently linked to bacterialadaptation to environmental stress in the form of pH,oxidative stress and heat shock (Williams et al., 2007;Humpel et al., 2010). While advantageous for environ-mental mycobacteria survival, this adaptive capability alsoproves to be important for pathogenic mycobacteria insidemacrophages (Russell, 2011). SigB is another ubiquitoussigma factor, implicated in control of the mycobacterialcell wall stress response (Manganelli et al., 1999). SigBregulates the expression of many pe_pgrs genes andsome ppe genes, which have been postulated to beinvolved in host-bacterium interaction at the cell wall (Leeet al., 2008; Fontan et al., 2009). SigD also regulatesmany pe_pgrs genes, and for the most part regulates onlype genes, with the exception of ppe19IV. SigD responds tothe human lung environment, modulating expression inresponse to starvation and hypoxia, and suppresses theexpression of multiple PE_PGRS proteins (Raman et al.,2004; Calamita et al., 2005). Intriguingly, both SigB andSigD appear to control the expression of the most recentlyevolved sublineage of pe/ppe genes found only in patho-genic mycobacteria and control the expression of genesessential for the stress response and pathogenicity of thebacteria in vivo (Calamita et al., 2005; Lee et al., 2008).This indicates that the PE/PPE proteins under theircontrol may also play a role in bacterial virulence.

Two-component regulatory systems (TCSs) are bacte-rial cell sensors, and in M. tuberculosis they contributeto bacterial adaptation to an intracellular lifestyle (Bretlet al., 2011). Several TCSs affect the expression ofpe/ppe genes. The most influential TCS in pe/ppe geneexpression is phoPR, controlling the expression ofat least 13 different pe/ppe genes (Fig. 1) (Walters et al.,2006). Furthermore, a recent study that mappedgenome-wide PhoP binding sites using ChIP-Seq iden-tified six pe/ppe targets (Solans et al., 2014). Thesestudies revealed an overrepresentation of sublineage IVmembers among the PhoP-dependent genes. In con-trast, the influence of the TCS MprAB on pe/ppe expres-sion is more widely observed in all sublineages of the pefamily, including the ancestral pe35I, as well as subline-ages II and IV of the ppe family, pointing to a moregeneral role in adaptation to stress, rather than specifi-cally to mycobacterial pathogenicity (He et al., 2006;Pang et al., 2007). Finally, DevRS has been implicatedin regulation of the M. tuberculosis dormancy response(Malhotra et al., 2009). DevRS controls cell wall pro-cesses and other intermediary metabolic processes thatare involved in adaptation to dormancy, and the regulonincludes the operonic pair pe18/ppe25IV and ppe51IV

(Majumdar et al., 2012).



The global nucleoid-associated transcriptional inhibitorLsr2 has been implicated in the transition to the stringentresponse (Gordon et al., 2010). Although not a regulatorper se, RelA has similarly been implicated in this adaptivepathway, and together with Lsr2 impacts on the expres-sion of a number of pe/ppe genes (Dahl et al., 2003;Gordon et al., 2010). Lsr2 has been associated with theexpression of all sublineage V ppe_mptr genes and withmultiple pe/ppe genes from other sublineages (Fig. 1).EspR is another nucleoid-associated regulator, whichregulates numerous cell wall components. ChIP-Seqanalysis revealed that EspR binds regions upstream of, orwithin, at least 30 pe/ppe genes, with overrepresentationof more recent pe/ppe sublineages; no sublineage I or IIgenes were identified and 24/30 of the pe/ppe targetswere from sublineage V (Blasco et al., 2012). The linkbetween the virulence factors RelA, Lsr2 and EspR andpe/ppe gene expression, in particular the more recentsublineages, further supports the idea that selectedpe/ppe genes play a role in mycobacterial virulence andpersistence mechanisms.

The second messenger cAMP is important for funda-mental physiological processes as well as mycobacterialvirulence. Multiple cAMP-dependent regulators of thepe/ppe family have been uncovered, including Rv3676, acAMP receptor protein predicted to bind upstream of ninepe/ppe genes, including five pe_pgrs genes (sublineageV) and three ppe genes of sublineage II (Bai et al., 2005).Interestingly, Bai et al. also predicted binding of Rv3676 tope15II. This gene is immediately upstream of ppe20II,which is downregulated in a Rv3676 deletion mutant(Rickman et al., 2005), providing support for the role ofcAMP-mediated regulation of pe/ppe transcription. Asecond ppe gene, ppe51V, was also downregulated in theRv3676 mutant, which was shown to be impaired for invitro and in vivo growth (Rickman et al., 2005). AnothercAMP-associated regulator, Rv1675c, is also importantfor intramacrophage gene regulation, and its regulonincludes pe_pgrs6V (Gazdik et al., 2009). While there iscurrently no definitive link, this suggests that there mightbe merit in exploring selected pe/ppe family members ascAMP-mediated virulence factors.

Localization and functional studies of PE/PPE proteins

Understanding how, where and which PE/PPE proteins aretrafficked in the mycobacterial cell and beyond couldprovide important clues to their function. A bioinformaticapproach exploiting statistical analysis of network topologyand gene ontology term enrichment suggested that at least29 PE/PPE proteins are associated with the ‘cell wall andcell processes’ functional category (Mazandu and Mulder,2012); such members are found in all but sublineage I(Table 1). There is increasing experimental evidence to

support PE/PPE association with the mycomembrane,or surface exposure at the host-pathogen interface(Sampson et al., 2001; Delogu et al., 2004). Several high-throughput proteomics studies have confirmed the produc-tion and localization of PE/PPE family members (Table 1).Interestingly, at least seven PE/PPE proteins have beendetected in culture filtrate (Table 1), suggesting theirrelease from the mycobacterial cell. Over 35 PE/PPEproteins have been detected in or on the mycobacterialmembrane and/or cell wall (Table 1). Culture filtrate, cellwall or membrane-associated PE/PPE family membershave been identified in all PE/PPE sublineages. This sug-gests that while the PE/PPE functions may have diversi-fied, their specific function is frequently dependent on cellwall or extracellular localization.

Recent attention has focused on the dissection ofPE/PPE domains involved in subcellular localization.The N-terminal PE domain of the well-characterizedPE_PGRS33V was shown to be necessary for proteinlocalization to the cell wall in M. marinum and M. tubercu-losis (Cascioferro et al., 2007; 2011; Zumbo et al., 2013).This function also exists in LipYV and PE_PGRS30V,whereby the N-terminal sequence enables secretion andthe C-terminal encodes the lipase activity of the protein(Daleke et al., 2011; Iantomasi et al., 2012). Recent workon PPE17IV suggests that the PE and PPE domains mayfulfill similar cell wall targeting roles (Dona et al., 2013).Further support for the importance of the PE domain inlocalizing PE_PGRS proteins to the cell wall was providedby examination of PE_PGRS30V (Chatrath et al., 2014).This study also suggested that the PGRS domain isinvolved in polar localization. This is intriguing in the light ofreports that the ESX-1 secretion machinery similarly showspolar localization, although direct interaction of the PGRSdomain with the ESX machinery has yet to be confirmed.

Several PE/PPE proteins are secreted via the type VII(ESX) secretion system (T7S) (Abdallah et al., 2006;2009; McNamara et al., 2012). ESX-1, one of the T7S,has been implicated in PPE68I secretion in M. marinum(Sani et al., 2010; Houben et al., 2012). Using an in silicoapproach to examine the architecture of the ESX-1system, the authors predicted that PPE68I was associ-ated with the mycomembrane and likely acts as a gatingprotein for the ESX-1 secretion system (Das et al., 2011).An M. marinum study demonstrates that the ESX-5protein EspG5 binds PPE-SVP family members as asecretion chaperone (Daleke et al., 2012). ESX-1 homo-logues of EspG5 are also known to facilitate PE/PPEprotein secretion and stability (Bottai et al., 2011). Thestructural basis for this has been suggested by recentstudies demonstrating that the PE25III/PPE41III pair inter-acts with EspG from the ESX-5 secretion system (Ekiertand Cox, 2014; Korotkova et al., 2014). Together, thesestudies suggest that EspG confers specificity and guides



Table 1. Summary of proteomic data indicating subcellular localization.

Rv number PE/PPE name Essentiala Functional predictionb Sublineagec

In vivo expression

(guinea pig

lung)d (days) Localization (proteomic studies)

Rv0096 PPE1 N Cell wall and cell processes II 90Rv0109 PE_PGRS1 N V 90Rv0124 PE_PGRS2 N V 30 and 90Rv0151c PE1 N V 30Rv0152c PE2 N V Membranee

Rv0159c PE3 N V Membranef

Rv0160c PE4 N V 30Rv0256c PPE2 N II Membranee

Rv0278c PE_PGRS3 Virulence, detoxification, adaptation V 30 and 90Rv0279c PE_PGRS4 N V 30 and 90Rv0280 PPE3 N II 90Rv0285 PE5 Y Cell wall and cell processes II CF, predicted SP confirmed

experimentallyg; CF and membrane

fraction, enriched in membrane

fraction, SP confirmedh; membranee

Rv0286 PPE4 Y Cell wall and cell processes II Membranee

Rv0297 PE_PGRS5 N V 90Rv0304c PPE5 N NA 30Rv0305c PPE6 N V Cytosol, cell wall and membranee

Rv0335c PE6 N Insertion sequences and phages V Cell walle

Rv0354c PPE7 N NA 90Rv0355c PPE8 N V 30 and 90Rv0388c PPE9 N IVRv0442c PPE10 N V 30 and 90Rv0453 PPE11 N II 30 Cytosole; CFg

Rv0532 PE_PGRS6 N VRv0578c PE_PGRS7 N V Membranee,h

Rv0742 PE_PGRS8 N VRv0746 PE_PGRS9 N V 30 and 90Rv0747 PE_PGRS10 N Lipid metabolism V 30 and 90Rv0754 PE_PGRS11 N V 30 and 90Rv0755c PPE12 N V 90 daysRv0832 PE_PGRS12 N V Membrane (not CF), SP confirmedh;

membranee

Rv0833 PE_PGRS13 N VRv0834c PE_PGRS14 N V 30 and 90Rv0872c PE_PGRS15 N V 30 and 90Rv0878c PPE13 N Cell wall and cell processes VRv0915c PPE14 N IVRv0916c PE7 N Intermediary metabolism and

respiration

IV

Rv0977 PE_PGRS16 N VRv0978c PE_PGRS17 N V 90Rv0980c PE_PGRS18 N VRv1039c PPE15 N Cell wall and cell processes IV 90Rv1040c PE8 N Cell wall and cell processes IVRv1067c PE_PGRS19 N V 30 and 90Rv1068c PE_PGRS20 VRv1087 PE_PGRS21 N V 90Rv1088 PE9 N V 90 Membranee

Rv1089 PE10 N VRv1091 PE_PGRS22 V 30 and 90Rv1135c PPE16 N VRv1168c PPE17 N Intermediary metabolism and

respiration

IV

Rv1169c lipX N III Membranef,h

Rv1172c PE12 N V Membrane, SP confirmedh; membranef

Rv1195 PE13 N IV WCL, but not CF or membrane, SP

confirmedh

Rv1196 PPE18 N Cell wall and cell processes IV 90 Cell walli; membranef

Rv1214c PE14 N Cell wall and cell processes V 90 Cell walli

Rv1243c PE_PGRS23 N V 30Rv1325c PE_PGRS24 N V 30 and 90Rv1361c PPE19 N Cell wall and cell processes IV 90Rv1386 PE15 N Intermediary metabolism and

respiration

II CFg, WCL, SP confirmedh; membranef,h

Rv1387 PPE20 N Cell wall and cell processes II Membrane, not CFh; membranef

Rv1396c PE_PGRS25 N V



Table 1. cont.


In vivo expression

(guinea pig


Rv1430 PE16 N Intermediary metabolism and

respiration

V

Rv1441c PE_PGRS26 N VRv1450c PE_PGRS27 V Cell wallRv1452c PE_PGRS28 N VRv1468c PE_PGRS29 N V 30 SP confirmedh; membranef,h

Rv1548c PPE21 N Lipid metabolism V 30Rv1646 PE17 N Intermediary metabolism and

respiration

V WCL, but not CF or membraneh

Rv1651c PE_PGRS30 N V 30 and 90Rv1705c PPE22 N IV 30Rv1706c PPE23 N Cell wall and cell processes IV 90Rv1753c PPE24 Y V 90Rv1759c wag22 N Cell wall and cell processes V 30 and 90 CFg

Rv1768 PE_PGRS31 N V 30 and 90Rv1787 PPE25 N IVRv1788 PE18 N Intermediary metabolism and

respiration

NA

Rv1789 PPE26 N IV Membranef,h

Rv1790 PPE27 IV 90Rv1791 PE19 IVRv1800 PPE28 N VRv1801 PPE29 N Cell wall and cell processes IV Cell walli

Rv1802 PPE30 N Cell wall and cell processes IV 30 and 90Rv1803c PE_PGRS32 V 30 and 90Rv1806 PE20 IV Membrane and WCL but not CFh

Rv1807 PPE31 Y (in vivo) IV WCL but not CFh; membranef,h

Rv1808 PPE32 N IV WCL but not CFh; membranef,h

Rv1809 PPE33 IV 90 WCL but not CFh; membranef,h

Rv1818c PE_PGRS33 N Intermediary metabolism and

respiration

V

Rv1840c PE_PGRS34 N Cell wall and cell processes VRv1917c PPE34 N V 30 and 90 Surface exposedj

Rv1918c PPE35 N V 30 and 90Rv1983 PE_PGRS35 N Cell wall and cell processes V 90Rv2098c PE_PGRS36 N Cell wall and cell processes VRv2099c PE21 VRv2107 PE22 IIIRv2108 PPE36 N IIIRv2123 PPE37 N IIRv2126c PE_PGRS37 N V 90Rv2162c PE_PGRS38 N Cell wall and cell processes V 30 and 90 Membranef

Rv2328 PE23 N V 90Rv2340c PE_PGRS39 N V 90 Membranee

Rv2352c PPE38 N Cell wall and cell processes IV 90 WCL, but not CF or membraneh;

membranef

Rv2353c PPE39 N NA Membranef

Rv2356c PPE40 Y Cell wall and cell processes V WCL, but not CF or membraneh

Rv2371 PE_PGRS40 N VRv2396 PE_PGRS41 N VRv2408 PE24 N VRv2430c PPE41 N III CFg; WCL, but not CF or membraneh

Rv2431c PE25 N III CFg; WCL, CF, membraneh; cell walli;

membranef

Rv2487c PE_PGRS42 N Regulatory V 30 and 90Rv2490c PE_PGRS43 N V 30 and 90Rv2519 PE26 N VRv2591 PE_PGRS44 N VRv2608 PPE42 N VRv2615c PE_PGRS45 N VRv2634c PE_PGRS46 N Cell wall and cell processes V 30 and 90Rv2741 PE_PGRS47 N V 90Rv2768c PPE43 N IV 30 and 90Rv2769c PE27 N IV 30Rv2770c PPE44 N IV 90Rv2853 PE_PGRS48 N V 30 and 90Rv2892c PPE45 N Intermediary metabolism and

respiration

IV 90

Rv3018c PPE46 N Cell wall and cell processes II



PE/PPE proteins to the ESX secretion systems (Ekiertand Cox, 2014; Korotkova et al., 2014).

While there has been intensive focus on T7S-mediatedsecretion, it is important to note that classical Secpathway signal sequences have also been identified insome PE and PPE proteins (McDonough & Braunstein,2008). Recent proteomic data indicate that predictedsignal peptidase I sites in at least seven PE/PPE proteins

are indeed cleaved, suggesting that these proteins maybe exported via the general secretory pathway (de Souzaet al., 2011). However, the relative contribution of theSecA, ESX and other secretion systems to PE/PPEsecretion has yet to be fully elucidated.

Given the association of several PE/PPE proteinswith the mycobacterial cell wall, it is unsurprising thatsome PE/PPE proteins have been implicated in struc-

Table 1. cont.


In vivo expression

(guinea pig


Rv3018A PE27A NA Membranef

Rv3021c PPE47 Y Cell wall and cell processes NARv3022c PPE48 Cell wall and cell processes IIRv3022A PE29 N NARv3097c lipY N V 90Rv3125c PPE49 N Cell wall and cell processes IV 90Rv3135 PPE50 N IVRv3136 PPE51 Cell wall and cell processes IV 30 Cell walli; membranef

Rv3144c PPE52 N V 90Rv3159c PPE53 N V 90Rv3343c PPE54 Y V 30 and 90Rv3344c PE_PGRS49 VRv3345c PE_PGRS50 V 30 and 90Rv3347c PPE55 N V 30 and 90Rv3350c PPE56 N V 30 and 90Rv3367 PE_PGRS51 N V 90Rv3388 PE_PGRS52 N V 30Rv3425 PPE57 N IIIRv3426 PPE58 III Membranee

Rv3429 PPE59 N IIIRv3477 PE31 N IV Membrane, lysate not CFh

Rv3478 PPE60 IV 90 Membranef

Rv3507 PE_PGRS53 N V 30 and 90Rv3508 PE_PGRS54 Y V 90Rv3511 PE_PGRS55 N VRv3512 PE_PGRS56 N V 30 and 90 Membranee

Rv3514 PE_PGRS57 Y V 30 and 90Rv3532 PPE61 N IVRv3533c PPE62 Intermediary metabolism and respiration VRv3539 PPE63 N Intermediary metabolism and respiration NA Membranee

Rv3558 PPE64 N VRv3590c PE_PGRS58 N Intermediary metabolism and respiration V 30 and 90Rv3595c PE_PGRS59 N V 90Rv3621c PPE65 N Intermediary metabolism and respiration IV 30 and 90Rv3622c PE32 N Cell wall and cell processes IVRv3650 PE33 N Information pathways VRv3652 PE_PGRS60 N VRv3653 PE_PGRS61 N VRv3738c PPE66 N NARv3739c PPE67 N IIRv3746c PE34 N IRv3812 PE_PGRS62 N Cell wall and cell processes V 30 and 90Rv3872 PE35 Y I CFg; CF but not membrane or WCLh; membranef

Rv3873 PPE68 Y (in vivo) I WCL, SP confirmedh; membranef,h

Rv3892c PPE69 N III 90Rv3893c PE36 N Cell wall and cell processes III

a. Based on Tuberculist annotation: http://tuberculist.epfl.ch/index.html, Y denotes essential in vitro, unless otherwise noted.b. Mazandu and Mulder (2012).c. Gey van Pittius et al. (2006).d. Kruh et al. (2010).e. Mawuenyega et al. (2005).f. Malen et al. (2011).g. Malen et al. (2007).h. de Souza et al. (2011).i. Wolfe et al. (2010).j. Sampson et al. (2001).CF, culture filtrate; WCL, whole cell lysate; SP, signal peptide.



http://tuberculist.epfl.ch/index.html

tural integrity and colony morphology of M. tuberculosis.PE_PGRS30V decreased colony size upon expression inthe nonpathogenic, fast-growing M. smegmatis and alsolocalized to the poles of the bacilli (Chatrath et al.,2011). Deletion of ppe38IV from M. marinum compro-mised structural integrity by altering the cell surface andpreventing biofilm formation (Dong et al., 2012). Disrup-tion of three ppe genes (all from sublineage V) viatransposon mutagenesis led to significantly increasedresistance to ampicillin, implicating these proteins inmaintaining the cell wall integrity of M. tuberculosis(Danilchanka et al., 2008).

Surface exposure or secretion into the extracellularmilieu may position PE/PPE proteins to interact directlywith host targets. There is some evidence that selectedfamily members may interact with host molecules, such ashost cell surface TLR2 (Basu et al., 2007; Nair et al.,2009; Bansal et al., 2010b; Tiwari et al., 2012; Bhat et al.,2013; Zumbo et al., 2013). Intriguingly, PPE41III wasdetected in the late endosome, detached from the myco-bacteria, suggesting that some family members may bedeliberately secreted into the phagosome (Abdallah et al.,2006). Intraphagosomal host targets of PE/PPE proteinshave yet to be identified, but could be an exciting avenuefor future research.

Recent reports have suggested an intriguing linkbetween the PE/PPE families and mycobacterial lipidmetabolism. Upon entry into a macrophage, M. tuberculo-sis undergoes physiological adaptations to the intracellu-lar environment, including the upregulation of lipidmetabolism genes (Rosas-Magallanes et al., 2007). Thebacterium scavenges host triacylglycerol (TAG) and incor-porates these fatty acids into either storage bodies ormycobacterial lipids (Daniel et al., 2011). Like many otherviral and bacterial pathogens, M. tuberculosis utilizes lipidinclusions, composed mostly of TAG, as a carbon sourceduring times of starvation, oxygen depletion or pathogenreactivation (Stehr et al., 2012). It was recently shown thata M. marinum mutant deficient in lipooligosaccharide syn-thesis is impaired in secretion of PE_PGRS proteins (vander Woude et al., 2012). Other work has shown that fol-lowing starvation, M. tuberculosis upregulates the expres-sion of lipase genes, including two members of the PEfamily, namely LipYV (PE_PGRS63V) and LipXIV (PE11IV)(Deb et al., 2006). LipYV was the first PE/PPE proteinfound to have enzymatic properties, namely TAG lipaseactivity, allowing it to cleave TAG into a diacylglycerol.LipYV is secreted by ESX-5 into the extracellular milieuwhere it scavenges TAG for a nutrient source (Dalekeet al., 2011). In silico analysis of PE/PPE functional motifsidentified a prominent serine α/β hydrolase fold in 10members of the protein family, all in sublineage V (Sultanaet al., 2011). From this list, PE16V has been experimentallyconfirmed as an esterase that functions at neutral pH in

the presence of salts (Sultana et al., 2013). Mazandu andMulder (2012) have further predicted that PE_PGRS10V

and PPE21V are also involved in lipid metabolism,although this has yet to be experimentally verified. Thesestudies point toward a role for the most recent PE/PPEsublineage in lipid metabolism, an important feature ofmycobacterialpathogenicity.

PE/PPE protein family and host immune response: acomplex relationship

In comparison to other pathogenic organisms, M. tuber-culosis does not contain nearly as many hypermutableantigens as one might expect (Comas et al., 2010;Russell, 2013). The PE/PPE protein family has long beenspeculated to contain variable antigens (Banu et al.,2002a), akin to antigenic variation in other organisms (vander Woude and Baumler, 2004). However, this remains anunresolved debate, and is unlikely to hold true for allfamily members.

In addition to the characteristic short, GC-rich PGRSrepeats, two PE_PGRS proteins (PE_PGRS17V andPE_PGRS18V) have been shown to possess tandemrepeats of 41–43 amino acid (aa) residues in theC-terminal variable regions (Adindla and Guruprasad,2003), which correspond to the AB repeats identified in cellsurface antigens of Methanosarcina mazei (Mayerhoferet al., 1995). Three PPE_MPTR proteins (PPE_MPTR24V,PPE_MPTR34V and PPE_MPTR35V) possess similartandem repeats of between 23 and 26 aa, which have beenshown to exhibit variable copy number in clinical isolate ofM. tuberculosis (Sampson et al., 2001). While this is typicalof antigenically variable domains in other pathogens, theimmunogenicity and in vivo significance of these variablerepeats have yet to be established. An extensive study ofpe_pgrs genes by Copin et al. concluded that T cell recog-nition does not play a significant role in diversifying selec-tion (Copin et al., 2014).

Closer examination of PE/PPE epitopes suggests thatthe highly immunogenic nature of selected family membersmay partly be driven by a high degree of immunogeniccross-reactivity. For example, epitopes found in PPE25IV

were identified in other members, such as PPE32IV,PPE22IV and PPE45IV (Sayes et al., 2012), and PE19IV

epitopes were also replicated in other sublineage IVmembers. Similarly, ex vivo responses to a large library ofpeptide pools representing 36 PE/PPE proteins demon-strated that the most immunogenic domains (as measuredby IFN-gamma response) of these proteins are the moreconserved N-terminal motifs (Vordermeier et al., 2012).This study also revealed recognition of potentially cross-reactive epitopes throughout infection, arguing againstthese proteins representing variable antigens.



Prior to the onset of the adaptive immune response, M.tuberculosis infection is established by entry into hostmacrophages (Kaufmann, 2001; Thi et al., 2012). SeveralPE/PPE proteins have been linked to inhibition and/oractivation of host macrophage activity (Fig. 2). The ESX-5secretion system and associated PE/PPE proteins areresponsible for some M. tuberculosis virulence and con-tribute to its ability to grow in a macrophage (Bottai et al.,2012). For example, PE_PGRS62V inhibits phagosomematuration and certain bactericidal mechanisms in vivo(Huang et al., 2012). PE_PGRS62V, PE5II, PPE15IV andPPE2II all inhibit reactive nitrogen species production(Tiwari et al., 2012; Bhat et al., 2013). Disrupting ppe38IV inM. marinum reduced both macrophage phagocytosis andvirulence in zebrafish (Dong et al., 2012). PE_PGRS30V

has also been associated with mycobacterial virulence,specifically by inhibiting the phagolysosomal fusion(Iantomasi et al., 2012). Deleting a ppe25IV homologuefrom a M. avium subspecies reduced intra-macrophagereplication and inhibited phagolysosomal fusion (Jha et al.,2010). PE5II, PE15II and PE4V have also recently beenidentified as proteins that increase mycobacterial survival

within the macrophage (Singh et al., 2012; Tiwari et al.,2012). A review of the immunomodulating effects ofPE/PPE proteins further provides evidence of these pro-teins’ ability to modulate the macrophage environment(Mukhopadhyay and Balaji, 2011). It is noteworthy that themany of the PE/PPE proteins implicated above are fromlineages outside the pathogen-specific sublineages; thissuggests that these proteins may have had a general rolein enabling mycobacterial adaptation to harsh environ-ments (pH and oxidative stress) and have since beenco-opted into aiding M. tuberculosis adaptation to themacrophage environment.

Examination of in vivo expression of 10 ppe_mptr genes(all belonging to sublineage V) in lung and spleen tissue ofM. tuberculosis-infected mice revealed no clear pattern inthe production of even closely related PPE proteins(Soldini et al., 2011). This study also showed that the sameppe_mptr genes are strongly upregulated when exposedto isoniazid and ethambutol. These drugs target the enzy-matic pathways leading to cell wall biosynthesis, providingindirect evidence of a role for PPE_MPTR proteins in thecell wall and possibly at the host-pathogen interface.

Early phagosome/bacterialreplication

Acidification/

Lysosome

TLR2

pH 6.5

pH 4.5

H+

Latephagosome

Phagocytosis

H+

phagosomematuration

Phagolysosome

RNSvia iNOS

APE_PGRS33VPPE38IV

PPE18IVPPE17IVPE_PGRS17VPE_PGRS11VPPE38IV

PE_PGRS30V

PE_PGRS62VPE5IIPE15IIPPE2IIPE4V

MAV_2928 (PPE25)IVPE_PGRS62V

M. tb

Host macrophage

A

BB

CC

DD

Fig. 2. PE/PPE proteins influence macrophage processing of Mycobacterium tuberculosis. Adapted from Kaufmann (2001). A representationof M. tuberculosis infection of a host macrophage, and the points at which bacterial PE/PPE proteins affect this process.A. Proteins in this group have been shown to inhibit phagocytosis of the bacilli (Basu et al., 2007; Dong et al., 2012).B. Proteins in this group bind TLR-2 and induce downstream proliferation of the immune system (Nair et al., 2009; Bansal et al., 2010b; Donget al., 2012; Dona et al., 2013).C. Proteins in this group stop mechanisms of nitrosative stress by inhibiting iNOS production of RNS (Huang et al., 2012; Singh et al., 2012;Tiwari et al., 2012).D. Proteins in this group inhibit some part of phagosome maturation, either acidification or phagolysosome fusion (Jha et al., 2010; Huanget al., 2012; Iantomasi et al., 2012).



Another in vivo study revealed the striking relative abun-dance of PE/PPE proteins detected during guinea piginfection, underlining their potential role in virulence (Kruhet al., 2010). Examination of the data reveals a bias towardexpression of sublineage V PE/PPE proteins during in vivoinfection (Table 1). Of the 35 PE/PPE proteins detected atboth early and late time points, 32 of these belong to themost recent sublineage V. Over 60% of the PE_PGRS andPPE_MPTR proteins are expressed in guinea pig lungs at90 days post infection. The precise role and host targets (ifany) of the proteins during infection have yet to be estab-lished, although several studies support the ability ofPE/PPE proteins to modulate the host environment. Forexample, in recombinant M. smegmatis, PPE37II interfereswith the pro-inflammatory response, decreasing the pro-duction of TNF-alpha, IL-6 and IL-1B (Daim et al., 2011).PE4V, PE5II and PE15II, expressed in recombinant M.smegmatis, upregulate anti-inflammatory cytokines (Singhet al., 2012; Tiwari et al., 2012). Many other PE/PPE pro-teins also induce anti-inflammatory cytokine signalling(Balaji et al., 2007; Basu et al., 2007; Nair et al., 2009;Bansal et al., 2010a,b; Beaulieu et al., 2010). Comprehen-sive reviews of how PE/PPE proteins modulate hostimmune responses can be found elsewhere (Sampson,2011; Akhter et al., 2012; Deng and Xie, 2012). Althoughtechnical variations in the numerous published studiesmake it difficult to compare and interpret the resultsobtained, it is evident that several family members have thecapacity to influence host immune responses.

Conclusion and future questions

From a genomic perspective, the pe/ppe gene familieshave expanded from a few ancestral genes in non-tuberculous mycobacteria to the present-day family com-prising multiple sublineages of genes, with the mostrecent being the highly polymorphic genes that arepresent only in pathogenic mycobacteria. Although hyper-variable genes are present in the most recent sublineages(IV and V) of the pe and ppe gene family, it is important tonote the differing degrees of variability even within thesublineages. Much published work has focused on pe andppe genes in sublineages IV and V, which could biasconclusions made. However, it is tempting to speculatethat the variation observed in some members of the mostrecent sublineages of both gene families could be linkedto a functional role in M. tuberculosis pathogenicity.

Examination of the transcriptional signature of thepe/ppe gene family reveals that the most recent subline-age genes are frequently under control of virulence-associated regulators, for example, SigD and SigB.Regulators of earlier sublineage pe/ppe genes, such asthe TCS mprAB, may have been present before thepathogenicity of mycobacteria arose. Correspondingly,

these genes may play less of a role in virulence and moreof a role in environmental adaptation, a necessity for thesaprophytic mycobacteria.

Functional studies demonstrate that the PE/PPE pro-teins frequently localize to the mycobacterial surface andare involved in mycobacterial secretion systems. Intrigu-ingly, this family contains lipolytic enzymes, a part of themycobacterial metabolome that has implications in its viru-lence. Together, these results demonstrate that PE/PPEproteins are relevant to M. tuberculosis virulence as keycomponents of the cell wall that interact with the host, likelyfor survival purposes in vivo. When considered in the lightof PE/PPE phylogeny, there are indications that PE/PPEsecretion may be sublineage specific, at least with regardto T7S-mediated secretion. PE/PPE proteins that havebeen implicated in cell wall integrity occur in sublineages IVand V. Notably, the presence of predicted and confirmedlipolytic enzymes in the PE/PPE protein families isrestricted to sublineage V of the PE and the PPE proteinphylogenies; this could be linked to the essential role thatlipid metabolism plays in M. tuberculosis virulence.

The PE/PPE protein family affects mycobacterial inter-actions with the innate immune system, specifically inhib-iting macrophage function. In addition, the protein familyhas the capacity to modulate the adaptive immuneresponse to facilitate its own survival. Mycobacteriumtuberculosis has evolved from environmental mycobacte-ria, and the PE/PPE family likely contains proteins inher-ited from environmental mycobacteria that M. tuberculosisnow utilizes for intracellular survival. Examination of adap-tive immune responses to PE/PPE proteins suggests thatthese proteins are not variable antigens by the typicaldefinition. There are several examples of conservedPE/PPE epitopes evoking host immune responses, withindications that it may indeed be beneficial to the pathogento do so. In vivo proteomics reveal that the majority of thePE/PPE proteins that potentially interact with the hostcome from sublineage V. This last sublineage of proteinsexhibits enzymatic and immunogenic functionality, and isthe only lineage associated with mycobacterial lipidmetabolism. This, together with their apparent abundanceduring in vivo infection, suggests that these most recentlyevolved family members play important roles inpathogenicity.

The PE/PPE protein family contributes to many aspectsof M. tuberculosis pathogenicity and should be consideredfor novel drug targeting or immune-modulatory strategies.However, to fully realize this potential, a better understand-ing of PE/PPE protein function is required. We thereforeneed to address several key questions and challenges.First, the existence of multiple copies of similar genes, aswell as the repetitive nature of some family members,imposes limitations on next-generation sequencing dataextraction; many alignment algorithms automatically



discard reads mapping to multiple locations, restricting theamount of reliable pe/ppe sequence data. Addressing thiswill require the use of sequencing platforms yielding longerread lengths and/or more advanced alignment algorithms.The multicopy nature of the families also hinders efforts toascertain function; the effect of knocking out or overex-pressing a single pe/ppe gene may be difficult to detectusing conventional methods due to functional redundancy.Next, given the mounting evidence that selected PE/PPEproteins are able to modulate host immune responses, itwould be of interest to comprehensively identify hosttargets of these proteins. Finally, as highlighted above, onlythree PE/PPE proteins have been structurally character-ized to date, hampering our understanding of the functionalconsequences of PE/PPE variation. Additional experimen-tally verified structures or more sophisticated predictivemodeling approaches are required. For these and otherchallenges, it is likely that computational approaches willplay an important role in providing an integrated view ofhow different family members co-ordinate their functionduring infection.

Acknowledgements

SLS is funded by the South African Research Chairs Initiativeof the Department of Science and Technology and theNational Research Foundation (NRF) of South Africa, awardnumber UID 86539. The content is solely the responsibility ofthe authors and does not necessarily represent the officialviews of the NRF. SF was supported by a Fulbright Programgrant sponsored by the Bureau of Educational and CulturalAffairs of the United States Department of State and admin-istered by the Institute of International Education.

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