Pathogenomics Analysis of Leishmania spp.: FlagellarGene Families of Putative Virulence Factors
DIANA M. OLIVEIRA,1 JOAO JOSE S. GOUVEIA,1 NILO B. DINIZ,1ANA CAROLINA L. PACHECO,1 ELTON JOSE R. VASCONCELOS,1MICHELY C. DINIZ,1 DANIEL A. VIANA,1 THIAGO D. FERREIRA,1
MARIANNA C. ALBUQUERQUE,1 DANIEL C. FORTIER,1 ALLAN R.S. MAIA,1LUIS A.C. COSTA,1 JOÃO OSMAR P. MELO,1 MARIA CRISTINA DA SILVA,1
CEZAR A. WALTER,1 JOSE O. FARIA,1 ADRIANA R. TOME,1MARCOS JOSE N. GOMES,2 SONIA M.P. OLIVEIRA,3
RAIMUNDO ARAÚJO-FILHO,4 RAIMUNDO B. COSTA,1 RODRIGO MAGGIONI,1and PROGENE, The Brazilian Northeast Genome Program5
ABSTRACT
The trypanosomatid flagellar apparatus contains conventional and unique features, whoseroles in infectivity are still enigmatic. Although the flagellum and the flagellar pocket arecritical organelles responsible for all vesicular trafficking between the cytoplasm and cellsurface, still very little is known about their roles in pathogenesis and how molecules get toand from the flagellar pocket. The ongoing analysis of the genome sequences and proteomeprofiles of Leishmania major and L infantum, Trypanosoma cruzi, T. brucei, and T. gambi-ensi (�www.genedb.org�), coupled with our own work on L. chagasi (as part of the BrazilianNortheast Genome Program—�www.progene.ufpe.br�), prompted us to scrutinize flagellargenes and proteins of Leishmania spp. promastigotes that could be virulence factors in leish-maniasis. We have identified some overlooked parasite factors such as the MNUDC-1 (a pro-tein involved in nuclear development and genomic fusion) and SQS (an enzyme of sterolbiosynthesis), among the described flagellar gene families. A database concerning the resultsof this work, as well as of other studies of Leishmania and its organelles, is available at�http://nugen.lcc.uece.br/LPGate�. It will serve as a convenient bioinformatics resource ongenomics and pathology of the etiological agents of leishmaniasis.
1Núcleo de Genômica e Bioinformática, Faculdade de Veterinária, Universidade Estadual do Ceara (UECE), Cam-pus do Itaperi, Fortaleza, Ceara, Brazil.
2Laboratorio de Computaçao Cientifica, Departamento de Estatística Computaçao, UECE, Campus do Itaperi, Fort-aleza, Ceara, Brazil.
3Setor de Analise Estatística, Departamento de Zootecnia, Centro de Ciências Agrárias, Universidade Federal doCeara (UFC), Campus do Pici, Fortaleza, Ceara, Brazil.
4Laboratorio de Patologia Humana, Centro de Ciências da Saúde, Universidade de Fortaleza (UNIFOR), EdsonQueiroz, Fortaleza, Ceara, Brazil.
5The Brazilian Northeast Genome Program (PROGENE—�www.progene.ufpe.br�) is a regional, multi-institutionalconsortium for sequencing and annotation of expressed sequence tags from Leishmania chagasi.
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INTRODUCTION
UNICELLULAR PARASITES of the Trypanosomatidae family employ sophisticated immune evasion strate-gies to invade and persist on host cells. As a member of this family, Leishmania spp. are obligate in-
tracellular protozoa that exist in two forms: a promastigote form (elongated cells with a long flagellum)that converts into amastigote (ovoid cells exhibiting a very short flagellum). These two life stages corre-spond to the transit of the organisms between the sand fly vector and the mammalian host. The character-istic shapes of these two developmental stages are maintained by an array of subpellicular microtubules thatunderlie the plasma membrane (McConville et al., 2002). The flagellum is responsible for the motility oftrypanosomatids and for their early interaction with the hosts, either by adhering to the insect digestive tract(Vickerman and Tetley, 1977), or initiating the contact with mammalian cells (De Souza, 1984). Try-panosomatids depend on this adhesion to survive and differentiate (reviewed in De Souza, 1989). Amastig-otes replicate within phagolysosomes—acidic parasitophorous vacuoles—of the macrophages. How theythrive in a hostile environment that is lethal to other microbes (Bogdan et al., 1996) is not exactly under-stood, but they probably rely upon their unique secretory organelles for invasion and other specialized func-tions. The nucleus of Leishmania spp. is centrally located and the cytoplasm contains randomly distributedribosomes and endoplasmic reticulum, while the Golgi complex is located in the anterior region, close tothe flagellar pocket. Some unique organelles of trypanosomatids are (1) the kinetoplast, which correspondsto a condensation of extranuclear DNA that lies within the unique ramified mitochondrion, and is localizedin front of the basal body that gives rise to the flagellum; (2) the glycosome; (3) the unusual tubular com-partment, termed the multivesicular tubule (MVT) in promastigotes; and (4) the acidocalcisome (Mullin etal., 2001; Weise et al., 2000; Ghedin et al., 2001). As extensively reviewed (Clayton et al., 1995; Overathet al., 1997), exchanges between the environment and parasite occur mainly via the specialized and deepplasma membrane invagination at the base of the flagellum, the flagellar pocket, where endo- and exocy-tosis is considered to occur exclusively (Balber, 1990; Webster and Russell, 1993).
The flagellum emerges from the flagellar pocket in the promastigote and contains the typical 9�2 pat-tern of microtubule doublets, as well as a highly elaborated network of filamentous structures connected tothe axoneme through small bridges forming a paracrystalline structure termed the paraflagellar (or parax-ial) rod (Gull, 1999). The intraflagellar cytoskeleton comprises, then, this canonical type 9�2 axoneme andthe unique paraflagellar rod (Freymuller and Camargo, 1981). The components of the axoneme are highlyconserved in eukaryotes, but the paraflagellar structure is different from any cytoskeletal structure of themammalian host cell (Gull, 1999). The paraxial structure contains two major proteins in all trypanosomatids:a slowly migrating protein, PFR1, ranging from 70 to 78 kDa, and a faster one, PFR2, ranging from 68 to73 kDa (Bastin et al., 1996). Ultrastructural studies suggest that the organization of the Leishmania pro-mastigote flagellar pocket is similar to that of Trypanosoma (Vickerman and Preston, 1976), withhemidesmosome-like flagellum-cell body attachment sites (Pimenta and De Souza, 1987). Because thehemidesmosome zone between plasma and flagellar membranes closes the pocket, the flagellar pocket is asemisecluded extracellular surface compartment. It is accessible to a range of proteins, including very largemacromolecular complexes, but not to cellular components of the mammalian host immune system (Overathet al., 1997). The assembly and maintenance of the internal microtubule axoneme requires the continuousimport of axoneme precursor proteins from the cytosol, as well as the removal of proteins generated byturnover of axonemal structures. The import and export of these proteins appear to be largely mediated byintraflagellar transport particles that move along the axonemal doublet microtubules just beneath the fla-gellar membrane (Kozminski et al., 1995; Rosenbaum and Witman, 2002; Cole, 2003) and are associatedwith either kinesin or dynein motor proteins, recycling kinesin and discarding axoneme proteins back to thecytosol (Tull et al., 2004).
The eukaryotic flagellum represents one of the most complex macromolecular structures found in anyorganism and contains more than 250 proteins (Dutcher, 1995). This surface organelle plays a key role inLeishmania motility and sensory reception, and it is essential for parasite migration, invasion and persis-tence on host tissues (Landfear and Ignatushchenko, 2001). Parasite survival within these environments re-quires the regulated surface transport of highly abundant coat glycoproteins and glycolipids (McConvilleet al., 2002), as well as a large number of other, less abundant plasma membrane transporters, surface en-
zymes, and receptors that are delivered to the cell surface via the flagellar pocket. The flagellar membraneis almost devoid of intramembranous particles that are poor in protein, but rich in sterols. The flagellarreservoir is not subtended by the subpellicular microtubule array and is thought to be the major site of ex-ocytosis of secretory cargo (glycoproteins, proteoglycans, and glycolipids) that forms the parasite protec-tive surface coats. The parasite surface is the primary interface of Leishmania with the host. It is coveredwith glycosylphosphatidylinositol (GPI)–anchored glycoproteins like lipophosphoglycan (LPG), gly-coinositolphospholipids, proteins such as gp63, gp46/PSA-2, proteophosphoglycans and with free GPI gly-colipids (McConville et al., 2002; Naderer et al., 2004).
One of the goals of the present work was to scrutinize genes and proteins of Leishmania spp. promastigotesthat are related to or responsible for the flagellar role in pathogenesis. Many studies have sought to iden-tify and characterize molecules that confer parasite resistance against the host immune response and a sig-nificant number of individual candidate genes/proteins have now been identified. The recent developmentof robust molecular genetic and proteomic approaches (Acestor et al., 2002; El Fakhry et al., 2002; Drum-melsmith et al., 2003, 2004), coupled with the ongoing analysis of the genome sequences of L. major, Linfantum, and L. chagasi, as well as the related genomes of Trypanosoma cruzi, T. brucei, and T. gambi-ensi, prompted us to study the milieu of genes and proteins within the flagellar compartments. Althoughthe flagellar apparatus comprises critical organelles for the biology of trypanosomatids, conducting all vesic-ular trafficking between the cytoplasm and cell surface (Overath et al., 1997), very little is currently knownabout how molecules get to and from the flagellar pocket. A second goal of this work was, then, to applycomputational biology tools in order to search for virulence factors in genomes, transcriptomes and pro-teomes of several Leishmania species. Here we present data that underscore some unforeseen or overlookedparasite factors such as the MNUDC-1 protein, involved in nuclear development and genomic fusion, andSQS (squalene synthase) gene, whose product is an enzyme that catalyses the first committed step of sterolbiosynthesis. Findings are displayed as flagellar-related gene families in Leishmania and their correlationto infective promastigotes. We have focused on the processes through which Leishmania selectively ex-presses molecules at the flagellar structures, an issue that has not been systematically analyzed and resolved.
MATERIALS AND METHODS
Leishmania species and gene sets
Characteristics of the intravectorial cycle (supra- and peri-pyloric), previously used to define the sub-genus group Leishmania, Viannia (Lainson and Shaw, 1987), and the classification suggested by Rioux etal. (1990) were utilized here to base a depiction chart that discriminates Leishmania species as pathogenic(infecting human/canine host) or non-pathogenic (infecting other hosts). The differences among the Leish-mania species in levels of virulence and in responses to the various chemotherapeutic regimens are re-markable. A list with 38 species of Leishmania, their original taxonomy using biochemical, particularly en-zymatic, characters, our distinction of pathogenic from non-pathogenic species, and their respective entriesin public databases can be seen in Table 1. For simple reasons of clinical and epidemiological importance,coupled with biological data availability, the following Leishmania species were used in this study: cha-gasi, major, infantum, mexicana, donovani, amazonensis, tropica, guyanensis, braziliensis and tarentolae(the latter as the non-pathogenic reference). We have categorized homologous genes into orthologs and par-alogs and also considered gene synteny, that is, the location of two or more genes on the same chromo-some. When the order of multiple orthologous genes is the same in two species, the genomic intervals arereferred to as conserved segments (also called “conserved linkages”). As gene orthology and map infor-mation can be obtained for a larger number of species (and also applied for Leishmania spp comparisons),it has made it easier to identify conserved segments between all related species of interest. Comparativeanalyses of genomic DNA from closely related species, such as these from the genus Leishmania, can iden-tify sequences that have changed and genomic rearrangements that have occurred in recent evolutionaryhistory (Frazer et al., 2003). We have, then, selected and compared orthologous sequences between Leish-mania species in search for gene differences between the organisms, believing that these differences might
List containing 38 different species of the genus Leishmania (Ross, 1903) and their subgenus (according to phenetic,phylogenetic, and cladistic classifications), depicting their respective geographic distribution, year of first description, in-fectivity to human/mammalian hosts, clinical manifestation, and number of registered entries in Entrez databases (NCBI)per organism.
Leishmania (L) Ross, 1903 or Viannia (V) Lainson and Shaw, 1987 or Lizard-infecting (Liz); Geographic distribution:Old World (OW) or New World/Americas (NW); Pathogenicity: P � pathogenic; N � non-pathogenic; Cutaneous � C;Visceral � V.
Entrez/NCBI records—number of entries
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have potential functional consequences and/or be responsible for traits that are unique to the referencespecies pathogenic mode of action.
Biological databases and bioinformatics tools
Sources. The underlying sequencing projects of whole genome shotgun strategy on L. major (�ftp://ftp.sanger.ac.uk/pub/databases/L.major_sequences�) and its proteomic analysis (�www.cri.crchul.ulaval.ca/pro-teome�), whole genome shotgun strategy on L. infantum (�www.sanger.ac.uk/Projects/L_infantum�) and onL. braziliensis (�ftp://ftp.sanger.ac.uk/pub/pathogens/L_braziliensis�) and our own L. chagasi promastigoteEST strategy (�http://nugen.lcc.uece.br/leishmaniaLogin.php�), plus all publicly available related datasets ofindividual or clusters of gene/protein data on Leishmania spp. (Entrez/RefSeq at NCBI at �www.ncbi.nlm.nih.gov/RefSeq�; EBI at �http://srs.ebi.ac.uk�, GeneDB (Hertz-Fowler et al., 2004) at �www.genedb.org�;PIR at �http://pir.georgetown.edu�; PDB at �http://mirrors.rcsb.org/SMS/index_m_mirror.html�; StructuralGenomics of Pathogenic Protozoa at �http://sgpp.org/�) were used as main sources for nucleotide and aminoacid sequences as input. Besides Leishmania spp, we have used publicly available data from other try-panosomatids (T. cruzi at �http://tcruzidb.org�, T. brucei and T. gambiensi), other eukaryotes (Chlamy-domonas reinhardtii, Plasmodium falciparum, Caenorhabditis elegans, Aspergillus fumigatus and Schisto-soma mansoni), and prokaryotic genomes (Shigella flexneri, Coxiella burnetti and Vibrio cholerae) forspecific genes of interest.
Tools. Publicly available open-source software was employed to identify candidate genes through databasesearches and gene prediction methods, provide instant access to relevant genomic information, and comparemultiple sequences to assess correlations and relationships. BLAST programs (Altschul et al., 1997) were widelyused for sequence similarity searches. All individual sequences or aligned clusters were built into a local data-base suitable for subsequent searches. The programs RepeatMasker (�http://ftp.genome.washington.edu/RM/RepeatMasker.html�), Genefinder (Wilson and Green, unpublished data), GLIMMER 2.0 (Salzberg et al., 1999),GlimmerM (Majoros et al., 2003) at �www.tigr.org/tdb/glimmerm/glmr_form.html�) and GENESCAN (Tiwariet al., 1997) were used for nucleotide comparison and gene prediction. For global analysis of protein sequences(either deduced or experimentally generated), we used MUSCLE (Edgar, 2004) for sequence alignment and theSTING Millenium suite (Higa et al., 2004), v. 2.3, at �http://mirrors.rcsb.org/SMS�. For comparative 3D mod-eling of proteins we employed Modeller 7 (Sali and Blundell, 1993).
Collection of database search results. Database search results for ESTs, GSS or WSG and individual re-mote sequence matches were included in our local dataset. For amino acid sequences, matches with a givenlevel of similarity were included, but matching subsequences with low complexity were filtered out usinglocal complexity statistics (Wootton and Federhen, 1996). Following manual evaluation and model cura-tion, genes and proteins were assigned as of interest based on the organism from which they originated(e.g., leishmanial, trypanosomatid, eukaryotic, prokaryotic), on their pathogenic role (e.g., adhesion, at-tachment, invasion) and on organellar or subcellular localization (e.g., flagellar pocket or membrane, ax-oneme, microtubule).
In silico survey
A flow chart detailing the procedure used is shown in Figure 1. Our scrutiny took alignments createdwith FASTA or BLAST as input and computed alignment tables providing hierarchical and successive cor-relations between each two sets of sequences to produce as output: (1) mapping of the target sequencesonto the L. major genome sequence, used here as a reference; (2) multiple sequence alignment of the querysequences aligned both to each other and to the reference genome; (3) a set of detected conserved regionsstored as pairs of possible pathogenic indices; and (4) a set of molecular relationships stored for manualevaluation. Since Leishmania genes are expected not to contain introns (Myler et al., 1999; Drummelsmithet al., 2003), and the signal sequences for trans-splicing and polyadenylation are poorly defined, consensusmethods have little utility for Leishmania gene prediction. In addition, �70% of the genes have no signif-icant similarity to existing genes in sequence databases, so extrinsic content sensing methods are of limited
use; leaving only intrinsic content sensing methods for possible use in gene prediction (Aggarwal et al., 2003).Given that the number of experimentally confirmed gene predictions in Leishmania is currently small, and manymethods use similar statistical approaches (Fickett, 1996), we had to extract a large number of consensus se-quences for each Leishmania species by examining for putative protein-coding open reading frames (ORFs) us-ing a combination of gene prediction tools with semi-automated procedures. The deduced amino acid sequences(from predicted genes) were, then, used as query sequences for local BLASTP (Altschul et al., 1997) searchesof the NCBI non-redundant protein database and the GeneDB database. Generally, hits with BLAST scores of�50 and e-values of �1 � 10�6 were considered potentially significant, although some exceptions were madeupon visual inspection of the alignments. Each protein sequence was then searched against numerous collec-tions of protein motifs and families, such as UniProt/Swiss-Prot version 45.2 (�www.ebi.ac.uk/swissprot/�),ProDom version 2004.1 �http://prodes.toulouse.inra.fr/prodom/current/html/home.php�), SMART version 4.0(�http://smart.embl-heidelberg.de�), Pfam version 16.0 and respective iPfam version (�www.sanger.ac.uk/Soft-ware/Pfam/�) and PROSITE release 18.40 (�www.expasy.org/prosite�). These searches were carried out usingvarious search algorithms, usually as web-based tools (�http://hits.isb-sib.ch/cgi-bin/PFSCAN�; �http://dna.stan-ford.edu/emotif/emotif-search.html�). BLAST searching of the database of Clusters of Orthologous Groups ofProteins (COGs) (�www.ncbi.nlm.nih.gov/COG�) was performed simultaneously, along with identification ofputative protein localization sites and transmembrane spanning regions using PSORT (Nakai and Horton, 1999)and TMHMM version 2.0 (�www.cbs.dtu.dk/services/TMHMM-2.0/�) (Krogh et al., 2001) Gene ontology (GO)terms (Ashburner et al., 2000) were assigned to the predicted proteins, based on their top matches to proteinswith GO annotations from SWISSPROT and TREMBL (�www.expasy.org/sprot�) and with AMIGO aftergeneDB access (�www.genedb.org/amigo/perl/go.cgi�). Coding portions of the base genome (L. major), as com-pared to sequences from other Leishmania species, found to be related to the flagellar structure, were first se-lected and, then, assigned to a distinct subset of data, where they were organized hierarchically into familiesand superfamilies of genes. A gene family was defined as a group of genes whose members have �50% pair-wise amino acid similarity and a superfamily as an alignable group of genes with similarity below this thresh-old (Thornton and DeSalle, 2000). Members of a given gene family in publicly available databases can be ob-tained by using local alignments as the Position-Specific Iterated (PSI)–BLAST (�www.ncbi.nlm.nih.gov/blast/�),for finding very distantly related members of a family that may be missed by single-pass similarity searchesproteins. For a putative prediction of organellar assignment to the flagellar location, we have used the TargetPv1.01 (�www.cbs.dtu.dk/services/TargetP�) (Emanuelsson et al., 2000) which predicts the subcellular locationof eukaryotic protein sequences based on the predicted presence of N-terminal presequences.
RESULTS AND DISCUSSION
Leishmania pathogenomics database
With data from biochemical, genomic and proteomic studies in several Leishmania species organized indatasets and with the multi-program platform described above, we started building up an organellarpathogenomics database for Leishmania spp., starting with the flagellum and flagellar pocket. Leishmaniaspp. potential virulence factors that are also components of the flagellar structure, or directly related to it,
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FIG. 1. Flow-chart detailing the steps employed in our searches. Data for an integrated Leishmania spp. localpathogenomics database, specifically related to flagellum and flagellar apparatus, were gathered from publicly avail-able data warehouses (NCBI, EBI, GeneDB), the PROGENE database (Brazilian Northeast Genome Program), and avariety of outside sources before being integrated in a relational PostGreSQL database and served to users on the In-ternet. Our analysis took as input any single DNA or protein sequence (or clusters containing EST sequences) from aparticular Leishmania species along with the equivalent sequences on L. major (as the reference genome). Our analy-sis produces as output a mapping of the selected sequences or clusters onto the reference genomic sequence, a multi-ple sequence alignment of the set of selected sequences aligned both to each other and to the reference genomic se-quence, a set of detected putative flagellar-related factors organized in gene families and a set of manually assigned,plausible relationships among the derived factors.
are depicted in Table 2. Relevant results of performed analyses were used to create a relational database(PosGreSQL) with all the information produced during the survey. A database concerning results of thiswork, as well as other databases comprising studies of Leishmania and its organelles, is available at �http://nugen.lcc.uece.br/LPGate� (Fig. 2). This web site will serve as a convenient bioinformatics resource on ge-nomics and pathology of the etiological agents of leishmaniasis.
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TABLE 2. CANDIDATE GENES AND PROTEINS FOR FLAGELLAR PATHOLOGICAL INVOLVEMENT IN LEISHMANIASIS
Leishmania flagellar-related Reference/virulence factors in Role on parasite accessionpromastigotes Site Species metabolism number
Paraflagellar rod protein Ax, Fr me, ch, ma flagellar integrity and cell Maga et al., 1999;(PRF-1, PRF-2) motility Mishra et al., 2003
Axonemal dynein heavy Ax, Fr ma, cha cell motility, trafficking Gls: 34875852,chain genes along microtubules 6706264,
33944957Membrane proteins
Putative transferrin receptor Fp do Internalization and Sengupta et al.,(46-kDa protein) degradation of 1999
hemoglobinReceptor adenylate cyclase Fr do Flagellum integrity Sanchez et al.,
1995Rab family of membrane- — — Endocytosis regulation Singh et al.,
associated proteins by vesicle fusion 2003Rab1 and Rab11 ? ma, cha ? NPR; Gls 1877404
and 11611729
Rab5 Fp, Fr do Hemoglobin endocytosis Singh et al.,2003AY337266
Rab7 ? amaz Late endocytic fusion Courret et al.,2002
Mitogen-activated proteinkinase kinase homologues:LmxMKK, FP, Fm mex, flagellar assembly and Li et al., 1996;
cell size Wiese et al., 2003
LchMKK FP, Fm cha Flagellar-length AJ243118Family of small myristoylated
proteins (SMPs)SMP-1 to 7 Fm ma ? Tull et al., 2004
The database is converted to various output formats, including the Flat File and Abstract Syntax Nota-tion 1 (ASN.1) versions. The organization of data files will include a release consisting of a set of ASCIItext files, containing sequence data for each species and organelle. Supplemental “index” files will even-tually be also supplied, containing comprehensive lists of all sources from which we retrieved author names,journal citations, gene names, and keywords, along with the accession numbers of the original records inwhich they can be found at GenBank or EBI. Files included in this database are as follows:
1. l_.idx - Index of the entries according to accession number.2. l_est.seq - EST (expressed sequence tag) sequence entries.3. l_gss.seq - GSS (genome survey sequence) sequence entries.4. l_htc.seq - HTC (high throughput cDNA sequencing) sequence entries.5. l_sts.seq - STS (sequence tagged site) sequence entries.6. l_aas.seq - AAS (aminoacid sequences) sequence entries.7. l_pep.seq - PEP (peptide fragments sequence) sequence entries.8. l_pro.seq - PRO (protein structures) sequence entries.9. l_may.seq - DOM (conserved domains) sequence entries.
10. l_jou.idx - Index of the entries according to journal citation.11. l_key.idx - Index of the entries according to keyword phrase.
There is a selected per-organism statistics with tables providing the number of entries for the 38 Leishma-nia species in the first release of the LPGateway (�http://nugen.lcc.uece.br/LPGate�), as well as the total in-formation for each species that will be presented for, each DNA/amino acid data sequence, as its ORFs/pro-
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TABLE 2. CANDIDATE GENES AND PROTEINS FOR FLAGELLAR PATHOLOGICAL
INVOLVEMENT IN LEISHMANIASIS (CONTINUED)
Leishmania flagellar-related Reference/virulence factors in Role on parasite accessionpromastigotes Site Species metabolism number
Enzymes on sterolsynthesis pathwaySqualene synthase (SQS)— FP, Fm, ma flagellar integrity, sterol Cotrim et al., 1999;
membrane-bound pm biosynthesis GI:3169689CYP51 (sterol 14-alpha- ? ma Sterol byosynthesis Lepesheva et al.,
demethylase) 2004S-adenosyl-l-methionine: memb do sterol methylation, Pourshafie et al.,
Meta gene familyMeta 1 FP do, ma, metacyclogenesis and Nourbakhsh et al.,
amaz, motility 1996Meta 2 ? ma, amaz metacyclogenesis and Ramos et al.,
motility 2004
Among a great number of known parasite virulence factors, there are many recognized as related to Leishmania spp.pathogenesis and already described, but not so many directly associated to the flagellar apparatus role in infection. Thefollowing list contains the selected Leishmania spp. flagellar genes/proteins most plausibly related to the promastigote in-fectiveness ability, as result of current searches in publicly available biological databases searches (December 2004).
L. major genome sequence database (located at GenDB) and all other available data from any organisms were searchedfor sequences that showed significant homology to genes/proteins involved in flagellum activity and that would be presentin any Leishmania spp. Abbreviations used: FP � flagellar pocket; Fm � flagellar membrane; Fr � flagellar reservoir; pm� plasma membrane; other membranes � memb; Mv � multivesicular tubule (MVT)-lysosome; ma � L. major; mex �L. mexicana; cha � L. chagasi; do � L. donovani; inf � L. infantum; trop � L. tropica; tar � L. tarentolae; amaz � L. ama-zonensis; NPR � not previously reported in Leishmania NPV � not previously reported as leishmanial virulence factor.
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tein, its database search results, predicted exons and identified repetitive elements, pathogenic indices orconserved domains into an HTML file of multiply aligned sequences. Hypertext links will point to data-base search results and their relevant records in the respective remote databases. A summary report withhypertext links to all data sequences of those sequencing/proteomic projects mentioned in Materials andMethods (Sources) and to entries in their respective database search results will also be generated. The hy-pertext files can, then, be studied as web pages using any computer program capable of browsing hyper-text files, to facilitate its exploration for multiple users.
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FIG. 2. Leishmania Pathogenomics Gateway (LPGateway) is a portal to the internal connections of ongoing genomesequencings of Leishmania spp. and information about BLAST searches, EST alignments, DNA and protein analyses,as well as gene models. This web resource aims at providing easily accessible data and relevant information to pathol-ogists interested in Leishmania spp. and leishmaniasis. Links are provided for other web sites (publicly available bio-logical data warehouses and databases and a variety of pertinent outside sources), and, through some of them, Inter-Pro, PROSITE and PFAM domains, and Smith-Waterman alignments to protein databases are displayed with a graphicalinterface. LPGateway is accessible at �http://nugen.lcc.uece.br/LPGate� and contains our local, relational PostGreSQLdatabase of L. chagasi ESTs panel, besides a currently under-construction whole organellar genomics database for Leish-mania spp. to be established to provide integration of databases and robust data retrieval on all species of Leishmaniaand their organelles in terms of pathogenesis.
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Flagellar analysis of protein/gene datasets
We analyzed complete published and available eukaryotic genomes, related (T. cruzi, T. brucei) or notrelated (C. reinhardtii, P. falciparum) to Leishmania, and prokaryotic genomes (S. flexneri and V. cholerae)for genes related to or associated with flagellar components (a complete list of flagellar related genes andproteins selected in our survey is presented in Table 2). The resulting database, as can be seen in Figure 1,was queried for those proteins most similar to flagellar proteins in any organism (and those Leishmaniaproteins most similar to bacterial or trypanosome flagellar proteins). This approach can capitalize on thesignificant evolutionary distance between different domains of life (bacteria and eukarya) with respect toconserved genes and proteins comprising organellar structures. Proteins that are annotated by SWISS-PROTas being encoded in an organelle functional or structurally related to the flagellar membranes, or contain-ing an organelle transit peptide according to TargetP (Emanuelsson et al., 2000) are specifically highlightedin the database; so if eukaryotic organellar genes, that have moved to the nucleus, tend to be most similarto bacterial genes (Reumann and Keegstra, 1999; Rujan and Martin, 2001), it is important to trace this pat-tern, since here we are interested on a organelle (the flagellum) whose component structures are highly con-served among trypanosomatids, but also conserved in distantly related organisms, such as C. reinhardtiiand human (as in tracheal epithelium and sperm cells). Note that there are obvious limitations to this analy-sis because it only detects primary sequence similarities detected by BLAST; it is not automated for iden-tification of other proteins of interest, even if highly conserved between all domains of life, requiring awhole start of procedures to initiate a new search; its effectiveness is limited by the number of known fla-gellar-related genes/proteins in public databases (although this tends to improve over time); and it is lim-ited by the accuracy of organellar transit peptide prediction algorithms.
Leishmania gene discovery
Eukaryotic gene-discovery can be most effectively accomplished through analysis of either whole genomesequencing or direct gene transcripts sequencing using cDNA libraries. The latter approach was the choiceat the Brazilian Northeast Genome Program (�www.progene.ufpe.br�) to generate ESTs from promastigotesof L. chagasi (Andrade et al., unpublished results). Comparison of ESTs to each other and to whole genomesequences can be extremely useful for gene discovery. Here we present results obtained with the in silicosearch for flagellar-related molecules. The flagellar pocket and flagellar membranes have long been recog-nized as morphologically separate domains that are component parts of the plasma membrane that surroundsthe entire trypanosomatid cell. The structural and functional specialization of these two membranes has beenunderscored by the identification of multiple proteins that are targeted selectively to each of these domains,and non-membrane proteins have also been identified that are targeted to the internal lumina of these or-ganelles. As depicted on Table 2, we have tried to discriminate, on the column SITES, this putative tar-geting. Investigations on the functions of these organelle-specific proteins should continue to shed light onthe unique biological activities of the flagellum and flagellar pocket. The functional assignment of thesegenes was inferred using the RPS-BLAST search against conserved domain databases and these genes werefurther classified according to their function and subcellular localization.
Identification of gene families functionally related to Leishmania flagellum
Transport and assembly of flagellar subunits continue throughout the life of the organelle due to the con-stant turnover of axonemal components at the flagellar tip. Continuous turnover produces disassembled pro-teins that must be removed from the flagellum that, in spite of its apparent stability, is a dynamic structurein which the microtubules and appended proteins are constantly turning over (Qin et al., 2004). In searchfor genomic and/or proteomic evidences of flagellar genes/proteins in Leishmania spp, we have used manyavailable databases, including EST databases (that have proven to be useful tools for detecting homologousgenes, and for gene identification, sometimes yielding the only unambiguous evidence for the existence ofa gene expression product). While many uses for ESTs have indeed been found, there is one feature of eu-karyotic genomes for which the establishment of these databases has proved particularly helpful: the nu-merous large families of homologous genes, many of which are functionally redundant (Gemund et al.,
2001). Hence, due to the availability of over 30,000 ESTs from L. chagasi (data not shown) and theirmatches being intrinsic to the gene prediction protocol here employed, we were able to distinguish the fol-lowing flagellar-related gene families in Leishmania spp.
A. The paraflagellar rod (PFR) protein family: Orthologs of L. chagasi, L. mexicana and L. major PFR-1D (accession no. AAO25623) and PFR-2C (accession no. AAB17719). PFR is essential for flagellar motil-ity in Leishmania promastigotes. However, it is absent from the attenuated flagellum of amastigotes, as re-viewed by Mishra et al. (2003) in a recent work that identified a PFR regulatory element (PRE). The cisPRE controls mRNA degradation by destabilizing the PFR2 mRNA in amastigotes. It is known that, forLeishmania, flagellar biogenesis must also accommodate synthesis of the PFR (Bastin et al., 1996). Magaet al. (1999) reported a comparison on PFR substructures of L. mexicana, and their results concluded thatPFR1, but not PFR2, interacts with axoneme attachment filaments. The authors, then, suggested the possi-bility that these two proteins might be located within discrete PFR substructures. Rosenbaum et al. (1999)described that, in the process of intraflagellar transport, rafts of protein components travel between the ax-oneme and the flagellar membrane and are dependent on the activity of motor proteins. Considering the rel-ative abundance of PFR genes and of axonemal components, as dynein gene families in all Leishmaniaspecies studied, together with plentiful evidences of their part on flagellum assembly, we realize that theseare crucial elements for this unique organelle and that stage-specific regulatory mechanisms employed byLeishmania might be directly related to them.
B. The axonemal dynein heavy chain family: Paralogs of L. chagasi and L. major Rattus norgevicus ax-onemal dynein heavy chain 7 (accession no. CAB65927) and to T. brucei dynein heavy chain putative gene(accession no. XP_340626). Recent studies have revealed the molecular structures and functions of a num-ber of axonemal components, including dyneins, which drive the force for flagellar movement, exerted bythe sliding of outer-doublet microtubules. This family represents the C-terminal region of dynein heavychain. The chain also contains ATPase activity and microtubule binding ability and acts as a motor for themovement of organelles and vesicles along microtubules. The dynein subunit consists of at least two heavychains and a number of intermediate and light chains. Dynein is directly involved in flagella movement. Itsabundant expression in Leishmania, together with PFR, might account for the intensive process of in-traflagellar transport, as required for promastigote metabolism (Rosenbaum et al., 1999), and for pro-mastigote differentiation to amastigote. This differentiation is a key step on leishmaniasis pathogenesis. In-traflagellar transport serves as a two-way transport system, to bring precursors to the tip for assembly andto return the products of axonemal turnover to the cell body (Qin et al., 2004). Such process probably as-sures that flagella movement, between the axoneme and the flagellar membrane, may also be responsiblefor the movement of some integral membrane proteins in the flagellar membrane (Snapp and Landfear,1999).
C. The Rab family of membrane-associated proteins: Orthologs of L. chagasi and L. major to T. bruceiRab7 (rab7 and rab1)/GTP binding protein gene (accession no. CAA68210) and small GTPase Rab11 (ac-cession no. AAG39034). Rabs are a family of small GTPases (Field et al., 1998) with multiple Rab iso-forms. For example, Rab5 was found uniquely on early endosomes (Bucci et al., 1992) and this localiza-tion suggested that Rabs act as specificity determinants in vesicular trafficking processes (Ferro-Novick andNovick, 1993). More recently, Singh et al. (2003) have localized Rab5 in the flagellar pocket of L. dono-
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FIG. 3. Multiple alignment of the amino acid sequences of squalene synthases (SQS) from different organisms. Thesequences for L. major SQS1 (gi 1389688), human (PDB 1EZFA) S. cerevisiae (gi 729468), and Oriza sativa (gi2463565) squalene synthases are shown, as compared to a query with a deduced amino acid sequence from an ESTcluster of L. chagasi (here named as LeSQS1 orthologue to L. major SQS). Amino acids identical and functional havebeen aligned, and the locations of conserved domains are numbered. Regions I/II and III/IV bear an aspartate-rich mo-tif (a probable binding site for diphosphate moiety of the prenyl substrates) (Cotrim et al., 1999), whereas region VI ishighly conserved and preceeds hydrophobic residues that may function as a membrane-binding domain. #Feature 1refers to the substrate binding pocket of the PDB structure 1EZFA.
vani (Table 2). In T. brucei, Rab11 (TbRAB11) is homologous to higher eukaryotic Rab proteins that me-diate deep recycling pathways; it is positioned close to the basal body, a microtubule organizing centre intrypanosomatids (Gull, 1999). Coordinate regulation of endomembrane structures and the basal body/kine-toplast is a common feature in trypanosomes, and is clearly seen for early endosomes (Field et al., 1998).It is potentially a mechanism for faithful organelle segregation. If TbRAB11 actually defines a new com-partment of the trypanosome endosomal system and its position between the kinetoplast and nucleus is con-sistent with an endosomal function, we might believe that the existence of Rab11 orthologs in L. chagasiand L. major can also indicate an essentially similar endosomal function. The presence of Rab7p on a highpercentage of early L. amazonenesis promastigote-containing phagosomes (70–80%) is consistent with theirability to fuse with late endocytic compartments, as a recent report indicate that this protein is importantfor late endosome/lysosome fusion events (Courret et al., 2002).
D. Gene family of small myristoylated proteins (SMPs). Biochemically identified and characterized in L.major by Tull et al. (2004), the phylogenetic analysis of the trypanosomatid SMP family revealed eightclusters. Of them, only SMP-1, SMP-2 and SMP-3 proteins are unique to L major and lack any clear or-thologs in T. brucei or T. cruzi. LmSMP-1 was shown by Tull et al. (2004) to be expressed in all pro-mastigotes stages containing an elongated flagellum, but not in the amastigote stage that contains a highlytruncated flagellum. SMP-1 was primarily localized on the inner leaflet of the flagellar membrane (Tull etal., unpublished results). Myristoylation and palmitoylation of SMP-1 was shown, by the same authors, tobe required for flagellar membrane targeting. They also have suggested that partial inhibition of sterol andsphingolipid biosynthesis interferes with both the biogenesis of new flagellum in log phase parasites, aswell as with the retraction of existing flagellum in stationary phase promastigotes. However, the flagellarlocalization of SMP-1 was not affected when sterol biosynthesis was perturbed with ketoconazole, despitecausing the massive distension of the flagellum membrane and the partial loss of internal axoneme struc-tures. These data suggested that flagellar membrane targeting of SMP-1 is not dependent on axonemal struc-tures and that alterations in flagellar membrane lipid composition disrupt axoneme extension. Such find-ings become more interesting when we consider the L. major (LMU30455) and L. chagasi ortholog tosqualene synthase gene (LeSQS1) whose sequence alignments are shown in Fig. 3. This eukaryotic farne-syl-diphosphate farnesyltransferase [PROSITE; PDOC00802] catalyzes the conversion of two molecules offarnesyl diphosphate into squalene and it is the first committed step in the cholesterol/ergosterol biosyn-thetic pathway (Schechter et al., 1994). As in fungi, Leishmania synthesizes ergosterol rather than choles-terol as its bulk membrane sterol (Goad et al., 1984). Here we have identified the L. chagasi ortholog to L.major SQS1 gene (Fig. 3). Cholesterol is known as a major constituent of eukaryotic membranes and playsa crucial role in cellular membrane organization, dynamics, function, and sorting (Pucadyil et al., 2004). Itis often found distributed non-randomly in domains in membranes and recent observations suggest that cho-lesterol exerts many of its actions by maintaining a specialized type of membrane domain, termed “lipidrafts”, in a functional state. Lipid rafts are enriched in cholesterol and sphingolipids, and have been thoughtto act as platforms through which signal transduction events are coordinated and pathogens gain entry toinfect host cells (Pucadyil et al., 2004). Leishmania are particularly rich in lipid-anchored surface mole-cules and lipid rafts are important in the differentiation of promastigotes, perhaps orchestrating the clearlyobservable reorganization of the plasma membrane during this process that leads to an activated metacyclicprimed for invasion (Denny and Smith, 2004).
FIG. 4. Comparison of a few NUDC proteins and the mNUDC-like protein of L. major. (A) Multiple sequence align-ment of six species—the L. major mNUDC-like protein with Aspergillus nidulans, T. brucei, T. cruzi human, and M.musculus. Residues that are conserved across all six species are indicated by the consensus line, whereas dashes rep-resent spaces inserted for maximum alignment. The overlined region corresponds to the PFAM domain (accession n.PF03593). (B) Graphical SMART representation of the similar group alignment. Publicly available proteins were alignedusing ClustalW. The PFAM domain nuclear movement protein (accession no. PF03593), highly conserved in NUDCsequences, is depicted. Intron positions (in human and murine representations) correspond to vertical lines, showing in-tron phase and exact position in amino acids (taken from Ensembl gene predictions). Domains, intrinsic features, andintrons were mapped onto the alignment with their positions adjusted according to gaps.
SQS is a membrane-bound enzyme in Leishmania promastigotes with a dual subcellular localization, be-ing almost evenly distributed between glycosomes and mitochondrial/microsomal vesicles (Urbina et al.,2002). The inhibition of SQS gene, demonstrated in T. cruzi by Braga et al. (2004), was able to evoke struc-tural changes that included the detachment of the plasma membrane from the cell body, forming blebs; thedetachment of the membrane lining the cell body and the flagellum from the subpellicular and axonemalmicrotubules; enlargement of the flagellar pocket and of a vacuole localized close to the flagellar pocket.These alterations were interpreted by the authors (Braga et al., 2004) as a consequence of the depletion ofessential parasite sterols induced by the experimental compounds and the concomitant alteration of the phys-ical properties of the parasite membranes. Since these changes were particularly associated to the flagellarapparatus, we were tempted to verify some correlations of a possible flagellar role for SQS functional ac-tivity in sterol biosynthesis in Leishmania too. Lepesheva et al. (2004) have reported new isoforms of CYP51(sterol 14�-demethylase), an essential enzyme in sterol biosynthesis in trypanosomes and L. major. The setof sterol biosynthetic enzymes in the protozoan genomes, together with available information about sterolcomposition of kinetoplastid cells, suggest a possible novel sterol biosynthetic pathway in Trypanosomati-dae (Lepesheva et al., 2004). The importance of an active sterol biosynthetic pathway in trypanosomatidsfor growth and viability has been demonstrated by imadazole- and triazole-based drugs (e.g., ketoconazoleand itraconazole), which inhibit the 14�-methylsterol 14-demethylase, and the allylamines (e.g., terbinafine),which inhibit squalene epoxidase (Ginger et al., 2001). Having in mind that disruption of sphingolipid and/orsterol biosynthesis in Leishmania has a profound affect on axoneme biogenesis or maintenance (Tull et al.,2004), we attributed a putative flagellar role for SQS genes found in L. major and L. chagasi (Table 2; Fig3). On Table 2, we present two other molecules, besides SQS, that are enzymes of the sterol biosyntheticpathway (CYP5 and the S-adenosyl-L-methionine:C-24-delta-sterol-methyltransferase) in Leishmania.
E. Peptidase M20/M25/M40 family: L. chagasi ortholog to C. burnetti [RSA 493] peptidase M20/M25/M40family (accession no. AAO89669). This family includes a range of zinc metallopeptidases belonging to sev-eral families in the peptidase classification. Family M20 is composed of glutamate carboxypeptidases andfamily M25 contains X-His dipeptidases. Metalloproteases are the most diverse of the four main types ofprotease, with more than 30 families identified to date (Rawlings and Barrett, 1995). This group of proteinscontains the metallopeptidases and non-peptidase homologues that belong to the MEROPS peptidase fam-ily M20 (Rawlings and Barrett, 1995), like for example, glutamate carboxypeptidases from Pseudomonasspp. (UniProt accession no. P06621) and from C. burnetii. The latter organism, like Leishmania, is a typi-cal pathogen of the acidified phagolysosome and a granuloma inducer. It has been argued that C. burnetiitargets the prohibitive (phagolysosomal) niche where it is under considerable oxidative and osmotic stress(Heinzen and Samuel, 2001) maybe because it has a number of genes associated with stress-response orvacuole detoxification systems or maybe by encoding an unusually high number of basic proteins. Althoughthis hypothesis of basic proteins of C. burnetii seems not to be applicable to Leishmania, the abundance ofleishmanial genes/proteins associated with stress-response or vacuole detoxification systems might accountfor the parasite ability to phagolysosomal persistence. Here we report a putative L. chagasi M20/M25/M40peptidase, whose similar sequences include Sphingomonas spp. polyaspartic acid (PAA) hydrolase-2 (Hi-raishi et al., 2003), an enzyme involved in tPAA biodegradation, and a putative peptidase belonging to theM20/M25/M40 family of proteins from Caulobacter crescentus CB15 (Hiraishi et al., 2003). These resultswere surprising, as the L. major genome does not seem to encode a similar peptidase. The L. chagasi pu-tative M20/M25/M40 peptidase gene will certainly require biochemical characterization to clarify its role.
F. mNudC-like protein (accession no. CAB95231): L. major ortholog to MNUDC protein of Mus mus-culus (accession no. CAA57201). We have noticed that one of the proteins identified in the L. major pro-teomic analysis (Drummelsmith et al., 2003) was the mNudC-like protein (accession no. CAB95231) withhigh similarity and 45.0% identity to MNUDC protein of Mus musculus (accession no. CAA57201). Se-quence alignments of mNudC proteins are depicted in Figure 4A, and conserved domains are shown in Fig-ure 4B. The Nud (Nuclear Division related, microtubule motor-associated protein) family of genes is knownas evolutionary conserved nuclear migration factors with roles in embryonic development, cell division andcell migration. NudC was first identified as a gene that regulates nuclear movement in the filamentous fun-
gus Aspergillus nidulans (Aumais et al., 2003), where NudC is required for nuclear distribution, cell walldeposition, colony growth and viability (Osmani et al., 1990; Chiu et al., 1997). Other nud loci encode ei-ther components or proposed regulators of the minus-end-directed microtubule (MT)–dependent motordynein and its activator dynactin (Morris, 2000). The mammalian homologues of NUD proteins includeLis-1 (the Miller-Dieker lissencephaly gene involved in a human neuronal migration disease), NUDF (Xi-ang et al., 1995) and the actin-related protein Arp1 (NUDK) (Xiang et al., 2000), which is a component ofdynactin. The high degree of sequence conservation in NudC from fungus to human suggests a functionthat has been conserved over evolution (Moreau et al., 2001), while now we add the NudC homologues ofL. major and L. chagasi to this list (Fig. 4).
Nud genes from A. nidulans exhibit amazing features that deserve an analysis in our Leishmania surveybecause the NudA gene sequence showed that it encodes a cytoplasmic dynein heavy chain (Xiang et al.,1994) and the sequence of the NudF protein is similar to the Lis-1 gene product with an identity of 42%throughout the whole protein (Xiang et al., 1995). Lis-1 gene regulates microtubule dynamics and interactswith the molecular motor cytoplasmic dynein and its cofactor dynactin, being necessary for pronuclear mi-gration during fertilization (Payne et al., 2003). Besides confirming an astonishing role for Lis-1, raisingevident implications for human reproduction, these relationships of genes and gene products on nuclearmovement (Payne et al., 2003) also raise a question when we consider, in a whole, the consistency ofmNudC-like protein in L. major proteome (Drummelsmith et al., 2003) and the trypanosomatid flagellarstructures displayed in such a beautifully intricate network of microtubules and dyneins.
CONCLUSION
We have focused on flagellate promastigotes because considerable differences exist between them andthe amastigotes, and a number of genes are specifically expressed in one form or the other, e.g. LdARL-3A,SMP-1, LmxMKK or PFR-2 (as can be seen in Table 2 that depicts only promastigote expressed molecules).The contribution of pathogen locomotion or movement to virulence is well documented for bacterial andviral pathogens, but in the case of protozoan pathogens (e.g., Leishmania spp.), the contribution of cellmotility to host–pathogen interactions has been largely unexplored (Hill, 2003). There are significant evi-dences of roles for the flagellum in the control of cell size, shape, polarity and division (cytokinesis) in try-panosomatids (Kohl et al., 2003). In this work, we have tried to distinguish Leishmania spp. flagellar vir-ulence factors and their organization in gene families. In doing that, we hope to have given new insightsto better characterize the pathogenic role of flagellum in promastigotes or in the promastigote-to-amastig-ote differentiation, a key event in leishmaniasis infection.
ACKNOWLEDGMENTS
This work was supported by the following Brazilian research funding agencies: CNPq, FINEP, BNB/FUN-DECI and FUNCAP (through individual grants to D.M.O., M.C.S., C.A.W., J.O.F., A.R.T., and R.B.C.,and collaborative financial support to PROGENE). We thank all our colleagues from the PROGENE con-sortium, including Paulo P. Andrade, Valdir Q. Balbino, and Ederson A. Kido at the Universidade Federalde Pernambuco.
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