Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites.Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp.The known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms.The EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence.  Babesia caballi and Theileria equi, transmitted by ixodid ticks such as Dermacentor nitens  and Rhipicephalus microplus (T. equi)[B. caballi and T. equi were diagnosed between 1961 and 1969 leading to an eradication campaign which lasted twenty-five years and cost twelve million dollars[T. equi in Florida[Amblyomma cajennense and D. variabilis, were identified as novel vectors in the 2009 Texas outbreak[Equine piroplasmosis of horses, mules, donkeys and zebras is caused by the tick-borne apicomplexan protozoan parasites T. equi),2. AlthoT. equi), the U.  dollars. The re- Florida and Texa Florida raised c Florida. The cauoutbreak. The re-Plasmodium as well as T. parva and T. annulata, and Babesia bovis causes of malaria, bovine theileriosis and babesiosis, respectively. The phylogenetic position of T. equi has been controversial, and the organism has been renamed several times[T. equi between B. bovis and Theileria spp.[Theileria may be appropriate for T. equi.Additional members of the phylum Apicomplexa, important to global human and animal health include the organisms in the genus al times. Moleculria spp.,10 and iT. annulata, transmission of T. equi to equids eventually results in lysis of erythrocytes and prolonged anemia. Anemia associated with T. parva occurs later during infection and is comparatively and clinically mild[T. parva and mononuclear phagocytes and B-lymphocytes by T. annulata lead to reversible cell transformation[T. equi has been reported[T. equi, unlike T. parva and T. annulata remains unresolved, and PBMC proliferation and/or transformation have not been associated with clinical equine piroplasmosis.Similar to bovine theileriosis caused by lly mild. Infectiormation. Infectireported,13. HoweT. equi infection is anemia and the associated erythrolysis is independent of parasite-specific immune responses[3 to 106 infected peripheral erythrocytes per ml/blood resulting in efficient acquisition and transmission by ticks[B. bovis[P. falciparum[T. brucei[T. equi. A candidate multigene gene family in T. equi encodes Equi Merozoite Antigens (EMAs), which are immunodominant for antibody, however, this family contains just 10 members, and lacks an apparent structural basis for variation. The reemergence and persistence of T. equi in the U. S. prompted this genomic research due to the lack of a vaccine to block infection or clinical disease and the need for additional chemotherapeutics aimed at eliminating persistent infection and tick-borne transmission risk.The primary clinical outcome of acute esponses. Resolutesponses. Persistby ticks. A hallm[B. bovis, PfEMP1 alciparum and VSG T. brucei. An analT. equi contains six molecules, including four chromosomes  , or B. bovis (137), it has far more unique genes (1985), which account for the increased size of the genome. The four species have 2,482 genes in common. T. equi has two rRNA operons, and 46 tRNA genes.The larger genome size of a) Table is also T. equi, the A+T rich (71%) 47.8 kb apicoplast genome is larger than those of other piroplasms, due primarily to expansion of the repertoires of three hypothetical genes  is actually cytochrome c oxidase subunit III (cox3)[cox3 sequences, T. equi could not conduct respiration without cox3, and these genes are the only candidate for this function. Our findings corroborate the linear structure of the mt genome and the long inverted terminal repeat structure[The mitochondrial (mt) genome is 9001 bp in length, longer than in other piroplasms and shows evidence of gene duplication and rearrangement. The I (cox3). Althougtructure.T. equi is similar to other piroplasms: most elements of the core pathways for energy production are present including glycolysis, pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. Glycolysis is fully intact with putative enzymes identified for each stage of the conversion of sugars to pyruvate for the production of energy, making carbon the primary energy source. However, since pyruvate dehydrogenase (EC 1.2.4.1) is missing from the T. equi genome (similar to B. bovis and other Theileria spp.), glycolysis does not seem to be coupled to the TCA cycle. Although the TCA cycle is intact, the lack of pyruvate dehydrogenase suggests that the primary function may be to generate precursors rather than produce energy. For example, succinyl CoA is a critical biosynthetic precursor for the synthesis of protoheme, used in cytochromes and many enzymes. Electron transport pathways in T. equi resemble those reported for P. falciparum[The predicted metabolic profile of alciparum. Lack ofDe novo purine biosynthesis is absent, however, unlike other piroplasms, hypoxanthine/guanine phosphoribosyl transferase (BEWA_017710) and adenine phosphoribosyltransferase (BEWA_017730) are present, indicating that purine salvage can occur, similar to P. falciparum. Fatty acid synthesis, heme biosynthesis and the shikimic acid pathway do not occur in the piroplasms, although these pathways are present in P. falciparum[Metabolic pathway similarities with other sequenced hemoparasites include the ability to synthesize pyrimidines, limited amino acid biosynthesis, and the lack of a urea cycle. alciparum-29.T. equi encodes dihydrofolate synthase  and a bifunctional dihydrofolate reductase-thymidylate synthase  and is predicted to carry out a limited folate biosynthetic pathway similar to Theileria spp[T. equi imports folate which is then modified into other compounds, chiefly nucleotide precursors. Folate biosynthesis predicts sensitivity to the drug pyrimethamine cycloguaryl, which has been observed in previous studies[T. equi DHFR-TS encodes the S125F mutation that reportedly confers pyrimethamine resistance in B. bovis and certain Plasmodium sp DHFR-TS[eria spp,28. Nota studies, even th DHFR-TS.B. bovis showing a markedly greater phospholipid composition in infected erythrocytes compared to uninfected erythrocytes[T. equi has an increased number of choline/ethanolamine kinase genes, even relative to B. bovis, which has a demonstrated increase in phosphatidylcholine relative to uninfected bovine erythrocytes[T. equi has been demonstrated to incorporate GPI anchors on membrane proteins[Phospholipid metabolism in apicomplexans is well-documented, and highlighted by studies in hrocytes. T. equihrocytes. Althougproteins. Approxiproteins.T. equi to have the most transporters of any hemoparasite genome sequenced to date[T. equi having 45 members of this family, compared to just 17 in T. parva, and 9 in B. bovis. Comparatively, Plasmodium falciparum has 16 members of the ABC family, most notably ABCB1 (MDR1), a known mediator of chloroquine and mefloquine resistance[T. equi, however specific mechanisms of resistance are unknown. Given the high number of ABC transporter family members in T. equi, including those of the MDR1 type and orthologues of the other known drug transport members ABCC1 and ABCG2, it is reasonable to hypothesize that ABC-mediated transport contributes to chemotherapeutic resistance in T. equi. Another expanded family is the Type II general secretory pathway, which contains 11 transporters, ~three times the number in the other hemoparasite genomes  about twice as many as other hemoparasites. The MFS transporters include drug efflux systems, organophosphate:phosphate exchangers and oligosaccharide:H+ symport permeases and are single-polypeptide secondary carriers capable only of transporting small solutes in response to chemiosmotic ion gradients[As noted earlier, the folate/biopterin family of transporters is increased (at 5), and thus, radients.T. equi and B. bovis, T. parva, or T. annulata. There are relatively few large regions that do not have syntenic matches in the other piroplasm genomes. Analysis of chromosome 1 demonstrated that these regions contain unique proteins (184), without Pfam hits or other functional assignment. The T. parva and T. annulata chromosomes are highly syntenic, and a much more fragmented pattern of synteny is seen between Theileria spp. and B. bovis[Theileria spp. and T. equi, suggesting more recent shared ancestry than for T. equi and B. bovis, where the blocks of synteny are more fragmented. T. equi chromosome 1 appears to be evolutionarily related to Thelieria spp. chromosomes 1 and 2. The ancestral Theileria chromosome 2 has split to provide elements of T. equi chromosomes 1 and 2. Approximately 2/3 of each of T. equi chromosomes 2 and 3 share synteny with Theileria spp. chromosomes 1 and 4, respectively. The remaining 1/3 of each of these chromosomes contains more of the B. bovis specific lineage sequences or unique sequences. With a few exceptions, T. equi chromosome 4 and is syntenic to both T. parva and T. annulata chromosome 3. Notably, the orthologous gene matches extend to the ends of each of the T. equi chromosomes demonstrating the lack of telomerically located species-specific gene families and repeats seen in other hemoparasites[Figure B. bovis,37. Largarasites,28.T. equi protein families that have at least 20 members revealed that most of these families are comprised of functional attributes similar to those represented in similar scans of B. bovis and Theileria spp. None of the T. equi families appear to contain a family of known immunodominant antigenically variable genes, nor are any of the families telomerically associated. Notable families are presented below.TribeMCL placed 2614 proteins in 334 families ranging in size from 2 to 356 members . The Tar genes were reported to be analogous to the Tpr genes of T. parva, however Family 3 does not share significant sequence identity with Tpr[Theileria is unknown, it is thought that these genes in T. parva are involved in the generation of diversity[Tpr repertoires appearing to be isolate specific[T. annulata, the T. equi Family 3 genes are distributed throughout all four chromosomes. Expressed Sequence Tags (ESTs) were found for only seven of the Family 3 genes suggesting that only a small number of these genes are expressed at a given time.Containing 109 members, this family has ~30% sequence conservation with a repeat gene family in with Tpr. Most meiversity, with thspecific. Like T.T. equi, Equi Merozoite Antigen 1 (EMA1). EMA1, a 34 kDa immunodominant protein, is used as the basis of a cELISA diagnostic test for T. equi, as this highly conserved antigen is recognized by sera from infected animals worldwide[T. equi proteins by serum from a horse challenged twice at a 2-month interval with T. equi revealed that in addition to EMA1, EMA2 has a GPI anchor and detectable antibody responses were limited to EMA1-3 proteins[ema1 was originally described as a single copy gene, genomic analysis has revealed a total of 10 genes in this family. The amino acid identity ranged from 17 to 55% in pairwise comparisons of family members. EMA2 was previously characterized as 50% identical to EMA1, and is also an immunodominant GPI-anchored protein. EMA1 is more highly expressed in blood stages of the parasite relative to EMA2 while EMA2 is more highly expressed in the tick salivary gland during transmission feeding than EMA1[T. annulata (TAMS1) and T. parva (mMPSA) but not in other Apicomplexa[ema family member, with the exception of BEWA_028210 and BEWA_047350, demonstrating that transcription in blood stage parasites takes place for most family members.Family 29 contains 10 members related to the best characterized protein in orldwide. EMA1 haproteins. Althoughan EMA1,41. A sicomplexa,43. At lema genes are predicted to encode proteins of ~30 kDa, with the remaining two encoding markedly differently sized proteins, one being truncated (BEWA_034050) and the other (BEWA_047350) being much longer and only having similarity in the C-terminal domain  or B. bovis ves1 genes (encoding VESA1) where evasion of the immune response through emergence of variable surface antigens is a well characterized mechanism of parasite persistence[The  evasion,44. Thersistence,45.T. annulata and T. parva as a stretch of 70 amino acids and was named “Frequently Associated IN Theileria”; subsequently, Pfam04385, the FAINT domain – a domain of unknown function (DUF529) was established and was found to be over represented in proteins predicted to be secreted[T. equi proteins may have multiple FAINT domains and are typically classified into several protein families. T. equi contains 271 proteins with 560 hits to Pfam04385. FAINT domains are not reported for B. bovis or P. falciparum. The FAINT domain containing genes are distributed throughout the T. equi genome.While not a protein family per se, the FAINT domain was originally detected in secreted,28. T. eT. equi, it has been reported to invade the lymphocyte and develop to a macroshizont stage[T. parva and T. annulata, the macroschizont life cycle can induce transformation of the infected cell[T. equi isolate used in this study infected equine lymphocytes/PBMCs, equine PBMCs were infected with sporozoites obtained from infected adult male Rhipicephalus microplus ticks. To verify infection of equine PBMC cultures, EMA-1 and EMA-2 monoclonal antibodies were tested by immunofluorescence and bound corresponding T. equi antigens within equine PBMCs  that do transform their host led us to search the T. equi genome for putative host cell transformation genes to see if these genes were retained in the genome. A set of genes[T. annulata and T. parva was used to explore the T. equi genome for orthologous genes, along with the EST expression data. T. parva and T. annulata express prohibitin in macro- and microschizonts, while T. equi expresses the ortholog (BEWA_014000) in erythrocyte-stage parasite ESTs. Prohibitin is reported to be a tumor suppressor that is involved in pathways that lead to immortalization, however, this gene was also identified in B. bovis, which does not undergo transformation[T. equi has several homologs of cyclophilins, which have been linked to transformation. This gene is found in B. bovis, T. parva and T. annulata, again, limiting the implication of this gene product as a host cell transforming factor. T. equi does not have a homolog of the Theileria schizont AT hook (TashAT) family of proteins. The AT hooks function as nuclear localization signals and DNA binding domains, and may function in cell transformation. Likewise, TashHN (host nucleus) and SuAT1 do not have T. equi orthologs. TashHN is expressed by Theileria macroschizonts that have lost the ability to form merozoites, while SuAT1 is a schizont protein with characteristics of the TashAT family, that can modulate the phenotype of cultured bovine macrophages by altering the expression profile of cytoskeletal proteins in a manner similar to that seen during infection. Although host cell transformation has not been reported for nt stage. In T. pted cell. To assuof genes identifiormation. Similarrap1) gene family are candidates for vaccine development in Babesia spp. Immunization of cattle with purified native RAP-1 of B. bigemina resulted in partially protective responses, and is one of three antigens in a recombinant vaccine developed in Australia[B. bovis RAP-1 failed to elicit protective immune responses in vaccine trials. The RAP-1 proteins of Babesia spp. are characterized by a signal peptide followed by a cysteine-rich region and several short conserved sequence motifs within the first 300 amino acids. Thus, the N-terminal region of all known Babesia RAP-1 proteins contains the “rap-1 domain”. The C-terminal regions are less conserved among Babesia species. B. bovis contains two identical rap1 genes in tandem and a shorter rap1 related protein gene (RRP) located ~30 kbp from the rap1 locus. Three rap1 genes were identified in T. parva and four in T. annulata. The general structure of rap1 in Theileria spp. is different from that in B. bovis, with the Theileria proteins containing more than one rap-1 domain. The T. equi rap1 locus contains two tandemly arranged genes: BEWA_037610 contains two rap-1 domains, and BEWA_037600 contains three rap-1 domains. Thus, the arrangement of rap-1 domains in T. equi resembles that found in other Theileria spp.Members of the rhoptry associated protein-1  is a microneme protein with surface exposed epitopes that has orthologs in hemoprotozoan Apicomplexa, as well as Toxoplasma gondii[in vitro, and has been protective in animal models[T. equi and B. bovis AMA-1 lacks the N-terminal extension found in other Theileria spp. AMA-1 has a set of conserved cysteine residues which may contribute to a conserved architecture through disulfide bonds. Plasmodium AMA-1 is polymorphic[T. equi before AMA-1 can be considered as a vaccine candidate for this species.ma gondii. AMA-1 al models-51. T. eymorphic, and thePlasmodium[T. equi proteins have low sequence similarity and it is unclear whether these would be effective vaccine candidates.Conserved hypothetical proteins BEWA_005690 and BEWA_005710 fall into a Cluster of Orthologous Groups (COG) with apicomplexan genes annotated as TRAP, sporozoite surface protein or microneme protein 2. Althouglasmodium, the T. Plasmodium that has a homolog in numerous species, including Leishmania and T. parva[B. bovis (BBOV_III006770) and T. equi (BEWA_7380). This target could provide a novel method of transmission blocking through gene knock out, thus preventing gamete fusion and tick transmission of a modified live vaccine. Membrane Occupation and Recognition Nexus protein (MORN1) seems to be related to cell division in all Apicomplexa studied, and functions in both asexual and sexual reproduction[Plasmodium, Theileria, and B. bovis, as well as Toxoplasma gondii and Cryptosporidium parvum. Heme Detoxification Protein (HDP) has a role in detoxifying heme that results from parasite metabolism of hemoglobin by polymerizing the heme into hemozoin[HAP2 is a protein essential for membrane fusion during zygote formation in  T. parva. A positoduction. A commohemozoin. BEWA_01T. parva p67 is an abundant sporozoite surface antigen, and along with its homolog from T. annulata (SPAG-1) has been shown to induce neutralizing antibody, and immunity to theileriosis and has been a target of vaccine development[B. bovis (BBOV_IV007750)[T. equi contains a gene at the same location (BEWA_015160). EST data show that BEWA_015160 is transcribed. The prospect that the genes occupying this position in syntenic loci have similar functions during parasite infection but tailored to different hosts makes them interesting candidates for vaccine studies.elopment. A positV007750),59 and TT. parva antigens that are the targets of CD8+ T lymphocyte responses in immune cattle have been designated Tp1,Tp2, Tp4,Tp5, Tp7 and Tp8[T. annulata. All of these vaccine candidates except hypothetical protein Tp1 have orthologs in T. equi and have been demonstrated to be transcribed in erythrocyte stages via EST analyses. While the significance of piroplasm stage expression is unclear, these T. equi proteins may have utility as vaccine components based on observations in T. parva.A set of six  and Tp8. Tp2 is Babesia equi as T. equi in 1998 due to the description of a lymphocytic stage for this parasite[T. equi was explored using 150 deduced amino acid sequences  analyses of individual sequences resulted in many different tree topologies; however, the four most commonly recovered topologies  consistently yielded the same estimates, which completely corroborate the trees generated by Bayesian inference and MP analyses of the concatenated datasets  of antigenic variation provided by the ema family isn’t yet known, we hypothesize that EMA family members provide antigenic variation and immune escape through a protein reassortment strategy that generates immune escape variants by creating novel heteromers of EMA on the cell surface. The genome sequence also revealed a 109 member gene family (Family 3); however these proteins have never been detected by immune serum, suggesting that they are not contributing to immune evasion. Genome sequencing allowed an exploration of the phylogenetic position of T. equi at a depth not previously feasible; demonstrating that this species clusters consistently with, and as a sister lineage to other Theileria spp. Genome sequences for closely related piroplasms may allow “fine tuning” of the T. equi phylogenetic position and appropriate naming of the lineage to which this species belongs. Finally, the genome sequence will provide an invaluable tool for researchers developing methods to control equine piroplasmosis.Characteristics of genes involved in immune evasion leading to persistence include immunodominance, highly variable domains, and membership in a multigene family. Within the phylum Apicomplexa,  evasion. An analAnimal experiments were approved by the Institutional Animal Care and Use Committee at University of Idaho, USA, in accordance with institutional guidelines based on the U.S. National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.Theileria equi USDA (Florida) stabilate (PPE: 0.64%) generated from infection of a spleen intact horse (H2) was inoculated intravenously into a splenectomized horse (H047). When the parasitemia reached 16% as determined by Giemsa stained blood smear examination, blood was collected into a flask containing glass beads for defibrination. The blood was washed three times with HBSS/10mM EDTA to remove the white blood cells (WBC). Erythrocytes were purified using histopaque 1077 following the manufacturer’s protocol. Microscopic examination determined WBC contamination to be ≪5 WBC per 1 ul of blood. T. equi genomic DNA was extracted from the purified erythrocytes using components of the Gentra Puregene DNA Purification System (Kit D-5000).in vitro PBMC infection, one thousand adult male Rhipicephalus microplus ticks were acquisition fed on a T. equi (Florida strain) merozoite stabilate-infected adult horse during ascending parasitemia for eight days (acquisition feeding). Following acquisition feeding, fed ticks were incubated for two days at 15°C with 94% relative humidity and a 12 hr photo period, and subsequently transmission fed on a naïve horse to induce final maturation of sporozoites into their infectious stage. On day seven of the transmission feeding, 240 live adult males were recovered and salivary gland pairs were dissected aseptically, and sporozoite extracts prepared using established methods. Briefly, collected salivary glands were washed in 0.5 ml of complete media  and crushed in a glass homogenizer to release sporozoites. The homogenate was then centrifuged at 300 g for 5 min and the supernatant containing sporozoites was collected and adjusted to 10 tick equivalents/ml. One ml of the sporozoite extract was inoculated into each of ten wells of a 24-well plate containing 2 × 106 PBMC from an uninfected horse. Starting on day nine post-inoculation, cell culture aliquots were cytospun and examined microscopically for infection following Diff-Quick staining.To obtain the sporozoites need for T. equi USDA (Florida) strain was used to construct a small and large insert plasmid library as well as a bacterial artificial chromosome library. A total of 100,835 high quality sequence reads (822 bp average read length) were generated and assembled using the Celera Assembler (http://sourceforge.net/projects/wgs-assembler/), resulting in 119 scaffolds consisting of 495 contigs. A BAC library was end sequenced to generate an additional 3023 reads which were used to confirm the assembly and for targeted sequencing in the closure phase. Gaps were closed by a combination of primer walking and transposon based or shotgun sequencing of medium insert clones, BAC clones or PCR products. The genome sequence has been deposited in GenBank under accession number ACOU00000000. A cDNA library was constructed using cultured infected erythrocytes (Florida strain) using the Creator SMART cDNA library construction kit (Clontech). cDNA in the size range of 0.3 -2kb was normalized using the Trimmer normalization kit (Evrogen). 7629 sequences (GenBank Accession #s HS032712 - HS040340) were generated and assembled into 2355 contigs using PASA[DNA from ing PASA.Genes encoding tRNA’s were identified using tRNA scan-SE. Gene moB. bovis[Functional annotation was as for B. bovis. In addiB. bovis and metaB. bovis. TranspoB. bovis. TribeMCB. bovis was usedhttp://sybil.sourceforge.net) was used to create an all-versus-all BLASTP search using the proteomes of T. equi, B. bovis, T. parva, T.annulata, P. falciparum, P. vivax, C. parvum and T. gondii. These outputs were subjected to Jaccard clustering, placing proteins into distinct clusters for each proteome. Clusters from different proteomes were linked based on best bidirectional BLASTP hits between them to provide Jf-COGs. A minimum block size of five with one gap was allowed in the analyses.Sybil  were defined using an in-house comparative pipeline, which starts with BLASTP among protein sequences within and across species. We used the BLOSUM62 matrix with expected value 10-5. Jaccard clustering was then performed twice, once to form within-species clusters of paralogous genes and a second time to derive a set of multi-species COGs. In the first case, we used an 80% identity cutoff over a minimum of 70% of the length of the smallest protein and a link score of 0.6. In the second, we set the identity cutoff at 50% over >70% length, and Jaccard coefficient cutoff of 0 for edge pruning. In order to minimize ambiguous amino acid homology assignments that can occur when indels are present, peptides were chosen that have the smallest protein size variation across all species  maximum parsimony (MP) with exhaustive tree search, and otherwise default parameters as implemented in PAUP v4.0b10, ii) BayT. orientalis does not transform it’s host cell and has been compared to T. parva and T. annulata to identify putative mediators of leukocyte transformation[B. microti phylogeny was analyzed using 316 single copy genes and found to clade separately to T. parva, T. annulata and B. bovis[Note: Two additional genome sequences of interest were published while this article was in review. ormation. B. micr B. bovis.Theileria; GPI: Glycosylphosphatidylinositol; MFS: Major Facilitator Superfamily; MP: Maximum Parsimony; mt: Mitochondrial; MORN: Membrane Occupation and Recognition Nexus; PBMC: Peripheral blood mononuclear cells; rap: Rhoptry associated protein; TCA: Tricarboxylic acid cycle; VESA: Variant erythrocyte surface antigen; WBC: White blood cells.ABC: ATP-binding cassette; AMA: Apical Membrane Antigen; COG: Cluster of Orthologous Groups; DHFR-TS: Dihydrofolate reductase-thymidylate synthase; EMA: Equi Merozoite Antigens; EST: Expressed sequence tag; FAINT: Frequently Associated IN The authors declare that they have no competing interests.LSK, MT, DRH, AD, JJG, JCS, VMN, RHM, DPK and KAB conceived and designed the experiments. LSK, MT, DRH, JDR, EC, AD, JJG, MGS, AOTL, HER, JCS, CES, and MWU performed the experiments. LSK, MT, DRH, JDR, EC, AD, JJG, MGS, AOTL, HER, JCS, CES, MWU, RHM, DPK, and KAB analyzed the data. LSK, DRH, DPK, and KAB wrote the paper. All authors read and approved the final manuscript.Figure. Depiction of the T. equi apicoplast genome gene arrangement, showing unidirectional coding of genes. Known enzymes shown in red, ribosomal proteins in green, rRNA sequences in yellow, and groupings of tRNA molecules in blue. Location of conserved hypothetical (gray), and hypothetical (black) protein-encoding genes are shown by arrows or bars. Members of the three expanded gene families are marked with either “*”, “¡” or “^” to indicate similar genes. The molecule is depicted as linear, though not experimentally demonstrated to be either circular or linear.Click here for fileTable. Nuclear encoded genes potentially targeted to the apicoplast.Click here for fileTable. GPI anchored proteins predicted by GPI-SOM.Click here for fileTable. Transporter comparison B.bovis, T. equi, and T.parva.Click here for fileTable. 30 largest protein families.Click here for fileFigure. Alignment of EMA family sequences. Residues highlighted in yellow are conserved among all family members, and those in blue conserved among the majority of family members. Dashes represent gaps introduced to accommodate non-conserved stretches of sequence.Click here for fileTable. Proteins used in phylogenetic analysis.Click here for fileFigure. Phylogenetic trees. A: Most frequently recovered trees from maximum parsimony analysis of 150 polypeptides conserved among eight taxa, showing the number of times tree recovered out of 210 total topologies. Value above the line represents the number times that branch was recovered out of total times whole topology was recovered , and value below the line is percentage bootstrap support for that branch out of 1000 replicates. The bootstrap support for the individual MP trees was calculated for each individual dataset, and averages across all of the individual trees are presented. B: Single most parsimonious tree estimated from the concatenated dataset of the 150 polypeptides. Taxon codes are Cp: Cryptosporidium parvum, Tg: Toxoplasma gondii, Pf: Plasmodium falciparum, Pv: Plasmodium vivax, Bb: Babesia bovis, Te: Thieleria equi, Ta: Theileria annulata, Tp: Theileria parva. C: Trees estimated with maximum likelihood using six different models of amino acid substitution. Branch support was assessed with 1000 bootstrap pseudoreplications.Click here for file


Hemileia vastatrix is the causal agent of coffee leaf rust, the most important disease of coffee Arabica. In this work, a 454-pyrosequencing transcriptome analysis of H. vastatrix germinating urediniospores (gU) and appressoria (Ap) was performed and compared to previously published in planta haustoria-rich (H) data. A total of 9234 transcripts were identified and annotated. Ca. 50% of these transcripts showed no significant homology to international databases. Only 784 sequences were shared by the three conditions, and 75% were exclusive of either gU (2146), Ap (1479) or H (3270). Relative transcript abundance and RT-qPCR analyses for a selection of genes indicated a particularly active metabolism, translational activity and production of new structures in the appressoria and intense signaling, transport, secretory activity and cellular multiplication in the germinating urediniospores, suggesting the onset of a plant-fungus dialogue as early as at the germ tube stage. Gene expression related to the production of carbohydrate-active enzymes and accumulation of glycerol in germinating urediniospores and appressoria suggests that combined lytic and physical mechanisms are involved in appressoria-mediated penetration. Besides contributing to the characterization of molecular processes leading to appressoria-mediated infection by rust fungi, these results point toward the identification of new H. vastatrix candidate virulence factors, with 516 genes predicted to encode secreted proteins. Hemileia vastatrix Berk & Broome is the major disease of Arabica cultivated coffees (Coffea arabica L.)  revealed that the majority (60%) had no homology in public genomic databases, representing potential coffee rust-specific genes. Nevertheless, H. vastatrix candidate effectors likely related to host infection and orthologous to other rust fungi, were identified among 382 predicted secreted proteins  was multiplied on its differential host plant . An in vitro method was used to produce germinating urediniospores and appressoria to ensure the generation of cDNA libraries with no contaminating plant sequences , including an in-solution DNase treatment following the manufacturer's instructions. RNA concentration and integrity were evaluated by spectrometry  and capillary electrophoresis  respectively. Following the SMARTer Pico PCR cDNA Synthesis Kit  protocol, cDNA were synthesized from 1 μg total RNA using SMARTScribe Reverse Transcriptase (Clontech) and amplified using Advantage 2 Polymerase (Clontech). cDNA fragments, which ranged between 500 and 3000 bp, were purified with the NucleoSpin Extract II kit  and their quality and concentration were evaluated by electrophoresis.www.454.com) at the Genoscope  following standard procedures recommended by Roche.For each sample, 20 μg cDNA was used for 454-pyrosequencing run on half of a picotitre plate on a Genome Sequencer FLX System using long-read GS FLX Titanium chemistry  was also employed. The relative abundance (Ra) of transcripts was calculated as the ratio between the number of 454 reads per contig and the length of the assembled contig  with default parameters. For comparative purposes, the MIRA 3.2 assembler (www.ncbi.nlm.nih.gov), the genome sequences of Melampsora larici-populina and Puccinia spp. ; the euKaryotic Orthologous Group (KOG) database ; the Phytopathogenic Fungi and Oomycete EST Database  and 20,567 (P. graminis f. sp. tritici) and 11,638 (P. triticina) from the Puccinia spp. transcript catalogue (http://www.broadinstitute.org/annotation/genome/puccinia_group/MultiDownloads.html) were also considered. Homology searches were performed using BLAST algorithms . For each search against a given database, only the best hit was considered. The assignment of 454-contig sequences into KOG functional categories was obtained using Reverse psi-BLAST  using default parameters. ORFs below 18 amino acids were not considered. A secretome bioinformatics pipeline was employed to define a tentative set of secreted proteins encoded by H. vastatrix transcripts, using SignalP v4.0  were predicted with the translation tool getorf from the European Molecular Biology Open Software Suite  of enzymes that degrade, modify, or create glycosidic bonds (carbohydrate-active enzymes—CAZymes) was investigated by blastp comparison of predicted polypeptides to the CAZymes database  is defined as τ = i is the expression profile component normalized by the maximal component value  and 1, 2, 3, 7, 14, and 21 days after inoculation for the compatible interaction between isolate 178a and the C. arabica genotype H147/1, as previously described  and appressoria (Ap) samples for Given that different 454-pyrosequencing data assemblers are available and are known to generate diverse results  to the NCBI nr nucleotide database using blastn (Supplementary Data In the absence of genomic information for H. vastatrix infected-coffee leaf samples (H library) and previously predicted as of plant origin . These 50 contigs  were incorporated in the present study, together with the 4415 fungal contigs initially identified in the H library, summing a total of 4465 H contigs  and P. graminis f. sp. tritici  , suggesting they may correspond to highly divergent or specific H. vastatrix genes. In fact, among these, only 3.2% have a specific KOG category assigned, with an overrepresentation of categories involved in cellular structure, nucleic acid activity and signaling .A total of 4040 transcripts  presented homologies against the NCBI non-redundant nucleotide database (Supplementary Data Ustilago maydis (21%), Giberella spp. (13%), and M. oryzae (13%) being the most represented species. Further, 588 transcripts showed homology to fungal pathogenicity-related genes listed in the PHI database (Supplementary Data M. oryzae (19%), Candida albicans (18%), and Cryptococcus neoformans (14%). About 70% of these 588 transcripts had specific KOG categories assigned, with categories such as “Cell cycle control, cell division, chromosome partitioning,” “Secondary metabolites biosynthesis, transport and catabolism” and “Signal transduction mechanisms” overrepresented as compared to KOG categories assigned to all genes  index  to the M. larici-populina or the P. graminis f. sp. tritici predicted secreted proteins  encoding predicted secreted proteins are orthologous of the rust transferred protein (RTP1) genes  identified in Melampsora lini   with homology to Mn-type superoxide dismutase, orthologs of the H. vastatrix transcripts to the carbohydrate-active enzymes (CAZymes) database . On the contrary, some transcript families found in H. vastatrix are absent from the genomes of M. larici-populina  or P. graminis f. sp. tritici (Glycoside Hydrolase families 51 and 92). Additionally, eight Glycoside Hydrolase family 7 genes were identified both in M. larici-populina and in P. graminis f. sp. tritici, but none in H. vastatrix transcripts. CAZyme transcripts were more frequently expressed in the gU library and less in H  with 25 different transcripts predicted in H. vastatrix, against 20 in M. larici-populina, 22 in P. graminis f. sp. tritici, and 19–25 in a selection of basidiomycetes , required for the development of penetration hyphae and the proliferation of the fungus  identified in the three H. vastatrix libraries, and one (04218) only identified in the H library but at higher Ra values.Among the 215 es Table . The traH. vastatrix, transcript 00156, orthologous of a hexokinase, is upregulated in Ap according to RT-qPCR results. Two transcripts (04402 and 04553) were identified orthologous of trehalose-6-phosphate synthase genes in M. larici-populina (gene 33497) and P. graminis f. sp. tritici (PGT_06208), and RT-qPCR results showed the accumulation of transcript 04402 in appressoria. H. vastatrix transcript 00704, an ortholog of a neutral trehalase (M. larici-populina gene 116200), was detected in the three libraries, RT-qPCR showing a peak of expression in the appressoria and at 21 days after inoculation, suggesting a close control of trehalose/trehalose-6-P levels at these stages.The availability of nutrients for the fungus is very scarce at the early stages of the infection process and energy must be obtained from urediniospore contents. Carbohydrate metabolism by glycolysis/tricarboxylic acid cycle (TCA)/glyoxylate shuttle and lipids metabolism seems to be crucial to the success of the penetration process  showed higher expression during germination and appressoria formation according both to 454 pyrosequencing and RT-qPCR results. Increased levels of these enzymes were also described in P. pachyrhizi at the appressorial stage , acyl-CoA dehydrogenase (transcript 01629), enoyl CoA hydratase (transcript 01055), 3-hydroxyacyl-CoA dehydrogenase (transcript 01628), and 3-ketoacyl-CoA thiolase (transcript 00191). A similar profile was detected for acyl-CoA oxidase (transcript 00622). RT-qPCR profiles for these transcripts further revealed a second peak of expression at 2 days for transcripts 00191 and 01628, and at 7 days for transcript 00175. Transcripts 01594, 01988 and 07400 are orthologs of M. oryzae carnitine acetyl-transferase gene (crat1), involved in transport of peroxisomal acetyl-CoA. M. oryzae deletion mutants for this gene show reduced appressoria melanisation, and are not able to elaborate penetration pegs or infection hyphae   (Supplementary Data cdc35) and adenylate cyclase (cac1) genes, respectively from Candida albicans , necessary for penetration and sporulation  in eukaryotic cells. Transcript 06565 shows homology to the Cryptococcus neoformans virulence-related gpa1 gene   , all of them in the gU library, indicative of a possible role in appressoria formation in H. vastatrix. RT-qPCR expression profiling further showed induction of transcripts 02022 and 07174 during early pre-penetration events  were identified in H. vastatrix .A comparison of M. larici-populina annotated genes that are >10-fold up-regulated in infected leaves as compared to urediniospores , 10 secreted proteins and six glycoside hydrolases , the carbohydrate-active enzymes (CAZy) database or the Transporter Classification Database (TCDB). Database comparisons further indicate that half of these transcripts (4707) present no significant homology to genomic or transcriptomic data from other rusts, potentially representing novel or very divergent H. vastatrix genes.In this study, 7894 contigs were obtained by 454 pyrosequencing of cDNA from H. vastatrix transcripts and comparison of their relative abundance in each of the three sampling stages suggest a particularly active metabolism, translational activity, production of new structures and signaling in appressoria and intense transport, secretory activity and cellular multiplication in germinating urediniospores (Figure H. vastatrix transcripts to genes known to be involved and/or required for pathogenicity in other fungal plant pathogens, namely in appressoria-mediated infection, enabled the identification of an array of putative pathogenicity factors, a large proportion of which are expressed as early as during germ-tube elongation. Also, while melanized cuticle-breaching appressoria have been thoroughly investigated over the last few decades, namely in M. oryzae and Colletotrichum spp. (Deising et al., Annotation of s Figure . TranscrThis early activation of signaling, transport and secretory pathways suggests a precocious plant-fungus dialogue, which is corroborated by the possible induction of an hypersensitive reaction in stomatal cells of some resistant coffee varieties as early as at the appressorial stage (Silva et al., This study was conceived and directed by Pedro Talhinhas, Helena G. Azinheira, Sébastien Duplessis, Maria do Céu Silva, and Diana Fernandez. The laboratorial experiments were conducted by Pedro Talhinhas, Helena G. Azinheira, Andreia Loureiro, Sílvia Tavares, and Anne-Sophie Petitot. 454-pyrosequencing was conducted by Julie Poulain and Corinne Da Silva. Bioinformatic analyses were conducted by Bruno Vieira, Emmanuelle Morin, and Octávio S. Paulo. Biological interpretation of bioinformatics analyses were conducted by Pedro Talhinhas, Helena G. Azinheira, Andreia Loureiro, Sílvia Tavares, Sébastien Duplessis, and Diana Fernandez. Pedro Talhinhas, Helena G. Azinheira, Andreia Loureiro, Sílvia Tavares, Dora Batista, Octávio S. Paulo, Sébastien Duplessis, Maria do Céu Silva, and Diana Fernandez wrote the paper. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-ethylmaleimide-sensitive factor attachment protein receptors), the regulators including SM (Sec1/Munc18) family proteins, Rab GTPases and exocyst subunits. Verticillium dahliae is a widespread soil fungus that causes disruptive vascular diseases on a wide range of plants. To date, no genes involved in vesicular fusion process have been identified and characterized in V. dahliae. The recent publication of the draft genome sequence of V. dahliae allowed us to conduct a genome-wide identification, phylogeny and expression profile of genes encoding vesicular fusion components. Using compared genomics and phylogenetic methods, we identified 44 genes encoding vesicle fusion components in the V. dahliae genome. According to the structural features of their encoded proteins, the 44 V. dahliae genes were classified into 22 SNAREs , 4 SM family proteins, 10 Rab GTPases and 8 exocyst proteins. Based on phylogeny and motif constitution analysis, orthologs of vesicle fusion component in filamentous fungi were generally clustered together into the same subclasses with well-supported bootstrap values. Analysis of the expression profiles of these genes indicated that many of them are significantly differentially expressed during vegetative growth and microsclerotia formation in V. dahliae. The analysis show that many components of vesicle fusion are well conserved in filamentous fungi and indicate that vesicle fusion plays a critical role in microsclerotia formation of smoke tree wilt fungus V. dahliae. The genome-wide identification and expression analysis of components involved in vesicle fusion should facilitate research in this gene family and give new insights toward elucidating their functions in growth, development and pathogenesis of V. dahliae.Vesicular trafficking plays a crucial role in protein localization and movement, signal transduction, and multiple developmental processes in eukaryotic cells. Vesicle fusion is the final and key step in vesicle-mediated trafficking and mainly relies on SNAREs (soluble  Saccharomyces cerevisiae), mammals, and plants have shown that vesicular trafficking plays a crucial role in protein localization and movement, signal transduction, and in multiple developmental processes Vesicular trafficking is a critical feature for eukaryotic cells, and represents the movement of cargo among between different cell organelles, and between the cell and surroundings. In eukaryotic cells, it is mediated by distinct exocytic and endocytic routes. Endocytosis is a process by which extracellular material is internalized; exocytosis is the secretion of cell synthesized products outside the cell N-ethylmaleimide-sensitive factor attachment protein receptors), tethering complexes, SM (Sec1/Munc18) family proteins and Rab GTPases S. cerevisiaePlasmodium falciparumArabidopsis thalianaCaenorhabditis elegansHomo sapiensAspergillus oryzaeProtein transport among intracellular compartments is mediated by carrier vesicles that bud from one organelle and fuse selectively with another Rhizopus oryzaeArabidopsis contains 57 potential Rab GTPases that are categorized into eight subfamilies (RabA to RabH) H. sapiensThe formation of fusion competent SNARE complexes are regulated by multiple protein families S. cerevisiae contains four members of the SM protein family Arabidopsis genome contains six members of the SM protein family A member of the SM protein family is essential for regulating SNARE proteins and SNARE-mediated membrane fusion by directly binding to SNAREs and preventing their entry into nonproductive or premature SNARE complexes trans-SNARE complexes In addition to Rabs and SM proteins, tethering factors are also essential for SNARE mediated membrane fusion by triggering the engagement of Verticillium dahliae Kleb. , an ubiquitous and important soil borne fungal pathogen, causes vascular wilt disease on over 400 host plants, including important agronomical, horticultural, ornamental and woody plants Cotinus coggygria, which is one of the most important plant species cultivated in landscape ecology, has been caused by V. dahliae, resulting in stunted growth of stems, early senescence of leaves, and severe mortality of trees, with seriously detrimental effects on the red leaf scenery in Beijing V. dahliae secretes a reservoir of phytotoxic metabolites that behave as elicitors, inducing phytoalexin formation. V. dahliae also produces phytotoxins, which trigger wilt symptoms and vascular discoloration V. dahliae comprise high-molecular-weight protein-lipopolysaccharide (PLP) complexes, glycoproteins and plant cell wall degrading enzymes, including pectinases, polysaccharidases, and proteinases, which contribute to the pathogenicity of the fungus V. dahliae elicitors During colonization of the vascular system, V. dahliaeIn addition, microsclerotia, melanized, multicellular, long-term survival structures, represent a significant developmental event in the life cycle and disease cycle of V. dahliae genome sequence has been reported recently, and the Verticillium genomes encode numerous secreted proteins and are similar to those of other plant pathogenic fungi such as Fusarium spp. and Magnaporthe oryzaeV. dahliae and other plant pathogenic fungi with completely sequenced genomes are feasible and important. Therefore, to investigate the number, phylogeny and expression profiles of genes responsible for the last step of vesicle trafficking pathways, vesicle fusion, we undertook a genome-wide identification of genes encoding SNAREs, Rabs, SM proteins and exocysts in this species and other filamentous fungi such as Fusarium spp, Sclerotinia sclerotiorum, M. oryzae and A. oryzae. We analyzed the phylogeny of these genes among fungal species. Finally, we reported the differential expression of the above gene families during hyphae, conidia and microsclerotia formation. These results provide important information on the reservoir of secretory proteins and will help to characterize their roles in the growth, development and pathogenesis of V. dahliae.Fortunately, a draft of the V. dahliae strain XS11, which is a single-spore culture isolated from a smoke-tree in Fragrant Hills Park, Beijing. Cultures were grown on potato dextrose agar. Hyphae were grown in liquid CM (complete medium) 6 spores/mL and inoculated in liquid basal medium (BM) by shaking culture for 12 h at 24°C, at 130 rpm, and collected by filtering through two layers of Miracloth. To prepare microsclerotia, conidia were spread over cellulose membranes overlayed onto BM agar , and grown in the dark at 24°C for 14 days.This experiment used S. cerevisiae were obtained from the SGD database (http://www.yeastgenome.org/) and respectively used to identify the homologs. With these genes as initial queries, systematic BLAST-searches were performed on the Verticillium genome database (http://www.broadinstitute.org/annotation/genome/verticillium_dahliae/Blast.html) . The amino acid sequences of predicted V. dahliae proteins showing similarity to yeast proteins were extracted. These sequences were subjected to Pfam motif analysis (http://pfam.sanger.ac.uk/), PROSITE analysis (http://prosite.expasy.org/), and to the SNARE Database (http://bioinformatics.mpibpc.mpg.de/snare/snareSubmitSequencePage.jsp), SM superfamily database 1.75 (http://supfam.org/SUPERFAMILY/cgi-bin/scop.cgi?sunid=56815), and RabDB (http://www.rabdb.org/browse/rabify/) or Rab Database (http://bioinformatics.mpibpc.mpg.de/rab/rabMainPage.jsp) to verify the presence of domains. All genes of V. dahliae were subjected to TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), check the presence of transmembrane regions.Amino acid sequences of SNARE, SM, Rab and exocyst proteins in Verticillium Genome structure display website, and partially confirmed by alignment of the gDNA and cDNA sequences amplified by PCR and by sequencing.The exon/intron organization of individual genes was illustrated using the V. dahliae genes encoding SNARE, SM, Rab and exocyst proteins to identify their counterparts in other available fungal genomes using tBLASTN: Fusarium genomes (http://www.broadinstitute.org/annotation/genome/fusarium_group/MultiHome.html), S. sclerotiorum genome (http://www.broadinstitute.org/annotation/genome/sclerotinia_sclerotiorum/MultiHome.html), M. oryzae genome (http://www.broadinstitute.org/annotation/genome/magnaporthe_comparative/Blast.html), and A. oryzae genome (http://www.broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html). The identified protein sequences were subjected to the above domain search procedures. Synteny and orthology were analyzed and classified by the Multiple Alignment Tool provided by NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi).We used yeast and The entire amino acid sequences of all SNARE, SM and Rab proteins were aligned using Clustal X (version 1.83) without masking unreliably aligned positions Total genomic DNA was extracted from each isolate by a modified CTAB (cetyl trimethylammonium bromide) method using cultures grown on CM with shaking at 150 rpm Total RNA was isolated from frozen tissues, such as hyphae (HY), conidia (CO), germinated conidia (GC), microsclerotia formation 60 h (MS1), microsclerotia formation 72 h (MS2), microsclerotia formation 96 h (MS3), and microsclerotia formation 14 d (MS4). The samples were ground to a fine powder, and extracted with TRIzol reagent  using procedures recommended by the supplier. For microsclerotia, cultures were grown as described above. The cellulose membrane was removed from the agar, quick-frozen in liquid nitrogen, ground to a fine powder, and then extracted using the TRIzol reagent. Total RNA was eluted in RNase-free water and checked by agarose electrophoresis.V. dahliae, preferentially from 3′ end of the gene using Primer 3 with default parameters (http://frodo.wi.mit.edu/primer3/). Two independent biological replicates and three technical replicates of each biological replicate were made for qRT-PCR analysis in vegetative growth stages and microsclerotia formation. The RNA samples were incubated at 37°C for 30 min with DNase I (RNase-free)  to remove DNA contamination before RNA reverse transcription. One microgram of RNA was then reverse transcribed into cDNA using an oligo (dT) primer and Invitrogen’s SuperScript III Reverse Transcriptase , according to the manufacturer’s instructions, and diluted 100-fold with nuclease-free water. Primer specificity was further confirmed by dissociation curve analysis obtained after real-time PCR. The expression of each gene in different RNA samples was normalized using the geometric average of the expression levels of two frequently used housekeeping genes, actin and beta-tubulin −ΔΔCt method. ΔΔCt = ΔCt treated sample – ΔCt hyphae or conidia sample (vegetative growth stages or microsclerotia formation were referred to as treatment). Values are the mean of two biological replicates, each with three technical replicates. Error bars indicate the standard deviation.qRT-PCR was carried out using SYBR-green fluorescence in an ABI7500 (Roche Applied Science). Gene-specific primers were designed for genes encoding SNAREs, SM proteins, Rabs and exocyst components of No specific permissions were required for field studies. The field studies did not involve endangered or protected species.Verticillium and other fungi, we performed a tBLASTN search against the Verticillium genomes using SNARE proteins in S. cerevisiae as queries and the resulting sequences were used as secondary queries. By removing the redundant sequences, 22 SNARE proteins were identified in V. dahliae. All candidates were manually analyzed using the Pfam program (http://pfam.sanger.ac.uk/) to verify the presence of domains. In addition, we further checked and confirmed SNARE motifs and groups in the SNARE Database (http://bioinformatics.mpibpc.mpg.de/snare/snareSubmitSequencePage.jsp). Finally, we designated Verticillium SNARE genes following the nomenclature of their orthologs in S. cerevisiae.To identify putative SNAREs and related proteins in V. dahliae, including intron number, transmembrane region and protein feature is listed  were categorized into Qa-type, 4 genes  were categorized into Qb-type, 6 genes  were categorized into Qc-type and one gene (VDAG_04327) encoding a 480-amimo acid protein which contains two SNARE domains was classified as mixed Qbc SNARE. Four genes  were categorized into R-type. The SNARE motif structure of SNAREs of V. dahliae is confined within a minimum sequence length of 53 residues, except for VdSft1 containing 44 amino acids.Fungal SNAREs have been categorized as either Q- or R-SNAREs, depending on whether they contribute a conserved glutamine or arginine, respectively s listed . The 22 Verticillium genomes permitted in silico analysis to identify the ORFs encoding putative SNARE proteins, we found that there are some errors of annotation of VdBet1 (VDAG_06787) and VdVam7 (VDAG_03579). Based on comparative genomics analysis, we performed PCR and RT-PCR analysis using gDNA and cDNA templates, respectively, extracted from V. dahliae XS1 strain. After confirmation by sequencing, the VdBet1 gene was confirmed to have three introns rather than one intron , as well as five fungal plant pathogens with available whole genome sequences, such as Fusarium oxysporum , Fusarium verticillioides , Fusarium graminearum  of wheat and barley), S. sclerotiorum , M. oryzae (rice blast fungus) and A. oryzae . After confirming the presence of SNARE motifs, we identified 22 genes encoding SNARE motif in each genome, and they can be categorized into Q- and R-types  have a characteristic Sec1 domain, identifying them as members of the SM family proteins.To identify SM family and Rab GTPases in M family . HoweverV. dahliae was mined in the genome by tBLASTN searching. By removing the redundant sequences, a total of 10 Rab family proteins  were identified, all of which contained a monomeric GTPases domain  were identified in the e genome . SimilarV. dahliae to detect the distribution of various domain structures. As displayed schematically in V. dahliae were identified and generated in this study. Qa-type SNARE proteins contain SynN (SyntaxinN) domains and a t-SNARE domain  domains. Sec9 is known to contain a tandem of SNARE domain, VaSec9 protein had one of the Qb and one of the Qc type . A MP phQc-type SNAREs have three classes of domain architectures, class 1 has a V-SNARE domain , class 2 has Syntaxin-6_N and T-SNARE domains (VdTlg1), and class 3 (comprising only VdVam7) harbors a V-SNARE domain and a PX domain as yet unidentified (the PX domain is missing due to incomplete sequence information) . The phyV. dahliae. VdSro7 contained two WD40 domain sand a lethal giant larvae domain  have exocyst complex subunit domains .V. dahliae, including hyphae (HY), conidia (CO) and germinated conidia (GC). All of the predicted genes were expressed in at least one vegetative growth stage  methods in the normal vegetative growth stages of th stage . The expth stage .V. dahliae. In our previous study, microsclerotia formation from conidia clearly represents four stages, such as microsclerotia initiation, middle phase , late phase (formation of large clusters of heavily pigmented cells) and mature microsclerotia . To investigate the expression profiles of SNARE, SM, Rab and exocyst proteins during microsclerotia formation, we conducted qRT-PCR assays of these genes plus VDH1, which was confirmed to be involved in microsclerotia development The formation of melanized, multicellular survival structures referred to as microsclerotia manifests a significant developmental event in the life cycle and disease cycle of V. dahliae. The total number of these genes was 44 and it is fewer than that of S. cerevisiae. We further compared the number of vesicle fusion components among other filamentous fungi, such as V. albo-atrum, Fusarium spp, S. sclerotiorum, M. oryzae and A. oryzae  bound for the Golgi apparatus . oryzae . The comV. dahliae genome. We re-annotated these genes, by comparing alignments of orthologs from Fusarium spp, S. sclerotiorum, M. oryzae and A. oryzae, and by identifying motifs in each protein. The exon/intron structure and amino acid sequences of the ORF of these 44 genes were screened manually. We cloned and sequenced full-length cDNAs and gDNAs of genes with annotation errors, and some of these errors were corrected. For example, three introns were verified in the VdBet1 gene, and the entire amino acid sequence was corrected. Unfortunately, even using 5′ rapid amplification of cDNA ends PCR, we could not clone the 5′ sequence encoding the PX domain of VdVam7 due to incomplete genome sequence information. Anyway, general and main components involved in vesicle fusion is correctly identified and represented in our study.Complete and accurate annotation of genes is an essential platform for further study of evolution and gene family function. We identified 22 SNAREs, 10 Rabs, 4 SM proteins and 8 exocyst subunits from 10535 annotated genes in V. dahliae genome, and certain secreted protein families were significantly expanded The secretome represents a catalog of the complete set of proteins secreted into the extracellular space for a given organism Ustilago maydis, loss of a t-SNARE Yup1 resulted in a polar distribution of wall components and morphological alterations M. oryzae, a MoVam7 deletion mutant affected vacuole formation and membrane trafficking Each vesicle tracking pathway can be divided into four essential steps that include vesicle budding, transport, tethering, and fusion V. dahliae allow the elucidation of the roles of vesicle trafficking components and, therefore, represent a challenging, but potentially valuable, basis for future functional studies. In our laboratory, the knockout VdSec22 gene significantly affected the production of melanin and extracellular substances . It is possible that further important secreted proteins (as effectors or suppressors) will be identified among mutants of vesicle trafficking pathways.The availability of the complete genomic sequence and an efficient genetic transformation system for V. dahliae. Loss of SNAREs in other fungi affect fungal growth and development, i.e. MoVam7 and MoSec22 of M. oryzae are regulate Conidiogenesis and growth V. dahliae.In the present study, the expression profiles of vesicle fusion components were examined using qRT-PCR assays. The expression of all the genes was detected during vegetative growth and nearly half of the genes were differentially expressed. For example, SNARE proteins VdSso1, VdSso2, VdGos1, VdSec20, VdVti1, VdUse1, VdSft1 and VdSec22 were significantly expressed during growth and development of V. dahliaeV. dahliae.The formation of melanized, long-term survival structures known as microsclerotia is an important developmental event and a significant hallmark in the life cycle of V. dahliae.The expression profiles of genes encoding vesicle fusion components during microsclerotia formation showed that 30 genes were significantly expressed. Interestingly, 19 genes were significantly upregulated, including 8 SNAREs. VdSec22 was highly expressed in MS initials, the loss of VdSec22 resulted in deficient of MS formation as well. Therefore, gene expression profiles of vesicle fusion would provide useful clues and help to understand molecular bases of protein secretion during darken microsclerotia and which roles of vesicle fusion machinery play in microsclerotia formation of Verticillium and other important filamentous fungi. We analyzed their phylogeny, and performed expression profiling of 44 genes under normal vegetative growth and during microsclerotia formation. These 44 genes encode 22 SNAREs, 4 SM proteins, 10 Rab proteins and 8 exocyst components in V. dahliae. These sequences and expression information reported herein will be useful for further investigations of the roles of genes encoding vesicle trafficking components in the growth, development and pathogenesis of V. dahliae. Many of these genes are significantly differentially expressed during vegetative growth and microsclerotia formation, strongly suggesting that their participation in vesicle trafficking processes are required for microsclerotia formation, especially melanin metabolism and secreted proteins. Combined with bioinformatics analyses, the genomic structure and protein sequence of VdBet1 were revised by RT-PCR and sequencing. Although the genome sequence of V. dahliae has been published, functional studies on V. dahliae genes lag behind. Analysis of one or several gene families, rather than one gene, will be important. Based on all the results presented here, we speculate that vesicle fusion plays a critical role in microsclerotia formation. Therefore, the genome-wide identification and expression analysis of components involved in vesicle fusion should facilitate research in this gene family and give new insights toward elucidating their functions. Studies on vesicle trafficking may contribute to the understanding of secretory proteins and their roles in growth, development and pathogenesis of V. dahliae.In this study, we identified a set of genes encoding the vesicle fusion components such as SNARE, SM, Rab and exocyst proteins in Table S1The complete set of SNAREs of V. dahlia.(DOC)Click here for additional data file.Table S2Predicted proteins of the SM family of V. dahlia.(DOC)Click here for additional data file.Table S3Predicted proteins of the Rab family of V. dahlia.(DOC)Click here for additional data file.Table S4Predicted proteins of Exocyst complex subunits of V. dahlia.(DOC)Click here for additional data file.Text S1The revision of VdBet1 (VDAG_06787.1) by PCR and sequencing.(TXT)Click here for additional data file.Dataset S1Complete list of the deduced amino acid sequences of SNAREs, SM proteins, Rabs and exocysts that were identified from Saccharomyces cerevisiae, Verticillium dahlia, Verticillium albo-atrum, Fusarium oxysporum, Fusarium verticillioides, Fusarium graminearum, Sclerotinia sclerotiorum, Magnaporthe oryzae, Aspergillus oryzae.(XLS)Click here for additional data file.


Yersinia enterocolitica strains responsible for mild gastroenteritis in humans are very diverse with respect to their metabolic and virulence properties. Strain W22703  was recently identified to possess nematocidal and insecticidal activity. To better understand the relationship between pathogenicity towards insects and humans, we compared the W22703 genome with that of the highly pathogenic strain 8081 , the only Y. enterocolitica strain sequenced so far.Yersinia genome island 1 (YGI-1) involved in tight adherence were identified to be common to the 8081 and W22703 genomes. However, sets of ~550 genes revealed to be specific for each of them in comparison to the other strain. The plasticity zone (PZ) of 142 kb in the W22703 genome carries an ancient flagellar cluster Flg-2 of ~40 kb, but it lacks the pathogenicity island YAPIYe, the secretion system ysa and yts1, and other virulence determinants of the 8081 PZ. Its composition underlines the prominent variability of this genome region and demonstrates its contribution to the higher pathogenicity of biotype 1B strains with respect to W22703. A novel type three secretion system of mosaic structure was found in the genome of W22703 that is absent in the sequenced strains of the human pathogenic Yersinia species, but conserved in the genomes of the apathogenic species. We identified several regions of differences in W22703 that mainly code for transporters, regulators, metabolic pathways, and defence factors.We used whole-genome shotgun data to assemble, annotate and analyse the sequence of strain W22703. Numerous factors assumed to contribute to enteric survival and pathogenesis, among them osmoregulated periplasmic glucan, hydrogenases, cobalamin-dependent pathways, iron uptake systems and the Yersinia enterocolitica strains, thus contributing novel data to the Y. enterocolitica pan-genome. This study also sheds further light on the strategies of this pathogen to cope with its environments.The W22703 sequence analysis revealed a genome composition distinct from other pathogenic  Yersinia currently comprises three human pathogens , and at least 14 species considered harmless for humans, namely Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. mollaretii, Y. rohdei, Y. ruckeri [Y. aleksiciae [Y. similis [Y. massiliensis [Y. entomophaga [Y. nurmii [Y. pekkanenii [Y. enterocolitica infection causes diarrhea, terminal ileitis, and mesenteric lymphadenitis, but not systemic infection, and often leads to secondary immunologically induced sequelae including erythema nodosum, reactive arthritis and Reiter's syndrome [Y. enterocolitica encompass six biotypes as differentiated upon biochemical tests [Yersinia virulence plasmid pYV [The genus eksiciae , Y. simi similis , Y. massomophaga , Y. nurm. nurmii  and Y. pkkanenii . Y. entesyndrome . The hetal tests . Biotypesmid pYV , whereassmid pYV . Biotypesmid pYV .Manduca sexta larvae upon oral infection, and that this insecticidal activity is correlated with the presence of the so-called pathogenicity island TC-PAIYe [tcaA, tcaB, tcaC and tccC with homology to TC genes of Photorhabdus luminescens. However, the absence of TC-PAIYe is not reflected by a loss of toxicity in case of subcutaneous infection, indicating the presence of yet unknown insecticidal determinants in Y. enterocolitica [We have recently shown that strain W22703  confers lethality towards nematodes and TC-PAIYe ,14. Thiscolitica .Y. enterocolitica, we have chosen to sequence the genome of the low-pathogenicity strain W22703. We report the annotation of this second genome sequence of a Y. enterocolitica strain, and a detailed comparative genome analysis of the W22703 genome with that of strain 8081, a representative of the highly pathogenic biotype 1B group. The data obtained provide novel insights into the biology, metabolism, adaptation strategies and evolutionary relationships of Y. enterocolitica.To investigate the genomic heterogeneity of the species Y. enterocolitica strain W22703 genome revealed a total number of 243656 reads with an average read length of 363. Assembly of 232502 reads resulted in 305 contigs larger and 705 contigs shorter than 1,000 base pairs (bp) with a median level of coverage in contigs > 5 kb of 16.49  and one specific prophage.ary 2011 -20. It bng Mauve  shows long Mauve  was not Y. enterocolitica strains compared here, but might constitute additional metabolic or virulence-associated properties contributing to the overall strain fitness. Twelve ROD present in strain W22703 are shown in Figure Yersinia strains, with the exception of contig 1280 that harbours phage related genes. The 8081 genes flanking the ROD might give additional information about the underlying recombination events. For example, a glycosyltransferase operon of 8081 (YE3070-YE3087) might have been replaced by a related operon on contig 1878 (or vice versa) that possibly contributes to O-antigen synthesis. A substitution of hypothetical genes by a non-homologous cluster of functionally unknown genes is observed in contig 1764. The ROD on contigs 1186, 1240, 1280 and 1973 obviously interrupt gene linearity with respect to the 8081 genome, indicating that loss and substitution, or insertion, of genes might have taken place in these cases. Contig 1280 harbours several phage-related genes and is therefore assumed to represent a second prophage region. Transposase genes were found in the 8081 genome between YE2773 and YE2779, a region covered by the LPS synthesis in W22703 (contig 1162), and a similar observation was made for the PTS encoding cluster on contig 1884. More functional details on these ROD are described below.We then searched for regions of difference (ROD) between the genome sequences of 8081 and W22703. By definition, those ROD genes do not belong to the core genome of the two Ye is characteristic for biotype 2-5 strains, but absent in biotype 1A and 1B strains with the exception of WA314  [tccC2 locus [While YGI-1, which is responsible for adhesion and includes a T4SS, is completely encoded on contig 1802, the high pathogenicity island (HPI) encoding yersiniabactin  is missiype O:8) . Anotherype O:8) . Beside C2 locus , no furtY. enterocolitica needs to overcome both cellular (hemocytic) and peptide-mediated components. A candidate of the former group is the exported repeats-in-toxin RtxA, a cytolytic toxin of approximately 3200 amino acids length encoded by contig 1867. It clusters with a putative Rtx activating protein, RtxC, and a peptide chain release factor 1, RtxH (Table Y. enterocolitica serotype O:3 strain and Y. kristensenii carry homologs of these proteins. The genome of W22703 encodes three two-partner secretion (TCS) systems involved in hemolysin release, one of which is absent in the 8081 genome. Three peptidases of W22703 might play a role in resistance towards antimicrobial peptides. W22703 also produces an antibacterial protein or bacteriocin that is absent in 8081. The pyocin locus on contig 1216 probably encodes killer proteins and a dual type immunity protein with domains similar to pyocin-E2 and colicin (S2). Its biological function, as well as that of a small toxic polypeptide (contig 1810), is yet unknown.To survive within its host organisms, ysa of 8081 is absent in W22703, this strain harbours another T3SS, which we termed ysa2, located on contigs 1804 and 1807  cluster yst1 and yst2 in 8081, only yst2 responsible for a general secretion pathway (GSP) is present in strain W22703. Contig 1170 encodes factors involved in conjugal DNA-transfer, namely TraD, a MobA/MobL protein, and a putative type IV prepilin that might contribute to LGT.Two distinct, chromosomally located type three secretion systems (T3SS) mark one of the most striking differences between the two genomes. While 7 Figure . PCR tarormation . The funAs iron is often a rate-limiting factor for pathogenic bacteria during infection, W22703 requires iron-scavenging systems for survival in the host. Beside the two iron and enterobactin transport systems within the PZ, those comprise a hemophore cluster for heme binding and uptake . In total, our analysis identified a higher number of putative transporters with respect to strain 8081.We also identified eight ABC transporters in W22703, two phosphotransferase systems (PTS), four permeases, two major facilitators, a sodium:bile acid symporter, and other transporters listed in Table et al. [Y. enterocolitica biotypes, and it is absent from Y. pestis and Y. pseudotuberculosis. Four contigs revealed to carry PZ genes. We linked contigs 1088/1891 and 1803/1802 due to the presence of truncated hypF and fepG genes, respectively, at their ends. The primer combination 5'-GTTTCTTTATGGGCGCG-3'/5'-TTGGCATGGAGGCCTG-3' hybridizing to the ends of contigs 1891 and 1803 resulted in a PCR product of approximately 1500 bp, thus allowing the linear reconstruction of the W22703-specific PZ  the T3SS ysa important for pathogenicity in an mouse oral infection model [yst1 required for full virulence [hyb-) locus, fecBCDE encoding an iron transporter, the ferric enterbactin transport system fepBDGC/fes, and proP encoding a betain/proline transporter involved in osmoprotection and osmoregulation.The plasticity zone of strain 8081 ranges from YE3447 to YE3644 and has a total length of approximately 199 kb with 186 coding sequences (CDS). It was defined by Thomson irulence . FurtherYersinia sequences available so far, is in doubt due to its low coding density.The recently identified flagellar cluster Flg-2  within tflg-1) of 8081 is present in W22703 , but only one of two type-1 fimbrial operons was found . The surface-exposed lipopolysaccharide (LPS) molecule constituting the serotype O:9 O-antigen is synthesized by the O-polysaccharide gene cluster (contig 1162) [Y. enterocolitica to human complement and polymyxin B has been described recently [The main flagella and chemotaxis gene cluster I (ig 1162) ; a seconrecently .Y. enterocolitica genomes compared here are involved in nitrogen metabolism. Examples are the capability to catalyse urease that is encoded by seven genes on contig 1225. The assimilation of the urease product ammonia for amino acid and nucleotide synthesis is then achieved by glutamine synthase. Two ornithine decarboxylases forming putrescine, and a putrescine/ornithine antiporter are encoded on the PZ and also contribute to amino acid metabolism.Several enzymatic activities common to both cel gene cluster for cellulose production (contig 1798) and the genes mdoC, mdoG and mdoH for osmoregulated periplasmic glucan (OPG) biosynthesis (contig 1967). Both the capability to produce cellulose and to synthesize OPGs have been lost or inactivated in Y. pestis and Y. pseudotuberculosis. OPG mutants exhibit deficiencies in virulence, biofilm formation and antibiotic resistance, as well as hypersensitivity towards bile salts [Like 8081, strain W22703 carries the le salts .12)-dependent manner is encoded on contigs 2012, 1667, 1476, 1555, 1999, and 1235, and the respective cob/cbi/pdu genes are collinear to the 8081 genes YE2707-YE2750. In line with the yersiniae core genome, ttr genes responsible for tetrathionate reduction are present (contig 1975), and the eut genes allowing B12-dependent ethanolamine utilization are absent. The mtn genes located on contig 1812 are involved in methionine salvage. This cobalamin-dependent pathway recycles methylthioadenosine derived from sperimidine, spermin and N-acylhomoserine lactone synthesis. The hydrogenases Hyd-4 encoded within the hyf locus  and Hyd-2 within the hyb locus  are absent in W22703.We identified few enzymes or capabilities that are missing in W22703 in comparison to 8081 , YE185 (YE1667-YE1693), YE200 (YE1799-YE1819) and YE250 (YE2292-YE2363) of strain 8081 are absent in strain W22703 that, however, carries another prophage of 37 CDS in contig 1796. Then, the Yersinia genome islands YGI-2, YGI-3 and YGI-4 [Whole genome comparison allows to follow the dynamic processes by which genomes separate from a common ancestor. In addition to genomic islands or clusters already mentioned above, the genomes of the two Y. enterocolitica. Adding, rearranging and reducing or losing DNA has been proposed as the general recipe for Yersinia genome evolution when eight less-pathogenic strains had been compared [The genome analysis and genome comparison performed here intended to contribute to a better understanding of the ecology, pathogenicity and evolution of compared .Y. enterocolitica [Several ROD shown in Figure colitica .ysa2) that is absent in the pathogenic species Y. pestis and Y. pseudotuberculosis, but present in apathogenic species such as Y. intermedia, Y. frederiksenii and Y. kristensenii, might play a role in the interaction with non-mammalian hosts. The TC proteins have been shown to be secreted upon activity of the plasmid-encoded T3SS of Y. pestis [ysa2 is a candidate for TcaA secretion by W22703. This finding supports the assumption that T3SS are not unique vehicles for delivering anti-vertebrate factors but ancient secretion systems for the transport of effector molecules across host membranes, with the potential to play a role in a wide range of bacteria-host interactions [ysa2 contributes to the pathosphere of yersiniae, a concept hypothesizing that all of the pathogenic genes shared by enteric bacteria form a "pool" [Other (virulence) regions absent in W22703 might have been acquired by 8081 after separation of both strains. The novel T3SS  is a nalidixic acid-resistant  restriction mutant (Res-Mod') isolated from strain W227 [-) was used. To avoid contaminations and to validate the strain cultured for DNA isolation, strain W22703 pYV- was streaked from a glycerol stock on Yersinia selective agar plates . A single colony was used for inoculation of Luria-Bertani (LB) broth  containing 20 μg ml-1 nalidixic acid, and the culture was grown for twelve hours at a selective temperature of 15°C. When the culture had reached stationary phase, aliquots were plated in parallel on LB and Yersinia seletive agar plates. PCRs targeting W22703-specific genes tcaA, tcaC and two genes of Flg-2 were performed as a further control.ain W227 . A plasm2EDTA, pH 7.4) containing 1 μl of RNase (10 mg/ml). Polymerase chain reactions (PCR) were carried out with Taq polymerase  and the following programme: one cycle at 95°C for 2 min; 30 cycles at 95°C for 10 sec, at the appropriate annealing temperature for 30 sec, at 72°C for 45 sec to 180 sec depending on the expected fragment length; one cycle at 72°C for 10 min. 4 μl of chromosomal DNA (100 ng ml-1) was used as template for PCR amplification, and the GeneRuler DNA mix (Fermentas) served as DNA ladder. For gap closure, the following oligonucleotides were used (targeted contigs): 5'-CAACATTAAATCACGAAGG-3'/5'-TTAGTACAAATACCGATGG-3' (1804/1807); 5'-GTTTCTTTATGGGCGCG-3'/5'-TTGGCATGGAGGCCTG-3' (1891/1803); 5'-TAACCTCTAGCGCGG-3'/5'-CCCCGATAGTTCTGG-3' (1088/1891).DNA and RNA manipulation was performed according to standard procedures . To isolY. enterocolitica genome sequence belongs to the category "Annotation-Directed Improvement". The EMBL accession numbers for the sequences reported in this paper are FR718488-FR718797. The raw sequence data files are deposited in the ENA trace archive as ERP000495. The annotated sequence is available under the URL address http://pedant.gsf.de.High throughput sequencing of a shotgun library was done on the GS FLX system  using the Titanium series with approximately 20-fold coverage, and assembly were performed by Eurofins MWG GmbH, Ebersberg, Germany. According to the newly defined standards for classification of genome sequences , the Y. http://pedant.gsf.de; [http://www.ncbi.nlm.nih.gov/BLAST/) to ascribe a protein function or domain.The PEDANT software system  was use.gsf.de; . Protein.gsf.de; . Biochem.gsf.de; , BRENDA .gsf.de; , and Mic.gsf.de;  database.gsf.de; , rRNA ho.gsf.de; . AdditioY. enterocolitica 8081  was performed using the Y. enterocolitica Blast Server from the Sanger Institute (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/yersinia). The criterion applied was an 80% identity of the amino acid sequence. Incomplete proteins encoded on contig ends were considered to be present in strain W22703 if the lacking sequence could be identified on another contig. Genome sequences of Yersinia strains were obtained from the NCBI database and compared using the homepage http://www.microbesonline.org/. Protein sequence alignment was done with the ClustalW program [Comparison with the genome sequence of  program . Phyloge program  and the  program  as referYersinia genome island.ABC transporter: ATP-binding cassette transporter; DMSO: dimethylsulfoxide; LGT: lateral gene transfer; PTS: phosphotransferase system; ROD: region(s) of difference; TCS: two-partner secretion system; T3SS: type three secretion system; T2SS: type two secretion system; TCS: two component system; YGI: TMF supervised the study, and drafted the manuscript. KB analysed the annotated genome, MS closed contig gaps, and TR was responsible for automatic genome sequence analysis and annotation. All authors read and approved the final manuscript.FR accession numbers of all W22703 contigsClick here for fileW22703 gene names, locus tags, and protein accession numbers.Click here for fileMauve-type genome alignment between the reference genome of strain 8081  and draft genome of strain W22703 . Red lines indicate chromosome and contig borders. Similar regions are indicated by frames and assigned to each other by connecting lines. The degree of sequence similarity is shown within each region as similarity plot.Click here for file


Endometrial cancer is the fourth most common malignancy in women, with most cases being classified as early stage endometrioid tumors that carry a favorable prognosis. The endometrial serous histological subtype (ESC), however, while only accounting for 10% of all endometrial cancers is responsible for a disproportionate number of deaths. Unlike the estrogen-dependent, well differentiated endometrioid tumors, which are commonly associated with a younger age of onset, ESCs are estrogen-independent and tend to present at an advanced stage and in older women. Treatment for ESC entails aggressive surgery and multimodal adjuvant therapy. In this review, we describe the clinical behavior, molecular aspects, and treatment strategies for ESC. Endometrial cancer is classified into two subtypes: Type I (endometrioid histology) and Type II  . Type I Endometrial serous adenocarcinoma (ESC) typically arises in postmenopausal women. The tumor bears histological similarity to ovarian serous adenocarcinoma. ESC has a high propensity for early lymphovascular invasion, as well as intraperitoneal and extra-abdominal spread. It commonly presents at an advanced stage. The overall 5-year survival rate is about 30% for all stages and the recurrence rate after surgery is extremely high 50–80%) . Several0% . SeveUnlike endometrioid adenocarcinomas which arise directly from atypical endometrial hyperplasia, the precursor lesion for ESC, endometrial glandular dysplasia and endometrial intraepithelial carcinoma (EIC), originates in atrophic endometrium ,11. ESC et al. reported a proposed model of ESC carcinogenesis [p53 and is associated with HER2/neu overexpression in ESC [Recently, Zheng ogenesis . Accordiogenesis . Loss ofn in ESC .ESCs histologically resemble ovarian serous adenocarcinomas. They contain fibrous stroma and show a papillary pattern of proliferation. Tumor cells are pleomorphic, have prominent nuclear atypia, high N/C ratios, distinctive nucleoli, and frequent nuclear fission. Sixty percent of ESCs contain psammoma bodies. Other pathognomonic cellular features include a ballooned or hobnail-like nucleus and stromal hyalinization.K-ras, PTEN, β-catenin, which are often mutated in Type I endometrioid adenocarcinomas, are rarely altered in ESCs [p53 is often observed [p53 mutation was 81.6% [PIK3CA is common in both endometrioid adenocarcinomas and ESCs [et al. reported that amplification of CCNE1 was 30.4% in ESCs [ARID1A, which was recently discovered by an exome sequencing study of ovarian clear cell carcinomas [PPP2R1A, another gene implicated in ovarian clear cell carcinomas, is frequently mutated in ESCs [et al. reported that oncofetal protein IMP3 may be a useful diagnostic marker in the assessment of endometrial cancers and their precursor lesions, particularly when the amount available tissue materials is limited and concern of type II cancer arises [Type I and Type II endometrial carcinomas also differ on a molecular basis. ESCs are not hormone-sensitive and therefore do not express ER or PR . K-ras,  in ESCs . Insteadobserved . Exomeseas 81.6% . Amplifias 81.6% ,16. Someas 81.6% ,16,17. Iand ESCs . Very re in ESCs . The novrcinomas ,20, is r in ESCs ,22. Prev in ESCs . Recentlr arises .Historically, the surgical approach for endometrial cancer has not been histology-specific. Deep myometrial invasion is the strongest predictor of extrauterine spread of disease in endometrioid adenocarcinomas and is a factor used to triage patients for lymphadenectomy. Invasion, however, does not predict nodal or intra-abdominal spread in ESC. Therefore, many have advocated extensive staging including pelvic and para-aortic lymphadenectomy and omentectomy even in ESCs with none or minimal myometrial invasion. As in ovarian cancer, optimal cytoreduction has been shown to improve outcomes in stage III and IV ESC .The benefit of adjuvant chemotherapy in completely resected stage Ia ESC is controversial; however, the recurrence rate in these patients is 14% without adjuvant treatment . In stagMultiple phase III randomized controlled trials, conducted by the GOG, support the efficacy of systemic chemotherapy in stage III/IV endometrial carcinoma. These regimens are summarized in A randomized phase III study, JGOG2043, is presently underway in Japan. Treatment arms are AP, DP, and TC. The study has reached its quota and is closed to accrual with results pending. Ultimately the results of this study will be used to develop histology-specific treatment protocols.In Europe and the United States, adjuvant pelvic radiotherapy is frequently employed for endometrial cancer patients with lymph node metastasis ,34. WhilESC is uncommon, but accounts for a disproportionate number of endometrial cancer deaths. Histology-specific treatment algorithms are needed to improve outcomes; however, the development of standardized protocols through randomized controlled trials has been hampered by the rarity of the subtype. Evaluation of the currently available retrospective and prospective data suggests that ESCs benefit from complete cytoreduction followed by adjuvant chemotherapy, regardless of stage. It is hoped that JGOG2043 will address key questions regarding adjuvant chemotherapy. Additional trials are also required to clarify the role of radiotherapy in ESCs.


We report a case of disseminated histoplasmosis in a 37-year-old male acquired immunodeficiency syndrome patient from south India. The patient presented with high-grade fever, cough, conjunctival nodule and papulonodular hyperpigmented skin lesions. Histology of skin lesions and conjunctival nodule showed numerous intracellular Periodic Acid Schiff-positive rounded yeast cells within macrophages. Bone marrow aspirate confirmed disseminated histoplasmosis. The patient showed dramatic response after starting treatment with Amphotercin B. Histoplasma capsulatum, a dimorphic fungus. The fungus has two variants. H. capsulatum var. capsulatum, which is more ubiquitous, generally causes a subclinical infection. Symptomatic disease presents as a spectrum ranging from chronic progressive pulmonary infection to acute fulminant, or chronic, disseminated histoplasmosis. Infections due to H. capsulatum var. duboisii are restricted to West Africa. The pulmonary and disseminated forms of histoplasmosis are very common in acquired immunodeficiency syndrome (AIDS) patients. H. capsulatum infection is uncommon in India, with only occasional case reports published in the literature. We report one such rare case of disseminated histoplasmosis.Histoplasmosis refers to a granulomatous infection caused by A 37-year-old male farmer, hailing from Puttur, Karnataka, a known case of AIDS on antiretroviral treatment, presented with high-grade fever, cough with expectoration of 6 weeks duration and multiple papulonodular lesions on the face, neck and chest of 2 weeks duration. The lesions were sudden in onset and were gradually progressive.On examination, he had multiple, discrete, mildly tender, hyperpigmented, papulonodular lesions, distributed over the face , neck, c3, differential count was neutrophils 81, lymphocytes 15, monocytes 2 and eosinophils 2 and his platelet count was 1.69 lakhs. Peripheral smear showed microcytic hypochromic red blood cells, anisopoikilocytosis, polychromasia and reduced leucocyte count and toxic granules with no evidence of hemolysis. The erythrocyte sedimentation rate was 140 mm/h. The CD4 count was 150/mL. Serum lactate dehydrogenase (LDH) was 842 IU/L. The Venereal Disease Research Laboratory (VDRL) test was non-reactive. Urine and stool examinations were within normal limits. Liver and renal function tests were normal. Blood culture was negative. Chest roentgenogram showed reticulonodular pattern with hilar lymphadenopathy. Sputum examination for Mycobacterium tuberculosis was negative.On investigation, his hemoglobin was 6.5 gm/dL, total count was 2220/mmH. capsulatum within macrophages [Leishman-stained bone marrow aspirate smears showed a reactive marrow with few extracellular and intracellular rophages .H. capsulatum infection. There was minimal inflammatory response around the macrophages.Skin biopsy specimen showed dermis with aggregates of numerous macrophages containing rounded to oval organisms surrounded by a clear space within the cytoplasm. The organisms were positive for Periodic Acid Schiff (PAS) stain, confirming H. capsulatum [Histopathological examination of the conjunctival specimen showed numerous macrophage aggregates with 2–4 mm PAS-positive yeast-like forms of psulatum .The patient was started on IV Amphotericin B at a dose of 1 mg/kg/day. Antiretroviral therapy with zidovudine, lamivudine and efavirenz was continued. He became afebrile within 48 h after starting treatment. The skin lesions and the conjunctival lesion healed within 10 days. He was then switched over to oral itraconazole 200 mg twice daily and discharged with an advice to continue the drug for his lifetime.H. capsulatum var. duboisii presents a natural cutaneous infection, whereas cutaneous dissemination is rare in H. capsulatum var. capsulatum infection and is mostly associated with AIDS. There are few cases of eye involvement in histoplasmosis reported in the past but isolated conjunctival involvement is very rare and thus far, to the best of our knowledge, there is only one case reported in the literature.[Histoplasmosis, acquired by inhalation of mycelial fragments and microconidia, is most often self-limiting but can nonetheless cause potentially lethal infection in patients with pre-existing conditions. It remains a frequent cause of opportunistic infection among patients whose immune system is impaired either by pharmaceutical agents or by human immunodeficiency virus (HIV) in endemic areas. Histoplasmosis can present as pulmonary histoplasmosis or progressive disseminated histoplasmosis (PDH). terature.In HIV-infected individuals, the risk factors for the development of histoplasmosis are CD4 count of <200 cells/μL, history of exposure to chicken coops and a known positive serology for complement fixing antibodies prior to illness.4 In the 6et al. indicate that histoplasmosis has no particular predilection to East India.[Histoplasmosis is sporadically reported from India and is considered to be endemic in certain areas of East India, but recest India.10 Our paThus, in the current scenario of AIDS pandemic, a high index of suspicion has to be maintained in diagnosing rare opportunistic infections like histoplasmosis even in non-endemic parts of the world. Serum LDH can be used as a diagnostic clue. A timely diagnosis and appropriate medical intervention results in a good prognosis.