Haemophilus parasuis can be either a commensal bacterium of the porcine respiratory tract or an opportunistic pathogen causing Glässer's disease, a severe systemic disease that has led to significant economical losses in the pig industry worldwide. We determined the complete genomic sequence of H. parasuis SH0165, a highly virulent strain of serovar 5, which was isolated from a hog pen in North China. The single circular chromosome was 2,269,156 base pairs in length and contained 2,031 protein-coding genes. Together with the full spectrum of genes detected by the analysis of metabolic pathways, we confirmed that H. parasuis generates ATP via both fermentation and respiration, and possesses an intact TCA cycle for anabolism. In addition to possessing the complete pathway essential for the biosynthesis of heme, this pathogen was also found to be well-equipped with different iron acquisition systems, such as the TonB system and ABC-type transport complexes, to overcome iron limitation during infection and persistence. We identified a number of genes encoding potential virulence factors, such as type IV fimbriae and surface polysaccharides. Analysis of the genome confirmed that H. parasuis is naturally competent, as genes related to DNA uptake are present. A nine-mer DNA uptake signal sequence (ACAAGCGGT), identical to that found in Actinobacillus pleuropneumoniae and Mannheimia haemolytica, followed by similar downstream motifs, was identified in the SH0165 genome. Genomic and phylogenetic comparisons with other Pasteurellaceae species further indicated that H. parasuis was closely related to another swine pathogenic bacteria A. pleuropneumoniae. The comprehensive genetic analysis presented here provides a foundation for future research on the metabolism, natural competence and virulence of H. parasuis. Haemophilus parasuis is a strictly swine pathogen. It is a non-motile, pleomorphic NAD-dependent coccobacillus belonging to the family Pasteurellaceae of the γ-proteobacteria H. parasuis is also an opportunistic etiologic agent that can cause serious systemic disease, namely Glässer's disease, which is characterized by fibrinous polyserositis, arthritis and meningitis, or acute pneumoniae and acute septicaemia The Gram-negative bacterium H. parasuis have been classified, with apparent differences in virulence H. parasuis strains exhibiting high heterogeneity at the molecular level H. parasuis serovar 4 predominates in North American herds, whereas serovar 5 and non-typeable strains are commonly isolated in many European countries Based on the heat-stable antigens and the gel diffusion test, 15 serovars of Pasteurellaceae have been identified in H. parasuis, including lipopolysaccharide (LPS), capsular polysaccharide, fimbriae, outer membrane proteins, neuraminidase and iron acquisition systems To date, many potential virulence-associated factors investigated in the family Haemophilus are publicly available . These include H. parasuis SH0165 (GenBank accession no. CP001321), H. ducreyi 35000HP (AE017143), H. influenzae Rd KW20 (L42023), 86-028NP (CP000057), PittEE (CP000671), PittGG (CP000672), Histophilus somni 129PT (CP000436) and 2336 (CP000947). Among these, H. influenzae Rd and H. somni 129PT are both nonpathogenic strains of human and bovine origin, respectively, and H. ducreyi 35000HP causes chancroid, a human sexually transmitted disease H. parasuis SH0165, a high virulent serovar 5 strain recovered from the lung of a diseased piglet in North China. Comparisons of the genomic components between H. parasuis SH0165 and other representative species within the family Pasteurellaceae were performed in detail. Our work focused on identifying the particular genes/pathways that play a role in carbon metabolism, natural competence, virulence and host colonization of H. parasuis.Currently, the complete genome sequences of eight strains from four species within the genus H. parasuis strain SH0165 was isolated in 2001 from the lung of a diseased piglet in North China and was identified as serovar 5. This bacterial strain was cultured on tryptic soy agar (TSA) plates supplemented with 5% bovine serum and 10 µg/ml nicotinamide adenine dinucleotide (NAD). Bacterial DNA was extracted and sequenced using a shotgun sequencing strategy, as described previously H. parasuis serovar 5 strain 29755 (NZ_ABKM00000000) was aligned to the complete genome of H. parasuis strain SH0165 (CP001321) using BLASTN (minimum sequence identity of 95% and expected threshold of 1e−5). Short DNA repeats in the genome sequences were searched with a Perl script repeat_finder (http://folk.uio.no/stephanf/repeat_finder.html) and graphical representations of the patterns in sequence conservation were created using WebLogo Glimmer3 H. somni 129PT (CP000436), H. influenzae Rd (L42023), H. ducreyi 35000HP (AE017143), Actinobacillus pleuropneumoniae JL03 (CP000687), A. succinogenes 130Z (CP000746), Aggregatibacter aphrophilus NJ8700 (CP001607), A. actinomycetemcomitans D11S-1 (CP001733), Pasteurella multocida Pm70 (AE004439) and Mannheimia succiniciproducens MBEL55E (AE016827), were retrieved from the GenBank database. Protein sequences from the draft genome assemblies of H. parasuis strain 29755 were also retrieved. In this study, orthologous pairs of proteins between the CDS sets of two genomes were defined if protein identity was above 45%, alignment coverage above 70% and E_value below 1e−20. Orthologs present in the complete genomes of ten Pasteurellaceae species were retrieved. These protein sequences were aligned using the program MUSCLE 3.6 via the bootstrap test of 1,000 replicates.All protein sequences from the complete genomes of H. parasuis SH0165 contains a single, circular chromosome that is 2,269,156 base pairs (bp) in length  was shared by both strains, accounting for 88% of the total length of the SH0165 genome. There were 1,714 orthologous pairs of protein-coding genes between both H. parasuis strains of serovar 5, and H. parasuis strain SH0165 has 317 genes absent in strain 29755. It is worth noting that one third (104 genes) of these unique genes were phage-related. The H. parasuis SH0165 genome had about 198 CDSs encoding proteins involved in phage functions or phage islands, accounting for approximately 10% of the total CDSs. The majority of phage-related genes were mainly located in three phage islands designated HP-P1 (68.4 kb ranging from HAPS0536 to HAPS0638), HP-P2  and HP-P3 . It has been suggested that the integration of phage elements, as a strategy of horizontal gene transfer, play a potentially important role in genetic diversity and virulence variations in many bacteria H. parasuis SH0165 genome may be also a putative contributor to virulence and inheritance differences.n length . The chrultocida . In compH. parasuis and other organisms within the family Pasteurellaceae showed that H. parasuis is more closely related to A. pleuropneumoniae , which also colonizes the upper respiratory tract of pigs Haemophilus species, with only 1,095 orthologs found in H. influenzae  transport complexes involved in the utilization of distinct sugars, including ribose , galactose  and maltose  , which can be devoted to the uptake of other sugars, including glucose , fructose , sucrose  and mannose . These confirmed genotypes support the previously observed biochemical patterns of sugar fermentation in H. parasuisAPS0237) . H. paraH. parasuis SH0165 genome, as genes edd and eda encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxygluconate 6-phosphate aldolase, respectively, are absent. By contrast, H. somni 129PT and H. influenzae Rd both have an active Entner–Doudoroff pathway responsible for the conversion of gluconate-6-phosphate to pyruvate H. parasuis SH0165 possessed an enzyme , encoded by the rbsK2 (HAPS1688) gene, that was homologous to the fructokinase CscK (307 aa) of Escherichia coli EC3132, which can catalyze the phosphorylation of fructose to fructose-6-phosphate H. influenzae Rd, H. somni 129PT and H. ducreyi 35000HP all lack this gene. Five H. parasuis genes coding for enzymes involved in the conversion of pyruvate were identified, which are necessary for ATP production through lactic acid fermentation , and for the generation of formate  and acetate . Besides fermentation, H. parasuis may conduct the entire oxidation of glucose derivatives aerobically to release energy through an intact TCA system  cycle, as well as the pentose phosphate pathway, were identified in the SH0165 genome . The EntA system . In contH. parasuis may perform not only aerobic respiration but also anaerobic respiration, as approximately 61 genes involved in the branched electron respiration transport chains were identified in the SH0165 genome , which is used for aerobic respiration in the presence of oxygen, H. parasuis SH0165 possesses an intact napFDAGHBC (HAPS1796–1790) operon encoding a putative periplasmic nitrate reductase responsible for the reduction of terminal electron acceptor nitrates in anaerobic environments 5 genome . BesidesH. parasuis SH0165 encodes about 94 genes related to regulatory functions  for growth, but NAD is not necessary for the in vitro growth of H. somni or H. ducreyinadV, which encodes the nicotinamide phosphoribosyltransferase responsible for NAD salvage, is present in the genomes of H. somni and H. ducreyi  H. parasuis can be explained genetically H. influenzae and H. ducreyi, H. parasuis can grow well in vitro without requiring additional supplementation of iron porphyrin (X factor) H. parasuis SH0165. An entire set of genes encoding enzymes that are responsible for the conversion of L-glutamate to protoheme were identified in our study  encoding coproporphyrinogen III oxidases (EC: 1.3.99.22) were identified in H. parasuis SH0165. Despite the identity between HemN1 and HemN2 only being 23%, they both belong to the HemN_C (PF06969) family whose members catalyze the conversion of coproporphyrinogen-III to protoporphyrinogen-IX Unlike ur study . NotablyhktE (HAPS2238) may support a catalase-positive phenotype ureABC encoding urease are all absent from the H. parasuis SH0165 genome, explaining the urease-negative characteristic of this species Otherwise, the SH0165 genome encodes almost all indispensable enzymes involved in the biosynthesis of amino acids, nucleotides, fatty acids and cofactors. The catalase encoded by H. influenzae and Mannheimia haemolyticaH. parasuis SH0165 genome also contains homologous genes encoding proteins involved in the assembly of the DNA uptake machinery. Outer membrane proteins ComE (HAPS2289) and PilA (HAPS2013) play a potential role in binding liberated DNA segments at the cell surface H. parasuis SH0165 genome, including comA (HAPS2285), dprA (HAPS1573) and comM (HAPS1740) H. parasuisMany bacteria are competent, indicating that they have the ability to actively acquire extracelluar DNA from the environment Pasteurellaceae species preferentially take up short DNA sequences, called uptake signal sequences (USS), that are overrepresented in their respective genomes H. influenzae, the highly redundant USS has the sequence AAGTGCGGT, and this is also the most common uptake sequence in the genomes of H. somni, A. actinomycetemcomitans, P. multocida and M. succiniciproducensH. parasuis SH0165, the most frequent nine-mer repeat is ACAAGCGGT, being present in 523 copies, whereas only 109 copies of the H. influenzae core USS were identified in H. parasuis. Moreover, the core USS of H. parasuis is identical to the most common nine-mer repeats previously identified in A. pleuropneumoniae and M. haemolyticaH. parasuis SH0165 genome. Two AT-rich motifs were observed downstream of the H. parasuis USSs and similar patterns of sequence conservation have been found in M. haemolytica and A. pleuropneumoniae, further confirming that these three microorganisms belong to the same evolutionary subclade It has been shown that H. parasuis after in vivo passage pilABCD coding for type IV fimbriae has been identified in a number of Gram-negative pathogens in the genera of Haemophilus, Actinobacillus, Pseudomonas, Vibrio and others H. parasuis SH0165 also possesses four type IV fimbrial genes encoding the major structural unit PilA (HAPS2013) and three biogenesis proteins PilBCD (HAPS2011–2009) for mediating bacterial adherence. The proteins encoded by these CDSs share 68%, 68%, 50% and 44% identity with the ApfABCD proteins of A. pleuropneumoniae HK361, respectively H. parasuis SH0165 also encodes a protein  that shares 54% similarity with Pseudomonas aeruginosa PilF (22.4 kDa), which is involved in type IV fimbrial biogenesis and twitching motility Fimbriae (namely pili) are a common virulence factor that mediate bacterial adherence to mucosal epithelia H. parasuis SH0165  in this bacterium, are considered a virulence factor associated with disseminated intravascular coagulation and thrombosis LPSs are the primary structural and functional components of the Gram-negative bacterial outer membrane, which can be recognized and targeted by the mammalian immune system lpxC, kdsA, lpxB, kdsB, lpxH, lpxK, lpxD, lpxA, kdtA, lpxM, kdsC and lpxL) and core oligosaccharide  are present and highly conserved among the genus Haemophilus. Furthermore, a 13,964 bp genomic region that is likely associated with O-antigen biogenesis was predicted in the H. parasuis SH0165 genome. Twelve putative CDSs were identified within this potential O-antigen region located between HAPS0039 and HAPS0052, and all of these share the same transcriptional orientation (H. parasuis O-antigen gene cluster had a G+C content (32.8%) lower than that of the SH0165 genome (40.0%). Nine of these CDSs could be divided into three categories according to their functional roles in the biogenesis of O-antigen neuA1 and wbgX); glycosyltransferase genes ; and O unit processing genes wzx, wzy and wzz involved in the assembly of O-polysaccharide.All genes coding for enzymes involved in the biosynthesis of LOS were identified in the SH0165 genome . Unsurprentation . Like mawzx (HAPS0041) gene of H. parasuis encodes an O-antigen flippase (398 aa) which has 12 predicted transmembrane helices and shares 48% identity and 69% similarity with a Wzx homologue (404 aa) from the Shigella boydii O-antigen gene cluster H. parasuis is the O-antigen polymerase gene, wzy. More experimental works are still required to verify the function of H. parasuis Wzy in the O-antigen-processing process.In general, O-unit-processing enzymes  in diverse Gram-negative bacteria show considerable sequence variation but share conserved topologies in membrane spanning regions neuA1 (HAPS0040) gene of H. parasuis SH0165 shares 63% identity and 81% similarity with NeuA (229 aa) of H. ducreyi 35000 H. parasuis, exhibits about 51% similarity and 31% identity with Lst (371 aa) of Neisseria meningitidis and has a glycosyltransferase family 52 domain (PF07922) bearing alpha-2,3-sialytransferase activity. Lst encodes a CMP-NeuNAc transferase that is responsible for the linkage α-NeuNAc-(2–3)-Gal H. ducreyi LOS H. parasuis may indicate that this organism can synthesize a sialylated LPS glycoform. In addition, a putative nucleotide sugar aminotransferase WbgX encoded by HAPS0046 exhibits 85% similarity and 72% identity to the Shigella sonnei homologue that is involved in the synthesis of 2-acetamino-4-amino-2,4,6-trideoxy-D-galactose (4n-FucNAc) As is well known, sialylated oligosaccharide units in bacterial polysaccharides play a role in evading the host immune response by mimicking the glycolipid components of mammalian tissues H. parasuis SH0165 O-antigen gene cluster encodes four glycosyltransferases that sequentially add diverse sugar residues to make an expected four-sugar repeating O-unit. Besides LsgB, WcfQ (272 aa) encoded by HAPS0045 belongs to the glycosyl transferase family 2  whose members can transfer different sugar donors to receptors. H. parasuis WcfQ is also 58% similar to the putative glycosyltransferase WcfQ (268 aa) involved in the type A capsular polysaccharide biosynthesis of Bacteroides fragilis 9343 wcwK (HAPS0044) encodes an enzyme (343 aa) that shares 35% identity (53% similarity) with a capsular polysaccharide phosphotransferase (335 aa) found in Streptococcus pneumoniaePlesiomonas shigelloides O17 O-antigen synthesis The H. parasuis SH0165 O-antigen chain cluster are homologous to the proteins WbfY, Wza, Wzb and Wzc encoded in the Vibrio vulnificus group 1 capsular polysaccharide operon, sharing 52%, 53%, 57% and 50% aa identity, respectively H. parasuis through the suppression subtractive hybridization of a highly virulent and a nonvirulent strain The deduced protein products  of the last four genes (HAPS0048–0051) in the H. parasuis SH0165 genome, which also has a low G+C content (28.4%), indicating that this DNA region may originate from a species other than H. parasuis. This cluster contains six putative sugar transferase genes, lbgB, rfaF2, lbgA, rfaG, wabH and lpsA, which appear to be unique to H. parasuis, with most of them being absent from other species within the genus Haemophilus , a complex cell surface glycolipid found in all Gram-negative enteric bacteria wec gene cluster in the E. coli K12 chromosome H. parasuis ECA-like related genes, wecA, wecB, wecC, rmlB, wecD, wecE, wecF, wzyE and wecG, are dispersed throughout the SH0165 chromosome  coding for a potential virulence factor, neuraminidase , was identified in the SH0165 genome and its product may function in H. parasuis colonization H. parasuis SH0165 genome contains approximately 37 genes encoding different proteases that may contribute to virulence  may be unique to H. parasuis. A clpPX  operon encodes the structural subunits of the ATP-dependent Clp protease which proteolysis-function has been reported to be pivotal to the secretion processes of Gram-negative pathogens lon (HAPS1011) encodes a cytoplasmic serine protease (800 aa) that shares 70% identity with Salmonella enterica serovar Typhimurium Lon protease (784 aa), a potential target of antimicrobial therapy A number of enzymes, including neuraminidase and proteases, may be important for the virulence of irulence . The majH. parasuis may be deficient in production of these molecules, as reflected by the lack of related biosynthetic genes H. parasuis SH0165 was found to encode at least 37 genes involved in the capture and utilization of iron from porcine transferrin, heme and heme–hemopexin  (HAPS1366–1364) and tonB2 (267 aa)–exbB2–exbD2 (HAPS2220–2222) were identified in the SH0165 genome, which are responsible for the formation of the TonB1 and TonB2 systems, respectively. Gene components of the TonB2 system have been described previously in H. parasuisA. pleuropneumoniaeH. parasuis TbpA and TbpB  have low sequence identity (28% and 42%) with H. influenzae Tbp1 and Tbp2 , respectively, but are highly homologous to the A. pleuropneumoniae TbpA and TbpB  proteins, sharing 93% and 87% identity, respectively H. parasuis and A. pleuropneumoniae preferentially bind porcine transferrin but not human transferrin negative bacteria H. parasuis seems to have various ABC transport systems involved in the uptake of extracellular iron, hemin and hemoprotein. A hemin transport system encoded by the hmuTUV operon (HAPS1572–1570) was identified in the SH0165 genome and was found to be homologous to a family of ABC transporters  that function in transporting free heme into the cytoplasm yfeABCD (HAPS1097–1100) and fbpABC (HAPS1129–1127) were found in A. actinomycetemcomitans and M. haemolytica, respectively, and their protein products make up periplasmic ABC transport complexes involved in iron acquisition H. parasuis, the periplasmic iron transport system FbpABC together with the transferrin outer membrane receptor complex TbpAB may form another iron uptake pathway, which is used by Neisseria species to transport ferric iron into the cell Besides the TonB-dependent systems, fhuCDBA operon involved in ferric hydroxamate uptake has been reported in strain Nagasaki of H. parasuis serovar 5 fhuC' (HAPS0095) and 3′ portion of fhuA' (HAPS0096), and lacking fhuDB. Genes hhdB' and hhdA' , encoding two components of a putative hemolysin export system, and cirA' (HAPS0477), encoding an iron transporter, are also likely to be non-functional due to frameshift mutations Although the intact H. parasuis strain SH0165, a highly virulent strain isolated in China, and further performed a comparative genomic analysis. Genomic and phylogenetic comparisons indicated that H. parasuis and A. pleuropneumoniae may derive from a recent common ancestor. Together with a full set of the relevant metabolic genes, we confirmed that H. parasuis generates ATP via both fermentation and respiration with an intact TCA cycle for anabolism. Furthermore, genes and operons related to bacterial virulence factors, such as surface polysaccharides, fimbriae and iron acquisition systems, were identified at the genomic level and compared with known virulence genes from other pathogens. A putative Wzy/Wzx-dependent biosynthetic pathway for O-chain polysaccharide was proposed and requires further experimental confirmation in H. parasuis. Our findings should provide a genetic foundation for future research into the mechanisms of pathogenesis of H. parasuis and will accelerate the development of safe and effective vaccines to prevent and control this severe swine disease.In this study, we reported the complete genome sequence of Table S1Genes encoding enzymes involved in central metabolism of H. parasuis SH0165 and orthologs present in genomes of five representative species within Pasteurellaceae.(XLS)Click here for additional data file.Table S2Genes coding for proteins related to regulatory functions of strain SH0165.(XLS)Click here for additional data file.Table S3Heme biosynthetic enzymes encoded in the genome of H. parasuis SH0165 and orthologs from five representative Pasteurellaceae genomes.(DOC)Click here for additional data file.Table S4Genes encoding enzymes with a role in surface polysaccharides of strain SH0165 and orthologs present in genomes of three representative Haemophilus spp.(DOC)Click here for additional data file.Table S5Putative protease-encoding genes in the H. parasuis SH0165 genome.(XLS)Click here for additional data file.Table S6Genes encoding proteins involved in iron metabolism of H. parasuis SH0165 and orthologous proteins from five representative genomes within Pasteurellaceae.(DOC)Click here for additional data file.


Staphylococcus saprophyticus is the second most frequent causative agent of community-acquired urinary tract infections (UTI), accounting for up to 20% of cases. A common feature of staphylococci is colonisation of the human skin. This involves survival against innate immune defenses including antibacterial unsaturated free fatty acids such as linoleic acid which act by disrupting bacterial cell membranes. Indeed, S. saprophyticus UTI is usually preceded by perineal skin colonisation.The Gram-positive bacterium S. saprophyticus, encoded on plasmid pSSAP2 of strain MS1146, which we termed S. saprophyticus surface protein F (SssF). The sssF gene is highly prevalent in S. saprophyticus clinical isolates and we demonstrate that the SssF protein is expressed at the cell surface. However, unlike all other characterised cell wall-anchored proteins of S. saprophyticus, we were unable to demonstrate a role for SssF in adhesion. SssF shares moderate sequence identity to a surface protein of Staphylococcus aureus (SasF) recently shown to be an important mediator of linoleic acid resistance. Using a heterologous complementation approach in a S. aureus sasF null genetic background, we demonstrate that SssF is associated with resistance to linoleic acid. We also show that S. saprophyticus strains lacking sssF are more sensitive to linoleic acid than those that possess it. Every staphylococcal genome sequenced to date encodes SssF and SasF homologues. Proteins in this family share similar predicted secondary structures consisting almost exclusively of α-helices in a probable coiled-coil formation.In this study we identified a previously undescribed 73.5 kDa cell wall-anchored protein of S. saprophyticus that contributes to resistance against the antibacterial effects of linoleic acid. SssF is a member of a protein family widely disseminated throughout the staphylococci.Our data indicate that SssF is a newly described and highly prevalent surface-localised protein of  Staphylococcus saprophyticus, a coagulase-negative staphylococcus, is the second most common causative agent of community-acquired urinary tract infection after Escherichia coli . When the sssF gene from S. saprophyticus MS1146 was introduced into S. aureus SH1000sasF, resistance to linoleic acid was also restored, demonstrating that SssF contributes to the survival of S. aureus in the presence of linoleic acid.Survival assays were carried out with a t Figure . In agreS. saprophyticus is a major cause of community-acquired UTI in young women. Knowledge of the virulence mechanisms of S. saprophyticus has advanced in recent years, particularly with the acquisition and analysis of whole genome sequence data. The majority of acknowledged virulence factors of S. saprophyticus are proteins tethered to the cell surface, which with the exception of the Ssp lipase [S. saprophyticus that we have termed SssF - the sixth surface protein described for this species.p lipase , are allp lipase ; UafA adp lipase ; SdrI bip lipase ,31 and Up lipase . Here wesssF gene was identified in the sequence of the pSSAP2 plasmid of S. saprophyticus MS1146 due to the presence of the canonical LPXTG sortase motif in the translated protein sequence. A copy of the sssF gene is also located on the pSSP1 plasmid of S. saprophyticus ATCC 15305 , an unidentified but partially characterised protein secreted by most staphylococci which detoxifies free fatty acids by esterifying them to an alcohol [S. aureus and S. epidermidis demonstrate optimal activity at pH 6.0, and have little activity at pH 7.4 [We found that the survival of  alcohol ,35. The t pH 7.4 . This ist pH 7.4 . RP-HPLCS. aureus [S. aureus reduces bacterial hydrophobicity when expressed at the cell surface under the cue of iron starvation to resist fatty acid membrane attack and also promotes fatty acid resistance of S. aureus in a volunteer human skin survival model [S. saprophyticus, and this corresponds with matching data for SasF and S. aureus [There are precedents for bacterial surface structures that provide protection against bactericidal free fatty acids. Gram-positive bacterial cell wall teichoic acids provide protection against free fatty acid mediated killing of . aureus . The Isdal model . Our stu. aureus .Streptococcus pyogenes and Bifidobacterium breve as fatty acid hydratases [No conserved motifs that might predict the functional residues of SssF-like proteins were identified. The observation that the SssF-like proteins are structurally related to myosin is noteworthy, especially in light of the recent characterisation of myosin cross-reactive antigens of dratases ,41. Thesdratases  and mammdratases  lipid-biS. saprophyticus is a uropathogen, but SssF is unlikely to have evolved to facilitate survival in the urinary tract. A common trait of staphylococci is skin colonisation. Staphylocidal free fatty acids  are present on human skin [S. saprophyticus in the events preceding urethral entry in community-acquired UTI - colonisation of perineal and periurethral tissue. This would account for the absence of SssF involvement in the mouse model of UTI, in which the inocula are delivered directly into the bladder.man skin  and are man skin . Furtherman skin , and is man skin . SssF masssF on a plasmid in both sequenced S. saprophyticus strains is intriguing, particularly as every other staphylococcal SssF-like protein is chromosomally encoded. It has been observed that many genes that are located on plasmids encode for traits which have extracellular functions [sssF falls into this category. Furthermore, plasmid genes have often been noted to confer selective advantage to the bacteria in some environmental niches but not others [sssF-like gene is known to commensally inhabit the skin, and this can be considered their main niche. S. saprophyticus, on the other hand, primarily resides in the genitourinary and gastrointestinal tracts [S. saprophyticus, sssF has been retained as an accessory gene required for survival on the skin during non-UTI periods. Nonetheless, it may still be the case that sssF is found on the chromosome of some S. saprophyticus strains.The location of unctions , and ssst others . Every pl tracts ,20. It iS. saprophyticus. We present here evidence that the S. saprophyticus SssF protein has a role in the protection against free fatty acid mediated killing, and that it is a member of a newly identified protein family broadly distributed throughout the Staphylococcus genus.SssF represents the fourth LPXTG motif-containing protein described in S. saprophyticus isolate collection used in this study is as previously described [S. saprophyticus ATCC 15305 was described previously [-1) as required. E. coli strains were cultivated in/on Luria-Bertani (LB) broth/agar supplemented with ampicillin (100 μg ml-1) as required.The bacterial strains and plasmids used in this study are listed in Table escribed . In addieviously . StaphylsssF (primers 1127 and 1128) were performed using Taq DNA polymerase (NEB) under the following conditions: 2 min at 94°C, 25 cycles of 15 s at 94°C, 30 s at 55°C, 20 s at 72°C, 1 cycle of 3 min at 72°C, 4°C hold. Primers were synthesised by Sigma and are listed in Table sssF gene was performed using Phusion Hot Start DNA Polymerase (Finnzymes).Genomic and plasmid DNA were isolated as previously described . PCR asssssF gene was identified in plasmid pSSAP2 of S. saprophyticus MS1146. The final pSSAP2 sequence was finished to Q40 standard with an average Sanger read depth of ~23 × coverage, which corresponds to an estimated number of four pSSAP2 plasmid copies per cell, based on the observed chromosomal read coverage (data not shown). Annotation of plasmid pSSAP2 was carried out manually using Artemis [S. saprophyticus plasmid pSSAP2 is available from the GenBank/EMBL/DDBJ database under accession number HE616681. The multiple alignment . The S. aureus SH1000 sasF gene was also interrupted with the TargeTron system as above, using primers 2065-2067 (Table S. aureus strain (RN4220), then electroporated into S. aureus SH1000 and induced to create the sasF mutant (SH1000sasF). For complementation of the S. saprophyticus MS1146 sssF mutation, the sssF gene was initially amplified from S. saprophyticus MS1146  were performed as described previously , using 1S. aureus linoleic acid survival assays were performed essentially as described by Kenny et al. [t test. S. saprophyticus survival assays were performed similarly, but with agar plates containing 5 mM linoleic acid, supplemented with 0.85 M NaCl.y et al. . BrieflySecondary structure and three-dimensional fold predictions were performed using PSI-PRED  and PhyrsssF gene, participated in the design of the study, performed sequence analysis, performed the preliminary SssF phenotypic experiments, performed the PCR prevalence screening, prepared the sssF antigen for antibody production, constructed the knockout mutants, performed the Western blots, prepared the samples for electron microscopy, performed the survival assays, and was the principal writer of the manuscript. TS performed the subcloning and transformations of S. saprophyticus and S. carnosus for the complementation of the S. saprophyticus MS1146 sssF mutant, and assisted in editing the manuscript. NLBZ prepared Figure sssF prevalence data and assisted in editing the manuscript. SAB co-directed the research and assisted in writing and editing the manuscript. MAS directed the research and assisted in writing and editing the manuscript. All authors read and approved the final manuscript.NPK identified the Table S1. Predicted protein-coding genes of pSSAP2.Click here for fileFigure S1. ClustalW alignment of the C-terminal 402 amino acid residues of S. saprophyticus MS1146 SssF protein (61% of entire sequence) with corresponding sequence from other staphylococcal SssF-like proteins, showing clusters of amino acid conservation. Only one representative protein from each species is shown. Sequences are sorted (in descending order) by similarity to S. saprophyticus MS1146 SssF sequence, which ranges from 31.1% (S. pseudintermedius HKU10-03) to 48.5% (S. carnosus TM300). Jalview was used to colour-code the alignment by percentage identity. The C-terminal sortase anchor motifs are indicated by a red box. GenBank accessions for the SssF-like proteins are as follows: S. carnosus TM300, CAL29334; S. capitis SK14, EEE48467; S. caprae C87, EFS16450; S. epidermidis RP62A, AAW53125; S. warneri L37603, EEQ79103; S. haemolyticus JCSC1435, BAE03665; S. hominis SK119, EEK11979; S. aureus NCTC 8325, ABD31969; S. lugdunensis HKU09-01, ADC86449; S. pseudintermedius HKU10-03, ADV06726.Click here for file


Streptomyces viridosporus strain T7A ATCC 39115, a plant biomass-degrading actinomycete. This bacterium is of special interest because of its capacity to degrade lignin, an underutilized component of plants in the context of bioenergy. It has a full complement of genes for plant biomass catabolism.We announce the availability of the genome sequence of  Streptomyces viridosporus strain T7A ATCC 39115, a soil-dwelling actinomycete  Joint Genome Institute (JGI) using a combination of Illumina (The draft genome sequence of Illumina  and 454 Illumina . The IllIllumina . The 454Illumina , or sequIllumina –19, by Phttp://img.jgi.doe.gov.The total genome size is 8,278,598 bp with a G+C content of 72.5%. Prodigal software  and the S. virdosporus or its complement of genes for plant catabolism could constitute a lignocellulose biorefinery.The genome contains numerous genes encoding homologs of enzymes that deconstruct plant biomass. COG annotation showed that 8.35% of the predicted proteins are involved in carbohydrate transport and metabolism. Genes encoding putative lignin-degrading enzymes, such as heme peroxidases, Dyp-type peroxidases, and catalases, were identified and are currently being analyzed. Pathways for the catabolism of lignin-derived aromatic compounds, such as protocatechuate, benzoate, and catechol, were also identified. Based on these findings, we anticipate that Streptomyces viridosporus has been deposited in GenBank under accession no. AJFD00000000.The genome sequence of


Oryza longistaminata, an AA genome type (2 n = 24), originates from Africa and is closely related to Asian cultivated rice (O. sativa L.). It contains various valuable traits with respect to tolerance to biotic and abiotic stress, QTLs with agronomically important traits and high ability to use nitrogen efficiently (NUE). However, only limited genomic or transcriptomic data of O. longistaminata are currently available.O. longistaminata root transcriptome using 454 pyrosequencing. One sequencing run using a normalized cDNA library from O. longistaminata roots adapted to low N conditions generated 337,830 reads, which assembled into 41,189 contigs and 30,178 singletons. By similarity search against protein databases, putative functions were assigned to over 34,510 uni-ESTs. Comparison with ESTs derived from cultivated rice collections revealed expressed genes across different plant species, however 16.7% of the O. longistaminata ESTs had not been detected as expressed in O. sativa. Additionally, 15.7% had no significant similarity to known sequences. RT-PCR and Southern blot analyses confirmed the expression of selected novel transcripts in O. longistaminata.In this study we present the first comprehensive characterization of the O. longistaminata. The generated sequence data are publicly available and will facilitate gene discovery in O. longistaminata and rice functional genomic studies. The large number of abundant of novel ESTs suggests different metabolic activity in O. longistaminata roots in comparison to O. sativa roots.Our results show that one run using a Genome Sequencer FLX from 454 Life Science/Roche generates sufficient genomic information for adequate de novo assembly of a large number of transcripts in a wild rice species,  Oryza sativa L.) is a staple food crop for about half of the world's population. In 2008, the total rice-harvested area and rough rice yield in the world were 155.7 million hectares and 661.8 million tons, respectively [International Rice Research Institute (IRRI) 2009]. However, the productivity of rice is severely affected by soil nitrogen nutrient deficiency worldwide. Commercially available urea fertilizer is the most widely used resource to meet a rice crop's nitrogen requirement, of which one third is lost through emission of greenhouse gasses and leaching, causing adverse environmental impacts [Rice  and 22 wild species with diverse ecological adaptation. These species are categorized into 10 recognizable genome types  [Oryza longistaminata chev. , broadly distributed throughout tropical Africa, is a perennial species with characteristics of long anthers, self-incompatibility, allogamy, strong rhizomes and high biomass production on poor soils. In spite of its overall inferior appearance, O. longistaminata has furnished genes for developing perennial rice [O. longistaminata in public databases, and no genome sequence is available.The genus nd HHKK) ,10. Wildnd HHKK) -14. Oryzial rice ,16 and fial rice . To makeO. sativa ssp. japonica cv. Nipponbare and the draft genome sequence of the O. sativa ssp. indica cv. 93-11 through a map-based sequencing strategy and through a whole-genome shotgun sequencing approach, respectively [O. minuta (BBCC genome) and 1,888 leaf FLcDNAs from the O. rufipogon (AA genome) [Sequencing and analysis of expressed sequence tags (ESTs) has become a primary strategy for functional genomic studies in plants including novel gene discovery, gene expression profiling, microarray and molecular marker development, and accurate genome annotation. After completing the full genome sequence of ectively ,19, much genome) ,21. EspeO. longistaminata was undertaken to provide an overview of O. longistaminata root transcriptome and thus a molecular basis for the identification of useful genes. As newly developed massively parallel 454 pyrosequencing allows rapid generation of sequence data and deep sequencing coverage with reducing labour and cost [O. longistaminata by 454 GS-FLX pyrosequencing technology. This led to the discovery of a huge amount of novel ESTs which will facilitate gene mining and provide a basis for comparative studies within the genus Oryza.Therefore, a comprehensive survey of ESTs in roots of and cost -24, we hO. longistaminata plants were clonally propagated and were adapted to low-nitrogen conditions in unfertilized soil for several months. Mature plants with high biomass production  to sequences of bacteria, fungi or metazoa, resulting in a total of 71,367 processed unique sequences. The length of contigs varied from 101 bp to 2082 bp with an average of 299 bp, and that of singlets ranged from 101 bp to 393 bp with an average of 215 bp  with average sequence length of 258 bp  from root cDNAs of O. longistaminata consensuses after assembly was shown in Table The majority of reads was in length of 201-300 bp (95% out of raw reads), which was consistent with the 454 GS-FLX sequencing capacity. The size distribution of The sequence data obtained were in a similar range as for other plant EST sequencing projects using this technology ,27, howeO. longistaminata to chromosomal and expressed sequences of O. sativa revealed a large set of two types of novel ESTs, those previously not found to be expressed by rice, and those not even detected in the rice genome. The ESTs were aligned to genomic sequences of two O. sativa varieties, japonica - type Nipponbare http://rgp.dna.affrc.go.jp/IRGSP/ and indica -type 93-11 http://rice.genomics.org.cn/rice/index2.jsp, by using the BLASTN program with an E-value cut-off of e-5. In total, 60,155 (84.3%) out of 71,367 sequence tags were anchored in rice genome. These sequences mapped on all the 12 rice chromosomes  cDNA collection, the indica cDNA database http://www.ncgr.ac.cn/ricd/, and the NCBI rice EST database. 83.3% matched to O. sativa genes found to be expressed previously.In order to assess how many O. sativa but not to predicted genes, 23% with genes of predicted functions, and 10% with genes encoding hypothetical proteins. This was also reflected in the lack of functional assignments (see below), as after in silico translation only for a small fraction (777) of these ESTs could be assigned according to Gene Ontology (GO). This emphasizes the power of the next generation sequencing approach to detect novel transcripts or even novel genes. As the O. sativa genome may still contain regions that are not fully annotated, our ESTs might indicate as yet unpredicted genes or UTRs that might be functional in O. sativa as well. On the other hand, O. longistaminata might express a special set of genes in comparison to O. sativa, due the particular conditions - being adapted to low availability of external nitrogen sources-, or due to the interspecies differences in expression.A large amount of ESTs  had previously not been detected as expressed. For most of them, we did not find homologies to predicted gene models: Inspection of the 30 longest ESTs showed that 67% shared sequence similarity with O. sativa chromosomes by homology search against genomic sequences. Among them, 250 matched the publicly available O. sativa mRNAs or ESTs. The remaining 10,962 sequence tags showed no significant sequence identity (cut- off e-5) with any rice genomic or expressed sequences in public database. Among these, only a very small number (740) had a significant hit in NCBI non-redundant (NR) nucleotide database or ESTs database. The remaining 10,222 ESTs may therefore represent novel genetic material present in O. longistaminata and other root-residing eukaryotes.As another category of novel ESTs, in total, 11,212 (15.7%) of 71,367 unique EST sequences could not be mapped to the -5. A majority (51.6%) of the O. longistaminata sequences did not match any known protein sequences. Most of the 10,962 novel sequence tags (15.4%) fell into this category. This can partly be attributed to the short length of most of these uni-ESTs, or a large fraction of the ESTs might represent untranslated regions. Mapping those uni-ETSs to rice gene models supported this assumption. https://www.gabipd.org/database/cgi-bin/GreenCards.pl.cgi.The consensus sequences were annotated for sequence similarities using the BLASTX translated sequence comparison against the NCBI non-redundant (NR) protein database. Among the 71,367 contigs and singlets, 34,510 (48.4%) had at least a significant alignment to exisiting gene models in the NR database at an E-value cut-off of ehttp://www.geneontology.org). Only to 25,448 O. longistaminata sequence tags GO numbers were assigned, however a broad range of GO categories was covered: the percentage distribution of GO terms is shown in Figure The unique ESTs were further classified into Molecular Function, Biological Process and Cellular Components, according to the standard Gene Ontology terms  was grown under nitrogen-limiting conditions in soil without nitrogen fertilizer in the phytotron in Bremen. The soil (from Camargue) had a low percentage of total nitrogen  and a high C/N ratio (25.5). The roots and leaves were harvested by snap-freezing in liquid nitrogen and prepared for RNA and DNA isolation, respectively. The seeds of O. longistaminata collected from Namibia were surface-sterilized [The erilized  and cultrogen 0,2% and a hThe RNA was extracted by the CTAB method described by Chang et al.  from soihttp://compbio.dfci.harvard.edu/tgi/software/ was applied to eliminate low quality sequences, poly A/T sequences, adaptor sequences. The cleaned sequences were subjected to the CAP3 program [Arabidopsis gene identifiers with the strongest BLASTx alignments to the corresponding O. longistaminata ESTs. Comparison of the distribution of cellular component, biological processes or molecular function obtained using GO annotation was done using the GOSlim program http://www.geneontology.org.Synthesis of cDNA and normalization for pyrosequencing was carried out by MWG  using RNA from roots of soil-grown plants without N-fertilizer. High quality polyA+ RNA was isolated from total RNA as template for first- and second-strand synthesis. By using a semirandom priming approach for both strands, an even shotgun-like distribution of cDNA fragments was achieved. The fragments were size-fractionated and normalised by denaturing and re-association. Approximately 10 μg of cDNAs were sheared by nebulisation and sequenced on a 454 GS-FLX pyrosequencing platform. A total of 337,830 raw reads were obtained. SeqClean software  program  for clushttp://www.gabipd.org/ under the accession Xa21_454, and at GenBank (dbEST acc. No. HS317469 - HS388835).The sequences are available at O. longistaminata, randomly selected sequences were used for expression analysis by RT-PCR (root RNA) and Southern blot (leaf DNA) analyses. About 100 ng total RNA was use to synthesize the first-strand cDNA by SuperScript™ II Reverse Transcriptase  with Oligo(dT)12-18 primers. Specific primer pairs for cDNA amplification were designed by Primer3 software [To validate the presence of novel ESTs detected by pyrosequencing in software  accordinsoftware .O. longistaminata root RNA isolation, RT-PCR and participated in sequence analyses, and drafted the manuscript; LH carried out Southern analysis and participated in sequence analyses; TH prepared the plant materials used in this study and co-designed the experiment; BR designed the experiment and assisted in the manuscript preparation. All authors read and approved the final manuscript.HY carried out O. longistaminata root ESTs.Source of Click here for fileCharacterization of O. longistaminata ESTs before and after assembly.Click here for fileSummary of component reads per assembly.Click here for fileConsensus sequences specifically matched to indica or japonica genome sequences.Click here for fileO. longistaminata ESTs analyzed by RT-PCR from cDNA of pot-grown O. longistaminata rice roots.Click here for fileAssembled pyrosequencing ESTs. The data represent all the assembled pyrosequencing ESTs.Click here for file


Trypanosoma brucei, like many protistan blood parasites, is crucial for mediating host-parasite interactions and is instrumental to the initiation, maintenance and severity of infection. Previous comparisons with the related trypanosomatid parasites T. cruzi and Leishmania major suggest that the cell-surface proteome of T. brucei is largely taxon-specific. Here we compare genes predicted to encode cell surface proteins of T. brucei with those from two related African trypanosomes, T. congolense and T. vivax. We created a cell surface phylome (CSP) by estimating phylogenies for 79 gene families with putative surface functions to understand the more recent evolution of African trypanosome surface architecture. Our findings demonstrate that the transferrin receptor genes essential for bloodstream survival in T. brucei are conserved in T. congolense but absent from T. vivax and include an expanded gene family of insect stage-specific surface glycoproteins that includes many currently uncharacterized genes. We also identify species-specific features and innovations and confirm that these include most expression site-associated genes (ESAGs) in T. brucei, which are absent from T. congolense and T. vivax. The CSP presents the first global picture of the origins and dynamics of cell surface architecture in African trypanosomes, representing the principal differences in genomic repertoire between African trypanosome species and provides a basis from which to explore the developmental and pathological differences in surface architectures. All data can be accessed at: http://www.genedb.org/Page/trypanosoma_surface_phylome.The cell surface of  Trypanosoma brucei) is a single-celled, vector-borne parasite that causes Human African Trypanosomiasis (or ‘sleeping sickness’) throughout sub-Saharan Africa and, along with related species T. congolense and T. vivax, a similar disease in wild and domestic animals. Together, the African trypanosomes have significant effects on human and animal health and associated costs for socio-economic development in Africa. Genes expressed on the trypanosome cell surface are instrumental in causing disease and sustaining infection by resisting the host immune system. Here we compare repertoires of genes with predicted cell-surface expression in T. brucei, T. congolense and T. vivax and estimate the phylogeny of each predicted cell-surface gene family. This ‘cell-surface phylome’ (CSP) provides a detailed analysis of species-specific gene families and of gene gain and loss in shared families, aiding the identification of surface proteins that may mediate specific aspects of pathogenesis and disease progression. Overall, the CSP suggests that each trypanosome species has modified its surface proteome uniquely, indicating that T. brucei, T. congolense and T. vivax have subtly distinct mechanisms for interacting with both vertebrate and insect hosts.The African trypanosome ( Trypanosoma spp. section Salivaria) are unicellular hemoparasites of vertebrates. They are transmitted by Tsetse flies (Glossina spp.) and cause endemic disease throughout sub-Saharan Africa. African trypanosomes include T. brucei which causes Human African Trypanosomiasis (‘sleeping sickness’) and, along with two related species T. congolense and T. vivax, a similar disease in domestic and wild animals (‘nagana’). Although the incidence of human disease has recently declined African trypanosomes ; serial replacement of VSG (i.e. antigenic variation) is a means of immune evasion and results in chronic infection VSG gene families VSG expression sites (ES) Expression Site-Associated Genes  but these are unrelated to VSG Leishmania promastigotes have a largely non-proteinaceous, lipophosphoglycan-based surface coat The first genomic comparisons between T. brucei, T. cruzi and L. major have physiological similarities associated with shared ancestry, the cell-surface architectures are highly divergent, reflecting the evolution of specific mechanisms for immune evasion and survival by each parasite T. brucei, T. cruzi and L. major diverged long ago; so surface features that appear exclusive when their genomes are compared are not necessarily species-specific, or diagnostic of the diseases they cause. In particular, it remains to be determined if the T. brucei-specific surface features identified from these initial comparisons are truly species- or disease-specific, or general features of all African trypanosomes. Comparisons between more closely related species are essential to resolving this issue.Hence, while T. congolense, the closest known relative of T. brucei, and T. vivax, a more distantly related species, and described the evolution of VSG genes in African trypanosomes T. vivax can cause hyperacute hemorrhagic disease in cattle typically with much higher mortality than other species T. brucei and T. congolense infect the midgut but then migrate to the salivary glands and proboscis respectively prior to transmission to the vertebrate. In contrast, T. vivax avoids the insect midgut, a feature that seems to facilitate wholly mechanical transmission and its colonization of Tsetse-free areas T. brucei has evolved human infectivity, probably on at least two occasions in east (T. b. rhodesiense) and west Africa (T. b. gambiense) respectively We recently reported the draft genome sequences for Cell surface-expressed gene families encode abundant proteins at the forefront of host-parasite interactions Trypanosoma brucei TREU927 T. congolense IL3000 and T. vivax Y486 T. cruzi CL Brener The African trypanosome cell surface phylome is a collection of phylogenies for gene families with predicted cell surface expression. The approach is summarized in T. brucei genes with cell surface motifs,  were extracted from the T. brucei 927 genome sequence. Genes annotated as ‘unlikely’ or with fewer than 100 codons were removed. Homologs to each T. brucei ‘surface’ gene were identified among all T. brucei, T. congolense, T. vivax and T. cruzi predicted genes using wuBLAST T. brucei genes (i.e. widespread gene families), the BLAST exercise was repeated for T. congolense and T. vivax genes to identify cases absent in T. brucei. Signal peptides were predicted using SignalP trans-membrane helices were predicted using TMHMM T. congolense genome that were separately assembled), of genes with known expression in mitochondrial, lysosomal or other internal membranes, and by combining families with overlapping homology. Surface-expressed families may have been omitted because they possess signal peptides, GPI anchors, or trans-membrane helices that cannot be reliably recognized by current methods, or because their 5′ or 3′ regions are mis-specified. Equally, spurious recognition of these domains in hypothetical proteins (mostly T. vivax families) cannot be excluded. Each family is given a ‘Fam’ number (0–81) as described in All T. bruceiT. congolenseT. vivaxT. vivax genome using SMALT Given that most species-specific genes are putative and encode hypothetical proteins, evidence in support of their coding status was gathered from three sources: i) transcriptomic studies of sk) and non-synonymous substitutions (ak) per site were calculated for each alignment using KaKs Calculator 2.0 Translated nucleotide sequences for each family were aligned in ClustalW T. cruzi sequences, or otherwise mid-point rooted. VSG phylogenies were estimated using alignments of selected, full-length sequences representative of global diversity under different conditions, as described previously Bayesian phylogenies were estimated using MrBayes v3.2.1 T. brucei, T. congolense], T. vivax], T. cruzi]) using NOTUNG 2.6 ρ, was calculated from the ratio of speciation duplications (i.e. nodes supporting orthologs in daughter species) to unilateral duplications , adjusted for gene family size. ρ reflects the degree of gene family turnover (combined incidence of gene gain and loss); high values of ρ indicate a phylogeny with minimal turnover, in which most lineages are represented by orthologs in all species. Low values indicate a phylogeny with high turnover, in which ancestral genes are frequently lost and replaced by novel duplicates, resulting in clades of species-specific in-paralogs and minimal orthology.The CSP contains phylogenies of gene families drawn from multiple species. We can infer historical gene duplications and losses from comparison of gene family phylogenies with the overlying species evolution Significant differences in evolutionary rate between two lineages were examined using relative rates tests RRTs; . NucleotESAG family, the index was calculated using PhiPack ESAGs sensu stricto and of homologous sequences from non-ES loci .Phylogenetic incompatibility describes the presence of multiple phylogenetic signals within a single sequence alignment and is the historical signature of recombination. The Pair-wise Homoplasy Index (PHI) detects incompatibility between sites and is robust in the presence of rate heterogeneity in vitro cultured bloodstream-stage and procyclic forms; in vivo cultured short stumpy bloodstream-stage; and in vivo cultured T. brucei bloodstream-stage). T. brucei Rab11 was used as a control to determine relative quantities of mRNA. The relative abundance of specific RNA was subsequently determined.To determine mRNA expression levels for a single Fam50 family member (Tb927.7.380), quantitative real-time polymerase chain reaction (qRT-PCR) was carried out on total RNA extracted using RNeasy Mini Kit (QIAGEN). cDNA was generated using SuperScript II reverse transcriptase according to the manufacturer's instructions. qRT-PCR was carried out using three different isolated mRNA samples from four life-cycle stages (T. brucei single marker bloodstream line cells were cultured in HMI-9 medium as described previously Fam1 (i.e. Tb927.6.1310) and Fam50 (i.e. Tb927.7.380) genes were synthesized by Eurogentec. Tb927.6.1310 is the most divergent of all Fam1 gene copies, so it was selected for the benefit of targeting a single copy gene in localization experiments. Tb927.7.380 is also one of five tandem copies, and was selected because it was expressed to the greatest level for all paralogs in qPCR analyses. http://www.genedb.org/Page/trypanosoma_surface_phylome.We estimated phylogenies using Maximum Likelihood and Bayesian methods for 79 gene families in African trypanosomes with known or predicted cell-surface location. This cell-surface phylome describes how these families have diversified during the evolution of African trypanosomes, and also identifies species-specific gene families, many of which remain uncharacterized. Taken together, the CSP shows that the cell-surface architecture evolved in the common ancestor of all African trypanosomatids, and has subsequently experienced subtle changes in individual lineages, suggesting the adaptation of their common inheritance. The CSP is described in a Venn diagram  and in Ttrans-sialidases) T. brucei; only 8/45 species-specific families  are characterized to some extent  and function is only well known for two (VSG and ESAG6/7). Naturally, many trypanosome cell-surface proteins perform basic functions that are constrained by selection, resulting in small species differences . However, a widespread family is not necessarily unchanged, and the phylogenies of several conserved families involved in host-parasite interaction indicate surface proteome differences between species that could have functional implications.The conserved elements of the CSP, at the centre of T. vivax, whole lineages have been lost, and on multiple occasions; for example among trans-sialidase genes (see Fam47 CSP page), there are no T. vivax orthologs to basal-branching lineages represented in T. brucei by Tb927.5.440 and Tb927.2.5280, which are otherwise widespread. Similarly, there are only three Major Facilitator Superfamily (MFS) transporters loci in T. vivax compared with six in T. brucei (see Fam58 CSP page), and no orthologs to the Proteins Associated with Differentiation (PAD) genes, one of which encodes a carboxylate transporter implicated in differentiation from vertebrate to insect life stages in T. brucei  is present in T. brucei, T. congolense and the outgroup T. cruzi, but is absent from T. vivax; . This coincides with the evolution of 11 MSP-C genes in T. vivax, a gene that is single-copy in all other species . However, while T. brucei has elaborated its nucleoside transporter lineage, producing four species-specific loci from a single-copy ancestral locus (probably Tb09.160.5480), T. congolense instead diversified its nucleobase transporter lineage, with 18 gene copies compared with three in T. brucei and five in T. vivax. This is not simply a difference in gene dosage, or an artifact of sequence assembly, since seven of these T. congolense-specific transporters (e.g. TcIL3000.0.12740) have a highly derived predicted protein sequence, lacking ∼130 amino acids from the 3′ end and displaying only 39% amino acid identity with the T. congolense chromosome 11 isoform (54% similarity), and which itself displays 54% identity and 66% similarity with its T. brucei ortholog. Therefore, these genes are predicted to encode proteins with signal peptides and eight trans-membrane helices, but lack the canonical C-terminus of the conserved nucleobase transporter including its GPI-anchor signal.The surface functional repertoire also diverges through gene gain, for example among Fam61 genes (nucleoside/nucleobase transporters), required to scavenge host purines and are functionally differentiated with respect to both parasite life stage and substrate ρ for each family, the ratio of orthology (DIV) to paralogy (DUP), corrected for gene family size, and where DIV is the incidence of gene divergence through speciation and DUP is the incidence of gene duplication, inferred through phylogenetic reconciliation (ρ = 0.67) and Fam65 (ρ = 0.73) possess high ρ values, indicating that most loci are retained in all species; for example, across 22 ABC transporter loci there are no unilateral gene duplications and only 7 gene losses . While these losses probably have functionally consequence, Fam56 and similar examples have a relatively constant gene complement.The combined effect of gene gains and losses, i.e. gene family turnover, is reflected in the topology of phylogenies. Typically, gene families predate contemporary genomes, and orthologs in each species of each ancestral gene form a clade in the phylogeny. Examples of this familiar pattern in trypanosomes naturally include structural or metabolic gene families displaying little innovation iliation . Familieρ<0.05, indicating that gene copies cluster more by species than by locus, i.e. recent paralogy rather than ancient orthology. Fam54 , Fam58  and Fam61  all display low ρ values due to T. brucei-specific expansions , which occurs against a general background of conservation. This cannot be said for phylogenies for other families, e.g. Fam46 (ρ = 0.013), Fam49 (ρ = 0.002), Fam50 (ρ = 0.003), Fam67 (ρ = 0.003), and Fam77 (ρ = 0.002), which consist of species-specific clades of highly similar, tandem duplicates, at one or a few conserved loci. Fam47  and Fam51  provide examples intermediate between the first and second patterns, with T. brucei and T. congolense possessing orthologs to conserved loci, while all T. vivax genes are monophyletic and hence lack orthology with other species.Conversely, many familiar cell surface components have T. bruceiT. congolense  are significantly longer than in T. brucei  or T. vivax . T. congolense cysteine peptidase B includes structural variants with distinct catalytic functionality T. brucei. Gene family diversification is a product of both gene duplication and sequence divergence T. brucei and is required for iron uptake ESAG6 and 7  at a strand-switch region on chromosome 7, probably representing a secondary transposition from an ES, and the Procyclin-Associated Genes , which are adjacent to the procyclin loci ESAG6 and 7 and the PAGs are homologous to the a-type VSGs  that are homologous to ESAG6/7, plus 31 genes (in Fam14) whose closest sequence match is to PAGs in T. brucei. We refer to both Fam14 and Fam15 as TFR-like genes. TFR-like genes and shows that the T. congolense genes are paraphyletic, that is, there are two clades (Fam14 and 15) each more closely related to sequences in T. brucei (PAG and ESAG6/7 respectively) than to each other. Given the homology between a-VSG and TFR genes, this shows that Fam14 and 15 are not a-VSG . These results suggest that an orthologous TFR is present in T. brucei and T. congolense but not T. vivax.The essential difference between I anchor . Second,T. brucei and T. congolense possess a third, highly abundant major surface glycoprotein expressed during the insect stage. These are known as BruceiAlanine-Rich Protein  in the CSP. We identified conserved sequence regions that unite these familiar families with other insect stage-specific genes and several hypothetical or uncharacterized genes  is the sister clade to BARP/GARP and may be present in T. vivax also (TvY486_11149440). The CongolenseEpimastigote-Specific Protein (CESP) gene family is expressed in T. congolense epimastigotes only, where it may have a role in adhesion to host surfaces CESP has a sister clade in T. brucei comprising a tandem gene array on chromosome 8 (marked ‘ii); notably, these genes may be preferentially expressed in insect salivary glands T. brucei epimastigotes. An ortholog to CESP may be present in T. vivax (i.e. TvY486_0016400), although the position of this gene is not robust.ed genes . The regGARP and CESP, the CSP identified two related subfamilies encoding hypothetical proteins, (marked ‘iii’ and ‘iv’), which comprise subtelomeric tandem gene arrays. Analysis of stage-defined T. congolense EST  is also preferentially expressed in procyclic cells based on transcriptome data iv’ comprises sequences on chromosome 7 in T. brucei (i.e. Tb927.7.360) and a single-copy ortholog in T. congolense (i.e. TcIL3000.0.02370). In transcriptomic studies of T. brucei, expression data for these genes was weak and inconclusive T. congolense indicated 13-fold higher expression of TcIL3000.0.02370 in epimastigotes over procyclics iv’ genes are expressed during the insect-to-vertebrate transition.In addition to ense EST  proteomitage see . Accordiiv’ in T. brucei, Tb927.7.380 was haemagglutinin (HA) epitope-tagged at the N-terminus  and expressed ectopically using a pDEX-577 inducible-expression vector. Protein expression was confirmed by Western blot  are present in T. congolense and we have previously argued that ESAG2 belongs to a widespread lineage most closely related to b-type VSG in T. congolenseT. congolense and T. vivax genomes contain homologous sequences to 9 of 12 ESAG families, while ESAG9 may have homologs in T. cruziESAG family shown in their individual CSP pages. First, ESAGs from multiple T. brucei strains are monophyletic and therefore, have a single origin; and second, with the exception of ESAG6/7, the sister clades to ESAGs are not orthologs in other species but chromosomal-internal genes in T. brucei. We interpret this as evidence for origins post-speciation, i.e. ESAGs are T. brucei-specific. Examining these closest relatives outside of the expression sites provides some indication of the origins of ESAGs, as demonstrated by Fam51, i.e. ESAG4 and the adenylate cyclases.. brucei . OrtholoTrans-membrane adenylate cyclases are conserved across Trypanosomatids T. bruceiESAG4 is one lineage expressed specifically in the bloodstream stage, and instrumental in inhibiting host innate immunity T. congolense and T. vivax genome sequences include 34 and 24 adenylate cyclase genes respectively. The adenylate cyclase phylogeny , which is syntenic. Relative rates tests show that the substitution rate of ESAG4 has accelerated significantly compared with Tb11.01.8820 . Furthermore, of 54 sites where Tb11.01.8820 and TcIL3000.11.16970 are conserved, but ESAG4 is derived (i.e. unambiguous ESAG4 apomorphies), 41 occur in the intracellular domain. While the adenylate cyclase catalytic domain is intracellular, the evolution of ESAG4 has not altered the 8 residues identified as important for catalytic function ESAG4 represents a T. brucei-specific expansion of adenylate cyclase genes, most likely initiated through the transposition of a conserved locus to the ES, and coinciding with derivation of the protein structures associated with signal transduction within the cell but not catalysis.hylogeny  shows thequences  shows thESAGs in their CSP pages suggests that, like ESAG4 and Tb11.01.8820, ESAGs themselves are T. brucei-specific but descended from conserved genes, typically members of multi-copy families with subtelomeric distributions in several species. For example, ESAG2 and ESAG6/7 were, as previously noted, derived from VSGESAG3- and ESAG5-like loci are on T. vivax contigs containing telomeric repeats (GenBank accessions HE578915 and HE578917), but not VSG. ESAG8, although not surface-expressed, is most closely related to two leucine-rich repeat protein (LRRP) genes (i.e. Tb927.1.3670 and Tb927.3.580), that are chromosome-internal and include nuclear localization signal and RING motifs, which are diagnostic of ESAG8T. brucei-specific, they are more closely related to conserved LRRP genes, suggesting that they may be progenitors of ESAG8. Finally, on the Fam3 CSP page, a structural comparison of ESAG11 and Invariant Surface Glycoprotein (ISG) sequences indicates that ESAG11 is homologous to ISG and so perhaps a highly modified derivative of these widespread surface proteins ESAG1 and ESAG12 appear to have no homology beyond T. brucei, suggesting that they have evolved de novo within the ES.The detail presented for other ESAGs, the CSP contains various species-specific genes  genes previously identified in T. bruceiT. brucei, although similar in structure to canonical VSG in T. congolenseVSG-like genes unique to T. brucei: Fam1.Besides ic genes . T. brucT. brucei 927, Fam1 comprises a polymorphic tandem array of 5 copies (0.1–3.2% nucleotide sequence divergence) at a strand-switch region on chromosome 6 , and tagged at the N-terminus of the mature protein with an HA-9 epitope . The HA T. congolense, Fam22 is the most abundant species-specific gene family with >100 copies. Fam22 genes are distributed throughout putative subtelomeric regions and are typically situated immediately downstream of VSG. T. congolense VSG 3′UTR's are too short,  than either other species, which may be expected given that T. vivax is the natural outgroup to T. brucei and T. congolense. None have any significant similarity with known protein structures and more transcriptomic and proteomic surveys will be required to confirm that these sequence families genuinely encode T. vivax-specific proteins. However, many of these putative gene families are abundant (e.g. Fam31 and Fam34 have 38 and 34 members respectively) and transcripts corresponding to several gene families are among bloodstream-form RNA-seq data  during various life-stages, also appears to have originated in the ancestral African trypanosome.The ancestor of ESAG6/7 are thought to have evolved from a-VSG variant antigens ESAG6/7 are T. congolense Fam15 genes, which do not encode any known variant antigens VSG in T. brucei, phylogenetic analysis of all VSG-like sequences [see Fam0 CSP pages] indicates that TFR-like sequences evolved from an a-VSG-like gene, (and further differentiated into ESAG6- and PAG-like genes), in the T. brucei/T. congolense ancestor, after separation from the lineage leading to T. vivax. While there are no TFR-like sequences in T. vivax, this does not preclude an analogous transferrin receptor in this species, since there is a large and structurally diverse a-VSG-like family  = 0.114, n = 21) are much shorter than distances among the T. congolense genes , implying a recent origin for ESAG7 from ESAG6 through the deletion of its C-terminus. We consider this to reflect rapid turnover post-speciation of TFR-like genes that evolved in the ancestor, rather than independent origins, which is less parsimonious. Indeed, the same pattern of reciprocal monophyly between species is seen in other phylogenies , but it is clearly unparsimonious to suggest recent origins for these widely conserved families. Gene turnover replaces ancestral-type genes with more derived types post-speciation resulting in concerted evolution, a process exacerbated by recombination among tandem gene duplicates ESAG6/7 routinely T. congolense subtelomeres is also apparent (data not shown). Given that this molecular evolution introduces phylogenetic artefacts, the Fam15 phylogeny need not refute the most parsimonious hypothesis that a TFR protein originated in the T. brucei/T. congolense ancestor.However, if the ESAGs in T. brucei, uniquely linked to the telomeric VSG expression site, is a principal example of species-specific genomic adaptation. In some cases we can identify the likely origin of ESAG lineages among chromosome-internal loci; ESAGs 3, 4, 5 and 10 are derived from conserved loci that can be located precisely ESAGs 2 and 6/7 are derived from variant antigen genes that evolved in the T. brucei/T. congolense ancestor ESAGs 8, 9 and 11 have more remote homology to conserved subtelomeric gene families, i.e. LRRPMASPISG (see Fam3 CSP page) respectively. This suggests a scenario in which genes with existing subtelomeric distributions (except ESAG10) and cell-surface roles (except ESAG8) were progressively compartmentalized into an independently-promoted telomeric locus, perhaps to provide a more precise regulatory environment.While the essential character of the cell surface was established in the ancestral trypanosome, this common inheritance has been adapted subsequently. The evolution of T. brucei, the evolution of the ES demonstrates how novel cell-surface genes are repeatedly derived from existing major surface glycoproteins, whose abundance seems to provide a reservoir of raw material for neofunctionalization. Although ESAG functions are obscure, ESAG phylogenies suggest that they are distinct from those of conserved genes from which ESAGs evolved and indispensable on an evolutionary timescale. ESAGs from different T. brucei strains are monophyletic (except ESAG3), indicating no frequent transposition of sequences between ES and non-ES loci. ESAG-related genes at chromosome-internal loci are not observed in the ES and do not recombine with ESAGs, despite very frequent recombination among ES and non-ES copies respectively ESAGs are not essential in the short term ESAG sequences sensu stricto and the telomeric ES has been preserved by selection over the long term, suggesting that ESAG and ESAG-like functions are distinct and non-redundant.Like the origin of Fam1 in ESAGs in T. brucei, but significant phenotypic differences, such as life cycle variation, could be due to relatively subtle differences in conserved gene families such as Fam50. Given that BARP, GARP and CESP are preferentially expressed in the epimastigote stage T. congolense and T. brucei indicate that subfamilies ‘iii’ and ‘iv’ are associated with insect mid-gut and salivary gland stages respectively T. brucei and T. congolense, T. vivax remains within the insect mouthparts after feeding; this could reflect the basal-branching position of T. vivax in the species phylogeny (i.e. T. vivax is plesiomorphic and never evolved a mid-gut stage) or secondary loss . T. vivax also has a relatively small Fam50 repertoire, lacking orthologs to three clades: BARP/GARP and subfamilies ‘iii’ and ‘iv’. These genes might have evolved in the T. brucei/T. congolense ancestor if T. vivax is plesiomorphic, in which case all T. vivax genes should branch towards the root. Yet two of five Fam50 lineages in T. vivax, (i.e. TvY486_0016400 and TvY486_1114940), are nested among the would-be T. brucei/T. congolense gains. Reconciliation of this topology with the species tree indicates that if functionality is absent in T. vivax, this is due to secondary loss, rather than T. brucei/T. congolense gain.The CSP emphasizes dramatic cases of gene gain such as T. conglolenseT. brucei expresses various glycoconjugates on their surfaces that only become apparent in null mutants that cannot express the major surface glycoprotein T. brucei is single-copy.Having systematically analyzed protein coding sequences for species differences, it is particularly important to remember that the cell-surface architecture comprises much more than the proteins encoded by the genes in the CSP and that non-proteinaceous elements, not least the surrounding glycocalyx composed of the carbohydrate moieties attached to membrane glycoproteins and glycolipids, might be equally important in determining phenotypic variation. Experimental studies of the cell-surface demonstrate that non-protein glycoconjugates could play an equal role in regulating host-parasite interactions, for example, a protease-resistant surface molecule (PRS) is known to dominate the surface of procyclic-stage MSP subtypes, cathepsin-L and B, or ESAG6-like and PAG-like TFR genes, is a strong indication of long-term functional differentiation and non-redundancy among paralogs. Truly species-specific genes represent adaptations of this shared inheritance and, in T. brucei, include almost all ESAGs as well as various GPI-anchored glycoproteins associated with strand-switch regions (Fam4-7). We anticipate that with improved genome assembly, species-specific genes, perhaps analogous to ESAGs, will be revealed in T. congolense and T. vivax also. To this extent, comparative genomics has met its objectives and the challenge now is to define how these unique genes and variants influence phenotypic differences in biology and disease.The essential character of genes expressed on African trypanosomes cell-surfaces was largely established in the common ancestor. Subsequently, prominent families have experienced rapid turnover of phylogenetic diversity, indicating both functional dynamism and redundancy. As we distinguish the functions of family members, we should be mindful of where orthology is absent and where it is retained; the latter, for example among Figure S1Flowchart describing how the cell-surface phylome was compiled.(EPS)Click here for additional data file.Figure S2trans-membrane helix (green) and adenyly cyclase catalytic Pfam domain (yellow) are marked on the Tb11.01.8820 sequence. ESAG4 apomorphies, i.e. characters that have changed in ESAG4 but remained constant in Tb11.01.8820 and its ortholog in T. congolense (TcIL3000.11.16970), are marked with an asterisk.Distribution of unambiguous, apomorphic characters in ESAG4. The figure shows an amino acid sequence alignment for four ESAG4 proteins and Tb11.01.8820, the most related non-ES homolog (at top). Identical residues are represented with a dot. Positions conserved in ESAG4 only are shaded red. The location of the predicted (EPS)Click here for additional data file.Table S1Gene families comprising the cell surface phylome.(DOCX)Click here for additional data file.


Trypanosoma cruzi has similarities with some intracellular bacterial taxa especially regarding calcium mobilization. This mechanism is not observed in other trypanosomatids, suggesting that the molecules involved in this type of cell invasion were a product of (1) acquisition by horizontal gene transfer (HGT); (2) secondary loss in the other trypanosomatid lineages of the mechanism inherited since the bifurcation Bacteria-Neomura (1.9 billion to 900 million years ago); or (3) de novo evolution from non-homologous proteins via convergent evolution. Similar to T. cruzi, several bacterial genera require increased host cell cytosolic calcium for intracellular invasion. Among intracellular bacteria, the mechanism of host cell invasion of genus Salmonella is the most similar to T. cruzi. The invasion of Salmonella occurs by contact with the host's cell surface and is mediated by the type III secretion system (T3SS) that promotes the contact-dependent translocation of effector proteins directly into host's cell cytoplasm. Here we provide evidence of distant sequence similarities and structurally conserved domains between T. cruzi and Salmonella spp T3SS proteins. Exhaustive database searches were directed to a wide range of intracellular bacteria and trypanosomatids, exploring sequence patterns for comparison of structural similarities and Bayesian phylogenies. Based on our data we hypothesize that T. cruzi acquired genes for calcium mobilization mediated invasion by ancient HGT from ancestral Salmonella lineages.The cell invasion mechanism of  Trypanosoma cruzi is a heteroxenic parasite and the causative agent of Chagas disease which represents an important public health problem in Latin America  the acquisition by horizontal gene transfer (HGT), (2) secondary loss in non-T. cruzi trypanosomatids, or (3) parallel or convergent evolution from non-homologous T. cruzi surface proteins.The protist ica WHO, . DiffereT. cruzi and Leishmania major genome  that promotes the contact-dependent translocation of effector proteins directly into host's cell cytoplasm , were used as BLASTN queries . New searches were performed in T. cruzi CL-Brener genome database (http://www.genedb.org/Homepage/Tcruzi in October/2009) using the nucleotide sequences from 57 strains of 11 genera and 28 intracellular bacterial species  obtained in the former search .Nucleotide sequences of genes encoding proteins SipD, SopB, SopD, and SopE2, present in all strains of genus T. cruzi CL-Brener protein database (http://www.genedb.org/Homepage/Tcruzi in September/2009). Only the sequences of proteins whose role in calcium mobilization during T. cruzi invasion is currently known were selected  str. EDL933, Salmonella enterica (serovar Typhi) str. CT18, Shigella flexneri (serotype 2a) str. 301, Pseudomonas aeruginosa PAO1, and Yersinia pestis CO92, downloaded from the Virulence Factors Database (http://www.mgc.ac.cn/VFs/ in March/2010) were also submitted to BLASTP (http://www.genedb.org/Homepage/Tcruzi in March/2010), being selected only the first 15 sequences according to their lower E-values. The amino acid consensus sequences of T. cruzi proteins retrieved from BLASTP, TcCLB.508221.420, TcCLB.510693.150, TcCLB.511089.90, and TcCLB.506611.20  were manually mapped and submitted again to BLASTP in the T. cruzi genome database GeneDB (http://www.genedb.org/Homepage/ in March/2010) and TriTrypDB—Esmeraldo-like and Non-Esmeraldo-like (http://tritrypdb.org/tritrypdb in April/2010), being selected only the first 15 non-redundant sequences according to their lower E-values , Euglena gracilis (txid3039) and Paramecium tetraurelia strain d4-2 (txid412030) (http://blast.ncbi.nlm.nih.gov/Blast.cgi in June/2011). Only the first 15 non-redundant sequences were selected.Amino acid sequence of S. typhi CT18  was downloaded from NCBI (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi in October/2011) and submitted to BLASTN algorithm in the L. major strain Friedlin, T. brucei strain 927 and T. cruzi strain CL Brener genome databases at GeneDB (http://www.genedb.org/Homepage in November/2011). Sequences encoding ubiquitous proteins such as heat shock and mitochondrial were discarded. Amino acid sequences of proteins SipD, SopB, SopD, and SopE2 of S. typhi were used as query in BLASTP searches in the genome database from L. major strain Friedlin and T. brucei strain 927 at GeneDB (http://www.genedb.org/Homepage in May/2012).Genome sequence of The amino acid sequences were aligned using ClustalX  for signal peptide (SP), transmembrane domains, function, and subcellular localization and Post-translational modifications such as N and O-glycosylation. Prediction of GPI-anchor sites (glycosylphosphatidylinositol) was performed by servers GPI-SOM  using The Sequence Manipulation Suite , using MrBayes v3.1.2 . The consensus trees were then used to determine the posterior probabilities values. All phylogenetic trees were then formatted with the FigTree v1.3.1 program (http://tree.bio.ed.ac.uk/software/figtree/).Phylogenetic trees were inferred from amino acid sequence alignments retrieved from BLASTP  and from alignments generated from database searches of different protists  were retrieved. Sequences whose in silico translation included frameshifts and/or unrelated amino acids, were excluded. Only amino acid sequences obtained by BLASTP were used for further analysis.Among all bacterial genera analyzed , positive BLASTN results were obtained only for genera S. typhi effector proteins SipD, SopB, SopD, and SopE2 against the L. major, T. brucei, and T. cruzi genome database, yielding 21, 24, and 42 sequences, respectively. From these sequences, we performed predictions to determine their possible locations and functions . We show that the number of T. cruzi amino acid sequences potentially involved in the invasion mechanism was superior to other trypanosomatids. Two sequences with the potential to be on the parasite surface were found both in L. major and in T. brucei . However, they were not analyzed further because they are classified as hypothetical or pseudogenes and because it is already known that both parasites do not mobilize intracellular calcium during invasion and thus cannot actively invade host cells . Among those, only the putative sequences of mucins and/or mucin associated surface proteins (MASP)  were selected because of their already known involvement with calcium mobilization during T. cruzi cell invasion . These searches revealed a considerable number of MASPs and mucins  to elevate intracellular calcium and consequently induce cytoskeleton rearrangement and remain inside vacuoles during the first stages of cell invasion  and MASPs  were aligned. As expected, due to the high rate of divergence among sequences, it resulted in few conserved blocks and positions embedded in highly divergent domains (data not shown). However, the mapping of local amino acid residues  resulted in an alignment with good quality   Table . SipD haT. cruzi and Salmonella proteins and also address the possible structural and functional properties shared by them, amino acid sequences were analyzed by different prediction methods. Searches for known sequence motifs and domains from manually curated databases using the amino acid sequences of proteins 420, 150, 90, and 20 from T. cruzi and the sequence of S. typhi SipD, showed that no characterized domains or motifs are present (data not shown). However, our predictions showed that SipD is part of the IpaD family, effector proteins from Shigella that share similar functional roles with SipD  between T. cruzi present potential cleavage sites in positions 21 and 22, 25 and 26, 26 and 27, and 24 and 25, respectively. More importantly, the fact that the possible signal sequences in these proteins remain outside amino acid blocks that aligns with SipD  the ingestion of blood contaminated with Salmonella spp. or some other T3SS intracellular bacteria by species of Triatominae and the insertion of bacterial genes into the T. cruzi genome or (2) insertions and/or gene exchange by endosymbiotic bacteria. We also do not exclude that other trypanosomatids lost their ability to invade since the Bacteria-Neomura bifurcation (secondary loss). Nevertheless, the occurrence of multiple HGT events from bacterial endosymbionts in plants to trypanosomatids described by Hannaert et al.   highly similar genes, and (c) genes whose phylogenies are incongruent with the relationships inferred from other genes in their respective genomes , proline (PRO), and threonine (THR)  Figure . Usually) Figure . TogetheT. cruzi and S. typhi genes in this study, both with approximately 51% GC content  and sequences obtained from database searches of different protists were aligned and submitted to Bayesian phylogenetic inferences. A total of six multiple alignments were generated (one for each T. cruzi proteins), comprising up to 36 sequences which included the S. typhi SipD, with up to 152 positions, and other 2 alignments, one comprising 179 sequences with 368 positions (different protists) and the other with 139 sequences and 444 positions (only trypanosomatids), obtained by searches in different protein databases. Apart from the phylogenetic inference obtained with the putative mucin 20, which showed a large polytomy (Figure S. typhi SipD and several T. cruzi proteins, with posterior probabilities above 0.79 (Figure T. cruzi, suggesting a possible homology and analogy of these MASPs with SipD.To obtain a congruent analysis that could establish evolutionary relationships between d Brown,  by perfoy Figure , all phy9 Figure . SipD, a9 Figure  was rece9 Figure  as MASP1Euglena gracilis, Paramecium tetraurelia, and Bodo saltans would cluster together with SipD (Figure Paramecium sequence, one Euglena sequence and a polytomus subclade including several trypanosomatids. For this analysis the Bayesian inference was used to obtain several phylogenies in two runs with convergent LnL scores after the burn-in, around 3 × 107 generations (Figure The phylogeny inferred using amino acid sequences of different protists was used to test if earlier branching organisms such as D Figure . A SipD T. cruzi with posterior probability 1.00 (Figure Salmonella spp to T. cruzi, because even with a large number of sequences from different trypanosomatids, SipD still clustered with T. cruzi sequences.To resolve the polytomy observed in the Bayesian tree in Figure 0 Figure . This reL. tarentolae expressing two different proteins of the MASP family trigger intracellular calcium transients in HeLa cells, presumably by injury to the cell membrane (Choi et al., Salmonella SipD and the HGT here proposed.The accuracy with which phylogenies can be reconstructed, and by which HGTs can be detected, depends on the degree of divergence (Gogarten et al., S. typhi to T. cruzi in early evolutionary history of T. cruzi. Because of the marginal sequence similarities involved and long divergence dates, our data cannot rule out extreme convergent evolution. Nevertheless, the acquisition of ancestral T3SS from Salmonella might have contributed to the pathogenicity and singular invasion mechanisms among trypanosomatids that allowed it to actively invade host cells.Our results are consistent with the hypothesis that genes involved in host cell invasion were horizontally transferred from 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.


Trichuris vulpis, while the respiratory parasites Eucoleus boehmi (syn. Capillaria boehmi) and Eucoleus aerophilus (syn. Capillaria aerophila) are rarely observed in pets. E. boehmi is reported for the first time in this study in north-western Italy with other Trichuridae. Dog faecal samples (270) were examined by flotation. E. boehmi (2.2%), E. aerophilus (4.4%) and T. vulpis (12.2%) were found; identification was done with measurements and through observation of morphological characters already known. The specific identification of E. boehmi was confirmed using scanning electron microscopy: its egg shell shows a dense network with a fine mesh, surrounding small pits, on the contrary E. aerophilus eggs present a thick mesh with wide depressions, while T. vulpis eggs surface is smooth.Dogs can be infected by several nematodes of the Trichuridae family. Trichuridae eggs are all similar, barrel shaped with polar plugs, and misdiagnosis among different species can occur. The most common species is  Trichuris vulpis , while the respiratory parasites Eucoleus boehmi (syn. Capillaria boehmi)  and Eucoleus aerophilus (syn. Capillaria aerophila)  are rarely reported in pets.Dogs can be infected by several nematodes belonging to the Trichuridae family. The most common species is the intestinal whipworm E. boehmi eggs are 54- 60 × 30-35 µm in size, contain a multicellular embryo that does not fill the egg, are clear to golden in colour and show a delicately pitted surface . Samples were examined by centrifugal flotation with zinc sulphate (s.g. 1.350).E. boehmi eggs were also examined using scanning electron microscopy (JEOL JSM 5410 SEM). For SEM, eggs were isolated by flotation in a zinc sulphate solution and sieved, following the technique described in Trichuridae eggs were identified on the basis of morphologic and morphometric characteristics described in the literature  were positive for . boehmi , 12  . Further