Candida, are commensal members of our microbiota, continuously interacting with epithelial cells. Transient and localised perturbations to the mucosal environment can facilitate the overgrowth of fungi, causing infection. This minireview will examine the direct and indirect mechanisms by which Candida species and epithelial cells interact with each other, and explore the factors involved in the central processes of adhesion, invasion, and destruction of host mucosal surfaces.A plethora of intricate and dynamic molecular interactions occur between microbes and the epithelial cells that form the mucosal surfaces of the human body. Fungi, particularly species of  Candida spp. There are over 150 spp. of Candida fungi, of which approximately 20 are known to be pathogenic to humans. While Candida spp. are typically commensal organisms, several factors, including alterations in the host immune status, microbial dysbiosis, lifestyle choices , and genetic predisposition can facilitate fungal overgrowth and a switch to pathogenic behaviour. Although Candida albicans is regarded as the most common of the Candida spp, C. glabrata, C. tropicalis, C. parapsilosis, and others are increasingly isolated from mucosal surfaces [Candida spp. and their continually evolving threat to human health is underpinned by the recent emergence of C. auris [The mucosal surfaces of the human body are colonised by a rich and diverse microbiota, placing them under relentless microbial scrutiny. Fungi are an important but often overlooked component of the microbiota ,2,3 thatsurfaces . Indeed,Candida spp. must first interact with a mucosal surface. Candida spp. use physical attributes and an array of proadhesive, proinvasive and damage-inducing factors to facilitate colonisation and infection, and in this minireview, we will discuss the role of these factors in Candida–epithelial interactions.In order to colonise a host, Candida spp. to epithelial cells is a complex, dynamic, and multifactorial process defined by the intimate association between components of the fungal cell wall and epithelial surface proteins.Microbial adhesion to epithelial surfaces is an essential prerequisite for mucosal colonisation and infection. The contact with the epithelium can be mediated through direct physical attachment or indirectly through physical association with co-colonising microbes and/or abiotic substrates. The site of fungal attachment can serve as a staging post for superficial, deep-seated, and systemic infections with high mortality in susceptible individuals. The adhesion of C. albicans yeast and host epithelium occur through the passive processes of hydrophobic and electrostatic attraction [C. albicans to adhere to epithelial cells [Initial interactions between traction . Changestraction  and the al cells ,8. Moreoal cells .Candida spp. undergo morphological switching from yeast to hyphae, and this morphological transition is a major virulence trait of some, but not all, Candida spp. For example, while C. albicans, C. dubliniensis, and C. tropicalis form hyphae, which adhere more strongly to epithelial cells than yeast cells [C. glabrata and C. parapsilosis do not form hyphae but are still adherent.Once in contact with the epithelium, some st cells , other sC. albicans Hyphal Wall Protein 1 (Hwp1p) is a key hypha-associated adhesin that facilitates epithelial cell attachment and is highly expressed during colonisation and infection of the oral mucosa [C. albicans to the oral epithelium [HWP1 results in reduced adhesion to oral epithelial cells and decreased virulence in a murine model of oropharyngeal candidiasis (OPC) [C. albicans Hwp2p has also been reported to be required for adhesion to HT-29 human epithelial cells in vitro [l mucosa ,12. The ithelium . These iithelium . Deletiois (OPC) . In addiin vitro .C. albicans Als (agglutinin-like sequence) proteins [C. albicans Als3p (hypha-associated) is a major epithelial adhesin that is strongly upregulated during epithelial infection in vitro [ALS3 gene reduces epithelial adhesion in vitro. Likewise, decreasing the expression of the ALS2 gene also reduces adhesion [ALS5, ALS6, or ALS7 genes was observed to increase adhesion [The proteins , comprisproteins . Als-depproteins , suggestproteins , and theproteins . C. albiin vitro , and disadhesion ,23. Inteadhesion , indicatadhesion ,25,26.C. parapsilosis, C. dubliniensis, and C. tropicalis although there is significant sequence divergence between spp. [C. parapsilosis homologue of C. albicans Als3p (CPAR2_404800) resulted in reduced adhesion to buccal epithelial cells in vitro and diminished pathogenicity in a murine model of urinary candidiasis [Putative homologues of Als proteins have also been identified in een spp. ,28 and ldidiasis , suggestC. albicans and C. dubliniensis differ considerably in terms of their pathogenicity, their genomes are remarkably similar [ALS gene family with the exception of ALS3, which is absent from C. dubliniensis [ALS3 for C. dubliniensis and expansion of a telomere-associated (TLO) gene family of transcriptional regulators within C. albicans) may account for the differences in pathogenicity between species [While  similar . The siminiensis . Indeed, species ,31.C. albicans Hyphally Regulated protein (Hyr1p) in epithelial adhesion is less clear, although a role in biofilm formation has been established. Disruption of the HYR1 gene leads to reduced virulence in vivo [C. albicans adhesion. For example, Eap1p (Enhanced Adherence to Polystyrene) is required for biofilm formation and adhesion to epithelial and fungal cells [SAP9 gene from C. albicans increases adhesion, while deletion of the SAP10 gene reduces adhesion [C. albicans to epithelial but not endothelial cells [The role of  in vivo . Other pal cells ,34,35, wadhesion . Overexpal cells .C. albicans, the main adhesion proteins mediating attachment of the non-filamentous fungus C. glabrata are Epithelial Adhesin (Epa) and Epa-like proteins. Epa1p of C. glabrata is a 1034-amino acid GPI-anchored, glucan cross-linked, cell wall-associated lectin required for robust, calcium-dependent adhesion to laryngeal and hamster ovary epithelial cells in vitro [EPA1 gene deletion mutant exhibited a 95% reduction in epithelial adhesion, identifying this factor as a major contributor to C. glabrata adherence. Indeed, Epa1p alone is sufficient to mediate adhesion to epithelial cells, as expression of EPA1 in Saccharomyces cerevisiae resulted in robust epithelial adhesion [In contrast to in vitro . Epa1p badhesion .C. glabrata rely on Epa1p for the majority of their epithelial adhesion, while others have minimal dependence. C. glabrata contains a number of EPA1 gene paralogues located at the subtelomeric regions of its genome [C. glabrata express differing repertoires of Epa adhesins [EPA gene family in C. glabrata (>20 EPA-like members), it is likely that a certain degree of functional redundancy may account for the differences in experimental observations. Indeed, a degree of molecular promiscuity is apparent in the adhesin domain of Epa1p, Epa6p, and Epa7p that facilitates attachment to a number of different carbohydrate ligands, particularly those that contain terminal galactose residues [Candida spp. to interact with epithelial cells are presented in Intriguingly, Epa1p-mediated adhesion to epithelial cells appears to be strain-dependent . Some sts genome , and difadhesins , some ofadhesins . Given tresidues ,44. CommCandida spp. can invade the epithelium in order to establish an infection. C. albicans can enter epithelial cells by a passive host-mediated process called induced endocytosis [C. albicans hyphae and non-filamentous C. glabrata occurs within 4 h of initial contact with epithelial cells [C. albicans invasins have been identified, Als3p and Ssa1p, both of which interact with the host epithelial receptor E-cadherin [C. albicans als3∆/∆ and ssa1∆/∆ gene deletion mutants exhibit reduced binding to, and invasion of epithelial cells and are attenuated for virulence in a murine model of OPC [Following colonisation, ocytosis ,46. Endoal cells ,48. Inducadherin ,50. Intecadherin . C. albil of OPC ,50,52.C. albicans, as siRNA knockdown of PDGF receptor beta (PDGFRB) or NEDD9 causes a reduction in endocytosis [C. albicans results in Src-mediated phosphorylation of EGFR and fungal internalisation, while inhibition of AhR was observed to reduce invasion and OPC severity [Induced endocytosis can also occur independently of epithelial E-cadherin through the interaction of fungal Als3p and Ssa1p with the epidermal growth factor receptor (EGFR/HER2) heterodimer expressed on epithelial cells  and throocytosis . More reocytosis . Activatseverity .Candida spp. exploit the process of induced endocytosis to avoid immune recognition. For instance, C. parapsilosis is efficiently endocytosed by endothelial cells which provide a means of escape from patrolling neutrophils [Intriguingly, some trophils . HoweverCandida spp. must therefore use an alternative mechanism to invade a tissue that does not readily support internalisation. This process is called active penetration. Active penetration of mucosal barriers requires a viable fungus and occurs by hyphal invasion through or between epithelial cells. Active penetration is dependent upon fungal attributes including hyphal turgor pressure, physical advancement of the hyphal tip, and secretion of factors such as hydrolytic enzymes that may facilitate the breaching of mucosal barriers by degrading host substrates. The C. albicans-secreted aspartic proteinase Sap5p can degrade E-cadherin, an important component of epithelial adherens junctions [C. albicans invasion and penetration, secreted lipases and phospholipases appear to play a limited role in these processes [C. parapsilosis has been observed to invade the connective tissue of human oral mucosa [C. parapsilosis and C. tropicalis also colonise and invade reconstituted human oral epithelium [The oral and vaginal mucosae are comprised of stratified layers, the outermost of which are terminally differentiated, nonproliferative, and therefore less likely to support fungal invasion through induced endocytosis. unctions , while Sunctions . Active unctions ,60. Whilrocesses . C. paral mucosa , althougithelium ,64 and bC. albicans hyphae occurs at all mucosal surfaces, but in the gut it is the sole driver of invasion across the epithelium [C. albicans to invade through the outermost layers of an epithelium in vivo. However, once the fungus has accessed the underlying proliferative layers of an epithelium, induced endocytosis may then occur to further facilitate invasion. While induced endocytosis and active penetration are therefore mechanistically distinct processes, both are nevertheless likely to be required to establish infection through stratified mucosal barriers in vivo.Active penetration of ithelium ,60. ActiCandida spp. is poorly understood, but recent studies have shed light on the signalling pathways activated by Candida, particularly C. albicans. C. albicans yeast cells weakly activate three key signalling pathways: all three MAPK (mitogen-activated protein kinase) pathways , the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway, and the PI3K (phosphatidylinositide 3-kinase) pathway [C. albicans hyphae activate the same signalling pathways, but there is a far stronger activation of MAPK signalling, with specific induction of the transcription factor c-Fos (via p38) [C. albicans hyphae also activate MKP1 (MAPK phosphatase 1) via the ERK1/2 pathway, which is known to regulate MAPK-mediated immune responses [The process of receptor-mediated epithelial recognition of  pathway ,66. Thisvia p38) . C. albiesponses . This coC. albicans to initiate these signalling mechanisms and induce immune responses are unclear. Epithelial cells express numerous pattern recognition receptors (PRRs) , C-type lectin receptors), including TLR2, TLR4, and dectin-1 [Candida challenge [C. albicans hyphae [C. glabrata, Rhizopus delemar, Aspergillus fumigatus, S. cerevisiae, and C. albicans yeast and hyphae can interact with the epithelial PRR Ephrin Type-A Receptor 2 (EphA2), activating signal transducer and activator of transcription 3 (STAT3) and MAPK signalling that culminates in proinflammatory antifungal responses [Candida infections [The epithelial receptors that interact with dectin-1 ,65,67,68hallenge ,66. Theshallenge ,70. Howes hyphae   proteins Rbt5p, Pga7p, and Csa1p also play a role in iron acquisition from host proteins [C. albicans, the CFEM family is expanded in C. parapsilosis [CFEM2 and CFEM3 genes of C. parapsilosis are critical for haem utilisation, whereas the CFEM6 gene is only partially required [In this regard, it is interesting to note that Als3p expressed on al cells . The C. proteins ,90. Comppsilosis . The CFErequired .C. albicans pH-regulated antigen 1 (Pra1p) is required to scavenge zinc from endothelial cells [Candida spp.  and increased adhesion of C. albicans to abiotic substrates [The ability to acquire and assimilate host micronutrients including copper and zinc has a profound influence on fungal pathogenicity . Indeed,al cells , while zbstrates . FactorsCandida–epithelial interactions. Adhesion of Candida spp. to a mucosal surface can be direct  but can also occur indirectly through specific interactions with other fungi and bacteria [It is becoming increasingly clear that the microbial communities that colonise mucosal surfaces have a profound influence on bacteria ,97.C. albicans can form heterotypic aggregates with C. glabrata and C. parapsilosis in vitro [C. glabrata during murine OPC is enhanced when co-infected with C. albicans or when added to a pre-established C. albicans infection [Candida can have a dramatic influence on the pathogenicity of a different fungal spp. Such interactions are not limited to other fungi, however. Streptococcus gordonii is a primary commensal coloniser of the oral cavity, and C. albicans Als3p can interact with the SspB adhesin of S. gordonii [S. gordonii [C. albicans and Staphylococcus aureus [The analysis of mixed fungal cultures using a magnetic bead-based adherence and aggregation assay revealed that in vitro . Infectinfection , stronglgordonii , while Agordonii . Als3p as aureus .Candida spp. with one another and with mucosal bacteria may serve to increase the likelihood of a successful Candida–epithelial interaction, although this is not always the case. For instance, the probiotic bacterium Lactobacillus rhamnosus GG induces a metabolic reprogramming of C. albicans causing reduced hyphal extension, adhesion to, and invasion of oral epithelial cells in vitro [C. albicans and mucosal bacteria can have variable outcomes in the context of mucosal interaction.The interaction of in vitro , demonstCandida species employ a diverse range of direct and indirect mechanisms that enable the interaction with host epithelial cells. Under suitably predisposing conditions, commensal colonisation of mucosal surfaces is followed by pathogenic infiltration and secretion of hydrolytic enzymes and toxins that reduce mucosal barrier function, facilitating disease progression.Candida spp. and between fungal strains of the same species have an undoubted impact on Candida–epithelial interactions. For instance, while C. albicans triggers more epithelial cell damage than other Candida spp., the extent of damage varies between strains [C. glabrata [C. albicans exhibit variable pathogenicity in animal models [Importantly, variations between different  strains , leading strains ,104. Likglabrata , while dl models ,106,107.Candida spp. and members of the resident microbiota can influence the mucosal interactions and has direct consequences on disease outcome. In response, the epithelial receptor-mediated recognition of Candida spp. activates a number of dynamic host signalling pathways that enable appropriate proinflammatory and immune defence mechanisms to be implemented. The environmental conditions greatly affect the outcome of any experiment, including Candida–host interactions. The precise factors that influence these interactions are unquestionably variable and complex.The physical contact between Candida–epithelial interactions remain to be explored in full. The majority of experimental data obtained thus far describes factors utilised by C. albicans, but there is an increasing need to identify the proteins used by other Candida spp. that influence epithelial interactions. It is likely that there are many more epithelial-specific receptors involved in Candida recognition that have yet to be identified and characterised. The signalling responses induced by fungal endocytosis have yet to be characterised in full, and the contribution of secreted factors to fungal pathogenicity remains an area of intense study. Indeed, while Candidalysin has been shown to contribute to both oral and vaginal infections in mice [Numerous aspects of  in mice ,108, theCandida fungi and mucosal surfaces, continued research will undoubtedly provide further clarity into the mechanisms that underpin this important host–pathogen interaction.While considerable progress has increased our understanding of the complex relationship that exists between


Previous investigations have shown that, during metazoan radiation, the exon-intron patterns of serpin superfamily in lineages pre- and postdating the split of vertebrates. Multiple intron gains were detected in a group of ray-finned fishes, once the canonical groups of vertebrate serpins had been established. In two genes, co-occurrence of non-standard introns was observed, implying that intron gains in vertebrates may even happen concomitantly or in a rapidly consecutive manner. DNA breakage/repair processes associated with genome compaction are introduced as a novel factor potentially favoring intron gain, since all non-canonical introns were found in a lineage of ray-finned fishes that experienced genomic downsizing.Here we investigated intron dynamics in the serpin genes of a lineage of ray-finned fishes, but not in any other vertebrates, suggesting that insertion rates for introns may be episodically increased. The co-occurrence of non-standard introns within the same gene discloses the possibility that introns may be gained simultaneously. The sequences flanking the intron insertion points correspond to the proto-splice site consensus sequence MAG↑N, previously proposed to serve as intron insertion site. The association of intron gains in the serpin superfamily with a group of fishes that underwent genome compaction may indicate that DNA breakage/repair processes might foster intron birth.Multiple intron acquisitions were identified in  Spliceosomal introns are key attributes of most eukaryotic genes. Their origin is still unclear, though descent from group II self-splicing introns seems to be likely ,2. All cvice versa, was proposed to foster functional diversification of serpins [The serpins are a superfamily of proteins that cover a highly divergent spectrum of functions ,13. The  serpins ,15.serpin genes are often arranged in tandem arrays and they constitute a substantial fraction of mammalian genomes. During diversification of vertebrates the superfamily has undergone considerable expansion [Serpin genes of basal metazoans, in contrast, are not generally intron-rich, and their exon-intron structures are not conserved along the lineages leading to vertebrates. Sporadic investigations of various species revealed radically different intron patterns in serpin genes, indicating that, during diversification of eumetazoans, massive changes in gene architectures have occurred [serpin genes from various vertebrates  and Branchiostoma floridae (B. floridae), representing a group of extant cephalochordates (phylum: Chordata). Experimental approaches disclosed several genes and cDNAs coding for serpins in B. lanceolatum, further superfamily members were detected by mining the genome of the closely related B. floridae [serpin genes (L1–L3) with distinctly different intron patterns were observed . The L2 genes contain introns at positions ~86b, 151c, 223b, 283c and 339c. The L3 genes exhibit common introns at positions 73b, 125b, ~175c and 339c, however, there are also introns that are unique to individual group members . Another intron in Bflor_Spn10 is located outside the conserved serpin scaffold (gene-specific numbering of position: 29a). There are several additional serpin-like sequences in the B. floridae genome, but it is currently not discernible whether they represent intact genes (not shown); however, it is clear that serpin genes from lancelets and vertebrates differ largely with respect to their exon-intron organizations. In fact, just a single intron location (position 339c) is shared. Apparently, major changes affecting the exon-intron patterns of serpin genes have occurred since the cephalochordate/vertebrate split.We first studied floridae . Altogetn genes L–L3 with n genes L–L3 with serpins in vertebrates, we turned to lampreys, a group of basal, jawless fishes. Lampreys, in sharp contrast with lancelets, depict at least four of the six canonical groups of vertebrate serpins. A survey of cDNA and genomic sequences from Lampetra fluviatilis  and from Petromyzon marinus  revealed representatives of groups V2, V4 and V6 . Members of groups V3 and V5 were not identified.Having established lancelets as appropriate outgroup for evaluating evolution of L. fluviatilis and P. marinus , among other features, definitely reveals this protein as member of the serpin superfamily.Inspection of lamprey serpin sequences disclosed the presence of angiotensinogen and heparin cofactor II (HCII), two prominent members of group V2. All known angiotensinogen proteins depict a conserved decapeptide sequence close to the N-terminus that, after controlled enzymatic cleavage, gives rise to formation of peptides (angiotensin I-IV) involved in blood pressure regulation and other important physiological processes . ClearlyP. marinus (see below). We also recognized a lamprey serpin exhibiting the exon-intron pattern of group V6 . Characteristic features of all HCII sequences are the highly conserved Arg/Lys-rich helix D that is involved in GAG binding and the acidic N-terminal extension that mediates GAG accelerated thrombin inhibition ,26. Thesollagens ,27. A haollagens  and its serpin genes, the dynamics of their exon-intron patterns was analyzed. The list of species investigated included: man, chicken , the clawed frog , and several fishes including Danio rerio (D. rerio), Oryzias latipes (O. latipes), Gasterosteus aculeatus (G. aculeatus), Tetraodon nigroviridis (T. nigroviridis), and Takifugu rubripes (T. rubripes). Several serpin genes, though clearly members of one of the six canonical groups, were found to deflect from the standard organizations. Like their counterparts in lampreys, angiotensinogen genes of tetrapods and D. rerio exhibit the canonical gene architecture of group V2, the T. rubripes orthologue, however, contains two extra introns that split the exonic sequences at positions 77c and 233c, respectively . Though also found in O. latipes and G. aculeatus, this intron here is not considered any further, due to its location outside the serpin scaffold. Examination of lamprey HCII also revealed unique introns. The 83c intron (α1-antitrypsin numbering) is embedded in a well-conserved region; its origin, however, is difficult to evaluate. The others (correctly predicted?) map to the N-terminal extension (gene-specific numbering of positions: 38b and 118c).The  mammals ,30, chicSpn_94a, with a surplus intron at position 94a . The origin of these genes is unclear; the unique surplus intron suggests that they share a common ancestor , all of which are equipped with the standard set of introns , which probably arose as a consequence of genome duplication events in the stem lineage of ray-finned fishes [Dan_HSP47_1, as indicated by the conserved gene order . In G. aculeatus, a second intact HSP47 homologue, Gast_HSP47_2, was identified. Dan_HSP47_2 and Gast_HSP47_2 are orthologous to each other, since they share a set of flanking markers ; Gast_HSP47_2, in contrast, merely possesses the default introns. These findings suggest that the novel introns, which are restricted to HSP47_1 orthologues, were acquired by group V6 during evolution of ray-finned fishes after divergence of the D. rerio lineage, though an intron loss scenario cannot be excluded. Two intron gains or a single, coupled intron gain event are/is sufficient to explain the exon-intron patterns found in group V6; the intron removal scenario, in contrast, requires multiple intron loss events. If such losses had occurred, they must have affected several taxa, including lampreys, tetrapods and fishes. Moreover, this scenario also demands that the same two introns (positions 36b and 102c) were always deleted in parallel, while all other introns were unaffected. The parallel emergence of introns at positions 77c and 233c in angiotensinogen genes , the only member of group V5, is a potent thrombin inhibitor in the presence of heparin. Among other characteristic features, AT orthologues are easily discernible through a highly conserved sequence centering around helix D . Intron 262c is a standard attribute of group V1 that is believed to share a common ancestor with group V5 [AT genes of tetrapods. We were not able to identify this gene in P. marinus; further tracing of the 262c intron was therefore not possible.In contrast with several intron gains, we detected a single case of probable intron loss during evolution of vertebrate group V5 . We therserpins from groups V3 and V4, deviations from the standard structures were not observed (not shown). All genes from group V3 analyzed featured introns at positions ~86a-90a, 167a, 230a, 290b, 323a, 352a, and 380a. The intron pattern characteristic of group V4  was also conserved.In T. rubripes [Spn_94a orthologues cannot be aligned without introducing gaps , referred to as proto-splice sites [serpins of ray-finned fishes  were purchased from the Alfred-Wegener-Institut für Polar- und Meeresforschung, Helgoland, Germany. European river lampreys (L. fluviatilis) were obtained from the Bundesforschungsanstalt für Fischerei, Hamburg, Germany.Adult lancelets (The isolation of poly(A)-RNA and genomic DNA, the synthesis of cDNA, PCR amplification of serpin cDNA fragments using various sets of degenerate primers, and cloning of 5'- and 3'-cDNA ends followed published procedures ,51. DNA serpins from human, chicken [X. tropicalis [ D. rerio [ G. aculeatus [O. latipes [T. rubripes [T. nigroviridis [P. marinus [). Sequences from the B. floridae genome were gathered from the JGI genome browser . EST and cDNA data mining included searches in the NCBI trace archive  and in the UCSC genome browser [Genomic data for  chicken , X. tropopicalis , D. reriD. rerio , G. aculculeatus , O. lati latipes , T. rubrrubripes  and T. noviridis  were extoviridis , or in t marinus , from Pr browser , applyin1-antitrypsin as described [i.e. positions 33 to 394 of human α1-antitrypsin) were considered.All intron positions predicted by gene models were examined visually, corrected and amended manually, if necessary. Whenever cDNA or EST sequences were available, intron positions were checked by means of GENEWISE . Proteinescribed . All int)) and with RepBase Censor  [Sequences of non-canonical introns were searched for repetitive elements with the RepeatMasker package (version 3.2.6; (ensor   using dePhylogenetic analysis was performed using the Neighbor-Joining method  conducteHR designed the study, performed part of data analyses, and wrote the paper. AK accomplished data analyses. KK, YW, CB, MAF, NP and OK generated data and contributed to data evaluation.Mapping of intron positions to aligned serpin sequences. Figure depicting intron positions of serpin genes mapped onto the aligned amino acid sequences.Click here for fileserpin genes analyzed in this study and their accession numbersList of . Table listing accession numbers of genes, cDNAs and ESTs investigated in this study.Click here for fileSpn_94a genesChromosomal gene order reveals orthology of . Figure showing chromosomal synteny of Spn_94a genes.Click here for fileserpin genesSequences of non-canonical introns from vertebrate . Figure depicting the sequences of non-standard introns.Click here for file


Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium and opportunistic human pathogen, the GacS/GacA two-component system positively controls the transcription of two sRNAs , which are crucial for the expression of genes involved in virulence. In the biocontrol bacterium Pseudomonas fluorescens CHA0, three GacA-controlled sRNAs  regulate the response to oxidative stress and the expression of extracellular products including biocontrol factors. RsmX, RsmY and RsmZ contain multiple unpaired GGA motifs and control the expression of target mRNAs at the translational level, by sequestration of translational repressor proteins of the RsmA family.Small RNAs (sRNAs) are widespread among bacteria and have diverse regulatory roles. Most of these sRNAs have been discovered by a combination of computational and experimental methods. In P. aeruginosa. Eight of these regions encode newly identified sRNAs. The intergenic region 1698 was found to specify a novel GacA-controlled sRNA termed RgsA. GacA regulation appeared to be indirect. In P. fluorescens CHA0, an RgsA homolog was also expressed under positive GacA control. This 120-nt sRNA contained a single GGA motif and, unlike RsmX, RsmY and RsmZ, was unable to derepress translation of the hcnA gene (involved in the biosynthesis of the biocontrol factor hydrogen cyanide), but contributed to the bacterium's resistance to hydrogen peroxide. In both P. aeruginosa and P. fluorescens the stress sigma factor RpoS was essential for RgsA expression.A combined computational and experimental approach enabled us to identify 14 intergenic regions encoding sRNAs in Pseudomonas species highlights the complexity of this global regulatory system and suggests that the mode of action of GacA control may be more elaborate than previously suspected. Our results also confirm that several GGA motifs are required in an sRNA for sequestration of the RsmA protein.The discovery of an additional sRNA expressed under GacA control in two  Escherichia coli, either fortuitously due to their abundance or by the observation of phenotypes conferred by their overexpression. Some abundant and stable sRNAs were found early in gel electrophoretic analysis. They include 4.5 S RNA, which is implicated in protein export; a component of RNase P, which participates in tRNA processing; and tmRNA, which has an important role in translational quality control [E. coli. Computational searches mainly focused on intergenic regions (IgRs) and were combined with predictions of promoters and of rho-independent transcription terminators [Bacillus subtilis [In bacteria, > 150 non-coding small RNAs (sRNAs) have been described [ control . The majminators ,4. The Qsubtilis .E. coli, the iron-containing superoxide dismutase SodB is regulated by iron availability via the sRNA RyhB, which base-pairs with sodB mRNA. This interaction blocks ribosome access and favors nucleolytic degradation of the mRNA [regulator of secondary metabolism) and CsrA (carbon storage regulator) can act as translational repressors; sRNAs having high affinity for these proteins are therefore able to relieve translational repression by sequestering them [Most bacterial sRNAs studied have regulatory roles in gene expression, occurring in many instances at a post-transcriptional level. In one type of post-transcriptional regulation, which is usually governed by the RNA chaperone Hfq in Gram-negative bacteria, sRNAs interact with specific mRNA targets, thereby modifying the accessibility of the Shine-Dalgarno sequence to the translational machinery and often altering the stability of the mRNA. For instance, in the mRNA . A seconing them .P. fluorescens, the sRNAs RsmX, RsmY and RsmZ were detected by their binding capacity to the regulatory protein RsmA [gacA mutation [E. coli) activates the transcription of these three sRNAs. When they are present in high concentrations, they titrate the RNA-binding proteins RsmA and its homolog RsmE, resulting in enhanced translational expression of genes involved in biocontrol of plant root diseases and in resistance to oxidative stress [P. aeruginosa, the Gac/Rsm system involves two sRNAs, RsmY and RsmZ [rhlI gene, which codes for the enzyme synthesizing the quorum sensing signal N-butanoyl-homoserine lactone [P. aeruginosa led to the discovery of two iron-regulated sRNAs, PrrF1 and PrrF2 [In pseudomonads, few sRNAs have been studied. In ein RsmA ,10, by tein RsmA , or by mmutation . The Gace stress ,14. Bioce stress . In P. aand RsmZ . As part lactone ,18. Furtnd PrrF2 . More rend PrrF2 .Pseudomonas spp., by applying the QRNA method to IgRs of various Pseudomonas spp., combined with a prediction of rho-independent terminators and, where appropriate, putative promoters. Eight sRNAs were newly identified in P. aeruginosa by Northern blotting experiments. By comparing sRNA expression in the wild-type with that in a gacA mutant, we discovered a novel GacA-controlled sRNA termed RgsA. We analyzed its regulation and involvement in biocontrol factor expression and oxidative stress response in P. fluorescens CHA0. Moreover, we show that RgsA expression in P. aeruginosa and P. fluorescens strictly depends on the stress sigma factor RpoS.We have begun to search for new GacA-regulated sRNAs in P. aeruginosa PAO1 on the criterion that sRNA genes should be located in IgRs that are larger than 50 bp. A total of 3168 IgRs fulfilled this condition. We then assumed that functional sRNA sequences should be conserved in closely related species [P. aeruginosa in the genomic sequences of five related pseudomonads . Only those PAO1 IgRs were selected that shared more than 65% sequence identity with a genomic sequence of at least one other pseudomonad. The alignments resulting from the BLAST program were then subjected to QRNA analysis [P. aeruginosa PAO1 genome annotations, sequences whose coding probabilities calculated by QRNA were found to be high were nevertheless retained for further analysis. By applying QRNA in this way, 162 out of the initial 3168 IgRs from P. aeruginosa PAO1 were retained  were preferentially chosen for Northern blot analysis. Cultures of P. aeruginosa PAO1 were grown in nutrient yeast broth (NYB) to exponential or stationary phase and total RNA was extracted. Northern blot analysis was carried out with digoxigenin (DIG)-labeled probes, each covering an entire IgR. Among 49 IgRs thus analyzed, 14 were reproducibly found to express sRNAs (Table ffs gene in IgR 888) [rnpB in IgR 2510) [P. aeruginosa (encoded by prrF1 and prrF2 in IgR 2667) [P. aeruginosa sRNAs described by Livny et al. [IgRs occurring in IgR 888) ,25, an RgR 2510) , and PrrgR 2667) . They wey et al. . In thatribC and ribD genes, is a homolog of sroG, a transcript resulting from the cleavage of a riboswitch element found upstream of ribB in E. coli [P. aeruginosa sroG homolog was slightly longer than the sroG transcript in E. coli (147 nt) [P. aeruginosa may be involved in regulation of riboflavin biosynthesis, by analogy with the homologous element in E. coli [E. coli RNA , except for 1559 sRNA, which was not tested in these alternative media. Six regions  produced more than one transcript  . The 231 E. coli ,29. In fP. aeruginosa gacA mutant PAO6281 by Northern blotting. The 1698 sRNA was the only transcript that showed a significantly decreased expression in the gacA mutant, compared with that in the wild-type PAO1 (data not shown). This 120-nt transcript was further characterized. It was virtually absent from strain PAO1 during the exponential phase, but was produced abundantly in the stationary phase. In the gacA mutant, the expression of the 1698 sRNA was about two-fold lower than in the wild-type .The expression of the 11 sRNAs shown in Figure e Figure , suggeste Figure . We therP. fluorescens CHA0  is conserved in all Pseudomonas strains sequenced to date. In E. coli, the tatD product exhibits Mg-dependent DNase activity, but is not required for protein transport by the Tat pathway, contrary to what was originally expected [The genomic context of the expected .rgsA regulation by GacA during growth, we constructed rgsAPAO1-lacZ and rgsACHA0-lacZ transcriptional fusions in plasmids pME7235 and pME7234, respectively. The primers used for these constructions . In conclusion, transcription of the rgsA gene is probably activated indirectly by GacA and directly by RpoS, which is assumed to bind to the -10 promoter element , no significant difference was observed, compared with the native context (CHA207 containing the empty vector pME6032). Similarly, the rgsACHA0 overexpressing plasmid pME7236 was unable to elevate hcnA'-'lacZ expression in the rsmXYZ triple mutant (CHA1145/pME7236), which was indistinguishable from the strain carrying the vector alone (CHA1145/pME6032)  was found in an unpaired region of the RgsA sRNA of strain CHA0 Figure . The Gacorescens . We therpression . We meast Figure . When rg) Figure . In a co5 Figure . In concproteins .P. fluorescens to stress imposed by hydrogen peroxide [rgsA deletion mutant of strain CHA0 and tested its survival after a 30-min exposure to 40 mM H2O2 in NYB with shaking. This experiment was carried out three times with triplicate cultures. Only 69 ± 16% of the mutant cells survived, compared with 94 ± 12% surviving wild type cells.  Thus, survival was significantly (P < 0.05) higher in the wild type than in the mutant. Overexpression of rgsA afforded no significant effect. These results suggest that RgsA may contribute to oxidative stress response.The Gac/Rsm cascade modulates the response of peroxide . We consP. aeruginosa and other Pseudomonas spp. The identification of four of these sRNAs can be considered as a validation of the method, as they are either widespread in bacteria  or have been previously described in P. aeruginosa  [et al. [et al. [et al. [et al. [P. fluorescens Pf0-1 and P. fluorescens Pf-5, and by considering only the most widely conserved candidates we also demanded a higher level of conservation than did Livny et al. [The screening procedure adopted in our study enabled us to find evidence for 15 sRNAs in , PrrF2) . Three a [et al. , and the [et al. , we obta [et al.  had used [et al.  were noty et al. .P. aeruginosa [RsmY and RsmZ, the RsmA-binding sRNAs previously described in ruginosa ,34, wereruginosa ), just bP. aeruginosa PAO1 and P. fluorescens CHA0 the Gac/Rsm cascade regulates the expression of exoproducts such as HCN. In these organisms, a double rsmY rsmZ and triple rsmX rsmY rsmZ mutant, respectively, have the same exoproduct phenotype as gacA mutants [rpoS expression and, consequently, of the response to oxidative stress [gacA mutants, by comparison with the wild-type strains  and its derivatives PAO1-rpoS [gacA::Ω-Sm/Sp) [P. fluorescens CHA0 [gacA::Kmr) [hcnA'-'lacZ) [rpoS) [rsmXYZ, hcnA'-'lacZ) [rgsA, this study). E. coli DH5α [-1 or tetracycline at 25 μg ml-1 (100 μg ml-1 for pseudomonads) were added. For some RNA extractions, strains were also grown in GGP medium [E. coli and P. aeruginosa and 30°C for P. fluorescens. Survival of stationary phase P. fluorescens was assayed in NYB following H2O2 stress as described previously [We used AO1-rpoS  and PAO6Ω-Sm/Sp) , P. fluoens CHA0 , CHA89 (cA::Kmr) , CHA207 '-'lacZ) , CHA815 '-'lacZ)  and CHA1oli DH5α  served fP medium  or in MMP medium . To monieviously . StatistDNA manipulations were carried out according to standard procedures . PlasmidlacZ fusion to the rgsA gene from P. fluorescens CHA0, a 510-bp fragment containing the rgsA sRNA promoter was amplified from chromosomal DNA by PCR using primers 1698Fus1 (EcoRI) and 1698Fus2 (BamHI) listed in Additional file R, Stratagene). After sequencing, the fragment was cloned into the shuttle vector pME6016 [rgsACHA0 promoter controlling the expression of the lacZ gene. Similarly, we constructed pME7235 carrying a transcriptional lacZ fusion to the rgsA gene from P. aeruginosa PAO1 using primers 1698Fus3 (EcoRI) and 1698Fus4 (BamHI)  and 1698Sur2 (HindIII) .To overexpress rgsA gene in the P. fluorescens CHA0 chromosome was made as follows. A 860-bp fragment containing the upstream region and the rgsA promoter was amplified by PCR with primers 1698DelE (EcoRI) and 1698Del3 (NcoI)  and 1698DelX (XbaI)  was introduced into CHA0, giving CHA1181.A deletion of the on; Tcr; ). E. col1/pME497 . In thisP. aeruginosa PAO1 genome having a length exceeding 50 bp. The corresponding 3168 sequences were listed in FASTA format and compared by BLASTn to the genome sequences of P. putida KT2440 [P. syringae pv. tomato DC3000 [P. fluorescens SBW25 [P. fluorescens Pf0-1) [P. fluorescens Pf-5 [Pseudomonas strain, were given as input data into the program QRNA [Our screening was limited to the IgRs of the a KT2440 , P. syrio DC3000 , P. fluons SBW25 , P. fluos Pf0-1) , and P. 70) binding sites (TTGACAN(17)TATAAT), RpoN (σ54) binding sites (TGGCACN(5)TTGCW, where W is A or T, based on Barrios et al. [38)-binding sites (TGN(0–2)CCATACT, according to Lacour et al. [P. aeruginosa PAO1 sequencing project site [FUZZNUC, a free access nucleic acid pattern search program , was uses et al. ) or for r et al. ). We user et al.  for multr et al.  to ident), RpoN σ binding  et al. [2, pH 7.5), before mixing with lysis buffer . DIG-labelled probes were obtained as follows: PCR fragments were synthesized with P. aeruginosa or P. fluorescens genomic DNA as the template and pairs of primers listed in the Additional file +, GE-Amersham) and revealed by hybridization with the DIG-labeled probes described above and exposure to a light-sensitive film . 5S rRNA served as loading control. For this purpose, a 5S-rDNA probe was synthesized with primers 5S-rRNA-1 and 5S-rRNA-2  was added to the cultures to avoid cell aggregation. Samples were taken during various growth phases and permeabilized with 5% toluene. β-Galactosidase activities were then measured according to the Miller method [r method .BLAST, Basic Local Alignment Search Tool; CTAB, cetyl-trimethyl-ammonium bromide; DEPC, di-ethyl-pyrocarbonate; DIG, digoxigenin; EDTA, ethylene-di-amine-tetraacetic acid; GGP, iron-limited medium; HCN, hydrogen cyanide; IgR, intergenic region; MME, minimal medium E; NYB, nutrient yeast broth; SRNA, small RNA; URS, upstream regulating sequence; Xgal, 5-bromo-4-chloro-3-indolyl-β-D-galactoside;NG designed and carried out experiments, and drafted manuscript; SH and TJ executed the bioinformatics search; CV, EK and CR performed Northern blots; DH directed research and wrote the manuscript.Intergenic regions. List of IgRs of Pseudomonas aeruginosa PAO1 giving a positive result in QRNA analysis.Click here for filePredicted coordinates of three sRNA genes. Figure showing the predicted coordinates of sRNA genes in IgRs 645, 1887 and 2315.Click here for filePrimers. Primers used in this study for Northern blots and recombinant DNA work.Click here for file


Brachyspira pilosicoli colonizes the large intestine of various species of birds and mammals, including humans. It causes “intestinal spirochetosis”, a condition characterized by mild colitis, diarrhea and reduced growth. This study aimed to sequence and analyse the bacterial genome to investigate the genetic basis of its specialized ecology and virulence.The anaerobic spirochete B. pilosicoli 95/1000 was sequenced, assembled and compared with that of the pathogenic Brachyspira hyodysenteriae and a near-complete sequence of Brachyspira murdochii. The B. pilosicoli genome was circular, composed of 2,586,443 bp with a 27.9 mol% G+C content, and encoded 2,338 genes. The three Brachyspira species shared 1,087 genes and showed evidence of extensive genome rearrangements. Despite minor differences in predicted protein functional groups, the species had many similar features including core metabolic pathways. Genes distinguishing B. pilosicoli from B. hyodysenteriae included those for a previously undescribed bacteriophage that may be useful for genetic manipulation, for a glycine reductase complex allowing use of glycine whilst protecting from oxidative stress, and for aconitase and related enzymes in the incomplete TCA cycle, allowing glutamate synthesis and function of the cycle during oxidative stress. B. pilosicoli had substantially fewer methyl-accepting chemotaxis genes than B. hyodysenteriae and hence these species are likely to have different chemotactic responses that may help to explain their different host range and colonization sites. B. pilosicoli lacked the gene for a new putative hemolysin identified in B. hyodysenteriae WA1. Both B. pilosicoli and B. murdochii lacked the rfbBADC gene cluster found on the B. hyodysenteriae plasmid, and hence were predicted to have different lipooligosaccharide structures. Overall, B. pilosicoli 95/1000 had a variety of genes potentially contributing to virulence.The genome of B. pilosicoli 95/1000 will facilitate functional genomics studies aimed at elucidating host-pathogen interactions and virulence.The availability of the complete genome sequence of  Brachyspira are anaerobic bacteria that colonize the large intestine of animals and birds Brachyspira hyodysenteriae, the agent of a major pig disease called swine dysentery, and Brachyspira pilosicoli, the cause of a condition known as intestinal (or colonic) spirochetosis. B. pilosicoli has a wider host range than B. hyodysenteriae, and colonizes a variety of species, including human beings B. pilosicoli are particularly common in intensively housed pigs and chickens: colonized individuals may develop focal areas of inflammation in the large intestine, with diarrhea and reduced rates of growth and production. Colonization with B. pilosicoli also occurs at a high prevalence rate in human beings living in crowded and unhygienic conditions, particularly in developing countries B. pilosicoli as a pathogen, it has not been extensively studied. Progress has been hampered by the spirochete's specialised growth requirements and slow growth rate, as well as by a lack of genomic information and an absence of means for genetic manipulation.Spirochetes of the genus B. pilosicoli is the attachment of the spirochete by one cell end to the luminal surface of enterocytes in the large intestine; with time, dense mats of these attached bacteria may form a “false brush-border” covering the whole luminal surface of the enterocytes B. pilosicoli has been shown to induce apoptosis, actin rearrangement and increased expression of interleukin-1ß (IL-1ß) and IL-8 in vivo.A characteristic feature of infection with B. hyodysenteriae strain WA1 Brachyspira murdochii type strain 56-150T (DSM 12563) in GenBank represent important opportunities to advance research on these Brachyspira species. B. murdochii is generally considered to be a harmless commensal in pigs, chickens and rats B. pilosicoli strain 95/1000 was sequenced and subjected to comparative genomic analysis, particularly in relation to the sequences of WA1 and 56-150T. As the sequence of the latter was incomplete some comparisons were not appropriate. The overall aim of this work was to enhance knowledge about B. pilosicoli and its relationships to the other Brachyspira species, particularly in regard to identifying the genetic basis of their different ecologies and pathogenic potentials.The recent publication of the genome sequence of Brachyspira pilosicoli strain 95/1000 (ATCC BAA-1826) was originally isolated in Western Australia from the diarrheic feces of a commercial pig with porcine intestinal spirochetosis B. hyodysenteriaehttp://www.454.com/). To finish the genome sequence, remaining gaps were closed by PCR walking between un-linked contiguous sequences Preparation of genomic DNA, library construction and sequencing was as previously described for B. pilosicoli 95/1000 and for the incomplete genome sequence of B. murdochii 56-150T obtained from GenBank (ABTG00000000), sequence analysis and annotation were as previously described for B. hyodysenteriae WA1 T and WA1 were generated using Freckle, an in-house development of the Dotter tool For both the newly sequenced B. pilosicoli 95/1000 has been deposited in GenBank . Annotations and functional assignments for B. pilosicoli 95/1000 and for B. murdochii 56-150T also can be accessed at the CCG website .The complete nucleotide sequence and annotation of B. pilosicoli 95/1000, together with those of B. hyodysenteriae WA1 and the near-complete genome of B. murdochii 56-150T are summarised in B. pilosicoli 95/1000 consisted of a single circular chromosome of 2,586,443 bp . The overall G+C content of the B. pilosicoli genome was 27.9 mol%. Unlike B. hyodysenteriae WA1, B. pilosicoli 95/1000 did not contain a ∼36 Kb plasmid. Based on the available sequence it also appeared unlikely that B. murdochii contained a similar plasmid. The B. pilosicoli genome encoded 2,338 genes, with an overall 85% coding region. In comparison, B. murdochii 56-150T had 3,055 predicted genes in the seven contigs that were available. The percent coding region was similar across the three genomes. The average size of the predicted genes in B. pilosicoli 95/1000 was 997 bp, and tentative function was assigned to 1,645 of these. The B. pilosicoli strain had 569 more functionally assigned genes than did B. murdochii 56-150T and 282 less than B. hyodysenteriae. B. pilosicoli 95/1000 had 655 genes with unknown function and this was somewhat fewer than for B. hyodysenteriae (704), but much fewer than the extremely high number of unmatched genes for B. murdochii . A total of 1,201 (51.4%) genes in B. pilosicoli had matches in the COG database and 1,410 (60.3%) had matches in the KEGG database (B. pilosicoli open reading frames (ORFs) screened, 244 were predicted to encode proteins with signal peptides and 48 were predicted to have transmembrane helices. Eleven genes encoded proteins predicted to have seven transmembrane helices.The general features of the genome of 6,443 bp , making database . Of thesBrachyspira species had similar numbers of tRNA genes, representing all 20 amino acids  gene being about 645 Kb from the other two genes. This rRNA gene organisation has been noted earlier; it distinguishes the Brachyspira species from other spirochetes, and presumably pre-dates speciation in the Brachyspira genus Treponema pallidum rRNA genes appear to be arranged in two typical rrn operons Borrelia species, with rrs separated from rrl and rrf by a small segment of DNA (∼4 Kb). In Borrelia burgdorferi the rrf-rrl cluster is duplicated and is found immediately adjacent to the rrs-rrl-rrf cluster Leptospira species possess two copies each of rrs and rrl and one copy of rrfLeptospira biflexa contains two copies of each rRNA gene Brachyspira species, B. pilosicoli is more distantly related to B. hyodysenteriae than is B. murdochii (The three no acids . As withurdochii .B. pilosicoli 95/1000 were predicted based on the position of dnaA (BP951000_0595), as bacterial chromosome replication origins are typically located near this gene. The putative oriC origin of replication was identified in an AT-rich intergenic region upstream of dnaA, in the vicinity of a cluster of hypothetical DnaA boxes. Similar putative oriCs with comparable DnaA boxes have been identified in other spirochete genomes  Cell motility, (U) Intracellular trafficking, secretion, and vesicular transport, (C) Energy production and conversion, (H) Co-enzyme transport and metabolism, and (I) Lipid transport and metabolism. On the other hand, B. hyodysenteriae had more genes functioning in (K) Transcription, (V) Defense mechanisms, (T) Signal transduction mechanisms, (O) Posttranslational modification, protein turnover, chaperones, (G) Carbohydrate transport and metabolism, (E) Amino acid transport and metabolism, (P) Inorganic ion transport and metabolism, and (Q) Secondary metabolites biosynthesis, transport and catabolism. B. murdochii had more genes involved with (L) Replication, recombination and repair, and (M) Cell wall/membrane/envelope biogenesis. It was noteworthy that B. pilosicoli had the largest number of genes involved in energy production and conversion, and this capacity may enhance its potential to colonize various hosts.The COG categories of the protein-coding genes are shown in B. hyodysenteriae lacks many genes for the biosynthesis of small molecules, and therefore must acquire these from the environment B. pilosicoli was predicted to contain substantially fewer genes involved in inorganic ion transport and metabolism (P) than both B. hyodysenteriae and B. murdochii  database at NCBI identified greatest similarities with B. hyodysenteriae and B. murdochii. However, as with B. hyodysenteriaeClostridium species (∼10%) and E. coli (∼5%). The three sequenced Brachyspira species contained a total of 1,087 conserved or “core” genes  were conserved in B. hyodysenteriae; therefore, most of the genome-inferred metabolic potential of B. hyodysenteriae described previously can be extrapolated to B. pilosicoli. Only an additional 99 genes were conserved between B. pilosicoli and B. murdochii, whereas an additional 311 were conserved between B. hyodysenteriae and B. murdochii. B. hyodysenteriae had 1,014 genes that were not shared with B. pilosicoli, whereas B. murdochii contained a remarkable 1,900 genes not found in B. pilosicoli. On this basis, B. pilosicoli seemed to be more similar to B. hyodysenteriae than to B. murdochii. A large proportion of the unique genes in both B. hyodysenteriae and B. murdochii were of unknown function , or encoding transcriptional regulators, transporters and predicted surface antigens, as well as forming a bacteriophage region. Selected differences found in B. pilosicoli that appear to be potentially significant are described below.Although the Brachyspira pilosicoli 95/1000 contained a 25 Kb region  encoding components of an integrated bacteriophage ; OrfG VSH-1 phage protein homolog (BP951000_1459); putative Lys - endolysin; glycoside hydrolase (BP951000_1461); putative RNA polymerase domain-containing protein (BP951000_1463); phage tail tape measure protein homolog, TP901 family (BP951000_1469); bacteriophage TP901-1 like family protein homolog (BP951000_1474); putative DNA packaging, Phage QLRG family protein (BP951000_1476); phage major capsid protein homolog, HK97 family (BP951000_1477); phage prohead protease (BP951000_1478); putative phage portal protein, HK97 family (BP951000_1479); DNA methylase N-4/N-6 domain protein homolog (BP951000_1480); putative phage terminase, small subunit, P27 family protein (BP951000_1481); and putative phage terminase, large subunit (BP951000_1482). The GC content in this bacteriophage region was 29.65%, which was slightly higher than the average for the whole genome (27.90%). It is not yet clear whether this integrated bacteriophage is functional, or capable of transferring genetic material between B. pilosicoli strains. If it is, it could be developed into a tool for genetic manipulation of the spirochete. By analogy with other prophages, it potentially also could be involved in lysogenic conversion, for example modifying the spirochete phenotype to enhance its virulence B. pilosicoli strains. B. hyodysenteriae WA1 also contained two genes predicted to be bacteriophage-associated: phage terminase large subunit  and integrase (BHWA1_02688), but these were distinct from the bacteriophage genes found in B. pilosicoli. A predicted integrase-recombinase protein (4083292.C14.orf00918) was found in B. murdochii, but it only had 25.61% identity at the protein level.riophage . The 29 B. pilosicoli 95/1000 was distinct from the smaller prophage-like gene transfer agent (GTA) that previously was described in the genomes of B. pilosicoli 95/1000 and in B. hyodysenteriae WA-1 B. hyodysenteriae B204 as VSH-1 B. intermedia HB60 B. pilosicoli 95/1000 B. murdochii 56-150T was found to have a cluster of 31 genes on contig 4083292.C21 (orf00108–orf00140) that included many of the described components of the GTA, as well as 17 hypothetical proteins. Genes for Hvp19/Hvp22, Hvp13, Hvp38, and Hvp28 were not identified in the partial genome of B. murdochii. Again, the known genes showed rearrangements from the order found in B. hyodysenteriae and B. pilosicoli, and it is unclear whether the B. murdochii GTA is functional.The novel bacteriophage region in B. pilosicoli 95/1000 and B. murdochii 56-150T differed from B. hyodysenteriae WA1 in that they possessed nine genes within a glycine reductase complex (grd cluster): this arrangement is similar to that in the anaerobic bacterium Clostridium sticklandiiB. pilosicoli included genes encoding the GrdX protein , thioredoxin reductase , glycine reductase , sarcosine reductase , glycine reductase complex selenoprotein B , two copies for the glycine/sarcosine/betaine reductase complex, component C, alpha subunit , and two copies for a sodium∶alanine symporter family protein (BP951000_1859 and BP951000_1860). In B.murdochii, the locus consisted of genes encoding putative glycine reductase complex component  thioredoxin reductase , glycine/sarcosine/betaine reductase component B alpha/beta subunit , two copies of glycine reductase complex selenoprotein A , two copies of selenoprotein B, glycine/betaine/sarcosine/D-proline reductase family , 3-oxoacyl-(acyl-carrier-protein) synthase III , and fatty acid/phospholipid biosynthesis enzyme .grdX preceded the thioredoxin/glycine reductase gene cluster in both B. pilosicoli and B. murdochii, and it is known to be co-transcribed in C. sticklandiitrxB were identified in the B. pilosicoli genome (BP951000_1651 and BP951000_1853). Other genes in B. pilosicoli and B. murdochii that were outside the cluster and which encoded proteins with predicted selenocysteine sites were thioredoxin  and glutathione peroxidase . Given the potential role of these selenoproteins in antioxidant functions, deficiency of selenium may have detrimental effects on their ability to withstand oxidative stress.ORF B. pilosicoli and B. murdochii therefore were predicted to be able to utilise glycine anaerobically as a sole source of carbon and energy, and hence have a distinct energy-conserving mechanism using an internal reaction in which glycine serves as an electron donor during oxidation by a glycine cleavage system, or as an electron acceptor when being reduced by glycine reductase. In contrast, the lack of this complex in B. hyodysenteriae suggested that it is unable to ferment glycine to act as a carbon and energy source. The difference between the species suggests that high levels of glycine might favor B. pilosicoli and B. murdochii populations over B. hyodysenteriae, either in vitro or in vivo. The glycine reductase complex is predicted to play an important role in allowing B. pilosicoli to successful colonize a broader range of host species than B. hyodysenteriae, and it also may confer advantages to B. murdochii. Clearly, further research is required to determine whether glycine does enhance growth of B. pilosicoli and B. murdochii, as predicted.The glycine reductase complex catalyses the reductive deamination of glycine to acetylphosphate and ammonia with the generation of ATP from ADP and orthophosphate grd cluster in B. hyodysenteriae may make it unable to synthesise glutathione and glutaredoxins, and therefore it was predicted to be less able to withstand oxidative stress than the other two species. Only two copies of a gene encoding thioredoxin reductase were identified in B. hyodysenteriae . The predicted ability of B. pilosicoli to rapidly respond to an oxidative stress or a redox insult was consistent with its reported ability to survive outside the host for a longer period than B. hyodysenteriaeB. hyodysenteriae, such as in the bloodstream of immunocompromised patients B. murdochii also has been isolated from extra-intestinal sites B. pilosicoli more adaptable in terms of its abilities to colonize the large intestines of a wide variety of host species, where environmental conditions are likely to vary.The absence of the B. pilosicoli 95/1000, whilst one putative copy was found in B. murdochii (4083292.C42.orf00884), and none in B. hyodysenteriae. Sulfatases play important roles in the cycling of sulfur in the environment, in the degradation of sulfated glycosaminoglycans and glycolipids in the lysosome, and in remodeling sulfated glycosaminoglycans in the extracellular space. The sulfatase genes potentially could encode for enzymes that modify gycosaminoglycans to generate binding sites required for attachment of B. pilosicoliThree copies of genes encoding sulfatases  were identified in fucT, BP951000_1232 and BP951000_1235) and beta-1, 3-galactosyltransferase (BP951000_1768) were identified in B. pilosicoli 95/1000: these are key enzymes in the biosynthesis of Lewis antigens, structures found on the surface of human erythrocytes and epithelial cells. The gastric pathogen Helicobacter pylori can express Lewis and related antigens in the O-chains of its surface lipopolysaccharide B. pilosicoli expresses Lewis antigens, and to investigate their potential involvement in the pathogenesis of infection by this spirochete species.A novel set of two copies of genes encoding alpha-1, 2-fucosyltransferase  were identified in B. pilosicoli 95/1000, but not in B. hyodysenteriae or B. murdochii. NanA proteins are produced by a wide variety of mucosal pathogens, and are a potential virulence factor in bacteria B. pilosicoli colonization or induce tissue damage.Three copies of a gene encoding a sialidase (neuraminidase) family protein homolog , and this was not present in B. hyodysenteriae. Aconitase is a tricarboxylic acid (TCA) cycle protein that catalyses the conversion of citrate to isocitrate, and, amongst other things, has been implicated in the virulence of Staphylococcus aureusBrachyspira species contained genes for a complete TCA cycle, this suggests that retention of the aconitase gene may have an important functional significance. Two other genes were located adjacent to the aconitase gene in both B. pilosicoli and B. murdochii, these being putative citrate synthase (BP951000_0368 and 4083292.C42.orf00523) and putative isocitrate dehydrogenase (BP951000_0369 and 4083292.C42.orf00525). They were not identified in B. hyodysenteriae WA1. The presence of aconitase implies that B. pilosicoli and B murdochii can utilise the TCA to produce glutamate, and this capacity is discussed later in the section on central metabolic pathways.B. pilosicoli 95/1000, compared to 303 in B. hyodysenteriae WA1 and 142 in B. murdochii 56-150T. The number of genes in the different categories for all three Brachyspira species is shown in B. murdochii was complicated by the fact that the genome was incomplete, and additional genes are likely to be identified once a complete genome sequence becomes available. Overall, apart from what is shown, the predicted gene products did not have significant similarities with those of other well-characterized toxins or adhesins described in the major species of enteropathogenic bacteria - such as in E. coli or Clostridium species. Nevertheless, it is important to remember that other virulence-associated genes are likely to exist amongst those that are currently recorded as encoding proteins that are hypothetical or of unknown function.Independent of the previous analysis, screening of all coding sequences (CDS) of the three species using Blast, SignalP, PSORT and TMHMM to look for similarities to genes reported to be associated with virulence in other bacteria identified a total of 235 genes with putative roles in virulence in B. hyodysenteriae, the most pathogenic of the three species, contained more genes involved with lipopolysaccharide biosynthesis, motility and chemotaxis, and adhesion and/or surface proteins than did the other two species. The latter two did not have more of any category of these potential virulence-associated genes, apart from the bacteriophage genes in B. pilosicoli.It was of interest that Brachyspira species is considered to contain lipoologosaccharide (LOS) rather than lipopolysaccharide, based on the lack of a typical Gram-negative ladder formation of repeated O-sugar components on silver-stained SDS-PAGE gels B. hyodysenteriaeBrachyspira species. LOS is also antigenic, and protective immunity to B. hyodysenteriae is specific to the LOS serogroup B. pilosicoli is less clear, as pigs experimentally infected with B. pilosicoli 95/1000 did not develop a systemic antibody response against the spirochete The outer envelope of the B. pilosicoli contained a set of 25 core genes involved in the biosynthesis of LOS  that are likely to be involved in O-antigen assimilation. A similar rfbBADC cluster was not found in B. pilosicoli or in B. murdochii, implying that they were unable to produce these antigens. Similar rfb gene clusters have been linked to virulence in numerous Gram negative bacteria Salmonella enterica serovars rfb cluster in the virulence of B. hyodysenteriae. It was interesting that despite the presence of genes encoding glycosyltransferases in this pathway, B. hyodysenteriae does not appear to produce ladder-like O-antigens Brachyspira species will be to make a detailed comparison of polysaccharide content and structures. It is unfortunate that no such structural data currently are available.s of LOS . This waBrachyspira species, allowing them to colonize specialized niches in the complex nutritional, physical and microbiological environment of the large intestine. Interestingly, a striking difference was found in the numbers of chemotaxis genes. B. hyodysenteriae contained 52 genes associated with chemotaxis, compared to 27 in B. pilosicoli and 39 in the partial genome of B. murdochii [B. pilosicoli having no mcpC genes. These differences are likely to be highly significant in relation to the chemotactic signals that the different species can respond to: they may explain the differences that have been observed in the attraction of B. pilosicoli and B. hyodysenteriae to mucin, and their tendency to occupy different local niches within the large intestine B. hyodysenteriae colonizes the lumen of the colon, but also penetrates deep into the colonic crypts, where it enters goblet cells. Although B. pilosicoli also can enter the crypts, unlike B. hyodysenteriae it penetrates the dense mucus layer at the crypt shoulders, and attaches to the underlying colonic enterocytes. As B. hyodysenteriae has a much more limited host range than B. pilosicoli, it seems paradoxical that it has a greater number and variety of methyl-accepting chemotaxis genes.The capacity for chemotactic responses and motility are extremely important for urdochii . The disB. pilosicoli contained 13 chemosensory transducer genes, and it was interesting that seven of these, and genes for five hypothetical proteins, were located within a cluster at a ∼5.7 Kb locus. This locus comprised cheB (BP951000_0460), cheD (BP951000_0461), cheR (BP951000_0462), cheW (BP951000_0463), cheB (BP951000_0464), cheY , and cheX (BP951000_0471). Similarly, six of the chemosensory transducer genes of B. hyodysenteriae were within one cluster at a locus of similar size. These genes encoded chemotaxis response regulator , chemotaxis histidine kinase , chemotaxis protein , chemotaxis protein methyltransferase , chemotaxis protein , and response regulator receiver modulated CheB methylesterase . The other chemosensory transduction genes were located elsewhere on the two genomes, and a similar clustering of che genes was not found in B. murdochii.Brachyspira strains . Such lipoproteins, where they are surface associated, are likely to be important as potentially targets for immune recognition and could be used as subunit vaccine candidates Brachyspira species was the presence of a gene encoding a peptidoglycan-associated outer membrane lipoprotein  in B. hyodysenteriae that was absent in B. pilosicoli. PAL is involved in maintenance of the integrity of the cell envelope As with other bacteria, predicted surface associated proteins and lipoproteins of the B. hyodysenteriae has been reported to have a set of variable surface proteins of about 39 KDa B. pilosicoli contained single copies of the genes for VspD, VspE and VspH located in different regions, while B. murdochii 56-150T had three copies of vspG, two of vspF and one of vspG. Differential expression of these genes may be involved in immune evasion, allowing persistence of the spirochetes B. pilosicoli 95/1000 contained three copies of bspA, predicted to encode a BspA-like surface protein. BspA-like proteins are expressed on the surface of many pathogenic bacteria: they may bind to fibronectin, stimulate a significant serological response Amongst the other outer membrane proteins, B. hyodysenteriae is strongly hemolytic, whereas B. pilosicoli and B. murdochii are weakly hemolytic B. hyodysenteriae is thought to be an important virulence trait B. hyodysenteriae WA1 tlyA, tlyB, tlyC, and acpP , ATP-dependent Clp protease proteolytic subunit , hemolysin C , hemolysin related protein , acyl carrier protein: contains beta-hemolysin , putative hemolysin III (BP951000_0424), and putative channel protein hemolysin III family protein (BP951000_0888). The eighth was a putative hemolysin (BP951000_2207) that was similar to a protein of Parabacteroides distasonis ATCC 8503, with ∼42.9% protein identity. B. pilosicoli lacked a gene with similarity to that encoding the new putative hemolysin of B. hyodysenteriae (BHWA1_00962). From this, it is possible that the new putative hemolysin identified in B. hyodysenteriae is responsible for its strongly hemolytic phenotype. Alternatively, it has been suggested that differences between B. hyodysenteriae and the weakly hemolytic Brachyspira species such as B. pilosicoli in the FabF ACP synthase and FabG ACP reductase proteins that flank the acyl carrier protein (HlyA) may result in different lipid moieties being attached to the HlyA proteins E. coliB. hyodysenteriae.Only single copies of B. hyodysenteriae WA1, B. pilosicoli 95/1000 and B. murdochii 56-150T contained similar numbers of genes predicted to encode proteases . The proteolytic capacity in all three species was linked with the large number of ORFs encoding enzymes involved in uptake and metabolism of amino acids. These proteases are likely to be required for survival in the intestinal environment, but also may be involved in induction of local destruction of host tissues, and hence contribute to virulence. A serine protease previously described in B. pilosicoli  B. murdochii 56-150T (ZP_04048074) and B. hyodysenteriae WA1 (YP_002721653).B. hyodysenteriaeB. pilosicoli lacked genes for the specialized secretion systems found in pathogenic Gram-negative bacteria, although it had genes for the Sec pathway and ABC-type transporters. Six sec genes were identified: secA (BP951000_0230), secY (BP951000_1006), secF (BP951000_1532), secD (BP951000_1533), secE (BP951000_1559) and secG (BP951000_1896), and there were 36 genes encoding ABC transporters . Ten flagella-associated genes that can form part of a type III secretary system were found in B. pilosicoli 95/1000, but needle associated genes that encode proteins required for the injection of toxins into the host cell were not identified.As with B. pilosicoli showed that it shared many metabolic capabilities with B. hyodysenteriae. The pathways for B. pilosicoli are shown in B. hyodysenteriae by examining B. hyodysenteriae, a complete set of genes for the non-oxidative pentose phosphate pathway, nucleotide metabolism and a respiratory electron transport chain were identified in B. pilosicoli and B. murdochii. It can be seen from the figures that some of the obvious ways in which B. pilosicoli differed from B. hyodysenteriae related to the presence of pathways associated with citrate and 2-oxo-glutarate, a pathway from acetyl-CoA to ethanol, and a lack of the rfbBADC genes located on the B. hyodysenteriae plasmid (predicted to be involved in O-antigen biosynthesis).A reconstruction of the central metabolic pathways of B. hyodysenteriae, B. pilosicoli and B. murdochii both appeared to have the capacity to use enzymes of the incomplete TCA to synthesize glutamate. Relevant predicted enzymes in the two species were putative citrate synthase , putative isocitrate dehydrogenase , aconitase (BP951000_0369 and 4083292.C42.orf00530), malate dehydrogenase , and two copies of fumarate hydratase in B. pilosicoli : fumA was not indentified in B. murdochii, possibly due to the genome being incomplete. This capacity emphasized the dual function of the TCA portion of the cycle: provision of NADH as part of the complete cycle in aerobes, and provision of alpha-oxaglutarate in aerobes and anaerobes. Aconitase allows the TCA cycle to function during periods of oxidative stress, and may play a role in mediating oxidative stress and regulating cell death B. pilosicoli and B. murdochii.Unlike B. pilosicoli and B. murdochii involved 2-oxoglutarate, succinyl-CoA and fumarate, precursors that could proceed in either the oxidative or reductive direction. Unlike B. hyodysenteriae, B. pilosicoli and B. murdochii also had a gene set for the production of ethanol from acetyl-CoA via a branched fermentation pathway. The significance of this difference between the species is unknown, but it is likely to influence overall metabolic flexibility.A key branch in the partial TCA cycle of B. pilosicoli can be distinguished from all the other Brachyspira species in that it lacks beta-glucosidase activity B. pilosicoli genome; however, a novel system for metabolizing multiple sugars, including beta-glucosides was identified that involved alpha-galactosidase/6-phospho-beta-glucosidase , PTS system, arbutin-like IIC component  and 6-phospho-alpha-glucosidase . These enzymes have specificity for 6-phospho-beta-D-glycoside substrates rather than the beta-D-glycoside substrates of beta-glucosidases Brachyspira species as they are known to regulate growth, adhesion and colonization in other bacterial species B. hyodysenteriae and B. murdochii had genes encoding phosphoenolpyruvate synthase, whereas B. pilosicoli did not appear to have the capacity to interconvert phosphoenolpyruvate (PEP) and pyruvate. Instead, it may utilize glycerate but not pyruvate for growth, and use pyruvate kinase  to catalyse the reverse conversion of PEP to pyruvic acid B. hyodysenteriae and B. murdochii possessed a complete set of genes for histidine metabolism, while B. pilosicoli did not. It is possible that B. pilosicoli has an alternative pathway for synthesizing histidine. It may however also obtain nutrients from the surrounding environment, particularly as the intestine is a rich source of amino acids, including histidine B. pilosicoli, and not in the other two species.B. hyodysenteriaeB. murdochii. In contrast, an incomplete gene set was identified in B. pilosicoli, with only the genes for (3R)-hydroxymyristoyl-(acyl carrier protein) dehydratase , malonyl CoA acyl carrier protein transacylase , and the previously mentioned locus comprising ACP synthase II , acyl carrier protein  and acyl reductase  being identified while FabZ provides the 3-hydroxymyristate moieties characteristic of lipid A. FabD is involved in elongation reactions in fatty acid synthesis. These findings suggested that B. pilosicoli does not incorporate carbon from simple carbon sources into fatty acids.A complete fatty acid biosynthesis gene set previously has been found in B. pilosicoli, a widespread but somewhat neglected enteric pathogen of humans and animals, was obtained and made available for analysis by the scientific community. The spirochete genome was shown to have basic similarities to the genomes of the related species B. hyodysenteriae and B. murdochii, but a number of distinct features were identified that may help to explain the different host ranges and colonization sites of the species. These included predicted differences in chemotactic responses, oxygen sensitivity, the use of some different substrates, the presence of different surface proteins, and predicted differences in LOS content and structure. The first description of genes for an apparently complete bacteriophage in B. pilosicoli also was made: this finding has important practical implications, since if the bacteriophage can be shown to be functional it could be used for genetic manipulation of B. pilosicoli. The current lack of such a genetic tool for B. pilosicoli is a constraint that limits functional studies of the genes that have been identified.In this study the complete genome sequence of Figure S1B. pilosicoli 95/1000 and B. hyodysenteriae WA1 displayed using the Artemis Comparison Tool. Regions of DNA:DNA similarity are joined by lines.Whole-genome comparisons between (2.24 MB TIF)Click here for additional data file.


Clonorchis sinensis and Opisthorchis viverrini, have a major impact on the health of tens of millions of humans throughout Asia. The greatest impact is through the malignant cancer ( = cholangiocarcinoma) that these parasites induce in chronically infected people. Therefore, both C. sinensis and O. viverrini have been classified by the World Health Organization (WHO) as Group 1 carcinogens. Despite their impact, little is known about these parasites and their interplay with the host at the molecular level. Recent advances in genomics and bioinformatics provide unique opportunities to gain improved insights into the biology of parasites as well as their relationships with their hosts at the molecular level. The present study elucidates the transcriptomes of C. sinensis and O. viverrini using a platform based on next-generation (high throughput) sequencing and advanced in silico analyses. From 500,000 sequences, >50,000 sequences were assembled for each species and categorized as biologically relevant based on homology searches, gene ontology and/or pathway mapping. The results of the present study could assist in defining molecules that are essential for the development, reproduction and survival of liver flukes and/or that are linked to the development of cholangiocarcinoma. This study also lays a foundation for future genomic and proteomic research of C. sinensis and O. viverrini and the cancers that they are known to induce, as well as novel intervention strategies.The two parasitic trematodes,  Clonorchis sinensis and Opisthorchis viverrini, have a serious impact on the health of tens of millions of people throughout Asia. The greatest impact, however, is through the malignant, untreatable cancer (cholangiocarcinoma) that these parasites induce in chronically infected people. These liver flukes are officially classified by the World Health Organization (WHO) as Group 1 carcinogens. In spite of their massive impact on human health, little is known about these parasites and their relationship with the host at the molecular level. Here, we provide the first detailed insight into the transcriptomes of these flukes, providing a solid foundation for all of the molecular/-omic work required to understand their biology, but, more importantly, to elucidate key aspects of the induction of cholangiocarcinoma. Although our focus has been on the parasites, the implications will extend far beyond the study of parasitic disease. Importantly, insights into the pathogenesis of the infection are likely to have major implications for the study and understanding of other cancers.The parasitic worms,  Clonorchis sinensis and Opisthorchis viverrini, which cause the diseases clonorchiasis and opisthorchiasis, respectively, represent a substantial public health problem in many parts of Asia Clonorchis sinensis is endemic predominantly in regions of China (including Hong Kong and Taiwan), Korea and North Vietnam O. viverrini is endemic throughout Thailand, the Lao People's Democratic Republic, Vietnam and Cambodia C. sinensis) and Khon Kaen province in Thailand (O. viverrini) O. felineus, is endemic in Siberia and eastern regions of the former USSR, and causes a similar disease and disease burden to O. viverrini and C. sinensisLiver flukes (Platyhelminthes: Digenea) include important food-borne eukaryotic pathogens of humans C. sinensis and O. viverrini are similar C. sinensis only) as intermediate hosts. Fish–eating ( = piscivorous) mammals, including humans, dogs and cats, act as definitive hosts, in which sexual reproduction occurs. Clonorchiasis and opisthorchiasis are prevalent in geographical regions where raw cyprinid fish (C. sinensis and O. viverrini) and/or shrimp (C. sinensis) are a staple of the diet of humans C. sinensis and O. viverrini infections in the etiology of cholangiocarcinoma, a malignant cancer of the bile ducts in humans which has a very poor prognosis. Indeed, C. sinensis and O. viverrini are both categorized by the International Agency for Research on Cancer (IARC) as Group 1 carcinogens The life cycles of O. viverrini is highly endemic, the incidence of cholangiocarcinoma is unprecedented In humans, the onset of cholangiocarcinoma occurs with chronic clonorchiasis or opisthorchiasis, associated with hepatobiliary damage, inflammation, periductal fibrosis and/or cellular responses to antigens from the infecting fluke via education programs), the ancient cultural custom to consume raw, undercooked or freshly pickled fish and shrimp persists in endemic areas O. viverrini-infected hamsters with praziquantel (400 mg per kg of live weight) has been shown to induce a dispersion of parasite antigens, resulting in adverse immunopathological changes as a result of oxidative and nitrative stresses following re-infection with O. viverriniC. sinensis and O. viverrini, there is substantial merit in searching for new intervention methods, built on a detailed understanding of the interplay between the parasites and their hosts as well as the biology of the parasites themselves at the molecular level. Furthermore, the characterization of the genes expressed in these parasites should assist in elucidating the molecular mechanisms by which clonorchiasis and opisthorchiasis (or the respective parasites) initiate and enhance the development of cholangiocarcinoma Currently, there is no effective chemotherapy to combat cholangiocarcinoma, such that intervention strategies need to rely on the prevention or treatment of liver fluke infection/disease. Although effective prevention could be readily achieved by persuading people to consume cooked fish and shrimp  are publicly available for C. sinensisO. viverriniTo date, most molecular biological research of socioeconomically important trematodes has focused on the human blood flukes, Fasciola hepatica using next-generation sequencing-bioinformatic platform has discovered numerous molecules of biological relevance, some of which are inferred to be involved in key biological processes or pathways that could serve as key targets for new trematocidal drugs or vaccines C. sinensis and O. viverrini, in order to provide essential resources for future genomic, proteomic, metabolomic and systems biological explorations of these important pathogens, and to underpin future efforts toward the improved intervention and control of cholangiocarcinoma.Some recent genomic, bioinformatic and proteomic studies C. sinensis) and the Khon Kaen province, Thailand (O. viverrini). Helminth-free inbred Syrian golden hamsters (Mesocricetus auratus) were infected with metacercariae of each species as described previously C. sinensis) to 42 (O. viverrini) days after infection, adult flukes were collected from the bile ducts of hamsters and cultured in vitro to allow the worms to regurgitate caecal contents using an established procedure Metacercariae were collected from naturally infected cyprinoid fish, using established methods C. sinensis and O. viverrini were characterized by 454 sequencing (Roche) from normalized, complementary DNA (cDNA) libraries  following the approach applied to F. hepaticaC. sinensis and O. viverrini, and polyadenylated (polyA+) RNA was then purified from 25 µg of pooled total RNA. First-strand cDNA synthesis of polyA+ RNA was primed using a hybrid, random hexamer (N6) oligonucleotide containing a specifically designed adapter (5′- TCGCAGTGAGTGACAGGCCA-3′) and transcribed using M-MLV H− reverse transcriptase. After RNA hydrolysis, a specifically designed adapter primer was attached to the 3′-end of the first-strand cDNA (5′-AGTCAGGACCTTGGCTGTCACTC-3′). The adapter sequences on both ends of the cDNA were then used to synthesize second-strand cDNA and amplify (18 cycles) the cDNA employing oligonucleotides complementary to the adapters by long and accurate PCR (LA-PCR) 5′- CCATCTCATCCCTGCGTGTCTCCGACTCAG -3′) and B (3′- CTGAGACTGCCAAGGCACACAGGGGATAGG -5′)  were added to the 5′- and 3′-ends of the cDNA, respectively. Normalization was conducted using one cycle of denaturation and reassociation of the cDNA. Reassociated double-stranded cDNA was separated from the remaining single-stranded cDNA  by purification on a hydroxylapatite column The transcriptomes of both in silico-assembly pipeline (Eurofins MWG Operon) was used to assemble de novo the sequence data for each C. sinensis and O. viverrini. High quality, base-called and clipped reads from each data set were extracted from the SFF-files and their contigs assembled using MIRA v.2.9 (http://chevreux.org/projects_mira.html) ftp://ftp.ncbi.nlm.nih.gov/blast/executables/), allowing sequences to cluster if they aligned across >60% of their length and shared >95% amino acid residue identity.An automated, C. sinensis and O. viverrini were each annotated using a semi-automated bioinformatic pipeline http://www.ncbi.nlm.nih.gov/est/) for C. sinensis , O. viverrini  and non-redundant sequence databases; ENSEMBL (http://www.ensembl.org/); SchistoDB (http://schistodb.net/schistodb20/) for S. mansoni; and the Shanghai Centre for Life Science & Biotechnology Information (http://lifecenter.sgst.cn/sjapathdb/data.html) for S. japonicum as well as a transcriptomic data set available for F. hepatica−05), moderate (<1E−15) and/or stringent (<1E−30) search strategies. Homologues were identified in other eukaryotic organisms using permissive, moderate and stringent search strategies. ORFs were predicted from the final transcriptomic data sets for C. sinensis and O. viverrini using ESTScan, and proteins were inferred from ORFs by conceptual translation. Predicted proteins were classified functionally using InterProScan C. sinensis and O. viverrini were individually classified according Gene Ontology (GO) categories and assigned parental (i.e. level 2) terms (http://www.geneontology.org/). Inferred proteins with homologues in organisms for which sequence data were available were subjected to analysis, utilizing KEGG-Orthology Based Annotation System (KOBAS) The transcriptome data sets for C. sinensis and O. viverrini using a previously described bioinformatic pipeline http://www.signalpeptide.de/), and the subset of proteins with known homologues  were retained and summarized based on the biochemical pathway inferred using KOBAS.Amino acid sequences were subjected to analysis using TMHMM (a membrane topology prediction program) C. sinensis  and O. viverrini  (http://www.ncbi.nlm.nih.gov/sra). BLASTn searches  revealed that most (92–97%) of sequences available in public databases for these flukes were contained within their respective data sets. As most (88–91%) sequences generated for each species were novel, only the present data were assembled  and 101,654  sequences were singletons and could thus not be assembled.More than 500,000 sequences were generated for each 3 bases) . Sequencbled see . The assC. sinensis sequences (415±363 bases) and 162,487 O. viverrini sequences (447±348 bases) were retained for further analyses. From the MIRA-assembled data, ORFs were predicted for 88,714 (66.1%) of C. sinensis sequences (383±371 bases) and 107,217 (66.0%) of O. viverrini sequences (389±355 bases). CAP3 clustered approximately half of these ORFs into ORF-enriched supercontigs, equating to 12,050 sequences (980±747 bases) for C. sinensis and 14,698 sequences (939±731 bases) for O. viverrini, with an average depth of coverage of 3.6–3.7 reads per supercontig for each species. For each species, the average G+C content (∼47±4%) was similar to the estimates for F. hepatica, a digenean trematode related to C. sinensis and O. viverrini−05) and protein  levels to potential host (M. auratus) molecules or microbial organisms were excluded. The ORFs of both clustered and unique sequences (singletons) were subjected to further analysis.In total, 134,301 C. sinensis and O. viverrini were each used to interrogate genomic databases  using BLASTx  O. viverrini sequences matched known proteins at a cut-off value of <1E−05  Saccharomyces cerevisiae (yeast) (ii) F. hepatica, S. mansoni and S. japonicum (trematodes) (iii) Caenorhabditis elegans (nematode) (iv) Drosophila melanogaster (insect), (v) Danio rerio, Gallus gallus, Xenopus tropicalis , and (vi) Homo sapiens and Mus musculus   and O. viverrini  had the highest homology to one another using the permissive , moderate  and stringent  search strategies. Both C. sinensis and O. viverrini shared greatest amino acid sequence similarity to proteins of other members of the Trematoda considered here, resulting in 14,526–27,103 sequence matches (28.6–53.4%) for the former and in 15,982–29,995 matches (26.0–48.8%) for the latter species . In agreement with the data available for Schistosoma spp. F. hepaticaC. sinensis and O. viverrini shared greater amino acid sequence similarity  to mammalian proteins  than to those of C. elegans .The transcriptomic data sets for g BLASTx . Of the f <1E−05 . Proteinmammals) . Protein−05) amongst representatives of the families Fasciolidae (F. hepatica), Schistosomatidae (S. mansoni) and Opisthorchiidae (C. sinensis or O. viverrini) , moderate  and stringent  search strategies ; 9,954–10,983 sequences (18–19.6%) had significant matches . A significant percentage  of the proteins predicted for the two Asian liver flukes were conserved across the eukaryotic model organisms considered. These molecules included actin-like proteins, alpha and beta-tubulins, dynein-1-alpha heavy chain, elongation factor EF-2, enolase, glycogen synthase 1, heat shock protein 70, nucleosome assembly protein 1-like protein and ubiquitin-activating enzyme E1 , of which most sequence matches  were to proteins inferred for S. cerevisiae  was shared  . More prrategies . Relativrevisiae .C. sinensis and O. viverrini with homology to those within non-redundant gene data sets  were clustered , the number of homologous sequences predicted to encode proteins was 1.4 to 2.4-fold greater than expected  GO terms assigned to the data sets for each F. hepaticaS. mansoni  were represented in the transcriptomic data sets of the two Asian flukes  cellular processes (35–36%), such as protein amino acid phosphorylation, translation and regulation of transcription; (ii) metabolic processes (33–34%), such as proteolysis, carbohydrate metabolic process and oxidation reduction; and, (iii) biological regulation processes (8%), such as regulation of transcription and signal transduction  biological pathway terms, respectively  and transmembrane motifs  were identified (C. sinensis and (particularly) O. viverrini were linked with biological pathways which are recognized or considered to be linked to carcinogenesis (<20 matches) in humans. These molecules include homologues to human proteins, such as calmodulin, c-Jun N-terminal kinase (JNK), laminin and the Ras family GTPase-Rap1, which, when deregulated, have tumorogenic potential Proteins inferred from the entified . Based oentified . Functioentified . The majentified . ES protentified . ES protC. sinensis and O. viverrini, with more than 50,000 unique sequences being identified for each species. More than 85% of the sequences generated from each of these transcriptomes (available viahttp://research.vet.unimelb.edu.au/gasserlab/index.html) were novel and thus represent a significant contribution to current databases C. sinensis and O. viverrini were inferred to be homologues, reflecting their relatively close biological S. japonicum, S. mansoni (Schistosomatidae), F. hepatica (Fasciolidae), C. sinensis and O. viverrini (Opisthorchiidae), the latter three species shared the greatest (29–31%) protein sequence homology. Interestingly, ∼70% of protein sequences in each of the data sets presented herein did not match those available in public databases and are interpreted to be specific to the biology of these liver flukes and their mode of existence in the hosts and, thus, could represent potential candidates for new drugs and/or vaccines. The considerable percentage of protein sequences predicted that have significantly greater similarity to those of mammals (20%) than nematodes (15%) is interesting, and in accordance with previous observations for parasitic trematodes Schmidtea mediterranea, also shares a high degree (60%) of sequence homology to the proteome of various vertebrates Brugia, Ecdysozoa) The integrated genomic-bioinformatic approach used in the present study permitted a deep exploration of the transcriptomes of both C. sinensis and O. viverrini, the numbers of sequences that clustered with genes from eukaryotic model organisms  was approximately two-fold greater than expected. An estimate of the number of proteins expressed by an organism relates to the number of genes present as well as transcriptional variation C. sinensis and O. viverrini are high quality drafts, and the assignment of molecules encoded in the transcriptomes to molecular functions and biological pathways reveals a substantial diversity of terms, comparable with those predicted for other parasitic trematodes, including S. mansoni and F. hepaticaThe present and future transcriptomic data sets, incorporating a wider array of free-living and parasitic invertebrates, should assist in identifying genes linked specifically to parasitism and also contribute significantly to our understanding of the evolution of the Metazoa. For each C. sinensis and O. viverrini form a solid foundation and present unique opportunities for studying the developmental and reproductive biology of these parasites, parasite-host interactions and the pathogenesis of the diseases that these flukes cause in humans and animals. Importantly, the annotated data sets should also assist in the testing of current theories regarding the molecular basis of the pathogenesis of cholangiocarcinoma induced by chronic clonorchiasis or opisthorchiasis in vitroO. viverrini secretes a granulin-like molecule, which causes host cells to proliferate and is thought to be intimately involved in the initiation of carcinogenesis C. sinensis and O. viverrini in the development of cholangiocarcinoma C. sinensis and O. viverrini, following mass spectrometric analyses F. hepaticaThe present transcriptomes inferred for C. sinensis and O. viverrini. However, there are surprisingly few transcriptional data for other developmental stages, with only 419 ESTs available for the metacercarial stage of C. sinensisO. viverrini. Currently, there are no sequence data for the other developmental stages  of these parasites. Future studies should now focus on the differential expression of genes through the multiple free-living and parasitic life history stages. The transcriptional data for adult C. sinensis and O. viverrini will underpin the characterization of transcriptional profiles of this stage, utilizing next-generation sequencing, microarray and/or quantitative real-time PCR analyses, which will have important implications for understanding development, reproduction as well as parasite-host interactions at the molecular level. Importantly, elucidating the transcriptomes of both immature and adult flukes provides the prospect of exploring immunopathological changes as well as carcinogenesis in humans at different stages of clonorchiasis and opisthorchiasis The transcriptomes defined herein represent the adult stage of each S. mansoni and F. hepaticaC. sinensis and O. viverrini as well as the determination of gene structures, prediction of alternative transcript splicing and the characterization of regulatory elements. Clearly, the future annotation of the genomes of these two parasites should also provide a foundation for the prediction of drug targets, based on an improved understanding of global biochemical pathways as well as genetic interactions C. sinensis and/or O. viverrini as well as diagnostic tools, particularly for the early diagnosis of cholangiocarcinoma. Given possible adverse effects of praziquantel treatment In conclusion, the present transcriptomic data will assist in fundamental molecular studies of development, reproduction and metabolic pathways by providing a foundation for new developments in functional genomics. Gene perturbation assays are available for