Comparative genomic analysis of three Leishmania species that cause diverse human disease.

Leishmania parasites cause a broad spectrum of clinical disease. Here we report the sequencing of the genomes of two species of Leishmania: Leishmania infantum and Leishmania braziliensis. The comparison of these sequences with the published genome of Leishmania major reveals marked conservation of synteny and identifies only approximately 200 genes with a differential distribution between the three species. L. braziliensis, contrary to Leishmania species examined so far, possesses components of a putative RNA-mediated interference pathway, telomere-associated transposable elements and spliced leader-associated SLACS retrotransposons. We show that pseudogene formation and gene loss are the principal forces shaping the different genomes. Genes that are differentially distributed between the species encode proteins implicated in host-pathogen interactions and parasite survival in the macrophage.

Decellularised human bone allograft from different anatomical sites as a basis for functionally stratified repair material for bone defects.

Tissue engineered bone solutions aim to overcome the limitations of autologous and allogeneic grafts. Decellularised tissues are produced by washing cellular components from human or animal tissue to produce an immunologically safe and biocompatible scaffold, capable of integration following implantation. A decellularisation procedure utilising low concentration sodium dodecyl sulphate (0.1% w/v) was applied to trabecular bone from human femoral heads (FH) and tibial plateaus (TP). Biological (histology, DNA quantification), biomechanical (compression testing) and structural (µCT) comparisons were made between decellularised and unprocessed cellular tissue. Total DNA levels of decellularised FH and TP bone were below 50 ng mg-1 dry tissue weight and nuclear material was removed. No differences were found between cellular and decellularised bone, from each anatomical region, for all the biomechanical and structural parameters investigated. Differences were found between cellular FH and TP and between decellularised FH and TP. Decellularised FH had a higher ultimate compressive stress, Young's modulus and 0.2% proof stress than decellularised TP (p = 0.001, 0.002, 0.001, Mann Whitney U test, MWU). The mineral density of cellular and decellularised TP bone was significantly greater than cellular and decellularised FH bone respectively (cellular: p = 0.001, decellularised: p < 0.001, MWU). The bone volume fraction and trabecular thickness of cellular and decellularised FH bone were significantly greater than cellular and decellularised TP bone respectively (cellular: p = 0.001, 0.005; decellularised: p < 0.001, <0.001, MWU). Characterisation of decellularised trabecular bone from different anatomical regions offers the possibility of product stratification, allowing selection of biomechanical properties to match particular anatomical regions undergoing bone graft procedures.

The genome of the protist parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

Decellularized Intervertebral Discs: A Potential Replacement for Degenerate Human Discs.

Intervertebral disc (IVD) degeneration is a major cause of back pain. Current surgical interventions have limitations. An alternative approach is to replace degenerated IVDs with a natural biological scaffold. The removal of cellular components from human IVDs should render them nonimmunogenic upon implantation. The aim of this initial proof of technical feasibility study was to develop a decellularization protocol on bovine IVDs with endplates (EPs) and assess protocol performance before application of the protocol to human IVDs with attached EP and vertebral bone (VB). A decellularization protocol based on hypotonic low concentration sodium dodecyl sulfate (0.1% w/v) with proteinase inhibitors, freeze/thaw cycles, and nuclease and sonication treatments was applied to IVDs. Histological, biochemical, and biomechanical comparisons were made between cellular and decellularized tissue. Cell removal from bovine IVDs was demonstrated and total DNA levels of the decellularized inner annulus fibrosus (iAF), outer annulus fibrosus (oAF), and EP were 40.7 (±11.4), 25.9 (±3.8), and 29.3 (±3.1) ng.mg-1 dry tissue weight, respectively (n = 6, ±95% confidence level [CL]). These values were significantly lower than in cellular tissue. No significant difference in DNA levels between bovine cellular and decellularized nucleus pulposus (NP) was found. Glycosaminoglycans (GAGs) were largely retained in the NP, iAF, and oAF. Cyclic compression testing showed sufficient sensitivity to detect an increase in stiffness of bovine IVD postdecellularization (2957.2 ± 340.8 N.mm-1) (predecellularization: 2685.4 ± 263.1 N.mm-1; n = 5, 95% CL), but the difference was within natural tissue variation. Total DNA levels for all decellularized tissue regions of human IVDs (NP, iAF, oAF, EP, and VB) were below 50 ng.mg-1 dry tissue weight (range: 2 ng.mg-1, iAF to 29 ng.mg-1, VB) and the tissue retained high levels of GAGs. Further studies to assess the biocompatibility and regenerative potential of decellularized human IVDs in vitro and in vivo are now required; however, proof of technical feasibility has been demonstrated and the retention of bone in the IVD samples would allow incorporation of the tissue into the recipient spine. Impact statement Intervertebral disc (IVD) degeneration is a major cause of back pain. Current surgical treatments have limitations and relatively poor outcomes. An implantable cell-free biological scaffold, which will not invoke adverse immune responses, has the potential to preserve the natural mobility of the patient's spine and be regenerated with endogenous cells, preventing further degeneration and improving surgical outcomes. This study demonstrates, for the first time, that it is possible to create a cell-free human IVD biological scaffold with attached bone using decellularization technology, the first step toward the development of an implantable regenerative device for IVD replacement.

Pseudogene accumulation in the evolutionary histories of Salmonella enterica serovars Paratyphi A and Typhi.

BACKGROUND: Of the > 2000 serovars of Salmonella enterica subspecies I, most cause self-limiting gastrointestinal disease in a wide range of mammalian hosts. However, S. enterica serovars Typhi and Paratyphi A are restricted to the human host and cause the similar systemic diseases typhoid and paratyphoid fever. Genome sequence similarity between Paratyphi A and Typhi has been attributed to convergent evolution via relatively recent recombination of a quarter of their genomes. The accumulation of pseudogenes is a key feature of these and other host-adapted pathogens, and overlapping pseudogene complements are evident in Paratyphi A and Typhi. RESULTS: We report the 4.5 Mbp genome of a clinical isolate of Paratyphi A, strain AKU_12601, completely sequenced using capillary techniques and subsequently checked using Illumina/Solexa resequencing. Comparison with the published genome of Paratyphi A ATCC9150 revealed the two are collinear and highly similar, with 188 single nucleotide polymorphisms and 39 insertions/deletions. A comparative analysis of pseudogene complements of these and two finished Typhi genomes (CT18, Ty2) identified several pseudogenes that had been overlooked in prior genome annotations of one or both serovars, and identified 66 pseudogenes shared between serovars. By determining whether each shared and serovar-specific pseudogene had been recombined between Paratyphi A and Typhi, we found evidence that most pseudogenes have accumulated after the recombination between serovars. We also divided pseudogenes into relative-time groups: ancestral pseudogenes inherited from a common ancestor, pseudogenes recombined between serovars which likely arose between initial divergence and later recombination, serovar-specific pseudogenes arising after recombination but prior to the last evolutionary bottlenecks in each population, and more recent strain-specific pseudogenes. CONCLUSION: Recombination and pseudogene-formation have been important mechanisms of genetic convergence between Paratyphi A and Typhi, with most pseudogenes arising independently after extensive recombination between the serovars. The recombination events, along with divergence of and within each serovar, provide a relative time scale for pseudogene-forming mutations, affording rare insights into the progression of functional gene loss associated with host adaptation in Salmonella.

Complete genome sequence of uropathogenic Proteus mirabilis, a master of both adherence and motility.

The gram-negative enteric bacterium Proteus mirabilis is a frequent cause of urinary tract infections in individuals with long-term indwelling catheters or with complicated urinary tracts (e.g., due to spinal cord injury or anatomic abnormality). P. mirabilis bacteriuria may lead to acute pyelonephritis, fever, and bacteremia. Most notoriously, this pathogen uses urease to catalyze the formation of kidney and bladder stones or to encrust or obstruct indwelling urinary catheters. Here we report the complete genome sequence of P. mirabilis HI4320, a representative strain cultured in our laboratory from the urine of a nursing home patient with a long-term (> or =30 days) indwelling urinary catheter. The genome is 4.063 Mb long and has a G+C content of 38.88%. There is a single plasmid consisting of 36,289 nucleotides. Annotation of the genome identified 3,685 coding sequences and seven rRNA loci. Analysis of the sequence confirmed the presence of previously identified virulence determinants, as well as a contiguous 54-kb flagellar regulon and 17 types of fimbriae. Genes encoding a potential type III secretion system were identified on a low-G+C-content genomic island containing 24 intact genes that appear to encode all components necessary to assemble a type III secretion system needle complex. In addition, the P. mirabilis HI4320 genome possesses four tandem copies of the zapE metalloprotease gene, genes encoding six putative autotransporters, an extension of the atf fimbrial operon to six genes, including an mrpJ homolog, and genes encoding at least five iron uptake mechanisms, two potential type IV secretion systems, and 16 two-component regulators.

The genome sequence of the fish pathogen Aliivibrio salmonicida strain LFI1238 shows extensive evidence of gene decay.

BACKGROUND: The fish pathogen Aliivibrio salmonicida is the causative agent of cold-water vibriosis in marine aquaculture. The Gram-negative bacterium causes tissue degradation, hemolysis and sepsis in vivo. RESULTS: In total, 4 286 protein coding sequences were identified, and the 4.6 Mb genome of A. salmonicida has a six partite architecture with two chromosomes and four plasmids. Sequence analysis revealed a highly fragmented genome structure caused by the insertion of an extensive number of insertion sequence (IS) elements. The IS elements can be related to important evolutionary events such as gene acquisition, gene loss and chromosomal rearrangements. New A. salmonicida functional capabilities that may have been aquired through horizontal DNA transfer include genes involved in iron-acquisition, and protein secretion and play potential roles in pathogenicity. On the other hand, the degeneration of 370 genes and consequent loss of specific functions suggest that A. salmonicida has a reduced metabolic and physiological capacity in comparison to related Vibrionaceae species. CONCLUSION: Most prominent is the loss of several genes involved in the utilisation of the polysaccharide chitin. In particular, the disruption of three extracellular chitinases responsible for enzymatic breakdown of chitin makes A. salmonicida unable to grow on the polymer form of chitin. These, and other losses could restrict the variety of carrier organisms A. salmonicida can attach to, and associate with. Gene acquisition and gene loss may be related to the emergence of A. salmonicida as a fish pathogen.

Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter.

The nucleobase-cation-symport-1 (NCS1) transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85-angstrom resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, 10 of which are arranged in two inverted repeats of five helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved, showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine transporter LeuT(Aa) and the galactose transporter vSGLT reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronized by the inverted repeat helices 3 and 8, providing the structural basis of the alternating access model for membrane transport.

Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis.

Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis.

Multidrug-resistant Salmonella enterica serovar paratyphi A harbors IncHI1 plasmids similar to those found in serovar typhi.

Salmonella enterica serovars Typhi and Paratyphi A cause systemic infections in humans which are referred to as enteric fever. Multidrug-resistant (MDR) serovar Typhi isolates emerged in the 1980s, and in recent years MDR serovar Paratyphi A infections have become established as a significant problem across Asia. MDR in serovar Typhi is almost invariably associated with IncHI1 plasmids, but the genetic basis of MDR in serovar Paratyphi A has remained predominantly undefined. The DNA sequence of an IncHI1 plasmid, pAKU_1, encoding MDR in a serovar Paratyphi A strain has been determined. Significantly, this plasmid shares a common IncHI1-associated DNA backbone with the serovar Typhi plasmid pHCM1 and an S. enterica serovar Typhimurium plasmid pR27. Plasmids pAKU_1 and pHCM1 share 14 antibiotic resistance genes encoded within similar mobile elements, which appear to form a 24-kb composite transposon that has transferred as a single unit into different positions into their IncHI1 backbones. Thus, these plasmids have acquired similar antibiotic resistance genes independently via the horizontal transfer of mobile DNA elements. Furthermore, two IncHI1 plasmids from a Vietnamese isolate of serovar Typhi were found to contain features of the backbone sequence of pAKU_1 rather than pHCM1, with the composite transposon inserted in the same location as in the pAKU_1 sequence. Our data show that these serovar Typhi and Paratyphi A IncHI1 plasmids share highly conserved core DNA and have acquired similar mobile elements encoding antibiotic resistance genes in past decades.

Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes.

Clostridium botulinum is a heterogeneous Gram-positive species that comprises four genetically and physiologically distinct groups of bacteria that share the ability to produce botulinum neurotoxin, the most poisonous toxin known to man, and the causative agent of botulism, a severe disease of humans and animals. We report here the complete genome sequence of a representative of Group I (proteolytic) C. botulinum (strain Hall A, ATCC 3502). The genome consists of a chromosome (3,886,916 bp) and a plasmid (16,344 bp), which carry 3650 and 19 predicted genes, respectively. Consistent with the proteolytic phenotype of this strain, the genome harbors a large number of genes encoding secreted proteases and enzymes involved in uptake and metabolism of amino acids. The genome also reveals a hitherto unknown ability of C. botulinum to degrade chitin. There is a significant lack of recently acquired DNA, indicating a stable genomic content, in strong contrast to the fluid genome of Clostridium difficile, which can form longer-term relationships with its host. Overall, the genome indicates that C. botulinum is adapted to a saprophytic lifestyle both in soil and aquatic environments. This pathogen relies on its toxin to rapidly kill a wide range of prey species, and to gain access to nutrient sources, it releases a large number of extracellular enzymes to soften and destroy rotting or decayed tissues.

The genome of Rhizobium leguminosarum has recognizable core and accessory components.

BACKGROUND: Rhizobium leguminosarum is an alpha-proteobacterial N2-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841. RESULTS: The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes (Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were over-represented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens. CONCLUSION: Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.

Complete genome sequence and lytic phase transcription profile of a Coccolithovirus.

The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Among the 472 predicted genes of the 407,339-base pair genome are a variety of unexpected genes, most notably those involved in biosynthesis of ceramide, a sphingolipid known to induce apoptosis. Uniquely for algal viruses, it also contains six RNA polymerase subunits and a novel promoter, suggesting this virus encodes its own transcription machinery. Microarray transcriptomic analysis reveals that 65% of the predicted virus-encoded genes are expressed during lytic infection of E. huxleyi.

Extensive DNA inversions in the B. fragilis genome control variable gene expression.

The obligately anaerobic bacterium Bacteroides fragilis, an opportunistic pathogen and inhabitant of the normal human colonic microbiota, exhibits considerable within-strain phase and antigenic variation of surface components. The complete genome sequence has revealed an unusual breadth (in number and in effect) of DNA inversion events that potentially control expression of many different components, including surface and secreted components, regulatory molecules, and restriction-modification proteins. Invertible promoters of two different types (12 group 1 and 11 group 2) were identified. One group has inversion crossover (fix) sites similar to the hix sites of Salmonella typhimurium. There are also four independent intergenic shufflons that potentially alter the expression and function of varied genes. The composition of the 10 different polysaccharide biosynthesis gene clusters identified (7 with associated invertible promoters) suggests a mechanism of synthesis similar to the O-antigen capsules of Escherichia coli.

Sequencing and analysis of the genome of the Whipple's disease bacterium Tropheryma whipplei.

BACKGROUND: Whipple's disease is a rare multisystem chronic infection, involving the intestinal tract as well as various other organs. The causative agent, Tropheryma whipplei, is a Gram-positive bacterium about which little is known. Our aim was to investigate the biology of this organism by generating and analysing the complete DNA sequence of its genome. METHODS: We isolated and propagated T whipplei strain TW08/27 from the cerebrospinal fluid of a patient diagnosed with Whipple's disease. We generated the complete sequence of the genome by the whole genome shotgun method, and analysed it with a combination of automatic and manual bioinformatic techniques. FINDINGS: Sequencing revealed a condensed 925938 bp genome with a lack of key biosynthetic pathways and a reduced capacity for energy metabolism. A family of large surface proteins was identified, some associated with large amounts of non-coding repetitive DNA, and an unexpected degree of sequence variation. INTERPRETATION: The genome reduction and lack of metabolic capabilities point to a host-restricted lifestyle for the organism. The sequence variation indicates both known and novel mechanisms for the elaboration and variation of surface structures, and suggests that immune evasion and host interaction play an important part in the lifestyle of this persistent bacterial pathogen.