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1.  Genome-Wide Saturation Mutagenesis of Burkholderia pseudomallei K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development 
mBio  2014;5(1):e00926-13.
ABSTRACT
Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 106 transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied.
IMPORTANCE
Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.
doi:10.1128/mBio.00926-13
PMCID: PMC3950516  PMID: 24520057
2.  Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis 
Open Biology  2013;3(5):130002.
Glycoconjugate-based vaccines have proved to be effective at producing long-lasting protection against numerous pathogens. Here, we describe the application of bacterial protein glycan coupling technology (PGCT) to generate a novel recombinant glycoconjugate vaccine. We demonstrate the conjugation of the Francisella tularensis O-antigen to the Pseudomonas aeruginosa carrier protein exotoxin A using the Campylobacter jejuni PglB oligosaccharyltransferase. The resultant recombinant F. tularensis glycoconjugate vaccine is expressed in Escherichia coli where yields of 3 mg l−1 of culture were routinely produced in a single-step purification process. Vaccination of BALB/c mice with the purified glycoconjugate boosted IgG levels and significantly increased the time to death upon subsequent challenge with F. tularensis subsp. holarctica. PGCT allows different polysaccharide and protein combinations to be produced recombinantly and could be easily applicable for the production of diverse glycoconjugate vaccines.
doi:10.1098/rsob.130002
PMCID: PMC3866875  PMID: 23697804
Francisella tularensis; vaccine; glycoconjugate; protein glycan coupling technology
3.  Evidence that Cd101 is an autoimmune diabetes gene in NOD mice 
We have previously proposed that sequence variation of the CD101 gene between NOD and C57BL/6 (B6) mice accounts for the protection from type 1 diabetes (T1D) provided by the Idd10 region, a <1 Mb region on mouse chromosome 3. Here, we provide further support for the hypothesis that Cd101 is Idd10 using haplotype and expression analyses of novel Idd10 congenic strains coupled to the development of a CD101 knockout mouse. Susceptibility to T1D was correlated with genotype-dependent CD101 expression on multiple cell subsets, including FoxP3+ regulatory CD4+ T cells, CD11c+ dendritic cells and Gr1+ myeloid cells. The correlation of CD101 expression on immune cells from four independent Idd10 haplotypes with the development of T1D supports the identity of Cd101 as Idd10. Since CD101 has been associated with T regulatory and antigen presentation cell functions, our results provide a further link between immune regulation and susceptibility to T1D.
doi:10.4049/jimmunol.1003523
PMCID: PMC3128927  PMID: 21613616
Rodent; Diabetes; Autoimmunity
4.  Identification of Cd101 as a susceptibility gene for Novosphingobium aromaticivorans - induced liver autoimmunity 
Environmental and genetic factors define the susceptibility of an individual to autoimmune disease. Although common genetic pathways affect general immunological tolerance mechanisms in autoimmunity, the effects of such genes could vary under distinct immune challenges within different tissues. Here we demonstrate this by observing that autoimmune type 1 diabetes (T1D) protective haplotypes at the susceptibility region 10 (Idd10) introgressed from chromosome 3 of B6 and A/J mice onto the NOD background increase the severity of autoimmune primary biliary cirrhosis (PBC) induced by infection with Novosphingobium aromaticivorans (N. aro), an ubiquitous alphaproteobacterium, when compared to mice having the NOD and NOD.CAST Idd10 T1D susceptible haplotypes. Substantially increased liver pathology in mice having the B6 and A/J Idd10 haplotypes correlates with reduced expression of CD101 on dendritic cells (DCs), macrophages and granulocytes following infection, delayed clearance of N. aro and the promotion of overzealous, IFN-γ- and IL-17-dominated T cell responses essential for the adoptive transfer of liver lesions. CD101-knockout mice generated on the B6 background also exhibit substantially more severe N.aro-induced liver disease correlating with increased IFN-γ and IL-17 responses compared to wild type mice. These data strongly support the hypothesis that allelic variation of the Cd101 gene, located in the Idd10 region, alters the severity of liver autoimmunity induced by N. aro.
doi:10.4049/jimmunol.1003525
PMCID: PMC3134939  PMID: 21613619
rodent; cytokines; dendritic cells; T cells; autoimmunity; costimulation; inflammation
5.  Incidental fleurodeoxyglucose uptake in the prostate 
The British Journal of Radiology  2010;83(995):902-903.
This commentary confirms the rarity of prostatic cancer associated with incidental prostatic fleurodeoxyglucose (FDG) uptake. The study adds to the literature by showing that even if a prostate lesion is FDG avid it is unlikely to be due to cancer. The commentary considers the management of incidental prostate FDG uptake on the basis of the available evidence.
doi:10.1259/bjr/28173921
PMCID: PMC3473722  PMID: 20965899
6.  Intra- and Interhost Evolutionary Dynamics of Equine Influenza Virus▿ §  
Journal of Virology  2010;84(14):6943-6954.
Determining the evolutionary basis of cross-species transmission and immune evasion is key to understanding the mechanisms that control the emergence of either new viruses or novel antigenic variants with pandemic potential. The hemagglutinin glycoprotein of influenza A viruses is a critical host range determinant and a major target of neutralizing antibodies. Equine influenza virus (EIV) is a significant pathogen of the horse that causes periodical outbreaks of disease even in populations with high vaccination coverage. EIV has also jumped the species barrier and emerged as a novel respiratory pathogen in dogs, canine influenza virus. We studied the dynamics of equine influenza virus evolution in horses at the intrahost level and how this evolutionary process is affected by interhost transmission in a natural setting. To this end, we performed clonal sequencing of the hemagglutinin 1 gene derived from individual animals at different times postinfection. Our results show that despite the population consensus sequence remaining invariant, genetically distinct subpopulations persist during the course of infection and are also transmitted, with some variants likely to change antigenicity. We also detected a natural case of mixed infection in an animal infected during an outbreak of equine influenza, raising the possibility of reassortment between different strains of virus. In sum, our data suggest that transmission bottlenecks may not be as narrow as originally perceived and that the genetic diversity required to adapt to new host species may be partially present in the donor host and potentially transmitted to the recipient host.
doi:10.1128/JVI.00112-10
PMCID: PMC2898244  PMID: 20444896
7.  Reciprocal Analysis of Francisella novicida Infections of a Drosophila melanogaster Model Reveal Host-Pathogen Conflicts Mediated by Reactive Oxygen and imd-Regulated Innate Immune Response 
PLoS Pathogens  2010;6(8):e1001065.
The survival of a bacterial pathogen within a host depends upon its ability to outmaneuver the host immune response. Thus, mutant pathogens provide a useful tool for dissecting host-pathogen relationships, as the strategies the microbe has evolved to counteract immunity reveal a host's immune mechanisms. In this study, we examined the pathogen Francisella novicida and identified new bacterial virulence factors that interact with different parts of the Drosophila melanogaster innate immune system. We performed a genome-wide screen to identify F. novicida genes required for growth and survival within the fly and identified a set of 149 negatively selected mutants. Among these, we identified a class of genes including the transcription factor oxyR, and the DNA repair proteins uvrB, recB, and ruvC that help F. novicida resist oxidative stress. We determined that these bacterial genes are virulence factors that allow F. novicida to counteract the fly melanization immune response. We then performed a second in vivo screen to identify an additional subset of bacterial genes that interact specifically with the imd signaling pathway. Most of these mutants have decreased resistance to the antimicrobial peptide polymyxin B. Characterization of a mutation in the putative transglutaminase FTN_0869 produced a curious result that could not easily be explained using known Drosophila immune responses. By using an unbiased genetic screen, these studies provide a new view of the Drosophila immune response from the perspective of a pathogen. We show that two branches of the fly's immunity are important for fighting F. novicida infections in a model host: melanization and an imd-regulated immune response, and identify bacterial genes that specifically counteract these host responses. Our work suggests that there may be more to learn about the fly immune system, as not all of the phenotypes we observe can be readily explained by its interactions with known immune responses.
Author Summary
To infect a host and survive attacks from the host immune system, bacteria express genes that allow them to counteract immune responses. By identifying these genes we can learn how hosts fight infections and how bacteria resist immune attacks. We identified Francisella novicida genes that interact with the fruit fly immune system by performing a genetic screen of bacterial mutants. We identified genes that when mutated cause the bacteria to grow poorly within the fly. Many of these genes were shown to help the bacteria survive oxidative stress, providing resistance to an immune response known as melanization. We then identified bacterial genes that interact with another branch of the immune system, the imd pathway, by performing a second screen in imd mutant flies. We identified bacterial mutants that cannot grow in wild-type flies but are rescued in imd mutants, indicating an interaction with this pathway. We followed up one example from this screen and found that mutants in the gene FTN_0869 grow normally inside cells, but cannot grow extracellularly. We found that this was due to being unable to resist previously unexplored aspects of the imd-regulated immune response that help fight off F. novicida infections.
doi:10.1371/journal.ppat.1001065
PMCID: PMC2928790  PMID: 20865166
8.  The genome of the simian and human malaria parasite Plasmodium knowlesi 
Nature  2008;455(7214):799-803.
Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the ‘kra’ monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia1,2. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated3, and it has a close phylogenetic relationship to Plasmodium vivax​4, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or ‘hypnozoite’ in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone5) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome4 and other sequenced Plasmodium genomes6-8. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs9, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.
doi:10.1038/nature07306
PMCID: PMC2656934  PMID: 18843368
9.  Comparative genomic analysis of three Leishmania species that cause diverse human disease 
Nature genetics  2007;39(7):839-847.
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 ∼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.
doi:10.1038/ng2053
PMCID: PMC2592530  PMID: 17572675
10.  Complete Genome Sequence of Uropathogenic Proteus mirabilis, a Master of both Adherence and Motility▿ †  
Journal of Bacteriology  2008;190(11):4027-4037.
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 (≥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.
doi:10.1128/JB.01981-07
PMCID: PMC2395036  PMID: 18375554
11.  Complete Genome of Acute Rheumatic Fever-Associated Serotype M5 Streptococcus pyogenes Strain Manfredo▿  
Journal of Bacteriology  2006;189(4):1473-1477.
Comparisons of the 1.84-Mb genome of serotype M5 Streptococcus pyogenes strain Manfredo with previously sequenced genomes emphasized the role of prophages in diversification of S. pyogenes and the close relationship between strain Manfredo and MGAS8232, another acute rheumatic fever-associated strain.
doi:10.1128/JB.01227-06
PMCID: PMC1797351  PMID: 17012393
12.  Drosophila eiger Mutants Are Sensitive to Extracellular Pathogens 
PLoS Pathogens  2007;3(3):e41.
We showed previously that eiger, the Drosophila tumor necrosis factor homolog, contributes to the pathology induced by infection with Salmonella typhimurium. We were curious whether eiger is always detrimental in the context of infection or if it plays a role in fighting some types of microbes. We challenged wild-type and eiger mutant flies with a collection of facultative intracellular and extracellular pathogens, including a fungus and Gram-positive and Gram-negative bacteria. The response of eiger mutants divided these microbes into two groups: eiger mutants are immunocompromised with respect to extracellular pathogens but show no change or reduced sensitivity to facultative intracellular pathogens. Hence, eiger helps fight infections but also can cause pathology. We propose that eiger activates the cellular immune response of the fly to aid clearance of extracellular pathogens. Intracellular pathogens, which can already defeat professional phagocytes, are unaffected by eiger.
Author Summary
We show that the gene eiger, which is the sole tumor necrosis factor homolog in the fruit fly, can play opposing roles in the fly's response to infections. Sometimes eiger contributes to the disease induced by an infection, while at other times it is required to fight an infection. Commonly, the fly's immune response is described as dividing microbes into two groups with Gram-positive bacteria and fungi lying in one group and Gram-negative bacteria lying in the other. Pathogenic bacteria can also be divided into two groups based on their behavior in eiger mutant flies, but these two groups differ from past descriptions. eiger tends to be required for the innate immune response against extracellular pathogens but tends to cause pathology during an infection with an intracellular pathogen. We suggest that eiger is required for innate immune responses that are effective at fighting extracellular pathogens but are wasteful or simply ineffective when fighting intracellular pathogens. We show here that the fly immune response is more complex than previously recognized and suggest new directions for studying pathogenesis in addition to innate immunity in the fly.
doi:10.1371/journal.ppat.0030041
PMCID: PMC1829408  PMID: 17381241
13.  Meningococcal Genetic Variation Mechanisms Viewed through Comparative Analysis of Serogroup C Strain FAM18 
PLoS Genetics  2007;3(2):e23.
The bacterium Neisseria meningitidis is commonly found harmlessly colonising the mucosal surfaces of the human nasopharynx. Occasionally strains can invade host tissues causing septicaemia and meningitis, making the bacterium a major cause of morbidity and mortality in both the developed and developing world. The species is known to be diverse in many ways, as a product of its natural transformability and of a range of recombination and mutation-based systems. Previous work on pathogenic Neisseria has identified several mechanisms for the generation of diversity of surface structures, including phase variation based on slippage-like mechanisms and sequence conversion of expressed genes using information from silent loci. Comparison of the genome sequences of two N. meningitidis strains, serogroup B MC58 and serogroup A Z2491, suggested further mechanisms of variation, including C-terminal exchange in specific genes and enhanced localised recombination and variation related to repeat arrays. We have sequenced the genome of N. meningitidis strain FAM18, a representative of the ST-11/ET-37 complex, providing the first genome sequence for the disease-causing serogroup C meningococci; it has 1,976 predicted genes, of which 60 do not have orthologues in the previously sequenced serogroup A or B strains. Through genome comparison with Z2491 and MC58 we have further characterised specific mechanisms of genetic variation in N. meningitidis, describing specialised loci for generation of cell surface protein variants and measuring the association between noncoding repeat arrays and sequence variation in flanking genes. Here we provide a detailed view of novel genetic diversification mechanisms in N. meningitidis. Our analysis provides evidence for the hypothesis that the noncoding repeat arrays in neisserial genomes (neisserial intergenic mosaic elements) provide a crucial mechanism for the generation of surface antigen variants. Such variation will have an impact on the interaction with the host tissues, and understanding these mechanisms is important to aid our understanding of the intimate and complex relationship between the human nasopharynx and the meningococcus.
Author Summary
Human surface tissues, including the skin and gut lining, are host to many different species of bacteria. N. meningitidis is a species of bacteria that is only found in humans where it is able to colonise mucosal surfaces of the nasopharynx (nose and throat). This association is normally harmless and at any one time around 15% of the population are carriers. Some strains of N. meningitidis can cause disease by invading the host tissue leading to septicaemia or meningitis. We aim to gain understanding of the mechanisms by which these bacteria cause disease by studying and comparing genomes from different strains. Here we describe specific genes and associated repetitive DNA sequences that are involved in variation of the bacterial cell surface. The repeat sequences encourage the swapping of genes that code for variant copies of cell surface proteins. The resulting variation of the bacterial cell surface appears to be important in the close interaction between host and bacteria and the potential for disease.
doi:10.1371/journal.pgen.0030023
PMCID: PMC1797815  PMID: 17305430
14.  Meningococcal Genetic Variation Mechanisms Viewed through Comparative Analysis of Serogroup C Strain FAM18 
PLoS Genetics  2007;3(2):e23.
The bacterium Neisseria meningitidis is commonly found harmlessly colonising the mucosal surfaces of the human nasopharynx. Occasionally strains can invade host tissues causing septicaemia and meningitis, making the bacterium a major cause of morbidity and mortality in both the developed and developing world. The species is known to be diverse in many ways, as a product of its natural transformability and of a range of recombination and mutation-based systems. Previous work on pathogenic Neisseria has identified several mechanisms for the generation of diversity of surface structures, including phase variation based on slippage-like mechanisms and sequence conversion of expressed genes using information from silent loci. Comparison of the genome sequences of two N. meningitidis strains, serogroup B MC58 and serogroup A Z2491, suggested further mechanisms of variation, including C-terminal exchange in specific genes and enhanced localised recombination and variation related to repeat arrays. We have sequenced the genome of N. meningitidis strain FAM18, a representative of the ST-11/ET-37 complex, providing the first genome sequence for the disease-causing serogroup C meningococci; it has 1,976 predicted genes, of which 60 do not have orthologues in the previously sequenced serogroup A or B strains. Through genome comparison with Z2491 and MC58 we have further characterised specific mechanisms of genetic variation in N. meningitidis, describing specialised loci for generation of cell surface protein variants and measuring the association between noncoding repeat arrays and sequence variation in flanking genes. Here we provide a detailed view of novel genetic diversification mechanisms in N. meningitidis. Our analysis provides evidence for the hypothesis that the noncoding repeat arrays in neisserial genomes (neisserial intergenic mosaic elements) provide a crucial mechanism for the generation of surface antigen variants. Such variation will have an impact on the interaction with the host tissues, and understanding these mechanisms is important to aid our understanding of the intimate and complex relationship between the human nasopharynx and the meningococcus.
Author Summary
Human surface tissues, including the skin and gut lining, are host to many different species of bacteria. N. meningitidis is a species of bacteria that is only found in humans where it is able to colonise mucosal surfaces of the nasopharynx (nose and throat). This association is normally harmless and at any one time around 15% of the population are carriers. Some strains of N. meningitidis can cause disease by invading the host tissue leading to septicaemia or meningitis. We aim to gain understanding of the mechanisms by which these bacteria cause disease by studying and comparing genomes from different strains. Here we describe specific genes and associated repetitive DNA sequences that are involved in variation of the bacterial cell surface. The repeat sequences encourage the swapping of genes that code for variant copies of cell surface proteins. The resulting variation of the bacterial cell surface appears to be important in the close interaction between host and bacteria and the potential for disease.
doi:10.1371/journal.pgen.0030023
PMCID: PMC1797815  PMID: 17305430
15.  Rapid and Sensitive Detection of Single Cryptosporidium Oocysts from Archived Glass Slides 
Journal of Clinical Microbiology  2006;44(9):3285-3291.
In this study we report on the development and application of a novel method for efficiently extracting and detecting single Cryptosporidium oocysts from archived glass slides. Laser capture microscopy was used to extract low numbers of oocysts from archived glass slides. Highly sensitive real-time PCR methods were then developed to enable the rapid detection and identification of Cryptosporidium oocysts from these samples. The method was applied to fecal smears stained with a variety of standard oocyst stains and water samples. This application, with samples derived from both public health and water service laboratories, highlighted the strong potential of this method to be used as a rapid high-throughput screening tool for the routine monitoring of Cryptosporidium and other medically important pathogens from clinical, veterinary, and environmental water samples. Importantly, the application of our protocol could be used to type Cryptosporidium and other pathogens from stored archived glass slides in public health and water service laboratories, providing vital epidemiological updates and helping to identify and trace pathogens and their routes of infection and ultimately improve their control.
doi:10.1128/JCM.00541-06
PMCID: PMC1594691  PMID: 16954262
16.  The Complete Genome Sequence and Comparative Genome Analysis of the High Pathogenicity Yersinia enterocolitica Strain 8081 
PLoS Genetics  2006;2(12):e206.
The human enteropathogen, Yersinia enterocolitica, is a significant link in the range of Yersinia pathologies extending from mild gastroenteritis to bubonic plague. Comparison at the genomic level is a key step in our understanding of the genetic basis for this pathogenicity spectrum. Here we report the genome of Y. enterocolitica strain 8081 (serotype 0:8; biotype 1B) and extensive microarray data relating to the genetic diversity of the Y. enterocolitica species. Our analysis reveals that the genome of Y. enterocolitica strain 8081 is a patchwork of horizontally acquired genetic loci, including a plasticity zone of 199 kb containing an extraordinarily high density of virulence genes. Microarray analysis has provided insights into species-specific Y. enterocolitica gene functions and the intraspecies differences between the high, low, and nonpathogenic Y. enterocolitica biotypes. Through comparative genome sequence analysis we provide new information on the evolution of the Yersinia. We identify numerous loci that represent ancestral clusters of genes potentially important in enteric survival and pathogenesis, which have been lost or are in the process of being lost, in the other sequenced Yersinia lineages. Our analysis also highlights large metabolic operons in Y. enterocolitica that are absent in the related enteropathogen, Yersinia pseudotuberculosis, indicating major differences in niche and nutrients used within the mammalian gut. These include clusters directing, the production of hydrogenases, tetrathionate respiration, cobalamin synthesis, and propanediol utilisation. Along with ancestral gene clusters, the genome of Y. enterocolitica has revealed species-specific and enteropathogen-specific loci. This has provided important insights into the pathology of this bacterium and, more broadly, into the evolution of the genus. Moreover, wider investigations looking at the patterns of gene loss and gain in the Yersinia have highlighted common themes in the genome evolution of other human enteropathogens.
Synopsis
The goal of this study was to catalogue all the genes encoded within the Y. enterocolitica genome to help us better understand how this bacterium and related bacteria cause different diseases. There are currently genome sequences (complete gene catalogues) available for two other members of this bacterial lineage, which cause dramatically different diseases: Y. pseudotuberculosis, like Y. enterocolitica, is a gut pathogen (enteropathogen) causing gastroenteritis in humans and animals. Yersinia pestis mostly resides within blood (circulating or in fleas following blood meals) and lymph tissue. It causes bubonic plague in humans and animals, and is historically known as “The Black Death.” A three-way comparison of these genomes revealed a patchwork of genes we have defined as being species- or disease-specific and genes that are common to all three Yersinia species. This has provided us with important information on shared gene functions that define the two enteropathogenic yersinias and those that differentiate them. This will help us to connect what we know about the Y. enterocolitica lifestyle within the gut to the disease it causes and its genetic makeup. We have also provided further evidence of gene-loss by Y. pestis as it has evolved from Y. pseudotuberculosis into a more acute systemic pathogen. Similar patterns of gene loss are seen in other important pathogens such as Salmonella enterica serovar Typhi.
doi:10.1371/journal.pgen.0020206
PMCID: PMC1698947  PMID: 17173484
17.  The Complete Genome Sequence and Comparative Genome Analysis of the High Pathogenicity Yersinia enterocolitica Strain 8081 
PLoS Genetics  2006;2(12):e206.
The human enteropathogen, Yersinia enterocolitica, is a significant link in the range of Yersinia pathologies extending from mild gastroenteritis to bubonic plague. Comparison at the genomic level is a key step in our understanding of the genetic basis for this pathogenicity spectrum. Here we report the genome of Y. enterocolitica strain 8081 (serotype 0:8; biotype 1B) and extensive microarray data relating to the genetic diversity of the Y. enterocolitica species. Our analysis reveals that the genome of Y. enterocolitica strain 8081 is a patchwork of horizontally acquired genetic loci, including a plasticity zone of 199 kb containing an extraordinarily high density of virulence genes. Microarray analysis has provided insights into species-specific Y. enterocolitica gene functions and the intraspecies differences between the high, low, and nonpathogenic Y. enterocolitica biotypes. Through comparative genome sequence analysis we provide new information on the evolution of the Yersinia. We identify numerous loci that represent ancestral clusters of genes potentially important in enteric survival and pathogenesis, which have been lost or are in the process of being lost, in the other sequenced Yersinia lineages. Our analysis also highlights large metabolic operons in Y. enterocolitica that are absent in the related enteropathogen, Yersinia pseudotuberculosis, indicating major differences in niche and nutrients used within the mammalian gut. These include clusters directing, the production of hydrogenases, tetrathionate respiration, cobalamin synthesis, and propanediol utilisation. Along with ancestral gene clusters, the genome of Y. enterocolitica has revealed species-specific and enteropathogen-specific loci. This has provided important insights into the pathology of this bacterium and, more broadly, into the evolution of the genus. Moreover, wider investigations looking at the patterns of gene loss and gain in the Yersinia have highlighted common themes in the genome evolution of other human enteropathogens.
Synopsis
The goal of this study was to catalogue all the genes encoded within the Y. enterocolitica genome to help us better understand how this bacterium and related bacteria cause different diseases. There are currently genome sequences (complete gene catalogues) available for two other members of this bacterial lineage, which cause dramatically different diseases: Y. pseudotuberculosis, like Y. enterocolitica, is a gut pathogen (enteropathogen) causing gastroenteritis in humans and animals. Yersinia pestis mostly resides within blood (circulating or in fleas following blood meals) and lymph tissue. It causes bubonic plague in humans and animals, and is historically known as “The Black Death.” A three-way comparison of these genomes revealed a patchwork of genes we have defined as being species- or disease-specific and genes that are common to all three Yersinia species. This has provided us with important information on shared gene functions that define the two enteropathogenic yersinias and those that differentiate them. This will help us to connect what we know about the Y. enterocolitica lifestyle within the gut to the disease it causes and its genetic makeup. We have also provided further evidence of gene-loss by Y. pestis as it has evolved from Y. pseudotuberculosis into a more acute systemic pathogen. Similar patterns of gene loss are seen in other important pathogens such as Salmonella enterica serovar Typhi.
doi:10.1371/journal.pgen.0020206
PMCID: PMC1698947  PMID: 17173484
18.  The genome of Rhizobium leguminosarum has recognizable core and accessory components 
Genome Biology  2006;7(4):R34.
The genome sequence of the α-proteobacterial N2-fixing symbiont of legumes, Rhizobium leguminosarum, is described, revealing a 'core' and an 'accessory' component.
Background
Rhizobium leguminosarum is an α-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.
doi:10.1186/gb-2006-7-4-r34
PMCID: PMC1557990  PMID: 16640791
19.  Construction and analysis of tag single nucleotide polymorphism maps for six human-mouse orthologous candidate genes in type 1 diabetes 
BMC Genetics  2005;6:9.
Background
One strategy to help identify susceptibility genes for complex, multifactorial diseases is to map disease loci in a representative animal model of the disorder. The nonobese diabetic (NOD) mouse is a model for human type 1 diabetes. Linkage and congenic strain analyses have identified several NOD mouse Idd (insulin dependent diabetes) loci, which have been mapped to small chromosome intervals, for which the orthologous regions in the human genome can be identified. Here, we have conducted re-sequencing and association analysis of six orthologous genes identified in NOD Idd loci: NRAMP1/SLC11A1 (orthologous to Nramp1/Slc11a1 in Idd5.2), FRAP1 (orthologous to Frap1 in Idd9.2), 4-1BB/CD137/TNFRSF9 (orthologous to 4-1bb/Cd137/Tnrfrsf9 in Idd9.3), CD101/IGSF2 (orthologous to Cd101/Igsf2 in Idd10), B2M (orthologous to B2m in Idd13) and VAV3 (orthologous to Vav3 in Idd18).
Results
Re-sequencing of a total of 110 kb of DNA from 32 or 96 type 1 diabetes cases yielded 220 single nucleotide polymorphisms (SNPs). Sixty-five SNPs, including 54 informative tag SNPs, and a microsatellite were selected and genotyped in up to 1,632 type 1 diabetes families and 1,709 cases and 1,829 controls.
Conclusion
None of the candidate regions showed evidence of association with type 1 diabetes (P values > 0.2), indicating that common variation in these key candidate genes does not play a major role in type 1 diabetes susceptibility in the European ancestry populations studied.
doi:10.1186/1471-2156-6-9
PMCID: PMC551616  PMID: 15720714
21.  The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129 
Nucleic Acids Research  2003;31(22):6516-6523.
Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium’s nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.
doi:10.1093/nar/gkg874
PMCID: PMC275568  PMID: 14602910
22.  Hepatic perfusion changes in patients with liver metastases: comparison with those patients with cirrhosis. 
Gut  1993;34(4):554-557.
Previous studies using dynamic scintigraphy have shown that the measurement of changes in hepatic perfusion may be exploited to detect liver metastases. Similar hepatic haemodynamic changes also occur in cirrhosis, however, thereby reducing the diagnostic power of the technique. The ability of duplex colour Doppler sonography (DCDS) to differentiate between the changes in liver perfusion in patients with cirrhosis and those with hepatic metastases was assessed. Hepatic arterial and portal venous blood flows were measured in 30 control subjects, 20 patients with cirrhosis, and 55 patients with overt liver metastases. The Doppler perfusion index (DPI) (the rate of hepatic arterial to total liver blood flow) and the congestive index (ratio of the cross sectional area of the vessel to time averaged velocity of blood flow in the vessel) of the hepatic artery (HCI) and portal vein (PCI) were calculated. The hepatic arterial blood flow of the cirrhotic and metastatic groups was significantly raised compared with that of controls, and the portal venous blood flow of the former groups were reduced (p < 0.0001). The DPIs of the cirrhotic and metastatic groups were therefore significantly raised compared with those of controls (p < 0.0001). No significant difference was noted in HCI values between the three groups. The PCI values of the cirrhotic group, however, were significantly raised compared with those of controls and patients with metastases (p < 0.0001). The data suggest that DCDS measurement of PCI may be of value in differentiating between the hepatic perfusion changes caused by cirrhosis and those resulting from hepatic metastases, thereby increasing the diagnostic power of this technique.
PMCID: PMC1374320  PMID: 8491406
23.  Radiological evaluation of the pancreas in malnutrition-related (phasic insulin dependent) diabetes mellitus. 
This study was undertaken to investigate pancreatic changes associated with phasic insulin dependent diabetes mellitus (PIDDM). Twelve PIDDM patients were studied. They were compared with groups of patients, 10 insulin dependent (IDDM), 10 non-insulin dependent (NIDDM), and 10 normal controls. Each group was matched for age, sex, and body mass index. For the study, the mean age was 56.7 +/- 2.5 years, mean body mass index 24.0 +/- 0.8, and mean duration of diabetes 14.2 +/- 2.2 years. Flat abdominal radiograph and ultrasonography were performed on each participant. The results suggest an increased echogenicity of the pancreas in the phasic insulin dependent group of patients.
PMCID: PMC2627002  PMID: 1994067
24.  The specific IgM response to Mycoplasma pneumoniae infection: interpretation and application to early diagnosis. 
Epidemiology and Infection  1987;99(3):685-692.
Specific IgM antibody production in patients with serologically proven Mycoplasma pneumoniae infection by the complement fixation (CF) test was studied using a mu-capture ELISA. Sera from 79% of patients were found to be IgM positive. Patients could be divided into two groups relating to the amount of specific antibody produced. High levels of specific IgM (greater than or equal to 10 units) were more commonly found in younger patients. Seventy-six per cent of patients under the age of 20 produced relatively high levels of IgM compared to 35% of patients over the age of 20. In contrast, the number of patients who produced low or undetectable levels of IgM (less than 10 units) was found to increase with age. This trend was found to be significant which suggests that low or undetectable levels of IgM may be due to reinfection with M. pneumoniae. Specific IgM was found to appear in the serum at approximately 7 days after the onset of symptoms, peaking at between 10 and 30 days, and then falling to undetectable levels at an estimated 12-26 weeks post onset of symptoms. Twenty-eight per cent of acute-phase sera (CF titres less than 256) from patients whose sera subsequently showed a fourfold or greater rise in M. pneumoniae CF antibody titre were IgM positive. Thus using mu-capture ELISA a diagnosis of M. pneumoniae infection may often be made more rapidly than by the complement fixation test.
PMCID: PMC2249255  PMID: 3123266
25.  Hepatic Folate in Man 
British Medical Journal  1966;1(5484):396-399.
PMCID: PMC1843551  PMID: 5904108

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