Lofthouse EK, Wheeler PR, Beste DJV, Khatri BL, Wu H, Mendum TA, Kierzek AM, McFadden J (2013) Systems-Based Approaches to Probing Metabolic Variation within the Mycobacterium tuberculosis Complex, PLOS ONE8(9)ARTN e75913 PUBLIC LIBRARY SCIENCE
Mendum TA, Chilima BZ, Hirsch PR (2000) The PCR amplification of non-tuberculous mycobacterial 16S rRNA sequences from soil., FEMS Microbiol Lett185(2)pp. 189-192
Non-tuberculous mycobacteria are free living saprophytic organisms commonly found in soil and water. Some are major causes of opportunistic infection, particularly in immuno-compromised patients, and may influence the efficacy of bacille Calmette-Guérin vaccinations. Many of these organisms are not amenable to culture, so information about their distribution is limited. PCR primers designed to amplify part of the mycobacterial 16S rRNA gene were applied to DNA extracted from cultured organisms and soil. The PCR products from soil contained sequences with similarity to slow growing mycobacteria similar to Mycobacterium lentiflavum, and to fast growing mycobacteria such as the xenobiotic degraders PYR-I and RJGII.
Mendum TA, Clark IM, Hirsch PR (2001) Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia., Antonie Van Leeuwenhoek79(2)pp. 189-197
Two Rhizobium leguminosarum biovar viceae bacteriophages with contrasting properties were isolated from a field site in which the survival of genetically modified R. leguminosarum inoculants had been monitored for several years. Inoculant strain RSM2004 was used as the indicator for phage isolation and propagation. One phage, RL1RES, was temperate and could not replicate in any of the 42 indigenous R. leguminosarum field isolates tested although nested PCR indicated that phage sequences were present in six of the isolates. The second phage, RL2RES, was virulent, capable of generalised transduction, contained DNA with modified cytosine residues, and was capable of infecting all field isolates tested although the GM inoculant strain CT0370 was resistant. Sequence with homology to RL2RES was detected by nested PCR in six of the 42 field-isolates. These were not the same isolates that showed homology to RL1RES. The implication of these findings for the survival of rhizobial inoculants, and the ecology of phages and their host bacteria, are discussed.
Mendum TA, Sockett RE, Hirsch PR (1999) Use of molecular and isotopic techniques to monitor the response of autotrophic ammonia-oxidizing populations of the beta subdivision of the class proteobacteria in arable soils to nitrogen fertilizer., Appl Environ Microbiol65(9)pp. 4155-4162
This study examined the effects of NH(4)NO(3) fertilizer on the size and activity of nitrifying, autotrophic, ammonia-oxidizing populations of the beta subdivision of the class Proteobacteria in arable soils. Plots under different long-term fertilizer regimes were sampled before and after NH(4)NO(3) additions, and the rates of nitrification were determined by (15)N isotopic pool dilution assays. Ammonia-oxidizing populations in the plots were quantified by competitive PCR assays based on the amoA and ribosomal 16S genes. Prior to fertilizer addition, ammonium concentrations and nitrification rates in the plots were comparatively low; ammonia-oxidizing populations were present at 10(4) to 10(5) gene copies g of soil(-1). Three days after the application of fertilizer, nitrification rates had risen considerably but the size of the ammonia-oxidizing population was unchanged. Six weeks after fertilizer treatment, ammonium concentrations and nitrification rates had fallen while the ammonia-oxidizing populations in plots receiving fertilizer had increased. The rapidity of the rise in nitrification rates observed after 3 days suggests that it results from phenotypic changes in the ammonia-oxidizing bacterial population. Associated increases in population sizes were only observed after 6 weeks and did not correlate directly with nitrifying activity. Phylogenetic analyses of PCR products from one of the plots revealed a population dominated by Nitrosospira-type organisms, similar to those prevalent in other soils.
Hirsch PR, Mauchline TH, Mendum TA, Kerry BR (2000) Detection of the nematophagous fungus Verticillium chlamydosporium in nematode-infested plant roots using PCR, Mycological Research104(4)pp. 435-439
PCR-based methods to detect Verticillium chlamydosporium on infected plant roots were developed. Arbitrary ERIC primers and those based on rRNA genes, to identify fungi grown in pure culture, were unsuitable for DNA extracted from nematode-infested roots, because of interference by plant and nematode DNA. A novel method utilizing specific primers designed from an amplified and cloned fragment of the V. chlamydosporium ²-tubulin gene was developed. Although it could not discriminate between different isolates of V. chlamydosporium, one primer set could identify the fungus on tomato roots infested with root-knot nematodes. The V. chlamydosporium ²-tubulin sequence data showed close homology to sequences from plant endophytic Acremonium and Epichloe species and the saprotrophic Trichoderma viride.
Minnikin DE, Taylor GM, Stewart GR, Mendum TA, Roffey S, Tucker K, Lee OY-C, Wu HHT, Besra GS, Cole ST (2014) Defining the origins and spread of leprosy using molecular biomarkers, AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY153pp. 186-187 WILEY-BLACKWELL
Newcombe J, Mendum TA, Ren CP, McFadden J (2013) Identification of the immunoproteome of the meningococcus by cell surface immunoprecipitation and MS., Microbiology160(Pt 2)pp. 429-438
Most healthy adults are protected from meningococcal disease by the presence of naturally acquired anti-meningococcal antibodies; however, the identity of the target antigens of this protective immunity remains unclear, particularly for protection against serogroup B disease. To identify the protein targets of natural protective immunity we developed an immunoprecipitation and proteomics approach to define the immunoproteome of the meningococcus. Sera from 10 healthy individuals showing serum bactericidal activity against both a meningococcal C strain (L91543) and the B strain MC58, together with commercially available pooled human sera, were used as probe antisera. Immunoprecipitation was performed with each serum sample and live cells from both meningococcal strains. Immunoprecipitated proteins were identified by MS. Analysis of the immunoproteome from each serum demonstrated both pan-reactive antigens that were recognized by most sera as well as subject-specific antigens. Most antigens were found in both meningococcal strains, but a few were strain-specific. Many of the immunoprecipitated proteins have been characterized previously as surface antigens, including adhesins and proteases, several of which have been recognized as vaccine candidate antigens, e.g. factor H-binding protein, NadA and neisserial heparin-binding antigen. The data demonstrate clearly the presence of meningococcal antibodies in healthy individuals with no history of meningococcal infection and a wide diversity of immune responses. The identification of the immunoreactive proteins of the meningococcus provides a basis for understanding the role of each antigen in the natural immunity associated with carriage and may help to design vaccination strategies.
Santangelo MP, McIntosh D, Bigi F, Armoa GRG, Campos ASD, Ruybal P, Dellagostin OA, McFaddend J, Mendum T, Gicquel B, Winter N, Farber M, Cataldi A (2007) Mycobacterium bovis BCG as a delivery system for the RAP-1 antigen from Babesia bovis, VACCINE25(6)pp. 1104-1113 ELSEVIER SCI LTD
Bigi F, Gioffre A, Klepp L, Santangelo MP, Velicovsky CA, Giambartolomei GH, Fossati CA, Romano MI, Mendum T, McFadden JJ, Cataldi A (2005) Mutation in the P36 gene of Mycobacterium bovis provokes attenuation of the bacillus in a mouse model, TUBERCULOSIS85(4)pp. 221-226 CHURCHILL LIVINGSTONE
Borsuk S, Seixas FK, Ramos DF, Mendum T, McFadden J, Dellagostin O (2012) Rational design of diagnostic and vaccination strategies for tuberculosis., Braz J Infect Dis16(1)pp. 68-73
The development of diagnostic tests which can readily differentiate between vaccinated and tuberculosis-infected individuals is crucial for the wider utilization of bacillus Calmette-Guérin (BCG) as vaccine in humans and animals. BCG_0092 is an antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both animals and humans infected with the tubercle bacilli. We carried out bioinformatics analyses of the BCG_0092 and designed a diagnostic test by using the predicted MHC class I epitopes. In addition, we performed a knockout of this gene by homologous recombination in the BCG vaccine strain to allow differentiation of vaccinated from infected individuals. For that, the flanking sequences of the target gene (BCG_0092)were cloned into a suicide vector. Spontaneous double crossovers, which result in wild type revertants or knockouts were selected using SacB. BCG_0092 is present only in members of the Mycobacterium tuberculosis complex. Eight predicted MHC class I epitopes with potential for immunological diagnosis were defined, allowing the design of a specific diagnostic test. The strategy used to delete the (BCG_0092) gene from BCG was successful. The knockout genotype was confirmed by PCR and by Southern blot. The mutant BCG strain has the potential of inducing protection against tuberculosis without interfering with the diagnostic test based on the use of selected epitopes from BCG_0092.
SELBITSCHKA W, JORDING D, NIEMAN S, SCHMIDT R, PUHLER A, MENDUM T, HIRSCH P (1995) CONSTRUCTION AND CHARACTERIZATION OF A RHIZOBIUM-LEGUMINOSARUM BIOVAR VICIAE STRAIN DESIGNED TO ASSESS HORIZONTAL GENE-TRANSFER IN THE ENVIRONMENT, FEMS MICROBIOLOGY LETTERS128(3)pp. 255-263 ELSEVIER SCIENCE BV
Borsuk S, Mendum TA, Fagundes MQ, Michelon M, Cunha CW, McFadden J, Dellagostin OA (2007) Auxotrophic complementation as a selectable marker for stable expression of foreign antigens in Mycobacterium bovis BCG, Tuberculosis87(6)pp. 474-480
Mycobacterium bovis BCG has the potential to be an effective live vector for multivalent vaccines. However, most mycobacterial cloning vectors rely on antibiotic resistance genes as selectable markers, which would be undesirable in any practical vaccine. Here we report the use of auxotrophic complementation as a selectable marker that would be suitable for use in a recombinant vaccine. A BCG auxotrophic for the amino acid leucine was constructed by knocking out the leuD gene by unmarked homologous recombination. Expression of leuD on a plasmid not only allowed complementation, but also acted as a selectable marker. Removal of the kanamycin resistance gene, which remained necessary for plasmid manipulations in Escherichia coli, was accomplished by two different methods: restriction enzyme digestion followed by re-ligation before BCG transformation, or by Cre-loxP in vitro recombination mediated by the bacteriophage P1 Cre Recombinase. Stability of the plasmid was evaluated during in vitro and in vivo growth of the recombinant BCG in comparison to selection by antibiotic resistance. The new system was highly stable even during in vivo growth, as the selective pressure is maintained, whereas the conventional vector was unstable in the absence of selective pressure. This new system will now allow the construction of potential recombinante vaccine strains using stable multicopy plasmid vectors without the inclusion of antibiotic resistance markers. © 2007 Elsevier Ltd. All rights reserved.
Mendum TA, Wu H, Kierzek AM, Stewart GR (2015) Lipid metabolism and type VII secretion systems dominate the genome scale virulence profile of Mycobacterium tuberculosis in human dendritic cells, BMC Genomics16(1)
© 2015 mendum et al.Background: Mycobacterium tuberculosis continues to kill more people than any other bacterium. Although its archetypal host cell is the macrophage, it also enters, and survives within, dendritic cells (DCs). By modulating the behaviour of the DC, M. tuberculosis is able to manipulate the host's immune response and establish an infection. To identify the M. tuberculosis genes required for survival within DCs we infected primary human DCs with an M. tuberculosis transposon library and identified mutations with a reduced ability to survive. Results: Parallel sequencing of the transposon inserts of the surviving mutants identified a large number of genes as being required for optimal intracellular fitness in DCs. Loci whose mutation attenuated intracellular survival included those involved in synthesising cell wall lipids, not only the well-established virulence factors, pDIM and cord factor, but also sulfolipids and PGL, which have not previously been identified as having a direct virulence role in cells. Other attenuated loci included the secretion systems ESX-1, ESX-2 and ESX-4, alongside many PPE genes, implicating a role for ESX-5. In contrast the canonical ESAT-6 family of ESX substrates did not have intra-DC fitness costs suggesting an alternative ESX-1 associated virulence mechanism. With the aid of a gene-nutrient interaction model, metabolic processes such as cholesterol side chain catabolism, nitrate reductase and cysteine-methionine metabolism were also identified as important for survival in DCs. Conclusion: We conclude that many of the virulence factors required for survival in DC are shared with macrophages, but that survival in DCs also requires several additional functions, such as cysteine-methionine metabolism, PGLs, sulfolipids, ESX systems and PPE genes.
Clark IM, Mendum TA, Hirsch PR (2002) The influence of the symbiotic plasmid pRL1JI on the distribution of GM rhizobia in soil and crop rhizospheres, and implications for gene flow., Antonie Van Leeuwenhoek81(1-4)pp. 607-616
The distribution of two genetically modified Rhizobium leguminosarum strains was investigated in the field. One, RSM2004, released in 1987, carries a TnS marker on its conjugative symbiotic plasmid (pSym). The second, CT0370, released at the same site in 1994, has a gusA gene integrated into its chromosome but no pSym. Plate counts indicated that the CT0370 population became established at a higher level than RSM2004. However, when peas, alfalfa and barley were grown, RSM2004 was found to outnumber CT0370 on all roots and by 100-fold on pea. Although the transfer of pSym from RSM2004 to CT0370 could be detected on plates and in microcosm studies with high inoculum densities, no transfer was detected in the field. Subsequent transfer of pSym from RSM2004 to CT0370 demonstrated that it conferred an advantage in the rhizosphere. In addition to increasing host fitness, plasmids may transfer, or mobilise other genetic elements, to other bacteria. This is more likely in sites such as the rhizosphere, where cells are active and numbers are high. The distribution of pSym and other genetic elements associated with rhizobia, in bacterial sub-populations from the soil and roots of the different plants, was investigated using PCR. The genetic elements studied were: ISRm3, an insertion element from Sinorhizobium meliloti; pSB 102, a broad host range mer plasmid; the Rhizobium nodC gene (carried on pSym) and plasmid replication origins repCI and repCII. As expected, ISRm3 was detected in rhizoflora cultured from alfalfa but not the other plants. The mer gene was ubiquitous but the transfer region of pSB 102 was not detected. The nodC and both repC primers amplified products from all the plants, giving further evidence for the occurrence of plasmids originating from Rhizobium in the rhizoflora of non-host plants. Despite the abundance of elements associated with transferable plasmids in rhizobia, none was detected in either inoculant strain.
Mendum TA, Schuenemann VJ, Roffey S, Taylor GM, Wu H, Singh P, Tucker K, Hinds J, Cole ST, Kierzek AM, Nieselt K, Krause J, Stewart GR (2014) Mycobacterium leprae genomes from a British medieval leprosy hospital: towards understanding an ancient epidemic.,BMC Genomics15
BACKGROUND: Leprosy has afflicted humankind throughout history leaving evidence in both early texts and the archaeological record. In Britain, leprosy was widespread throughout the Middle Ages until its gradual and unexplained decline between the 14th and 16th centuries. The nature of this ancient endemic leprosy and its relationship to modern strains is only partly understood. Modern leprosy strains are currently divided into 5 phylogenetic groups, types 0 to 4, each with strong geographical links. Until recently, European strains, both ancient and modern, were thought to be exclusively type 3 strains. However, evidence for type 2 strains, a group normally associated with Central Asia and the Middle East, has recently been found in archaeological samples in Scandinavia and from two skeletons from the medieval leprosy hospital (or leprosarium) of St Mary Magdalen, near Winchester, England. RESULTS: Here we report the genotypic analysis and whole genome sequencing of two further ancient M. leprae genomes extracted from the remains of two individuals, Sk14 and Sk27, that were excavated from 10th-12th century burials at the leprosarium of St Mary Magdalen. DNA was extracted from the surfaces of bones showing osteological signs of leprosy. Known M. leprae polymorphisms were PCR amplified and Sanger sequenced, while draft genomes were generated by enriching for M. leprae DNA, and Illumina sequencing. SNP-typing and phylogenetic analysis of the draft genomes placed both of these ancient strains in the conserved type 2 group, with very few novel SNPs compared to other ancient or modern strains. CONCLUSIONS: The genomes of the two newly sequenced M. leprae strains group firmly with other type 2F strains. Moreover, the M. leprae strain most closely related to one of the strains, Sk14, in the worldwide phylogeny is a contemporaneous ancient St Magdalen skeleton, vividly illustrating the epidemic and clonal nature of leprosy at this site. The prevalence of these type 2 strains indicates that type 2F strains, in contrast to later European and associated North American type 3 isolates, may have been the co-dominant or even the predominant genotype at this location during the 11th century.
Mendum TA, Hirsch PR (2002) Changes in the population structure of ²-group autotrophic ammonia oxidising bacteria in arable soils in response to agricultural practice, Soil Biology and Biochemistry34(10)pp. 1479-1485
Autotrophic ammonia oxidising bacteria of the ²-group proteobacteria are important in the global nitrogen cycle, as they are responsible for the majority of ammonia oxidation in arable soil. This study examined the effects of agricultural practices on the community structure of autotrophic ammonia oxidising populations in arable plots that receive either NH4NO3, farmyard manure plus NH4NO3, or no nitrogen. The 16S rRNA genes of autotrophic ammonia oxidisers were PCR amplified from DNA extracted from these soils, and the products hybridised to oligonucleotide probes specific for each of the previously recognised phylogenetically distinct groups of autotrophic ammonia oxidising bacteria. Only 16S rDNA sequences from group 3 and group 4 organisms were detected in the PCR products. Products from the plot that received NH4NO3 fertiliser were dominated by group 3 sequences, while those that received no nitrogen fertiliser were dominated by group 4 sequences. Ploughing of the plots was associated with an increased abundance of group 3 organisms particularly in plots that had previously received NH4NO3 fertiliser. These data were related to both the size and activity of the ammonia oxidising populations, as determined by a competitive PCR assay based upon the amoA gene, and by 15N isotopic pool dilution, respectively. © 2002 Elsevier Science Ltd. All rights reserved.
Mendum TA, Sockett RE, Hirsch PR (1998) The detection of Gram-negative bacterial mRNA from soil by RT-PCR, FEMS Microbiology Letters164(2)pp. 369-373
The method described extracts high quality RNA from a clay-rich soil for which other published extraction protocols have failed. Transcript encoding the enzyme ²-glucuronidase could be reliably detected by reverse transcriptase polymerase chain reaction in soil seeded with a genetically modified Rhizobium leguminosarum containing the uidA gene. Copyright (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
BACKGROUND: Neisseria meningitidis is an important human commensal and pathogen that causes several thousand deaths each year, mostly in young children. How the pathogen replicates and causes disease in the host is largely unknown, particularly the role of metabolism in colonization and disease. Completed genome sequences are available for several strains but our understanding of how these data relate to phenotype remains limited. RESULTS: To investigate the metabolism of N. meningitidis we generated and selected a representative Tn5 library on rich medium, a minimal defined medium and in human serum to identify genes essential for growth under these conditions. To relate these data to a systems-wide understanding of the pathogen's biology we constructed a genome-scale metabolic network: Nmb_iTM560. This model was able to distinguish essential and non-essential genes as predicted by the global mutagenesis. These essentiality data, the library and the Nmb_iTM560 model are powerful and widely applicable resources for the study of meningococcal metabolism and physiology. We demonstrate the utility of these resources by predicting and demonstrating metabolic requirements on minimal medium such as a requirement for PEP carboxylase, and by describing the nutritional and biochemical status of N. meningitidis when grown in serum, including a requirement for both the synthesis and transport of amino acids. CONCLUSIONS: This study describes the application of a genome scale transposon library combined with an experimentally validated genome-scale metabolic network of N. meningitidis to identify essential genes and provide novel insight to the pathogen's metabolism both in vitro and during infection.
Schuenemann VJ, Singh P, Mendum TA, Krause-Kyora B, Jäger G, Bos KI, Herbig A, Economou C, Benjak A, Busso P, Nebel A, Boldsen JL, Kjellström A, Wu H, Stewart GR, Taylor GM, Bauer P, Lee OY, Wu HH, Minnikin DE, Besra GS, Tucker K, Roffey S, Sow SO, Cole ST, Nieselt K, Krause J (2013) Genome-wide comparison of medieval and modern Mycobacterium leprae., Science341(6142)pp. 179-183
Leprosy was endemic in Europe until the Middle Ages. Using DNA array capture, we have obtained genome sequences of Mycobacterium leprae from skeletons of five medieval leprosy cases from the United Kingdom, Sweden, and Denmark. In one case, the DNA was so well preserved that full de novo assembly of the ancient bacterial genome could be achieved through shotgun sequencing alone. The ancient M. leprae sequences were compared with those of 11 modern strains, representing diverse genotypes and geographic origins. The comparisons revealed remarkable genomic conservation during the past 1000 years, a European origin for leprosy in the Americas, and the presence of an M. leprae genotype in medieval Europe now commonly associated with the Middle East. The exceptional preservation of M. leprae biomarkers, both DNA and mycolic acids, in ancient skeletons has major implications for palaeomicrobiology and human pathogen evolution.
Borsuk S, Mendum TA, Fagundes MQ, Michelon M, Cunha CW, McFadden J, Dellagostin OA (2007) Auxotrophic complementation as a selectable marker for stable expression of foreign antigens in Mycobacterium bovis BCG, TUBERCULOSIS87(6)pp. 474-480 CHURCHILL LIVINGSTONE
Cullen DW, Nicholson PS, Mendum TA, Hirsch PR (1998) Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction., J Appl Microbiol84(6)pp. 1025-1034
Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn5; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strain provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g-1 soil in soil-extracted DNA.
BACKGROUND: Mycobacterium tuberculosis continues to kill more people than any other bacterium. Although its archetypal host cell is the macrophage, it also enters, and survives within, dendritic cells (DCs). By modulating the behaviour of the DC, M. tuberculosis is able to manipulate the host's immune response and establish an infection. To identify the M. tuberculosis genes required for survival within DCs we infected primary human DCs with an M. tuberculosis transposon library and identified mutations with a reduced ability to survive. RESULTS: Parallel sequencing of the transposon inserts of the surviving mutants identified a large number of genes as being required for optimal intracellular fitness in DCs. Loci whose mutation attenuated intracellular survival included those involved in synthesising cell wall lipids, not only the well-established virulence factors, pDIM and cord factor, but also sulfolipids and PGL, which have not previously been identified as having a direct virulence role in cells. Other attenuated loci included the secretion systems ESX-1, ESX-2 and ESX-4, alongside many PPE genes, implicating a role for ESX-5. In contrast the canonical ESAT-6 family of ESX substrates did not have intra-DC fitness costs suggesting an alternative ESX-1 associated virulence mechanism. With the aid of a gene-nutrient interaction model, metabolic processes such as cholesterol side chain catabolism, nitrate reductase and cysteine-methionine metabolism were also identified as important for survival in DCs. CONCLUSION: We conclude that many of the virulence factors required for survival in DC are shared with macrophages, but that survival in DCs also requires several additional functions, such as cysteine-methionine metabolism, PGLs, sulfolipids, ESX systems and PPE genes.
Beste DJ, Nöh K, Niedenführ S, Mendum TA, Hawkins ND, Ward JL, Beale MH, Wiechert W, McFadden J (2013) (13)C-Flux Spectral Analysis of Host-Pathogen Metabolism Reveals a Mixed Diet for Intracellular Mycobacterium tuberculosis., Chem Biol20(8)pp. 1012-1021 Elsevier
Whereas intracellular carbon metabolism has emerged as an attractive drug target, the carbon sources of intracellularly replicating pathogens, such as the tuberculosis bacillus Mycobacterium tuberculosis, which causes long-term infections in one-third of the world's population, remain mostly unknown. We used a systems-based approach-(13)C-flux spectral analysis (FSA) complemented with manual analysis-to measure the metabolic interaction between M. tuberculosis and its macrophage host cell. (13)C-FSA analysis of experimental data showed that M. tuberculosis obtains a mixture of amino acids, C1 and C2 substrates from its host cell. We experimentally confirmed that the C1 substrate was derived from CO2. (13)C labeling experiments performed on a phosphoenolpyruvate carboxykinase mutant revealed that intracellular M. tuberculosis has access to glycolytic C3 substrates. These findings provide constraints for developing novel chemotherapeutics.
Neisseria meningitidis causes meningococcal disease, a global life threatening illness with annual incidences of between 1 and 1000 per 100,000 population. Humans are the only known host with approximately 10% of people having asymptomatic nasopharyngeal carriage at any one time. Thus, the ability of meningococci to attach, invade, and grow in the epithelium is crucial for both its commensal and pathogenic properties. In the rare event that meningococci cross the epithelium into the bloodstream, disease may occur. In order to better understand the mechanisms of meningococcal pathogenesis, transposon mutagenesis was used to identify bacterial genes involved in epithelial cell adherence and internalization as well as traversal of the epithelial barrier.
Three epithelial cell lines of respiratory origin, A549 cells, 16HBE14o- cells and Detroit 562 cells were used to examine N. meningitidis L91543 (C:2a:P1.2, ST-11; ET-37) pathogenesis. First, adhesion, invasion and traversal assays were optimized for bacterial uptake to enable the maximum number of mutants to be tested and to avoid stochastic loss from the transposon library. Since the highest level of meningococci adherence and invasion was observed using 16HBE14o- cells, this cell line was chosen for subsequent traversal assays, where an intact epithelial barrier was established on Transwell® membrane inserts. Epithelial barrier integrity was assessed by measuring transepithelial electrical resistance (TEER), permeability of the marker protein, 70 kDa Dextran, and by examining the distribution of the tight junction proteins, occludin and ZO-1, by immunofluorescence.
Next, transposon mutagenesis libraries comprising of approx. 14,500 N. meningitidis L91543 mutants, were used to probe meningococcal interactions with 16HBE14o- epithelial cells. Illumina sequencing of amplified transposon junctions was performed on DNA extracted from both input and output pools obtained from the various assays. Comparative analysis of input/output pools showed reduced fitness, not only of genes associated with type IV pili, but mainly of genes involved in metabolism especially nucleotide and amino acid metabolism. Genes involved in membrane transport, regulatory functions and cellular processes also showed reduced fitness. The function of putative genes of interest was validated by generating insertion knockout mutants and testing them independently for their ability to alter meningococcal-epithelial cell interactions. The knockout mutant assays showed 67-100% agreement to the Tn-Seq analysis prediction. Based on the knock out mutant assays as well the Tn-Seq prediction we can conclude that type IV pili, nucleotide biosynthesis, glucose and amino acid metabolism, as well as resistance to antimicrobial peptide are critical for meningococcal interaction with epithelial cells.
Basu Piyali, Sandhu Noor, Bhatt Apoorva, Singh Albel, Balhana Ricardo, Gobe Irene, Crowhurst Nicola A, Mendum Thomas, Gao Liang, Ward Jane L, Beale Michael H, McFadden Johnjoe, Beste Dany (2018) The anaplerotic node is essential for the intracellular survival of Mycobacterium tuberculosis,Journal of Biological Chemistry293(15)pp. 5695-5704
American Society for Biochemistry and Molecular Biology
Enzymes at the phosphoenolpyruvate (PEP)?pyruvate?oxaloacetate or anaplerotic (ANA) node control the metabolic flux to glycolysis, gluconeogenesis, and anaplerosis. Here we used genetic, biochemical, and 13C isotopomer analysis to characterize the role of the enzymes at the ANA node in intracellular survival of the world's most successful bacterial pathogen, Mycobacterium tuberculosis (Mtb). We show that each of the four ANA enzymes, pyruvate carboxylase (PCA), PEP carboxykinase (PCK), malic enzyme (MEZ), and pyruvate phosphate dikinase (PPDK), performs a unique and essential metabolic function during the intracellular survival of Mtb. We show that in addition to PCK, intracellular Mtb requires PPDK as an alternative gateway into gluconeogenesis. Propionate and cholesterol detoxification was also identified as an essential function of PPDK revealing an unexpected role for the ANA node in the metabolism of these physiologically important intracellular substrates and highlighting this enzyme as a tuberculosis (TB)-specific drug target. We show that anaplerotic fixation of CO2 through the ANA node is essential for intracellular survival of Mtb and that Mtb possesses three enzymes (PCA, PCK, and MEZ) capable of fulfilling this function. In addition to providing a back-up role in anaplerosis we show that MEZ also has a role in lipid biosynthesis. MEZ knockout strains have an altered cell wall and were deficient in the initial entry into macrophages. This work reveals that the ANA node is a focal point for controlling the intracellular replication of Mtb, which goes beyond canonical gluconeogenesis and represents a promising target for designing novel anti-TB drugs.
We report on a Mycobacterium leprae genome isolated from the remains of an individual with lepromatous leprosy that were excavated from a 7th century Hungarian cemetery. We determined that the genome was from a SNP type 3K0 M. leprae strain, a lineage that diverged early from other M. leprae lineages. This is one of the earliest 3K0 M. leprae genomes to be sequenced to date. A number of novel SNPs as well as SNPs characteristic of the 3K0 lineage were confirmed by conventional PCR and Sanger sequencing. Recovery of accompanying human DNA from the burial was poor, particularly when compared to that of the pathogen. Modern 3K0 M. leprae strains have only been isolated from East Asia and the Pacific and so these findings require new scenarios to describe the origins and routes of dissemination of leprosy during antiquity that have resulted in the modern phylogeographical distribution of M. leprae.
Mendum Tom A., Chandran Aneesh, Williams Kerstin, Vordermeier H. Martin, Villarreal-Ramos Bernardo, Wu H., Singh Albel, Smith Alex A., Butler Rachel E., Prasad Aravind, Bharti Neeraj, Banerjee Ruma, Kasibhatla Sunitha M., Bhatt Apoorva, Stewart Graham R., McFadden Johnjoe (2019) Transposon libraries identify novel Mycobacterium bovis BCG genes involved in the dynamic interactions required for BCG to persist during in vivo passage in cattle,BMC Genomics20431pp. 1-13
BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG?s efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host?s immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria.
To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host.
These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host?s innate immune system to allow it to persist, a property that is important for its protective efficacy.
Sexually transmitted diseases (STDs) can be important drivers of population dynamics because of their negative effects on reproduction.
However, screening for STDs, especially in wildlife populations, is widely neglected. Using the promiscuous, polygynandrous European badger (Meles meles) as a model, we investigated the presence and prevalence of herpesviruses (HVs) in a wild, high-density population and assessed potential differences in somatic fitness and female reproductive condition between infected and uninfected individuals. We collected n=98 genital swabs from 71 females (51 adults and 20 cubs) and 27 males (26 adults and 1 cub) during spring and summer 2015. Using a PCR specific for a mustelid ±-HV, all genital-swab samples tested negative. In a panherpes PCR, a ³-HV was found in 55% (54/98; 39 adults and 15 cubs), identified as mustelid gammaherpesvirus 1 (MusGHV-1) using DNA sequencing. This contrasts with the results of a previous study, which reported MusGHV-1 in 98% (354/361) of blood samples taken from 218 badgers in the same population using PCR. The detection of MusHV-1 in the female reproductive tract strongly indicates the potential for a horizontal and, likely also a vertical, route of transmission. Our results suggest a potential linkage of genital HVs and impaired future reproductive success in females, but because reproductive failure can have many reasons in badgers, the causative link of this negative relationship remains to be investigated.
Mycobacterium bovis is the causative agent of bovine tuberculosis and the predominant cause of zoonotic tuberculosis in people. Bovine tuberculosis occurs in farmed cattle but also in a variety of wild animals, which form a reservoir of infection. Although direct transmission of tuberculosis occurs between mammals, the low frequency of contact between different host species and abundant shedding of bacilli by infected animals suggests an infectious route via environmental contamination. Other intracellular pathogens that transmit via the environment deploy strategies to survive or exploit predation by environmental amoebae. To explore if M. bovis has this capability, we investigated its interactions with the soil and dung-dwelling amoeba, Dictyostelium discoideum. We demonstrated that M. bovis evades phagocytosis and destruction by D. discoideum and actively transits through the amoeba using the ESX-1 Type VII Secretion System as part of a programme of mechanisms, many of which have been co-opted as virulence factors in the mammalian host. This capacity of M. bovis to utilise an environmental stage between mammalian hosts may enhance its transmissibility. In addition, our data provide molecular evidence to support an evolutionary role for amoebae as training grounds for the pathogenic M. tuberculosis complex.
New approaches are needed to control leprosy, but understanding of
the biology of the causative agent Mycobacterium leprae remains rudimentary, principally because the pathogen cannot be grown in axenic culture. Here, we applied
13C isotopomer analysis to measure carbon metabolism of M. leprae in its primary
host cell, the Schwann cell. We compared the results of this analysis with those of a
related pathogen, Mycobacterium tuberculosis, growing in its primary host cell, the
macrophage. Using 13C isotopomer analysis with glucose as the tracer, we show that
whereas M. tuberculosis imports most of its amino acids directly from the host macrophage, M. leprae utilizes host glucose pools as the carbon source to biosynthesize
the majority of its amino acids. Our analysis highlights the anaplerotic enzyme phosphoenolpyruvate carboxylase required for this intracellular diet of M. leprae, identifying this enzyme as a potential antileprosy drug target.
Nitrogen metabolism of Mycobacterium tuberculosis(Mtb) is crucial for the survival of this important pathogen in its primary human host cell, the macrophage, but little is known about the source(s) and their assimilation within this intracellular niche. Here, we have developed 15N-flux spectral ratio analysis(15N-FSRA) to explore Mtb?s nitrogen metabolism; we demonstrate that intracellular Mtb has access to multiple amino acids in the macrophage, including glutamate, glutamine, aspartate, alanine, glycine,and valine; and we identify glutamine as the pre-dominant nitrogen donor. Each nitrogen source is uniquely assimilated into specific amino acid pools,indicating compartmentalized metabolism during intracellular growth. We have discovered that serine is not available to intracellular Mtb, and we show that a serine auxotroph is attenuated in macrophages. This work provides a systems-based tool for exploring the nitrogen metabolism of intracellular pathogens and highlights the enzyme phosphoserine transaminase as an attractive target for the development of novel anti-tuberculosis therapies.
Taylor G. Michael, Murphy Eileen M., Mendum Thomas, Pike Alistair W.G, Linscott Bethan, Wu Huihai, O'Grady Justin, Richardson Hollian, O'Donovan Edmond, Troy Carmelita, Stewart Graham (2018) Leprosy at the edge of Europe?Biomolecular,
isotopic and osteoarchaeological findings
from medieval Ireland,PLOS One
Public Library of Science
Relatively little is known of leprosy in Medieval Ireland; as an island located at the far west of
Europe it has the potential to provide interesting insights in relation to the historical epidemiology of the disease. To this end the study focuses on five cases of probable leprosy identified in human skeletal remains excavated from inhumation burials. Three of the individuals
derived from the cemetery of St Michael Le Pole, Golden Lane, Dublin, while single examples were also identified from Ardreigh, Co. Kildare, and St Patrick?s Church, Armoy, Co.
Antrim. The individuals were radiocarbon dated and examined biomolecularly for evidence
of either of the causative pathogens, M. leprae or M. lepromatosis. Oxygen and strontium
isotopes were measured in tooth enamel and rib samples to determine where the individuals
had spent their formative years and to ascertain if they had undertaken any recent migrations. We detected M. leprae DNA in the three Golden Lane cases but not in the probable
cases from either Ardreigh Co. Kildare or Armoy, Co. Antrim. M. lepromatosis was not
detected in any of the burals. DNA preservation was sufficiently robust to allow genotyping
of M. leprae strains in two of the Golden Lane burials, SkCXCV (12-13th century) and
SkCCXXX (11-13th century). These strains were found to belong on different lineages of
the M. leprae phylogenetic tree, namely branches 3 and 2 respectively. Whole genome
sequencing was also attempted on these two isolates with a view to gaining further information but poor genome coverage precluded phylogenetic analysis. Data from the biomolecular study was combined with osteological, isotopic and radiocarbon dating to provide a
comprehensive and multidisciplinary study of the Irish cases. Strontium and oxygen isotopic
analysis indicate that two of the individuals from Golden Lane (SkCXLVIII (10-11th century)
and SkCXCV) were of Scandinavian origin, while SkCCXXX may have spent his childhood
in the north of Ireland or central Britain. We propose that the Vikings were responsible for
introducing leprosy to Ireland. This work adds to our knowledge of the likely origins of leprosy in Medieval Ireland and will hopefully stimulate further research into the history and spread of this ancient disease across the world.
Metabolism underpins the pathogenic strategy of the causative agent of TB, Mycobacterium tuberculosis (Mtb), and therefore metabolic pathways have recently re-emerged as attractive drug targets. A powerful approach to study Mtb metabolism as a whole, rather than just individual enzymatic components, is to use a systems biology framework, such as a Genome-Scale Metabolic Network (GSMN) that allows the dynamic interactions of all the components of metabolism to be interrogated together. Several GSMNs networks have been constructed for Mtb and used to study the complex relationship between the Mtb genotype and its phenotype. However, the utility of this approach is hampered by the existence of multiple models, each with varying properties and performances. Here we systematically evaluate eight recently published metabolic models of Mtb-H37Rv to facilitate model choice. The best performing models, sMtb2018 and iEK1011, were refined and improved for use in future studies by the TB research community.
Bovine tuberculosis is an important animal health problem and the predominant cause of zoonotic tuberculosis worldwide. It results in serious economic burden due to losses in productivity and the cost of control programmes. Control could be greatly improved by the introduction of an efficacious cattle vaccine but the most likely candidate, BCG, has several limitations including variable efficacy. Augmentation of BCG with a subunit vaccine booster has been shown to increase protection but the selection of antigens has hitherto been left largely to serendipity. In the present study, we take a rational approach to identify the protective antigens of BCG, selecting a BCG transposon mutant library in naïve and BCG-vaccinated cattle. Ten mutants had increased relative survival in vaccinated compared to naïve cattle, consistent with loss of protective antigen targets making the mutants less visible to the BCG immune response. The immunogenicity of three putative protective antigens, BCG_0116, BCG_0205 (YrbE1B) and BCG_1448 (PPE20) was investigated using peptide pools and PBMCs from BCG vaccinated cattle. BCG vaccination induced PBMC to release elevated levels of IP10, IL-17a and IL-10 in response to all three antigens. Taken together, the data supports the further study of these antigens for use in subunit vaccines.