Biology subjects

Langford, P. R.

Publications and source records attributed to Langford, P. R..

4 recordsLinked to original sources

bioRxiv2024

PluMu - a Mu-like bacteriophage infecting Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia, an economically important lung disease of pigs. In draft genomes of two Cypriot clinical A. pleuropneumoniae isolates (MIDG3457 and MIDG3459), we previously identified single genomic regions with homology to Mu-like bacteriophage and presented preliminary evidence of active phage. Here, updated Phastest genomic analysis identified two loci in both MIDG3457 and MIDG3459 that were predicted to encode proteins with high homology to, and whose organization was characteristic of, Mu-like phages. Phylogenetically, the closest matches were with Mannheimia Vb and Glaesserella SuMu phages. Phastest scored the loci as "complete", indicating they produced active phage. PCR amplification of the Mu-like phage c and tail genes from DNase-treated polyethylene glycol 8000 (PEG)-precipitated supernatants of MIDG3457 and MIDG3459 (grown in either Brain Heart Infusion-NAD or Graces Insect Medium-NAD broth) indicated the presence of intact virions. The phages from MIDG3457 and MIDG3459 were named PluMu 3457-1, 3457-2, and PluMu 3459-1 and PluMu 3459-2, respectively. Transmission electron microscopy (TEM) of PEG-precipitated supernatants of broth-grown MIDG3459 identified virions with icosahedral heads and tails, consistent with other Mu-like phages. We conclude that MIDG3459 produces an active Mu- like phage.

microbiology↗

bioRxiv2022

Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that comprise distinct phasevarions

ABSTRACTActinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase- variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferase, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase variants results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

microbiology↗

bioRxiv2019

Identification of reduced host transcriptomic signatures for tuberculosis and digital PCR-based validation and quantification

Recently, host whole blood gene expression signatures have been identified for diagnosis of tuberculosis (TB). Absolute quantification of the concentrations of signature transcripts in blood have not been reported, but would facilitate the development of diagnostic tests.\n\nTo identify minimal transcript signatures, we applied a novel transcript selection procedure to microarray data from African adults comprising 536 patients with TB, other diseases (OD) and latent TB (LTBI), divided into training and test sets. Signatures were validated using reverse transcriptase (RT) - digital PCR (dPCR).\n\nA four-transcript signature (GBP6, TMCC1, PRDM1, ARG1) measured using RT-dPCR distinguished TB patients from those with OD (area under the curve (AUC) 93.8% (CI95% 82.2 - 100%). A three-transcript signature (FCGR1A, ZNF296, C1QB) differentiated TB from LTBI (AUC 97.3%, CI95%: 93.3 - 100%), regardless of HIV.\n\nThese signatures have been validated across platforms and across samples offering strong, quantitative support for their use as diagnostic biomarkers for TB.

molecular biology↗

bioRxiv2018

Rationally designed mariner vectors to allow functional genomic analysis of Actinobacillus pleuropneumoniae and other bacteria by transposon-directed insertion-site sequencing (TraDIS)

Transposon Directed Insertion Sequencing (TraDIS) is a high-throughput method for mapping insertion sites in large libraries of transposon mutants. The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide. In this study, we generated two novel mariner vectors, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), in order to facilitate TraDIS identification of conditionally essential genes in Actinobacillus pleuropneumoniae and other bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and Pasteurella multocida that showed a near random distribution of insertions around the respective chromosomes. A preliminary screen of 5000 mutants each identified 8 and 15 genes, respectively, that are required for growth under anaerobic conditions.

microbiology↗