Imperial College London

ProfessorPaulLangford

Faculty of MedicineDepartment of Infectious Disease

Professor of Paediatric Infectious Diseases
 
 
 
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Contact

 

+44 (0)20 7594 3359p.langford Website

 
 
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Location

 

236Wright Fleming WingSt Mary's Campus

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Summary

 

Publications

Publication Type
Year
to

229 results found

Xu J, Chen L, Pang S, Zhang Q, Deng S, Zhu J, Chen X, Langford PR, Huang Q, Zhou R, Li Let al., 2024, HylS', a fragment of truncated hyaluronidase of Streptococcus suis, contributes to immune evasion by interaction with host complement factor C3b., Virulence, Vol: 15

Pathogenic bacteria have evolved many strategies to evade surveillance and attack by complements. Streptococcus suis is an important zoonotic pathogen that infects humans and pigs. Hyaluronidase (HylA) has been reported to be a potential virulence factor of S. suis. However, in this study, it was discovered that the genomic region encoding HylA of the virulent S. suis strain SC19 and other ST1 strains was truncated into four fragments when aligned with a strain containing intact HylA and possessing hyaluronidase activity. As a result, SC19 had no hyaluronidase activity, but one truncated HylA fragment, designated as HylS,' directly interacted with complement C3b, as confirmed by western ligand blotting, pull-down, and ELISA assays. The deposition of C3b and membrane attack complex (MAC) formation on the surface of a HylS'-deleted mutant (ΔhylS') was significantly increased compared to wild-type SC19. In human sera and whole blood, ΔhylS' survival was significantly reduced compared to that in SC19. The resistance of ΔhylS' to macrophages and human polymorphonuclear neutrophil PMNs also decreased. In a mouse infection model, ΔhylS' showed reduced lethality and lower bacterial load in the organs compared to that of SC19. We conclude that the truncated hyaluronidase HylS' fragment contributes to complement evasion and the pathogenesis of S. suis.

Journal article

Wang L, Sun J, Zhao J, Bai J, Zhang Y, Zhu Y, Zhang W, Wang C, Langford PR, Liu S, Li Get al., 2024, A CRISPR-Cas12a-based platform facilitates the detection and <i>serotyping of Streptococcus suis </i>serotype 2, TALANTA, Vol: 267, ISSN: 0039-9140

Journal article

da Silva GC, Rosa J, de Castro A, Barbosa E, Clarindo W, Mantovani H, Li Y, Bosse J, Langford P, Bazzolli Det al., 2023, Identification of novel small RNAs in extracellular vesicles produced by Actinobacillus pleuropneumoniae, Frontiers in Microbiology, Vol: 14, ISSN: 1664-302X

Extracellular vesicle (EV) production by bacteria is an important mechanism for microbial communication and host-pathogen interaction. EVs of some bacterial species have been reported to contain nucleic acids. However, the role of small RNAs (sRNAs) packaged in EVs is poorly understood. Here, we report on the RNA cargo of EVs produced by the pig pathogen Actinobacillus pleuropneumoniae, the causal agent of porcine pleuropneumonia, a disease which causes substantial economic losses to the swine industry worldwide. The EVs produced by aerobically and anaerobically grown bacteria were only slightly different in size and distribution. Total cell and outer membrane protein profiles and lipid composition of A. pleuropneumoniae whole cell extracts and EVs were similar, although EVs contained rough lipopolysaccharide compared to the smooth form in whole cells. Approximately 50% of Galleria mellonella larvae died after the injection of EVs. RNAseq, RT-PCR, protection from nuclease degradation, and database searching identified previously described and 13 novel A. pleuropneumoniae sRNAs in EVs, some of which were enriched compared to whole cell content. We conclude that A. pleuropneumoniae EVs contain sRNAs, including those known to be involved in virulence, and some with homologs in other Pasteurellaceae and/or non-Pasteurellaceae. Further work will establish whether the novel sRNAs in A. pleuropneumoniae EVs play any role in pathogenesis.

Journal article

Abouelhadid S, Atkins ER, Kay EJ, Passmore IJ, North SJ, Lehri B, Hitchen P, Bakke E, Rahman M, Bossé JT, Li Y, Terra VS, Langford PR, Dell A, Wren BW, Cuccui Jet al., 2023, Development of a novel glycoengineering platform for the rapid production of conjugate vaccines, Microbial Cell Factories, Vol: 22, Pages: 1-13, ISSN: 1475-2859

Conjugate vaccines produced either by chemical or biologically conjugation have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccine assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel biotechnological platform that overcomes the limitations of the current conjugate vaccine design method(s). As a model, we focused our design on a leading bioconjugation method using N-oligosaccharyltransferase (OTase), PglB. The installation of MAGIC led to at least twofold increase in glycoconjugate yield via MAGIC when compared to conventional N-OTase based bioconjugation method(s). Then, we improved MAGIC to (a) allow rapid installation of glycoengineering component(s), (b) omit the usage of antibiotics, (c) reduce the dependence on protein induction agents. Furthermore, we show the modularity of the MAGIC platform in performing glycoengineering in bacterial species that are less genetically tractable than the commonly used Escherichia coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against serious bacterial pathogens. We anticipate the utility of the MAGIC platform could enhance vaccines production due to its compatibility with virtually any bioconjugation method, thus expanding vaccine biopreparedness toolbox.

Journal article

Siris S, Gladstone CA, Guo Y, Patel R, Pinder CL, Shattock RJ, McKay PF, Langford PR, Bidmos Fet al., 2023, Increasing human monoclonal antibody cloning efficiency with whole-cell modified Immunoglobulin-Capture Assay (mICA), Frontiers in Immunology, Vol: 14, Pages: 1-15, ISSN: 1664-3224

Expression cloning of fully human monoclonal antibodies (hmAbs) is seeing powerful utility in the field of vaccinology, especially for elucidating vaccine-induced B-cell responses and novel vaccine candidate antigen discovery. Precision of the hmAb cloning process relies on efficient isolation of hmAb-producing plasmablasts of interest. Previously, a novel immunoglobulin-capture assay (ICA) was developed, using single protein vaccine antigens, to enhance the pathogen-specific hmAb cloning output. Here, we report a novel modification of this single-antigen ICA using formalin-treated, fluorescently stained whole cell suspensions of the human bacterial invasive pathogens, Streptococcus pneumoniae and Neisseria meningitidis. Sequestration of IgG secreted by individual vaccine antigen-specific plasmablasts was achieved by the formation of an anti-CD45-streptavidin and biotin anti-IgG scaffold. Suspensions containing heterologous pneumococcal and meningococcal strains were then used to enrich for polysaccharide- and protein antigen-specific plasmablasts, respectively, during single cell sorting. Following application of the modified whole-cell ICA (mICA), ~61% (19/31) of anti-pneumococcal polysaccharide hmAbs were cloned compared to 14% (8/59) obtained using standard (non-mICA) methods – representing a ~4.4-fold increase in hmAb cloning precision. A more modest ~1.7-fold difference was obtained for anti-meningococcal vaccine hmAb cloning; ~88% of hmAbs cloned via mICA versus ~53% cloned via the standard method were specific for a meningococcal surface protein. VDJ sequencing revealed that cloned hmAbs reflected an anamnestic response to both pneumococcal and meningococcal vaccines; diversification within hmAb clones occurred by positive selection for replacement mutations. Thus, we have shown successful utilisation of whole bacterial cells in the ICA protocol enabling isolation of hmAbs targeting multiple disparate epitopes, thereby increasing the power of approache

Journal article

Zhang Q, Peng L, Han W, Chen H, Tang H, Chen X, Langford PR, Huang Q, Zhou R, Li Let al., 2023, The morphology and metabolic changes of Actinobacillus pleuropneumoniae during its growth as a biofilm, BMC Veterinary Research, Vol: 54, Pages: 1-18, ISSN: 1746-6148

Actinobacillus pleuropneumoniae is an important swine respiratory pathogen. Previous studies have suggested that growth as a biofilm is a natural state of A. pleuropneumoniae infection. To understand the survival features involved in the biofilm state, the growth features, morphology and gene expression profiles of planktonic and biofilm A. pleuropneumoniae were compared. A. pleuropneumoniae in biofilms showed reduced viability but maintained the presence of extracellular polymeric substances (EPS) after late log-phase. Under the microscope, bacteria in biofilms formed dense aggregated structures that were connected by abundant EPS, with reduced condensed chromatin. By construction of Δpga and ΔdspB mutants, polymeric β-1,6-linked N-acetylglucosamine and dispersin B were confirmed to be critical for normal biofilm formation. RNA-seq analysis indicated that, compared to their planktonic counterparts, A. pleuropneumoniae in biofilms had an extensively altered transcriptome. Carbohydrate metabolism, energy metabolism and translation were significantly repressed, while fermentation and genes contributing to EPS synthesis and translocation were up-regulated. The regulators Fnr (HlyX) and Fis were found to be up-regulated and their binding motifs were identified in the majority of the differentially expressed genes, suggesting their coordinated global role in regulating biofilm metabolism. By comparing the transcriptome of wild-type biofilm and Δpga, the utilization of oligosaccharides, iron and sulfur and fermentation were found to be important in adhesion and aggregation during biofilm formation. Additionally, when used as inocula, biofilm bacteria showed reduced virulence in mouse, compared with planktonic grown cells. Thus, these results have identified new facets of A. pleuropneumoniae biofilm maintenance and regulation.

Journal article

Nahar N, Tram G, Jen FE-C, Phillips ZN, Weinert LA, Bosse JT, Jabbari JS, Gouil Q, Du MRM, Ritchie ME, Bowden R, Langford PR, Tucker AW, Jennings MP, Turni C, Blackall PJ, Atack JMet al., 2023, Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions, Nucleic Acids Research, Vol: 51, Pages: 3240-3260, ISSN: 0305-1048

Actinobacillus 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 methyltransferases, 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 in A. pleuropneumoniae 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.

Journal article

Li Y, Bosse J, Stringer O, Hennig-Pauka I, Mortensen P, Langford Pet al., 2023, Detection of novel Actinobacillus pleuropneumoniae serovars by multiplex PCR: a cautionary tale, Microbiology Spectrum, Vol: 11, Pages: 1-2, ISSN: 2165-0497

Journal article

Asai M, Li Y, Newton S, Robertson B, Langford Pet al., 2023, Galleria mellonella-intracellular bacteria pathogen infection models: the ins and outs, FEMS Microbiology Reviews, Vol: 47, Pages: 1-32, ISSN: 0168-6445

Galleria mellonella (greater wax moth) larvae are used widely as surrogate infectious disease models, due to ease of use and the presence of an innate immune system functionally similar to that of vertebrates. Here, we review G. mellonella–human intracellular bacteria pathogen infection models from the genera Burkholderia, Coxiella, Francisella, Listeria, and Mycobacterium. For all genera, G. mellonella use has increased understanding of host–bacterial interactive biology, particularly through studies comparing the virulence of closely related species and/or wild-type versus mutant pairs. In many cases, virulence in G. mellonella mirrors that found in mammalian infection models, although it is unclear whether the pathogenic mechanisms are the same. The use of G. mellonella larvae has speeded up in vivo efficacy and toxicity testing of novel antimicrobials to treat infections caused by intracellular bacteria: an area that will expand since the FDA no longer requires animal testing for licensure. Further use of G. mellonella–intracellular bacteria infection models will be driven by advances in G. mellonella genetics, imaging, metabolomics, proteomics, and transcriptomic methodologies, alongside the development and accessibility of reagents to quantify immune markers, all of which will be underpinned by a fully annotated genome.

Journal article

Li Y, Ma B, Hua K, Gong H, He R, Luo R, Bi D, Zhou R, Langford PR, Jin Het al., 2023, PPNet: Identifying functional association networks by phylogenetic profiling of prokaryotic genomes, Microbiology Spectrum, Vol: 11, ISSN: 2165-0497

Identification of microbial functional association networks allows interpretation of biological phenomena and a greater understanding of the molecular basis of pathogenicity and also underpins the formulation of control measures. Here, we describe PPNet, a tool that uses genome information and analysis of phylogenetic profiles with binary similarity and distance measures to derive large-scale bacterial gene association networks of a single species. As an exemplar, we have derived a functional association network in the pig pathogen Streptococcus suis using 81 binary similarity and dissimilarity measures which demonstrates excellent performance based on the area under the receiver operating characteristic (AUROC), the area under the precision-recall (AUPR), and a derived overall scoring method. Selected network associations were validated experimentally by using bacterial two-hybrid experiments. We conclude that PPNet, a publicly available (https://github.com/liyangjie/PPNet), can be used to construct microbial association networks from easily acquired genome-scale data. IMPORTANCE This study developed PPNet, the first tool that can be used to infer large-scale bacterial functional association networks of a single species. PPNet includes a method for assigning the uniqueness of a bacterial strain using the average nucleotide identity and the average nucleotide coverage. PPNet collected 81 binary similarity and distance measures for phylogenetic profiling and then evaluated and divided them into four groups. PPNet can effectively capture gene networks that are functionally related to phenotype from publicly prokaryotic genomes, as well as provide valuable results for downstream analysis and experiment testing.

Journal article

Bidmos FA, Gladstone CA, Langford PR, 2023, Reverse vaccinology, Molecular Medical Microbiology, Third Edition, Pages: 2023-2038, ISBN: 9780128186190

Vaccines have long been considered as a crucial control measure for infectious diseases. The initial development of effective antigen (subunit)-based vaccines against pathogens was of limited efficiency. Only a handful of candidate antigens were studied at a time, and relatively few became components of successful vaccines. Thus the traditional vaccine development process from laboratory bench (candidate discovery and testing) to bedside (licensing and usasge) was slow, and particularly unsuitable for a rapid response to emergent epidemics or pandemics. This chapter provides a detailed description, with examples, of the high-throughput approach known as reverse vaccinology, and related methods, aimed at rapidly identifying antigens suitable for use in subunit vaccines.

Book chapter

Menikou S, McArdle AJ, Li MS, Kaforou M, Langford PR, Levin Met al., 2023, A Proteomics-Based Method for Identifying Antigens within Immune Complexes, Advances in Medical Imaging, Detection, and Diagnosis, Pages: 1099-1118, ISBN: 9789814877466

The binding of Immune Complexes (ICs) to FcγRs may lead to either activation or suppression of inflammatory cells depending on the type of FCGR gene expressed, and IC-induced cellular activation or suppression has been documented in many disease processes. ICs may also precipitate in tissues, leading to local inflammation or organ damage, influx of inflammatory cells and complement activation. Methods for IC detection are typically based on their physicochemical properties, such as their size, antibody class, affinity for specific cellular receptors and susceptibility to phagocytosis by macrophages [13]. A variety of methods are available for isolation of ICs or identifying elements associated with but not directly identifying ICs and are summarized in Menikou et al. Full gene names for UniProt proteins were determined by InterMineR. Individual proteins were classified using string matching as keratin, trypsin, complement, albumin, fibrinogen, immunoglobulin, other, contaminant and influenza.

Book chapter

Di Blasio S, Clarke M, Hind CK, Asai M, Laurence L, Benvenuti A, Hassan M, Semenya D, Man DK-W, Horrocks V, Manzo G, Van Der Lith S, Lam C, Gentile E, Annette C, Bosse J, Li Y, Panaretou B, Langford PR, Robertson BD, Lam JKW, Sutton JM, McArthur M, Mason AJet al., 2023, Bolaamphiphile analogues of 12-bis-THA Cl2 are potent antimicrobial therapeutics with distinct mechanisms of action against bacterial, mycobacterial, and fungal pathogens., mSphere, Vol: 8, Pages: 1-20, ISSN: 2379-5042

12-Bis-THA Cl2 [12,12'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications. IMPORTANCE While we face an acute threat from antibiotic resistant bacteria and a lack of new classes of antibiotic, there are many effective antimicrobials which have limited application due to concerns regar

Journal article

Cunha da Silva G, Rossi CC, Nogueira Rosa J, Moreno Sanches N, Lopes Cardoso D, Li Y, Witney A, Gould K, Pereira Fontes P, Callaghan A, Bosse J, Langford P, Bazzolli Det al., 2022, Identification of small RNAs associated with RNA chaperone Hfq reveals a new stress response regulator in Actinobacillus pleuropneumoniae, Frontiers in Microbiology, Vol: 13, ISSN: 1664-302X

The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.

Journal article

Asai M, Li Y, Spiropoulos J, Cooley W, Everest D, Kendall S, Martin C, Robertson B, Langford P, Newton Set al., 2022, Galleria mellonella as an infection model for the virulent Mycobacterium tuberculosis H37Rv, Virulence, Vol: 13, Pages: 1543-1557, ISSN: 2150-5594

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a leading cause of infectious disease mortality. Animal infection models have contributed substantially to our understanding of TB, yet their biological and non-biological limitations are a research bottleneck. There is a need for more ethically acceptable, economical, and reproducible TB infection models capable of mimicking key aspects of disease. Here we demonstrate and present a basic description of how Galleria mellonella (the greater wax moth, Gm) larvae can be used as a low cost, rapid and ethically more acceptable model for TB research. This is the first study to infect Gm with the fully virulent MTB H37Rv, the most widely used strain in research. Infection of Gm with MTB resulted in a symptomatic lethal infection, the virulence of which differed from both attenuated Mycobacterium bovis BCG and auxotrophic MTB strains. The Gm-MTB model can also be used for anti-TB drug screening, although CFU enumeration from Gm is necessary for confirmation of mycobacterial load reducing activity of the tested compound. Furthermore, comparative virulence of MTB isogenic mutants can be determined in Gm. However, comparison of mutant phenotypes in Gm against conventional models must consider the limitations of innate immunity. Our findings indicate that Gm will be a practical, valuable and advantageous additional model to be used alongside existing models to advance tuberculosis research.

Journal article

Luan T, Wang L, Luan H, Zhang Y, Langford P, Liu S, Zhang W, Li Get al., 2022, A CRISPR/Cas12a-assisted rapid detection platform by biosensing the apxIVA of Actinobacillus pleuropneumoniae, Frontiers in Microbiology, Vol: 13, ISSN: 1664-302X

Actinobacillus pleuropneumoniae is an important respiratory pig pathogen that causes substantial losses in the worldwide swine industry. Chronic or subclinical infection with no apparent clinical symptoms poses a challenge for preventing transmission between herds. Rapid diagnostics is important for the control of epidemic diseases. In this study, we formulated an A. pleuropneumoniae species-specific apxIVA-based CRISPR/Cas12a-assisted rapid detection platform (Card) that combines recombinase polymerase amplification (RPA) of target DNA and subsequent Cas12a ssDNase activation. Card has a detection limit of 10 CFUs of A. pleuropneumoniae, and there is no cross-reactivity with other common swine pathogens. The detection process can be completed in 1 h, and there was 100% agreement between the conventional apxIVA-based PCR and Card in detecting A. pleuropneumoniae in lung samples. Microplate fluorescence readout enables high-throughput use in diagnostic laboratories, and naked eye and lateral flow test readouts enable use at the point of care. We conclude that Card is a versatile, rapid, accurate molecular diagnostic platform suitable for use in both laboratory and low-resource settings.

Journal article

Zhang Q, Tang H, Yan C, Han W, Peng L, Xu J, Chen X, Langford PR, Bei W, Huang Q, Zhou R, Li Let al., 2022, The metabolic adaptation in response to nitrate Is critical for Actinobacillus pleuropneumoniae growth and pathogenicity under the regulation of NarQ/P, Infection and Immunity, Vol: 90, Pages: 1-17, ISSN: 0019-9567

Nitrate metabolism is an adaptation mechanism used by many bacteria for survival in anaerobic environments. As a by-product of inflammation, nitrate is used by the intestinal bacterial pathogens to enable gut infection. However, the responses of bacterial respiratory pathogens to nitrate are less well understood. Actinobacillus pleuropneumoniae is an important bacterial respiratory pathogen of swine. Previous studies have suggested that adaptation of A. pleuropneumoniae to anaerobiosis is important for infection. In this work, A. pleuropneumoniae growth and pathogenesis in response to the nitrate were investigated. Nitrate significantly promoted A. pleuropneumoniae growth under anaerobic conditions in vitro and lethality in mice. By using narQ and narP deletion mutants and single-residue-mutated complementary strains of ΔnarQ, the two-component system NarQ/P was confirmed to be critical for nitrate-induced growth, with Arg50 in NarQ as an essential functional residue. Transcriptome analysis showed that nitrate upregulated multiple energy-generating pathways, including nitrate metabolism, mannose and pentose metabolism, and glycerolipid metabolism via the regulation of NarQ/P. Furthermore, narQ, narP, and its target gene encoding the nitrate reductase Nap contributed to the pathogenicity of A. pleuropneumoniae. The Nap inhibitor tungstate significantly reduced the survival of A. pleuropneumoniae in vivo, suggesting that Nap is a potential drug target. These results give new insights into how the respiratory pathogen A. pleuropneumoniae utilizes the alternative electron acceptor nitrate to overcome the hypoxia microenvironment, which can occur in the inflammatory or necrotic infected tissues.

Journal article

Wu T, Jia L, Lei S, Jiang H, Liu J, Li N, Langford PR, Liu H, Lei Let al., 2022, Host HSPD1 translocation from mitochondria to the cytoplasm induced by streptococcus suis Serovar 2 enolase mediates apoptosis and loss of blood–brain barrier integrity, Cells, Vol: 11, Pages: 1-16, ISSN: 2073-4409

Streptococcus suis serovar 2 (S. suis serovar 2) is a zoonotic pathogen that causes meningitis in pigs and humans, and is a serious threat to the swine industry and public health. Understanding the mechanism(s) by which S. suis serovar 2 penetrates the blood–brain barrier (BBB) is crucial to elucidating the pathogenesis of meningitis. In a previous study, we found that expression of the virulence factor enolase (Eno) by S. suis serovar 2 promotes the expression of host heat shock protein family D member 1 (HSPD1) in brain tissue, which leads to the apoptosis of porcine brain microvascular endothelial cells (PBMECs) and increased BBB permeability, which in turn promotes bacterial translocation across the BBB. However, the mechanism by which HSPD1 mediates Eno-induced apoptosis remains unclear. In this study, we demonstrate that Eno promotes the translocation of HSPD1 from mitochondria to the cytoplasm, where HSPD1 binds to β-actin (ACTB), the translocated HSPD1, and its interaction with ACTB led to adverse changes in cell morphology and promoted the expression of apoptosis-related proteins, second mitochondria-derived activator of caspases (Smac), and cleaved caspase-3; inhibited the expression of X-linked inhibitor of apoptosis protein (XIAP); and finally promoted cell apoptosis. These results further elucidate the role of HSPD1 in the process of Eno-induced apoptosis and increased BBB permeability, increasing our understanding of the pathogenic mechanisms of meningitis, and providing a framework for novel therapeutic strategies.

Journal article

Stringer OW, Li Y, Bosse JT, Forrest MS, Hernandez-Garcia J, Tucker AW, Nunes T, Costa F, Mortensen P, Velazquez E, Penny P, Rodriguez Manzano J, Georgiou P, Langford Pet al., 2022, Rapid detection of A. pleuropneumoniae from clinical samples using recombinase polymerase amplification, Frontiers in Veterinary Science, Vol: 9, ISSN: 2297-1769

Actinobacillus pleuropneumoniae (APP) is the causative agent of porcine pleuropneumonia, resulting in high economic impact worldwide. There are currently 19 known serovars of APP, with different ones being predominant in specific geographic regions. Outbreaks of pleuropneumonia, characterized by sudden respiratory difficulties and high mortality, can occur when infected pigs are brought into naïve herds, or by those carrying different serovars. Good biosecurity measures include regular diagnostic testing for surveillance purposes. Current gold standard diagnostic techniques lacksensitivity (bacterial culture), require expensive thermocycling machinery (PCR) and are time consuming (culture and PCR). Here we describe the development of an isothermal point-of-care diagnostic test - utilizing recombinase polymerase amplification (RPA) for the detection of APP,targeting the species-specific apxIVA gene. Our APP-RPA diagnostic test achieved a sensitivity of 10 copies/µL using a strain of APP serovar 8, which is the most prevalent serovar in the UK. Additionally, our APP-RPA assay achieved a clinical sensitivity and specificity of 84.3% and 100%, respectively,across 61 extracted clinical samples obtained from farms located in England and Portugal. Using a small subset (n = 14) of the lung tissue samples, we achieved a clinical sensitivity and specificity of 76.9% and 100%, respectively) using lung imprints made on FTA cards tested directly in the APP- RPA reaction. Our results demonstrate that our APP-RPA assay enables a suitable rapid and sensitive screening tool for this important veterinary pathogen.

Journal article

Stringer O, Li Y, Bosse J, Langford Pet al., 2022, JMM Profile: Actinobacillus pleuropneumoniae: a major cause of lung disease in pigs but difficult to control and eradicate, Journal of Medical Microbiology, Vol: 71, ISSN: 0022-2615

The Gram-negative bacterium Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia in pigs, its only known natural host. Typical symptoms of peracute disease include fever, apathy and anorexia, and time from infection to death may only be 6 h. Severe lung lesions result from presence of one or two of the ApxI-III toxins. Control is through good husbandry practice, vaccines and antibiotic use. Culture and presence of the species-specific apxIV gene by PCR confirms diagnosis, and identification of serovar, of which 19 are known, informs on appropriate vaccine use and epidemiology.

Journal article

Srijuntongsiri G, Mhoowai A, Samngamnim S, Assavacheep P, Bossé JT, Langford PR, Posayapisit N, Leartsakulpanich U, Songsungthong Wet al., 2022, Novel DNA Markers for Identification of Actinobacillus pleuropneumoniae, Microbiology Spectrum, Vol: 10, ISSN: 2165-0497

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 - 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico, as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [Actinobacillus] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA, nusG, sppA, xerD, ybbN, ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae (covering all 19 serovars), but not to four closely related Actinobacillus species, four [Actinobacillus] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease. IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-spe

Journal article

Tang H, Zhang Q, Han W, Wang Z, Pang S, Zhu H, Tan K, Liu X, Langford PR, Huang Q, Zhou R, Li Let al., 2022, Identification of FtpA, a Dps-like protein involved in anti-oxidative stress and virulence in Actinobacillus pleuropneumoniae, Journal of Bacteriology, Vol: 204, Pages: 1-12, ISSN: 0021-9193

Bacteria have evolved a variety of enzymes to eliminate endogenous or host-derived oxidative stress factors. The Dps protein, first identified in Escherichia coli, contains a ferroxidase center and protects bacteria from reactive oxygen species damage. There is a lack of knowledge of the role of Dps-like proteins in bacterial pathogenesis. Actinobacillus pleuropneumoniae causes pleuropneumonia, a respiratory disease of swine. The A. pleuropneumoniae ftpA gene is up-regulated during a shift to anaerobiosis, in biofilms and, as found in this study, also by H2O2. An A. pleuropneumoniae ftpA deletion mutant (△ftpA) had increased H2O2 sensitivity, less intracellular viability in macrophages, and decreased virulence in a mouse infection model. Expression of ftpA in an E. coli dps mutant restored wild-type resistance to H2O2. FtpA possesses a conserved ferritin domain containing a ferroxidase site. Recombinant rFtpA bound and oxidized Fe2+ reversibly. Under aerobic conditions, compared with the wild-type strain, the viability of an △ftpA mutant was reduced after extended culture, transition from anaerobic to aerobic conditions, and upon supplementation with Fenton reaction substrates. Under anaerobic conditions, additional H2O2 resulted in a more severe growth defect of △ftpA than under aerobic conditions. Therefore, by oxidizing and mineralizing Fe2+, FtpA alleviates oxidative damage mediated by intracellular Fenton reactions. Furthermore, by mutational analysis, two residues were confirmed to be critical for Fe2+ binding and oxidization, as well as for A. pleuropneumoniae H2O2 resistance. Taken together, this study demonstrates that A. pleuropneumoniae FtpA is a Dps-like protein, playing critical roles in oxidative stress resistance and virulence. IMPORTANCE As a ferroxidase, Dps of Escherichia coli can protect bacteria from reactive oxygen species damage, but its role in bacterial pathogenesis has received little attention. In this study, FtpA of the swine respiratory p

Journal article

da Silva G, Gonçalves O, Rosa J, França K, Bosse JT, Santana M, Langford P, Bazzolli Det al., 2022, Mobile genetic elements drive antimicrobial resistance gene spread in Pasteurellaceae species, Frontiers in Microbiology, Vol: 12, Pages: 1-14, ISSN: 1664-302X

Mobile genetic elements (MGEs) and antimicrobial resistance (AMR) drive important ecological relationships in microbial communities and pathogen-host interaction. In this study, we investigated the resistome-associated mobilome in 345 publicly available Pasteurellaceae genomes, a large family of Gram-negative bacteria including major human and animal pathogens. We generated a comprehensive dataset of the mobilome integrated into genomes, including 10,820 insertion sequences, 2,939 prophages, and 43 integrative and conjugative elements. Also, we assessed plasmid sequences of Pasteurellaceae. Our findings greatly expand the diversity of MGEs for the family, including a description of novel elements. We discovered that MGEs are comparable and dispersed across species and that they also co-occur in genomes, contributing to the family's ecology via gene transfer. In addition, we investigated the impact of these elements in the dissemination and shaping of AMR genes. A total of 55 different AMR genes were mapped to 721 locations in the dataset. MGEs are linked with 77.6% of AMR genes discovered, indicating their important involvement in the acquisition and transmission of such genes. This study provides an uncharted view of the Pasteurellaceae by demonstrating the global distribution of resistance genes linked with MGEs.

Journal article

Bidmos F, Bossé J, Langford P, 2022, Preface

Book

Langford P, Stringer O, Li Y, Bosse Jet al., 2021, Application of the MISTEACHING(S) disease susceptibility framework to Actinobacillus pleuropneumoniae to identify research gaps: an exemplar of a veterinary pathogen, Animal Health Research Reviews, Vol: 22, Pages: 120-135, ISSN: 1466-2523

Historically, the MISTEACHING (microbiome, immunity, sex, temperature, environment, age, chance, history, inoculum, nutrition, genetics) framework to describe the outcome of host-pathogen interaction, has been applied to human pathogens. Here, weshow, using Actinobacillus pleuropneumoniaeas an exemplar, that the MISTEACHING framework can be applied to a strict veterinary pathogen enabling the identification of major research gaps, and the formulation of hypotheses whose study will lead to a greater understanding of pathogenic mechanisms, and/or improved prevention/therapeutic measures. We also suggest that the MISTEACHING framework should be extended with the inclusion of a “strain” category, to become MISTEACHINGS. We conclude that the MISTEACHINGS framework can be applied to veterinary pathogens, whether they be bacteria, viruses, or parasites, and hope to stimulate others to use it to identify research gaps and to formulate hypotheses worthy of study with their own pathogens.

Journal article

Bossé JT, Li Y, Leanse LG, Zhou L, Chaudhuri RR, Peters SE, Wang J, Maglennon GA, Holden MTG, Maskell DJ, Tucker AW, Wren BW, Rycroft AN, Langford PR, Maskell DJ, Tucker AW, Peters SE, Weinert LA, Wang J, Luan S-L, Chaudhuri RR, Rycroft AN, Maglennon GA, Beddow J, Wren BW, Cuccui J, Terra VS, Bossé JT, Li Y, Langford PRet al., 2021, Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS), Animal Diseases, Vol: 1

Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). 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, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines.

Journal article

Bosse J, Li Y, Cohen LM, Stegger M, Angen Ø, Lacouture S, Gottschalk M, Lei L, Koene M, Kuhnert P, Bandara AB, Inzana TJ, Holden MTG, Harris D, Oshota O, Maskell DJ, Tucker AW, Wren BW, Rycroft AN, Langford PRet al., 2021, Complete genome for Actinobacillus pleuropneumoniae serovar 8 reference strain 405: comparative analysis with draft genomes for different laboratory stock cultures indicates little genetic variation, Microbial Genomics, Vol: 7, ISSN: 2057-5858

We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain.

Journal article

Cohen LM, Bossé JT, Stegger M, Li Y, Langford PR, Kielland C, Klem TB, Gulliksen SM, Ranheim B, Grøntvedt CA, Angen Øet al., 2021, Comparative genome sequence analysis of actinobacillus pleuropneumoniae serovar 8 isolates from Norway, Denmark, and the United Kingdom indicates distinct phylogenetic lineages and differences in distribution of antimicrobial resistance genes, Frontiers in Microbiology, Vol: 12, Pages: 1-13, ISSN: 1664-302X

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a disease of major impact on pig health, welfare, and productivity globally. Serovar 8 (APP) is the predominant clinical serovar in Norway and the United Kingdom (UK), and has been isolated from clinical cases in Denmark. The primary objective of this study was to characterize the genetic variability of isolates of A. pleuropneumoniae APP8 in the Norwegian population. The secondary objectives were to determine the within-host variability of APP8; to compare the APP8 bacterial populations in Norway, Denmark, and the UK, including antimicrobial resistance (AMR) gene profiles and to assess the effect of national differences in antimicrobial drug use and restricted animal movement on the occurrence of resistance. Isolates of APP8 from the UK (n=67), Denmark (n=22), and Norway (n=123) collected between 1983 and 2020 were compared using whole genome sequencing. To investigate genetic variability within individual hosts, an additional 104 APP8 isolates from the lungs of six Norwegian pigs were compared. Very low within-host variation was observed (≤ 2 single nucleotide polymorphisms). The phylogeny of 123 Norwegian APP8 isolates from 76 herds revealed some within-herd genetic variation, but substantial geographical clustering. When inferring the relatedness of the three international APP8 collections, the topology highlighted the existence of two distinct monophyletic branches characterized by the Norwegian and UK isolates, respectively. Three Danish isolates were scattered across the UK branch, whereas the remaining 19 Danish isolates clustered in two monophyletic groups nested in the Norwegian branch. Coalescence analysis, performed to estimate the divergences from a common ancestor, indicated a last common ancestor several centuries ago. The phylogenetic analyses also revealed striking differences in occurrence of AMR genes, as these were 23-times more prevalent among the UK isolates

Journal article

Stringer O, Bosse J, Lacoutre S, Gottschalk M, Fodor L, Angen Ø, Velazquez E, Penny P, Lei L, Langford P, Li Yet al., 2021, Rapid detection and typing of Actinobacillus pleuropneumoniae serovars directly from clinical samples: combining FTA® card technology with multiplex-PCR, Frontiers in Veterinary Science, Vol: 8, Pages: 1-9, ISSN: 2297-1769

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is highly contagious and responsible for high morbidity, mortality and economic losses in the swine industry worldwide, but quick serotyping and diagnosis are still not widely available. In this study, we sought to validate the use of Whatman FTA® cards for collection and processing of A. pleuropneumoniae isolates, or porcine lung tissue samples, for direct use in diagnostic multiplex PCRs.We have optimized the processing of 3 mm discs punched from FTA® cards loaded with cultured A. pleuropneumoniae, or imprinted on lesioned regions of lung tissue, with only three distilled water washes before addition into our APP-mPCR assay for rapid, low-cost identification and serotyping. DNA captured on FTA® cards generated the same diagnostic PCR results as DNA extracted using commercial kits for 85 A. pleuropneumoniae clinical isolate cultures and 22 lung samples. Additionally, bacterial DNA bound to FTA® cards was detectable by PCR after six months of storage at 37°C.This study provides simple, efficient, rapid and practical sample processing for detection and molecular serotyping of A. pleuropneumoniae.

Journal article

Bao C, Liu B, Zhu R, Xiao J, Li Z, Jiang H, Wang B, Langford PR, Fei R, Li N, Lei Let al., 2021, IFN-γ<SUP>-/-</SUP> Mice Resist Actinobacillus pleuropneumoniae Infection by Promoting Early Lung IL-18 Release and PMN-I Accumulation, INFECTION AND IMMUNITY, Vol: 89, ISSN: 0019-9567

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