Publications
130 results found
Whittington AM, Turner FS, Baark F, et al., 2023, An acidic microenvironment in Tuberculosis increases extracellular matrix degradation by regulating macrophage inflammatory responses, PLoS Pathogens, Vol: 19, Pages: 1-25, ISSN: 1553-7366
Mycobacterium tuberculosis (M.tb) infection causes marked tissue inflammation leading to lung destruction and morbidity. The inflammatory extracellular microenvironment is acidic, however the effect of this acidosis on the immune response to M.tb is unknown. Using RNA-seq we show that acidosis produces system level transcriptional change in M.tb infected human macrophages regulating almost 4000 genes. Acidosis specifically upregulated extracellular matrix (ECM) degradation pathways with increased expression of Matrix metalloproteinases (MMPs) which mediate lung destruction in Tuberculosis. Macrophage MMP-1 and -3 secretion was increased by acidosis in a cellular model. Acidosis markedly suppresses several cytokines central to control of M.tb infection including TNF-α and IFN-γ. Murine studies demonstrated expression of known acidosis signaling G-protein coupled receptors OGR-1 and TDAG-8 in Tuberculosis which are shown to mediate the immune effects of decreased pH. Receptors were then demonstrated to be expressed in patients with TB lymphadenitis. Collectively, our findings show that an acidic microenvironment modulates immune function to reduce protective inflammatory responses and increase extracellular matrix degradation in Tuberculosis. Acidosis receptors are therefore potential targets for host directed therapy in patients.
Gordon SB, Sichone S, Chirwa AE, et al., 2023, Practical considerations for a TB controlled human infection model (TB-CHIM); the case for TB-CHIM in Africa, a systematic review of the literature and report of 2 workshop discussions in UK and Malawi, Wellcome Open Research, Vol: 8, Pages: 1-28, ISSN: 2398-502X
Background: Tuberculosis (TB) remains a major challenge in many domains including diagnosis, pathogenesis, prevention, treatment, drug resistance and long-term protection of the public health by vaccination. A controlled human infection model (CHIM) could potentially facilitate breakthroughs in each of these domains but has so far been considered impossible owing to technical and safety concerns. Methods: A systematic review of mycobacterial human challenge studies was carried out to evaluate progress to date, best possible ways forward and challenges to be overcome. We searched MEDLINE (1946 to current) and CINAHL (1984 to current) databases; and Google Scholar to search citations in selected manuscripts. The final search was conducted 3 rd February 2022. Inclusion criteria: adults ≥18 years old; administration of live mycobacteria; and interventional trials or cohort studies with immune and/or microbiological endpoints. Exclusion criteria: animal studies; studies with no primary data; no administration of live mycobacteria; retrospective cohort studies; case-series; and case-reports. Relevant tools (Cochrane Collaboration for RCTs and Newcastle-Ottawa Scale for non-randomised studies) were used to assess risk of bias and present a narrative synthesis of our findings. Results: The search identified 1,388 titles for review; of these 90 were reviewed for inclusion; and 27 were included. Of these, 15 were randomised controlled trials and 12 were prospective cohort studies. We focussed on administration route, challenge agent and dose administered for data extraction. Overall, BCG studies including fluorescent BCG show the most immediate utility, and genetically modified Mycobacteria tuberculosis is the most tantalising prospect of discovery breakthrough. Conclusions: The TB-CHIM development group met in 2019 and 2022 to consider the results of the systematic review, to hear presentations from many of the senior authors whose work had been reviewed and to consider b
Asai M, Li Y, Newton S, et 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.
Gordon SB, Sichone S, Chirwa AE, et al., 2023, Practical considerations for a TB controlled human infection model (TB-CHIM); the case for TB-CHIM in Africa, a systematic review of the literature and report of 2 workshop discussions in UK and Malawi, Wellcome Open Research, Vol: 8, Pages: 71-71
<ns4:p><ns4:bold>Background:</ns4:bold> Tuberculosis (TB) remains a major challenge in many domains including diagnosis, pathogenesis, prevention, treatment, drug resistance and long-term protection of the public health by vaccination. A controlled human infection model (CHIM) could potentially facilitate breakthroughs in each of these domains but has so far been considered impossible owing to technical and safety concerns.</ns4:p><ns4:p> <ns4:bold>Methods:</ns4:bold> A systematic review of mycobacterial human challenge studies was carried out to evaluate progress to date, best possible ways forward and challenges to be overcome. We searched MEDLINE (1946 to current) and CINAHL (1984 to current) databases; and Google Scholar to search citations in selected manuscripts. The final search was conducted 3<ns4:sup>rd </ns4:sup>February 2022. Inclusion criteria: adults ≥18 years old; administration of live mycobacteria; and interventional trials or cohort studies with immune and/or microbiological endpoints. Exclusion criteria: animal studies; studies with no primary data; no administration of live mycobacteria; retrospective cohort studies; case-series; and case-reports. Relevant tools (Cochrane Collaboration for RCTs and Newcastle-Ottawa Scale for non-randomised studies) were used to assess risk of bias and present a narrative synthesis of our findings.</ns4:p><ns4:p> <ns4:bold>Results:</ns4:bold> The search identified 1,388 titles for review; of these 90 were reviewed for inclusion; and 27 were included. Of these, 15 were randomised controlled trials and 12 were prospective cohort studies. We focussed on administration route, challenge agent and dose administered for data extraction. Overall, BCG studies including fluorescent BCG show the most immediate utility, and genetically modified <ns4:italic>Mycobacteria tuberculosis</ns4:italic> is the most tantalising prospect of disco
Di Blasio S, Clarke M, Hind CK, et 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
Asai M, Li Y, Spiropoulos J, et 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.
Weng Y, Shepherd D, Liu Y, et al., 2022, Inhibition of the Niemann-Pick C1 protein is a conserved feature of multiple strains of pathogenic mycobacteria, Nature Communications, Vol: 13, Pages: 1-16, ISSN: 2041-1723
Mycobacterium tuberculosis (Mtb) survives and replicates within host macrophages (MΦ) and subverts multiple antimicrobial defense mechanisms. Previously, we reported that lipids shed by pathogenic mycobacteria inhibit NPC1, the lysosomal membrane protein deficient in the lysosomal storage disorder Niemann-Pick disease type C (NPC). Inhibition of NPC1 leads to a drop in lysosomal calcium levels, blocking phagosome-lysosome fusion leading to mycobacterial survival. We speculated that the production of specific cell wall lipid(s) that inhibit NPC1 could have been a critical step in the evolution of pathogenicity. We therefore investigated whether lipid extracts from clinical Mtb strains from multiple Mtb lineages, Mtb complex (MTBC) members and non-tubercular mycobacteria (NTM) inhibit the NPC pathway. We report that inhibition of the NPC pathway was present in all clinical isolates from Mtb lineages 1, 2, 3 and 4, Mycobacterium bovis and the NTM, Mycobacterium abscessus and Mycobacterium avium. However, lipid extract from Mycobacterium canettii, which is considered to resemble the common ancestor of the MTBC did not inhibit the NPC1 pathway. We conclude that the evolution of NPC1 inhibitory mycobacterial cell wall lipids evolved early and post divergence from Mycobacterium canettii-related mycobacteria and that this activity contributes significantly to the promotion of disease.
Rao KU, Henderson DI, Krishnan N, et al., 2021, A broad spectrum anti-bacterial peptide with an adjunct potential for tuberculosis chemotherapy, Scientific Reports, Vol: 11, ISSN: 2045-2322
Alternative ways to prevent and treat infectious diseases are needed. Previously, we identified a fungal peptide, NZX, that was comparable to rifampicin in lowering M. tuberculosis load in a murine tuberculosis (TB) infection model. Here we assessed the potential synergy between this cationic host defence peptide (CHDP) and the current TB drugs and analysed its pharmacokinetics. We found additive effect of this peptide with isoniazid and ethambutol and confirmed these results with ethambutol in a murine TB-model. In vivo, the peptide remained stable in circulation and preserved lung structure better than ethambutol alone. Antibiotic resistance studies did not induce mutants with reduced susceptibility to the peptide. We further observed that this peptide was effective against nontuberculous mycobacteria (NTM), such as M. avium and M. abscessus, and several Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In conclusion, the presented data supports a role for this CHDP in the treatment of drug resistant organisms.
Asai M, Sheehan G, Li Y, et al., 2021, Innate immune responses of Galleria mellonella to Mycobacterium bovis BCG challenge identified using proteomic and molecular approaches, Frontiers in Cellular and Infection Microbiology, Vol: 11, ISSN: 2235-2988
The larvae of the insect Galleria mellonella, have recently been established as a non-mammalian infection model for the Mycobacterium tuberculosis complex (MTBC). To gain further insight into the potential of this model, we applied proteomic (label-free quantification) and transcriptomic (gene expression) approaches to characterise the innate immune response of G. mellonella to infection with Mycobacterium bovis BCG lux over a 168 h time course. Proteomic analysis of the haemolymph from infected larvae revealed distinct changes in the proteome at all time points (4, 48, 168 h). Reverse transcriptase quantitative PCR confirmed induction of five genes (gloverin, cecropin, IMPI, hemolin, and Hdd11), which encoded proteins found to be differentially abundant from the proteomic analysis. However, the trend between gene expression and protein abundance were largely inconsistent (20%). Overall, the data are in agreement with previous phenotypic observations such as haemocyte internalization of mycobacterial bacilli (hemolin/β-actin), formation of granuloma-like structures (Hdd11), and melanization (phenoloxidase activating enzyme 3 and serpins). Furthermore, similarities in immune expression in G. mellonella, mouse, zebrafish and in vitro cell-line models of tuberculosis infection were also identified for the mechanism of phagocytosis (β-actin). Cecropins (antimicrobial peptides), which share the same α-helical motif as a highly potent peptide expressed in humans (h-CAP-18), were induced in G. mellonella in response to infection, giving insight into a potential starting point for novel antimycobacterial agents. We believe that these novel insights into the innate immune response further contribute to the validation of this cost-effective and ethically acceptable insect model to study members of the MTBC.
Alzahabi KH, Usmani O, Georgiou TK, et al., 2020, Approaches to treating tuberculosis by encapsulating metal ions and anti-mycobacterial drugs utilizing nano- and microparticle technologies, Emerging Topics in Life Sciences, Vol: 4, Pages: 581-600, ISSN: 2397-8554
Tuberculosis (TB) is caused by a bacterial infection that affects a number of human organs, primarily the lungs, but also the liver, spleen, and spine, causing key symptoms of fever, fatigue, and persistent cough, and if not treated properly, can be fatal. Every year, 10 million individuals become ill with active TB resulting with a mortality approximating 1.5 million. Current treatment guidelines recommend oral administration of a combination of first-line anti-TB drugs for at least 6 months. While efficacious under optimum conditions, ‘Directly Observed Therapy Short-course’ (DOTS) is not without problems. The long treatment time and poor pharmacokinetics, alongside drug side effects lead to poor patient compliance and has accelerated the emergence of multi-drug resistant (MDR) organisms. All this, combined with the limited number of newly discovered TB drugs to treat MDR-TB and shorten standard therapy time, has highlighted the need for new targeted drug delivery systems. In this respect, there has been recent focus on micro- and nano-particle technologies to prepare organic or/and metal particles loaded with TB drugs to enhance their efficacy by targeted delivery via the inhaled route. In this review, we provide a brief overview of the current epidemiology of TB, and risk factors for progression of latent stage tuberculosis (LTBI) to the active TB. We identify current TB treatment regimens, newly discovered TB drugs, and identify studies that have used micro- or nano-particles technologies to design a reliable inhalation drug delivery system to treat TB more effectively.
McMaster D, Courtenay M, Santucci C, et al., 2020, Consensus-based antimicrobial resistance and stewardship competencies for UK undergraduate medical students, JAC-Antimicrobial Resistance, Vol: 2, Pages: 1-8, ISSN: 2632-1823
BackgroundIn the UK there is limited coverage of antimicrobial stewardship across postgraduate curricula and evidence that final year medical students have insufficient and inconsistent antimicrobial stewardship teaching. A national undergraduate curriculum for antimicrobial resistance and stewardship is required to standardize an adequate level of understanding for all future doctors.ObjectivesTo provide a UK national consensus on competencies for antimicrobial resistance and stewardship for undergraduate medical education.MethodsUsing the modified Delphi method over two online survey rounds, an expert panel comprising leads for infection teaching from 25 UK medical schools reviewed competency descriptors for antimicrobial resistance and stewardship education.ResultsThere was a response rate of 100% with all 28 experts who agreed to take part completing both survey rounds. Following the first-round survey, of the initial 55 descriptors, 43 reached consensus (78%). The second-round survey included the 12 descriptors from the first round in which agreement had not been reached, four amended descriptors and 12 new descriptors following qualitative feedback from the panel members. Following the second-round survey, a total of 58 consensus-based competency descriptors within six overarching domains were identified.ConclusionsThe consensus-based competency descriptors defined here can be used to inform standards, design curricula, develop assessment tools and direct UK undergraduate medical education.
Asai M, Li Y, Spiropoulos J, et al., 2020, A novel biosafety level 2 compliant tuberculosis infection model using a ΔleuDΔpanCD double auxotroph of Mycobacterium tuberculosis H37Rv and Galleria mellonella, Virulence, Vol: 11, Pages: 811-824, ISSN: 2150-5594
Mammalian infection models have contributed significantly to our understanding of the host-mycobacterial interaction, revealing potential mechanisms and targets for novel antimycobacterial therapeutics. However, the use of conventional mammalian models such as mice, are typically expensive, high maintenance, require specialised animal housing, and are ethically regulated. Furthermore, research using Mycobacterium tuberculosis (MTB), is inherently difficult as work needs to be carried out at biosafety level 3 (BSL3). The insect larvae of Galleria mellonella (greater wax moth), have become increasingly popular as an infection model, and we previously demonstrated its potential as a mycobacterial infection model using Mycobacterium bovis BCG. Here we present a novel BSL2 complaint MTB infection model using G. mellonella in combination with a bioluminescent ΔleuDΔpanCD double auxotrophic mutant of MTB H37Rv (SAMTB lux) which offers safety and practical advantages over working with wild type MTB. Our results show a SAMTB lux dose dependent survival of G. mellonella larvae and demonstrate proliferation and persistence of SAMTB lux bioluminescence over a 1 week infection time course. Histopathological analysis of G. mellonella, highlight the formation of early granuloma-like structures which matured over time. We additionally demonstrate the drug efficacy of first (isoniazid, rifampicin, and ethambutol) and second line (moxifloxacin) antimycobacterial drugs. Our findings demonstrate the broad potential of this insect model to study MTB infection under BSL2 conditions. We anticipate that the successful adaptation and implementation of this model will remove the inherent limitations of MTB research at BSL3 and increase tuberculosis research output.
Jégouzo SAF, Nelson C, Hardwick T, et al., 2020, Mammalian lectin arrays for screening host–microbe interactions, Journal of Biological Chemistry, Vol: 295, Pages: 4541-4555, ISSN: 0021-9258
Many members of the C-type lectin family of glycan-binding receptors have been ascribed roles in the recognition of microorganisms and serve as key receptors in the innate immune response to pathogens. Other mammalian receptors have become targets through which pathogens enter target cells. These receptor roles have often been documented with binding studies involving individual pairs of receptors and microorganisms. To provide a systematic overview of interactions between microbes and the large complement of C-type lectins, here we developed a lectin array and suitable protocols for labeling of microbes that could be used to probe this array. The array contains C-type lectins from cow, chosen as a model organism of agricultural interest for which the relevant pathogen–receptor interactions have not been previously investigated in detail. Screening with yeast cells and various strains of both Gram-positive and -negative bacteria revealed distinct binding patterns, which in some cases could be explained by binding to lipopolysaccharides or capsular polysaccharides, but in other cases they suggested the presence of novel glycan targets on many of the microorganisms. These results are consistent with interactions previously ascribed to the receptors, but they also highlight binding to additional sugar targets that have not previously been recognized. Our findings indicate that mammalian lectin arrays represent unique discovery tools for identifying both novel ligands and new receptor functions.
Singh Khara J, Mojsoska B, Mukherjee D, et al., 2020, Ultra-short antimicrobial peptoids show propensity for membrane activity against multi-drug resistant Mycobacterium tuberculosis, Frontiers in Microbiology, Vol: 11, Pages: 1-11, ISSN: 1664-302X
Tuberculosis (TB) results in both morbidity and mortality on a global scale. With drug resistance on the increase, there is an urgent need to develop novel anti-mycobacterials. Thus, we assessed the anti-mycobacterial potency of three novel synthetic peptoids against drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis in vitro using Minimum Inhibitory Concentration, killing efficacy and intracellular growth inhibition assays, and in vivo against mycobacteria infected BALB/c mice. In addition, we verified cell selectivity using mammalian cells to assess peptoid toxicity. The mechanism of action was determined using flow cytometric analysis, and microfluidic live-cell imaging with time-lapse microscopy and uptake of propidium iodide. Peptoid BM 2 demonstrated anti-mycobacterial activity against both drug sensitive and MDR M. tuberculosis together with an acceptable toxicity profile that showed selectivity between bacterial and mammalian membranes. The peptoid was able to efficiently kill mycobacteria both in vitro and intracellularly in murine RAW 264.7 macrophages, and significantly reduced bacterial load in the lungs of infected mice. Flow cytometric and time lapse fluorescence microscopy indicate mycobacterial membrane damage as the likely mechanism of action. These data demonstrate that peptoids are a novel class of antimicrobial which warrant further investigation and development as therapeutics against TB.
Subbarao S, Sanchez-Garrido J, Krishnan N, et al., 2020, Genetic and pharmacological inhibition of inflammasomes reduces the survival of Mycobacterium tuberculosis strains in macrophages, Scientific Reports, Vol: 10, ISSN: 2045-2322
Mycobacterium tuberculosis infection causes high rates of morbidity and mortality. Host-directed therapy may enhance the immune response, reduce tissue damage and shorten treatment duration. The inflammasome is integral to innate immune responses but over-activation has been described in tuberculosis (TB) pathology and TB-immune reconstitution syndrome. Here we explore how clinical isolates differentially activate the inflammasome and how inflammasome inhibition can lead to enhanced bacterial clearance. Wild-type, Nlrp3−/−/Aim2−/−, Casp1/11−/− and Asc−/− murine bone-marrow derived macrophages (BMDMs) were infected with laboratory strain M. tuberculosis H37Rv or clinical isolates from various lineages. Inflammasome activation and bacterial numbers were measured, and pharmacological inhibition of NLRP3 was achieved using MCC950. Clinical isolates of M. tuberculosis differed in their ability to activate inflammasomes. Beijing isolates had contrasting effects on IL-1β and caspase-1 activation, but all clinical isolates induced lower IL-1β release than H37Rv. Our studies suggest the involvement of NLRP3, AIM2 and an additional unknown sensor in IL-1β maturation. Pharmacological blockade of NLRP3 with MCC950 reduced bacterial survival, and combined treatment with the antimycobacterial drug rifampicin enhanced the effect. Modulating the inflammasome is an attractive adjunct to current anti-mycobacterial therapy that warrants further investigation.
Asai M, Li Y, Singh Khara J, et al., 2019, Galleria mellonella: a novel infection model for screening potential anti-mycobacterial compounds against members of the Mycobacterium tuberculosis complex, Frontiers in Microbiology, Vol: 10, ISSN: 1664-302X
Drug screening models have a vital role in the development of novel antimycobacterial agents which are urgently needed to tackle drug-resistant tuberculosis (TB). We recently established the larvae of the insect Galleria mellonella (greater wax moth) as a novel infection model for the Mycobacterium tuberculosis complex. Here we demonstrate its use as a rapid and reproducible screen to evaluate antimycobacterial drug efficacy using larvae infected with bioluminescent Mycobacterium bovis BCG lux. Treatment improved larval survival outcome and, with the exception of pyrazinamide, was associated with a significant reduction in in vivo mycobacterial bioluminescence over a 96 hour period compared to the untreated controls. Isoniazid and rifampicin displayed the greatest in vivo efficacy and survival outcome. Thus G. mellonella, infected with bioluminescent mycobacteria, can rapidly determine in vivo drug efficacy, and has the potential to significantly reduce and/or replace the number of animals used in TB research.
Asai M, Li Y, Khara J, et al., 2019, Use of the invertebrate Galleria Mellonella as an infection model to study the Mycobacterium tuberculosis complex, Jove-Journal of Visualized Experiments, Vol: 148, ISSN: 1940-087X
Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world’s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging.Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.
Roy RB, Sambou B, Uhia I, et al., 2019, An auto-luminescent fluorescent BCG whole blood assay to enable evaluation of paediatric mycobacterial responses using minimal blood volumes, Frontiers in Pediatrics, Vol: 7, ISSN: 2296-2360
Introduction: Understanding protective human immunity against mycobacteria is critical to developing and evaluating new vaccines against tuberculosis. Children are the most susceptible population to infection, disease, and death from tuberculosis, but also have the strongest evidence of BCG-inducible protection. Limited amounts of blood can be obtained for research purposes in paediatrics and therefore there is a need for high-yield, low-volume, human immunology assays.Methods: We transformed BCG Danish with plasmids encoding luciferase full operon derived from Photorhabdus luminescens together with Green Fluorescent Protein and antibiotic selection markers. We characterised the luminescent and fluorescent properties of this recombinant BCG strain (BCG-GFP-LuxFO) using a luminometer and flow cytometry and developed a paediatric whole blood in vitro infection model.Results: Luminescence of BCG-GFP-LuxFO correlated with optical density (Spearman Rank Correlation coefficient r = 0.985, p < 0.0001) and colony forming units (CFUs) in liquid culture medium (r = 0.971, p < 0.0001). Fluorescence of BCG-GFP-LuxFO in paediatric whole blood was confirmed by flow cytometry in granulocytes and monocytes 1 h following infection. Luminescence of BCG-GFP-LuxFO in whole blood corresponded with CFUs (r = 0.7123, p < 0.0001).Conclusion: The BCG-GFP-LuxFO assay requires 225 μL whole blood per sample, from which serial luminescence measurements can be obtained, together with biochemical analysis of supernatants and cellular assay applications using its fluorescent properties. This offers the opportunity to study human-mycobacterial interactions using multiple experimental modalities with only minimal blood volumes. It is therefore a valuable method for investigating paediatric immunity to tuberculosis.
Tenland E, Pochert A, Krishnan N, et al., 2019, Effective delivery of the anti-mycobacterial peptide NZX in mesoporous silica nanoparticles, PLoS ONE, Vol: 14, ISSN: 1932-6203
BackgroundIntracellular delivery of antimicrobial agents by nanoparticles, such as mesoporous silica particles (MSPs), offers an interesting strategy to treat intracellular infections. In tuberculosis (TB), Mycobacterium tuberculosis avoids components of the immune system by residing primarily inside alveolar macrophages, which are the desired target for TB therapy.Methods and findingsWe have previously identified a peptide, called NZX, capable of inhibiting both clinical and multi-drug resistant strains of M. tuberculosis at therapeutic concentrations. In this study we analysed the potential of MSPs containing NZX for the treatment of tuberculosis. The MSPs released functional NZX gradually into simulated lung fluid and the peptide filled MSPs were easily taken up by primary macrophages. In an intracellular infection model, the peptide containing particles showed increased mycobacterial killing compared to free peptide. The therapeutic potential of peptide containing MSPs was investigated in a murine infection model, showing that MSPs preserved the effect to eliminate M. tuberculosis in vivo.ConclusionsIn this study we found that loading the antimicrobial peptide NZX into MSPs increased the inhibition of intracellular mycobacteria in primary macrophages and preserved the ability to eliminate M. tuberculosis in vivo in a murine model. Our studies provide evidence for the feasibility of using MSPs for treatment of tuberculosis.
Arthur PK, Amarh V, Cramer P, et al., 2019, Characterization of two new multidrug-resistant strains of mycobacterium smegmatis: tools for routine in vitro screening of novel anti-mycobacterial agents, Antibiotics, Vol: 8, ISSN: 2079-6382
Mycobacterium tuberculosis is a pathogen of global public health concern. This threat is exacerbated by the emergence of multidrug-resistant and extremely-drug-resistant strains of the pathogen. We have obtained two distinct clones of multidrug-resistant Mycobacterium smegmatis after gradual exposure of Mycobacterium smegmatis mc² 155 to increasing concentrations of erythromycin. The resulting resistant strains of Mycobacterium smegmatis exhibited robust viability in the presence of high concentrations of erythromycin and were found to be resistant to a wide range of other antimicrobials. They also displayed a unique growth phenotype in comparison to the parental drug-susceptible Mycobacterium smegmatis mc² 155, and a distinct colony morphology in the presence of cholesterol. We propose that these two multidrug-resistant clones of Mycobacterium smegmatis could be used as model organisms at the inceptive phase of routine in vitro screening of novel antimicrobial agents targeted against multidrug-resistant Mycobacterial tuberculosis.
O'Connor G, Krishnan N, Fagan-Murphy A, et al., 2019, Inhalable poly(lactic-co-glycolic acid) (PLGA) microparticles encapsulating all-trans-Retinoic acid (ATRA) as a host-directed, adjunctive treatment for Mycobacterium tuberculosis infection, European Journal of Pharmaceutics and Biopharmaceutics, Vol: 134, Pages: 153-165, ISSN: 0939-6411
Ending the tuberculosis (TB) epidemic by 2030 was recently listed in the United Nations (UN) Sustainable Development Goals alongside HIV/AIDS and malaria as it continues to be a major cause of death worldwide. With a significant proportion of TB cases caused by resistant strains of Mycobacterium tuberculosis (Mtb), there is an urgent need to develop new and innovative approaches to treatment. Since 1989, researchers have been assessing the anti-bacterial effects of the active metabolite of vitamin A, all trans-Retinoic acid (ATRA) solution, in Mtb models. More recently the antibacterial effect of ATRA has been shown to regulate the immune response to infection via critical gene expression, monocyte activation and the induction of autophagy leading to its application as a host-directed therapy (HDT). Inhalation is an attractive route for targeted treatment of TB, and therefore we have developed ATRA-loaded microparticles (ATRA-MP) within the inhalable size range (2.07 ± 0.5 µm) offering targeted delivery of the encapsulated cargo (70.5 ± 2.3%) to the site of action within the alveolar macrophage, which was confirmed by confocal microscopy. Efficient cellular delivery of ATRA was followed by a reduction in Mtb growth (H37Ra) in THP-1 derived macrophages evaluated by both the BACT/ALERT® system and enumeration of colony forming units (CFU). The antibacterial effect of ATRA-MP treatment was further assessed in BALB/c mice infected with the virulent strain of Mtb (H37Rv). ATRA-MP treatments significantly decreased the bacterial burden in the lungs alongside a reduction in pulmonary pathology following just three doses administered intratracheally. The immunomodulatory effects of targeted ATRA treatment in the lungs indicate a distinct yet effective mechanism of action amongst the formulations. This is the first study to-date of a controlled release ATRA treatment for TB suitable for inhalation that offers improved targeting of a HDT, retains antib
Tenland E, Krishnan N, Rönnholm A, et al., 2018, A novel derivative of the fungal antimicrobial peptide plectasin is active against Mycobacterium tuberculosis, Tuberculosis, Vol: 113, Pages: 231-238, ISSN: 1472-9792
Tuberculosis has been reaffirmed as the infectious disease causing most deaths in the world. Co-infection with HIV and the increase in multi-drug resistant Mycobacterium tuberculosis strains complicate treatment and increases mortality rates, making the development of new drugs an urgent priority. In this study we have identified a promising candidate by screening antimicrobial peptides for their capacity to inhibit mycobacterial growth. This non-toxic peptide, NZX, is capable of inhibiting both clinical strains of M. tuberculosis and an MDR strain at therapeutic concentrations. The therapeutic potential of NZX is further supported in vivo where NZX significantly lowered the bacterial load with only five days of treatment, comparable to rifampicin treatment over the same period. NZX possesses intracellular inhibitory capacity and co-localizes with intracellular bacteria in infected murine lungs. In conclusion, the data presented strongly supports the therapeutic potential of NZX in future anti-TB treatment.
Li Y, Spiropoulos J, Cooley J, et al., 2018, Galleria mellonella - a novel infection model for the Mycobacterium tuberculosis complex, Virulence, Vol: 9, Pages: 1126-1137, ISSN: 2150-5594
Animal models have long been used in tuberculosis research to understand disease pathogenesis and to evaluate novel vaccine candidates and anti-mycobacterial drugs. However, all have limitations and there is no single animal model which mimics all the aspects of mycobacterial pathogenesis seen in humans. Importantly mice, the most commonly used model, do not normally form granulomas, the hallmark of tuberculosis infection. Thus there is an urgent need for the development of new alternative in vivo models. The insect larvae, Galleria mellonella has been increasingly used as a successful, simple, widely available and cost-effective model to study microbial infections. Here we report for the first time that G. mellonella can be used as an infection model for members of the M. tuberculosis complex. We demonstrate a dose-response for G. mellonella survival infected with different inocula of bioluminescent, Mycobacterium bovis BCG lux, and demonstrate suppression of mycobacterial luminesence over 14 days. Histopathology staining and transmission electron microscopy of infected G. mellonella phagocytic haemocytes show internalization and aggregation of M. bovis BCG lux in granuloma-like structures, and increasing accumulation of lipid bodies within M. bovis BCG lux over time, characteristic of latent tuberculosis infection. Our results demonstrate that G. mellonella can act as a surrogate host to study the pathogenesis of mycobacterial infection and shed light on host-mycobacteria interactions, including latent tuberculosis infection
Fox KA, Kirwan DE, Whittington AM, et al., 2018, Platelets regulate pulmonary inflammation and tissue destruction in tuberculosis, American Journal of Respiratory and Critical Care Medicine, Vol: 198, Pages: 245-255, ISSN: 1073-449X
RATIONALE: Platelets may interact with the immune system in tuberculosis (TB) to regulate human inflammatory responses that lead to morbidity and spread of infection. OBJECTIVES: To identify a functional role of platelets in the innate inflammatory and matrix degrading response in TB. METHODS: Markers of platelet activation were examined in plasma from 50 TB patients pre-treatment, and 50 controls. 25 patients were followed longitudinally. Platelet-monocyte interactions were studied in a co-culture model infected with live, virulent Mycobacterium tuberculosis (M.tb) and dissected using qPCR, Luminex multiplex arrays, matrix degradation assays and colony counts. Immunohistochemistry detected CD41 expression in a pulmonary TB murine model and secreted platelet factors were measured in bronchoalveolar lavage fluid (BALF) from 15 TB patients and matched controls. MEASUREMENTS AND MAIN RESULTS: Five of six platelet-associated mediators were upregulated in plasma of TB patients compared to controls, with concentrations returning to baseline by day 60 of treatment. Gene expression of the monocyte collagenase MMP-1 was upregulated by platelets in M.tb infection. Platelets also enhanced M.tb-induced MMP-1 and -10 secretion which drove Type I collagen degradation. Platelets increased monocyte IL-1 and IL-10 and decreased IL-12 and monocyte-derived chemokine (MDC, also known as CCL-22) secretion, as consistent with an M2 monocyte phenotype. Monocyte killing of intracellular M.tb was decreased. In the lung, platelets were detected in a TB mouse model and secreted platelet mediators were upregulated in human BALF, and correlated with MMP and IL-1β concentrations. CONCLUSIONS: Platelets drive a pro-inflammatory, tissue-degrading phenotype in TB.
Man DK-W, Kanno T, Manzo G, et al., 2018, Rifampin- or Capreomycin-Induced Remodeling of the Mycobacterium smegmatis Mycolic Acid Layer Is Mitigated in Synergistic Combinations with Cationic Antimicrobial Peptides, MSPHERE, Vol: 3, ISSN: 2379-5042
The mycobacterial cell wall affords natural resistance to antibiotics. Antimicrobial peptides (AMPs) modify the surface properties of mycobacteria and can act synergistically with antibiotics from differing classes. Here, we investigate the response of Mycobacterium smegmatis to the presence of rifampin or capreomycin, either alone or in combination with two synthetic, cationic, α-helical AMPs that are distinguished by the presence (D-LAK120-HP13) or absence (D-LAK120-A) of a kink-inducing proline. Using a combination of high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) metabolomics, diphenylhexatriene (DPH) fluorescence anisotropy measurements, and laurdan emission spectroscopy, we show that M. smegmatis responds to challenge with rifampin or capreomycin by substantially altering its metabolism and, in particular, by remodeling the cell envelope. Overall, the changes are consistent with a reduction of trehalose dimycolate and an increase of trehalose monomycolate and are associated with increased rigidity of the mycolic acid layer observed following challenge by capreomycin but not rifampin. Challenge with D-LAK120-A or D-LAK120-HP13 induced no or modest changes, respectively, in mycomembrane metabolites and did not induce a significant increase in the rigidity of the mycolic acid layer. Furthermore, the response to rifampin or capreomycin was significantly reduced when these were combined with D-LAK120-HP13 and D-LAK120-A, respectively, suggesting a possible mechanism for the synergy of these combinations. The remodeling of the mycomembrane in M. smegmatis is therefore identified as an important countermeasure deployed against rifampin or capreomycin, but this can be mitigated and the efficacy of rifampin or capreomycin potentiated by combining the drug with AMPs.
Uhia I, Priestman M, Joyce G, et al., 2018, Analysis of ParAB dynamics in mycobacteria shows active movement of ParB and differential inheritance of ParA, PLoS ONE, Vol: 13, Pages: 1-20, ISSN: 1932-6203
Correct chromosomal segregation, coordinated with cell division, is crucial for bacterial survival, but despite extensive studies, the mechanisms underlying this remain incompletely understood in mycobacteria. We report a detailed investigation of the dynamic interactions between ParA and ParB partitioning proteins in Mycobacterium smegmatis using microfluidics and time-lapse fluorescence microscopy to observe both proteins simultaneously. During growth and division, ParB presents as a focused fluorescent spot that subsequently splits in two. One focus moves towards a higher concentration of ParA at the new pole, while the other moves towards the old pole. We show ParB movement is in part an active process that does not rely on passive movement associated with cell growth. In some cells, another round of ParB segregation starts before cell division is complete, consistent with initiation of a second round of chromosome replication. ParA fluorescence distribution correlates with cell size, and in sister cells, the larger cell inherits a local peak of concentrated ParA, while the smaller sister inherits more homogeneously distributed protein. Cells which inherit more ParA grow faster than their sister cell, raising the question of whether inheritance of a local concentration of ParA provides a growth advantage. Alterations in levels of ParA and ParB were also found to disturb cell growth.
Uhia I, Krishnan N, Robertson BD, 2018, Characterising resuscitation promoting factor fluorescent-fusions in mycobacteria, BMC Microbiology, Vol: 18, ISSN: 1471-2180
Background Resuscitation promoting factor proteins (Rpfs) are peptidoglycan glycosidases capable of resuscitating dormant mycobacteria, and have been found to play a role in the pathogenesis of tuberculosis. However, the specific roles and localisation of each of the 5 Rpfs in Mycobacterium tuberculosis remain mostly unknown. In this work our aim was to construct fluorescent fusions of M. tuberculosis Rpf proteins as tools to investigate their function.Results We found that Rpf-fusions to the fluorescent protein mCherry are functional and able to promote cell growth under different conditions. However, fusions to Enhanced Green Fluorescent Protein (EGFP) were non-functional in the assays used and none were secreted into the extracellular medium, which suggests Rpfs may be secreted via the Sec pathway. No specific cellular localization was observed for either set of fusions using time-lapse video microscopy.ConclusionsWe present the validation and testing of five M. tuberculosis Rpfs fused to mCherry, which are functional in resuscitation assays, but do not show any specific cellular localisation under the conditions tested. Our results suggest that Rpfs are likely to be secreted via the Sec pathway. We propose that such mCherry fusions will be useful tools for the further study of Rpf localisation, individual expression, and function. KeywordsRpfs, mycobacteria, tuberculosis, fluorescent fusions, microscopy.
Butler RE, Krishnan N, Garcia-Jimenez W, et al., 2017, Susceptibility of M. tuberculosis-infected host cells to phospho-MLKL driven necroptosis is dependent on cell type and presence of TNFα., Virulence, Vol: 8, Pages: 1820-1832, ISSN: 2150-5594
An important feature of Mycobacterium tuberculosis pathogenesis is the ability to control cell death in infected host cells, including inhibition of apoptosis and stimulation of necrosis. Recently an alternative form of programmed cell death, necroptosis, has been described where necrotic cell death is induced by apoptotic stimuli under conditions where apoptotic execution is inhibited. We show for the first time that M. tuberculosis and TNFα synergise to induce necroptosis in murine fibroblasts via RIPK1-dependent mechanisms and characterized by phosphorylation of Ser345 of the MLKL necroptosis death effector. However, in murine macrophages M. tuberculosis and TNFα induce non-necroptotic cell death that is RIPK1-dependent but independent of MLKL phosphorylation. Instead, M. tuberculosis-infected macrophages undergo RIPK3-dependent cell death which occurs both in the presence and absence of TNFα and involves the production of mitochondrial ROS. Immunocytochemical staining for MLKL phosphorylation further demonstrated the occurrence of necroptosis in vivo in murine M. tuberculosis granulomas. Phosphorylated-MLKL immunoreactivity was observed associated with the cytoplasm and nucleus of fusiform cells in M. tuberculosis lesions but not in proximal macrophages. Thus whereas pMLKL-driven necroptosis does not appear to be a feature of M. tuberculosis-infected macrophage cell death, it may contribute to TNFα-induced cytotoxicity of the lung stroma and therefore contribute to necrotic cavitation and bacterial dissemination.
Shahrezaei V, Robertson B, Thomas P, et al., 2017, Mycobacteria modify their cell size control under sub-optimal carbon sources, Frontiers in Cell and Developmental Biology, Vol: 5, ISSN: 2296-634X
The decision to divide is the most important one that any cell must make. Recent single cell studies suggest that most bacteria follow an “adder” model of cell size control, incorporating a fixed amount of cell wall material before dividing. Mycobacteria, including the causative agent of tuberculosis Mycobacterium tuberculosis, are known to divide asymmetrically resulting in heterogeneity in growth rate, doubling time, and other growth characteristics in daughter cells. The interplay between asymmetric cell division and adder size control has not been extensively investigated. Moreover, the impact of changes in the environment on growth rate and cell size control have not been addressed for mycobacteria. Here, we utilize time-lapse microscopy coupled with microfluidics to track live Mycobacterium smegmatis cells as they grow and divide over multiple generations, under a variety of growth conditions. We demonstrate that, under optimal conditions, M. smegmatis cells robustly follow the adder principle, with constant added length per generation independent of birth size, growth rate, and inherited pole age. However, the nature of the carbon source induces deviations from the adder model in a manner that is dependent on pole age. Understanding how mycobacteria maintain cell size homoeostasis may provide crucial targets for the development of drugs for the treatment of tuberculosis, which remains a leading cause of global mortality.
Khara JS, Obuobi S, Wang Y, et al., 2017, Disruption of drug-resistant biofilms using de novo designed short α-helicalantimicrobial peptides with idealized facial amphiphilicity, Acta Biomaterialia, Vol: 57, Pages: 103-114, ISSN: 1878-7568
The escalating threat of antimicrobial resistance has increased pressure to develop novel therapeutic strategies to tackle drug-resistant infections. Antimicrobial peptides have emerged as a promising class of therapeutics for various systemic and topical clinical applications. In this study, the de novo design of α-helical peptides with idealized facial amphiphilicities, based on an understanding of the pertinent features of protein secondary structures, is presented. Synthetic amphiphiles composed of the backbone sequence (X1Y1Y2X2)n, where X1 and X2 are hydrophobic residues (Leu or Ile or Trp), Y1 and Y2 are cationic residues (Lys), and n is the number repeat units (2 or 2.5 or 3), demonstrated potent broad-spectrum antimicrobial activities against clinical isolates of drug-susceptible and multi-drug resistant bacteria. Live-cell imaging revealed that the most selective peptide, (LKKL)3, promoted rapid permeabilization of bacterial membranes. Importantly, (LKKL)3 not only suppressed biofilm growth, but effectively disrupted mature biofilms after only 2 h of treatment. The peptides (LKKL)3 and (WKKW)3 suppressed the production of LPS-induced pro-inflammatory mediators to levels of unstimulated controls at low micromolar concentrations. Thus, the rational design strategies proposed herein can be implemented to develop potent, selective and multifunctional α-helical peptides to eradicate drug-resistant biofilm-associated infections.
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