Imperial College London

Dr Masanori Asai

Faculty of MedicineDepartment of Infectious Disease

NC3Rs Training Fellow
 
 
 
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m.asai16

 
 
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224Wright Fleming WingSt Mary's Campus

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Summary

 

Publications

Publication Type
Year
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11 results found

Emma B, Asai M, Gabant P, Wigneshweraraj Set al., 2023, Enhancing the antibacterial function of probiotic Escherichia coli Nissle: when less is more, Applied and Environmental Microbiology, Vol: 89, ISSN: 0099-2240

Probiotic bacteria confer multiple health benefits, including preventing the growth, colonization, or carriage of harmful bacteria in the gut. Bacteriocins are antibacterial peptides produced by diverse bacteria, and their production is tightly regulated and coordinated at the transcriptional level. A popular strategy for enhancing the antibacterial properties of probiotic bacteria is to retrofit them with the ability to overproduce heterologous bacteriocins. This is often achieved from non-native constitutive promoters or in response to host or pathogen signal from synthetic promoters. How the dysregulated overproduction of heterologous bacteriocins affects the fitness and antibacterial efficacy of the retrofitted probiotic bacteria is often overlooked. We have conferred the prototypical probiotic Escherichia coli strain Nissle (EcN) the ability to produce microcin C (McC) from the wild-type promoter and two mutant promoters that allow, relative to the wild-type promoter, high and low amounts of McC production. This was done by introducing specific changes to the sequence of the wild-type promoter driving transcription of the McC operon while ensuring that the modified promoters respond to native regulation. By studying the transcriptomic responses and antibacterial efficacy of the retrofitted EcN bacteria in a Galleria mellonella infection model of enterohemorrhagic E. coli, we show that EcN bacteria that produce the lowest amount of McC display the highest antibacterial efficacy with little-to-none undesired collateral impact on their fitness. The results highlight considerations researchers may take into account when retrofitting probiotic bacteria with heterogenous gene products for therapeutic, prophylactic, or diagnostic applications.

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

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

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

Asai M, Sheehan G, Li Y, Robertson B, Kavanagh K, Langford P, Newton Set 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.

Journal article

Asai M, Li Y, Spiropoulos J, Cooley W, Everest D, Robertson BD, Langford PR, Newton SMet 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.

Journal article

Asai M, Li Y, Singh Khara J, Robertson B, Langford P, Newton Set 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.

Journal article

Lynskey NN, Jauneikaite E, Li H-K, Zhi X, Turner CE, Mosavie M, Pearson M, Asai M, Lobkowicz L, Chow JY, Parkhill J, Lamagni T, Chalker V, Sriskandan Set al., 2019, Emergence of dominant toxigenic M1T1 Streptococcus pyogenes clone during increased scarlet fever activity in England: a population-based molecular epidemiological study, Lancet Infectious Diseases, Vol: 19, Pages: 1209-1218, ISSN: 1473-3099

BackgroundEngland is experiencing scarlet fever activity unprecedented in modern times. In 2016, England’s scarlet fever seasonal rise coincided with an unexpected elevation in invasive Streptococcus pyogenes infections. We describe the molecular-epidemiological investigation of these events and emergence of a new emm1 lineage.Methods We analysed changes in S. pyogenes emm-genotypes, and notifications of scarlet fever and invasive disease 2014-2016 using regional (North-West London) and national (England and Wales) data. We analysed genomes of 135 non-invasive and 552 invasive emm1 isolates from 2009-2016, and compared 2800 global emm1 sequences. Expression of Streptococcal pyrogenic exotoxin (Spe)A by sequenced non-invasive emm1 isolates was quantified.FindingsCoincident with national increases in scarlet fever and invasive disease notifications, emm1 S. pyogenes increased significantly, from 19% (28/147) of upper respiratory tract isolates in MarchMay 2015, to 32·6% (47/144) in the same period 2016 in North-West London (χ2 (1df)=7·024, p=0·008), and from 31% (183/587) of invasive isolates in March-May 2015, to 41·9% (267/637)in the same period 2016 nationally (χ2 (1df)=15·16, p=0·0001). Sequences of emm1 isolates from 2009-2016 demonstrated emergence of a new emm1 lineage (M1UK), with overlap of pharyngitis, scarlet fever, and invasive M1UK strains, that could be genotypically distinguished from pandemic emm1 isolates (M1global). Compared with M1global, median expression of SpeA increased 9-fold in M1UK isolates (M1global, median=20·9 ng/ml, IQ range=21.3; (M1UK, median=190·2 ng/ml, IQ3 range 31.5; Mann-Whitney p<0·001). M1UK expanded nationally to represent 84% (252/299) of all emm1 genomes in 2016; phylogenetic analysis of published datasets identified single M1UK isolates in Denmark and USA.

Journal article

Asai M, Li Y, Khara J, Gladstone C, Robertson B, Langford P, Newton Set 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.

Journal article

Li Y, Spiropoulos J, Cooley J, Khara J, Gladstone C, Asai M, Bosse J, Robertson B, Newton SM, Langford Pet 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

Journal article

Buongiorno J, Turner S, Webster G, Asai M, Shumaker AK, Roy T, Weightman A, Schippers A, Lloyd KGet al., 2017, Interlaboratory quantification of Bacteria and Archaea in deeply buried sediments of the Baltic Sea (IODP Expedition 347), FEMS MICROBIOLOGY ECOLOGY, Vol: 93, ISSN: 0168-6496

Journal article

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