Imperial College London

DrGeraldLarrouy-Maumus

Faculty of Natural SciencesDepartment of Life Sciences

Senior Lecturer
 
 
 
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Contact

 

+44 (0)20 7594 7463g.larrouy-maumus

 
 
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Location

 

3.42Flowers buildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
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79 results found

Furniss RCD, Kaderabkova N, Barker D, Bernal P, Maslova E, Antwi AA, McNeil HE, Pugh HL, Dortet L, Blair JM, Larrouy-Maumus GJ, McCarthy RR, Gonzalez D, Mavridou DAet al., 2022, Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding., Elife, Vol: 11

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse β-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.

Journal article

Hamilton C, Olona A, Leishman S, MacDonald-Ramsahai K, Cockcroft S, Larrouy-Maumus G, Anand Pet al., 2021, NLRP3 inflammasome priming and activation are regulated by a novel phosphatidylinositol-dependent mechanism, Publisher: bioRxiv

Imbalance in lipid homeostasis is associated with discrepancies in immune signalling and is tightly linked to metabolic disorders. The diverse ways in which lipids impact immune signalling, however, remain ambiguous. The phospholipid phosphatidylinositol (PI), which is implicated in numerous immune disorders, is chiefly defined by its phosphorylation status. By contrast, the significance of the two fatty acid chains attached to the PI remains unknown. Here, by employing a mass-spectrometry-based assay, we demonstrate a role for PI acyl group chains in regulating both the priming and activation steps of the NLRP3 inflammasome in mouse macrophages. In response to NLRP3 stimuli, cells deficient in ABC transporter ABCB1, which effluxes lipid derivatives, revealed defective inflammasome activation. Mechanistically, Abcb1-deficiency shifted the total PI configuration exhibiting a reduced ratio of short-chain to long-chain PI-acyl lipids. Consequently, Abcb1-deficiency resulted in rapid degradation of TIRAP, the TLR adaptor protein which binds PI(4,5)-phosphate. Moreover, this accompanied increased NLRP3 phosphorylation at the Ser293 position and blunted inflammasome activation. Exogenously supplementing WT cells with linoleic acid, but not arachidonic acid, reconfigured PI acyl chains. Accordingly, linoleic acid supplementation increased TIRAP degradation, elevated NLRP3 phosphorylation, and abrogated inflammasome activation. Altogether, our study reveals a novel metabolic-inflammatory circuit which contributes to calibrating immune responses.

Working paper

Boonyasiri A, Myall AC, Wan Y, Bolt F, Ledda A, Mookerjee S, Weiße AY, Turton JF, Abbas H, Prakapaite R, Sabnis A, Abdolrasouli A, Malpartida-Cardenas K, Miglietta L, Donaldson H, Gilchrist M, Hopkins KL, Ellington MJ, Otter JA, Larrouy-Maumus G, Edwards AM, Rodriguez-Manzano J, Didelot X, Barahona M, Holmes AH, Jauneikaite E, Davies Fet al., 2021, Integrated patient network and genomic plasmid analysis reveal a regional, multi-species outbreak of carbapenemase-producing Enterobacterales carrying both blaIMP and mcr-9 genes

<jats:title>Abstract</jats:title><jats:p>The incidence of carbapenemase-producing Enterobacterales (CPE) is rising globally, yet Imipenemase (IMP) carbapenemases remain relatively rare. This study describes an investigation of the emergence of IMP-encoding CPE amongst diverse Enterobacterales species between 2016 and 2019 in patients across a London regional hospital network.</jats:p><jats:p>A network analysis approach to patient pathways, using routinely collected electronic health records, identified previously unrecognised contacts between patients who were IMP CPE positive on screening, implying potential bacterial transmission events. Whole genome sequencing of 85 Enterobacterales isolates from these patients revealed that 86% (73/85) were diverse species (predominantly <jats:italic>Klebsiella</jats:italic> spp, <jats:italic>Enterobacter</jats:italic> spp, <jats:italic>E. coli</jats:italic>) and harboured an IncHI2 plasmid, which carried both <jats:italic>bla</jats:italic><jats:sub>IMP</jats:sub> and the putative mobile colistin resistance gene <jats:italic>mcr-9</jats:italic>. Detailed phylogenetic analysis identified two distinct IncHI2 plasmid lineages, A and B, both of which showed significant association with patient movements between four hospital sites and across medical specialities.</jats:p><jats:p>Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of <jats:italic>bla</jats:italic><jats:sub>IMP</jats:sub>CPE, which remained unidentified during standard microbiology and infection control investigations. With whole genome sequencing (WGS) technologies and large-data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Analysing outbreaks at the plasmid level reveal

Journal article

Humphrey M, Larrouy-Maumus GJ, Furniss RCD, Mavridou DA, Sabnis A, Edwards AMet al., 2021, Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic, MICROBIOLOGY-SGM, Vol: 167, ISSN: 1350-0872

Journal article

Larrouy-Maumus G, Katy J, katheryn H, laurent D, markus K, Filloux A, Plesiat Pet al., 2021, Detection of colistin resistance in Pseudomonas aeruginosa using the MALDIxin test on the routine MALDI Biotyper Sirius mass spectrometer, Frontiers in Microbiology, Vol: 12, ISSN: 1664-302X

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in Pseudomonas aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-L-arabinose (L-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of L-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant Pseudomonas aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240-247) and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1 hour, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant Pseudomonas aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.

Journal article

Larrouy-Maumus G, Broda A, Drobniewski F, Khor MJ, kostrzewaet al., 2021, An improved method for rapid detection of Mycobacterium abscessus complex based on species-specific lipids fingerprint by routine MALDI-TOF, Frontiers in Chemistry, Vol: 9, Pages: 1-7, ISSN: 2296-2646

Rapid diagnostics of bacterial infection is the key to successful recovery and eradication of the disease. Currently, identification of bacteria is based on the detection of highly abundant proteins, mainly ribosomal proteins, by routine MALDI-TOF mass spectrometry. However, relying solely on proteins is limited in subspecies typing for some pathogens. This is the case for, for example, the mycobacteria belonging to the Mycobacterium abscessus (MABS) complex, which is classified into three subspecies, namely, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Being able to detect bacteria accurately and rapidly at the subspecies level could not only reliably identify the pathogen causing the disease but also enable better antibiotic stewardship. For instance, M. abscessus subsp. abscessus and M. abscessus subsp. bolletii possess a functional erm41 (erythromycin ribosomal methylation gene 41) gene, whilst M. abscessus subsp. massiliense does not, resulting in differences in macrolide antibiotic (e.g., clarithromycin and azithromycin) susceptibilities. This presents a challenge for physicians when designing an appropriate treatment regimen. To address this challenge, in addition to proteins, species-specific lipids have now been considered as a game changer in clinical microbiology diagnostics. However, their extraction can be time-consuming, and analysis requires the use of apolar toxic organic solvents (e.g., chloroform). Here, we present a new method to accurately detect species and subspecies, allowing the discrimination of the mycobacteria within the MABS complex and relying on the use of ethanol. We found that a combination of the matrix named super-DHB with 25% ethanol with a bacterial suspension at McFarland 20 gave robust and reproducible data, allowing the discrimination of the bacteria within the MABS complex strains tested in this study (n = 9). Further investigations have to be conducted to validate the metho

Journal article

Humphrey M, Larrouy-Maumus GJ, Furniss RCD, Mavridou DAI, Sabnis A, Edwards AMet al., 2021, Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic

<jats:title>Abstract</jats:title><jats:p>Colistin is a polymyxin antibiotic of last resort for the treatment of infections caused by multi-drug resistant Gram-negative bacteria. By targeting lipopolysaccharide (LPS), the antibiotic disrupts both the outer and cytoplasmic membranes, leading to lysis and bacterial death. Colistin resistance in <jats:italic>Escherichia coli</jats:italic> occurs via mutations in the chromosome or the acquisition of mobilised colistin resistance (<jats:italic>mcr</jats:italic>) genes. Both these colistin resistance mechanisms result in chemical modifications to the LPS, with positively charged moieties added at the cytoplasmic membrane before the LPS is transported to the outer membrane. We have previously shown that MCR-1-mediated LPS modification protects the cytoplasmic but not the outer membrane from damage caused by colistin, enabling bacterial survival. However, it remains unclear whether this observation extends to colistin resistance conferred by other <jats:italic>mcr</jats:italic> genes, or resistance due to chromosomal mutations. Using a panel of clinical <jats:italic>E. coli</jats:italic> that had acquired <jats:italic>mcr</jats:italic> -1, -1.5, -2, -3, -3.2 or -5, or had acquired polymyxin resistance independently of <jats:italic>mcr</jats:italic> genes, we found that almost all isolates were susceptible to colistin-mediated permeabilisation of the outer, but not cytoplasmic, membrane. Furthermore, we showed that permeabilisation of the outer membrane of colistin resistant isolates by the polymyxin is in turn sufficient to sensitise bacteria to the antibiotic rifampicin, which normally cannot cross the LPS monolayer. These findings demonstrate that colistin resistance in <jats:italic>E. coli</jats:italic> is typically due to protection of the cytoplasmic but not outer membrane from colistin-mediated damage, regardless of th

Journal article

Vivian T, Yi L, Ashleigh C, Larrouy-Maumus Get al., 2021, Metabolomics in infectious diseases and drug discovery, Molecular Omics, Vol: 17, Pages: 376-393, ISSN: 2515-4184

Metabolomics has emerged as an invaluable tool that can be used along with genomics, transcriptomics and proteomics to understand host–pathogen interactions at small-molecule levels. Metabolomics has been used to study a variety of infectious diseases and applications. The most common application of metabolomics is for prognostic and diagnostic purposes, specifically the screening of disease-specific biomarkers by either NMR-based or mass spectrometry-based metabolomics. In addition, metabolomics is of great significance for the discovery of druggable metabolic enzymes and/or metabolic regulators through the use of state-of-the-art flux analysis, for example, via the elucidation of metabolic mechanisms. This review discusses the application of metabolomics technologies to biomarker screening, the discovery of drug targets in infectious diseases such as viral, bacterial and parasite infections and immunometabolomics, highlights the challenges associated with accessing metabolite compartmentalization and discusses the available tools for determining local metabolite concentrations.

Journal article

Gonzalo X, Broda A, Drobniewski F, Larrouy-Maumus Get al., 2021, Performance of lipid fingerprint-based MALDI-ToF for the diagnosis of mycobacterial infections, Clinical Microbiology and Infection, Vol: 27, Pages: 912.e1-912.e5, ISSN: 1198-743X

ObjectivesBacterial diagnosis of mycobacteria is often challenging because of the variability of the sensitivity and specificity of the assay used, and it can be expensive to perform accurately. Although matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has become the workhorse of clinical laboratories, the current MALDI methodology (which is based on cytosolic protein profiling) for mycobacteria is still challenging due to the number of steps involved (up to seven) and potential biosafety concerns. Knowing that mycobacteria produce surface-exposed species-specific lipids, we here hypothesized that the detection of those molecules could offer a rapid, reproducible and robust method for mycobacterial identification.MethodsWe evaluated the performance of an alternative methodology based on characterized species-specific lipid profiling of intact bacteria, without any sample preparation, by MALDI MS; it uses MALDI-time-of-flight (ToF) MS combined with a specific matrix (super-2,5-dihydroxybenzoic acid solubilized in an apolar solvent system) to analyse lipids of intact heat-inactivated mycobacteria. Cultured mycobacteria are heat-inactivated and loaded directly onto the MALDI target followed by addition of the matrix. Acquisition of the data is done in both positive and negative ion modes. Blinded studies were performed using 273 mycobacterial strains comprising both the Mycobacterium tuberculosis (Mtb) complex and non-tuberculous mycobacteria (NTMs) subcultured in Middlebrook 7H9 media supplemented with 10% OADC (oleic acid/dextrose/catalase) growth supplement and incubated for up to 2 weeks at 37°C.ResultsThe method we have developed is fast (<10 mins) and highly sensitive (<1000 bacteria required); 96.7% of the Mtb complex strains (204/211) were correctly assigned as MTB complex and 91.7% (22/24) NTM species were correctly assigned based only on intact bacteria species-specific lipid profiling by MALDI-ToF MS.ConclusionsIntact bacter

Journal article

Larrouy-Maumus G, 2021, Shotgun bacterial lipid A analysis using routine MALDI-TOF mass spectrometry., Mass Spectrometry-Based Lipidomics, Editors: Hsu, Pages: 275-283

Detection of bacterial lipids and particularly the lipid A, the lipid anchor of the lipopolysaccharide, can be very challenging and requires a certain level of expertise. Here, this chapter describes a straightforward and simple method for the analysis of bacterial lipid A. In addition, such approach, lipid fingerprint, has the potential to be applied to other bacteria such as mycobacteria.

Book chapter

Borah K, Mendum TA, Hawkins ND, Ward JL, Beale MH, Larrouy-Maumus G, Bhatt A, Moulin M, Haertlein M, Strohmeier G, Pichler H, Forsyth VT, Noack S, Goulding CW, McFadden J, Beste DJet al., 2021, Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis, MOLECULAR SYSTEMS BIOLOGY, Vol: 17, ISSN: 1744-4292

Journal article

Sabnis A, Haggard K, Kloeckner A, Becce M, Evans L, Furniss R, Mavridou D, Stevens M, Murphy R, Davies J, Clarke T, Edwards Aet al., 2021, Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane, eLife, Vol: 10, Pages: 1-26, ISSN: 2050-084X

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

Journal article

Wu C-H, Rismondo J, Morgan RML, Shen Y, Loessner MJ, Larrouy-Maumus G, Freemont PS, Grundling Aet al., 2021, Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth, Journal of Biological Chemistry, Vol: 296, Pages: 1-14, ISSN: 0021-9258

UTP-glucose-1-phosphate uridylyltransferases are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UTP-glucose-1-phosphate uridylyltransferases are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When WT and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen-limited growth condition.

Journal article

Solntceva V, Kostrzewa M, Larrouy-Maumus G, 2021, Detection of species-specific lipids by routine MALDI TOF mass spectrometry to unlock the challenges of microbial identification and antimicrobial susceptibility testing, Frontiers in Cellular and Infection Microbiology, Vol: 10, ISSN: 2235-2988

MALDI-TOF mass spectrometry has revolutionized clinical microbiology diagnostics by delivering accurate, fast, and reliable identification of microorganisms. It is conventionally based on the detection of intracellular molecules, mainly ribosomal proteins, for identification at the species-level and/or genus-level. Nevertheless, for some microorganisms (e.g., for mycobacteria) extensive protocols are necessary in order to extract intracellular proteins, and in some cases a protein-based approach cannot provide sufficient evidence to accurately identify the microorganisms within the same genus (e.g., Shigella sp. vs E. coli and the species of the M. tuberculosis complex). Consequently lipids, along with proteins are also molecules of interest. Lipids are ubiquitous, but their structural diversity delivers complementary information to the conventional protein-based clinical microbiology matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) based approaches currently used. Lipid modifications, such as the ones found on lipid A related to polymyxin resistance in Gram-negative pathogens (e.g., phosphoethanolamine and aminoarabinose), not only play a role in the detection of microorganisms by routine MALDI-TOF mass spectrometry but can also be used as a read-out of drug susceptibility. In this review, we will demonstrate that in combination with proteins, lipids are a game-changer in both the rapid detection of pathogens and the determination of their drug susceptibility using routine MALDI-TOF mass spectrometry systems.

Journal article

McCarthy RR, Larrouy-Maumus GJ, Meiqi Tan MGC, Wareham DWet al., 2021, Antibiotic Resistance Mechanisms and Their Transmission in Acinetobacter baumannii., Adv Exp Med Biol, Vol: 1313, Pages: 135-153, ISSN: 0065-2598

The discovery of penicillin over 90 years ago and its subsequent uptake by healthcare systems around the world revolutionised global health. It marked the beginning of a golden age in antibiotic discovery with new antibiotics readily discovered from natural sources and refined into therapies that saved millions of lives. Towards the end of the last century, the rate of discovery slowed to a near standstill. The lack of discovery is compounded by the rapid emergence and spread of bacterial pathogens that exhibit resistance to multiple antibiotic therapies and threaten the sustainability of global healthcare systems. Acinetobacter baumannii is an opportunistic pathogen whose prevalence and impact has grown significantly over the last 20 years. It is recognised as a barometer of the antibiotic resistance crisis due to the diverse array of mechanisms by which it can become resistant.

Journal article

Wu C-H, Rismondo J, Morgan RML, Shen Y, Loessner MJ, Larrouy-Maumus G, Freemont PS, Gründling Aet al., 2020, Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth, Publisher: Cold Spring Harbor Laboratory

<jats:title>Abstract</jats:title><jats:p>UTP-glucose-1-phosphate uridylyltransferases (UGPases) are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In <jats:italic>Bacillus subtilis</jats:italic> 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The <jats:italic>B. subtilis</jats:italic> UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and <jats:italic>gtaB</jats:italic> mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UGPases, are present in <jats:italic>B. subtilis</jats:italic>. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated <jats:italic>in vitro</jats:italic> using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a <jats:italic>B. subtilis gtaB</jats:italic> mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase <jats:italic>in vivo</jats:italic>. When wild-type and mutant <jats:italic>B. subtilis</jats:italic> strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing <jats:italic>yngB</jats:italic> and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with g

Working paper

Saromi K, England P, Tang W, Kostrzewa M, Corran A, Woscholski R, Larrouy-Maumus Get al., 2020, Rapid glycosyl-inositol-phospho-ceramide fingerprint from filamentous fungal pathogens using the MALDI Biotyper Sirius system, RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Vol: 34, ISSN: 0951-4198

Journal article

Yong HY, Larrouy-Maumus G, Zloh M, Smyth R, Ataya R, Benton CM, Munday MRet al., 2020, Early detection of metabolic changes in drug-induced steatosis using metabolomics approaches, RSC ADVANCES, Vol: 10, Pages: 41047-41057

Journal article

Yi L, Rebollo-Ramirez S, Larrouy-Maumus G, 2020, Metabolomics reveals that the cAMP receptor protein regulates nitrogen and peptidoglycan synthesis in Mycobacterium tuberculosis, RSC Advances: an international journal to further the chemical sciences, Vol: 10, Pages: 26212-26219, ISSN: 2046-2069

Mycobacterium tuberculosis requires extensive sensing and response to environment for its successful survival and pathogenesis, and signalling by cyclic adenosine 3′,5′-monophosphate (cAMP) is an important mechanism. cAMP regulates expression of target genes via interaction with downstream proteins, one of which is cAMP receptor protein (CRP), a global transcriptional regulator. Previous genomic works had identified regulon of CRP and investigated transcriptional changes in crp deletion mutant, however a link to downstream metabolomic events were lacking, which would help better understand roles of CRP. This work aims at investigating changes at metabolome level in M. tuberculosis crp deletion mutant combining untargeted LC-MS analysis and 13C isotope tracing analysis. The results were compared with previously published RNA sequencing data. We identified increasing abundances of metabolites related to nitrogen metabolism including ornithine, citrulline and glutamate derivatives, while 13C isotope labelling analysis further showed changes in turnover of these metabolites and amino acids, suggesting regulatory roles of CRP in nitrogen metabolism. Upregulation of diaminopimelic acid and its related genes also suggested role of CRP in regulation of peptidoglycan synthesis. This study provides insights on metabolomic aspects of cAMP-CRP regulatory pathway in M. tuberculosis and links to previously published transcriptomic data drawing a more complete map.

Journal article

Larrouy-Maumus G, Dortet L, Filloux A, bonnin, le hello, bonnet, kostrzewaet al., 2020, Detection of colistin resistance in Salmonella enterica using MALDIxin test on the routine MALDI Biotyper Sirius mass spectrometer, Frontiers in Microbiology, Vol: 11, Pages: 1-6, ISSN: 1664-302X

Resistance to polymyxins in most Gram-negative bacteria arises from chemical modifications to the lipid A portion of their lipopolysaccharide (LPS) mediated by chromosomally-encoded mutations or the recently discovered plasmid-encoded mcr genes that have further complicated the landscape of colistin resistance. Currently, minimal inhibitory concentration (MIC) determination by broth microdilution, the gold standard for the detection of polymyxin resistance, is time consuming (24 hours) and challenging to perform in clinical and veterinatryveterinary laboratories. Here we present the use of the MALDIxin to detect colistin resistant Salmonella enterica using the MALDxin test on the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system.

Journal article

Giraud-Gatineau A, Coya JM, Maure A, Biton A, Thomson M, Bernard EM, Marrec J, Gutierrez MG, Larrouy-Maumus G, Brosch R, Gicquel B, Tailleux Let al., 2020, The antibiotic bedaquiline activates host macrophage innate immune resistance to bacterial infection, eLife, Vol: 9, ISSN: 2050-084X

Antibiotics are widely used in the treatment of bacterial infections. Although known for their microbicidal activity, antibiotics may also interfere with the host's immune system. Here, we analyzed the effects of bedaquiline (BDQ), an inhibitor of the mycobacterial ATP synthase, on human macrophages. Genome-wide gene expression analysis revealed that BDQ reprogramed cells into potent bactericidal phagocytes. We found that 579 and 1,495 genes were respectively differentially expressed in naive- and M. tuberculosis-infected macrophages incubated with the drug, with an over-representation of lysosome-associated genes. BDQ treatment triggered a variety of antimicrobial defense mechanisms, including phagosome-lysosome fusion, and autophagy. These effects were associated with activation of transcription factor EB, involved in the transcription of lysosomal genes, resulting in enhanced intracellular killing of different bacterial species that were naturally insensitive to BDQ. Thus, BDQ could be used as a host-directed therapy against a wide range of bacterial infections.

Journal article

Furniss RCD, Kostrzewa M, Mavridou DAI, Larrouy-Maumus Get al., 2020, The clue is in the lipid A: Rapid detection of colistin resistance, PLoS Pathogens, Vol: 16, ISSN: 1553-7366

Journal article

Larrouy-Maumus G, Shahrezaei V, tang W, england P, KOSTRZEWA Met al., 2020, Discrimination of bovine milk from non-dairy milk by lipids fingerprinting using routine matrix-assisted laser desorption ionization mass spectrometry, Scientific Reports, Vol: 10, ISSN: 2045-2322

An important sustainable development goal for any country is to ensure food security by producing a sufficient and safe food supply. This is the case for bovine milk where addition of non-dairy milks such as vegetables (e.g., soya or coconut) has become a common source of adulteration and fraud. Conventionally, gas chromatography techniques are used to detect key lipids (e.g., triacylglycerols) has an effective read-out of assessing milks origins and to detect foreign milks in bovine milks. However, such approach requires several sample preparation steps and a dedicated laboratory environment, precluding a high throughput process. To cope with this need, here, we aimed to develop a novel and simple method without organic solvent extractions for the detection of bovine and non-dairy milks based on lipids fingerprint by routine MALDI-TOF mass spectrometry (MS). The optimized method relies on the simple dilution of milks in water followed by MALDI-TOF MS analyses in the positive linear ion mode and using a matrix consisting of a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid (super-DHB) solubilized at 10 mg/mL in 70% ethanol. This sensitive, inexpensive, and rapid method has potential for use in food authenticity applications.

Journal article

Otter J, Brophy K, Palmer J, Harrison N, Riley J, Williams D, Larrouy-Maumus Get al., 2020, Smart surfaces to tackle infection and antimicrobial resistance, Briefing Paper

Report

Bottai D, Frigui W, Sayes F, Di Luca M, Spadoni D, Pawlik A, Zoppo M, Orgeur M, Khanna V, Hardy D, Mangenot S, Barbe V, Medigue C, Ma L, Bouchier C, Tavanti A, Larrouy-Maumus G, Brosch Ret al., 2020, TbD1 deletion as a driver of the evolutionary success of modern epidemic Mycobacterium tuberculosis lineages, NATURE COMMUNICATIONS, Vol: 11, ISSN: 2041-1723

Journal article

Hamilton C, Larrouy-Maumus G, Anand P, 2020, Phosphatidylinositol Acyl Chains Configure TLR-Dependent Priming and Activation of the NLRP3 Inflammasome, BioRxiv 2020

Abstract Lipids are important in establishing cellular homeostasis by conducting varied functions including relay of extracellular signals. Imbalance in lipid homeostasis results in metabolic diseases, and is tightly connected to discrepancies in immune signalling. The phosphorylation status of the lipid second messenger phosphatidylinositol phosphates is implicated in key physiological functions and pathologies. By contrast, little is known as to how phosphatidylinositol (PI) lipid acyl chains contribute to cellular processes. Here, by employing a mass-spectrometry-based method, we show a role for PI acyl group chains in regulating NLRP3 inflammasome activation in cells lacking ABC transporter ABCB1. In response to canonical stimuli, Abcb1 -/- cells revealed defective priming and activation of the NLRP3 inflammasome owing to blunted TLR-dependent signalling. Cellular lipidomics demonstrated that ABC transporter deficiency shifted the total PI balance such that Abcb1 -/- cells exhibited reduced ratio of the short-chain to long-chain acyl chain lipids. Changes in PI acyl chain configuration accompanied diminished levels of ganglioside GM1, a marker of cholesterol-rich membrane microdomains, in deficient cells. Strikingly, this was not due to differences in the expression of enzymes that either synthesize PI or are involved in acyl chain remodelling. Our study thus suggests an important role for PI lipid chains in priming and activation of the NLRP3 inflammasome thereby highlighting the metabolic regulation of immune responses.

Journal article

Dortet L, Broda A, bernabeu S, GLUPCZYNSKI Y, bogaerts P, bonnin R, naas T, Filloux A, Larrouy-Maumus Get al., 2020, Optimization of the MALDIxin test for the rapid identification of colistin resistance in Klebsiella pneumoniae using MALDI-TOF-MS, Journal of Antimicrobial Chemotherapy, Vol: 75, Pages: 110-116, ISSN: 0305-7453

Background. With the dissemination of carbapenemase producers, a revival of colistin was observed for the treatment of infections caused by multidrug-resistant Gram-negatives. Unfortunately, the increasing usage of colistin led to the emergence of resistance. In Klebsiella pneumoniae, colistin resistance arises through addition of L-arabinose-4N (L-Ara4N) or phosphoethanolamine (pEtN) on the native lipid A. The underlying mechanisms involve numerous chromosome-encoded genes or the plasmid-encoded phosphoethanolamine transferase MCR. Currently, detection of colistin resistance is time consuming since it still relies on MIC determination by broth microdilution. Recently, a rapid diagnostic test based on MALDI-TOF detection of modified lipid A was developed (the MALDIxin test) and tested on Escherichia coli and Acinetobacter baumannii.Objectives. Optimize the MALDIxin test for the rapid detection of colistin resistance in Klebsiella pneumoniae.Methods. This optimization consists on an additional mild-acid hydrolysis of 15 min in 1% acetic acid. The optimized method was tested on a collection of 81 clinical K. pneumoniae isolates including 49 colistin resistant strains among which 45 correspond to chromosome-encoded resistance, 3 MCR-related resistance and one isolate harbouring both mechanisms.Results. The optimized method allowed the rapid (< 30 min) identification of L-Ara4N and pEtN modified lipid A of K. pneumoniae which are known to be the real triggers of polymyxin resistance. In the same time, it discriminates between chromosome-encoded and MCR-related polymyxin resistance.Conclusions. The MALDIxin test has the potential to become an accurate tool for the rapid diagnostic of colistin resistance in clinically-relevant Gram negative bacteria.

Journal article

Furniss C, Dortet L, Bolland W, drews O, sparbier K, bonnin R, Filloux A, kostrzewa M, Mavridou D, Larrouy-Maumus Get al., 2019, Detection of colistin resistance in Escherichia coli using the MALDI Biotyper Sirius mass spectrometry system, Journal of Clinical Microbiology, Vol: 57, Pages: 1-7, ISSN: 0095-1137

Polymyxin antibiotics are a last-line treatment for multidrug-resistant Gram-negative bacteria. However, the emergence of colistin resistance, including the spread of mobile mcr genes, necessitates the development of improved diagnostics for the detection of colistin-resistant organisms in hospital settings. The recently developed MALDIxin test enables detection of colistin resistance by MALDI-TOF mass spectrometry in less than 15 minutes, but is not optimized for the mass spectrometers commonly found in clinical microbiology laboratories. In this study, we adapted the MALDIxin test for the MALDI Biotyper Sirius MALDI-TOF mass spectrometry system (Bruker Daltonics). We optimized the sample preparation protocol using a set of 6 MCR-expressing Escherichia coli clones and validated the assay with a collection of 40 E. coli clinical isolates, including 19 confirmed MCR producers, 12 colistin-resistant isolates which tested negative for commonly encountered mcr genes (i.e. likely chromosomally-resistant isolates) and 9 polymyxin-susceptible isolates. We calculated Polymyxin resistance ratio (PRR) values from the acquired spectra; a PRR value of zero, indicating polymyxin susceptibility, was obtained for all colistin-susceptible E. coli isolates, whereas positive PRR values, indicating resistance to polymyxins, were obtained for all resistant strains independent of the genetic basis of resistance. Thus, we report a preliminary feasibility study showing that an optimized version of the MALDIxin test, adapted for the routine MALDI Biotyper Sirius, provides an unbiased, fast, reliable, cost-effective and high-throughput way of detecting colistin resistance in clinical E. coli isolates.

Journal article

Potron A, Vuillemenot J-B, Puja H, Triponney P, Bour M, Valot B, Amara M, Cavalie L, Bernard C, Parmeland L, Reibel F, Larrouy-Maumus G, Dortet L, Bonnin RA, Plesiat Pet al., 2019, ISAba1-dependent overexpression of eptA in clinical strains of Acinetobacter baumannii resistant to colistin, JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY, Vol: 74, Pages: 2544-2550, ISSN: 0305-7453

Journal article

Tang W, Ranganathan N, Shahrezaei V, Larrouy-Maumus Get al., 2019, MALDI-TOF mass spectrometry on intact bacteria combined with a refined analysis framework allows accurate classification of MSSA and MRSA., PLoS ONE, Vol: 14, Pages: 1-16, ISSN: 1932-6203

Fast and reliable detection coupled with accurate data-processing and analysis of antibiotic-resistant bacteria is essential in clinical settings. In this study, we use MALDI-TOF on intact cells combined with a refined analysis framework to demonstrate discrimination between methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus. By combining supervised and unsupervised machine learning methods, we firstly show that the mass spectroscopy data contains strong signal for the clustering of MSSA and MRSA. Then we concentrate on applying supervised learning to extract and verify the important features. A new workflow is proposed that allows for extracting a fixed set of reference peaks so that any new data can be aligned to it and hence consistent feature matrices can be obtained. Also note that by doing so we are able to examine the robustness of the important features that have been found. We also show that appropriate size of the benchmark data, appropriate alignment of the testing data and use of an optimal set of features via feature selection results in prediction accuracy over 90%. In summary, as proof-of-principle, our integrated experimental and bioinformatics study suggests a novel intact cell MALDI-TOF to be of great promise for fast and reliable detection of MRSA strains.

Journal article

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