Biology subjects

Larrouy-Maumus, G.

Publications and source records attributed to Larrouy-Maumus, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Colistin resistance-mediated lipopolysaccharide modification in Klebsiella pneumoniae modulates host inflammatory response

Resistance to the polymyxin antimicrobial colistin in Gram-negative bacteria is associated with a modification of the immunogenic lipid A moiety of the lipopolysaccharide (LPS). Chromosomal and plasmid-borne colistin resistance results in the addition of L-Ara4N and pEtN groups to lipopolysaccharide (LPS), respectively. Here, using THP-1 cells, we studied the impact of different LPS modifications of Klebsiella pneumoniae in stimulating host immune response. K. pneumoniae clinical isolates were screened for colistin resistance using broth microdilution (BMD) and the MALDIxin test. LPS was extracted from colistin-resistant isolates and used to stimulate differentiated THP-1 cells. Luminex cytokine assay measured the immune induction via a panel of proinflammatory cytokines. Out of a collection of 72 clinical K. pneumoniae, eight (11.1%) exhibited phenotypic colistin resistance with a minimum inhibitory concentration (MIC) of 8 to 64 mg/L. In total, five isolates possessed genes associated with polymyxin resistance; three isolates had a mutation in the pmrB gene, and two were mcr-8.1 positive. MALDIxin demonstrated that all eight phenotypic colistin-resistant isolates elaborated peaks at m/z 1,955 and m/z 2,193, indicating an L-Ara4N group of LPS modification. For two mcr-8.1 positive isolates, LPS had a pEtN group. The LPS modification positively correlated with colistin MIC (correlation coefficient, r= 0.6 and R2= 0.4). Compared to the native structure, LPS modification was associated with greater production of IL-1{beta}, IL-6, and CXCL-8 (p<0.001). The pEtN-conjugated LPS triggered a significantly greater production of TNF-, IL-6, and CXCL-8 compared to L-Ara4N (p<0.05). This study reveals that the colistin MIC value can significantly predict lipid A modification in clinical K. pneumoniae, and differences in resistance-mediated lipid A modification result in variation in the immunological response. This study highlights the potential of dynamic host-pathogen interaction in the context of colistin resistance.

microbiology↗

bioRxiv2026

CysK2 couples copper sensing to metabolic and redox adaptation in Mycobacterium tuberculosis

Copper is increasingly recognized as a host-derived cue encountered by Mycobacterium tuberculosis and other microbes during infections, yet the magnitude, intracellular distribution, and physiological consequences of this exposure remain incompletely understood. Here, we combined high-resolution imaging, transcriptomic profiling, intracellular reporter assays, isotope tracing, and mouse infection models to define how M. tuberculosis responds to physiologically relevant copper levels during macrophage infection. NanoSIMS analysis showed that copper reaches intracellular bacilli and accumulates in discrete phosphorus-rich foci in bacteria. Exposure to physiological copper concentrations in vitro triggered a highly specific transcriptional response dominated by the copper-inducible CsoR and RicR regulons. The RicR-regulated gene cysK2 gene, encoding the S-sulfocysteine synthase CysK2, was one of the most strongly induced loci. In infected macrophages, cysK2 expression was modulated by extracellular copper availability, host copper transport pathways, and hypoxia. In vitro, an H37Rv cysK2-deficient mutant showed reduced amino acid biosynthesis in response to copper exposure. In vivo, the mutant was impaired in long-term persistence in mice and displayed a higher oxidation status. Together, these findings identify CysK2 as a copper-responsive metabolic effector that couples host-derived copper sensing to redox homeostasis and intracellular adaptation in M. tuberculosis. IMPORTANCESuccessful infection by Mycobacterium tuberculosis depends on its ability to detect and adapt to host-imposed changes in the phagosomal environment. Our work shows that copper contributes to this adaptation not simply by imposing toxicity, but by triggering a metabolic response that helps sustain bacterial fitness within host cells. We identify CysK2 as a key component of this response, linking copper sensing to sulfur metabolism, redox balance, and persistence during infection. These findings shift the view of copper from a purely antimicrobial factor to a host-derived environmental signal that remodels M. tuberculosis physiology, and they uncover a new mechanism by which the pathogen maintains intracellular survival.

microbiology↗

bioRxiv2026

Significant increase in root exudation of 2'-deoxymugineic acid (DMA) as a response to zinc deficiency in rice

1O_LIZinc (Zn) deficiency limits rice productivity and poses a risk to human health, particularly in populations reliant on rice-based diets. Although rice germplasm exhibits wide variation in Zn-deficiency tolerance, the underlying physiological mechanisms remain poorly resolved. Evidence across the literature for Zn-deficiency-induced secretion of 2'-deoxymugineic acid (DMA) is inconsistent. This study clarifies the role of DMA secretion as a Zn-deficiency stress response. C_LIO_LIWe developed and validated a sensitive LC-ESI-Q-TOF-MS method for selective detection of DMA in rice root exudates. Five rice genotypes with contrasting Zn-deficiency tolerance were grown hydroponically and DMA secretion measured. C_LIO_LIZn-deficiency increased DMA exudation across all genotypes, with sensitive genotypes also showing higher secretion compared with control, supporting DMAs role as a general response to Zn stress rather than being restricted to efficient genotypes. C_LIO_LIFold-change responses exceeded previous studies, likely due to more severe stress exposure. Our results confirm that DMA secretion is induced under Zn-deficiency in rice as part of the micronutrient stress response. However, the lack of increased Zn uptake indicates that additional tolerance mechanisms are involved. These findings reconcile inconsistencies in the literature and position DMA secretion as an important, but not exclusive, component of Zn-deficiency adaptation in rice. C_LI

plant biology↗

bioRxiv2026

Menaquinone depletion resensitises bedaquiline-resistant tuberculosis

Tuberculosis remains a leading cause of global mortality, and rising bedaquiline resistance threatens the effectiveness of current drug-resistant treatment regimens. Bedaquiline resistance typically arises through mutations in Rv0678 that upregulate drug efflux and confer cross-resistance to multiple drug classes. Here, we identify and optimise a chemical series targeting MenG, a central enzyme in the menaquinone biosynthesis pathway, yielding potent bactericidal inhibitors with in vivo efficacy. Strikingly, MenG inhibition restored bedaquiline susceptibility in efflux-mediated resistant strains, an effect confirmed in vivo where combination therapy achieved a 99.8% reduction in bacterial burden compared with bedaquiline alone. Potentiation also extended to pretomanid and other key agents. Disruption of upstream menaquinone and shikimate pathway enzymes produced similar resensitisation, establishing these pathways as tractable targets for restoring drug susceptibility in Mycobacterium tuberculosis. These findings provide a novel strategy to overcome bedaquiline resistance and strengthen future regimens for efflux-mediated drug-resistant tuberculosis.

microbiology↗

bioRxiv2025

Rv3400 is a phosphoglucomutase required for trehalose metabolism in Mycobacterium tuberculosis

Mycobacterium tuberculosis is a global killer causing over a million deaths from tuberculosis (TB) every year. It is therefore a major burden on human health. To reduce the deadly impact of TB, we need a better understanding of the strategies used by M. tuberculosis to adapt its metabolism to survive and persist in the human host. This will help us design better strategies for TB treatment and control. Previous enzymological studies have reported the Mtb rv3400 gene as encoding a {beta}-phosphoglucomutase; however, its role in M. tuberculosis metabolism was not investigated. In the present study, we show that deletion of rv3400 leads to a 30-fold increase in {beta}-D-glucose 1-phosphate, confirming its primary function as a {beta}-phosphoglucomutase. Additionally, deletion of rv3400 leads to a growth defect when trehalose is the sole carbon source indicting that this enzyme is required for optimal use of trehalose, an essential disaccharide in mycobacteria. ImportanceTrehalose metabolism plays a corner stone in Mycobacterium tuberculosis physiology and virulence. Hence, a better understanding of the metabolism of this essential disaccharide is required to develop novel strategies to eradicate Tuberculosis. Here we report on the characterisation of the strain lacking a {beta}-phosphoglucomutase, encoded by the gene rv3400. We show that deletion of rv3400 leads to an increase of {beta}-D-glucose 1-phosphate and a growth defect when using trehalose as sole carbon source. Taken together, the data presented here provide evidence that Rv3400 is required for the catabolism of trehalose.

microbiology↗

bioRxiv2025

Pleiotropic functions of glutathione in the adaptive response to long-term nitrogen starvation in Escherichia coli

Nitrogen (N) is essential for bacterial growth, and adaptation to N starvation involves extensive reprogramming of gene expression. A hallmark subcellular feature in long-term N starved Escherichia coli cells is the presence of biomolecular condensates of the major bacterial RNA regulator Hfq. The Hfq condensates, which accumulate gradually during N starvation, contribute to adaptation by modulating RNA metabolism and central metabolic pathways. Metabolites play central roles in stress responses, often acting as modulators of protein function to support survival and recovery. Glutathione (GSH), a universal stress protectant, has broad roles in bacterial stress adaptation, yet its function during N starvation remains unexplored. Using a GSH-deficient mutant ({Delta}gshAB), we show that GSH is required for optimal survival and recovery from prolonged N starvation. We reveal that GSH regulates the temporal dynamics of Hfq condensation and dissipation during starvation and recovery, respectively, via an as-yet unknown mechanism. Notably, these two functions of GSH appear mutually exclusive, highlighting its pleiotropic role in the adaptive response to N starvation that potentially extends its canonical function as a stress protectant. IMPORTANCENitrogen is a vital nutrient that bacteria need to grow. When nitrogen becomes scarce, bacteria must quickly adjust how their genes are used to survive. In Escherichia coli, one of the key changes during long-term nitrogen starvation is the formation of tiny structures inside the cell called Hfq condensates, which help manage genetic information flow and metabolism. Small molecules called metabolites play important roles in helping bacteria cope with stress, and one such molecule, glutathione (GSH), is known to protect cells under various stress conditions. However, its role during nitrogen starvation is not known. In this study, we used a mutant strain of E. coli that cannot produce GSH and found that these bacteria struggle to survive and recover from nitrogen starvation. We also discovered that GSH helps control when and how Hfq condensates form and disappear. Although, these two functions of GSH seem to be unrelated, our study highlights the versatile role of GSH in helping bacteria adapt to nitrogen stress.

microbiology↗

bioRxiv2025

VadK, a Non-Canonical Kinase that Regulates the Methylcitrate Cycle and is Essential for Mycobacterium tuberculosis Virulence

The evolution of new enzymatic functions is constrained and guided by the architecture of an organisms metabolic and regulatory networks as well as by environmental constraints. Here, we identify a previously uncharacterized kinase that has evolved from pyruvate phosphate dikinase (PPDK). Through biochemical and systems-level analyses, we show that this enzyme, encoded by Rv1127c in Mycobacterium tuberculosis (Mtb), has diverged from its ancestral role in central carbon metabolism to function as a histidine kinase in pathogenic mycobacteria and related species. We designate this enzyme Virulence Associated DiKinase (VadK), reflecting its ability to autophosphorylate and role in coordinating metabolism and virulence. VadK is essential for the utilization of exogenous carbon sources critical for survival within the host and is required for Mtb pathogenicity in murine models of tuberculosis. Furthermore, VadK interacts with key enzymes of the methylcitrate cycle, and 13C-metabolic flux analysis indicates that it fine-tunes flux through this pathway, with elevated flux proving growth limiting. Together, these findings identify VadK as a previously unrecognized regulatory kinase that integrates metabolic control with virulence in Mtb, revealing a new facet of metabolic regulation in bacterial pathogenesis and a potential target for therapeutic intervention.

microbiology↗

bioRxiv2025

Polymyxin heteroresistance in Klebsiella oxytoca

Antibiotic heteroresistance presents a growing public health concern since the phenotype is associated with treatment failure and is hard to detect using conventional diagnostic testing. In this study, we characterized polymyxin B heteroresistance in a collection of clinical and environmental isolates of Klebsiella oxytoca, an opportunistic pathogen associated with hospital-acquired infections. All six of the isolates tested exhibited heteroresistance, indicated by a 16-fold difference between the highest non-inhibitory concentration (2 {micro}g/mL) and the minimum inhibitory concentration (32-64 {micro}g/mL) observed in population analysis profile (PAP) assays. Heteroresistance was found to be due to the presence of a stable sub-population of resistant bacteria and was unaffected by growth phase or the presence of host antimicrobial factors present in human serum. MALDI-TOF analysis revealed L-Ara4N modifications of the lipid A of resistant sub-populations. This pilot study identifies polymyxin heteroresistance in K. oxytoca may complicate treatment of infections caused by this organism.

microbiology↗

bioRxiv2025

RfaH is Essential for Virulence and Adaptive Responses in Yersinia pseudotuberculosis Infection

We previously demonstrated that increased expression of the gene encoding transcriptional antiterminator RfaH during Yersinia pseudotuberculosis transcriptional reprogramming necessary for adapting to persistent infection. RfaH is known to regulate expression of the O-antigen biosynthesis operon in Y. pseudotuberculosis. In this study, we examined the role of RfaH in virulence, bacterial physiology under infection-relevant stress conditions, and determined the RfaH regulon in Y. pseudotuberculosis. We employed a mouse infection model and phenotypic assays to test RfaHs role in virulence and physiology, as well as RNA sequencing, including O-antigen biosynthesis-deficient strains. Our findings demonstrate that loss of rfaH significantly attenuates virulence, reducing the capacity of Y. pseudotuberculosis to establish persistent infection. RfaH expression is increased during the stationary growth phase and under various stress conditions, such as high osmolarity and temperature. Functional assays revealed that the{Delta} rfaH strain displayed defects in swimming and increased clumping, indicating altered surface properties affecting motility. Transcriptomic analysis showed that the absence of rfaH led to downregulation of genes involved in virulence besides O-antigen biosynthesis operon, suggesting RfaHs critical role in virulence and host adaptation. Notably, we identified a hypothetical non-coding RNA encoded within the 5-UTR of the O-antigen biosynthesis operon, which may regulate gene expression of the urease operon in Y. pseudotuberculosis. Collectively, our findings suggest that RfaH is essential for the virulence and adaptive capacity of Y. pseudotuberculosis to colonize the host. This study provides insights into regulatory mechanisms that facilitate bacterial survival in hostile environments and highlights the importance of RfaH and its regulatory targets in the pathogenesis of Y. pseudotuberculosis. Author SummaryFor bacterial pathogens to establish infection and persist in the host, they must adapt to harsh environments and fine-tune gene expression accordingly. The transcriptional antiterminator RfaH plays a pivotal role in regulating key genes essential for adaptation and virulence in Y. pseudotuberculosis. In this study, we explored the function of RfaH in bacterial physiology, stress responses, and infection dynamics. Using a mouse infection model, we found that loss of RfaH significantly reduced virulence and impaired the pathogens ability to establish persistent infection. Notably, RfaH expression increased under stress conditions, such as high osmolarity and temperature, underscoring its role in bacterial adaptation. On the other hand, the absence of RfaH led to motility defects and enhanced bacterial aggregation, suggesting alterations in surface properties. Transcriptomic analysis revealed that RfaH influences a broader set of genes beyond the O-antigen biosynthesis operon, including virulence factors critical for host adaptation. Additionally, we identified a potential non-coding RNA within the 5'-UTR of the O-antigen biosynthesis operon, which may regulate urease operon. Overall, our findings establish RfaH as a key regulator of Y. pseudotuberculosis virulence, shedding light on the molecular mechanisms that enable bacterial survival in challenging environments.

microbiology↗

bioRxiv2024

Anemia and tissue hypoxia are major determinants of malarial hypelactatemia

Hyperlactatemia, a key marker of severe malaria, is closely linked to increased mortality, though the exact mechanisms remain unclear. It may result from increased lactate production due to tissue hypoxia or reduced lactate clearance from organ dysfunction. This study used Plasmodium yoelii 17XL (Py17XL) murine model of severe malaria, which closely mimics hyperlactatemia seen in human cases, to investigate the contributions of severe anemia and infection-related organ dysfunction to hyperlactatemia. Non-infectious anemia models were also included for comparison. Anemia was found to elevate lactate in both malaria-infected and non-infectious models, but Py17XL infected mice showed higher lactate levels, indicating that anemia alone doesnt fully explain hyperlactatemia. Evidence of tissue hypoxia, particularly in the liver, kidney, and gut, was seen with hypoxyprobe staining and upregulated hypoxia-inducible factor 1-alpha (HIF-1), suggesting that hypoxia drives increased glycolysis and lactate production. Impaired lactate clearance may also play a role, as infected mice showed signs of liver and kidney dysfunction accompanied by reduced clearance of 13C3-labeled sodium-L-Lactate. Whole blood transfusion combined with artesunate significantly improved lactate clearance compared to artesunate alone, underscoring the importance of addressing anemia in treatment. A link between intestinal damage and hyperlactatemia was suggested by correlations between trefoil factor 3 (TFF3), a marker of gut injury, and lactate levels in human samples. Our findings highlight the multifactorial origin of hyperlactatemia in malaria, driven primarily by anemia and tissue hypoxia, pointing to the need for therapies targeting both aspects to reduce mortality in severe cases.

pathology↗

bioRxiv2024

Degradation of PET plastic with engineered environmental bacteria

Polyethylene terephthalate (PET) is one of the most widely used plastic materials in the food and textile industry. Consequently, post-consumer PET waste is a common environmental pollutant that leaks into the environment in the form of macro and microplastics with concerning health impacts. There is a pressing need to identify novel and sustainable solutions to process the abundance of PET waste contributing to this pollution. While there is extensive research into enzymes able to hydrolyse PET in vitro, a similar solution for discarded or difficult-to-collect PET based on whole-cell microbial catalysts is missing. In this work we report the engineering of environmental bacteria to use PET as a growth substrate. This was achieved by isolating a strain of Pseudomonas umsongensis able to use the PET monomer terephthalate as carbon source, engineering the strain to effectively secrete the high-activity PET hydrolase PHL7 through the addition of a recombinant TAT secretion leader sequence, and enhancing the bioavailability of PET by transforming it into an amorphous and macroporous structure by pre-treatment with an organic solvent. Our findings demonstrate the direct microbial consumption of PET, which could lead to improved and more sustainable upcycling strategies for this plastic.

synthetic biology↗

bioRxiv2023

ATP burst is the dominant driver of antibiotic lethality in Mycobacteria

AbstractAntibiotic-tolerant bacteria, due to their unique physiology, are refractory to antimicrobial killing and pose challenges for infection control. Incomplete knowledge of how bactericidal antibiotics work, limits our understanding of partial resistance due to phenotypic tolerance in mycobacteria, a driver for developing genetic resistance. Using proteomics, 13C isotopomer analysis, genetic and biochemical assays, we investigated the physiological response of M. smegmatis challenged with aminoglycoside and fluoroquinolone antibiotics. Two distinct classes of antibiotics elicited remarkably similar responses and increased flux through the TCA cycle, causing enhanced respiration, ROS generation, and ATP burst. We observed that excessive ATP levels and not ROS, dominantly contributes to cidality, which may in part be, conferred by sequestration of divalent metal ions by ATP. Consequently, 13C isotope tracing indicated TCA cycle flux deviation from its oxidative arm as a bacterial adaptive mechanism, which also included activated intrinsic resistance and a higher propensity to develop antibiotic resistance. Our study provides a new understanding of the intricate mechanisms of antibiotic-induced cell death and expands the current paradigm for antibiotic action.

microbiology↗

bioRxiv2023

Antimicrobial peptide glatiramer acetate targets Pseudomonas aeruginosa lipopolysaccharides to breach membranes without altering lipopolysaccharide modification

Antimicrobial peptides (AMPs) are key components of innate immunity across all kingdoms of life. Both natural and synthetic AMPs are receiving renewed attention in the efforts to combat the antimicrobial resistance (AMR) crisis and the loss of antibiotic efficacy. The gram-negative pathogen Pseudomonas aeruginosa is one of the most concerning infectious bacteria in AMR, particularly in people with cystic fibrosis (CF) where respiratory infections are difficult to eradicate and are associated with increased morbidity and mortality. Cationic AMPs exploit the negative charge of lipopolysaccharides (LPS) on P. aeruginosa to bind to and disrupt the bacterial membrane(s) and cause lethal damage. P. aeruginosa modifies its LPS, via environmental or genetic factors, to neutralise the charge of the cell and evade AMP killing. Free-LPS is also a component of CF sputum, as is anionic extracellular DNA (eDNA), each of which can bind AMPs by electrostatic interaction. Both free LPS and eDNA also feed into pro-inflammatory cycles. Glatiramer acetate (GA) is a random peptide co-polymer of glycine, lysine, alanine, and tyrosine and used as drug in the treatment of multiple sclerosis (MS); we have previously shown GA to be an AMP which synergises with tobramycin against P. aeruginosa from CF, functioning via bacterial membrane disruption. Here, we demonstrate direct binding and sequestration/neutralisation of P. aeruginosa LPS in keeping with GAs ability to disrupt the outer membrane. Binding and neutralisation of eDNA was also seen. At CF-relevant concentrations, however, neither strongly inhibited membrane disruption by GA. Furthermore, in both type strains and clinical CF isolates of P. aeruginosa, exposure to GA did not result in increased modification of the Lipid A portion of LPS or in increased expression of genetically encoded systems involved in AMP sensing and LPS modification. With this low selective pressure on P. aeruginosa for known AMP resistance mechanisms, the potential to neutralise pro-inflammatory CF sputum components, as well as the previously described enhancement of antibiotic function, GA is a promising candidate for drug repurposing.

microbiology↗

bioRxiv2023

Comparative genomics of carbapenemase-producing Morganella spp.

BackgroundMorganella are opportunistic pathogens involved in various infections. In Morganella, intrinsic resistance to multiple antibiotics including colistin combined with the emergence of carbapenemase-producers (CP) strongly limits the antimicrobial armamentarium. MethodsFrom 2013 to 2021, 172 highly drug-resistant (XDR) Morganella isolates from 8 European countries and Canada, two reference strains from the Pasteur Institute collection and two susceptible isolates were characterized by WGS, antimicrobial susceptibility testing and biochemical tests. Complete genomes from Genbank (n=103) were included for genomic analysis. Intrinsic resistance mechanism to polymyxins was deciphered by combining genetic analysis with mass spectrometry on the lipid A. FindingsMorganella could be separated into 4 species named M. psychrotolerans, M. sibonii, M. morganii and a new species represented by a unique strain. Morganella morganii included two subspecies: M. morganii subsp. morganii (the most prevalent) and M. morganii subsp. intermedius. Intrinsic resistance to tetracycline and conservation of metabolic pathways correlated this refined taxonomy. CP were mostly identified among five high-risk clones of M. morganii subsp. morganii. A single nucleotide polymorphism (SNP) cut-off of 100 was used to decipher outbreaks involving this species. Cefepime-zidebactam and ceftazidime-avibactam were the most potent antimicrobials towards the 172 XDR Morganella spp. isolates of our collection (including 145 CP) except for metallo-{beta}-lactamase-producers. The intrinsic resistance to polymyxins corresponds to the addition of L-Ara4N on the lipid A. InterpretationThis global characterization of the widest collection of XDR Morganella spp. highlighted the need to clarify the taxonomy, deciphered intrinsic resistance mechanisms and paved the way for further genomic comparisons. FundingNone RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSOn January 28th 2022, we have searched for the terms "Morganella" and "carbapenemase" in all published reports available in PubMed with no language restriction. We identified a total of 43 articles and most of them (41/43) corresponded to a report of a single isolate of carbapenemase-producing Morganella morganii. Only one article aimed to decipher the antimicrobial susceptibility on a collection of Proteus, Providencia and Morganella isolated from global hospitalized patients with intra-abdominal and urinary tract infections. However, this collection only included 7 M. morganii isolates. On March 2021, when we finished the inclusions in our collection, only 104 genomes of Morganella spp. were available in the NCBI database. Since September 2021, very few reports were published on carbapenemase-producing Morganella with the exception of a study from Xiang G et al. reported 40 multi-drug resistant M. morganii isolates recovered from three hospitals in China from 2014 to 2020. Unfortunately, this collection included only two carbapenemase-producing M. morganii isolates (one OXA-48 and one IMP-1). A report of KPC-producing M. morganii in Japan and a longitudinal study of carbapemase-producing Enterobactrales in Taiwan that did not focused on Morganella. We also searched in PubMed for the terms Morganella sibonii" or "Morganella psychrotolerans" in all published reports with no language restrictions. Our search identified a total of 20 articles. None of them was related to antimicrobial resistance and no study deciphered the Morganella spp. epidemiology on clinical isolates. Added values of this studyThis global characterization involved the widest collection of Morganella spp. isolates ever reported (barely doubling the number of Morganella spp. genomes in Genbank). In addition, 145 isolates of this worldwide collection made of 172 multidrug resistant Morganella spp. were carbapenemase producers for which therapeutic alternatives are scarce due to intrinsic resistance to last resort molecules, such as polymyxin. First, we found that cefepime-zidebactam and ceftazidime-avibactam were the most potent antimicrobials towards XDR Morganella spp. isolates except for metallo-{beta}-lactamase-producers. Then, we observed that carbapenemase-encoding genes were present in different Morganella species highlighting necessary changes in the taxonomy. Morganella genus could be divided into 4 species named M. psychrotolerans, M. sibonii, M. morganii and a new species represented by a unique strain. Morganella morganii includes two subspecies: M. morganii subsp. morganii (the most prevalent) and M. morganii subsp. intermedius. We demonstrated that this refined taxonomy correlated with the intrinsic resistance to tetracycline, which was found only in M. sibonii, as well as several metabolic pathways (e.g. trehalose assimilation, type III (T3SS) and type IV secretion system (T6SS), etc....). In addition, we highlighted five "high-risk" clones of carbapenemase-producing M. morganii subsp. morganii that have already disseminated worldwide. Combining whole genome sequencing (WGS) data with epidemiological investigations, we demonstrated that a cut-off of 100 single nucleotide polymorphisms (SNPs) could be used to discriminate clonally-related from sporadic independent isolates. This information is of the utmost importance since WGS is now considered as the reference method to identify and follow outbreaks. The intrinsic resistance of Morganella spp. to polymyxins was well-known but the underlying mechanism was unclear. Here, we demonstrated that the addition of L-Ara4N on the lipid A of Morganella is involved. Implications of all the available evidenceThe identification of "high-risk" clones among highly-drug resistant Morganella spp. paves the way of future investigations to better understand and hopefully limit the spread of these bugs. Additionally, our results identified new components and virulence factors of some Morganella species (e.g. T6SS and T3SS in M. sibonii) that deserve further investigation since they might be implicated in the bacterial lifestyle of this genus.

microbiology↗

bioRxiv2022

A serine-folate metabolic unit controls resistance and tolerance of infection

Immune activation drives metabolic change in most animals. Immune-induced metabolic change is most conspicuous as a driver of pathology in serious or prolonged infection, but it is normally expected to be important to support immune function and recovery. Many of the signalling mechanisms linking immune detection with metabolic regulation, and their specific consequences, are unknown. Here, we show that Drosophila melanogaster respond to many bacterial infections by altering expression of genes of the folate cycle and associated enzymes of amino acid metabolism. The net result of these changes is increased flow of carbon from glycolysis into serine and glycine synthesis and a shift of folate cycle activity from the cytosol into the mitochondrion. Immune-induced transcriptional induction of astray and Nmdmc, the two most-induced of these enzymes, depends on Dif and foxo. Loss of astray or Nmdmc results in infection-specific immune defects. Our work thus shows a key mechanism that connects immune-induced changes in metabolic signalling with the serine-folate metabolic unit to result in changed immune function.

immunology↗

bioRxiv2022

A universal stress protein acts as a metabolic rheostat controlling carbon flux in mycobacteria

Universal stress proteins (USPs) are ubiquitous amongst prokaryotes and have empirically determined roles in adaptation to stress, while their specific biochemical function in cellular processes is opaque. The most frequently encountered USP is a small ~15kDa protein comprised of a single domain (PF00582). In Mycobacterium tuberculosis Rv1636 is the sole single domain protein amongst its 10 USPs. We were unable to delete rv1636 from M. tuberculosis, supporting previous data that it is essential for growth. We deleted its orthologue, MS3811, from M. smegmatis, and in exponential phase competition experiments we observed that the advantage switched between wildtype and the mutant depending on the growth environment. In rich medium the wildtype had the competitive advantage, but in a defined medium with a single carbon source the competitive advantage switched, with the mutant rapidly taking over the culture. We hypothesised the USP is regulating metabolic flux, with its deletion leading to increased flux and more rapid turn-over of central-carbon metabolites. We tested this by performing 13C-stable isotope tracing, using [U-13C6] glucose as sole carbon source for growth of the parental and deletion strains. The findings were unequivocal; deletion of the USP led to an increase in label incorporation in central carbon metabolites and a profound change in the isotopologue distribution. Furthermore, in vivo protein-crosslinking provided evidence that Rv1636 interacts directly with key central metabolic enzymes, including the glycolytic enzymes pyruvate kinase, pyruvate dehydrogenase, pyruvate synthase. We propose the mycobacterial single domain USP acts as a metabolic rheostat to regulate central carbon metabolism. ImportanceThere are more than 62,000 universal stress proteins (USPs) found mostly in prokaryotes, but very few examples where the role of a USP is unequivocally linked to a specific biological or biochemical function. For the first time we were able to assign a role in metabolic regulation to a bacterial single domain universal stress protein. We demonstrate a mycobacterial USP regulates flux through central catabolic pathways, and our evidence, based on 13C-stable isotope tracing, indicates that the USP acts as a brake on carbon flux, and we assign to it a role as a metabolic rheostat.

microbiology↗

bioRxiv2021

Lipid mediated inhibition of Niemann-Pick C1 protein is an evolutionary conserved feature of multiple Mycobacterium lineages and non-tubercular mycobacteria.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and is a major cause of human morbidity and mortality. Crucially, Mtb can persist and replicate within host macrophages (M[FE]) and subvert multiple antimicrobial defense mechanisms. How this is achieved is incompletely understood. Previously, we reported that lipids shed by persistent mycobacteria inhibit NPC1, the lysosomal protein deficient in most cases of the rare, inherited lysosomal storage disorder Niemann-Pick disease type C (NPC). Inhibition of NPC1 leads to a drop in lysosomal calcium levels blocking phagosome-lysosome fusion and thereby leads to mycobacterial persistence. Studies of mycobacterial lineages have identified events during mycobacterial evolution that result in the acquisition of persistence. We speculated that the production of specific cell wall lipid(s) capable of inhibiting NPC1 activities could have been a critical step in the evolution of pathogenicity. In this study, we have therefore investigated whether lipid extracts from clinical Mtb strains representative of multiple Mtb lineages, members of the Mtb complex (MTBC) and selected non-tubercular mycobacteria (NTM) inhibit the NPC pathway. We have found that the ability to inhibit the NPC pathway was present in all clinical isolates studied from Mtb lineages 1, 2 and 4. We also found that lipids from MTBC member, Mycobacterium bovis and the NTM, Mycobacterium abscessus and Mycobacterium avium also inhibited the NPC pathway. However, when lipids were assayed from Mycobacterium canettii (M. canettii), a smooth tubercle mycobacterium, which is considered to resemble the common ancestor of the MTBC no inhibition of the NPC1 pathway was detected. We therefore conclude that the evolution of mycobacterial cell wall lipids that inhibit the NPC pathway evolved early and post divergence from M. canettii related mycobacteria and NPC1 inhibition significantly contributes to the ability of these pathogens to persist and cause disease. Authors summaryMycobacterium tuberculosis (Mtb) actively modifies the hostile intracellular environment of host cells to create a niche in which it can persist and replicate. How this is accomplished is incompletely understood. We previously reported an unexpected phenotypic similarity between cells infected with pathogenic mycobacteria and those of the rare, neurodegenerative lysosomal storage disorder, Niemann-Pick disease type C (NPC). Mechanistically, we showed that pathogenic mycobacteria shed lipids that inhibit NCP1, the lysosomal membrane protein that is dysfunctional in NPC and thereby establish conditions that favour their survival. In this study, we have investigated the phylogenetic distribution of lipids that inhibit NPC1. We have screened lipid extracts from multiple clinical mycobacterial isolates representing different Mtb lineages, other members of the Mtb complex and non-tuberculous mycobacteria for their ability to induce NPC phenotypes. We detected activity in all of the groups tested, but not from Mycobacterium canettii, which is believed to resemble the ancestral species. These data indicate that targeting of NPC1 to achieve intracellular persistence evolved early in the evolution of the Mtb complex, but after divergence from the presumed ancestral species.

microbiology↗

bioRxiv2021

Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding

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 {beta}-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. IMPACT STATEMENTDisruption of disulfide bond formation sensitizes resistant Gram- negative bacteria expressing {beta}-lactamases and mobile colistin resistance enzymes to currently available antibiotics.

microbiology↗