Biology subjects

Martinez, J. L.

Publications and source records attributed to Martinez, J. L..

At least 19 recordsLinked to original sources

bioRxiv2026

Exploiting outer-membrane protein promiscuity to induce transient collateral sensitivity via efflux pump competition

In the fight against antimicrobial resistance, the identification of robust collateral sensitivity (CS) patterns could form the basis of successful sequential and combinatorial antimicrobial therapies. While most of the focus has been devoted to study stable CS due to mutations, CS can be transiently induced in Pseudomonas aeruginosa using dequalinium chloride (DC), which increases the expression of the efflux operon mexCD-oprJ. This leads to transient CS to the aminoglycoside tobramycin, whose molecular mechanism remains unclear. Using a combination of experimental results, mechanistic mathematical models and structural simulations, we show that the inactivation of NfxB by DC not only increases mexCD expression but also reduces the effective amount of the MexXY-OprM aminoglycosides efflux pump. Our data suggest that, under conditions of high MexCD production, this efflux pump can outcompete MexXY for the outer-membrane protein OprM, thereby inducing CS to tobramycin. Bayesian fits accurately reproduce measured mexCD expression and minimal inhibitory concentration (MIC) shifts across DC doses. Model predictions were validated in P. aeruginosa PA14 mutants lacking functional outer-membrane channels. Loss of OprJ preserves DC-induced CS to tobramycin, whereas loss of OprM abolishes it. These results identify competition for OprM as the proximal cause of DC-induced, transient CS and provide a generalizable framework for eliciting CS by perturbing shared components of multidrug efflux systems. This strategy suggests immediate avenues to enhance aminoglycoside efficacy while minimizing selection for stable resistance.

microbiology↗

bioRxiv2025

The Antimicrobial Gut Resistome of the Wayampi reveals a shared background of antibiotic and metal resistance genes with industrialised populations, underscoring the robust-yet-fragile architecture of human gut microbiomes.

BackgroundMetagenomics enables detailed profiling of antimicrobial resistance genes, but most studies focus solely on antibiotic resistance genes (ARGs), use low-sensitivity methods, and rarely investigate populations with minimal anthropogenic pollution. We analyzed fecal resistomes of 95 Wayampi individuals, an Indigenous community in remote French Guiana, using a targeted metagenomic capture platform covering 8,667 resistance genes to antibiotics (ARGs), metals (MRGs), and biocides (BRGs) (PMID: 29335005). Resistome profiles were compared with Europeans to assess population-level differences. ResultsARG richness was similar between groups (259 in Wayampi vs. 264 in Europeans, 159 shared), but MRGs + BRGs (BAC) gene richness was signifcantly higher in Wayampi (3,670 vs. 2,039). Most genes appeared in a minority of individuals (mean 5% for ARGs, 2% for BAC), but several ARGs for tetracyclines [tet(32), tet(40), tet(O), tet(Q), tet(W), tet(X), tetAB(P)], aminoglycosides (ant6'-I, aph3-III), macrolides (ermB, ermF, mefA), and sulfonamides (sul2) were present in all individuals. Tetracycline resistance genes predominated overall, while beta-lactam resistance genes were more common in Wayampi, and genes conferring resistance to aminoglycosides, amphenicols, and folate inhibitors were more frequent in Europeans. Among MRGs, copper and arsenic resistance genes prevailed, followed by those for zinc, iron, cobalt, and, nickel. Up to 76% of Wayampiis carried acquired MRGs for copper (pcoABCDRS and tcrB), silver (silACFPRS), arsenic (ars), and mercury (mer) detoxification. Shannon diversity indices were similar for ARGs, MRGs, and BRGs, but composition and evenness differed significantly. UMAP and ADONIS analyses distinguised cohorts by ARG profiles (p < 0.001), but not MRGs or BRGs. Correlation analysis revealed conserved gene-sharing networks and introgression of acquired ARGs and MRGs in both gut microbiomes. ConclusionsThe diverse and balanced Wayampi resistome reflect a less perturbed microbiome compared to industrialized populations, and reveals a background of core and shell acquired ARGs and MRGs, consistent with the "robust-yet-fragile" architecture of scale-free networks. The patchy yet resilient distribution of resistance genes reflects varying levels of conserved gene sharing highways probably shaped by long-term microbial-human evolution, and support a broader view of acquired antimicrobial resistance.

microbiology↗

bioRxiv2025

Single-nucleus analysis reveals dysregulated oxidative phosphorylation in Down syndrome basal forebrain at birth

INTRODUCTIONBasal forebrain cholinergic neurons (BFCNs) are integral to learning, attention, and memory, and are prone to degeneration in Down syndrome (DS), Alzheimers disease, and other neurodegenerative diseases. However, the mechanisms that lead to the degeneration of these neurons are not known. METHODSSingle-nucleus gene expression and ATAC sequencing were performed on postmortem human basal forebrain from unaffected control and DS tissue samples at 0-2 years of age (n=4 each). RESULTSSequencing analysis of postmortem human basal forebrain identifies gene expression differences in DS early in life. Genes encoding proteins associated with energy metabolism pathways, specifically oxidative phosphorylation and glycolysis, and genes encoding antioxidant enzymes are upregulated in DS BFCNs. DISCUSSIONMultiomic analyses reveal that energy metabolism may be disrupted in DS BFCNs by birth. Increased oxidative phosphorylation and the accumulation of reactive oxygen species byproducts may be early contributors to DS BFCN neurodegeneration.

neuroscience↗

bioRxiv2024

A new type of AmpC β-lactamases defined by PIB-1, a metal-dependent carbapenem-hydrolyzing β-lactamase, from Pseudomonas aeruginosa: structural and functional analysis.

Antibiotic resistance is one of most important health concerns nowadays. {beta}-lactamases are the most important resistance determinants. Based on their structural and functional characteristics {beta}-lactamases are grouped in four categories. AmpC {beta}-lactamases are cephalosporinases presenting a set of highly conserved residues. Here we crystallized PIB-1, a Pseudomonas aeruginosa chromosomally-encoded {beta}-lactamase. Its crystal structure shows it is an AmpC {beta}-lactamase, although the number of conserved residues is low. Functional analysis showed that PIB-1 is able to degrade carbapenems but not the typical substrate of AmpC {beta}-lactamases, cephalosporins. Besides, the catalytic activity of PIB-1 increases in the presence of metal ions. Metals do not bind to the active center and increase the degradation of the antibiotic. They induce the formation of trimers. This suggests that the oligomer is more active than the monomer. While PIB-1 is structurally an AmpC {beta}-lactamase, the low sequence conservation, substrate profile and its metal-dependence, prompts us to position this enzyme as the founder of a new group inside the AmpC {beta}-lactamases. Consequently, the diversity of AmpC {beta}-lactamases might be wider than expected.

biochemistry↗

bioRxiv2023

Efflux pump mutations in Pseudomonas aeruginosa cause low-level clinical resistance and high-level tolerance to antibiotics in patients

Pseudomonas aeruginosa is one of the microorganisms with high-risk regarding antimicrobial resistance, since it has an overwhelming capacity to acquire antibiotic resistance, mainly by mutations during persistent lung infections. Mutations in mexZ, encoding the local negative regulator of genes encoding the MexXY efflux pump, are very frequently acquired at early stages of P. aeruginosa infections, while they are rarely selected for in vitro. Although traditionally related to resistance to the first-line drug tobramycin, caused by the overproduction of the aminoglycosides MexXY efflux pump, mutations in mexZ are actually associated with low levels of aminoglycosides resistance when determined in the clinical microbiology laboratory. This very moderate but frequent phenotype suggests that these mutations may shape the infection process, beyond causing conventional resistance. Here we investigated the colonization strategy of a mexZ mutant, compared to a wild-type strain, in a human airway infection model. We observed that the mexZ mutant tends to accumulate inside the epithelial cell layer. This behaviour allows bacteria to colonise the epithelium while being more protected against diverse antibiotics. The altered colonization phenotype was caused by the overexpression of lecA, a Quorum Sensing regulated gene encoding a lectin involved in P. aeruginosa tissue invasiveness. lecA upregulation was underlied by the competition for the shared porin, OprM, between the overproduced MexXY and the MexAB efflux pump, responsible for extruding Quorum Sensing molecules. These findings suggest that standardised antimicrobial susceptibility determined in the clinic may be misleading because antibiotic resistance often depends on the infection environment.

microbiology↗

bioRxiv2023

Rewiring Saccharomyces cerevisiae metabolism for optimised Taxol(R) precursors production

Saccharomyces cerevisiae has been recognised as a convenient host for the production of early precursors to the Taxol(R) anticancer drug. Recent studies have highlighted the harmful impact of oxidative stress as a result of the activity of Taxol(R) first cytochrome P450-reductase enzymes (Taxus spp. CYP725A4-POR). Here, we evolved a new oxidative stress-tolerant yeast strain on galactose, which led to a three-fold higher titre of the CYP725A4 enzyme substrate, taxadiene. We comprehensively analysed the performance of the evolved and parent strain in galactose-limited chemostat cultures before and during oxidative stress induction. Integrating the transcriptomics and metabolite profiling data in an enzyme-constrained genome scale model enabled a more accurate prediction of changes that occurred to biological pathways as a response to/consequence of evolution and oxidative stress. The analyses showed a better performance of the evolved strain with improved respiration and reduced overflow metabolites production. The strain was robust to re-introduction of the oxidative stress, potentially due to the cross-protection mechanism, which contributed to likely better heme, flavin and NADPH availability for an optimal expression of CYP725A4 and POR in yeast. The increased level of taxadiene production has potentially occurred due to the antioxidant properties of taxadiene or as a mechanism to overcome the toxicity of geranylgeranyl diphosphate, the precursor to taxadiene synthase. HighlightsO_LIThe antioxidant properties of taxadiene promotes its production in Saccharomyces cerevisiae C_LIO_LIS. cerevisiae ALE on H2O2 and galactose regulates Flavin, iron and NADPH metabolism as well as carbon and protein recycling pathways through cross-protection and anticipation mechanisms C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/543533v1_fig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1758c64org.highwire.dtl.DTLVardef@543681org.highwire.dtl.DTLVardef@c67339org.highwire.dtl.DTLVardef@b6c56e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Graphical abstract of the study. Figure was created with BioRender.com. C_FIG

synthetic biology↗

bioRxiv2023

Role of Lactate in the Regulation of Transcriptional Activity of Breast Cancer-Related Genes and Epithelial-to-Mesenchymal Transition Proteins: A Comparison of MCF7 and MDA-MB-231 Cancer Cell Lines

The Warburg Effect is characterized by accelerated glycolytic metabolism and lactate production and under fully aerobic conditions is a hallmark of cancer cells. Recently, we have demonstrated the role of endogenous, glucose-derived lactate as an oncometabolite which regulates gene expression in the estrogen receptor positive (ER+) MCF7 cell line cultivated in glucose media. Presently, with the addition of a triple negative breast cancer (TNBC) cell line, MDA-MB-231, we further confirm the effect of lactate on gene expression patterns and extend results to include lactate effects on protein expression. As well, we report effects of lactate on the expression of E-cadherin and vimentin, proteins associated with epithelial-to-mesenchymal transition (EMT). Endogenous lactate regulates the expression of multiple genes involved in carcinogenesis. In MCF7 cells, lactate increased the expression of EGFR, VEGF, HIF-1a, KRAS, MIF, mTOR, PIK3CA, TP53, and CDK4 as well as decreased the expression of ATM, BRCA1, BRCA2, E2F1, MET, MYC, and RAF mainly after 48h of exposure. On the other hand, in the MDA-MB-231 cell line, lactate increased the expressions of PIK3CA, VEGF, EGFR, mTOR, HIF-1, ATM, E2F1, TP53 and decreased the expressions of BRCA1, BRCA2, CDK4, CDK6, MET, MIF, MYC, and RAF after 48h of exposure. In response to endogenous lactate, changes in protein expression of representative genes corroborated changes in mRNA expressions. Finally, lactate exposure decreased E-cadherin protein expression in MCF7 cells and increased vimentin expression in MDA-MB-231 cells. Further-more, by genetically silencing LDHA in MCF7 cells, we show suppression of protein expression of EGFR and HIF-1, while full protein expression occurred under glucose and glucose + exogenous lactate exposure. Hence, endogenous, glucose-derived lactate, and not glucose, elicited changes in gene and protein expression levels. In this study, we demonstrate that endogenous lactate produced under aerobic conditions (Warburg Effect) elicits important changes in gene and protein expression in both ER+ and TNBC cell lines. The widespread regulation of multiple genes by lactate and involves those involved in carcinogenesis including DNA repair, cell growth, proliferation, angiogenesis, and metastasis. Furthermore, lactate affected the expression of two relevant EMT biomarkers, E-cadherin and vimentin, which could contribute to the complex process of EMT and a shift towards a more mesenchymal phenotype in the two cancer cell lines studied.

cancer biology↗

bioRxiv2023

Host space, not energy or symbiont size, constrains feather mite abundance across passerine bird species

Comprehending symbiont abundance among host species is a major ecological endeavour, and the metabolic theory of ecology has been proposed to understand what constraints symbiont populations. We parameterized metabolic theory equations to predict how bird species body size and the body size of their feather mites relate to mite abundance according to four potential energy (microbial abundance, uropygial gland size) and space constraints (wing area, number of feather barbs). Predictions were compared with the empirical scaling of feather mite abundance from 26,604 birds of 106 passerine species, using phylogenetic modelling and quantile regression. Feather mite populations were strongly constrained by host space (number of feather barbs) and not energy. Moreover, feather mite species body size was unrelated to their abundance or to the body size of their host species. We discuss the implications of our results for our understanding of the bird-feather mite system and for symbiont abundance in general.

ecology↗

bioRxiv2022

Improving Therapeutic Protein Secretion in the Probiotic Yeast Saccharomyces boulardii using a Multifactorial Engineering Approach

The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sbs innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e., to the expression cassette of the secreted protein) and trans- (i.e., to the Sb genome) that enhance Sbs ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by 6-fold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 - 463 mg/L. Then, guided by prior knowledge of S. cerevisiaes secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of >10-fold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.

synthetic biology↗

bioRxiv2022

Trivalent NDV-HXP-S vaccine protects against phylogenetically distant SARS-CoV-2 variants of concern in mice

Equitable access to vaccines is necessary to limit the global impact of the coronavirus disease 2019 (COVID-19) pandemic and the emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. In previous studies, we described the development of a low-cost vaccine based on a Newcastle Disease virus (NDV) expressing the prefusion stabilized spike protein from SARS-CoV-2, named NDV-HXP-S. Here, we present the development of next-generation NDV-HXP-S variant vaccines, which express the stabilized spike protein of the Beta, Gamma and Delta variants of concerns (VOC). Combinations of variant vaccines in bivalent, trivalent and tetravalent formulations were tested for immunogenicity and protection in mice. We show that the trivalent preparation, composed of the ancestral Wuhan, Beta and Delta vaccines, substantially increases the levels of protection and of cross-neutralizing antibodies against mismatched, phylogenetically distant variants, including the currently circulating Omicron variant.

immunology↗

bioRxiv2022

High-throughput screening of a Debaryomyces hansenii library for potential candidates with improved stress tolerance and wider carbon utilisation capabilities

Industrial biotechnology comprises the manufacturing of bulk chemicals and high-value end-products from renewable feedstocks, thus it presents a valuable aspect in the present transition from traditional-resource demanding manufacturing to sustainable solutions. The non-conventional yeast Debaryomyces hansenii encompasses halotolerant characteristics that ensures its use in industrial applications, and hence, its industrial importance. For this purpose, a comprehensive and holistic understanding of its behaviour and response to abiotic stresses is essential. Through high-throughput screening methods, using advanced robotics and automation devices, the present study enlightens intraspecies behavioural characteristics of novel D. hansenii strains in response to sodium, as well as their ability to tolerate abiotic stress in semi-controlled micro-fermentations and spot-test studies. A significantly improved performance under those abiotic stresses was observed under the presence of 1M NaCl. Moreover, a positive and summative effect on growth was also found in pH 4 and high salt content. Our results align with previous findings suggesting the halophilic (and not just halotolerant) behaviour of D. hansenii, which is now extensive to all the D. hansenii strains included in this study. Strain-specific differential responses to the presence of sodium were also observed, with some strains exerting a more notable induction by the presence of salt than the standard strain (CBS767). Furthermore, our study provides indications of the use of D. hansenii in industrial bioprocesses based on lignocellulosic biomass and non-lignocellulosic feedstocks.

microbiology↗

bioRxiv2021

Nanoscopic resolution within a single imaging frame

Mean-Shift Super Resolution (MSSR) is a principle based on the Mean Shift theory that extends spatial resolution in fluorescence images, beyond the diffraction limit. MSSR works on low- and high-density fluorophore images, is not limited by the architecture of the detector (EM-CCD, sCMOS, or photomultiplier-based laser scanning systems) and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image series, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other analytical super resolution image approaches. Altogether, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

bioinformatics↗

bioRxiv2021

Evolution under low antibiotic concentrations: a risk for the selection of Pseudomonas aeruginosa multidrug resistant mutants in nature

BACKGROUNDAntibiotic pollution of non-clinical environments might have a relevant impact on human health if resistant pathogens are selected. However, this potential risk is often overlooked, since drug concentrations in nature are usually below their minimal inhibitory concentrations (MICs). Albeit, antibiotic resistant bacteria can be selected even at sub-MIC concentrations, in a range that is dubbed the sub-MIC selective window, which depends on both the antibiotic and the pathogen. OBJECTIVESDetermine the sub-MIC selective windows of seven antibiotics of clinical relevance in the opportunistic pathogen Pseudomonas aeruginosa and evaluate the risk for selecting resistant mutants in nature, based on published data about the amount of antimicrobials detected in natural environments. METHODSWe conducted evolution experiments of P. aeruginosa PA14 in presence of sub-MIC concentrations of ceftazidime, amikacin, levofloxacin, ciprofloxacin, tetracycline, polymyxin B or imipenem, and measured drug susceptibility of the evolved populations. RESULTSSub-MIC selective window of quinolones was the largest, and the ones of polymyxin B and imipenem, the narrowest. Clinically relevant multidrug resistant (MDR) mutants (presenting MICs above EUCAST clinical breakpoints) arose within the sub-MIC selective windows of the majority of antibiotics tested, being these phenotypes probably mediated by efflux pumps' activity. DISCUSSIONOur data show that the concentration of antibiotics reported in aquatic ecosystems -colonizable by P. aeruginosa- are, in occasions, higher than the ones able to select MDR mutants. This finding has implications for understanding the role of different ecosystems and conditions in the emergence of antibiotic resistance from a One-Health point of view. Further, it highlights the importance of delineating the sub-MIC selective windows for drugs of clinical value in pathogens with environmental niches, in order to evaluate the health risks due to antibiotic pollution of natural ecosystems and ultimately tackle antibiotic resistance.

microbiology↗

bioRxiv2020

A wide-ranging Pseudomonas aeruginosa PeptideAtlas build: a useful proteomic resource for a versatile pathogen.

Pseudomonas aeruginosa is an important opportunistic pathogen with high prevalence in nosocomial infections. This microorganism is a good model for understanding biological processes such as the quorum-sensing response, the metabolic integration of virulence, the mechanisms of global regulation of bacterial physiology, and the evolution of antibiotic resistance. Till now, P. aeruginosa proteomic data, although available in several on-line repositories, were dispersed and difficult to access. In the present work, proteomes of the PAO1 strain grown under very different conditions and from diverse cellular compartments have been analyzed and joined to build the Pseudomonas PeptideAtlas. This resource is a comprehensive mass spectrometry-derived peptide and inferred protein database with 71.3% coverage of the total predicted proteome of P. aeruginosa PAO1. This is the highest published coverage among the eight bacterial PeptideAtlas datasets currently available. The proteins in the Pseudomonas PeptideAtlas cover 84% of metabolic proteins, 71% of proteins involved in genetic information processing, 72% of proteins responsible for environmental information processing, more than 80% of proteins related to quorum sensing and biofilm formation, and 81% of proteins responsible for antimicrobial resistance. It exemplifies a necessary tool for targeted proteomics studies, system-wide observations, and cross-species observational studies. Here we describe how this resource was built and some of the physiologically important proteins of this pathogen. SignificancePseudomonas aeruginosa is among the most versatile bacterial pathogens. Studies of its proteome are very important as they can reveal virulence factors and mechanisms of antibiotic resistance. The construction of a proteomic resource such as the PeptideAtlas enables targeted proteomics studies, system-wide observations, and cross-species observational studies.

microbiology↗

bioRxiv2020

The analysis of the role of MexAB-OprM on quorum sensing homeostasis shows that the apparent redundancy of Pseudomonas aeruginosa multidrug efflux pumps allows keeping the robustness and the plasticity of this intercellular signaling network

Multidrug efflux pumps are key determinants for antibiotic resistance. Besides contributing to intrinsic resistance, their overexpression is frequently a cause of the increased resistance acquired during therapy. In addition to their role in resistance to antimicrobials, efflux pumps are ancient and conserved elements with relevant roles in different aspects of the bacterial physiology. It is then conceivable that their overexpression might cause a burden that will be translated into a fitness cost associated with the acquisition of resistance. In the case of Pseudomonas aeruginosa, it has been stated that overexpression of different efflux pumps is linked to the impairment of the quorum sensing (QS) response. Nevertheless, the causes of such impairment are different for each analyzed efflux pump. In this study, we performed an in-depth analysis of the QS-mediated response of a P. aeruginosa antibiotic resistant mutant that overexpresses MexAB-OprM. Although previous work claimed that this efflux pump extrudes the QS signal 3-oxo-C12-HSL, we show otherwise. Our results suggest that the observed attenuation in the QS response when overexpressing this pump is related to a reduced availability of intracellular octanoate, one of the precursors of the biosynthesis of alkyl quinolone QS signals. The overexpression of other P. aeruginosa efflux pumps has been shown to also cause a reduction in intracellular levels of QS signals or their precursors impacting on these signaling mechanisms. However, the molecules involved are distinct for each efflux pump, indicating that they can differentially contribute to the P. aeruginosa quorum sensing homeostasis. ImportanceThe success of bacterial pathogens to cause disease relies on their virulence capabilities as well as in their resistance to antibiotic interventions. In the case of the important nosocomial pathogen Pseudomonas aeruginosa, multidrug efflux pumps participate in the resistance/virulence crosstalk since, besides contributing to antibiotic resistance, they can also modulate the quorum sensing (QS) response. We show that mutants overexpressing the MexAB-OprM efflux pump, present an impaired QS response due to the reduced availability of the QS signal precursor octanoate, not because they extrude, as previously stated, the QS signal 3-oxo-C12-HSL. Together with previous studies, this indicates that, although the consequences of overexpressing efflux pumps are similar (impaired QS response), the mechanisms are different. This apparent redundancy of RND efflux systems can be understood as a P. aeruginosa strategy to keep the robustness of the QS regulatory network and modulate its output in response to different signals.

microbiology↗

bioRxiv2020

A Physiological Characterization in Controlled Bioreactors Reveals a Novel Survival Strategy for Debaryomyces hansenii at High Salinity and Confirms its Halophilic Behavior

Debaryomyces hansenii is traditionally described as a halotolerant non-conventional yeast, being the model organism for the study of osmo- and salt tolerance mechanisms in eukaryotic systems for the past 30 years. However, unravelling of D. hanseniis biotechnological potential has always been difficult due to the persistent limitations in the availability of efficient molecular tools described for this yeast. Additionally, there is a lack of consensus and contradictory information along the recent years that limits a comprehensive understanding of its central carbon metabolism, mainly due to a lack of physiological studies in controlled and monitored environments. Moreover, there is controversy about the diversity in the culture conditions (media composition, temperature and pH among others) used by different groups, which makes it complicated when trying to get significant conclusions and behavioural patterns. In this work, we present for the first time a physiological characterization of D. hansenii in batch cultivations using highly instrumented and controlled lab-scale bioreactors. Our findings contribute to a more complete picture of the central carbon metabolism and the external pH influence on the yeast ability to tolerate high Na+ and K+ concentrations. Finally, the controversial halophilic/halotolerant character of this yeast is further clarified.

microbiology↗

bioRxiv2019

The inactivation of enzymes belonging to the central carbon metabolism, a novel mechanism of developing antibiotic resistance

Fosfomycin is a bactericidal antibiotic, analogous to phosphoenolpyruvate (PEP) that exerts its activity by inhibiting the activity of MurA. This enzyme catalyzes the first step of peptidoglycan biosynthesis, the transfer of enolpyruvate from PEP to uridine-diphosphate-N-acetylglucosamine. Fosfomycin is increasingly used in the last years, mainly for treating infections caused by Gram-negative multidrug resistant bacteria as Stenotrophomonas maltophilia, an opportunistic pathogen characterized by its low susceptibility to antibiotics of common use. The mechanisms of mutational resistance to fosfomycin in S. maltophilia were studied in the current work. None of the mechanisms so far described for other organisms, which include the production of fosfomycin inactivating enzymes, target modification, induction of alternative peptidoglycan biosynthesis pathway and the impaired entrance of the antibiotic, are involved in the acquisition of such resistance by this bacterial species. Rather the unique cause of resistance in the studied mutants is the mutational inactivation of different enzymes belonging to the Embden-Meyerhof-Parnas central metabolism pathway. The amount of intracellular fosfomycin accumulation did not change in any of these mutants showing that neither the inactivation nor the transport of the antibiotic were involved. Transcriptomic analysis also showed that the mutants did not present changes in the expression level of putative alternative peptidoglycan biosynthesis pathway genes neither any related enzyme. Finally, the mutants did not present an increased PEP concentration that might compete with fosfomycin for its binding to MurA. Based on these results, we describe a completely novel mechanism of antibiotic resistance based on the remodeling of S. maltophilia metabolism.\n\nSignificanceAntibiotic resistance (AR) has been largely considered as a specific bacterial response to an antibiotic challenge. Indeed, its study has been mainly concentrated in mechanisms that affect the antibiotics (mutations in transporters, the activity of efflux pumps and antibiotic modifying enzymes) or their targets (i.e.: target mutations, protection or bypass). Usually, AR-associated metabolic changes were considered to be a consequence (fitness costs) and not a cause of AR. Herein, we show that strong alterations in the bacterial metabolism can also be the cause of AR. In the study here presented, Stenotrophomonas maltophilia acquires fosfomycin resistance through the inactivation of glycolytic enzymes belonging to the Embden-Meyerhof-Parnas. Besides resistance to fosfomycin, this inactivation also impairs the bacterial gluconeogenic pathway. Together with previous work showing that AR can be under metabolic control, our results provide evidence that AR is intertwined with the bacterial metabolism.

microbiology↗

bioRxiv2019

Novel inducers of the expression of multidrug efflux pumps that trigger Pseudomonas aeruginosa transient antibiotic resistance

The study of the acquisition of antibiotic resistance (AR) has mainly focused in inherited processes, namely mutations and acquisition of AR genes. However, inducible, non-inheritable AR has received less attention and most information in this field derives from the study of antibiotics as inducers of their associated resistance mechanisms. Less is known about non-antibiotic compounds or situations that can induce AR during infection. Multidrug resistance efflux pumps are a category of AR determinants characterized by the tightly regulation of their expression. Their contribution to acquired AR relies in their overexpression. Herein we analyzed potential inducers of the expression of the chromosomally-encoded Pseudomonas aeruginosa clinically-relevant efflux pumps, MexCD-OprJ and MexAB-OprM. For this purpose, we developed a set of luxCDABE-based P. aeruginosa biosensor strains, which allows the high-throughput analysis of compounds able of modifying the expression of these efflux pumps. Using these strains, we analyzed a set of 240 compounds present in Biolog Phenotype Microarrays. Several inducers of the expression of the genes that encode these efflux pumps were found. The study focused in dequalinium chloride, procaine and atropine, compounds that can be found in clinical settings. Using real-time PCR, we confirmed that these compounds indeed induce the expression of mexCD-oprJ. In addition, P. aeruginosa presents lower susceptibility to ciprofloxacin (a MexCD-OprJ substrate) when dequalinium chloride, procaine or atropine are present. This work emphasizes the need of studying compounds that can trigger transient AR during antibiotic treatment, a phenotype difficult to discover using classical susceptibility tests.

microbiology↗