Biology subjects

Bueno, E.

Publications and source records attributed to Bueno, E..

13 recordsLinked to original sources

bioRxiv2026

Deconstructing a behavioral state: parallel neural integrators control distinct features of an aversive behavioral state in C. elegans

Transient sensory stimuli can trigger long-lasting changes in behavior and cognition. For example, repeated aversive experiences can lead to persistent changes in arousal and mood. Specialized integrator circuits can store information about recent experiences, but how they are embedded within the architecture of the brain to control internal brain states remains poorly understood. We find that C. elegans accumulates evidence of past aversive experiences over minutes to generate a scalable behavioral state consisting of multiple behavioral changes. Using a brain-wide imaging screen, we identified a set of neurons that integrate aversive sensory stimuli over different timescales, from seconds to minutes. Neuronal perturbations show that these integrator neurons are critical for the aversive behavioral state. They control distinct behavioral features, such as changes in locomotion speed or increased responsiveness to subsequent aversive stimuli. Moreover, the integrator neurons function in parallel to one another, utilize different mechanisms to generate persistent neural activity, and signal through different transmitters. Our result show how a behavioral state can be decomposed into distinct behavioral features that map onto different neural integrators that act in parallel. Combined action of the integrators then generates the full set of behaviors that comprise the global state.

neuroscience↗

bioRxiv2024

Neural Sequences Underlying Directed Turning in C. elegans

Complex behaviors like navigation rely on sequenced motor outputs that combine to generate effective movement. The brain-wide organization of the circuits that integrate sensory signals to select and execute appropriate motor sequences is not well understood. Here, we characterize the architecture of neural circuits that control C. elegans olfactory navigation. We identify error-correcting turns during navigation and use whole-brain calcium imaging and cell-specific perturbations to determine their neural underpinnings. These turns occur as motor sequences accompanied by neural sequences, in which defined neurons activate in a stereotyped order during each turn. Distinct neurons in this sequence respond to sensory cues, anticipate upcoming turn directions, and drive movement, linking key features of this sensorimotor behavior across time. The neuromodulator tyramine coordinates these sequential brain dynamics. Our results illustrate how neuromodulation can act on a defined neural architecture to generate sequential patterns of activity that link sensory cues to motor actions.

neuroscience↗

bioRxiv2024

Deep Neural Networks to Register and Annotate the Cells of the C. elegans Nervous System

Aligning and annotating the heterogeneous cell types that make up complex cellular tissues remains a major challenge in the analysis of biomedical imaging data. Here, we present a series of deep neural networks that allow for automatic non-rigid registration and cell identification, developed in the context of freely moving and deforming invertebrate nervous systems. A semi-supervised learning approach was used to train a C. elegans registration network (BrainAlignNet) that aligns pairs of images of the bending C. elegans head with single pixel-level accuracy. When incorporated into an image analysis pipeline, this network can link neurons over time with 99.6% accuracy. This network could also be readily purposed to align neurons from the jellyfish Clytia hemisphaerica, an organism with a vastly different body plan and set of movements. A separate network (AutoCellLabeler) was trained to annotate >100 neuronal cell types in the C. elegans head based on multi-spectral fluorescence of genetic markers. This network labels >100 different cell types per animal with 98% accuracy, exceeding individual human labeler performance by aggregating knowledge across manually labeled datasets. Finally, we trained a third network (CellDiscoveryNet) to perform unsupervised discovery of >100 cell types in the C. elegans nervous system: by comparing multi-spectral imaging data from many animals, it can automatically identify and annotate cell types without using any human labels. The performance of CellDiscoveryNet matched that of trained human labelers. These tools should be immediately useful for a wide range of biological applications and should be straightforward to generalize to many other contexts requiring alignment and annotation of dense heterogeneous cell types in complex tissues.

neuroscience↗

bioRxiv2024

Staphylococcus aureus counters organic acid anion-mediated inhibition of peptidoglycan cross-linking through robust alanine racemase activity

Weak organic acids are commonly found in host niches colonized by bacteria, and they can inhibit bacterial growth as the environment becomes acidic. This inhibition is often attributed to the toxicity resulting from the accumulation of high concentrations of organic anions in the cytosol, which disrupts cellular homeostasis. However, the precise cellular targets that organic anions poison and the mechanisms used to counter organic anion intoxication in bacteria have not been elucidated. Here, we utilize acetic acid, a weak organic acid abundantly found in the gut to investigate its impact on the growth of Staphylococcus aureus. We demonstrate that acetate anions bind to and inhibit D-alanyl-D-alanine ligase (Ddl) activity in S. aureus. Ddl inhibition reduces intracellular D-alanyl-D-alanine (D-Ala-D-Ala) levels, compromising staphylococcal peptidoglycan cross-linking and cell wall integrity. To overcome the effects of acetate-mediated Ddl inhibition, S. aureus maintains a substantial intracellular D-Ala pool through alanine racemase (Alr1) activity and additionally limits the flux of D-Ala to D-glutamate by controlling D-alanine aminotransferase (Dat) activity. Surprisingly, the modus operandi of acetate intoxication in S. aureus is common to multiple biologically relevant weak organic acids indicating that Ddl is a conserved target of small organic anions. These findings suggest that S. aureus may have evolved to maintain high intracellular D-Ala concentrations, partly to counter organic anion intoxication. SignificanceUnder mildly acidic conditions, weak organic acids like acetic acid accumulate to high concentrations within the cytosol as organic anions. However, the physiological consequence of organic anion accumulation is poorly defined. Here we investigate how the acetate anion impacts S. aureus. We show that acetate anions directly bind Ddl and inhibit its activity. The resulting decrease in intracellular D-Ala-D-Ala pools impacts peptidoglycan integrity. Since acetate is a weak inhibitor of Ddl, mechanisms that maintain a high intracellular D-Ala pools are sufficient to counter the effect of acetate-mediated Ddl inhibition in S. aureus.

microbiology↗

bioRxiv2024

Pathogen infection induces sickness behaviors by recruiting neuromodulatory systems linked to stress and satiety in C. elegans

When animals are infected by a pathogen, peripheral sensors of infection signal to the brain to coordinate a set of adaptive behavioral changes known as sickness behaviors. While the pathways that signal from the periphery to the brain have been intensively studied in recent years, how central circuits are reconfigured to elicit sickness behaviors is not well understood. Here we find that neuromodulatory systems linked to stress and satiety are recruited upon infection to drive sickness behaviors in C. elegans. Upon chronic infection by the bacterium Pseudomonas aeruginosa PA14, C. elegans decrease their feeding behavior, then display reversible bouts of quiescence, and eventually die. The ALA neuron and its neuropeptides FLP-7, FLP-24, and NLP-8, which control stress-induced sleep in uninfected animals, promote the PA14-induced feeding reduction. However, the ALA neuropeptide FLP-13 instead acts to delay quiescence and death in infected animals. Cell-specific genetic perturbations show that the neurons that release FLP-13 to delay quiescence in infected animals are distinct from ALA. A brain-wide imaging screen reveals that infection-induced quiescence involves ASI and DAF-7/TGF-beta, which control satiety-induced quiescence in uninfected animals. Our results suggest that a common set of neuromodulators are recruited across different physiological states, acting from distinct neural sources and in distinct combinations to drive state-dependent behaviors.

neuroscience↗

bioRxiv2023

Metabolic reprogramming and flux to cell envelope precursors in a pentose phosphate pathway mutant increases MRSA resistance to β-lactam antibiotics

Central metabolic pathways controls virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to {beta}-lactam antibiotics, particularly in chemically defined media with glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased {beta}-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. Further evidence of the pleiotropic effect of the pgl mutation was reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Reduced binding of wheat germ agglutinin (WGA) to pgl was indicative of lower wall teichoic acid/lipoteichoic acid levels or altered teichoic acid structures. Mutations in the vraFG or graRS loci reversed the increased OX resistance phenotype and restored WGA binding to wild-type levels. VraFG/GraRS was previously implicated in susceptibility to cationic antimicrobial peptides and vancomycin, and these data reveal a broader role for this multienzyme membrane complex in the export of cell envelope precursors or modifying subunits required for resistance to diverse antimicrobial agents. Altogether our study highlights important roles for the PPP and VraFG/GraRS in {beta}-lactam resistance, which will support efforts to identify new drug targets and reintroduce {beta}-lactams in combination with adjuvants or other antibiotics for infections caused by MRSA and other {beta}-lactam resistant pathogens. Author summaryHigh-level resistance to penicillin-type ({beta}-lactam) antibiotics significantly limits the therapeutic options for patients with MRSA infections necessitating the use of newer agents, for which reduced susceptibility has already been described. Here we report for the first time that the central metabolism pentose phosphate pathway controls MRSA resistance to penicillin-type antibiotics. We comprehensively demonstrated that mutation of the PPP gene pgl perturbed metabolism in MRSA leading to increased flux to cell envelope precursors to drive increased antibiotic resistance. Moreover, increased resistance was dependent on the VraRG/GraRS multienzyme membrane complex previously implicated in resistance to antimicrobial peptides and vancomycin. Our data thus provide new insights on MRSA mechanisms of {beta}-lactam resistance, which will support efforts to expand the treatment options for infections caused by this and other antimicrobial resistant pathogens.

microbiology↗

bioRxiv2023

Dissecting the Functional Organization of the C. elegans Serotonergic System at Whole-Brain Scale

Serotonin controls many aspects of animal behavior and cognition. But how serotonin acts on its diverse receptor types in neurons across the brain to modulate global activity and behavior is unknown. Here, we examine how serotonin release from a feeding-responsive neuron in C. elegans alters brain-wide activity to induce foraging behaviors, like slow locomotion and increased feeding. A comprehensive genetic analysis identifies three core serotonin receptors that collectively induce slow locomotion upon serotonin release and three others that interact with them to further modulate this behavior. The core receptors have different functional roles: some induce behavioral responses to sudden increases in serotonin release, whereas others induce responses to persistent release. Whole-brain calcium imaging reveals widespread serotonin-associated brain dynamics, impacting different behavioral networks in different ways. We map out all sites of serotonin receptor expression in the connectome, which, together with synaptic connectivity, helps predict serotonin-associated brain-wide activity changes. These results provide a global view of how serotonin acts at defined sites across a connectome to modulate brain-wide activity and behavior.

neuroscience↗

bioRxiv2022

Brain-wide representations of behavior spanning multiple timescales and states in C. elegans

Changes in an animals behavior and internal state are accompanied by widespread changes in activity across its brain. However, how neurons across the brain encode behavior and how this is impacted by state is poorly understood. We recorded brain-wide activity and the diverse motor programs of freely-moving C. elegans and built probabilistic models that explain how each neuron encodes quantitative features of the animals behavior. By determining the identities of the recorded neurons, we created, for the first time, an atlas of how the defined neuron classes in the C. elegans connectome encode behavior. Many neuron classes have conjunctive representations of multiple behaviors. Moreover, while many neurons encode current motor actions, others encode recent actions. Changes in behavioral state are accompanied by widespread changes in how neurons encode behavior, and we identify these flexible nodes in the connectome. Our results provide a global map of how the cell types across an animals brain encode its behavior.

neuroscience↗

bioRxiv2022

Transient glycolytic complexation of arsenate enhances resistance in the enteropathogen Vibrio cholerae

The ubiquitous presence of toxic arsenate (AsV) in the environment has virtually raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that V. cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress- related systems to counter ROS production caused by intracellular AsV. Characterization of the var mutants suggested these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions.

microbiology↗

bioRxiv2022

Candidate undecaprenyl phosphate translocases enable conditional microbial fitness and pathogenesis

The mechanisms that enable adaptation of peptidoglycan, the structural unit of the bacterial cell wall, to shifting extracellular conditions such as pH remain largely unknown. Here, we identify a DUF368-containing membrane protein in the cholera pathogen Vibrio cholerae that is critical for pathogenesis and alkaline fitness. V. cholerae and Staphylococcus aureus lacking their cognate DUF368-containing protein have pH-dependent cell wall defects consistent with surface accumulation of undecaprenyl phosphate (C55-P), an essential lipid carrier for the biogenesis of peptidoglycan and other key bacterial cell surface polymers. In both species, DUF368-containing proteins exhibit synthetic genetic interactions with putative transporters from the DedA family, suggesting these proteins represent complementary long-sought C55-P translocases that enable envelope maintenance functions critical for microbial fitness within and outside the host. One-Sentence SummaryDUF368-containing and DedA-family proteins are undecaprenyl phosphate transporter candidates and are required for bacterial alkaline fitness and pathogenesis.

microbiology↗

bioRxiv2021

Mutation of lipoprotein processing pathway gene lspA or inhibition of LspA activity by globomycin increases MRSA resistance to -lactam antibiotics

The Staphylococcus aureus cell envelope comprises numerous components, including peptidoglycan (PG), wall teichoic acids (WTA), lipoteichoic acids (LTA), targeted by antimicrobial drugs. MRSA resistance to methicillin is mediated by the mecA-encoded {beta}-lactam-resistant transpeptidase, penicillin binding protein 2a (PBP2a). However, PBP2a-dependent {beta}-lactam resistance is also modulated by the activity of pathways involved in the regulation or biosynthesis of PG, WTA or LTA. Here, we report that mutation of the lipoprotein signal peptidase II gene, lspA, from the lipoprotein processing pathway, significantly increased {beta}-lactam resistance in MRSA. Mutation of lgt, which encodes diacylglycerol transferase (Lgt) responsible for synthesis of the LspA substrate did not impact {beta}-lactam susceptibility. Consistent with previous reports, lgt and lspA mutations impaired growth in chemically defined media, but not in complex broth. MRSA exposure to the LspA inhibitor globomycin also increased {beta}-lactam resistance. Mutation of lgt in an lspA background restored {beta}-lactam resistance to wild type. The lspA mutation had no effect on PBP2a expression, PG composition or autolytic activity indicating a potential role for WTA or LTA. The lspA and lgt mutants exhibited marginally increased resistance to the D-alanine pathway inhibitor D-cycloserine. In addition, mutation of lgt and multicopy lspA expression, but not mutation of lspA, significantly increased susceptibility to the lipoteichoic acid synthase inhibitor Congo red revealing complex interplay between lipoprotein processing mutations and the expression/stability of cell surface glycopolymers. These findings indicate that accumulation of the LspA substrate, diacylglyceryl lipoprotein, increases MRSA resistance to {beta}-lactam antibiotics through impacts on cell envelope components other than PG.

microbiology↗

bioRxiv2020

The spore of the beans: Spatially explicit models predict coffee rust spread in fragmented landscapes

Landscape structure influences the spread of plant pathogens, primarily by affecting pathogen dispersal. Coffee leaf rust (Hemileia vastatrix), a fungal disease that causes heavy economic losses in the coffee industry, is likely to be affected by landscape structure via dispersal of its wind-borne spores. Previous studies have found positive associations between leaf rust incidence and the proportion of pasture cover, suggesting that deforestation may facilitate rust spore dispersal. We explored this idea by modeling the spread of rust transmission in simulated landscapes. Specifically, we modeled within-patch transmission using a probabilistic cellular automata model, and between-patch transmission using a random walk with spore movement inhibited by forest canopy cover. We used this model to understand how the spread of coffee rust is affected by: 1) clustering of coffee plants, 2) clustering of deforestation, and 3) proportion of landscape deforestation. We found that clustering of coffee plants is the primary driver of rust transmission, affecting the likelihood and severity of rust outbreak. Deforestation is important in landscapes with high clustering of coffee: rust outbreaks are more severe in landscapes with a higher proportion of deforested areas, and more variable in landscapes where deforested areas are more evenly dispersed throughout the landscape.

ecology↗

bioRxiv2020

Genetic dissection of the fermentative and respiratory contributions supporting Vibrio cholerae hypoxic growth

Both fermentative and respiratory processes contribute to bacterial metabolic adaptations to low oxygen tension (hypoxia). In the absence of O2 as a respiratory electron sink, many bacteria utilize alternative electron acceptors such as nitrate (NO3-). During canonical NO3- respiration, NO3- is reduced in a stepwise manner to N2 by a dedicated set of reductases. Vibrio cholerae, the etiological agent of cholera, only requires a single periplasmic NO3- reductase (NapA) to undergo NO3- respiration, suggesting that the pathogen possesses a non-canonical NO3- respiratory chain. Here, we used complementary transposon-based screens to identify genetic determinants of general hypoxic growth and NO3- respiration in V. cholerae. We found that while the V. cholerae NO3- respiratory chain is primarily composed of homologues of established NO3- respiratory genes, it also includes components previously unlinked to this process, such as the Na+-NADH dehydrogenase Nqr. The ethanol-generating enzyme AdhE was shown to be the principal fermentative branch required during hypoxic growth in V. cholerae. Relative to single adhE or napA mutant strains, a V. cholerae strain lacking both genes exhibited severely impaired hypoxic growth in vitro and in vivo. Our findings reveal the genetic bases for interactions between disparate energy production pathways that support pathogen fitness in shifting conditions. Such metabolic specializations in V. cholerae and other pathogens are potential targets for antimicrobial interventions. IMPORTANCEBacteria reprogram their metabolism in environments with low oxygen levels (hypoxia). Typically, this occurs via regulation of two major, but largely independent, metabolic pathways-fermentation and respiration. Here, we found that the diarrheal pathogen Vibrio cholerae has a respiratory chain for NO3- that consists largely of components found in other NO3- respiratory systems, but also contains several proteins not previously linked to this process. Both AdhE-dependent fermentation and NO3- respiration were required for efficient pathogen growth in both laboratory conditions and in an animal infection model. These observations provide genetic evidence for fermentative-respiratory interactions and identify metabolic vulnerabilities that may be targetable for new antimicrobial agents in V. cholerae and related pathogens.

microbiology↗