Biology subjects

Cesar, S.

Publications and source records attributed to Cesar, S..

5 recordsLinked to original sources

bioRxiv2025

CRISPR/Cas9-mediated elimination of the LMNA c.745C>G pathogenic mutation enhances survival and cardiac function in LMNA-associated congenital muscular dystrophy

LMNA-associated congenital muscular dystrophy is a currently incurable rare genetic disorder characterized by early-onset muscle weakness, dilated cardiomyopathy and respiratory failure, resulting from mutations in the LMNA gene. In this study, we assessed the potential of a CRISPR-mediated strategy to eliminate the mutant allele Lmna c.745C>T, p.R249W using a mutation specific guide (sg745T). Results from R249W-mutation-carrying cellular models showed specific activity of the Cas9/sg745T complex towards the mutant allele. This property varied depending on the concentration of CRISPR components, with a loss of specificity observed with increased dosage. We tested this strategy in vivo using adeno-associated virus delivery in LmnaR249W mice. Despite being associated with a modest CRISPR activity, this therapeutic approach resulted in a 10% increase in the survival of R249W homozygous mice. Interestingly, a similar CRISPR activity improved the cardiac pathology developed by Lmna+/R249W animals, significantly extending their median survival. These results represent the first therapeutic validation of a CRISPR/Cas9-mediated gene editing strategy for the treatment of LMNA-associated congenital muscular dystrophy.

bioengineering↗

bioRxiv2020

Starvation induces shrinkage of the bacterial cytoplasm

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. colis inner membrane from the cell wall. Shrinkage was accompanied by a [~]17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon-source quality. Robust recovery from starvation required the Tol-Pal system, highlighting the importance of envelope coupling during recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic. Significance statementBacterial cells constantly face nutrient fluctuations in their natural environments. While previous studies have identified gene expression changes upon nutrient depletion, it is much less well known how cellular morphology and cytoplasmic properties respond to shifts in nutrient availability. Here, we discovered that switching fast-growing Escherichia coli cells to nutrient-free conditions results in substantial shrinkage of the inner membrane away from the cell wall, especially at the new pole. Shrinkage was primarily driven by loss of cytoplasmic water contents. Shrinkage was also exhibited by cells naturally entering stationary phase, highlighting its biological relevance across starvation conditions. The membrane-spanning Tol-Pal system was critical for robust entry into and recovery from shrinkage, indicating the importance of cell-envelope homeostasis in surviving nutrient starvation.

microbiology↗

bioRxiv2020

Bacterial respiration during stationary phase induces intracellular damage that leads to dormancy

Most bacteria frequently encounter nutrient-depleted conditions, necessitating regulatory mechanisms that alter cellular physiology and allow for survival of starvation. Here, we show that regrowth of Escherichia coli from prolonged stationary phase upon encountering fresh nutrients is heterogeneous, with one subpopulation suddenly regrowing after a delay (dormancy) and another of nongrowing cells that represented an increasing fraction as the culture aged. Moreover, a sizeable fraction of cells rejuvenated immediately, even when the inoculum was from very old cultures. The size of the dormant and nongrowing subpopulations depended on the time cells had endured stationary phase, as opposed to time-dependent changes to the medium. Regrowth of dormant cells was correlated with the dissolution of polar phase-bright foci that likely represent aggregates of damage, and a deep-learning algorithm was able to distinguish cellular fates based on a single stationary-phase image. Growth restarted in dormant cells after the upregulation of chaperones and DNA repair enzymes, and deletion of the chaperone DnaK resulted in compromised stationary-phase cell morphology and higher incidence of non-growing cells. A mathematical model of damage accumulation and division-mediated partitioning was in quantitative agreement with experimental data, including the small population of cells capable of immediate regrowth even in old cultures. Cells that endured stationary-phase without the ability to respire all immediately and homogeneously regrew in fresh nutrients, indicating that respiration in stationary phase is the driver of dormancy. These findings establish the importance of intracellular damage control when nutrients are sparse, and repair when nutrients are plentiful.

microbiology↗

bioRxiv2020

Environmental and physiological factors affecting high-throughput measurements of bacterial growth

Bacterial growth in nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multi-well plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth. Abstract ImportanceHow starved bacteria adapt to and multiply in replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allows for accurate growth-rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli. Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic-acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

microbiology↗

bioRxiv2020

Bacterial evolution in high osmolarity environments

Bacteria must maintain a cytosolic osmolarity higher than that of their environment in order to take up water. High osmolarity environments therefore present a formidable stress to bacteria. To explore the evolutionary mechanisms by which bacteria adapt to high osmolarity environments, we selected Escherichia coli in media with a variety of osmolytes and concentrations for 250 generations. Adaptation was osmolyte-dependent, with sorbitol stress generally resulting in increased fitness in conditions with higher osmolarity, while selection in high concentrations of proline resulted in increased fitness specifically on proline. Consistent with these phenotypes, sequencing of the evolved populations showed that passaging in proline resulted in specific mutations in an associated metabolic pathway that increases the ability to utilize proline for growth, while evolution in sorbitol resulted in mutations in many different genes that generally improve growth in high osmolarity conditions at the expense of growth at low osmolarity. High osmolarity decreased growth rate but increased mean cell volume compared with growth on proline as the sole carbon source, demonstrating that osmolarity-induced changes in growth rate and cell size follow an orthogonal relationship from the classical Growth Law relating cell size and nutrient quality. Isolates from a sorbitol-evolved population that capture the likely temporal sequence of mutations revealed by metagenomic sequencing demonstrate a tradeoff between growth at high and low osmolarity. Our study highlights the utility of experimental evolution for dissecting complex cellular networks and environmental interactions, particularly in the case of behaviors that can involve both specific and general metabolic stressors. ImportanceFor bacteria, maintaining higher internal solute concentrations than the environment allows cells to take up water. As a result, survival is challenging in high osmolarity environments. To investigate how bacteria adapt to high osmolarity environments, we evolved Escherichia coli in a variety of high osmolarity solutions for hundreds of generations. We found that evolved populations adopted different strategies to improve their growth depending on the osmotic passaging condition, either generally adapting to high osmolarity conditions or better metabolizing the osmolyte as carbon source. Single-cell imaging demonstrated that enhanced fitness was coupled to faster growth, and metagenomic sequencing revealed mutations that reflect growth tradeoffs across osmolarities. Our study demonstrates the utility of long-term evolution experiments for probing adaptation during environmental stress.

microbiology↗