Biology subjects

Anderson, S. E.

Publications and source records attributed to Anderson, S. E..

12 recordsLinked to original sources

bioRxiv2025

Enzymatic Activity of PBP1B Compensates for β-lactam Mediated Inhibition of PBP2 in Cells Overproducing ppGpp

The alarmone (p)ppGpp (ppGpp) accumulates in response to starvation and other stress, leading to inhibition of multiple biosynthetic pathways and, at high concentrations, suppression of bacterial growth. Growth suppression by ppGpp is implicated in the formation of persister cells, which survive antibiotic challenge only to regrow once the drug is removed. However, there is also evidence that low levels of ppGpp contribute to resistance to certain cell wall-active antibiotics in actively growing cells. To characterize ppGpps contribution to antibiotic resistance, we measured MICs of a panel of {beta}-lactams in actively growing Escherichia coli cells overexpressing a ppGpp synthase (relA*). Cells engineered to modestly overproduce ppGpp exhibited up to 64-fold increases in resistance to PBP2-targeting {beta}-lactams only, with mecillinam the most dramatically affected. Resistance required the transcription factor DksA and the class A penicillin binding protein (PBP) PBP1B. PBP1B variants defective for transpeptidase activity, glycosyltransferase activity, or both were incapable of mediating resistance, suggesting the full enzymatic activity of PBP1B is required for resistance. Transcriptomics revealed that ppGpp overproduction leads to increased expression of lpoB, which encodes an activator of PBP1B. LpoB was required for mecillinam resistance, with an lpoB deletion mutant exhibiting a loss of ppGpp-dependent resistance. Resistance was partially lost in an lpoB deletion strain expressing an LpoB-bypass variant of PBP1B (mrcB*). Together these results support a model wherein overproduction of ppGpp favors cell wall synthesis by PBP1B, in part via increased expression of lpoB, allowing PBP1B to substitute for PBP2 when it is inhibited by {beta}-lactams. ImportanceAntimicrobial resistance is an increasing global health threat, but the molecular mechanisms underlying resistance remain incompletely understood. This work clarifies the role of the alarmone ppGpp in mediating antibiotic resistance in the model bacterium Escherichia coli. Elevated levels of ppGpp were found to cause resistance to {beta}-lactam antibiotics targeting the cell wall synthesis enzyme PBP2. Resistance required transcriptional regulation by ppGpp and enzymatic activity of the cell wall enzyme PBP1B. This work suggests that ppGpp causes resistance in part by increasing expression of lpoB, which encodes an activator of PBP1B. Because ppGpp levels are controlled by nutritional conditions, this work suggests that nutritional availability could impact the efficacy of antibiotics. Furthermore, this study adds to a number of studies demonstrating that cell wall synthesizing PBP enzymes can compensate for one another under certain conditions, leading to {beta}-lactam resistance.

microbiology↗

bioRxiv2025

Aberrant Fibro-Adipogenic Progenitor Subpopulations Drive Volumetric Muscle Loss-Induced Fibrosis

Volumetric muscle loss (VML) injuries result in chronic fibrosis, inflammation, and persistent functional deficits. Fibro-adipogenic progenitor (FAP) cells are a heterogeneous, muscle-resident stromal cell population that play a crucial role in muscle regeneration, but also contribute to fibrosis in muscle disease. The role of FAPs in VML is not well established and may be critical target to ensure functional muscle regeneration after VML. We utilized a VML model in the mouse quadriceps to study the location, secretome, surface marker distribution, gene expression, and single-cell transcriptional profile of FAPs after VML. After VML, a subpopulation of FAPs highly expressed {beta}1-integrin and were elevated in the post-VML muscle tissue; these FAPs had increased fibrotic gene expression and increased myofibroblast differentiation potential. Transforming growth factor-{beta}1 (TGF-{beta}1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) were identified as secreted proteins from VML derived FAPs that produced both pro-fibrotic and anti-myogenic signaling. These data establish an aberrant FAP sub-population that are elevated in VML injury and provides novel targets for future scarless muscle regeneration in VML.

pathology↗

bioRxiv2025

Dietary manipulation of intestinal microbes prolongs survival in a mouse model of Hirschsprung disease

Enterocolitis is a common and potentially deadly manifestation of Hirschsprung disease (HSCR) but disease mechanisms remain poorly defined. Unexpectedly, we discovered that diet can dramatically affect the lifespan of a HSCR mouse model (Piebald lethal, sl/sl) where affected animals die from HAEC complications. In the sl/sl model, diet alters gut microbes and metabolites, leading to changes in colon epithelial gene expression and epithelial oxygen levels known to influence colitis severity. Our findings demonstrate unrecognized similarity between HAEC and other types of colitis and suggest dietary manipulation could be a valuable therapeutic strategy for people with HSCR. AbstractHirschsprung disease (HSCR) is a birth defect where enteric nervous system (ENS) is absent from distal bowel. Bowel lacking ENS fails to relax, causing partial obstruction. Affected children often have "Hirschsprung disease associated enterocolitis" (HAEC), which predisposes to sepsis. We discovered survival of Piebald lethal (sl/sl) mice, a well-established HSCR model with HAEC, is markedly altered by two distinct standard chow diets. A "Protective" diet increased fecal butyrate/isobutyrate and enhanced production of gut epithelial antimicrobial peptides in proximal colon. In contrast, "Detrimental" diet-fed sl/sl had abnormal appearing distal colon epithelium mitochondria, reduced epithelial mRNA involved in oxidative phosphorylation, and elevated epithelial oxygen that fostered growth of inflammation-associated Enterobacteriaceae. Accordingly, selective depletion of Enterobacteriaceae with sodium tungstate prolonged sl/sl survival. Our results provide the first strong evidence that diet modifies survival in a HSCR mouse model, without altering length of distal colon lacking ENS. HighlightsO_LITwo different standard mouse diets alter survival in the Piebald lethal (sl/sl) mouse model of Hirschsprung disease, without impacting extent of distal colon aganglionosis (the region lacking ENS). C_LIO_LIPiebald lethal mice fed the "Detrimental" diet had many changes in colon epithelial transcriptome including decreased mRNA for antimicrobial peptides and genes involved in oxidative phosphorylation. Detrimental diet fed sl/sl also had aberrant-appearing mitochondria in distal colon epithelium, with elevated epithelial oxygen that drives lethal Enterobacteriaceae overgrowth via aerobic respiration. C_LIO_LIElimination of Enterobacteriaceae with antibiotics or sodium tungstate improves survival of Piebald lethal fed the "Detrimental diet". C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/637436v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@d95251org.highwire.dtl.DTLVardef@1ab58caorg.highwire.dtl.DTLVardef@5260b0org.highwire.dtl.DTLVardef@49ce42_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Rapid cyclic stretching induces synthetic, proinflammatory phenotypes in cultured human intestinal smooth muscle, with the potential to alter signaling to adjacent bowel cells

Background and AimsBowel smooth muscle experiences mechanical stress constantly during normal function, and pathologic mechanical stressors in disease states. We tested the hypothesis that pathologic mechanical stress could alter transcription to induce smooth muscle phenotypic class switching. MethodsPrimary human intestinal smooth muscle cells (HISMCs), seeded on electrospun aligned poly-{varepsilon}-caprolactone nano-fibrous scaffolds, were subjected to pathologic, high frequency (1 Hz) uniaxial 3% cyclic stretch (loaded) or kept unloaded in culture for 6 hours. Total RNA sequencing, qRT-PCR, and quantitative immunohistochemistry defined loading-induced changes in gene expression. NicheNet predicted how differentially expressed genes might impact HISMCs and other bowel cells. ResultsLoading induced differential expression of 4537 genes in HISMCs. Loaded HISMCs had a less contractile phenotype, with increased expression of synthetic SMC genes, proinflammatory cytokines, and altered expression of axon guidance molecules, growth factors and morphogens. Many differentially expressed genes encode secreted ligands that could act cell-autonomously on smooth muscle and on other cells in the bowel wall. DiscussionHISMCs demonstrate remarkably rapid phenotypic plasticity in response to mechanical stress that may convert contractile HISMCs into proliferative, fibroblast-like cells or proinflammatory cells. These mechanical stress-induced changes in HISMC gene expression may be relevant for human bowel disease.

bioengineering↗

bioRxiv2024

Improving Functional Muscle Regeneration in Volumetric Muscle Loss Injuries by Shifting the Balance of Inflammatory and Pro-Resolving Lipid Mediators

Severe tissue loss resulting from extremity trauma, such as volumetric muscle loss (VML), poses significant clinical challenges for both general and military populations. VML disrupts the endogenous tissue repair mechanisms, resulting in acute and unresolved chronic inflammation and immune cell presence, impaired muscle healing, scar tissue formation, persistent pain, and permanent functional deficits. The aberrant healing response is preceded by acute inflammation and immune cell infiltration which does not resolve. We analyzed the biosynthesis of inflammatory and specialized pro-resolving lipid mediators (SPMs) after VML injury in two different models; muscle with critical-sized defects had a decreased capacity to biosynthesize SPMs, leading to dysregulated and persistent inflammation. We developed a modular poly(ethylene glycol)-maleimide hydrogel platform to locally release a stable isomer of Resolvin D1 (AT-RvD1) and promote endogenous pathways of inflammation resolution in the two muscle models. The local delivery of AT-RvD1 enhanced muscle regeneration, improved muscle function, and reduced pain sensitivity after VML by promoting molecular and cellular resolution of inflammation. These findings provide new insights into the pathogenesis of VML and establish a pro-resolving hydrogel therapeutic as a promising strategy for promoting functional muscle regeneration after traumatic injury.

bioengineering↗

bioRxiv2023

The transcription factor DksA exerts opposing effects on cell division depending on the presence of ppGpp

Bacterial cell size is a multifactorial trait that is influenced by variables including nutritional availability and the timing of cell division. Prior work revealed a negative correlation between the alarmone (p)ppGpp (ppGpp) and cell length in Escherichia coli, suggesting that ppGpp may promote assembly of the division machinery (divisome) and cytokinesis in this organism. To clarify this counterintuitive connection between a starvation induced stress response effector and cell proliferation, we undertook a systematic analysis of growth and division in E. coli cells defective in ppGpp synthesis and/or engineered to overproduce the alarmone. Our data indicate that ppGpp acts indirectly on divisome assembly through its role as a global mediator of transcription. Loss of either ppGpp (ppGpp0) or the ppGpp-associated transcription factor DksA led to increased average length, with ppGpp0 mutants also exhibiting a high frequency of extremely long filamentous cells. Using heat-sensitive division mutants and fluorescently labeled division proteins, we confirmed that ppGpp and DksA are cell division activators. We found that ppGpp and DksA regulate division through their effects on transcription, although the lack of known division genes or regulators in available transcriptomics data strongly suggests that this regulation is indirect. Surprisingly, we also found that DksA inhibits division in ppGpp0 cells, contrary to its role in a wild-type background. We propose that the ability of ppGpp to switch DksA from a division inhibitor to a division activator helps tune cell length across different concentrations of ppGpp. ImportanceCell division is a key step in the bacterial lifecycle that must be appropriately regulated to ensure survival. This work identifies the alarmone ppGpp as a general regulator of cell division, extending our understanding of the role of ppGpp beyond a signal for starvation and other stress. Even in nutrient replete conditions, basal levels of ppGpp are essential for division to occur appropriately and for cell size to be maintained. This study establishes ppGpp as a "switch" that controls whether the transcription factor DksA behaves as a division activator or inhibitor. This unexpected finding enhances our understanding of the complex regulatory mechanisms employed by bacteria to coordinate division with diverse aspects of cell growth and stress response. Because division is an essential process, a better understanding the mechanisms governing assembly and activation of the division machinery could contribute to the development of novel therapeutics to treat bacterial infections.

microbiology↗

bioRxiv2021

Identifying dysregulated immune cell subsets following critical volumetric muscle loss with pseudo-time trajectories

Volumetric muscle loss (VML) results in permanent functional deficits and remains a substantial regenerative medicine challenge. A coordinated immune response is crucial for timely myofiber regeneration, however the immune response following VML has yet to be fully characterized. Here, we leveraged dimensionality reduction and pseudo-time analysis techniques to elucidate the cellular players underlying a functional or pathological outcome as a result of subcritical or critical VML in the murine quadriceps, respectively. We found that critical VML presented with a sustained presence of M2-like and CD206hiLy6Chi hybrid macrophages whereas subcritical defects resolved these populations. These macrophage subsets may contribute to fibrogenesis in critical VML, especially in the presence of TGF-{beta}. Furthermore, several T cell populations were significantly elevated in critical VML compared to subcritical injuries. Specifically, there was a significant increase of CD127+ T cells at days 3 and 7, and upregulated CD127 expression may indicate aberrant IL-7 signaling in critical VML. These results demonstrate a dysregulated immune response in critical VML that is unable to resolve the chronic inflammatory state and transition to a pro-regenerative microenvironment. These data provide important insights into potential therapeutic strategies which could reduce the immune cell burden and pro-fibrotic signaling characteristic of VML.

immunology↗

bioRxiv2020

Regenerating motor neurons prime muscle stem cells for myogenesis by enhancing protein synthesis and mitochondrial bioenergetics

Throughout life, skeletal muscle, the arbiter of voluntary movements, is maintained by a population of skeletal muscle-dedicated stem cells, called muscle satellite cells (MuSCs). Similar to other adult stem cells, the function of MuSCs is tightly coordinated by the cellular and acellular components of their microenvironment, or the niche. While the processes that control the coupling of neurotransmission and muscle contraction have been well characterized, little is known on the reciprocal crosstalk between neural cells and MuSCs within the muscle microenvironment. Here, we report that mild peripheral nerve injury enhances MuSC myogenic function and muscle regeneration by synergistically augmenting MuSC mitochondrial bioenergetics and upregulating anabolic protein synthesis pathways. We also demonstrate that chronic disruption or degeneration of neuromuscular synapses, such as in muscular dystrophy and biological aging, abolishes MuSC and motor neuron interactions, causing significant deficits in muscle regeneration following injury. These results underscore the importance of neuromuscular junction and neural network as an essential niche of MuSCs. Determining the significance of MuSC-nerve interactions and their functional outcomes, as well as the possibility of modulating these connections, have important implications for our understanding of neuromuscular disease pathology and development of therapeutic interventions. HighlightsO_LIMild peripheral nerve injury increases muscle stem cell bioavailability of healthy muscle. C_LIO_LINerve perturbation stimulates myogenesis by enhancing protein synthesis and mitochondrial metabolism in young, healthy muscle. C_LIO_LISynergistic crosstalk within neuromuscular niche boosts muscle regeneration in young, healthy muscle. C_LIO_LIPositive influences from the neural network on muscle stem cells are abolished in pathological denervation manifested in dystrophic and aging muscle. C_LI

cell biology↗

bioRxiv2020

Transplantation of muscle stem cell mitochondria rejuvenates the bioenergetic function of dystrophic muscle

Mitochondrial dysfunction has been implicated in various pathologies, including muscular dystrophies. During muscle regeneration, resident stem cells, also known as muscle satellite cells (MuSCs), undergo myogenic differentiation to form de novo myofibers or fuse to existing syncytia. Leveraging this cell-cell fusion process, we postulated that mitochondria stemming from MuSCs could be transferred to myofibers during muscle regeneration to remodel the mitochondrial network and restore bioenergetic function. Here, we report that dystrophic MuSCs manifest significant mitochondrial dysfunction and fuse with existing dystrophic myofibers to propagate mitochondrial dysfunction during muscle repair. We demonstrate that by transplanting healthy donor MuSCs into dystrophic host muscle, the mitochondrial network (reticulum) and bioenergetic function can be rejuvenated. Conversely, when bioenergetically-compromised donor MuSCs are transplanted, improvements in mitochondrial organization and bioenergetic function were ablated in the dystrophic recipient. Overall, these data reveal a unique role of muscle stem cells as an essential regulator of myofiber mitochondrial homeostasis and a potential therapeutic target against mitochondrial myopathies.

cell biology↗

bioRxiv2019

Characterization of a β-lactamase that contributes to intrinsic β-lactam resistance in Clostridioides difficile

Clostrididioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive spore former that is highly resistant to {beta}-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to {beta}-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of {beta}-lactam resistance in C. difficile are not fully understood. Our data reinforce prior evidence that C. difficile produces a {beta}-lactamase, which is a common {beta}-lactam resistance mechanism found in other bacterial species. We identified an operon encoding a lipoprotein of unknown function and a {beta}-lactamase that was greatly induced in response to several classes of {beta}-lactam antibiotics. An in-frame deletion of the operon abolished {beta}-lactamase activity in C. difficile strain 630{Delta}erm and resulted in decreased resistance to the {beta}-lactam ampicillin. We found that the activity of this {beta}-lactamase, herein named BlaD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a co-transcribed lipoprotein, BlaX, aids in BlaD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of the blaXD operon in response to {beta}-lactams. These results provide support for the function of a {beta}-lactamase in C. difficile antibiotic resistance, and reveal the unique roles of a co-regulated lipoprotein and reducing environment in {beta}-lactamase activity.\n\nIMPORTANCEClostridioides difficile is an anaerobic, gastrointestinal human pathogen. One of the highest risk factors for contracting C. difficile infection is antibiotic treatment, which causes microbiome dysbiosis. C. difficile is resistant to {beta}-lactam antibiotics, the most commonly prescribed class of antibiotics. C. difficile produces a recently discovered {beta}-lactamase, which cleaves and inactivates numerous {beta}-lactams. In this study, we report the contribution of this anaerobic {beta}-lactamase to ampicillin resistance in C. difficile, as well as the transcriptional regulation of the gene, blaD, by a BlaRI system. In addition, our data demonstrate co-transcription of blaD with blaX, which encodes a membrane protein of previously unknown function. Furthermore, we provide evidence that BlaX enhances {beta}-lactamase activity in a portion of C. difficile strains. This study demonstrates a novel interaction between a {beta}-lactamase and a membrane protein in a Gram-positive pathogen, and due to the anaerobic nature of the {beta}-lactamase activity, suggests that more {beta}-lactamases are yet to be identified in other anaerobes.

microbiology↗

bioRxiv2019

Wnt7a-releasing synthetic hydrogel enhances local skeletal muscle regeneration and muscle stem cell engraftment

Skeletal muscle possesses efficient ability to regenerate upon minor injuries, but its capacity to regenerate is severely compromised with traumatic injuries and muscle-associated diseases. Recent evidence suggests that skeletal muscle regeneration can be accelerated by transplantation of muscle satellite cells (MuSCs) or treatment with promyogenic factors, such as Wingless-type MMTV Integrated 7a (Wnt7a) protein. Although direct intramuscular injection is the simplest method to deliver MuSCs and Wnt7a for regenerative therapy, direct injection is not viable in many clinical cases where the structural integrity is severely compromised. To address this challenge, we engineered a synthetic poly(ethylene glycol) (PEG)-based hydrogel to facilitate the co-delivery of pro-myogenic factors, such as Wnt7a, and MuSCs to skeletal muscles affected by severe trauma or muscular dystrophies. Wnt7a release rate can be controlled by modulating the polymer density of the hydrogel, and this release rate can be further accelerated through the proteolytic degradation of the hydrogel. Treating cryo-injured tibialis anterior (TA) muscles with Wnt7a-loaded hydrogels resulted in an accelerated regenerative response, measured by increased muscle fiber cross-sectional area, bulk TA mass, and number of Pax7+ MuSCs at the injury site, compared to the TA muscles treated with Wnt7a-free hydrogels. Co-delivery of Wnt7a and primary MuSCs using the synthetic hydrogel to the cryo-injured TA muscles significantly increased cellular migration during the engraftment process. This work provides a synthetic biomaterial platform for advancing treatment strategies of skeletal muscle trauma and diseases, specifically in conditions where direct intramuscular injection may be challenging.

bioengineering↗

bioRxiv2018

Critical Limb Ischemia Induces Remodeling of Skeletal Muscle Motor Unit and Myonuclear- and Mitochondrial-Domains

Critical limb ischemia, the most severe form of peripheral artery disease, leads to extensive damage and alterations in skeletal muscle homeostasis. Although recent developments towards revascularization therapies have been introduced, there has been limited research into treatments for ischemic myopathy. To elucidate the regenerative mechanism of the muscle stem cell and its niche components in response to ischemic insults, we explored interactions between the vasculature, motor neuron, muscle fiber, and the muscle stem cell. We first investigated changes in the neuromuscular junction and motor neuron innervation following a surgical hindlimb ischemia model of critical limb ischemia in mice. Along with previous findings that support remodeling of the neuromuscular junction, we report that ischemic injury also causes significant alterations to the myofiber through a muscle stem cell-mediated increase of myonuclei number per fiber, a concomitant decrease in myonuclear domain size, and an increase in relative mitochondrial content per myonucleus. These results indicate that as a regenerative response to critical limb ischemia, myofibers exhibit myonuclear expansion to allow enhanced transcriptional support and an increase in mitochondrial content for bioenergetic need of the energy-demanding tissue regeneration.

physiology↗