@article{sarma_catella_san pedro_xiao_durmusoglu_menegatti_crook_magness_hall_2023, title={Design of 8-mer peptides that block <i>Clostridioides difficile</i> toxin A in intestinal cells}, volume={6}, ISSN={["2399-3642"]}, url={https://doi.org/10.1038/s42003-023-05242-x}, DOI={10.1038/s42003-023-05242-x}, abstractNote={Abstract Infections by Clostridioides difficile , a bacterium that targets the large intestine (colon), impact a large number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can be delivered to the gut and inhibit the biocatalytic activity of these toxins represent a promising therapeutic strategy to prevent and treat C. diff . infection. We describe an approach that combines a Pep tide B inding D esign (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in colon epithelial cells. One peptide, SA1, is found to block TcdA toxicity in primary-derived human colon (large intestinal) epithelial cells. SA1 binds TcdA with a K D of 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR).}, number={1}, journal={COMMUNICATIONS BIOLOGY}, author={Sarma, Sudeep and Catella, Carly M. and San Pedro, Ellyce T. and Xiao, Xingqing and Durmusoglu, Deniz and Menegatti, Stefano and Crook, Nathan and Magness, Scott T. and Hall, Carol K.}, year={2023}, month={Aug} }
 @article{durmusoglu_al'abri_li_islam williams_collins_martinez_crook_2023, title={Improving therapeutic protein secretion in the probiotic yeast Saccharomyces boulardii using a multifactorial engineering approach}, volume={22}, ISSN={["1475-2859"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85161032784&partnerID=MN8TOARS}, DOI={10.1186/s12934-023-02117-y}, abstractNote={The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb's 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 Sb's 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 sixfold (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 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae's 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 > tenfold 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.}, number={1}, journal={MICROBIAL CELL FACTORIES}, author={Durmusoglu, Deniz and Al'Abri, Ibrahim and Li, Zidan and Islam Williams, Taufika and Collins, Leonard B. and Martinez, Jose L. and Crook, Nathan}, year={2023}, month={Jun} }
 @misc{heavey_durmusoglu_crook_anselmo_2022, title={Discovery and delivery strategies for engineered live biotherapeutic products}, volume={40}, ISSN={["1879-3096"]}, DOI={10.1016/j.tibtech.2021.08.002}, abstractNote={The physiological microenvironment of the gut influences the efficacy of live biotherapeutic products (LBPs) and both discovery and delivery strategies can be used to overcome physiological challenges in the gut. Multi-omics illuminates colonization mechanisms of nonengineered LBPs to inspire engineering strategies. Functional genomics generates and tests engineered LBPs in a high-throughput manner to provide improved strains. Pharmaceutical formulations can be used to control the interactions between LBPs and their physiological microenvironment, creating modular technologies and approaches that can be applied to all LBPs. Genetic engineering approaches can improve LBP delivery through overcoming physiological challenges, enabling molecular interactions with host surfaces, controlling therapeutic functions in response to local physiological cues. Genetically engineered microbes that secrete therapeutics, sense and respond to external environments, and/or target specific sites in the gut fall under an emergent class of therapeutics, called live biotherapeutic products (LBPs). As live organisms that require symbiotic host interactions, LBPs offer unique therapeutic opportunities, but also face distinct challenges in the gut microenvironment. In this review, we describe recent approaches (often demonstrated using traditional probiotic microorganisms) to discover LBP chassis and genetic parts utilizing omics-based methods and highlight LBP delivery strategies, with a focus on addressing physiological challenges that LBPs encounter after oral administration. Finally, we share our perspective on the opportunity to apply an integrated approach, wherein discovery and delivery strategies are utilized synergistically, towards tailoring and optimizing LBP efficacy. Genetically engineered microbes that secrete therapeutics, sense and respond to external environments, and/or target specific sites in the gut fall under an emergent class of therapeutics, called live biotherapeutic products (LBPs). As live organisms that require symbiotic host interactions, LBPs offer unique therapeutic opportunities, but also face distinct challenges in the gut microenvironment. In this review, we describe recent approaches (often demonstrated using traditional probiotic microorganisms) to discover LBP chassis and genetic parts utilizing omics-based methods and highlight LBP delivery strategies, with a focus on addressing physiological challenges that LBPs encounter after oral administration. Finally, we share our perspective on the opportunity to apply an integrated approach, wherein discovery and delivery strategies are utilized synergistically, towards tailoring and optimizing LBP efficacy. components or appendages of bacteria that facilitate adhesion or adherence to other cells or to surfaces. the inability of an organism to synthesize a particular compound required for its own growth, requiring an external supply of that compound for survival. the prevention of engineered microbes from entering, being metabolically active in, or growing in environments outside of the host. a protective film primarily composed of polysaccharides, proteins, nucleic acids, and lipids that is secreted by a microbe and enables microbial adherence to surfaces. an organism that contains and supports the genetic components encoding for a desired engineered function. when microbes continuously grow and maintain metabolic activity in/on a host. a gene, or set of genes, that enable a microbe to colonize a host. intra- and interspecies exchange of nutrients. the amenability of a microbe for genetic manipulation. a water-containing gel composed of a network of crosslinked polymer chains. a pair of first-order nonlinear differential equations, frequently used to describe the dynamics of biological systems in which multiple species interact. interaction between the host and the microbiota that can determine fate and function of the microbe and the disease state of the host. a particle between 1 and 1000 μm in size, often composed of biocompatible lipids and/or polymers, which can encapsulate drug molecules or microbes. the position of a species within an ecosystem encompassing both the physical and environmental factors required for survival and the interactions with other species. the time course of a drug moving through the distinct compartments of the body. a sequence of DNA that controls the expression level of downstream coding regions. a molecule that signals the presence of related microbes nearby; often induces the expression of virulence-related genes. sets of macromolecules (e.g., proteins, RNA) that interact to control the level of expression of various genes in an organism. a DNA sequence that influences the rate of transcription of nearby genes. microbe associated with the host without implication of benefit or harm. capacity for a microbe to be transformed with foreign genetic material.}, number={3}, journal={TRENDS IN BIOTECHNOLOGY}, author={Heavey, Mairead K. and Durmusoglu, Deniz and Crook, Nathan and Anselmo, Aaron C.}, year={2022}, month={Mar}, pages={354–369} }
 @article{jensen_deichmann_ma_vilandt_schiesaro_rojek_lengger_eliasson_vento_durmusoglu_et al._2022, title={Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts}, volume={13}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-022-33961-y}, abstractNote={G protein-coupled receptors (GPCRs) enable cells to sense environmental cues and are indispensable for coordinating vital processes including quorum sensing, proliferation, and sexual reproduction. GPCRs comprise the largest class of cell surface receptors in eukaryotes, and for more than three decades the pheromone-induced mating pathway in baker's yeast Saccharomyces cerevisiae has served as a model for studying heterologous GPCRs (hGPCRs). Here we report transcriptome profiles following mating pathway activation in native and hGPCR-signaling yeast and use a model-guided approach to correlate gene expression to morphological changes. From this we demonstrate mating between haploid cells armed with hGPCRs and endogenous biosynthesis of their cognate ligands. Furthermore, we devise a ligand-free screening strategy for hGPCR compatibility with the yeast mating pathway and enable hGPCR-signaling in the probiotic yeast Saccharomyces boulardii. Combined, our findings enable new means to study mating, hGPCR-signaling, and cell-cell communication in a model eukaryote and yeast probiotics.}, number={1}, journal={NATURE COMMUNICATIONS}, author={Jensen, Emil D. and Deichmann, Marcus and Ma, Xin and Vilandt, Rikke U. and Schiesaro, Giovanni and Rojek, Marie B. and Lengger, Bettina and Eliasson, Line and Vento, Justin M. and Durmusoglu, Deniz and et al.}, year={2022}, month={Oct} }
 @article{durmusoglu_catella_purnell_menegatti_crook_2021, title={Design and in situ biosynthesis of precision therapies against gastrointestinal pathogens}, volume={23}, ISSN={["2468-8673"]}, DOI={10.1016/j.cophys.2021.06.007}, abstractNote={Gastrointestinal pathogens employ a variety of mechanisms to damage host tissue, acquire nutrients, and evade treatment. To supplement broad-spectrum antimicrobials, there has been increasing interest in designing molecules that target specific taxa and virulence processes. Excitingly, these antivirulence therapies may be able to be synthesized by gut-resident microbes, thereby enabling delivery of these drugs directly to the spatial and temporal site of infection. In this review, we highlight recent progress in our understanding of small molecules that inhibit specific virulence mechanisms. We additionally discuss emerging methods to discover pathogen-specific and mechanism-specific peptides and small proteins. Finally, we cover recent demonstrations of probiotics engineered to produce antimicrobials in response to pathogen-specific cues in the gut. Collectively, these advances point to an emerging integrative approach to treatment of gastrointestinal diseases, comprising microbiologists, peptide chemists, and synthetic biologists.}, journal={CURRENT OPINION IN PHYSIOLOGY}, author={Durmusoglu, Deniz and Catella, Carly M. and Purnell, Ethan F. and Menegatti, Stefano and Crook, Nathan C.}, year={2021}, month={Oct} }
 @article{durmusoglu_al'abri_collins_cheng_eroglu_beisel_crook_2021, title={In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii}, volume={10}, ISSN={["2161-5063"]}, url={https://doi.org/10.1021/acssynbio.0c00562}, DOI={10.1021/acssynbio.0c00562}, abstractNote={Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 °C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (β-carotene) and drug (violacein) titers. We found that S. boulardii colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1-2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled β-carotene synthesis (194 μg total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional β-carotene recovered in feces was 56-fold higher than the β-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by S. boulardii living in the mammalian gut and provides a set of tools for modulating these titers.}, number={5}, journal={ACS SYNTHETIC BIOLOGY}, publisher={American Chemical Society (ACS)}, author={Durmusoglu, Deniz and Al'Abri, Ibrahim S. and Collins, Scott P. and Cheng, Junrui and Eroglu, Abdulkerim and Beisel, Chase L. and Crook, Nathan}, year={2021}, month={May}, pages={1039–1052} }
 @article{al'abri_durmusoglu_crook_2021, title={What E. coli knows about your 1-year-old infant: Antibiotic use, lifestyle, birth mode, and siblings}, volume={29}, ISSN={["1934-6069"]}, DOI={10.1016/j.chom.2021.05.006}, abstractNote={The infant gut microbiota is shaped by diverse environmental exposures that alter its composition and can enrich antimicrobial resistance genes (ARGs). In this issue of Cell Host & Microbe, Li et al. (2021) studied the causes, spread, and dynamics of ARGs and their relationship with asthma-associated microbiota in Danish children. The infant gut microbiota is shaped by diverse environmental exposures that alter its composition and can enrich antimicrobial resistance genes (ARGs). In this issue of Cell Host & Microbe, Li et al. (2021) studied the causes, spread, and dynamics of ARGs and their relationship with asthma-associated microbiota in Danish children. The gut microbiota is fundamental to human health, but we lack a clear understanding of how it matures and how environmental factors modulate this process (Robertson et al., 2019Robertson R.C. Manges A.R. Finlay B.B. Prendergast A.J. The Human Microbiome and Child Growth - First 1000 Days and Beyond.Trends Microbiol. 2019; 27: 131-147Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). In the last decade, research has focused on understanding how the development of the gut microbiota changes in response to perturbations, especially in early life. We have learned that factors such as delivery mode, sex, exposure to antibiotics, rural versus urban lifestyle, hospitalization, and age significantly influence microbiota assembly, and consequently, human health (Stokholm et al., 2018Stokholm J. Blaser M.J. Thorsen J. Rasmussen M.A. Waage J. Vinding R.K. Schoos A.-M.M. Kunøe A. Fink N.R. Chawes B.L. et al.Maturation of the gut microbiome and risk of asthma in childhood.Nat. Commun. 2018; 9: 141Crossref PubMed Scopus (220) Google Scholar). However, the impact of these perturbations on the gut resistome is less well understood. The resistome is the collection of antibiotic resistance genes (ARGs) present in a microbial community. The overuse of antibiotics is associated with a low microbiota maturity, which can lead to metabolic disorders, malnutrition, infections, and even colon cancer (Langdon et al., 2016Langdon A. Crook N. Dantas G. The effects of antibiotics on the microbiome throughout development and alternative approaches for therapeutic modulation.Genome Med. 2016; 8: 39Crossref PubMed Scopus (437) Google Scholar). In addition, antibiotic treatment can lead to an increased abundance of microbes containing ARGs, facilitating future transfer of ARGs to pathogens and making treatment of infections more difficult (Ferreiro et al., 2018Ferreiro A. Crook N. Gasparrini A.J. Dantas G. Multiscale Evolutionary Dynamics of Host-Associated Microbiomes.Cell. 2018; 172: 1216-1227Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The high density of microbes in the gut enables horizontal gene transfer and makes the study of the gut resistome particularly important. Although we know that factors such as birth mode, breastfeeding, and antibiotic use impact resistome development, we still don’t fully understand how important other environmental factors are and how they are interconnected (Stokholm et al., 2020Stokholm J. Thorsen J. Blaser M.J. Rasmussen M.A. Hjelmsø M. Shah S. Christensen E.D. Chawes B.L. Bønnelykke K. Brix S. et al.Delivery mode and gut microbial changes correlate with an increased risk of childhood asthma.Sci. Transl. Med. 2020; 12: eaax9929Crossref PubMed Scopus (29) Google Scholar). In particular, there exists a gap in our understanding of resistome development between very early life (<1 year) (Gasparrini et al., 2016Gasparrini A.J. Crofts T.S. Gibson M.K. Tarr P.I. Warner B.B. Dantas G. Antibiotic perturbation of the preterm infant gut microbiome and resistome.Gut Microbes. 2016; 7: 443-449Crossref PubMed Scopus (57) Google Scholar, Gasparrini et al., 2019Gasparrini A.J. Wang B. Sun X. Kennedy E.A. Hernandez-Leyva A. Ndao I.M. Tarr P.I. Warner B.B. Dantas G. Persistent metagenomic signatures of early-life hospitalization and antibiotic treatment in the infant gut microbiota and resistome.Nat. Microbiol. 2019; 4: 2285-2297Crossref PubMed Scopus (81) Google Scholar) and adulthood (Schwartz et al., 2020Schwartz D.J. Langdon A.E. Dantas G. Understanding the impact of antibiotic perturbation on the human microbiome.Genome Med. 2020; 12: 82Crossref PubMed Scopus (48) Google Scholar), especially in healthy individuals. To fill this gap, Li et al., 2021Li X. Stokholm J. Brejnrod A. Vestergaard G.A. Russel J. Trivedi U. Thorsen J. Gupta S. Hjelmsø M.H. Shah S.A. et al.The infant gut resistome associates with E. coli, environmental exposures, gut microbiome maturity, and asthma-associated bacterial composition.Cell Host Microbe. 2021; 29 (this issue): 975-987Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar explored how diverse environmental factors shaped the resistomes of 662 1-year-old Danish children and the relationship of their resistomes to longer-term health impacts (Li et al., 2021Li X. Stokholm J. Brejnrod A. Vestergaard G.A. Russel J. Trivedi U. Thorsen J. Gupta S. Hjelmsø M.H. Shah S.A. et al.The infant gut resistome associates with E. coli, environmental exposures, gut microbiome maturity, and asthma-associated bacterial composition.Cell Host Microbe. 2021; 29 (this issue): 975-987Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The authors identified 409 ARGs in this cohort, with 167 of them conferring resistance to multiple antibiotics. Interestingly, ARG abundance was bimodal, with one cluster of children exhibiting more abundant and diverse ARGs in their gut microbiome than the other (Figure 1). To understand the drivers of this phenomenon, the authors investigated the species that comprised each cluster. This analysis revealed that the abundance of E. coli determined cluster membership, with individuals in the “ARG high” group having a much greater abundance of intestinal E. coli. This finding agrees with previous studies that found that the microbiotas of antibiotic-treated preterm infants are dominated by Gammaproteobacteria (Gasparrini et al., 2016Gasparrini A.J. Crofts T.S. Gibson M.K. Tarr P.I. Warner B.B. Dantas G. Antibiotic perturbation of the preterm infant gut microbiome and resistome.Gut Microbes. 2016; 7: 443-449Crossref PubMed Scopus (57) Google Scholar, Gasparrini et al., 2019Gasparrini A.J. Wang B. Sun X. Kennedy E.A. Hernandez-Leyva A. Ndao I.M. Tarr P.I. Warner B.B. Dantas G. Persistent metagenomic signatures of early-life hospitalization and antibiotic treatment in the infant gut microbiota and resistome.Nat. Microbiol. 2019; 4: 2285-2297Crossref PubMed Scopus (81) Google Scholar). Removing E. coli from the analyzed samples resulted in a 10-fold reduction in ARG richness in the “ARG high” group. This work therefore prioritizes further study on the ability of intestinal E. coli to mobilize ARGs to other members of the microbiota, perhaps serving as an important “node” for ARG “traffic” in the large intestine. The authors then wondered whether the “E. coli-high” cluster was persistent across age. However, when they measured the abundance of E. coli in samples from the same individuals collected at 1 week, 1 month, 4 years, 5 years, or 6 years of age, they found no difference in E. coli levels between each cluster. This indicates that the abundance of E. coli, and perhaps ARG abundance as a consequence, is mainly influenced by transient factors such as seasons, antibiotic exposure, or ecological dynamics in the microbiota. To understand how these ARGs were acquired, the authors then examined the correlation between ARG abundance and patient metadata. Interestingly, even infants that were not treated with any antibiotics had ARGs in their microbiomes, which led the researchers to investigate whether other environmental exposures had an impact. They found that the most prominent factors in shaping the distribution of ARGs were the presence of older siblings, living environment, mode of delivery, time since antibiotic treatment, and antibiotic use 40 days before childbirth. Other factors, such antibiotic usage frequency, living in an apartment, and usage of antibiotics by pregnant mothers in the first two trimesters had a smaller role. Comparing the effects of antibiotics to other factors revealed that while all factors affect the distribution of ARGs, antibiotic use was unique because it did not change the microbiota composition. Potential explanations for this counterintuitive finding include horizontal transfer of ARGs among gut microbes in response to antibiotic exposure, or the presence of antibiotic-resistant subpopulations with the same taxonomic assignments. It is noteworthy that not all environmental exposures propel the gut microbiota to inherit the same ARGs or at the same rate. Therefore, it is possible that minimizing childhood exposure into the most influential factors may protect against ARG proliferation in the gut microbiome. Finally, the authors investigated the relationship between ARG carriage, microbiota composition, and asthma, finding that high ARG abundance was associated with a microbiota composition that increases the risk of later asthma. Additionally, samples with abundant ARGs also had lower microbiota maturity, which is a risk factor for a variety of metabolic and immunological disorders. Although more studies are needed to fully elucidate the associations between ARGs, microbiota composition, asthma, and other diseases, this study supports a microbial route to identify asthma-prone individuals. Li et al., 2021Li X. Stokholm J. Brejnrod A. Vestergaard G.A. Russel J. Trivedi U. Thorsen J. Gupta S. Hjelmsø M.H. Shah S.A. et al.The infant gut resistome associates with E. coli, environmental exposures, gut microbiome maturity, and asthma-associated bacterial composition.Cell Host Microbe. 2021; 29 (this issue): 975-987Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar shed light on ARGs in the healthy developing gut in remarkable detail, finding that distribution of ARGs in the gut is strongly bimodal and largely based on the abundance of E.coli. It would be very fascinating to determine whether this clustering occurs in other parts of the world, or in other age groups. Additionally, the authors showed that different environmental factors can have different effects on the acquisition of ARGs, thereby setting priorities for future ARG mitigation. Interestingly, high ARG richness was correlated with low microbiota maturity and a high risk of asthma later in life, agreeing with prior studies in the nasopharyngeal microbiome (Teo et al., 2015Teo S.M. Mok D. Pham K. Kusel M. Serralha M. Troy N. Holt B.J. Hales B.J. Walker M.L. Hollams E. et al.The infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development.Cell Host Microbe. 2015; 17: 704-715Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). By elucidating the risk factors for high rates of ARG acquisition during development, this study facilitates efforts to combat the rise of antimicrobial-resistant pathogens and provides a roadmap to studying these important questions in other cohorts. The infant gut resistome associates with E. coli, environmental exposures, gut microbiome maturity, and asthma-associated bacterial compositionLi et al.Cell Host & MicrobeApril 21, 2021In BriefIn this comprehensive analysis of antibiotic resistance genes (ARGs) distribution in the infant gut, Li et al. show that E. coli is an extremely important reservoir of ARGs. They also reveal associations between infant gut resistome and environmental factors, gut microbiome maturation, and bacteria associated with later development of asthma. Full-Text PDF Open Archive}, number={6}, journal={CELL HOST & MICROBE}, author={Al'Abri, Ibrahim S. and Durmusoglu, Deniz and Crook, Nathan}, year={2021}, month={Jun}, pages={854–855} }