@article{foley_walker_stewart_o'flaherty_gentry_patel_beaty_allen_pan_simpson_et al._2023, title={Bile salt hydrolases shape the bile acid landscape and restrict Clostridioides difficile growth in the murine gut}, volume={3}, ISSN={["2058-5276"]}, DOI={10.1038/s41564-023-01337-7}, abstractNote={Bile acids (BAs) mediate the crosstalk between human and microbial cells and influence diseases including Clostridioides difficile infection (CDI). While bile salt hydrolases (BSHs) shape the BA pool by deconjugating conjugated BAs, the basis for their substrate selectivity and impact on C. difficile remain elusive. Here we survey the diversity of BSHs in the gut commensals Lactobacillaceae, which are commonly used as probiotics, and other members of the human gut microbiome. We structurally pinpoint a loop that predicts BSH preferences for either glycine or taurine substrates. BSHs with varying specificities were shown to restrict C. difficile spore germination and growth in vitro and colonization in pre-clinical in vivo models of CDI. Furthermore, BSHs reshape the pool of microbial conjugated bile acids (MCBAs) in the murine gut, and these MCBAs can further restrict C. difficile virulence in vitro. The recognition of conjugated BAs by BSHs defines the resulting BA pool, including the expansive MCBAs. This work provides insights into the structural basis of BSH mechanisms that shape the BA landscape and promote colonization resistance against C. difficile.}, journal={NATURE MICROBIOLOGY}, author={Foley, Matthew H. and Walker, Morgan E. and Stewart, Allison K. and O'Flaherty, Sarah and Gentry, Emily C. and Patel, Shakshi and Beaty, Violet V. and Allen, Garrison and Pan, Meichen and Simpson, Joshua B. and et al.}, year={2023}, month={Mar} }
 @article{o'flaherty_cobian_barrangou_2023, title={Impact of Pomegranate on Probiotic Growth, Viability, Transcriptome and Metabolism}, volume={11}, ISSN={["2076-2607"]}, url={https://doi.org/10.3390/microorganisms11020404}, DOI={10.3390/microorganisms11020404}, abstractNote={Despite rising interest in understanding intestinal bacterial survival in situ, relatively little attention has been devoted to deciphering the interaction between bacteria and functional food ingredients. Here, we examined the interplay between diverse beneficial Lactobacillaceae species and a pomegranate (POM) extract and determined the impact of this functional ingredient on bacterial growth, cell survival, transcription and target metabolite genesis. Three commercially available probiotic strains (Lactobacillus acidophilus NCFM, Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum Lp-115) were used in growth assays and flow cytometry analysis, indicating differential responses to the presence of POM extract across the three strains. The inclusion of POM extract in the growth medium had the greatest impact on L. acidophilus cell counts. LIVE/DEAD staining determined significantly fewer dead cells when L. acidophilus was grown with POM extract compared to the control with no POM (1.23% versus 7.23%). Whole-transcriptome analysis following exposure to POM extract showed markedly different global transcriptome responses, with 15.88% of the L. acidophilus transcriptome, 19.32% of the L. rhamnosus transcriptome and only 2.37% of the L. plantarum transcriptome differentially expressed. We also noted strain-dependent metabolite concentrations in the medium with POM extract compared to the control medium for punicalagin, ellagic acid and gallic acid. Overall, the results show that POM extract triggers species-specific responses by probiotic strains and substantiates the rising interest in using POM as a prebiotic compound.}, number={2}, journal={MICROORGANISMS}, author={O'Flaherty, Sarah and Cobian, Natalia and Barrangou, Rodolphe}, year={2023}, month={Feb} }
 @article{chamberlain_o'flaherty_cobian_barrangou_2022, title={Metabolomic Analysis of Lactobacillus acidophilus, L. gasseri, L. crispatus, and Lacticaseibacillus rhamnosus Strains in the Presence of Pomegranate Extract}, volume={13}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2022.863228}, abstractNote={Lactobacillus species are prominent inhabitants of the human gastrointestinal tract that contribute to maintaining a balanced microbial environment that positively influences host health. These bacterial populations can be altered through use of probiotic supplements or via dietary changes which in turn affect the host health. Utilizing polyphenolic compounds to selectively stimulate the growth of commensal bacteria can have a positive effect on the host through the production of numerous metabolites that are biologically active. Four Lactobacillus strains were grown in the presence of pomegranate (POM) extract. Two strains, namely, L. acidophilus NCFM and L. rhamnosus GG, are commonly used probiotics, while the other two strains, namely, L. crispatus NCK1351 and L. gasseri NCK1342, exhibit probiotic potential. To compare and contrast the impact of POM on the strains' metabolic capacity, we investigated the growth of the strains with and without the presence of POM and identified their carbohydrate utilization and enzyme activity profiles. To further investigate the differences between strains, an untargeted metabolomic approach was utilized to quantitatively and qualitatively define the metabolite profiles of these strains. Several metabolites were produced significantly and/or exclusively in some of the strains, including mevalonate, glutamine, 5-aminoimidazole-4-carboxamide, phenyllactate, and fumarate. The production of numerous discrete compounds illustrates the unique characteristics of and diversity between strains. Unraveling these differences is essential to understand the probiotic function and help inform strain selection for commercial product formulation.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Chamberlain, MaryClaire and O'Flaherty, Sarah and Cobian, Natalia and Barrangou, Rodolphe}, year={2022}, month={May} }
 @article{foley_o'flaherty_allen_rivera_stewart_barrangou_theriot_2021, title={Lactobacillus bile salt hydrolase substrate specificity governs bacterial fitness and host colonization}, volume={118}, ISSN={["1091-6490"]}, url={https://doi.org/10.1073/pnas.2017709118}, DOI={10.1073/pnas.2017709118}, abstractNote={Significance The transformation of bile acids (BAs) by the gut microbiota is increasingly recognized as an important factor shaping host health. The prerequisite step of BA metabolism is carried out by bile salt hydrolases (BSHs), which are encoded by select gut and probiotic bacteria. Despite their prevalence, the utility of harboring a bsh is unclear. Here, we investigate the role of BSHs encoded by Lactobacillus acidophilus and Lactobacillus gasseri . We show that BA type and BSH substrate preferences affect in vitro and in vivo growth of both species. These findings contribute to a mechanistic understanding of bacterial survival in various BA-rich niches and inform future efforts to leverage BSHs as a therapeutic tool for manipulating the gut microbiota.}, number={6}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Foley, Matthew H. and O'Flaherty, Sarah and Allen, Garrison and Rivera, Alissa J. and Stewart, Allison K. and Barrangou, Rodolphe and Theriot, Casey M.}, year={2021}, month={Feb} }
 @article{klotz_goh_o'flaherty_johnson_barrangou_2020, title={Deletion of S-Layer Associated Ig-Like Domain Protein Disrupts the Lactobacillus acidophilus Cell Surface}, volume={11}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2020.00345}, abstractNote={Bacterial surface-layers (S-layers) are crystalline arrays of repeating proteinaceous subunits that coat the exterior of many cell envelopes. S-layers have demonstrated diverse functions in growth and survival, maintenance of cell integrity, and mediation of host interactions. Additionally, S-layers can act as scaffolds for the outward display of auxiliary proteins and glycoproteins. These non-covalently bound S-layer associated proteins (SLAPs) have characterized roles in cell division, adherence to intestinal cells, and modulation of the host immune response. Recently, IgdA (LBA0695), a Lactobacillus acidophilus SLAP that possesses a Group 3 immunoglobulin (Ig)-like domain and GW (Gly-Tryp) dipeptide surface anchor, was recognized for its high conservation among S-layer-forming lactobacilli, constitutive expression, and surface localization. These findings prompted its selection for examination within the present study. Although IgdA and corresponding orthologs were shown to be unique to host-adapted lactobacilli, the Ig domain itself was specific to vertebrate-adapted species suggesting a role in vertebrate adaptation. Using a counterselective gene replacement system, igdA was deleted from the L. acidophilus NCFM chromosome. The resultant mutant, NCK2532, exhibited a visibly disrupted cell surface which likely contributed to its higher salt sensitivity, severely reduced adhesive capacity, and altered immunogenicity profile. Transcriptomic analyses revealed the induction of several stress response genes and secondary surface proteins. Due to the broad impact of IgdA on the cellular physiology and probiotic attributes of L. acidophilus, identification of similar proteins in alternative bacterial species may help pinpoint next-generation host-adapted probiotic candidates.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Klotz, Courtney and Goh, Yong Jun and O'Flaherty, Sarah and Johnson, Brant and Barrangou, Rodolphe}, year={2020}, month={Mar} }
 @article{foley_o'flaherty_barrangou_theriot_2019, title={Bile salt hydrolases: Gatekeepers of bile acid metabolism and host-microbiome crosstalk in the gastrointestinal tract}, volume={15}, ISSN={["1553-7374"]}, url={https://doi.org/10.1371/journal.ppat.1007581}, DOI={10.1371/journal.ppat.1007581}, abstractNote={Research on bile acids has increased dramatically due to recent studies demonstrating their ability to significantly impact the host, microbiome, and various disease states [1–3]. Although these liver-synthesized molecules assist in the absorption and digestion of dietary fat in the intestine, their reabsorption and recirculation also gives them access to peripheral organs [4] (Fig 1A). Bile acids serve as substrates for bile acid receptors (BARs) found throughout the body that control critical regulatory and metabolic processes and therefore represent an important class of bioactive molecules [5]. Despite the importance of bile acids to host health, there remain gaps in our knowledge about the bacterial enzymes driving their composition and modification.






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Fig 1

Bile salt hydrolases act on circulating conjugated bile acids in the gut-liver axis.
(A) Bile acids synthesized in the liver and stored in the gall bladder enter the small intestine through the duodenum where they reach millimolar concentrations. The majority of bile acids (95%) are reabsorbed in the ileum and recirculate to the liver through the portal vein. The remaining population transit to the colon as they continue to be reabsorbed, and a small (<5%) amount exit through the feces. Recirculating bile acids access host tissues outside the intestines to impart systemic effects on host physiology. (B) BSHs cleave the amide bond in conjugated bile acids to open up the bile acid pool to increased complexity. The gut microbiota performs additional chemistry on deconjugated bile acids to generate the secondary bile acid pool, which can undergo enterohepatic circulation and be reconjugated in the liver. These transformations are illustrated to the right as conjugated CA is deconjugated, subjected to 7 α-dehydroxylation to become DCA, and subsequently reconjugated. (C) Monomeric BSH overlay from Bifidobacterium longum (PDB ID 2HEZ), Enteroccocus faecalis (PDB ID 4WL3), Lactobacillus salivarius (PDB ID 5HKE), and Clostridium perfringens (PDB ID 2BJF). Hydrolyzed TDCA in the CpBSH active site is coordinated by several loops that contain the most variation in the peptide backbone compared to the other structures. BSH, bile salt hydrolase; CA, cholic acid; CpBSH, C. perfringens BSH; DCA,; TDCA, taurodeoxycholic acid; PDB ID, Protein Data Bank ID.}, number={3}, journal={PLOS PATHOGENS}, author={Foley, Matthew H. and O'Flaherty, Sarah and Barrangou, Rodolphe and Theriot, Casey M.}, editor={Knoll, Laura J.Editor}, year={2019}, month={Mar} }
 @article{o'flaherty_crawley_theriot_barrangou_2018, title={The Lactobacillus Bile Salt Hydrolase Repertoire Reveals Niche-Specific Adaptation}, volume={3}, ISSN={["2379-5042"]}, url={https://doi.org/10.1128/mSphere.00140-18}, DOI={10.1128/msphere.00140-18}, abstractNote={Various Lactobacillus species have been reported to deconjugate bile acids in the gastrointestinal tract (GIT) through the action of bile salt hydrolase (BSH) proteins. This function contributes to altering the gut microbiota composition and bile metabolism and detoxification and to lowering cholesterol levels. Here, we investigated the Lactobacillus BSH repertoire across 170 sequenced species. We used hidden Markov models to distinguish between BSH and closely related penicillin-V acylase (PVA) proteins. Even though BSH and PVA proteins have very different target substrates, they share high sequence similarity and are often misannotated. We determined that 82/170 (48.24%) species encoded PVA proteins, 39/170 (22.94%) species encoded BSH proteins, and 8/170 (4.71%) species encoded both BSH and PVA proteins, while 57/170 (33.53%) species encoded neither. Mapping the occurrence of BSH-encoding species onto a phylogenetic tree revealed that BSH-encoding lactobacilli primarily adopt the vertebrate-adapted lifestyle but not the environmental or plant-associated subsets. Phylogenetic analysis of the BSH sequences revealed two distinct clades, several conserved motifs, and the presence of six previously reported active-site residues. These data will guide future mechanistic studies of BSH activity and contribute to the development and selection of BSH-encoding Lactobacillus strains with therapeutic potential.IMPORTANCE Bile acids play an integral role in shaping the gut microbiota and host physiology by regulating metabolic signaling, weight gain, and serum cholesterol and liver triglyceride levels. Given these important roles of bile acids, we investigated the presence of bile salt hydrolase (BSH) in Lactobacillus genomes representing 170 different species, determined strain- and species-specific patterns of occurrences, and expanded on the diversity of the BSH repertoire in this genus. While our data showed that 28% of Lactobacillus species encode BSH proteins, these species are associated mainly with vertebrate-adapted niches, demonstrating selective pressure on lactobacilli to evolve to adapt to specific environments. These new data will allow targeted selection of specific strains of lactobacilli and BSH proteins for future mechanistic studies to explore their therapeutic potential for treating metabolic disorders.}, number={3}, journal={MSPHERE}, publisher={American Society for Microbiology}, author={O'Flaherty, Sarah and Crawley, Alexandra Briner and Theriot, Casey M. and Barrangou, Rodolphe}, editor={Ellermeier, Craig D.Editor}, year={2018} }
 @misc{hidalgo-cantabrana_o'flaherty_barrangou_2017, title={CRISPR-based engineering of next-generation lactic acid bacteria}, volume={37}, ISSN={["1879-0364"]}, DOI={10.1016/j.mib.2017.05.015}, abstractNote={The advent of CRISPR-based technologies has opened new avenues for the development of next-generation food microorganisms and probiotics with enhanced functionalities. Building off two decades of functional genomics studies unraveling the genetic basis for food fermentations and host-probiotic interactions, CRISPR technologies offer a wide range of opportunities to engineer commercially-relevant Lactobacillus and Bifidobacteria. Endogenous CRISPR-Cas systems can be repurposed to enhance gene expression or provide new features to improve host colonization and promote human health. Alternatively, engineered CRISPR-Cas systems can be harnessed to genetically modify probiotics and enhance their therapeutic potential to deliver vaccines or modulate the host immune response.}, journal={CURRENT OPINION IN MICROBIOLOGY}, publisher={Elsevier BV}, author={Hidalgo-Cantabrana, Claudio and O'Flaherty, Sarah and Barrangou, Rodolphe}, year={2017}, month={Jun}, pages={79–87} }
 @article{selle_goh_johnson_sarah_andersen_barrangou_klaenhammer_2017, title={Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus}, volume={8}, DOI={10.3389/fmicb.2017.00553}, abstractNote={Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.}, journal={Frontiers in Microbiology}, publisher={Frontiers Media SA}, author={Selle, Kurt and Goh, Yong J. and Johnson, Brant R. and Sarah, O’Flaherty and Andersen, Joakim M. and Barrangou, Rodolphe and Klaenhammer, Todd R.}, year={2017}, month={Apr} }
 @article{klotz_o'flaherty_goh_barrangou_2017, title={Investigating the Effect of Growth Phase on the Surface-Layer Associated Proteome of Lactobacillus acidophilus Using Quantitative Proteomics}, volume={8}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2017.02174}, abstractNote={Bacterial surface-layers (S-layers) are semi-porous crystalline arrays that self-assemble to form the outermost layer of some cell envelopes. S-layers have been shown to act as scaffolding structures for the display of auxiliary proteins externally. These S-layer associated proteins have recently gained attention in probiotics due to their direct physical contact with the intestinal mucosa and potential role in cell proliferation, adhesion, and immunomodulation. A number of studies have attempted to catalogue the S-layer associated proteome of Lactobacillus acidophilus NCFM under a single condition. However, due to the versatility of the cell surface, we chose a multiplexing-based approach with the intention of accurately contrasting multiple conditions. In this study, a previously described lithium chloride isolation protocol was used to release proteins bound to the L. acidophilus S-layer during logarithmic and early stationary growth phases. Protein quantification values were obtained via TMT (tandem mass tag) labeling combined with a triple-stage mass spectrometry (MS3) method. Results showed significant growth stage-dependent alterations to the surface-associated proteome while simultaneously highlighting the sensitivity and reproducibility of the technology. Thus, this study establishes a framework for quantifying condition-dependent changes to cell surface proteins that can easily be applied to other S-layer forming bacterium}, journal={FRONTIERS IN MICROBIOLOGY}, publisher={Frontiers Media SA}, author={Klotz, Courtney and O'Flaherty, Sarah and Goh, Yong Jun and Barrangou, Rodolphe}, year={2017}, month={Nov} }
 @article{johnson_o'flaherty_goh_carroll_barrangou_klaenhammer_2017, title={The S-layer Associated Serine Protease Homolog PrtX Impacts Cell Surface-Mediated Microbe-Host Interactions of Lactobacillus acidophilus NCFM}, volume={8}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2017.01185}, abstractNote={Health-promoting aspects attributed to probiotic microorganisms, including adhesion to intestinal epithelia and modulation of the host mucosal immune system, are mediated by proteins found on the bacterial cell surface. Notably, certain probiotic and commensal bacteria contain a surface (S-) layer as the outermost stratum of the cell wall. S-layers are non-covalently bound semi-porous, crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (SLPs). Recent evidence has shown that multiple proteins are non-covalently co-localized within the S-layer, designated S-layer associated proteins (SLAPs). In Lactobacillus acidophilus NCFM, SLP and SLAPs have been implicated in both mucosal immunomodulation and adhesion to the host intestinal epithelium. In this study, a S-layer associated serine protease homolog, PrtX (prtX, lba1578), was deleted from the chromosome of L. acidophilus NCFM. Compared to the parent strain, the PrtX-deficient strain (ΔprtX) demonstrated increased autoaggregation, an altered cellular morphology, and pleiotropic increases in adhesion to mucin and fibronectin, in vitro. Furthermore, ΔprtX demonstrated increased in vitro immune stimulation of IL-6, IL-12, and IL-10 compared to wild-type, when exposed to mouse dendritic cells. Finally, in vivo colonization of germ-free mice with ΔprtX led to an increase in epithelial barrier integrity. The absence of PrtX within the exoproteome of a ΔprtX strain caused morphological changes, resulting in a pleiotropic increase of the organisms' immunomodulatory properties and interactions with some intestinal epithelial cell components.}, journal={FRONTIERS IN MICROBIOLOGY}, publisher={Frontiers Media SA}, author={Johnson, Brant R. and O'Flaherty, Sarah and Goh, Yong Jun and Carroll, Ian and Barrangou, Rodolphe and Klaenhammer, Todd R.}, year={2017}, month={Jun} }
 @article{celebioglu_ejby_majumder_kobler_goh_thorsen_schmidt_o'flaherty_abou hachem_lahtinen_et al._2016, title={Differential proteome and cellular adhesion analyses of the probiotic bacterium Lactobacillus acidophilus NCFM grown on raffinose - an emerging prebiotic}, volume={16}, ISSN={["1615-9861"]}, DOI={10.1002/pmic.201500212}, abstractNote={Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT-29 cells increased three-fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D-DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress-related protein increasing in a range of +2.1 - +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to -2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α-galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β-galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host.}, number={9}, journal={PROTEOMICS}, author={Celebioglu, Hasan Ufuk and Ejby, Morten and Majumder, Avishek and Kobler, Carsten and Goh, Yong Jun and Thorsen, Kristian and Schmidt, Bjarne and O'Flaherty, Sarah and Abou Hachem, Maher and Lahtinen, Sampo J. and et al.}, year={2016}, month={May}, pages={1361–1375} }
 @article{o'flaherty_klaenhammer_2016, title={Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus}, volume={82}, ISSN={["1098-5336"]}, DOI={10.1128/aem.01533-16}, abstractNote={ABSTRACT Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic-cell-targeting peptide. A chromosomal region downstream of lba0889 carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids. IMPORTANCE Clostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is preferred over plasmid-based expression systems since expressing antigens from a chromosomal location confers an advantage to the vaccine strains by eliminating the antibiotic maintenance required for plasmids and negates issues with plasmid instability that would result in loss of the antigen. Lactic acid bacteria, including Lactobacillus acidophilus , have shown potential for mucosal vaccine delivery, as L. acidophilus is bile and acid tolerant, allowing transit through the gastrointestinal tract where cells interact with host epithelial and immune cells, including dendritic cells. In this study, we successfully expressed C. botulinum and B. anthracis antigens in the probiotic L. acidophilus strain NCFM. Both antigens were highly expressed individually or in tandem from the chromosome of L. acidophilus .}, number={20}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={O'Flaherty, Sarah and Klaenhammer, Todd R.}, year={2016}, month={Oct}, pages={6091–6101} }
 @article{sanozky-dawes_selle_o'flaherty_klaenhammer_barrangou_2015, title={Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri}, volume={161}, ISSN={["1350-0872"]}, DOI={10.1099/mic.0.000129}, abstractNote={Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 5′-NTAA-3′ flanking the 3′ end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.}, journal={MICROBIOLOGY-SGM}, author={Sanozky-Dawes, Rosemary and Selle, Kurt and O'Flaherty, Sarah and Klaenhammer, Todd and Barrangou, Rodolphe}, year={2015}, month={Sep}, pages={1752–1761} }
 @article{call_goh_selle_klaenhammer_o'flaherty_2015, title={Sortase-deficient lactobacilli: effect on immunomodulation and gut retention}, volume={161}, ISSN={["1350-0872"]}, DOI={10.1099/mic.0.000007}, abstractNote={Surface proteins of probiotic microbes, including Lactobacillus acidophilus and Lactobacillus gasseri, are believed to promote retention in the gut and mediate host–bacterial communications. Sortase, an enzyme that covalently couples a subset of extracellular proteins containing an LPXTG motif to the cell surface, is of particular interest in characterizing bacterial adherence and communication with the mucosal immune system. A sortase gene, srtA, was identified in L. acidophilus NCFM (LBA1244) and L. gasseri ATCC 33323 (LGAS_0825). Additionally, eight and six intact sortase-dependent proteins were predicted in L. acidophilus and L. gasseri, respectively. Due to the role of sortase in coupling these proteins to the cell wall, ΔsrtA deletion mutants of L. acidophilus and L. gasseri were created using the upp-based counterselective gene replacement system. Inactivation of sortase did not cause significant alteration in growth or survival in simulated gastrointestinal juices. Meanwhile, both ΔsrtA mutants showed decreased adhesion to porcine mucin in vitro. Murine dendritic cells exposed to the ΔsrtA mutant of L. acidophilus or L. gasseri induced lower levels of pro-inflammatory cytokines TNF-α and IL-12, respectively, compared with the parent strains. In vivo co-colonization of the L. acidophilus ΔsrtA mutant and its parent strain in germ-free 129S6/SvEv mice resulted in a significant one-log reduction of the ΔsrtA mutant population. Additionally, a similar reduction of the ΔsrtA mutant was observed in the caecum. This study shows for the first time that sortase-dependent proteins contribute to gut retention of probiotic microbes in the gastrointestinal tract.}, journal={MICROBIOLOGY-SGM}, author={Call, Emma K. and Goh, Yong Jun and Selle, Kurt and Klaenhammer, Todd R. and O'Flaherty, Sarah}, year={2015}, month={Feb}, pages={311–321} }
 @article{o'flaherty_2014, title={Probiotic-mediated modulation of host gene expression}, DOI={10.1201/b16439-5}, journal={Interactive Probiotics}, author={O'Flaherty, S.}, year={2014}, pages={81–98} }
 @article{johnson_selle_o'flaherty_goh_klaenhammer_2013, title={Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM}, volume={159}, ISSN={["1350-0872"]}, DOI={10.1099/mic.0.070755-0}, abstractNote={Bacterial surface (S-) layers are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. The S-layer-forming bacterium Lactobacillus acidophilus NCFM expresses three major Slps: SlpA (46 kDa), SlpB (47 kDa) and SlpX (51 kDa). SlpA has a demonstrated role in adhesion to Caco-2 intestinal epithelial cells in vitro, and has been shown to modulate dendritic cell (DC) and T-cell functionalities with murine DCs. In this study, a modification of a standard lithium chloride S-layer extraction revealed 37 proteins were solubilized from the S-layer wash fraction. Of these, 30 have predicted cleavage sites for secretion, 24 are predicted to be extracellular, six are lipid-anchored, three have N-terminal hydrophobic membrane spanning regions and four are intracellular, potentially moonlighting proteins. Some of these proteins, designated S-layer associated proteins (SLAPs), may be loosely associated with or embedded within the bacterial S-layer complex. Lba-1029, a putative SLAP gene, was deleted from the chromosome of L. acidophilus. Phenotypic characterization of the deletion mutant demonstrated that the SLAP LBA1029 contributes to a pro-inflammatory TNF-α response from murine DCs. This study identified extracellular proteins and putative SLAPs of L. acidophilus NCFM using LC-MS/MS. SLAPs appear to impart important surface display features and immunological properties to microbes that are coated by S-layers.}, journal={MICROBIOLOGY-SGM}, author={Johnson, Brant and Selle, Kurt and O'Flaherty, Sarah and Goh, Yong Jun and Klaenhammer, Todd}, year={2013}, month={Nov}, pages={2269–2282} }
 @article{o'flaherty_klaenhammer_2012, title={Influence of Exposure Time on Gene Expression by Human Intestinal Epithelial Cells Exposed to Lactobacillus acidophilus}, volume={78}, ISSN={["0099-2240"]}, DOI={10.1128/aem.00504-12}, abstractNote={Analysis of global temporal gene expression by human intestinal cells when exposed to Lactobacillus acidophilus revealed induction of immune-related pathways and NF-κB target genes after a 1-h exposure, compared to a 4- or 8-h exposure. Additionally, an L. acidophilus derivative expressing covalently bound flagellin resulted in increased induction of il8, cxc1, and cxcl2 compared to the parent L. acidophilus.}, number={14}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={O'Flaherty, Sarah and Klaenhammer, Todd R.}, year={2012}, month={Jul}, pages={5028–5032} }
 @article{mohamadzadeh_pfeiler_brown_zadeh_gramarossa_managlia_bere_sarraj_khan_pakanati_et al._2011, title={Regulation of induced colonic inflammation by Lactobacillus acidophilus deficient in lipoteichoic acid}, volume={108}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1005066107}, abstractNote={Imbalance in the regulatory immune mechanisms that control intestinal cellular and bacterial homeostasis may lead to induction of the detrimental inflammatory signals characterized in humans as inflammatory bowel disease. Induction of proinflammatory cytokines (i.e., IL-12) induced by dendritic cells (DCs) expressing pattern recognition receptors may skew naive T cells to T helper 1 polarization, which is strongly implicated in mucosal autoimmunity. Recent studies show the ability of probiotic microbes to treat and prevent numerous intestinal disorders, including Clostridium difficile -induced colitis. To study the molecular mechanisms involved in the induction and repression of intestinal inflammation, the phosphoglycerol transferase gene that plays a key role in lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus NCFM (NCK56) was deleted. The data show that the L. acidophilus LTA-negative in LTA (NCK2025) not only down-regulated IL-12 and TNFα but also significantly enhanced IL-10 in DCs and controlled the regulation of costimulatory DC functions, resulting in their inability to induce CD4 + T-cell activation. Moreover, treatment of mice with NCK2025 compared with NCK56 significantly mitigated dextran sulfate sodium and CD4 + CD45RB high T cell-induced colitis and effectively ameliorated dextran sulfate sodium-established colitis through a mechanism that involves IL-10 and CD4 + FoxP3 + T regulatory cells to dampen exaggerated mucosal inflammation. Directed alteration of cell surface components of L. acidophilus NCFM establishes a potential strategy for the treatment of inflammatory intestinal disorders.}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Mohamadzadeh, Mansour and Pfeiler, Erika A. and Brown, Jeffrey B. and Zadeh, Mojgan and Gramarossa, Matthew and Managlia, Elizabeth and Bere, Praveen and Sarraj, Bara and Khan, Mohammad W. and Pakanati, Krishna Chaitanya and et al.}, year={2011}, month={Mar}, pages={4623–4630} }
 @article{goh_goin_o'flaherty_altermann_hutkins_2011, title={Specialized adaptation of a lactic acid bacterium to the milk environment: the comparative genomics of Streptococcus thermophilus LMD-9}, volume={10}, ISSN={["1475-2859"]}, DOI={10.1186/1475-2859-10-s1-s22}, abstractNote={Abstract Background Streptococcus thermophilus represents the only species among the streptococci that has “Generally Regarded As Safe” status and that plays an economically important role in the fermentation of yogurt and cheeses. We conducted comparative genome analysis of S. thermophilus LMD-9 to identify unique gene features as well as features that contribute to its adaptation to the dairy environment. In addition, we investigated the transcriptome response of LMD-9 during growth in milk in the presence of Lactobacillus delbrueckii ssp. bulgaricus , a companion culture in yogurt fermentation, and during lytic bacteriophage infection. Results The S. thermophilus LMD-9 genome is comprised of a 1.8 Mbp circular chromosome (39.1% GC; 1,834 predicted open reading frames) and two small cryptic plasmids. Genome comparison with the previously sequenced LMG 18311 and CNRZ1066 strains revealed 114 kb of LMD-9 specific chromosomal region, including genes that encode for histidine biosynthetic pathway, a cell surface proteinase, various host defense mechanisms and a phage remnant. Interestingly, also unique to LMD-9 are genes encoding for a putative mucus-binding protein, a peptide transporter, and exopolysaccharide biosynthetic proteins that have close orthologs in human intestinal microorganisms. LMD-9 harbors a large number of pseudogenes (13% of ORFeome), indicating that like LMG 18311 and CNRZ1066, LMD-9 has also undergone major reductive evolution, with the loss of carbohydrate metabolic genes and virulence genes found in their streptococcal counterparts. Functional genome distribution analysis of ORFeomes among streptococci showed that all three S. thermophilus strains formed a distinct functional cluster, further establishing their specialized adaptation to the nutrient-rich milk niche. An upregulation of CRISPR1 expression in LMD-9 during lytic bacteriophage DT1 infection suggests its protective role against phage invasion. When co-cultured with L. bulgaricus , LMD-9 overexpressed genes involved in amino acid transport and metabolism as well as DNA replication. Conclusions The genome of S. thermophilus LMD-9 is shaped by its domestication in the dairy environment, with gene features that conferred rapid growth in milk, stress response mechanisms and host defense systems that are relevant to its industrial applications. The presence of a unique exopolysaccharide gene cluster and cell surface protein orthologs commonly associated with probiotic functionality revealed potential probiotic applications of LMD-9.}, journal={MICROBIAL CELL FACTORIES}, author={Goh, Yong Jun and Goin, Caitlin and O'Flaherty, Sarah and Altermann, Eric and Hutkins, Robert}, year={2011}, month={Aug} }
 @article{o'flaherty_klaenhammer_2011, title={The Impact of Omic Technologies on the Study of Food Microbes}, volume={2}, ISBN={["978-0-8243-4902-8"]}, ISSN={["1941-1421"]}, DOI={10.1146/annurev-food-030810-110338}, abstractNote={The advent of the molecular biology era in the 1950s and the subsequent emergence of new technologies positively impacted on all areas of biology. New discoveries in molecular biology and experimental tools were developed over the next 60 years that have revolutionized the study of food microbiology. Previously, food microbiology relied on classic microbiology techniques, which had remained relatively unchanged since the discoveries of Louis Pasteur in the 1800s. More recently, new advances resulting in “omic” technologies have exploded the areas of genomics, transcriptomics, and proteomics and revealed many fundamental processes driven by both pathogens and commensals. This review outlines advances in omic technologies and how these have impacted food microbiology through providing examples of recently published landmark work.}, journal={ANNUAL REVIEW OF FOOD SCIENCE AND TECHNOLOGY, VOL 2}, author={O'Flaherty, Sarah and Klaenhammer, Todd R.}, year={2011}, pages={353–371} }
 @article{sarah j. o'flaherty_klaenhammer_2010, title={Functional and phenotypic characterization of a protein from Lactobacillus acidophilus involved in cell morphology, stress tolerance and adherence to intestinal cells}, volume={156}, ISSN={["1465-2080"]}, DOI={10.1099/mic.0.043158-0}, abstractNote={Structural components of the cell surface have an impact on some of the beneficial attributes of probiotic bacteria. In silico analysis of the L. acidophilus NCFM genome sequence revealed the presence of a putative cell surface protein that was predicted to be a myosin cross-reactive antigen (MCRA). As MCRAs are conserved among many probiotic bacteria, we used the upp -based counterselective gene replacement system, designed recently for use in L. acidophilus , to determine the functional role of this gene (LBA649) in L. acidophilus NCFM. Phenotypic assays were undertaken with the parent strain (NCK1909) and deletion mutant (NCK2015) to assign a function for this gene. The growth of NCK2015 (ΔLBA649) was reduced in the presence of lactate, acetate, porcine bile and salt. Adhesion of NCK2015 to Caco-2 cells was substantially reduced for both stationary-phase (∼45 % reduction) and exponential-phase cells (∼50 % reduction). Analysis of NCK2015 by scanning electron microscopy revealed a longer cell morphology after growth in MRS broth compared to NCK1909. These results indicate a role for LBA649 in stress tolerance, cell wall division and adherence to Caco-2 cells.}, journal={MICROBIOLOGY-SGM}, author={Sarah J. O'Flaherty and Klaenhammer, Todd R.}, year={2010}, month={Nov}, pages={3360–3367} }
 @article{o'flaherty_klaenhammer_2010, title={The role and potential of probiotic bacteria in the gut, and the communication between gut microflora and gut/host}, volume={20}, ISSN={["1879-0143"]}, DOI={10.1016/j.idairyj.2009.11.011}, abstractNote={Recent research efforts have focused on understanding the interactions of probiotic bacteria with commensal gut bacteria and the human host as a means to determine mechanisms of probiotic functionality that contribute to their beneficial attributes. Our growing understanding of the intrinsic interactions between probiotic and commensal bacteria and between the milieu of bacteria and the host tissues of the gastrointestinal tract (GIT) has been facilitated by the use of ‘omic’ technologies. Surveys of bacterial inhabitants in the GIT using sequencing technologies have demonstrated the complexities of this human organ which varies between different populations and individuals, such as diet. In addition, transcriptomics have rapidly facilitated an insight into the complex communication between bacteria (commensal and probiotic) and the GIT. This review outlines the recent important advances in this exciting area of research, which has led to a greater understanding of the critical interface between gut microbiota, probiotic bacteria and the host.}, number={4}, journal={INTERNATIONAL DAIRY JOURNAL}, author={O'Flaherty, Sarah and Klaenhammer, Todd R.}, year={2010}, month={Apr}, pages={262–268} }
 @article{goh_azcarate-peril_o'flaherty_durmaz_valence_jardin_lortal_klaenhammer_2009, title={Development and Application of a upp-Based Counterselective Gene Replacement System for the Study of the S-Layer Protein SlpX of Lactobacillus acidophilus NCFM}, volume={75}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.02502-08}, abstractNote={ABSTRACT In silico genome analysis of Lactobacillus acidophilus NCFM coupled with gene expression studies have identified putative genes and regulatory networks that are potentially important to this organism's survival, persistence, and activities in the gastrointestinal tract. Correlation of key genotypes to phenotypes requires an efficient gene replacement system. In this study, use of the upp -encoded uracil phosphoribosyltransferase (UPRTase) of L. acidophilus NCFM was explored as a counterselection marker to positively select for recombinants that have resolved from chromosomal integration of pORI-based plasmids. An isogenic mutant carrying a upp gene deletion was constructed and was resistant to 5-fluorouracil (5-FU), a toxic uracil analog that is also a substrate for UPRTase. A 3.0-kb pORI-based counterselectable integration vector bearing a upp expression cassette, pTRK935, was constructed and introduced into the Δ upp host harboring the pTRK669 helper plasmid. Extrachromosomal replication of pTRK935 complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. This host background provides a platform for a two-step plasmid integration and excision strategy that can select for plasmid-free recombinants with either the wild-type or mutated allele of the targeted gene in the presence of 5-FU. The efficacy of the system was demonstrated by in-frame deletion of the slpX gene (LBA0512) encoding a novel 51-kDa secreted protein associated with the S-layer complex of L. acidophilus . The resulting Δ slpX mutant exhibited lower growth rates, increased sensitivity to sodium dodecyl sulfate, and greater resistance to bile. Overall, this improved gene replacement system represents a valuable tool for investigating the mechanisms underlying the probiotic functionality of L. acidophilus .}, number={10}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Goh, Yong Jun and Azcarate-Peril, M. Andrea and O'Flaherty, Sarah and Durmaz, Evelyn and Valence, Florence and Jardin, Julien and Lortal, Sylvie and Klaenhammer, Todd R.}, year={2009}, month={May}, pages={3093–3105} }
 @misc{ventura_o'flaherty_claesson_turroni_klaenhammer_sinderen_paul w. o'toole_2009, title={Genome-scale analyses of health-promoting bacteria: probiogenomics}, volume={7}, ISSN={["1740-1534"]}, DOI={10.1038/nrmicro2047}, number={1}, journal={NATURE REVIEWS MICROBIOLOGY}, author={Ventura, Marco and O'Flaherty, Sarah and Claesson, Marcus J. and Turroni, Francesca and Klaenhammer, Todd R. and Sinderen, Douwe and Paul W. O'Toole}, year={2009}, month={Jan}, pages={61–U77} }
 @article{azcarate-peril_altermann_goh_tallon_sanozky-dawes_pfeiler_o'flaherty_buck_dobson_duong_et al._2008, title={Analysis of the genome sequence of Lactobacillus gasseri ATCC 33323 reveals the molecular basis of an autochthonous intestinal organism}, volume={74}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.00054-08}, abstractNote={ABSTRACT This study presents the complete genome sequence of Lactobacillus gasseri ATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives, L. johnsonii NCC 533 (34%) and L. acidophilus NCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-like Siphoviridae phage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome of L. gasseri that were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche. L. gasseri encodes two restriction and modification systems, which may limit bacteriophage infection. L. gasseri also encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that of B. caccae ATCC 43185, a bacterial species isolated from human feces. In addition, L. gasseri encoded the highest number of putative mucus-binding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other human L. gasseri strains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.}, number={15}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Azcarate-Peril, M. Andrea and Altermann, Eric and Goh, Yong Jun and Tallon, Richard and Sanozky-Dawes, Rosemary B. and Pfeiler, Erika A. and O'Flaherty, Sarah and Buck, B. Logan and Dobson, Alleson and Duong, Tri and et al.}, year={2008}, month={Aug}, pages={4610–4625} }
 @article{klaenhammer_altermann_pfeiler_buck_goh_o'flaherty_barrangou_duong_2008, title={Functional genomics of probiotic Lactobacilli}, volume={42}, ISSN={["0192-0790"]}, DOI={10.1097/MCG.0b013e31817da140}, abstractNote={Lactic acid bacteria (LAB) have been used in fermentation processes for millennia. Recent applications such as the use of living cultures as probiotics have significantly increased industrial interest. Related bacterial strains can differ significantly in their genotype and phenotype, and features from one bacterial strain or species cannot necessarily be applied to a related one. These strain or family-specific differences often represent unique and applicable traits. Since 2002, the complete genomes of 13 probiotic LABs have been published. The presentation will discuss these genomes and highlight probiotic traits that are predicted, or functionally linked to genetic content. We have conducted a comparative genomic analysis of 4 completely sequenced Lactobacillus strains versus 25 lactic acid bacterial genomes present in the public database at the time of analysis. Using Differential Blast Analysis, each genome is compared with 3 other Lactobacillus and 25 other LAB genomes. Differential Blast Analysis highlighted strain-specific genes that were not represented in any other LAB used in this analysis and also identified group-specific genes shared within lactobacilli. Lactobacillus-specific genes include mucus-binding proteins involved in cell-adhesion and several transport systems for carbohydrates and amino acids. Comparative genomic analysis has identified gene targets in Lactobacillus acidophilus for functional analysis, including adhesion to mucin and intestinal epithelial cells, acid tolerance, bile tolerance, and quorum sensing. Whole genome transcriptional profiling of L. acidophilus, and isogenic mutants thereof, has revealed the impact of varying conditions (pH, bile, carbohydrates) and food matrices on the expression of genes important to probiotic-linked mechanisms.}, number={8}, journal={JOURNAL OF CLINICAL GASTROENTEROLOGY}, publisher={Ovid Technologies (Wolters Kluwer Health)}, author={Klaenhammer, Todd R. and Altermann, Eric and Pfeiler, Erika and Buck, Brock Logan and Goh, Yong-Jun and O'Flaherty, Sarah and Barrangou, Rodolphe and Duong, Tri}, year={2008}, month={Sep}, pages={S160–S162} }