Progress 10/01/19 to 09/30/20
Outputs
Target Audience:This project serves research scientists, veterinary practitioners, graduate students, postdoctoral fellows, farm managers, and equine industry stakeholders. Changes/Problems:The COVID-19 pandemic has significantly impacted our research landscape and timeline. We are currently performing studies outlined in this proposal under University guidelines andrestrictions limiting the number of personnel and on campus research activities. What opportunities for training and professional development has the project provided?During the current review period, a Doctor of Veterinary Medicine/PhD dual degree graduate student, a postdoctoral scholar, and two undergraduate research assistants were trained in basic and applied research. How have the results been disseminated to communities of interest?Results of these studies have been presented in regional academic seminars with a diverse audience of farm managers, equine practitioners, research scientists, graduate and professional students, and equine industry stakeholders. What do you plan to do during the next reporting period to accomplish the goals?We plan to submit the results from studies performed in Aims 1-3 for publication in peer-reviewed journals. We are also pursuing additional extramural funding to support studies outlined in Aims 1 and 2 (Grayson-Jockey Research Foundation, submitted October 2020). During the next reporting period, we plan to execute several in vitro studies aimed at analyzing our R. equi transposon mutant library. We will specifically focus on identifying and characterizing mutants that are unable to colonize equine airway organoids in co-culture with host-derived immune cells. For Specific Aim 3, we will continue to pursue identification of novel chemical scaffolds that reverse R. equi antimicrobial resistance and modulate pathogenesis. For example, we will test whether combinations of heterocyclic 2-pyridones can synergistically interfere with bacterial cell viability or macrophage colonization in conjunction with existing antimicrobial therapies.
Impacts
What was accomplished under these goals?
Please note that research progress was significantly impacted by the COVID-19 pandemic. Our laboratory has recently re-opened to allow bench work with reduced personnel. Many pathogenic bacteria have the remarkable ability to infect multiple hosts and distinct sites within the body. Rhodococcus equi, a soil-dwelling bacterium, is the most common cause of severe bronchopneumonia in young foals. Additionally, R. equi has been associated with a wide variety of diseases in other animals, and it is recognized as an emerging zoonotic pathogen that affects immunocompromised humans. Although R. equi has been widely studied, we lack a complete understanding of the bacterial factors that are required for host colonization and pathogenicity. The proposed research will uncover novel bacterial factors that are required for equine infection and respiratory disease, and will pinpoint decisive microbial checkpoints that can be targeted for development of innovative therapeutics to treat and prevent equine rhodococcal infection. Specific Aim 1: Identify R. equi genes required for colonization of the equine lung. The primary objective of this specific aim is to construct a robust R. equi transposon mutant library that will be exploited in foal challenge studies and in vitro assays. We previously optimized protocols to generate a library of R. equitransposon insertion mutants usingin vivo transposition mediated by theTn5 transposome. We have constructed a pilot transposon insertion library consisting of approximately 425 individual transposon mutants.In initial screens, we identified transposon insertion mutants that exhibit altered phenotypes and growth characteristics compared to the parental wild-type (WT) strain. To assess whether individual transposon insertion mutants are nutritional auxotrophs, we will evaluate the ability of individual mutants to replicate in minimal medium. Auxotrophic mutants that fail to replicate in minimal medium (representing mutants with probable impaired fitness in the equine host) will be selected for further analysis as potential vaccine strains. Currently, we are working to map the transposon insertion site within individual mutants of intertest. We have also developed new in vitro models of equine lung and tracheal tissue that will be used to assess R. equi pathogenicity and host colonization. Studies to engineer equine airway organoids were spearheaded by postdoctoral scholar Dr. Cecily Wood. Thus far, we have generated equine lung and tracheal organoids that can be passaged in three-dimensional (3D) cell culture and transitioned to two-dimensional systems that become fully differentiated within approximately 21 days. We have shown that bronchioalveolar stem cells and basal cells differentiate into various lunglineages, develop ciliated cells that coordinate beating of mucus, and exhibit characteristics of the native horse airway. Using this innovative system, we have performed studies to demonstrate that differentiated airway cells support equine influenza A (EIA) and equine herpes virus 1 (EHV-1) infection. We have maintained passage of our 3D airway organoid systems for approximately 1 year, demonstrating the ability of equine organoids to self-renew and expand. These studies have been incorporated into a manuscript that is currently in preparation for peer-reviewed publication.This unique resource provides a renewable, robust system that mimics the native equine airway and enables high-impact equine respiratory infectious disease pathogenesis studies in vitro. Specific Aim 2. Delineate genetic requirements for R. equi replication within macrophage-like cells. In concert with studies outlined in Aim 1, we are focusing our efforts on characterizing the biogenesis and function of R. equi cytoadhesive pili. Using electron microscopy, we have identified protein pilus systems that are assembled in response to host cell contact and appear to play a role in bacterial adhesion to the host cell and biofilm formation. In complementary experiments, we are characterizing the R. equi cell surface using biochemical labeling and purification coupled to mass spectrometry to identify cell surface appendages required for host cell interactions. These studies are being conducted by an undergraduate student in the Agricultural and Medical Biotechnology program (Erin McMahon), and formed the basis of her ABT395 Independent Study in Biotechnology project completed in the Spring of 2020. Using membrane-impermeable biotin labeling and purification techniques, we identified several R. equi cell surface proteins that potentially mediate interactions with host cells. Comparison of cell surface proteins produced by bacteria cultured under various conditions revealed several proteins and nutrient transport systems (potassium transporters and putative permease proteins) that are differentially localized depending on environmental factors. Additionally, we identified components of the cytoadhesive Rhodococcus pilus (Rpl) system on the bacterial cell surface. We are currently undertaking genetic-based approaches to delineate Rpl pilus protein-protein interactions in order to build a model of R. equi pilus architecture. These studies will characterize pathogenesis-related cell surface structures that can be targeted for disruption by novel anti-virulence compounds. Specific Aim 3. Identify chemical scaffolds that disrupt R. equi interactions with host cells. We have recently characterized a class of synthetic small molecule inhibitors that disarm virulence mechanisms in diverse bacterial pathogens, and we predict that similar chemical scaffolds will interfere with R. equi replication and survival. Using virulent R. equi, we have evaluated the capacity of several synthetic small moleculesto prevent R. equi replication in pure bacterial culture or within macrophage-like cells. Inspired by studies performed in the closely-related Mycobacterium tuberculosis, we conducted experiments to analyze whether synthetic small molecules could reverse antimicrobial resistance in R. equi. We have identified several chemical scaffolds that render multi-drug resistant R. equi susceptible to sub-inhibitory concentrations of clarithromycin. We are currently pursuing studies to identify small molecule mechanism(s) of action in synergistic combination with existing antimicrobial therapies. Additionally, we have characterized silver nanoparticle compounds that significantly reduce R. equi viability.The results of these investigations are included in a manuscript that is currently in preparation. In collaboration with investigators at the University of Kentucky Veterinary Diagnostic Lab, we are also performing studies to identify new therapeutic combinations that effectively eliminate R. equi from the equine respiratory tract and protected intracellular sites. These studies are currently being conducted by a DVM/PhD dual-degree student (Lynn Leedhanachoke, DVM). We have identified several chemotherapies and antimicrobial combination therapies that target and effectively kill both extracellular and intracellular R. equi at effective concentrations achievable in equine and human plasma. These studies have also identified several alternate antibiotic combinations for treating R. equi clinical isolates exhibiting multi-drug resistance. Our studies will ultimately provide the basis for the new therapeutic strategies that can be immediately deployed in the clinical setting to treat and rhodococcal disease. Individuals:Carrie L. Shaffer, PhD (PI),Lynn Leedhanachoke, DVM (PhD Student),Cecily R. Wood, PhD (Postdoctoral Scholar),Erin McMahon (undergraduate research assistant),Rachel Klueppel (undergraduate research assistant)
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Erol E, Shaffer CL, Lubbers BV (2020 in revision). Synergistic combination of clarithromycin and doxycycline reduces the emergence of antimicrobial resistance in Rhodococcus equi. Equine Veterinary Journal (in revision).
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2020
Citation:
Boudaher, E. (2020) Evaluation of Rhodococcus equi susceptibility to silver nanoparticle antimicrobials. Master of Science Thesis, University of Kentucky, Lexington, KY.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Anderson SL, Achilles SL, Wooton KE, Wisnieski L, Hunt JA, Shaffer CL (2020, under review). The impact of gel nail polish application on log reduction of bacterial viability following a surgical hand scrub. Veterinary Surgery (under review).
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:This project serves research scientists, veterinary practitioners, graduate students, postdoctoral fellows, farm managers, and equine industry stakeholders. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the current review period, a graduate student, postdoctoral scholar, an undergraduate research assistant, and a visiting Doctor of Veterinary Medicine student were trained in basic and applied research. How have the results been disseminated to communities of interest?Results of these studies have been presented in regional academic seminars with a diverse audience of farm managers, equine practitioners, research scientists, graduate and professional students, and equine industry stakeholders. What do you plan to do during the next reporting period to accomplish the goals?We plan to submit the results from studies performed in Aim 3 for publication in a peer-reviewed journal. We are also pursuing additional extramural funding to support the studies in Aims.1 and 2. During the next reporting period, we plan to execute several in vitro studies aimed at analyzing our R. equi transposon mutant library. We will specifically focus on identifying and characterizing mutants that are unable to colonize equine airway organoids in co-culture with host-derived immune cells. For Specific Aim 3, we will continue to pursue identification of novel chemical scaffolds that affect R. equi viability and pathogenesis. For example, we will test whether combinations of heterocyclic 2-pyridones can synergistically interfere with bacterial cell viability in conjunction with existing antimicrobial therapies.
Impacts
What was accomplished under these goals?
Many pathogenic bacteria have the remarkable ability to infect multiple hosts and distinct sites within the body. Rhodococcus equi, a soil-dwelling bacterium, is the most common cause of severe bronchopneumonia in young foals. Additionally, R. equi has been associated with a wide variety of diseases in other animals, and it is recognized as an emerging zoonotic pathogen that affects immunocompromised humans. Although R. equi has been widely studied, we lack a complete understanding of the bacterial factors that are required for host colonization and pathogenicity. The proposed research will uncover novel bacterial factors that are required for equine infection and respiratory disease, and will pinpoint decisive microbial checkpoints that can be targeted for development of innovative therapeutics to treat and prevent equine rhodococcal infection. Specific Aim 1: Identify R. equi genes required for colonization of the equine lung. The primary objective of this specific aim is to construct a robust R. equi transposon mutant library in a well-characterized, clinical strain that will be exploited in foal challenge studies and in vitro assays. We selected the fully-sequenced R. equi strain 103S for construction of a transposon insertion mutant library. In the previous year, we have optimized bacterial cell competency for in vivo transposition mediated by a Tn5 transposome. Use of an in vivo transposome allows for the random introduction of a kanamycin resistance cassette into target DNA without the requirement of additional bacterial factors to facilitate the transposition event. The transposome consists of a stable complex formed between the Tn5 transposase enzyme and the transposon harboring the Tn903 kanamycin-resistance gene (previously shown to be functional in R. equi) flanked by hyperactive 19-basepair mosaic end Tn5 recognition sequences. We have constructed a pilot transposon insertion library consisting of approximately 425 individual transposon mutants. In initial screens, we identified transposon insertion mutants that exhibit altered colony pigmentation phenotypes and growth characteristics compared to the parental wild-type (WT) strain. To assess whether individual transposon insertion mutants are nutritional auxotrophs, we will evaluate the ability of individual mutants to replicate in minimal medium. Auxotrophic mutants that fail to replicate in minimal medium (representing mutants with probable impaired fitness in the equine host) will be selected for further analysis as potential vaccine strains. Currently, we are working to map the transposon insertion site within individual mutants of intertest. These experiments are being conducted by an undergraduate student in the Agricultural and Medical Biotechnology program. We are prioritizing the development of protocols that will generate super-competent R. equi cells that will be used to construct a more comprehensive, saturated transposon mutant (requiring the generation of approximately 20,000 individual transposon insertion mutants to reach 4x genomic coverage). We expect that a robust transposon library can be generated by combining mutants resulting from several independent rounds of transposition in R. equi. Generation of a saturated library will allow us to advance animal challenge models that will uncover multiple R. equi genes and genetic pathways that are required for efficient colonization of the equine lung. We have also developed new in vitro models of equine lung and tracheal tissue that will be used to assess R. equi pathogenicity and host colonization. Thus far, we have generated equine distal lung and tracheal organoids that can be passaged in three-dimensional cell culture. These organoids differentiate into various lineages of equine lung tissue, develop ciliated cells that coordinate beating of mucus in the organoid lumen, and exhibit characteristics of the native horse airway. To our knowledge, our work represents the first description of equine respiratory system organoids - this unique resource provides a renewable, robust system that mimics the native equine lung and enables high-impact R. equi pathogenesis studies in vitro. Specific Aim 2. Delineate genetic requirements for R. equi replication within macrophage-like cells. In concert with studies outlined in Aim 1, we are characterizing the R. equi cell surface using biochemical labeling and purification coupled to mass spectrometry. These studies are designed to identify bacterial machinery and cell surface appendages required for host cell interactions. We are currently focusing our efforts on characterizing the biogenesis and function of R. equi cytoadhesive pili. Using electron microscopy, we have identified protein pilus systems that are assembled in response to host cell contact and appear to play a role in bacterial adhesion to the host cell and biofilm formation. We are currently undertaking genetic-based approaches to delineate pilus protein-protein interactions in order to build a model of R. equi pilus architecture. These studies will characterize pathogenesis-related cell surface structures that can be targeted for disruption by novel anti-virulence compounds. Specific Aim 3. Identify chemical scaffolds that disrupt R. equi interactions with host cells. We have recently characterized a class of synthetic small molecule inhibitors that disarm virulence mechanisms in diverse bacterial pathogens, and we predict that similar chemical scaffolds will interfere with R. equi replication and survival. Using virulent R. equi, we have evaluated the capacity of several synthetic small molecules and metal-based compounds to prevent R. equi replication in pure bacterial culture or within macrophage-like cells. While we did not identify a synthetic small molecule that interferes with R. equi viability in our initial screen, we have identified silver nanoparticle compounds that significantly reduce R. equi viability. We determined that silver nanoparticle complexes kill R. equi in pure culture within four to six hours of treatment. Our studies also demonstrate that silver nanoparticles are acutely toxic to mouse macrophage-like cells and equine pulmonary endothelial cells. We determined that silver nanoparticle complexes induce rapid apoptosis and secondary necrosis in host cell lines. We are currently pursuing studies aimed at resolving the mechanism underlying metal toxicity in R. equi. The results of these investigations are included in a manuscript that will be submitted for publication in early 2020, and will be published in aMasters of Science thesis in 2020(Elizabeth Boudaher). In collaboration with investigators at the University of Kentucky Veterinary Diagnostic Lab, we are also performing studies to identify new therapeutic combinations that effectively eliminate R. equi from protected intracellular sites. We have identified several chemotherapies that target and effectively kill both extracellular and intracellular R. equiat bioavailable concentrations inequine/human plasma.These studies have also identified several alternativeantibiotics for treating clinical isolates of multidrug (rifamin and macrolide) resistant R. equi. Our studies will ultimately provide the basis for the new therapeutic strategies that can be immediately deployed in the clinical setting to treat and prevent rhodococcal disease. Individuals: Carrie L. Shaffer, PhD (PI) Elizabeth Boudaher (Master of Science student) Cecily R. Wood, PhD (Postdoctoral Scholar) Erin McMahon (Agricultural and Medical Biotechnology undergraduate research assistant)
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Boudaher, E and CL Shaffer (2019). Inhibiting bacterial secretion systems in the fight against antibiotic resistance. MedChemComm 10(5):682-692. (PMID: 31741728)
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Scare JA, AE Steuer, CL Shaffer, P Slusarewicz, A Mousley, MK Nielsen (2018). Long live the worms: methods for maintaining and assessing the viability of intestinal stages of Parascaris spp. in vitro. Parasitology 18:1-9. (PMID: 30561286)
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Progress 02/21/18 to 09/30/18
Outputs
Target Audience:This project serves research scientists, veterinary practitioners, graduate students, postdoctoral fellows, farm managers, and equine industry stakeholders. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the current review period, a PhD graduate student, undergraduate Agricultural Biotechnology students, and a visiting Doctor of Veterinary Medicine student were trained in basic and applied research. How have the results been disseminated to communities of interest?Results of these studies have been presented in regional academic seminars to a diverse audience of farm managers, equine practitioners, research scientists, graduate and professional students, and equine industry stakeholders. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to execute several in vitro studies aimed at analyzing our R. equi transposon mutant library. We will specifically focus on identifying and characterizing mutants that are unable to replicate within host immune cells. In parallel, we will evaluate R. equi transcriptional activity by RNAseq in order to delineate global genetic regulation patterns that are altered in response to various environmental stimuli and during interaction with equine cells. For Specific Aim 3, we will continue to pursue identification of novel chemical scaffolds that affect R. equi viability and pathogenesis. For example, we will test whether combinations of heterocyclic 2-pyridones can synergistically interfere with bacterial cell viability in conjunction with existing antimicrobial therapies.
Impacts
What was accomplished under these goals?
Many pathogenic bacteria have the remarkable ability to infect multiple hosts and distinct sites within the body. Rhodococcus equi, a soil-dwelling bacterium, is the most common cause of severe bronchopneumonia in young foals. Additionally, R. equi has been associated with a wide variety of diseases in other animals, and it is recognized as an emerging zoonotic pathogen that affects immunocompromised humans. Although R. equi has been widely studied, we lack a complete understanding of the bacterial factors that are required for host colonization and pathogenicity. The proposed research will uncover novel bacterial factors that are required for equine infection and respiratory disease, and will pinpoint decisive microbial checkpoints that can be targeted for development of innovative therapeutics to treat and prevent equine rhodococcal infection. Specific Aim 1: Identify R. equi genes required for colonization of the equine lung. The primary objective of this specific aim is to construct a robust R. equi transposon mutant library in a well-characterized, clinical strain that will be exploited in foal challenge studies and in vitro assays. We selected the fully-sequenced R. equi strain 103S for construction of a transposon insertion mutant library. In the previous year, we have optimized bacterial cell competency for in vivo transposition mediated by a Tn5 transposome. Use of an in vivo transposome allows for the random introduction of a kanamycin resistance cassette into target DNA without the requirement of additional bacterial factors to facilitate the transposition event. The transposome consists of a stable complex formed between the Tn5 transposase enzyme and the transposon harboring the Tn903 kanamycin-resistance gene (previously shown to be functional in R. equi) flanked by hyperactive 19-basepair mosaic end Tn5 recognition sequences. We have constructed a pilot transposon insertion library consisting of approximately 425 individual transposon mutants. In initial screens, we identified transposon insertion mutants that exhibit altered colony pigmentation phenotypes and growth characteristics compared to the parental wild-type (WT) strain. To assess whether individual transposon insertion mutants are nutritional auxotrophs, we will evaluate the ability of individual mutants to replicate in minimal medium. Auxotrophic mutants that fail to replicate in minimal medium (representing mutants with probable impaired fitness in the equine host) will be selected for further analysis as potential vaccine strains. Currently, we are working to map the transposon insertion site within individual mutants of intertest. These experiments are being conducted by a second year PhD student, Elizabeth Boudaher. We are prioritizing the development of protocols that will generate super-competent R. equi cells that will be used to construct a more comprehensive, saturated transposon mutant (requiring the generation of approximately 20,000 individual transposon insertion mutants to reach 4x genomic coverage). We expect that a robust transposon library can be generated by combining mutants resulting from several independent rounds of transposition in R. equi. Generation of a saturated library will allow us to advance animal challenge models that will uncover multiple R. equi genes and genetic pathways that are required for efficient colonization of the equine lung. Specific Aim 2. Delineate genetic requirements for R. equi replication within macrophage-like cells. Thus far, we have identified several transposon insertion mutants that exhibit altered capacities to replicate in J774 murine macrophage-like cells compared to the WT parental strain. Using in vitro cell culture infection model and gentamicin protection assays, we have identified serval mutants that exhibit a decreased capacity to replicate in macrophage-like cells. For each transposon mutant exhibiting severe intracellular survival and replication defects, transposon insertion sites will be mapped using a combination of transposon-specific PCR and sequencing to identify the regions of flanking R. equi DNA. For all R. equi mutants that fail to replicate within macrophage-like cells, we will evaluate in vitro growth characteristics under a variety of environmental conditions, evaluate VapA status by immunoblot analysis, and evaluate intracellular replication phenotypes within equine alveolar macrophages obtained by bronchoalveolar lavage. When completed, these studies will identify bacterial genes that are required for host immune cell parasitization. Thus, these studies will reveal important biological information about the intracellular biology of R. equi that can potentially be targeted in new therapeutic intervention or prevention strategies. Specific Aim 3. Identify chemical scaffolds that disrupt R. equi interactions with host cells. We have recently characterized a class of synthetic small molecule inhibitors that disarm virulence mechanisms in diverse bacterial pathogens, and we predict that similar chemical scaffolds will interfere with R. equi replication and survival. Using fully-virulent WT R. equi, we have evaluated the capacity of several synthetic small molecules and metal-based compounds to prevent R. equi replication in pure bacterial culture or within macrophage-like cells. While we have not identified a synthetic small molecule that can interfere with R. equi viability in culture or within macrophage-like cells, we have evaluated a silver-based compound that significantly reduces R. equi viability. We are currently pursuing studies aimed at resolving the mechanism underlying metal toxicity in R. equi. These experiments are being conducted by Elizabeth Boudaher. Ultimately, this work will identify new chemical scaffolds that prevent key steps in R. equi pathogenesis and will provide the basis for the development of new therapeutics to treat rhodococcal disease. Individuals: Carrie L. Shaffer, PhD (PI) Elizabeth Boudaher (PhD student)
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Scare JA, AE Steuer, CL Shaffer, P Slusarewicz, A Mousley, MK Nielsen (2018). Long live the worms: methods for maintaining and assessing the viability of intestinal stages of Parascaris spp. in vitro. Parasitology 18:1-9. (PMID: 30561286)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
New approaches to study equine infectious disease. University of Kentucky Equine Forum, College of Agriculture, Food, and Environment, Lexington, KY, 2018.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
In vivo structures of a bacterial nanomachine revealed by electron cryotomography. MedVetPathogens, Prato, Italy, 2018. (Invited Plenary Speaker)
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