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Translational Science

Cory

Biography

Theodore CoryTheodore Cory, PharmD, PhD, is an Assistant Professor in the Department of Clinical Pharmacy. He completed his PharmD at Drake University, and a PhD in the Pharmaceutical Sciences at the University of Kentucky College of Pharmacy.  Following the completion of his PhD, he completed a post-doctoral fellowship in Clinical Pharmacology at the Antiviral Pharmacology Laboratory at the University of Nebraska Medical Center, under the direction of Dean Courtney Fletcher.

Contact:
College of Pharmacy Building Rm. 342
Phone: 901.448.7216
Email: tcory1@uthsc.edu

Lab Description

The Cory laboratory is dedicated to improving outcomes in HIV+ individuals by developing strategies to increase the concentrations of drugs used to fight HIV in cells in locations where drug concentrations may be insufficient to prevent replication of the virus.  These sites include the brain, lymph nodes, and secondary lymphoid tissues. While CD4+ T cells are the primary target of HIV, macrophages are infected early, and remain an important infected cell population. These two host cells interact in lymph nodes and secondary lymphoid tissue.  Macrophages exist in two phenotypically dissimilar polarized subsets, the classically activated (M1) phenotype, which is pro-inflammatory and involved in the destruction of intracellular pathogens, and the alternatively activated (M2) phenotype, which is anti-inflammatory and involved in tissue repair.  The role of these two subsets of macrophages in HIV is uncertain, as is the disposition of antiretrovirals in the cells.  Our goal is to define the mechanisms by which intracellular antiretroviral concentrations are altered in macrophage subsets, and the effect of this on viral replication and spread by focusing on developing strategies to modulate the expression and function of drug transporters on macrophages.

Additionally, we are investigating strategies to increase drug concentrations in the presence of drugs of abuse, including nicotine and alcohol.  These two drugs are commonly used in HIV+ individuals, and are strongly associated with worsened outcomes in these individuals.  

Projects

Projects in our laboratory include assessing the expression of drug efflux transporters in polarized macrophages, including P-glycoprotein and MRP1, and how these alterations effect the intracellular concentrations of antiretrovirals and viral production from infected cells. 

Additionally, we assess how nicotine or ethanol use alters transporter expression in polarized macrophages.  Both alcohol and nicotine are commonly abused by individuals with HIV, and are associated with worsened outcomes in these individuals.  These is a high importance to determine strategies to improve outcomes in individuals with HIV who either smoke or abuse alcohol.

Selected Publications
  1. Cory, TJ, He H, Winchester LC, Kumar S, Fletcher CV. Alterations in P-Glycoprotein Expression and Function between Macrophage Subsets.  Pharmaceutical Research.  2016:33(11):2713-2721.
  2. Cory TJ, Midde NM, Rao PSS, Kumar S. Investigational Reverse Transcriptase Inhibitors for the Treatment of HIV.  Expert Opinion on Investigational Drugs.  2015:24(9):1219-1228.
  3. Cory TJ, Winchester L, Robbins BL, Fletcher CV. A Rapid Spin Through Oil Results in Higher Cell-Associated Concentrations of Antiretrovirals Compared with Conventional Cell Washing.    2015:7(12):1447-1455.
  4. Cory TJ, Schacker TW, Stevenson M, Fletcher CV.  Overcoming Pharmacologic Sanctuaries.  Current Opinion in HIV and AIDS.  2013:8(3):190-195.  PMC3677586
Lab Personnel

Graduate Student:  Ying Mu 
Collaborator:  Santosh Kumar

 

Fortwendel

Lab Description

How do fungal pathogens sense and respond to the host environment during the initiation and progression of invasive disease? What signal transduction networks and protein interactions mediate fungal pathogenic fitness, promoting survival in the presence of host defenses? The overall goal of my lab is to delineate the molecular mechanisms that support fungal growth and promote virulence in the human lung environment. Invasive fungal infections are increasing in incidence and the associated high mortality rates highlight the need for a deeper understanding of the pathogenic mechanisms underlying these infections. Aspergillus fumigatus, the causative agent of invasive pulmonary aspergillosis, is the predominant mould pathogen of immunocompromised patients. Our lab utilizes molecular, biochemical and cellular biology techniques to address questions of A. fumigatus growth and pathogenesis in vitro and within the model host. Our current work is focused in three main areas:

1) Ras-mediated invasive growth mechanisms

Ras proteins orchestrate multiple fungal morphogenetic processes in pathogenic fungi. Regulation of these processes by Ras plays an essential role in fungal virulence, making fungal Ras signaling an invaluable tool for probing fungal pathogenesis and identifying new targets for novel therapeutics. Although many aspects of Ras signaling pathways are often considered too highly conserved to serve as antimicrobial targets in eukaryotic pathogens, we have identified novel, fungal-specific protein domains that define fundamental differences between fungal and human Ras proteins. We are currently working to fully characterize these differences, generating a new paradigm for the regulation of Ras signaling in lower eukaryotes. Because Ras signaling is essential for fungal virulence, identification and characterization of fundamental differences between human and Ras pathways carries the long-term potential of uncovering novel antifungal therapies.

2) Targeting protein localization for antifungal therapy

In most eukaryotes, including other yeast-like fungi, sensing external changes requires protein networks that begin at the cell periphery, often beginning with G-protein coupled receptors. Information is then sent to internal signal transduction pathways, usually headed by small trimeric G-proteins or monomeric GTPase proteins that are peripherally associated with cellular membranes via post-translational prenylation. Therefore, the networks orchestrated by the precise sub-cellular localization of proteins via the prenyltransferase enzymes, farnesyltransferase and geranylgeranyltransferase, are predicted to be major regulators of fungal pathogenic fitness. We, and others, have shown that loss of these enzyme complexes leads to reduced virulence in multiple pathogenic fungi. However, the underlying mechanisms are largely uncharacterized. We are currently exploring networks regulated by prenyltransferase complexes to uncover novel ways in which pathogenic fungi sense and respond to the host environment to promote disease.

3) Systematic functional analysis of the A. fumigatus kinome

Reversible protein phosphorylation regulates almost all eukaryotic processes and, on average, about 30% of cellular proteins are modified by phosphorylation. Although no systematic analysis has yet been accomplished in A. fumigatus, the relatively few protein kinases that have been characterized play diverse roles in cellular stress responses and virulence. Kinases are considered the second largest protein class currently functioning as drug targets, as their inhibition can be readily accomplished by small molecules. Unfortunately, the vast majority of A. fumigatus protein kinases remain unstudied. We have successfully adapted a novel CRISPR/Cas9-based mutational approach for use in wild type strains of A. fumigatus. Our preliminary work show that this facile system increases the typically low levels of gene targeting in wild type A. fumigatus to as high as 90%. With this new tool, we are systematically deleting and functionally analyzing all putative protein kinases in A. fumigatus. We expect to discover multiple, novel contributions of protein kinases to the pathobiology of invasive aspergillosis. The information generated by completion of this work will support future applications exploring novel aspects of A. fumigatus virulence.

Diagram of research work.

Jarrod R. Fortwendel, PhD
Assistant Professor
Department of Clinical Pharmacy and Translational Science
College of Pharmacy
811 Madison Ave,
Memphis, TN 38163
Labs: Room 325/327
Office: Room 343
Phone: 901-448-5140
Email: jfortwen@uthsc.edu

Selected Publications
  1. Norton TS, Al Abdallah Q, Hill AM, Lovingood RV, and Fortwendel JR. The Aspergillus fumigatus farnesyltransferase β-subunit, Ram1, mediates growth, virulence and antifungal susceptibility. Virulence. 1-16. doi: 10.1080/21505594.2017.1328343. 2017.
  2. Al Abdallah Q, Norton TS, Hill AM, LeClaire LL and Fortwendel JR. A Fungus-Specific Protein Domain Is Essential for RasA-Mediated Morphogenetic Signaling in Aspergillus fumigatus. mSphere. 1(6). pii: e00234-16. 2016.
  3. LeClaire LL and Fortwendel JR. Differential Support of Aspergillus fumigatus Morphogenesis by Yeast and Human Actins. PLoS One. 10(11): e0142535. doi: 10.1371/journal.pone.0142535. 2015.
  4. Fortwendel JR. Orchestration of morphogenesis in filamentous fungi: conserved roles for Ras signaling networks. Fung Biol Rev. 29: 54-62. 2015.
  5. Al Abdallah Q and Fortwendel JR. Exploration of Aspergillus fumigatus Ras pathways for novel antifungal drug targets. Front Microbiol. 6:128: doi: 10.3389/fmicb.2015.00128. 2015.
  6. Norton TS and Fortwendel JR. Control of Ras-mediated signaling in Aspergillus fumigatus. Mycopathologia. 178: 325-30. 2014.
  7. Juvvadi PR, Gehrke C, Fortwendel JR, Lamoth F, Soderblom EJ, Cook EC, Hast MA, Asfaw YG, Moseley MA, Creamer TP, Steinbach WJ. Phosphorylation of Calcineurin at a novel serine-proline rich region orchestrates hyphal growth and virulence in Aspergillus fumigatus. PLoS Pathog. 9(8):e1003564. doi: 10.1371/journal.ppat. 1003564. 2013.
  8. Fortwendel JR. Ras mediated signal transduction and virulence in human pathogenic fungi. Fungal Genomics Biol. 2(1): 105. 2012.
  9. Lamoth F, Juvvadi PR, Fortwendel JR, and Steinbach WJ.  Heat-shock protein 90 (Hsp90) is required for conidiation and cell wall integrity in Aspergillus fumigatus. Eukaryot. Cell. 11:1324-32. 2012.
  10. Fortwendel JR, Juvvadi PR, Rogg LE, Asfaw YG, Burns KA, Randell SH, and Steinbach WJ.  Plasma membrane localization is required for RasA-mediated polarized morphogenesis and virulence of Aspergillus fumigatus. Eukaryot. Cell. 11:966-77. 2012.
Lab Personnel

Wenbo Ge 
Qusai Al Abdallah, PhD  
Adela Martin Vicente, PhD 
Ana Camila Souza, PhD   

Senior Research Assistant 
Postdoctoral Fellow
Postdoctoral Fellow
Postdoctoral Fellow

 

Laizure-Parker

Lab Description

Robert ParkerThe Laizure-Parker lab focuses on understanding mechanisms affecting the variability in metabolism and disposition of drugs metabolized by the human carboxylesterase (CES) enzymes. In humans, two carboxylesterases, carboxylesterase-1 (CES1) and carboxylesterase-2 (CES2), found primarily in the liver and intestine respectively, play an important role in the biotransformation of a growing number of widely prescribed medications used to treat many common disorders (Table).

Factors affecting the CES activity would be expected to markedly alter the pharmacokinetics and clinical effects of substrate drugs. One key factor that could affect catalytic activity is drug interactions that inhibit CES function.  The importance of inhibition of drug metabolism in medication safety and efficacy is well established for drugs that undergo metabolism by cytochrome P450 enzymes. In distinct contrast, little is known about the potential for CES to serve as a target for metabolic inhibition mediated by drug interactions.   

Steven LaizureHow drug-induced metabolic inhibition affects the safety and effectiveness of CES substrate drugs is a major gap in our understanding of adverse drug events. During the last 20 years there has been an increase of prescribed CES substrate drugs; however our knowledge of the mechanisms, clinical importance and toxicity of drug interactions with these enzymes has yet to advance beyond in vitro metabolic studies. We are interested in exploring how inhibition of CES by drug interactions affects the metabolism, disposition, and response to medications hydrolyzed by these enzymes. 

Laizure-Parker LabTo address these questions, our laboratory uses a combination of in vitro metabolic studies, in vivo models, human studies, and modeling. We have identified a number of potent CES inhibitors including alcohol (ethanol), diltiazem, verapamil, aripiprazole, loperamide, and rivastigmine.


Current Funding:
Contact Info:
University of Tennessee Health Science Center
Department of Clinical Pharmacy
College of Pharmacy
881 Madison Ave.,
Memphis, TN 38163
Lab: Room 311; Office: Room 346 (Parker) and Laizure (358)
Phone: 901-448-7143 (Parker) and 901-448-6310 (Laizure)
Email: rparker@uthsc.edu, claizure@uthsc.edu

Center for Pediatric Pharmacokinetics and Therapeutics

Lab Personnel

Dr. Feng Chen (Postdoctoral fellow)

 

Palmer

Research Interests 

Glen PalmerAn estimated 1.5 million people die each year from invasive fungal infections, and many millions more are afflicted by debilitating mucosal and subcutaneous mycoses. Current antifungal therapies have serious deficiencies including poor efficacy, limited spectrum of activity, patient toxicity and the emergence of resistant fungi. Consequently, mortality rates are disturbingly high. A major obstacle to developing effective new antifungal drugs is the fundamental similarity between the cells of these eukaryotic pathogens and their mammalian host. This presents a challenge in devising therapeutic agents with pathogen selective toxicity. A major long-term goal of my research program is to identify and validate new target proteins that can provide a basis to develop efficacious new antifungal therapies. Current investigations within my lab include the discovery and development of new classes of antifungal agents that target either: 1). The integrity of a sub-cellular organelle called the fungal vacuole; 2). Fungal fatty acid biosynthesis; and 3) aromatic amino acid biosynthesis. As part of these studies we have devised several high-throughput (HTP) chemical screening assays to identify compounds that target these cellular functions. This includes a new and broadly applicable type of target based whole-cell screen (TB-WCS) that combines the benefits of both traditional target-based and cell-based chemical screens into a single HTP assay. We anticipate our TB-WCS approach to chemical screening will greatly enhance the speed and efficiency with which new pre-therapeutic leads, with a defined mechanism of action can be identified. Through these efforts, I have become increasingly excited about the enormous potential of applying yeast based systems (which are highly amenable to HTP approaches) to the discovery of new pharmacotherapies that target human disease related proteins.

Glen E. Palmer
Associate Professor, Clinical Pharmacy
PhD Genetics, University of Leicester, United Kingdom
BSc. Genetics, University of Sheffield, United Kingdom
Email: gpalmer5@uthsc.edu
Office: 901.448.3744

Current Lab Members

Lab manager - Tracy Peters M.S.
Postdoctoral researchers – Helene Tournu Ph.D; Arielle Butts Ph.D; Arturo Luna-Tapia Ph.D
Research Associate – Kathy Barker Ph.D
Graduate Students – Christian DeJarnette B.S.; Parker Reitler B.S.

Selected Publications
  1. Tournu, H., Carroll, J., Latimer, B., Dragoi, A., Dykes, S., Cardelli, J., Peters, T.L., Eberle, K.E., and Palmer, G.E. (2017). Identification of small molecules that disrupt vacuolar function in the pathogen Candida albicans. Accepted for publication in PLoS One.
  2. Luna-Tapia, A., Peters, B.M., Tournu, H., and Palmer, G.E. (2017). An azole tolerant endosomal trafficking mutant of Candida albicans is susceptible to azole treatment in a mouse model of vaginal candidiasis. Accepted for publication in Antimicrobial Agents and Chemotherapy.
  3. Luna-Tapia, A., Kerns, M., Eberle, K.E., Jursic, B.S., and Palmer, G.E. (2015). Trafficking through the late endosome significantly impacts Candida albicans tolerance of the azole antifungals. Antimicrobial Agents and Chemotherapy, 59: 2410-20. PMCID:
  4. Luna-Tapia, A., Tournu, H., Peters, T., and Palmer, G.E. (2016). Endosomal trafficking defects can induce calcium dependent azole tolerance in Candida albicans. Antimicrobial Agents and Chemotherapy, Sep 19: AAC.01034-16. [Epub ahead of print].
  5. Luna-Tapia, A., Peters, B.M., Eberle, K.E., Kerns, M.E., Foster, T.P., Marrero, L., Noverr, M.C., Fidel, P.L. Jr, Palmer, G.E. (2015). ERG2 and ERG24 are required for normal vacuolar physiology as well as Candida albicans pathogenicity in a murine model of disseminated but not vaginal candidiasis. Eukaryotic Cell, 14: 1006-1016. PMCID:

 

Peters

Description

The Peters lab has two main foci of research: 1) the host and fungal molecular mechanisms responsible for the immunopathogenesis of vulvovaginal candidiasis and 2) quorum sensing and toxin regulation during fungal-bacterial intra-abdominal infection.

Members and Collaborators

Current Members:

Kathy Barker, PhD (Research Associate; joint CPET Researcher)
David Lowes, PhD (Research Associate
Marjoleine Willems, PhD (Postdoc)
Emily Sansevere, PhD (Postdoc)
Olivia Todd, BS (Graduate Student, IPBS)

Previous Members:
Junyan Liu, MS (visiting student, South China University of Technology)
Winter Bruner, BS (technician)

Collaborators:
Glen Palmer, PhD (UTHSC)
Julian Naglik, PhD (King’s College London)
Mairi Noverr, PhD (LSUHSC)
Paul Fidel, PhD (LSUHSC)
Vinnie Bruno, PhD (UMB)

Projects

Candida albicans, an opportunistic human fungal pathogen, is the leading causative agent of vulvovaginal candidiasis (VVC) and presents major quality of life issues for women worldwide. It is estimated that nearly every woman of childbearing age will be afflicted by VVC at least once in her lifetime. Although these treatments are typically effective at reducing organism burden, static function of azole activity, fungal recalcitrance to clearance, and lack of comprehensive understanding of disease pathology necessitates further insight into the host and fungal factors that contribute to vaginitis immunopathology. 

[1] We are interested in exploring virulence mechanisms utilized by C. albicans, including the fungal toxin Candidalysin, to activate inflammasome signaling at the vaginal mucosa. We are also focused on determining the downstream signaling events relevant to disease pathogenesis, including the protective role of innate IL-17 signaling at the vaginal mucosa. [2] We are also currently testing sulfonylurea drugs as repurposed adjunctive therapeutic agents to more quickly arrest symptomatic disease.

Peters LabPolymicrobial intra-abdominal infection:

 

[3] Microorganisms rarely exist as single species communities but instead exist within multi-species consortia where mutually beneficial, parasitic, and antagonistic interactions may develop. However, relatively little is known about the functional consequences of these interactions as they relate to health and disease.

We aim to determine the complex inter-microbial signaling events that mediate infectious synergism observed during intra-abdominal infection with the ubiquitous bacterial pathogen Staphylococcus aureus and the fungus C. albicans. Current studies are focused on identifying activation of the S. aureus agr-quorum sensing system and downstream toxins as key pathways contributing to lethal infection. This polymicrobial intra-abdominal infection serves as an excellent model system for determining microbe-microbe induced virulence gene regulation in vivo. Identification of virulence determinants may serve as rationale for selection of vaccine candidates to reduce lethality clinically associated with fungal-bacterial intra-abdominal infection.

Funding

K22AI110541 (PI: Peters)
1R21AI127942 (PI: Peters, pending NoA)
R01AI116025 (PI: Noverr/ sub: Peters)
UTHSC Pocket CORNET award (PI: Cox/Peters)
UTHSC New Grant Support (PI: Peters)
UTHSC Startup funds
Center for Pediatric Pharmacokinetics and Therapeutics

Selected Publications
  1. Richardson JP, Willems HME, Moyes DL, Shoaie S, Tan SL, Barker KS, Palmer GE, Hube B, Naglik JR*, Peters BM*. Candidalysin drives epithelial signaling, neutrophil recruitment, and immunopathology at the vaginal mucosa. ACCEPTED. Infect Immun. *, denotes authors of equal contribution. Nov 2017.
  1. Willems HME, Bruner WS, Barker KS, Liu J, Palmer GE, Peters BM. Overexpression of Candida albicans Secreted Aspartyl Proteinases 2 or 5 is not sufficient for exacerbation of immunopathology in a murine model of vaginitis. Infect Immun. Jul 2017. [Epub ahead of print] 
  1. Peters BM, Luna-Tapia A, Tournu H, Rybak JM, Rogers PD, Palmer GE. An azole tolerant endosomal trafficking mutant of Candida albicans is susceptible to azole treatment in a mouse model of vaginal candidiasis. Antimicrob Agents Chemother. Mar 2017 May 24;61(6).
  1. Lown L, Peters BM, Walraven CJ, Noverr MC, Lee SA. An optimized lock solution containing micafungin, ethanol, and doxycycline inhibits Candida albicans and mixed albicans-Staphylococcus aureus biofilms. PLoS One. 2016 Jul 18;11(7)e0159225.
  1. Nash EE, Peters BM, Lilly EA, Noverr MC, Fidel PL Jr. A murine model of Candida glabrata vaginitis shows no evidence of an inflammatory immunopathogenic response. PLoS One. 2016 Jan 25; 11(1):e0147969. PMCID: PMC4726552.
  1. Nash E, Peters BM, Fidel Jr. PL, Noverr MC. Morphology-independent virulence of Candida species during polymicrobial intra-abdominal infections with Staphylococcus aureus. Infect Immun. 2015 Oct 19;84(1):90-8.
  1. Luna-Tapia A*, Peters BM*, Eberle KE, Kerns ME, Marrero L, Noverr MC, Fidel Jr. PL, Palmer GE. ERG2 and ERG2 encode the targets of the morpholine antifungals in Candida albicans and are required for pathogenicity in a mouse model of disseminated but not vaginal candidiasis. *authors share joint authorship of equal contribution. Eukaryot Cell. 2015 Oct; 14(10):1006-16.
  1. Bruno VM, Shetty AC, Yano J, Fidel Jr PL, Noverr MC, Peters BM. Transcriptomic analysis of vulvovaginal candidiasis identifies a role for the NLRP3 inflammasome. 2015. Apr 21;6(2). pii: e00182-15.
  1. Schlect LM*, Peters BM*, Harris ML, Jabra-Rizk MA, Shirtliff ME. Systemic Staphylococcus aureus infection mediated by Candida albicans hyphal invasion of mucosal tissue. *authors share joint authorship of equal contribution. Microbiology. 2015 Jan; 16(Pt 1): 168-81.
  1. Peters BM, Noverr MC, Fidel Jr. PL. Candida vaginitis: when opportunism knocks, the host responds. PLoS Pathog. 2014 Apr 3;10(4):e1003965.
  1. Yano J, Palmer GE, Eberle KE, Peters BM, Vogl T, Mckenzie AN, Fidel Jr. PL. Vaginal epithelial cell-derived S100 alarmins induced by albicans via pattern recognition receptor interactions is sufficient but not necessary for the acute neutrophil response during experimental vaginal candidiasis. Infect Immun. 2014 Feb;82(2):783-92.
  1. Peters BM, Palmer GE, Nash AK, Lilly EA, Fidel Jr. PL, Noverr MC. Fungal morphogenetic pathways are required for the hallmark inflammatory response during CandidaInfect Immun. 2014 Feb;82(2):532-43.
  1. Peters BM and Noverr MC. Candida albicans-Staphylococcus aureus polymicrobial peritonitis modulates host innate immunity. Infect Immun. 2013 Jun;81(6):2178-89.
  1. Peters BM, Ovchinnikova ES, Krom BP, Schlecht LM, Zhou H, Hoyer LL, van der Mei HC, Busscher HJ, Jabra-Rizk MA, Shirtliff ME. Candida albicans hyphal adhesin Als3 mediates interspecies interactions with Staphylococcus aureus during polymicrobial biofilm growth. 2012 Dec;158(Pt 12):2975-86. PMCID: not yet available.
  1. Peters BM, O’May GA, Jabra-Rizk MA, Costerton JW, Shirtliff ME. Polymicrobial interactions: impact on pathogenesis and human disease. Clin Microbiol Rev. 2012 Jan; 25(1):193-213. PMCID: PMC3255964.
  1. Peters BM, Jabra-Rizk MA, Leid JG, Costerton JW, Shirtliff ME. Microbial interactions and differential protein expression within Candida albicans-Staphylococcus aureus polymicrobial biofilms.  FEMS Immunol Med Microbiol. Aug:59(3):493-503, 2010. PMCID: 

 

Rogers

Lab Description

dave p rogersThe overarching long-term goal of the Rogers lab is to improve the safety and efficacy of antifungal pharmacotherapy.  My interest in this area is driven by insights gained as an infectious diseases clinical pharmacist into the significant limitations that exist with regard to the treatment of serious fungal infections.  Indeed, treatment of such infections is limited to only three antifungal classes.  The polyene amphotericin B is effective for many fungal infections, but its use is hampered by significant infusion-related reactions and nephrotoxicity.  It is also only available for intravenous administration.  The triazole antifungals are effective and in some cases superior, yet much less toxic, inexpensive, and available both orally and intravenously.  Unfortunately resistance has emerged which limits the utility of this antifungal class. The echinocandins, such as caspofungin, are particularly useful for invasive candidiasis, but lack utility against other fungal pathogens and are only available for intravenous administration. Moreover, resistance to this antifungal class has begun to emerge, particularly in the fungal pathogen Candida glabrata.  It must also be underscored that no new antifungal drug classes are on the horizon.  Novel strategies are therefore urgently needed to preserve, improve, and expand the current antifungal armamentarium.

Rogers LabFor almost two decades our primary focus has been understanding the molecular and cellular basis of resistance to the triazole class of antifungal agent in pathogenic fungi.  A long-term interest of my laboratory has been the use of genome-wide technologies to study antifungal stress responses in Candida species.  We used microarray and proteomic analysis to identify changes in the gene expression and proteomic profiles of these organisms in response to the various classes of antifungal agents.  This revealed both general and specific responses, some of which aligned with the mechanisms of action of these agents, and gave insight into factors that influence antifungal susceptibility (such as the azole-induction of the Cdr1 transporter). We also used this approach for genome-wide analysis of azole antifungal resistance in Candida species, which has provided insight into this process (1-4). 

My laboratory, working in collaboration with the laboratory of Joachim Morschhauser, discovered the transcriptional regulator Mrr1 and demonstrated that activating mutations in this transcription factor gene result in up-regulation of the Mdr1 transporter and fluconazole resistance in clinical isolates of C. albicans. In further work we have delineated the regulon of this transcriptional regulator and identified other regulators required for its activity (5-8).  Working again in collaboration with the Morschhauser laboratory, we discovered that activating mutations in the transcription factor Upc2 leads to up-regulation of the gene encoding the azole target (ERG11), and increased azole resistance in clinical isolates.  We have shown that this is a common and important mechanism of resistance among clinical isolates, identified additional regulators required for its activity, and have found it to be essential for azole resistance in clinical isolates exhibiting the major resistance mechanisms (9-12). More recently we have delineated the contribution of the putative lipid translocase Rta3 in azole resistance in this organism (13).

Our work has also explored the problem of triazole resistance in other fungal species.  Working in collaboration with the laboratory of Thomas Edlind, we discovered that activating mutations in the transcription factor Pdr1 were responsible for azole resistance in C. glabrata. This led to further work by our group elucidating the role of this transcription factor, as well as the transcription factor Upc2, in azole antifungal resistance in this important Candida species (14-17).  More recently we have begun to dissect this process in other non-albicans Candida species as well as the important fungal pathogen Aspergillus fumigatus (18, 19).  Currently my research program maintains three focus areas: 1) Understanding the genetic and molecular basis of triazole antifungal resistance in Candida albicans, 2) Dissecting the Upc2A transcriptional pathway, protein interaction partners, and genetic network to overcome fluconazole resistance in Candida glabrata, and 3) Delineating the genetic and molecular basis of triazole resistance in the fungal pathogen Aspergillus fumigatus

David Rogers, PharmD, PhD, FCCP
First Tennessee Endowed Chair of Excellence in Clinical Pharmacy
Vice-chair for Research
Director, Clinical and Experimental Therapeutics
Co-Director, Center for Pediatric Pharmacokinetics and Therapeutics
Professor of Clinical Pharmacy and Pediatrics

Projects

1) Novel Azole Resistance Mechanisms in Candida albicans

A critical barrier to progress in overcoming azole antifungal resistance in Candida albicans is the lack of a complete understanding of its molecular and genetic basis because the known mechanisms of resistance do not fully explain resistance observed among many clinical isolates. Our goal is to advance the treatment of Candida infections by identifying novel azole resistance mechanisms that can be exploited to ultimately overcome this problem. Our central hypothesis is that azole resistance in clinical isolates of C. albicans is multifactorial and involves complex genetic changes that 1) alter azole target binding, 2) activate transcriptional programs that impart resistance, and 3) reduce azole uptake. Our objectives are to 1) delineate the effects of clinically relevant mutations in ERG11, alone and in combination, on the activity of its gene product, fitness, and azole susceptibility, 2) determine the clinical significance of novel Zn(2)Cys6 transcription factors (ZCFs) that influence azole susceptibility, and 3) to discover the determinants of reduced azole import and their contribution to azole resistance in clinical isolates. Our preliminary data suggest that different ERG11 mutations diversely affect sterol demethylase activity, including alterations of catalytic efficiency, target binding kinetics, and reaction velocity. We have also observed that artificial activation of a distinct set of ZCFs in C. albicans increases azole resistance. We have identified azole-resistant clinical isolates that exhibit transcriptional profiles consistent with activation of these ZCFs and that contain candidate activating mutations in these ZCF genes. Finally, we have demonstrated that C. albicans takes up fluconazole by energy-independent facilitated diffusion. We have observed that some azole resistant isolates exhibit reduced fluconazole uptake. In Aim 1 of this proposal we will undertake genetic, microbiologic, and biochemical studies to dissect the effects of single and combinatorial mutations in ERG11 on sterol demethylase susceptibility, substrate affinity, azole binding, catalytic activity, and fitness. In Aim 2 we will undertake genetic and microbiologic studies to determine if and how mutations found in the genes encoding novel ZCFs in resistant clinical isolates result in their activation and increased azole resistance. In Aim 3 we will determine the mechanism of azole antifungal import and its contribution to azole resistance in clinical isolates of C. albicans. Our approach is innovative as we will determine for the first time precisely how mutations in ERG11 influence enzyme activity, dissect combinations of mutations, and determine the impact of such mutations on fitness of C. albicans. This work also explores novel mechanisms of azole resistance. The proposed research is significant as it will provide the understanding needed to ultimately overcome azole resistance through the development of improved azoles, interference with activated ZCFs, and enhancement of azole uptake. By fully understanding the genetic basis of azole resistance it will be possible to eventually develop non-culture based strategies to rapidly and accurately detect azole resistance in clinical isolates.

2) Upc2A: A Central Regulator and “Achilles’ Heel” of Fluconazole Resistance in Candida glabrata   

There is a significant gap in knowledge concerning the molecular and cellular underpinnings of triazole resistance in the important fungal pathogen Candida glabrata and how such resistance might be overcome. Our long-term goal is to improve the treatment of Candida infections by overcoming resistance to the triazole class of antifungals. Our overall objective in the present application is to identify the target genes directly regulated by this transcription factor, its protein interaction partners, and the genes that interact with UPC2A in the pathogenic fungus Candida glabrata. Our preliminary data demonstrate that loss of Upc2A function in both wild-type and triazole resistant isolates results in increased susceptibility to sterol biosynthesis inhibitors, including a reduction in fluconazole minimum inhibitory and minimum fungicidal concentrations and enhanced fluconazole activity by time-kill analysis. Our findings indicate that Upc2A is a key regulator of ergosterol biosynthesis as well as other unknown processes and is essential for resistance to fluconazole in C. glabrata. The Upc2A pathway therefore represents a potential co-therapeutic target for enhancing fluconazole activity against this inherently resistant species and restoring and preserving this class of antifungal for the treatment of invasive Candidiasis. In Aim 1 we will identify Upc2A target genes using transcriptional profiling (RNA-seq) and ChIP-seq, and we will then determine which target genes influence susceptibility to fluconazole by targeted gene disruption. In Aim 2 we will identify Upc2A interaction partner proteins using tandem affinity purification (TAP) and will determine which of these are essential for Upc2A activity under fluconazole exposure and which of these influence fluconazole susceptibility using targeted gene disruption. In Aim 3 of this proposal we will undertake screens of a transposon insertion mutant library as well as a recently developed deletion mutant library for genes that interact with, and are required for, Upc2 activation by sterol biosynthesis inhibition in order to identify and characterize the Upc2A genetic interaction network. The proposed studies are innovative as they uniquely focus on interference of activity of the transcription factor Upc2A as a strategy for circumventing fluconazole resistance in C. glabrata. Moreover, our approach is innovative as we will for the first time make use of a comprehensive set of genomic tools and techniques designed for yeast research and apply them to clinical isolates of the fungal pathogen C. glabrata. The proposed research is significant as it will provide new knowledge that can ultimately be exploited to overcome triazole resistance in this inherently resistant species of Candida and restore and preserve the use of this antifungal class for serious Candida infections. 

3) Novel Mechanisms of Triazole Resistance in Aspergillus fumigatus

A critical barrier to overcoming triazole resistance in Aspergillus fumigatus is the significant lack of understanding of its genetic and molecular basis because the known mechanisms of resistance do not fully explain resistance observed among many clinical isolates. Our goal is to advance the treatment of Aspergillus infections by understanding the genetic and molecular basis of triazole resistance, which will ultimately be exploited to overcome this problem. Our central hypothesis is that triazole resistance in clinical isolates of A. fumigatus is multifactorial and involves complex genetic changes that alter 1) triazole target enzyme binding and enzyme function, 2) sterol biosynthesis and its transcriptional activation, and 3) triazole efflux and import. Our objectives are to 1) delineate the effects of clinically relevant mutations in cyp51A on the activity of its gene product and on triazole susceptibility, 2) identify genetic and molecular determinants that influence triazole susceptibility through altered sterol biosynthesis and its transcriptional activation, and 3) identify the efflux and import transporters that participate in triazole resistance and determine how they are regulated. Our preliminary data suggest that mutations in cyp51A among triazole resistant clinical isolates are common, and that some influence susceptibility to multiple triazoles, whereas others do not. We have also observed mutations in sterol biosynthesis genes among resistant isolates that were not observed in susceptible isolates. Our preliminary data implicate these genes as contributing to this phenotype. Six isolates in our collection exhibit up-regulation of cyp51A that cannot be explained. Our preliminary data point to mutations that activate the srbA transcriptional pathway. Finally, our preliminary data implicate several transporters that may contribute to non-Cyp51A-medited triazole resistance through either increased triazole efflux or reduced triazole import. In Aim 1 we will undertake genetic, microbiologic, and biochemical studies to dissect the effects of mutations in cyp51A on sterol demethylase activity, susceptibility, and fitness. In Aim 2 we will undertake genetic and microbiologic studies to determine if triazole resistance is due in part to genetic mutations that alter sterol biosynthesis and its transcriptional activation. In Aim 3 we will determine whether non-Cyp51A-mediated triazole resistance is mediated in part by altered expression of drug transporters. Our approach is innovative as we will determine for the first time the genetic and biochemical effects of clinically relevant mutations in cyp51A on triazole susceptibility and fitness. Our proposed work also explores novel mechanisms of triazole resistance in this pathogen such as alteration in, and upregulation of, sterol biosynthesis, increased triazole efflux mediated by novel transporters, and reduced triazole import. The proposed research is significant as it will provide the understanding needed to ultimately design the next generation of triazoles and sterol biosynthesis inhibitors and will make possible the development of strategies to overcome triazole resistance, preserve the utility of the triazole antifungals, and more efficiently predict clinical treatment failure and success.

Selected Publications
  1. Hoehamer CF, Cummings ED, Hilliard GM, Rogers PD. Changes in the proteome of Candida albicans in response to azole, polyene, and echinocandin antifungal agents. Antimicrob Agents Chemother. 2010 May;54(5):1655-64. PMID: 20145080.
  2. Liu TT, Lee RE, Barker KS, Lee RE, Wei L, Homayouni R, Rogers PD. Genome-wide expression profiling of the response to azole, polyene, echinocandin, and pyrimidine antifungal agents in Candida albicans. Antimicrob Agents Chemother. 2005 Jun;49(6):2226-36. PMID: 15917516.
  3. Hooshdaran MZ, Barker KS, Hilliard GM, Kusch H, Morschhäuser J, Rogers PD. Proteomic analysis of azole resistance in Candida albicans clinical isolates. Antimicrob Agents Chemother. 2004 Jul;48(7):2733-5. PMID: 15215138.
  4. Rogers PD, Barker KS. Genome-wide expression profile analysis reveals coordinately regulated genes associated with stepwise acquisition of azole resistance in Candida albicans clinical isolates. Antimicrob Agents Chemother. 2003 Apr;47(4):1220-7. PMID: 12654650.
  5. Schubert S, Popp C, Rogers PD, Morschhäuser J. Functional dissection of a Candida albicans zinc cluster transcription factor, the multidrug resistance regulator Mrr1. Eukaryot Cell. 2011 Aug;10(8):1110-21. PMID: 21685320.
  6. Mogavero S, Tavanti A, Senesi S, Rogers PD, Morschhäuser J. Differential requirement of the transcription factor Mcm1 for activation of the Candida albicans multidrug efflux pump MDR1 by its regulators Mrr1 and Cap1. Antimicrob Agents Chemother. 2011 May;55(5):2061-6. PMID: 21343453.
  7. Dunkel N, Blass J, Rogers PD, Morschhäuser J. Mutations in the multi-drug resistance regulator MRR1, followed by loss of heterozygosity, are the main cause of MDR1 overexpression in fluconazole-resistant Candida albicans Mol Microbiol. 2008 Aug;69(4):827-40. PMID: 18577180.
  8. Morschhäuser J, Barker KS, Liu TT, BlaB-Warmuth J, Homayouni R, Rogers PD. The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans. PLoS Pathog. 2007 Nov;3(11):e164. PMID: 17983269.
  9. Vasicek EM, Berkow EL, Flowers SA, Barker KS, Rogers PD. UPC2 is universally essential for azole antifungal resistance in Candida albicans. Eukaryot Cell. 2014 Jul;13(7):933-46. PMID: 24659578.
  10. Flowers SA, Barker KS, Berkow EL, Toner G, Chadwick SG, Gygax SE, Morschhäuser J, Rogers PD. Gain-of-function mutations in UPC2 are a frequent cause of ERG11 upregulation in azole-resistant clinical isolates of Candida albicans. Eukaryot Cell. 2012 Oct;11(10):1289-99. PMID: 22923048.
  11. Heilmann CJ, Schneider S, Barker KS, Rogers PD, Morschhäuser J. An A643T mutation in the transcription factor Upc2p causes constitutive ERG11 upregulation and increased fluconazole resistance in Candida albicans. Antimicrob Agents Chemother. 2010 Jan;54(1):353-9. PMID: 19884367.
  12. Dunkel N, Liu TT, Barker KS, Homayouni R, Morschhäuser J, Rogers PD. A gain-of-function mutation in the transcription factor Upc2p causes upregulation of ergosterol biosynthesis genes and increased fluconazole resistance in a clinical Candida albicans Eukaryot Cell. 2008 Jul;7(7):1180-90. PMID: 18487346.
  13. Whaley SG, Tsao S, Weber S, Zhang Q, Barker KS, Raymond M, Rogers PD. The RTA3 gene, encoding a putative lipid translocase, influences the susceptibility of Candida albicans to fluconazole. Antimicrob Agents Chemother. 2016 Sep 23;60(10):6060-6. PMID: 27480868
  14. Whaley SG, Caudle KE, Vermitsky JP, Chadwick SG, Toner G, Barker KS, Gygax SE, Rogers PD. UPC2A is required for high-level azole antifungal resistance in Candida glabrata. Antimicrob Agents Chemother. 2014 Aug;58(8):4543-54. PMID: 24867980.
  15. Caudle KE, Barker KS, Wiederhold NP, Xu L, Homayouni R, Rogers PD. Genomewide expression profile analysis of the Candida glabrata Pdr1 regulon. Eukaryot Cell. 2011 Mar;10(3):373-83. PMID: 21193550.
  16. Rogers PD, Vermitsky JP, Edlind TD, Hilliard GM. Proteomic analysis of experimentally induced azole resistance in Candida glabrata. J Antimicrob Chemother. 2006 Aug;58(2):434-8. PMID: 16735426.
  17. Vermitsky JP, Earhart KD, Smith WL, Homayouni R, Edlind TD, Rogers PD. Pdr1 regulates multidrug resistance in Candida glabrata: gene disruption and genome-wide expression studies. Mol Microbiol. 2006 Aug;61(3):704-22. PMID: 16803598.
  18. Berkow EL, Manigaba K, Parker JE, Barker KS, Kelly SL, Rogers PD. Multidrug transporters and alterations in sterol biosynthesis contribute to azole antifungal resistance in Candida parapsilosis. Antimicrob Agents Chemother. 2015 Oct;59(10):5942-50. PMID: 26169412
  19. Rybak JM, Dickens CM, Parker JE, Caudle K, Manigaba K, Whaley SG, Nishimoto A, Luna-Tapia A, Roy S, Zhang Q, Barker KS, Palmer GE, Sutter TR, Homayouni R, Wiederhold NP, Kelly SL, Rogers PD. Loss of C-5 sterol desaturase activity results in increased resistance to azole and echinocandin antifungals in a clinical isolate of Candida parapsilosis. Antimicrob Agents Chemother. 2017 Jun 19. pii: AAC.00651-17. doi: 10.1128/AAC.00651-17. [Epub ahead of print] PMID: 28630186
Members and Collaborators

Lab Members:
P. David Rogers, PharmD, PhD, FCCP – Principal Investigator
Qing Zhang – Laboratory Manager
Kathy Barker, PhD – Research Specialist
Sarah G. Whaley, PharmD – Graduate Student, Pharmaceutical Sciences
Andrew T. Nishimoto, PharmD –  Graduate Student, Pharmaceutical Sciences
Jeffery M. Rybak, PharmD – Graduate Student, Integrated Biomedical Sciences
Laura Doorley – Graduate Student, Integrated Program in Biomedical Sciences
Yu Li – Graduate Student, Integrated Program in Biomedical Sciences 

Key Collaborators:
Joachim Morschhäuser, PhD – Universität Würzburg        
Steven Kelly, PhD, DSc – Swansea University
Scott Moye-Rowley, PhD – University of Iowa
Damian Krysan, MD, PhD – University of Rochester
Theodore White, PhD – University of Missouri Kansas City
Nathan Wiederhold, PharmD – University of Texas Health Science Center

 

Last Published: Nov 7, 2017