Resume

• 2009

  Molecular Mycology Course: Current Approaches to Fungal Pathogenesis

  Training in different molecular methods used to study the human\nfungal pathogens Candida

  Aspergillus

  and Cryptococcus

  and \nthe models used to uncover the mechanisms that underlie fungal \ndiseases and their treatment.

  Mycology

  Marine Biological Laboratory

  Woods Hole

• 2002

  Ph.D

  Cellular and Molecular Biology

  University of Wisconsin-Madison

• 1997

  Spanish

  Norwegian

  Chemistry

  and Norwegian

  St. Olaf College

  Bachelor of Arts (B.A.)

  Chemistry and Norwegian

  St. Olaf College

• Mycology

  RNA isolation

  Microscopy

  recombinant DNA technology

  PCR

  RT-PCR

  Molecular Cloning

  University Teaching

  DNA

  Western Blotting

  Confocal Microscopy

  Protein Purification

  Microbiology

  Fluorescence Microscopy

  Molecular Biology

  Genetics

  Protein Expression

  Biology

  Molecular Genetics

  qPCR

  Bcr1 functions downstream of Ssd1 to mediate antimicrobial peptide resistance in Candida albicans.

  In order to colonize the host and cause disease

  Candida albicans must avoid being killed by host defense peptides. Previously

  we determined that the regulatory protein Ssd1 governs antimicrobial peptide resistance in C. albicans. Here

  we sought to identify additional genes whose products govern susceptibility to antimicrobial peptides. We discovered that a bcr1Δ/Δ mutant

  like the ssd1Δ/Δ mutant

  had increased susceptibility to the antimicrobial peptides

  protamine

  RP-1

  and human β defensin-2. Homozygous deletion of BCR1 in the ssd1Δ/Δ mutant did not result in a further increase in antimicrobial peptide susceptibility. Exposure of the bcr1Δ/Δ and ssd1Δ/Δ mutants to RP-1 induced greater loss of mitochondrial membrane potential and increased plasma membrane permeability than with the control strains. Therefore

  Bcr1 and Ssd1 govern antimicrobial peptide susceptibility and likely function in the same pathway. Furthermore

  BCR1 mRNA expression was downregulated in the ssd1Δ/Δ mutant

  and the forced expression of BCR1 in the ssd1Δ/Δ mutant partially restored antimicrobial peptide resistance. These results suggest that Bcr1 functions downstream of Ssd1. Interestingly

  overexpression of 11 known Bcr1 target genes in the bcr1Δ/Δ mutant failed to restore antimicrobial peptide resistance

  suggesting that other Bcr1 target genes are likely responsible for antimicrobial peptide resistance. Collectively

  these results demonstrate that Bcr1 functions downstream of Ssd1 to govern antimicrobial peptide resistance by maintaining mitochondrial energetics and reducing membrane permeabilization.

  Bcr1 functions downstream of Ssd1 to mediate antimicrobial peptide resistance in Candida albicans.

  Candida species cause frequent infections owing to their ability to form biofilms — surface-associated microbial communities — primarily on implanted medical devices. Increasingly

  mechanistic studies have identified the gene products that participate directly in the development of Candida albicans biofilms

  as well as the regulatory circuitry and networks that control their expression and activity. These studies have uncovered new mechanisms and signals that govern C. albicans biofilm development and associated drug resistance

  thus providing biological insight and therapeutic foresight.

  Genetic control of Candida albicans biofilm development

  As obligate intracellular parasites

  viruses expertly modify cellular processes to facilitate their replication and spread

  often by encoding genes that mimic the functions of cellular proteins while lacking regulatory features that modify their activity. We show that the human cytomegalovirus UL97 protein has activities similar to cellular cyclin–cyclin-dependent kinase (CDK) complexes. UL97 phosphorylated and inactivated the retinoblastoma tumor suppressor

  stimulated cell cycle progression in mammalian cells

  and rescued proliferation of Saccharomyces cerevisiae lacking CDK activity. UL97 is not inhibited by the CDK inhibitor p21 and lacks amino acid residues conserved in the CDKs that permit the attenuation of kinase activity. Thus

  UL97 represents a functional ortholog of cellular CDKs that is immune from normal CDK control mechanisms

  Phosphorylation of Retinoblastoma Protein by Viral Protein with Cyclin-Dependent Kinase Function

  Sen1p in Saccharomyces cerevisiae is a Type I DNA/RNA helicase. Mutations in the helicase domain perturb accumulation of diverse RNA classes

  and Sen1p has been implicated in 3′ end formation of non‐coding RNAs. Using a combination of global and candidate‐specific two hybrid screens

  eight proteins were identified that interact with Sen1p. Interactions with three of the proteins were analyzed further: Rpo21p(Rpb1p)

  a subunit of RNA polymerase II

  Rad2p

  a deoxyribonuclease required in DNA repair

  and Rnt1p (RNase III)

  an endoribonuclease required for RNA maturation. For all three interactions

  the two‐hybrid results were confirmed by co‐immunoprecipitation experiments. Genetic tests designed to assess the biological significance of the interactions indicate that Sen1p plays functionally significant roles in transcription and transcription‐coupled DNA repair. To investigate the potential role of Sen1p in RNA processing and to assess the functional significance of the Sen1p/Rnt1p interaction

  we examined U5 snRNA biogenesis. We provide evidence that Sen1p functions in concert with Rnt1p and the exosome at a late step in 3′ end formation of one of the two mature forms of U5 snRNA but not the other. The protein–protein and protein–RNA interactions reported here suggest that the DNA/RNA helicase activity of Sen1p is utilized for several different purposes in multiple gene expression pathways.

  Multiple protein/protein and protein/RNA interactions suggest roles for yeast DNA/RNA helicase Sen1p in transcription

  transcription‐coupled DNA repair and RNA processing

  The Saccharomyces cerevisiae SEN1 gene codes for a nuclear-localized superfamily I helicase. SEN1 is an ortholog of human SETX (senataxin)

  which has been implicated in the neurological disorders ataxia-ocular apraxia type 2 and juvenile amyotrophic lateral sclerosis. Pleiotropic phenotypes conferred by sen1 mutations suggest that Sen1p affects multiple steps in gene expression. Sen1p is embedded in a protein–protein interaction network involving direct binding to multiple partners. To test whether the interactions occur independently or in a dependent sequence

  we examined interactions with the RNA polymerase II subunit Rpb1p

  which is required for transcription

  and Rnt1p

  which is required for 3′-end maturation of many noncoding RNAs. Mutations were identified that impair one of the two interactions without impairing the other interaction. The effects of the mutants on the synthesis of U5 small nuclear RNA were analyzed. Two defects were observed

  one in transcription termination and one in 3′-end maturation. Impairment of the Sen1p–Rpb1p interaction resulted in a termination defect. Impairment of the Sen1p–Rnt1p interaction resulted in a processing defect. The results suggest that the Sen1p–Rpb1p and Sen1p–Rnt1p interactions occur independently of each other and serve genetically separable purposes in targeting Sen1p to function in two temporally overlapping steps in gene expression.

  Sen1p Performs Two Genetically Separable Functions in Transcription and Processing of U5 Small Nuclear RNA in Saccharomyces cerevisiae

  Teun Boekhout

  M. ElGindi

  Saad Taj-Aldeen

  OBJECTIVES: \nWe identified Candida spp isolated from lower respiratory tract secretions obtained from cystic fibrosis (CF) patients

  by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)

  with the aim of determining the most prevalent causative agent. We also sought to determine their adhesive properties in order to understand their biology related to CF.\nMETHODS: \nTwenty-five clinical samples were collected from a cohort of 20 CF patients. Twenty-six isolates of Candida spp were isolated and identified by MALDI-TOF MS method. Adherence assays were performed using the Fluxion BioFlux 200

  a flow apparatus that allows for the visualization of adhering cells.\nRESULTS: \nMALDI-TOF MS analysis revealed C. dubliniensis to be the most prevalent species (n=18

  69%)

  followed by C. albicans (n=4)

  C. tropicalis (n=3)

  and C. glabrata (n=1). C. dubliniensis showed the strongest adherence under constant flow when compared to the other species of Candida. In the majority of cases

  C. dubliniensis was isolated in combination with Pseudomonas aeruginosa and Staphylococcus aureus. C. dubliniensis appears to be able to survive in the CF lung and coexist with bacteria.\nCONCLUSIONS: \nThe data presented here show that the presence of C. dubliniensis in the lower airways of CF patients may be related to increased adherence properties.

  High prevalence of Candida dubliniensis in lower respiratory tract secretions from cystic fibrosis patients may be related to increased adherence properties.

  An understanding of gene function often relies upon creating multiple kinds of alleles. Functional analysis in Candida albicans

  a major fungal pathogen

  has generally included characterization of mutant strains with insertion or deletion alleles and over-expression alleles. Here we use in C. albicans another type of allele that has been employed effectively in the model yeast Saccharomyces cerevisiae

  a \"Decreased Abundance by mRNA Perturbation\" (DAmP) allele (Yan et al.

  2008). DAmP alleles are created systematically through replacement of 30 noncoding regions with nonfunctional heterologous sequences

  and thus are broadly applicable. We used a DAmP allele to probe the function of Sun41

  a surface protein with roles in cell wall integrity

  cell-cell adherence

  hyphal formation

  and biofilm formation that has been suggested as a possible therapeutic target (Firon et al.

  2007; Hiller et al.

  2007; Norice et al.

  2007). A SUN41-DAmP allele results in approximately 10-fold reduced levels of SUN41 RNA

  and yields intermediate phenotypes in most assays. We report that a sun41Δ/Δ mutant is defective in biofilm formation in vivo

  and that the SUN41-DAmP allele complements that defect. This finding argues that Sun41 may not be an ideal therapeutic target for biofilm inhibition

  since a 90% decrease in activity has little effect on biofilm formation in vivo. We anticipate that DAmP alleles of C. albicans genes will be informative for analysis of other prospective drug targets

  including essential genes.

  Application of the systematic “DAmP” approach to create a partially defective C. albicans mutant

  Rpb1p

  the largest subunit of S. cerevisiae RNA polymerase II

  contains a repetitive structure called the C-terminal domain (CTD). The CTD serves as a scaffold for the regulated association and dissociation of more than a hundred proteins involved in RNA synthesis. Phosphorylation of two serine residues (Ser2 and Ser5) in the repeating units of the CTD change dynamically during the pre-initiation

  initiation

  elongation

  and termination of transcription to control the binding and release of transcriptional components. A modification of the well established yeast two-hybrid assay for protein-protein interactions is described that detects interactions between phosphorylated forms of the CTD and proteins whose interactions with the CTD depend on phosphorylation. The efficacy of the approach was established by first showing that two-hybrid fusions containing the CTD are phosphorylated at Ser2 and Ser5 residues. Interactions between the CTD and three known CTD-binding proteins were analyzed. The results suggest that the modified two-hybrid system accurately assays CTD-binding and provides a new and convenient assay for CTD-binding proteins.

  Detecting phosphorylation-dependent interactions with the C-terminal domain of RNA polymerase II subunit rpb1p using a yeast two-hybrid assay

  Nonsense-mediated mRNA decay (NMD) performs two functions in eukaryotes

  one in controlling the expression level of a substantial subset of genes and the other in RNA surveillance. In the vast majority of genes

  nonsense mutations render the corresponding transcripts prone to surveillance and subject to rapid degradation by NMD. To examine whether some classes of nonsense transcripts escape surveillance

  we asked whether NMD acts on mRNAs that undergo subcellular localization prior to translation. In Saccharomyces cerevisiae

  wild-type ASH1 mRNA is one of several dozen transcripts that are exported from the mother-cell nucleus during mitotic anaphase

  transported to the bud tip on actin cables

  anchored at the bud tip

  and translated. Although repressed during transport

  translation is a prerequisite for NMD. We found that ash1 nonsense mutations affect transport and/or anchoring independently of NMD. The nonsense transcripts respond to NMD in a manner dependent on the position of the mutation. Maximal sensitivity to NMD occurs when transport and translational repression are simultaneously impaired. Overall

  our results suggest a model in which ash1 mRNAs are insensitive to NMD while translation is repressed during transport but become sensitive once repression is relieved.

  Nonsense-Mediated Decay of ash1 Nonsense Transcripts in Saccharomyces cerevisiae

  Cell-substrate adherence is a fundamental property of microorganisms that enables them to exist in biofilms. Our study focuses on adherence of the fungal pathogen Candida albicans to one substrate

  silicone

  that is relevant to device-associated infection. We conducted a mutant screen with a quantitative flow-cell assay to identify thirty transcription factors that are required for adherence. We then combined nanoString gene expression profiling with functional analysis to elucidate relationships among these transcription factors

  with two major goals: to extend our understanding of transcription factors previously known to govern adherence or biofilm formation

  and to gain insight into the many transcription factors we identified that were relatively uncharacterized

  particularly in the context of adherence or cell surface biogenesis. With regard to the first goal

  we have discovered a role for biofilm regulator Bcr1 in adherence

  and found that biofilm regulator Ace2 is a major functional target of chromatin remodeling factor Snf5. In addition

  Bcr1 and Ace2 share several target genes

  pointing to a new connection between them. With regard to the second goal

  our findings reveal existence of a large regulatory network that connects eleven adherence regulators

  the zinc-response regulator Zap1

  and approximately one quarter of the predicted cell surface protein genes in this organism. This limited yet sensitive glimpse of mutant gene expression changes had thus defined one of the broadest cell surface regulatory networks in C. albicans.

  Portrait of Candida albicans Adherence Regulators

  The Saccharomyces cerevisiae SEN1 gene codes for a nuclear

  ATP-dependent helicase which is embedded in a complex network of protein-protein interactions. Pleiotropic phenotypes of mutations in SEN1 suggest that Sen1 functions in many nuclear processes

  including transcription termination

  DNA repair

  and RNA processing. Sen1

  along with termination factors Nrd1 and Nab3

  is required for the termination of noncoding RNA transcripts

  but Sen1 is associated during transcription with coding and noncoding genes. Sen1 and Nrd1 both interact directly with Nab3

  as well as with the C-terminal domain (CTD) of Rpb1

  the largest subunit of RNA polymerase II. It has been proposed that Sen1

  Nab3

  and Nrd1 form a complex that associates with Rpb1 through an interaction between Nrd1 and the Ser5-phosphorylated (Ser5-P) CTD. To further study the relationship between the termination factors and Rpb1

  we used two-hybrid analysis and immunoprecipitation to characterize sen1-R302W

  a mutation that impairs an interaction between Sen1 and the Ser2-phosphorylated CTD. Chromatin immunoprecipitation indicates that the impairment of the interaction between Sen1 and Ser2-P causes the reduced occupancy of mutant Sen1 across the entire length of noncoding genes. For protein-coding genes

  mutant Sen1 occupancy is reduced early and late in transcription but is similar to that of the wild type across most of the coding region. The combined data suggest a handoff model in which proteins differentially transfer from the Ser5- to the Ser2-phosphorylated CTD to promote the termination of noncoding transcripts or other cotranscriptional events for protein-coding genes.

  Interactions of Sen1

  Nrd1

  and Nab3 with Multiple Phosphorylated Forms of the Rpb1 C-Terminal Domain in Saccharomyces cerevisiae

  Jonathan

  Finkel

  Carnegie Mellon University in Qatar

  University of Detroit Mercy

  American Type Culture Collection

  Carnegie Mellon University

  Started from the ground up a government funded research laboratory that studied biofilm formation and adherence of a diverse set of pathogenic species isolated from the clinical setting.I lectured upper-level courses including Genetics

  Microbiology

  Molecular Biology

  and introductory Biology courses. I was a research mentor to 9 undergraduate students. Once a month I lead a journal discussion group

  with the area of discussion being determined by the students.

  Carnegie Mellon University in Qatar

  Carnegie Mellon University

  Carnegie Mellon University Pittsburgh

  Postdoctoral Fellow

  Laboratory of Aaron P. Mitchell

  Carnegie Mellon University

  Pittsburgh\n\tStudy of the genetic and molecular mechanisms governing C. albicans cell adherence and biofilm formation

  Postdoctoral Fellow

  Greater Detroit Area

  I am an Assistant Professor of Biology at the University of Detroit. In addition to teaching responsibilities

  I am continuing my research into biofilm formation by Candida albicans.

  Assistant Professor

  University of Detroit Mercy

  Manassas

  Virginia

  I am the Mycologist in charge of identifying and depositing medically relevant fungi in the NIAID BEI repository. This program aims to aid in the study of infectious diseases by providing essential reagents and strains to registered members free of charge. We will process

  QC and Mail free of charge. Suggestions for deposits are very welcome.

  Mycologist Scientist

  American Type Culture Collection

  American Society of Microbiology

  Mycological Society of America

  Genetic Society of America