One more amitochondriate. Trichomonads. Trichomonas vaginalis. Trichomonads. Class Trichomonada All parasitic. Family Trichomonadidae

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1 One more amitochondriate Trichomonads Trichomonads Class Trichomonada All parasitic Family Trichomonadidae Trichomonas tenax Human - mouth Trichomonas vaginalis Human - urogenital Tritrichomonas foetus Bovine - urogenital Trichomonas vaginalis Worldwide distribution (cosmopolitan) 7.4 million cases/yr in US (2007) 200 milion cases/yr worldwide Trichomoniasis - vaginitis Variable symptoms Females - only ~ 15 are asymptomatic Asymptomatic, to mild or moderate infections, to extreme vaginitis 50-75% abnormal discharge (frothy whitish, yellowish or greenish) 50% experience painful intercourse vaginal erythema - (2%) strawberry cervix Males % are asymptomatic minor urethral discharge

2 Trichomonas and HIV Transmission T. vaginalis amplifies HIV transmission The pathology caused by Trichomonas infection includes: - punctate hemorrhages - infiltration of white blood cells (including lymphocytes and macrophages) Trichomonas in an HIV Positive Person Trichomonas in an HIV Negative Person Hemorrhages allow ready access of HIV Infiltration of white blood cells provides cellular targets for HIV Trichomonas makes an HIV negative person more susceptible to HIV Trichomonas makes an HIV + person more infectious Hemorrhages allow leakage of free virus Infiltration of white blood cells includes HIV infected leucocytes This results in higher concentration of HIV in the infected area Epidemiological studies in Africa indicate a 23 fold increase in HIV transmission in people infected with T. vaginalis Trichomonad Life Cycle - Direct One form - trophozoite Binary fission Direct Transmission Direct contact with the parsite Classified as a STD Diagnosis Clinical presentation: discharge, strawberry cervix Visualization of the motile parasites in vaginal secretions (45-60% sensitivity) Culture of parasites in special media. (InPouch). This method will identify up to 95% of infections. Antigen detection test from Genzyme Corp. ( % sensitivity) Oligonucleotide probe test (80-90% sensitivity) PCR-based tests OSOM Trichomonas Rapid Test A rapid test for the detection of Trichomonas Vaginalis Easy - to - Run - Dipstick simple, one reagent. Accurate - 95% agreement against Composite Reference Standard (culture and wet mount). Unaffected by other common infections. InPouch, Biomed Diagnostics. A two-chambered plastic bag culture system to improve

3 Treatment METRONIDAZOLE: 2g orally in a single dose (97% cure rate) Brand name: Flagyl. Sexual partner should be treated TINIDAZOLE: recently licensed for use in the USA. Useful for resistant infections. (Really??) Sexual education helps prevent the spread of this disease Similarities and Differences G. lamblia E.histolytica T. vaginalis Morphology Flagellate Amoeba Flagellate/Amoeba Life Stages Trophozoite Trophozoite Trophozoite Only Resistant Cyst Resistant Cyst No Cyst Form Host Many Mammals Human Only Human Only Including Humans Transmission Ingestion of Cyst Ingestion of Cyst Sexual Intercourse Disease Diarrhea Diarrhea Vaginitis, PROM Manifestations Dysentery Liver Absess Amitochondriate Comparison Early Diverging Eukaryote Lumen-dwelling Extracellular Parasite Treatment via Metronidazole Microaerophilic Pyruvate fermentation No Classical Mitochondria

4 Genome sequenced in 2007 Highest coding capacity among Eukaryotes Massively expanded gene families One of the most repetitive genomes sequenced Large number of lateral gene transfers Newly discovered metabolic pathways Multiple ferredoxin genes Genome Sequenced 2007 Dense glycocalyx composed of LPG Genes for LPG biosynthetic pathway Monosaccharide rhamnose absent in the human Largest family of membrane proteins - BspA-like Similar to leucine rich adherence proteins in bacteria Much more work is required to understand the roles of these surface proteins. Cellular Microbiology (2008) Galectin-1 on cervical epithelial cells is a receptor for the sexually transmitted human parasite Trichomonas vaginalis Cheryl Y. Okumura, Linda G. Baum and Patricia J. Johnson Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA.

5 Galectin-1 binding T. vaginalis Cervical epithelium LPG Gal-1 Gal-1 in cervical epithelia (CEC) is involved in parasite cross-linking Gal-1 is the first identified receptor in CE that binds T. vaginalis Monosacharides in LPG interact with gal-1 homodimer Trichomonad structure Anterior nucleus Anterior flagella T. vaginalis - 4 Tritrichomonas foetus - 3 Recurrent flagellum Costa Axostyle Amitochondriate?? Contain a typical golgi Lack peroxisomes Lack mitochondria Granules associated with axostyle and costa Hydrogenosomes Abundant double membrane organelles Not a microbody!

6 The Hydrogenosome Hydrogenosome Anaerobic equivalent to mito Pyruvate fermentation ATP, CO 2, Acetate, H 2 Differences with mitochondria Morphology - no cristae No oxidative phosphorylation No DNA Evolution of mitochondria Drug target! Christian de Duve (relationship?) HYDROGENOSOME MITOCHONDRION Exploring Cells Light Microscopy - localization Electron Microscopy - localization Genetics - Phenotypic changes Exploring cells with a centrifuge! Founders of Modern Cell Biology Keith Porter National Medal of Science 1976 If I have seen further it is by standing on the shoulders of giants - Isaac Newton

7 Subcellular Fractionation Methodologies developed in the 50s Development of the ultracentrifuge Instrumental in the discovery of: Lysosomes - turned 55 in 2011! Peroxisomes Hydrogenosomes - will turn 40 in 2013 Ability to correlate the sedimented enzymatic activities with similar kinetics; could be attributed to structure(s) observed via EM. Subcellular Fractionation The ultimate reductionist approach 80s- 90s - popularity of this method declined Why? 21st century - ia this still a valid approach? Organellar Proteomics!! Think about Johnson Rev2011- severla proteomic studies Cell Separation Techniques Cell Extract Cell Homogenate N M C Differential Centrifugation Differential Gradient Centrifugation

8 Important Concepts/Assumptions Different sedimentation coefficients Polydispersity of a population Looked at the distribution of single enzymes across the fractionation (Histograms) Postulate of biochemical homogeneity large particles contain same biochemical composition - just more of it Postulate of single location One enzyme - one location Quality Control Balance sheet that contains: Recorded volumes of fractions Distribution of protein among the fractions Distribution of marker enzymes - proteins Latency of those marker enzymes Confirmation via EM studies substrate Histogram Analysis 1st introduced by de Duve Glucose-6-phosphatase enriched in ER fragments Relative specific activity N M P N-nuclear M-mitochondrial P-microsomal S-supernatant S % protein

9 Histogram Analysis 1st used by de Duve Relative specific activity N M P Cytochrome oxidase enriched in mitochondria N-nuclear M-mitochondrial P-microsomal S-supernatant S % protein Original data De duve (1955) Organelles (structures) are separated by their physical properties. Problem: some of these compartments share physical properties. Pattern I Pattern II Pattern III Original data De duve (1959) N-nuclear M-heavy mitochondrial L-light mitochondrial Polydispersity P-microsomal S-supernatant Possible problems: breakage of particles adsorption of soluble parts leakage from particles Much more fundamental: How to separate populations that display polydispersity

10 Density Gradient Centrifugation Different buoyant densities of organelles Linear gradient of viscous solution Purification of organelle of interest from other similar sized particles Lysosomes Hydrogenosomes Discovered by Lindmark and Muller, 1973 Rockefeller University Worked down the hall from C. de Duve! Purified Hydrogenosomes Hydrogenosomal Metabolism Glycolysis malate NADH pyruvate pyruvate CO 2 NFO 2Fd PFO (cytosol) NAD+ H2 Hydrogenase 2Fd- 2H+ acetyl CoA ASCT succinate CoA acetate succinyl-coa Pi STK ATP ADP

11 Hydrogenosomal/Mitochondrial Comparison Only Mitochondrial Shared Only Hydrogenosomal PDC complex TCA cycle Cytochromes Cytochrome oxidase DNA Cardiolipin F 0 F 1 ATPase Malic enzyme Ferrodoxins Adenylate kinase ATP/ADP exchanger Superoxide dismutase PFO Hydrogenase Oxidative phosphorylation vs. substrate level phosphorylation Carbohydrate Metabolism is Eukaryotes Hexose Hexose Hexose Pyruvate Pyruvate Pyruvate + O 2 CO 2 + Acetate CO 2 + H 2 + Acetate CO 2 + H 2 O No compartmentalization Hydrogenosomal compartmentalization Mitochondrial compartmentalization Substrate level phosphorylation Substrate level phosphorylation Oxidative phosphorylation Synthesis of ATP Oxidative phosphorylation - coupling of ATP formation to the respiratory chain (electron transport, membrane associated, O 2 as final e- acceptor). As electrons move through complexes, a proton gradient is generated which drives ATP formation. Chemiosmotic theory - P. Mitchell, Substrate level phosphorylation - direct phosphorylation of ADP via the transfer from a high-energy intermediate.

12 Origin of Hydrogenosomes (Mitosomes?) 1. a) Conversion of Mitochondria HYD b) Common ancestor with mitochondria? HYD 2. Independent Origin α-proteobacterium? - anaerobic bacterium HYD Approaches: Organelles - origins and biogenesis (1) Conduct phylogenetic analyses of similar proteins Hsp70 Fd Hsp60 Isc subunits (2) Examine protein targeting to the organelle matrix protein targeting membrane protein targeting (3) Characterize membrane/translocation components These components could have evolved as the endosymbiont was converted to organelle. Reveals evolutionary history. Comparison of Biogenesis Nuclear encoded preproteins synthesized on cytosolic ribosomes. Kept in a translocation competent form by cytosolic chaperones Related TOM and TIM components. Similar energetic requirements Matrix chaperones and processing protein. Similar targeting complex to the inner membrane. Hsp90 Hsp70 Review mitochondrial targeting if you are unfamiliar. Translocase of the outer membrane - TOM Translocase of the inner membrane - TIM Mitochondrial processing peptidase - MPP

13 Protein sorting and targeting signals Co-translational targeting of secretory proteins via the RER ER targeting N-terminal cleaved peptide amino acids (aa) Basic & hydrophobic enriched Lysosomal targeting Mannose-6-phosphate (sugar moiety, not removed) Post-translational targeting of organellar proteins via cytosolic chaperones Nuclear targeting Basic, internal sequence (not cleaved) Often bipartite Peroxisomal targeting C-terminal peptide (not cleaved) 3 aa (Ser Lys Leu; SKL) Other signals used also Mitochondrial Matrix targeting N-terminal cleaved peptide aa Rich in Arg, Leu, Ser Methodology to study processes Molecular Biology Tools Manipulate organism Cloning methodologies Cell Biology Tools Observe the organism Specific localization via tags, antibodies Biochemistry Tools Cell fractionation Proteomics Molecular Engineering Can changes the properties of any ORF that one wants to study Visualization introduce a tag Mutations Overexpression vs. endogenous expression levels Targeting Signal ORF ORF ORF ORF ORF GFP HA-epitope GFP GFP GFP Necessary and Sufficient

14 Transfection of DNA Electroporation of DNA Electrical discharge Can be used for various cell types Emperically determine conditions for each cell type Reversible destabilization of the cell membrane Transient formation of membrane pores Potentiates uptake of DNA Confocal Microscopy Significant advancement Single point of light emission that can scan across the specimen Spatial filtering techniques to eliminate out-of-focus light Digital cameras Three-dimensional renderings of images Comparison of Images Background fluorescence Sharper fluorescent images Single section Optical sectioning

15 Co-localization studies Importance of appropriate markers Different colored fluorophores are used IscS Mitotracker DAPI Merged Images Protein Import Studies in organello translocation of pfd into isolated hydrogenosomes 35 SDS-PAGE S - pfd pfd Fd 37 o C, 15 Proteinase K ATP + ATP Trap precursor using ATP depleted assay 35 S - pfd SDS-PAGE pfd Fd 37 o C, 15 Proteinase K No ATP - ATP Common features of mitochondrial and hydrogenosomal biogenesis established using protein targeting assays and biochemistry NUCLEUS AAAA mrna Synthesis of preproteins on cytosolic ribosomes with mitochondriallike presequence CYTOSOL X PREPROTEIN Interaction with cytosolic chaperones NEM-sensitive MITOCHONDRION Hsp70 cpn60 MPP cpn10 TOM TIM Presequence-dependent translocation ATP, temperature, ΔΨ-dependence Presequence cleavage (MPP) Similar presequences Mt-type Hsp 70 Chaperonins, cpn60/cpn10 Common membrane carriers

16 Giardia Mitosome Localization Key mitochondrial markers localize to the mitosome: Cpn60, Hsp70, IscS, IscU, Fd Mitochondrial Diversity Chicken Neocallimastix T. foetus 100 nm 50 nm Entamoeba - mitosome microsporidian - mitosome Giardia - mitosome Origins via Endosymbiosis Aerobic α-proteobacterium prokaryote gave rise to present day mitochondria. Are hydrogenosomes and mitosomes of anaerobic protists derived from the same proto-mitochondrion? Evidence for: accumulating evidence for several proteins that are currently found in mitochondria - Current dogma - mitochondria and related organelles arose just once in evolution Proteins of Fe-S cluster formation. Scenario A Common ancestor Scenario B degenerate mitochondrion invoke lateral gene transfer from anaerobic prokaryotes facultative organism?

17 Reconstructing Evolution Mitochondrial evolution well established endosymbiotic theory α-proteobacterium - Rickettsia prowazekii Hydrogenosomal evolution No DNA (NOW 1 example 2005, Nyctotherus!) Several proteins similar to mitochondria Mitosome evolution No DNA Few proteins identified similar to mitochondria Reconstructing Evolution Embley and Martin (2006) Nature 440:623 Organellar Comparison