“Take Two and Call Me in the Morning” A Case Study in Cell Structure and Function

1. “Take Two and Call Me in the Morning”A Case Study in Cell Structure and Function by Peggy Brickman University of Georgia

2. Part I - Mysterious Illness • Well, Becky thought, being a dorm counselor for freshmen was not going to be that bad. She got a free room for the year and the food was plentiful - free steaks last week at an outdoor BBQ followed by a hay ride in a horse-drawn wagon in their welcome celebration. • But, then again, it wasn’t perfect: she had ended up covered in bug bites; some of the students got sick from eating steak that was burned on the outside and raw in the middle; the horses had mucked up the courtyard; and pigeons had roosted on the dorm roof. • At least tonight the students were finally settling in and quieting down, she mused.

3. Part I, continued… • The quiet was shattered a few minutes later when one of the other counselors, Ann, yelled through her door: • “Becky, we’ve got a problem. One of the students found a homeless kitten, and the girl has been keeping her in her room. I only found out because the girl, Ellie, just came to my room complaining of being sick. I felt sick too when I saw the mess that kitten made. I thought cats were born housebroken, but I guess not.” • “Anyway, now I think Ellie might really be sick,” Ann continued. “She’s feverish and says she’s going to throw up.”

4. Part I, continued • “What do you want me to do?” Becky asked. • “I’m freaking out!” Ann answered. “Forget about the mess, just help me figure out what to tell them at the health center. I don’t know what she’s been exposed to. Or what we’ve been exposed to for that matter! This is the second girl this week with aches, fever, and nausea.” • “My Mom sent me a bunch of medicine,” Becky answered. “I’ll make a list of where we’ve been, what we’ve eaten, and what we’ve possibly been exposed to. Then we can start taking something right away to keep from getting it, too.”

5. Becky’s Task • The Health Center will be using the differences between organisms to diagnose and treat Ellie. • In the next 2 minutes, list the clues in the story that help you identify how Ellie could have contracted a disease with flu-like symptoms. Come up with possible suspects (organisms) that could cause her to be sick.

6. Your Task • Becky did an Internet search and found 5 possible suspects that could be causing Ellie’s illness. • During this class session we will investigate the differences between them. • Organisms are usually distinguished by the characteristics you listed in your homework answers to Table 1. Add any details you missed so that when you hear the results of the Health Center tests you will be able to figure out what was making Ellie sick. • Fill in possible drug treatments in Table 2.

7. Ellie’s Diagnosis • Initial Identification: The Health Center collected blood samples from Ellie and observed her cells under a microscope. They identified foreign structures with DNA and outer membranes. The cells were gram negative and about 1/10 the size of her cells. • “Ah, ha!” said Becky. “That matches one of my suspects. I knew those were a health hazard. I just need to re-check the size thing. This internet chart compares our cells to viruses and stuff.”

8. 1 meter (m) = ~3 feet 1 meter (m) = 1000 millimeter (mm) 1 millimeter (mm) = 1000 micrometer (µm) (smallest size distinguished by naked eye) 1 micrometer (µm) = 1000 nanometer (nm) (only seen with light microscope) 1 mm poppy seed = (1000 µm/mm) = 1000 µm 1 mm 10-3 Cells like ours 10-4 10-5 Bacteria 1 µm 10-6 meters 10-7 Viruses 10-8 Proteins Atoms 10-9 1 nm Metric Review

9. Becky’s Internet Search Results – List of Suspects Suspect 1: Coxiella burnetii causes Q-fever. Coxiella are often found in livestock and are excreted in milk, urine, and feces. Infection occurs 2-3 weeks after inhalation of barnyard dust. They are0.3-0.5 µm gram-negative bacterium (prokaryotes) that must invade and reside inside human cells to cause infection. 1µm

10. Prokaryotes • Unicellular • Reproduce asexually • Composition • Protected interior (cytoplasm) that contains genetic material (one circle of DNA) as well as complexes of protein enzymes to carry out necessary functions of gathering energy, manufacturing proteins (ribosomes), etc.

11. Prokaryotes • Size • 0.2-10 micrometer (µm) • Composition • Phospholipid membrane, many contain cell wall composed of peptidoglycan (positive for chemical Gram stain), those with little or no peptidoglycan called Gram negative (like Coxiella).

12. CQ1: “That’s great,” Becky said. “My Mom sent me 3 different antibiotics to kill bacteria.” Given the description of Ellie’s test results, which antibiotic will definitely NOT work: A: Amoxicillin, Penicillin, and other ß-lactams • Blocks the enzyme that normally creates links in peptidoglycan molecules. B: Streptomycin • Blocks prokaryotic ribosomes. C: Ciprofloxacin hydrochloride (Cipro) • Blocks bacterial DNA gyrase enzyme needed to counteract excessive twisting of DNA that occurs when circles of DNA are unwound to be copied into DNA or RNA.

13. 1 mm 10-3 Cells like ours 10-4 10-5 Bacteria meters 1 µm 10-6 10-7 Viruses 10-8 Proteins Atoms 10-9 1 nm CQ2: “Wait a minute!” Ann said. “The doctor said the blobs in Ellie’s blood were 1/10th the size of her cells. Could they be Coxiella?” A: Yes B: No

14. Part II: Microscope Analysis Becky and Ann talked together outside the student’s room at the student health center the next morning. “You’re right!” Becky exclaimed after viewing photographs of Ellie’s blood up close. “I wish I hadn’t started taking the antibiotics. The little crescent shaped structures that I thought were the bacteria may not be. When you zoom in on them, they show up clearly in the electron micrograph on the right. They aren’t too big to be bacteria, but they aren’t too small to be mitochondria or some kind of protozoan parasite.” “Wait a minute,” Ann replied. “The things on the right are the pathogens? Look at their insides, they can’t be bacteria.” “Why not?” Becky asked.

15. Part II, Continued 1µm

16. CQ3: “Well,” Becky admitted, “there should be differences between Ellie’s cells and the little blobs they saw. Otherwise, it might mean one of my other suspects is the cause. These are some of the structures normally found in all cells.” “No,” Ann answered, “one isn’t.” Which structure is NOT found in all cells? A: Cytoplasm B: DNA C: Outer phospholipid membrane D: Ribosomes E: Membrane-bound organelles

17. EukaryotesProkaryotes

18. Becky’s Internet Search Results – List of Suspects Eukaryote - Suspect 2: Cryptococcus neoformans 2.5-10 µm encapsulated fungus found in decaying pigeon or chicken droppings. Inhaled as spores that eventually spread to the brain causing meningoencephalitis. Has a black pigmented layer that can be seen sometimes on bird seed.

19. Becky’s Internet Search Results – List of Suspects Eukaryote - Suspect 3: Toxoplasma gondii • 4-6 µm single-celled protozoan parasite of mammals & birds. • Most likely acquired through ingesting cysts in undercooked meat.

20. Becky’s Internet Search Results – List of Suspects Suspect 3: Toxoplasma gondii • Usually no symptoms, but can cause flu-like complaints. • Sexual life cycle occurs in cats, so infection can follow contact with cat feces. • See QuickTime movies • “Invasion 4” and “Escape 2” at • http://www.uvm.edu/microbiology/videos_ward.php?id=23

21. Eukaryotes • Uni- or multicellular. • Reproduce asexually & sexually. • Composition: • Genetic material (long linear strands of DNA chromosomes) especially isolated and enclosed in membrane (nucleus) • Some have cell walls (plants have cellulose, fungi ß-glucan)

22. Eukaryotes • Size 10-100 (µm) • Composition: • Phospholipid membrane outside, as well as inside. • Interior membranes separate functions such as gathering and transforming cellular energy and manufacturing macromolecules.

23. Eukaryotic Organelles Animal Cell Plant Cell

24. Eukaryotic Organelles Endoplasmic reticulum Nucleus Mitochondrion Golgi Chloroplast

25. CQ4: Becky’s Anti-Eukaryotic Medicines: • Pyrimethamine, Sulfonamides: Interfere with enzymes used to make the folic acid needed to make thymine and uracil nucleotides. • Polyenes combine with a component of fungal and some bacterial membranes, disrupt and break them. • One of these drugs specifically affects one of the two eukaryotic suspects. Which test of Ellie’s blood would help you tell which eukaryotic suspect she was infected with? • A: Presence of DNA. • B: Presence of ß-glucan-containing cell walls. C: Presence of cellulose. D: Presence of peptidoglycan cell walls.

26. Part III: Viruses • Becky and Ann are back at the dorm waiting for the results of more tests. • “I’ve also got some tamiflu,” Becky volunteered. “I mean, what if those cells in the picture aren’t really making her sick. Maybe she just has the plain old flu.” • “What do you mean?” Ann asked. What’s the difference?” • “Flu is a virus,” Becky answers. “I’ve actually got two suspects that are viruses. They’re probably the most different from the prokaryotes and eukaryotes. They’re not even cells.”

27. Becky’s Internet Search Results – List of Suspects Electron micrographs • Influenza Virus: Spread primarily through respiratory droplets from sneezing or coughing. Virus has single strand of RNA surrounded by phospholipids/protein envelope (80-120nm). • West Nile Virus: Spread by mosquitoes that have previously fed on infected birds. 20% of infected people show symptoms. Single stranded RNA, phospholipid/protein envelope (50nm).

28. Viruses • Not cells • Cannot reproduce alone • hijacks a host cell to replicate itself. • Composition • Outer shell: repetitive protein often inserted into a lipid envelope (responsible for recognition and infection of host cell.)

29. Viruses • Size • Smallest Organisms (50nm) • 100 times smaller than bacteria • Composition • Protected interior that contains genetic material (DNA or RNA) with important protein enzymes required for duplication.

30. Virus hijacking host system

31. Tamiflu: Blocks neuraminidase enzyme made by all influenza A strains (cause the “flu” and avian flu.) Viruses are unable to remove sticky sialic acid, and can’t escape.

32. Take a few minutes to complete the homework table comparing viruses, bacteria, and eukaryotes. Table 1

33. CQ5: Match the description with the suspect. Use your responses to Table 1 to identify the row below that best describes West Nile Virus.

34. Part IV: DNA Analysis “Well, it isn’t viral,” Becky said, closing her cell phone. “No neuraminidase. But, they found some foreign DNA with the sequence: AACGTGGTCGTT. The closest match is a gene used to make ribosomes (rDNA). They are searching a huge DNA database of sequences to find the organism that has the closest match.”

35. A: Ellie’s nucleus ATGGTCTCAATG B: Ellie’s mitochondria TTGGTCCGTCAG C: Coxiella bacteria TTGGTCGGTCAG D: Toxoplasma nucleus AACGTGGTAGTT E: Cryptococcus nucleus ATGGTGGCAATG CQ6: Foreign DNA sequence isolated from Ellie: ACGTGGTCGTT. Which sequence is the best match with this foreign DNA?

36. Strange Similarities “What’s weird is that Ellie’s mitochondrial DNA matches the Coxiella sequence so closely.” Ellie’s mitochondria TTGGTCCGTCAG Coxiella bacteria TTGGTCGGTCAG “That makes sense in a way,” Ann answered. “There is a lot of evidence that points to mitochondria being descendents of gram-negative bacteria just like Coxiella.” “What type of evidence?” Becky asked.

37. N prokaryote N C Eukaryotic Organelles & Endosymbiosis Strange similarities: Chloroplasts and mitochondria are the same size as prokaryotes. Both have circular DNA without histones with similar sequence to photosynthetic bacteria (cyanobacteria) and obligate intracellular rickettsia bacteria. Both divide like prokaryotes.

38. N prokaryote N C Eukaryotic Organelles & Endosymbiosis Strange similarities: Have their own protein synthesis machinery (ribosomes) more like bacteria than eukaryotes (sensitivity to Streptomycin). Inner membrane of mitochondria contains unusual phospholipid characteristic of bacterial membranes.

39. Similarities Used to Group Organisms Single-celled eukaryotes 2 billion years ago First prokaryotes 3.75 billion years ago

40. CQ7: Which letter best defines the place mitochondria would take next to their closest relatives on this family tree of living organisms? A B C D E

41. Finale: Ellie’s Prognosis “Well, Ellie’s responding well to the pyrimethamines that the doctors prescribed,” Becky commented to Ann while checking her email a few days later at the dorm. “Yeah, and we’re lucky the cipro we took couldn’t harm our cells,” Ann replied. “We were so wrong! I’m never self-medicating again. Do you think we should warn the other students. They might have had contact with the kitten, too.” “We don’t know if it was from cat poop,” Becky answered. “I learned that something like 25-40% of American adults are already infected with Toxoplasma gondii, and not because of their cats—usually it’s from eating raw meat. Plus, apparently the oocysts in fresh cat poop aren’t infectious for a couple of days. So, if you scoop the box right away you don’t have to worry.” “So now I have to know how often the cat box is cleaned?! I don’t think I’m cut out for this job!” Ann moaned.