Biotechnology: Eukaryotes and Gene Products

• Lecture 12 is on Biotechnology and the Eukaryotes: Fungi, Algae, Protozoa, and Helminths

Making a Gene Product in Bacteria

• E. coli are frequently used to make a gene product
• Advantages of using E. coli include it being easily grown and having a known genome
• Disadvantages of using E. coli include that it produces endotoxins and does not secrete its protein products
• Harvesting product by purifying from lysed cells is inefficient and expensive using E. coli

Making a Gene Product in Yeast and Other Cells

• Saccharomyces cerevisiae (yeast) is an option for making a gene product
• Advantages of using yeast include it being easily grown and having a larger genome than bacteria
• Yeast can express eukaryotic genes easily and continuously secrete the desired product
• Plant cells and whole plants may be used to express eukaryotic genes easily
• Plants are easily grown, large-scale, and low-cost
• Mammalian cells may be used to express eukaryotic genes easily
• Products made in mammalian cells can be used for medical use, although they are harder to grow

Therapeutic Applications: Gene Silencing

• Gene silencing is a therapeutic application
• Small interfering RNAs (siRNAs) bind to mRNA which is then destroyed by RNA-induced silencing complex (RISC)
• RNA interference (RNAi) inserts DNA encoding siRNA into a plasmid and transferred into a cell
• Hereditary diseases where abnormal proteins are produced have been treated with RNAI

Therapeutic Applications: Gene Editing

• CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated enzymes are found in bacteria and archaea
• CRISPR destroys foreign DNA
• Small RNA complementary to the desired target is attached to CAS enzyme (CRISPR-associated enzyme) and DNA to be inserted
• Guide RNA-CAS is inserted to a target cell
• Inside the cell, guide RNA binds complementary DNA, CAS cuts target DNA to inactivate gene
• Inserted DNA can be incorporated into the cell

Eukaryotic Pathogens- Medical Significance

• More than 2 billion people suffer from parasitic diseases
• An additional 500 million people are infected with intestinal protozoa and helminths
• A parasite is an organism that lives in or on a host and derives nutrients at the host's expense

Eukaryotic Pathogens

• Fungi, protozoa, and helminths organisms cause diseases in humans
• Most fungi, protozoa, and helminths require microscopic examination
• Fungi are cultured on laboratory media
• Infections caused by eukaryotes are difficult to treat because humans have eukaryotic cells
• Arthropods that transmit infectious diseases are called vectors
• Arthropod-borne diseases such as West Nile encephalitis are best controlled by limiting exposure to the arthropod

Fungi

• Most fungi are not pathogenic to humans and animals
• Serious fungal infections are occurring increasingly in patients with compromised immune systems in health care settings
• Fungal diseases in plants cost > $1 billion annually
• Fungi have beneficial functions:
• Decompose dead plant matter
• Symbiotic fungi help plants absorb minerals
• Serve as a source of food (mushrooms, bread)
• Used in the production of drugs (penicillin)

Comparing Bacterial and Fungal Components

• Fungi are eukaryotic, while Bacteria are prokaryotic
• Fungi have sterols present in the cell membrane, bacteria have sterols absent, except in Mycoplasma
• Fungi have glucans, mannans, and chitin in the cell wall, while Bacteria have peptidoglycan (no peptidoglycan in fungi)
• Fungi undergo sexual and asexual reproduction via spore formation, while bacteria can only undergo asexual reproduction via endospores
• Spores in bacteria are not for reproduction
• Fungi are limited to heterotrophic metabolism
• Aerobic and facultatively anaerobic
• Bacterial metabolism can be heterotrophic or autotrophic
• Aerobic, facultatively anaerobic, or anaerobic

Fungi Identification

• Yeasts are identified using biochemical tests
• Multicellular fungi are identified based on physical appearance

Characteristics of Fungi Vegetative Structures

• Vegetative structures are composed of cells involved in catabolism and growth vs reproduction (spores)
• Molds and fleshy fungi are types of fungi
• The fungal thallus (body) consists of hyphae filaments
• Septate hyphae contain cross-walls and are one-nucleus cell-like units
• Spore-reproductive structure
• Coenocytic hyphae do not contain septa
• Vegetative hyphae obtain nutrients while aerial hyphae are involved with reproduction

Mycelium

• Hyphae grow to form a filamentous mass called mycelium which is visible to the unaided eye

Characteristics of Fungi Vegetative Structures: Yeast

• Yeasts are nonfilamentous and unicellular
• Budding yeasts divide unevenly
• Fission yeasts divide evenly
• Dimorphic fungi have two forms of growth and are found in pathogenic species
• Mold (vegetative and aerial hyphae)
• Yeast (budding)
• 37°C yeastLike
• 25°C moldlike

Fungi Reproduction

• Filamentous fungi reproduce asexually by fragmentation of their hyphae
• Yeast can reproduce asexually by budding or fission
• Fungi can reproduce sexually and asexually via the formation of spores that detach from the parent and germinates
• Asexual spores are genetically identical to the parent
• Sexual spores have genetic characteristics of both parental strains
• In sexual reproduction, opposite mating strains of same fungal species are involved

Environmental and Nutritional Adaptations of Fungi

• Fungi grow better at pH of 5, this pH is typically too acidic for bacteria
• Molds are mostly aerobic
• Yeasts are mostly facultative anaerobes
• Fungi grow in high sugar and salt concentration, and are resistant to osmotic pressure
• Fungi can grow in low moisture content
• Fungi can metabolize complex carbohydrates
• Fungi grow on bathroom walls, leather, paper

Fungal Diseases

• Mycosis is fungal infection
• Types of mycoses depend on tissues involved and mode of entry:
• Systemic mycoses: deep within the body, affects several tissues and organs
• Inhaled spores; not contagious
• Cutaneous mycoses: affect hair, epidermis, and nails; contagious
• Subcutaneous mycoses: beneath the skin
• Opportunistic mycoses: fungi harmless in normal habitat but pathogenic in a compromised host
• E.g. Broad-spectrum antibiotics suppressed immune system
• Are often difficult to treat

Fungi in Biotechnology

• Aspergillus niger: production of citric acid
• Aspergillus terreus: statins that inhibit cholesterol synthesis
• Saccharomyces cerevisiae: bread, wine, hepatitis B vaccine
• Taxomyces: taxol, anticancer drug
• Tolypocladium inflatum: cyclosporine-prevent transplant rejections
• Coniothyrium minitans: feeds on crop fungi; alternative to chemical pesticides

Protozoa

• Protozoa are unicellular eukaryotes that inhabit water and soil
• Over 50,000 species exist
• Some protozoa are normal microbiota in animals
• Some protozoa cause disease in humans and animals
• Plasmodium species: cause malaria
• 4th leading cause of death in children in Africa
• Protozoa have complex life cycles

Protozoa Life Cycle

• The feeding and growing form is a trophozoite
• Asexual reproduction: budding, fission (Schizogony multiple fission-many offspring cells)
• Sexual reproduction
• Some protozoa produce a cyst to survive unfavorable conditions
• Lack of food, moisture, oxygen
• Allows survival within host until new host is reached
• Enables parasitic protozoa to survive outside of host

Characteristics of Protozoa

• Some protozoa transport food across the plasma membrane
• Many have an outer protective pellicle and require specialized structures to take in food
• Ciliates wave cilia toward mouthlike cytosome
• Amoebae phagocytize food
• Food is digested in vacuoles and waste is eliminated through plasma membrane and an anal pore
• Protozoa use flagella, cilia, or pseudopods to move

Apicomplexa

• Apicomplexa is a phylum of protozoa
• Nonmotile
• Obligate intracellular parasites
• Complex life cycles: transmission between different hosts
• Toxoplasma gondii are transmitted by cats and cause fetal infections
• Plasmodium causes malaria

Plasmodium

• Plasmodium causes malaria (600,000 deaths/year)
• Sexually reproduces in the gut of Anopheles mosquito
• A mosquito injects a sporozoite (infective stage) into its bite, and the sporozoite undergoes schizogony in the liver producing merozoites
• Merozoites infect red blood cells, forming a ring stage inside the cell
• Red blood cells rupture, and merozoites infect new red blood cells OR develop into male/female forms to be picked up by mosquitos
• Waste products cause fever and chills

Helminths

• Helminths are parasitic worms
• Two phyla:
• Platyhelminthes (flatworms)
• Nematoda (roundworms)
• Platyhelminthes and Nematoda are multicellular eukaryotic animals
• They are specialized to live in hosts
• Helminths may lack digestive system, yet absorb nutrients from their host's food, body fluids and tissue
• Reduced nervous system: consistent environment in their host
• Reduced or lacking locomotion: transferred from host to host
• Complex reproductive system: produce many eggs that infect a suitable host

Life Cycle of Parasitic Helminths

• Dioecious helminths:
• Have separate male and female individuals
• Reproduction only occurs when in the same host
• Monoecious (hermaphroditic) helminths:
• Have male and female reproductive systems in one animal
• Egg Larva(e)→Adult
• The adult stage of a parasitic helminth is found in the definitive host
• Each larval stage of a parasitic helminth requires an intermediate host

Platyhelminths-Flatworms

• Trematodes (flukes) are flat and leaf-shaped
• They have a ventral and oral sucker for attachment
• Absorb food through cuticle covering
• Paragonimus spp. are lung fluke
• Schistosoma are blood fluke

Platyhelminths-Flatworm

• Cestodes (tapeworms):
• Use scolex (head) that has suckers for attachment
• Absorb food through cuticle
• Proglottids are body segments and contain male and female reproductive organs
• Continually produced by the scolex neck Mature proglottids are bags of eggs infective to proper host

Nematodes- Roundworms

• Roundworms are cylindrical with a complete digestive system
• Most are dioecious
• Can be free-living species and parasitic species
• Adult Enterobius vermicularis lives in large intestines of humans
• Eggs are deposited in the anus by females
• Eggs are excreted and ingested by new host

Arthropods as Vectors

• Arthropods are animals with segmented bodies, hard external skeletons, and jointed legs
• Vectors are arthropods that carry pathogenic microorganisms
• Vectors reside on an animal while feeding
• Mechanical transmission: flies transfer pathogens on their body
• Biological transmission: pathogen multiplies in the vector
• Definitive host

Q1: Quiz Question

• Which of the following are not found in fungi cell walls?
• Correct Answer: Peptidoglycan

Q2: Quiz Question

• Where does Plasmodium undergo sexual reproduction?
• Correct Answer: Anopheles gut