Micro. lecture exam 3 - 9

transformation

the process of importing free DNA into bacterial cells

• many bacteria naturally import DNA fragments and plasmids released from nearby dead cells via transformation

• other bacteria do not import DNA efficiently (CaCl2 + heat shock) (electroporation- using a brief electrical pulse to shoot DNA across the membrane by creating temporary pores)

conjugation

involves cell to cell contact, DNA donor cells (F+) produce a protein tube called a conjugation (sex) pilus, donor sends DNA through tube to recipient cells (F-)

• Hfr conjugation

transduction

the movement of DNA from one bacterial cell to another via a virus

• viruses that infect bacteria are called bacteriophages (phages)

• generalized: the phage capsid mistakenly fills with a fragment of bacterial DNA instead of phage DNA

• specialized: the phage genome initially integrates into a specific DNA sequence in the host genome

bacterial immune system

restriction and modification

• system will attack foreign DNA without attacking its own genome, involves two parts

• 1) enzymatic cleavage, restriction of foreign DNA

• 2) protective methylation (a chemical modification) of self DNA

clustered regularly interspaced short palindromic repeats (CRISPR)

captures pieces of DNA from failed phage attacks and integrates them into the CRISPR locus on the bacterial chromosome, serves as memory of the original attack

taxonomy

describing distinct life forms and organizing them into different categories with shared traits

• classification: taxa

• phylogeny: evolution

• nomenclature

identifying a microbe: dichotomous keys

difficult for bacteria that cannot be seen without a microscope, most conclusive way is to sequence part of all of its genome

• in clinical practice, DNA-based methods are being used more and more

• practically, identification is based on a combination of phylogeny and phenotypic traits

• multi-test biochemicals

yeast and fungi

▪ Single-celled fungi are known as yeasts. (multicellular fungi can be mushrooms, molds)

▪ They are capable of sexual and asexual reproduction. (asexual is typically budding)

filamentous fungi

• Fungi hyphae (can have no breaks - non-septake or have breaks - septake) extend and form branches, producing a mycelium.

• Chitin in cell walls - typical of fungi

Ameba’s

two major groups:

• lobed ameba’s with large, bulky pseudopods

• filamentous ameba’s with thin, needlelike pseudopods

• other diseases involve different species of ameba: primary amebic meningoencephalitis (Naegleria fowleri), corneal infection also known as keratitis (Acanthamoeba)

ciliate’s

cilia covered, heterotrophic protists that consume algae and smaller protists and are in turn consumed by amebas

• for sexual reproduction a ciliate undergoes conjugation (allows for genetic diversity- a sexually reproducing system)

Apicomplexans

a major group of parasites of humans and other animals

• apical complex is a specialized structure that facilitates entry of the parasite into a host cell

• ex: malaria (plasmodium spp., mosquito vector), toxoplasmosis (Toxoplasmosis gondii, food-borne and congenital transmission)

Hemoflagellates

obligate parasites that have a single flagellum, some of which are called trypanosomes (carried in blood and can cross the BBB, but most medication cannot cross the BBB)

• sleeping sickness (T. bruceli), Chagas disease (T. cruzi), and leishmaniasis (L. donovani)

• flagellated parasites (Metamonads): Giardia lamblia, often carried by beavers and racoons

Green and Red algae

protists that conduct photosynthesis by using chloroplasts (non-pathogenic)

Green algae:

 Chlorophyta group

 Photosynthetic autotrophs

 Unicellular and multicellular

Red algae:

 Rhodophyta

 Phycoerythrin pigment - gives it the darker color

 Photosynthetic

• algae face the issue of desiccation rather than photosynthesis

dinoflagellates

algae derived from secondary endosymbiosis, a major group of marine algae, essential to marine food chains

• responsible for red tide

Helminths

parasitic worms

 Nematodes (roundworms) are cylindrical (hence the term “roundworm”), with a digestive tube that ends in an anus. Reproductive forms are mostly hermaphroditic.

 Trematodes (flukes) are oval-shaped flatworms, with a digestive tube that ends in a cecum (no outlet). They are hermaphrodites.

 Cestodes (tapeworms) are parasitic flatworms, absorbing nutrients through their skin. The sucker head structure grows a long “tail” of segments containing male and female reproductive structures.

pinworms

small, white nematodes of the species Enterobius vermicularis

• makes you scratch a lot and it causes constant re-infection

• often seen in younger children

Roundworms

Ascaris lumbricoides

 Eggs ingested from soil - can also contract from being barefoot

 Worms increase in size in the digestive tract.

 Effects may be lethal.

• Worms may also invade the lungs or other organs, causing life-threatening damage.

trematodes (flukes)

Flatworms (Platyhelminthes phylum)

Internalized mouth

Pharynx

Digestive tube that ends in one or more pouches called ceca.

• Because the worm lacks an anus, it must expel its wastes back out of the mouth.

• Usually has two suckers, one near the mouth and one on the ventral side of the body

cestodes: tapeworms

 Transmitted as larvae in uncooked meat

 Different species are found in pork (Taenia solium), beef (Taenia saginata), and fish (Diphyllobothrium).

 Composed of a head and successive segments

 May grow 2–15 meters in length

Arthropods

Invertebrate animals with an exoskeleton and jointed appendages

 Many are free-living; some are ectoparasites.

Suck host blood or inject eggs into hosts

Ectoparasites may be vectors of infectious diseases.

 Include insects and arachnids (spiders, mites, and ticks)

Arachnids, mites, and ticks

• A: have eight legs

• M: cause mage (in animals) and scabies (in animals or humans)

• T: transmit pathogens that cause lyme disease and other bacterial infections, babesiosis, flaviviruses

virus and viral genome

a noncellular particle with a genome contained by a capsid

 Viruses take over a cell to manufacture progeny—they require a host cell to reproduce.

 Bacteriophages are used routinely as cloning vectors, small genomes into which foreign

genes can be inserted and cloned for gene technology.

 In natural environments, viruses fill important niches in all ecosystems.

viruses that replicate in different host cells (vocabulary)

viruses use a relatively small number of virus-encoded proteins to commander the metabolism of their hosts (antiviral agents are hard to discover, have severe side effects, and mutate faster than bacteria)

▪ Bacteriophages (phages)- viruses that infect bacteria. - know how to draw and label

▪ Virion – progeny viruses

▪ Virus particles – viruses that infect eukaryotes.

▪ Host range – the various hosts that a virus or phage can infect

• Tends to be small and specific to cell type

viral genomes

• potential genomes: DNA and RNA, single stranded or double stranded

• capsid: keeps the viral genome intact outside the host cell (equivalent to plasma membrane)

• envelope: external to capsid

• Baltimore model: classification system based on genotype (need to know how we group viruses- DNA material)

viral structure: capsid and envelope

Icosahedral capsids

• radial symmetry: based on an icosahedron, a polyhedron with 20 identical triangular faces

• ex: herpes simplex virus (HSV)

Filamentous

• helical symmetry, helical tube around the genome, generate a flexible filament

• ex: tobacco mosaic virus, Ebola virus

Envelope

• optional, derived from the host nuclear or endoplasmic reticulum membrane, may contain spike proteins

complex tailed bacteriophages

head: icosahedral protein package, contains genetic material

tail: injects genetic material into host cells (ex: bacteriophage T4)

Amorphous or complex viruses

No symmetrical form

 Flexible “core wall”

• Contains genetic material

 Viral envelope

• Studded with spike proteins

 Examples:

• Vaccinia virus

• Smallpox virus

prions

prions: infectious proteins

 Transmission: Forced misfolding of normal proteins by prion (PrPc)

 Mode of Transmission:

Consumption of prion infected material (generally meat)

 Targeted Host: Species specific (usually)

 Genome: None

 Inherited or transmissible

• Creutzfeldt-Jakob

• "Mad cow disease"

• Scrapie - associated with sheep

• Kuru

viroids vs prions

Viroids - virus-like infectious agents where the genome is itself the entire infectious

particle

• No protective capsid

• Targeted Host: Plants.

• Genome: RNA

Prions - infectious proteins

• Transmission: Forced misfolding of normal proteins by prion

• Mode of Transmission: Consumption of prion infected material (generally meat)

• Targeted Host: Species specific (usually)

• Genome: None

viral diversity and evolution

Antigenic drift: rapid mutation

 Leads to accumulation of random mutations, which then leads to viruses whose mutant proteins are no longer recognized by host antibodies.

• New strains of viruses that cause serious disease.

 New influenza strains requiring repeated immunization

• Segments from different strains reassort by coinfection of a host cell.

• Reassortment between human and avian strains causes antigenic shift.

different levels the virus evolves at

1) Within a host community

• Viruses evolve to preferentially infect different species.

• Example: equine herpes in horses versus murine herpes in mice

2) Within a viral species population

• Strains evolve that vary in infectivity and virulence.

• Closely related viruses may cause diseases that are similar (herpes simplex viruses 1 and 2) or different (varicella-zoster versus cytomegalovirus).

3) Within an individual organism

• Viruses evolve variants that resist therapeutic agents.

• Example: Hepatitis C and HIV evolve into diverse strains that infect different tissues within the host.

Bacteriophage replication and culture - all viral replication cycles must achieve the following

1) Host recognition and attachment

• Contact and adherence

2) Genome entry

• Gene expression

3) Assembly of progeny virions

• Expression and virion assembly

4) Exit and transmission

• Budding or cell lysis

Lytic infection vs Lysogenic infection

1) Virulent phage

▪ Reproduce entirely by the lytic cycle

2) Temperate phage (prophage)

▪ Can undergo lysogeny

▪ Prophage – viral genome integrates into host genome

3) Lysis (rupture of host cell)

papillomavirus: DNA genome

 Human papillomavirus

 In the United States, HPV infects 80% of adults.

• Cervical cancer–HPV link was discovered by Harald zur Hausen, which earned him the 2008 Nobel Prize in Physiology.

 HPV strains are highly infectious.

• Sexual contact, including oral contact leading to throat cancers

 Protection

• Condoms—partial protection

• Gardasil vaccine—greater protection

• 1) Can be administered for girls and boys ages 11–12, or up to age 26 for those not vaccinated sooner

• 2) Gives protection against four prevalent strains linked to cervical cancer

Viruses can insert oncogenes which are genes that cause cancer -> oncogenesis (the

development of cancer)

SARS-CoV-2 Coronavirus structure

▪ Contains core particle of RNA packaged in proteins (nucleoprotein complexes linked by RNA) with a surrounding envelope and spike proteins.

▪ "Crown" of spike proteins inspired the name corona (Latin for crown).

• spike proteins used to select for the specific cells the virus will enter, can undergo antigenic shift

influenza virus structure

▪ No fixed capsid

▪ Genome

 (–) sense RNA

 Eight chromosome segments

▪ Enveloped

 Hemagglutinin (HA) - clumping blood around infected cells

 Neuraminidase (NA)

▪ RNA-dependent RNA polymerase

 Used for synthesis

• spike proteins

influenza replication

 Attachment

• HA spike

 Entry

• Endocytosis

 Replication

• RNA-dependent RNA polymerase

 Assembly

 Budding

• Neuraminidase (NA)

HIV and AIDS

 Causative Agent: HIV

 Disease: AIDS

 Prevalence: 1:140 adults (15-49yo)

 Target: Helper T cells

 Prognosis: No treatment -> death due to opportunistic infections

• Treatment -> prolongs life but does not cure disease

 Preventative treatment, called preexposure prophylaxis (PrEP), can prevent infection and transmission.

• Reverse transcriptase

HIV structure

 Lentivirus

• Retrovirus

 Capsid

• Conical-icosahedral

 Enveloped

• Spike proteins

 Reverse transcriptase

• Enzyme

• RNA template to synthesize DNA

• High error rate

engineered lentiviral vectors treat cancer

 The exceptional ability of lentiviruses to deliver their genomes into a human genome makes them the most promising source of vectors for gene therapy.

 Lentivectors have been devised to modify T cells to treat certain forms of leukemia.

• Chimeric antigen receptor T cell (CAR-T) therapy is approved by the FDA to treat several forms of B-cell leukemia and multiple myeloma.

growing bacteria in culture media

 Microbes in nature usually exist in complex, multispecies communities. For detailed laboratory studies, however, they must be grown separately in pure culture—that is, as a single species.

 Bacterial culture media can be liquid or solid.

• Liquid medium allows bacteria to move freely.

• Solid medium is useful to separate mixtures of different organisms

obtaining pure cultures

1) solid media

• bacterial cells form colonies on solid media with agar added to make a firm surface

• isolation streaking allows for separation of colonies into pure cultures

2) pure colonies can also be isolated using the spread plate technique

-tenfold dilutions of the original culture are spread on agar plates

• early dilutions will show confluent growth

• each colony on the agar plate represents one viable organism

growth in synthetic and complex media

 Bacteria can be grown in a precisely defined synthetic medium that contains only known

components.

 A minimal medium is a synthetic medium in which the components are limited to only

those nutrients the organism needs to grow.

 Bacteria can also be grown in a nutrient-rich but less defined complex medium, also

called rich medium.

 Enriched media can be used to grow fastidious, or fussy, organisms that have a very specific set of nutritional requirements beyond basic media

selective and differential media

 Selective media

• Compounds in the media prevent some types of bacteria from growing, favoring the growth of one specific type.

 Differential media

• Species grow equally well, but compounds in the media are metabolized differently, often distinguished by a color indicator.

 Example: MacConkey medium

• Selective agents are bile salts and crystal violet, which prevent the growth of bacteria other than Gram-negative enteric bacteria.

• Differential agent is lactose, which some bacteria ferment (pink) and others do not.

distinguishing the living from the dead

▪ Plated (growth) methods count only live cells.

▪ Determining the total number of cells (dead or alive)

• Direct cell counts

• Indirect cell count: by fluorescence-activated cell sorter (FACS) or by measuring turbidity with a spectrophotometer