Microbiology Exam 3

gene therapy

use of DNA to treat diseases

replace defective or missing genes or remove genes

vaccines

use capsid

Victoria Gray

first person to have stem cells used for CRISPR to help her sickle cell

CRISPr as gene editing

harvest HSPCs from patient

Ex vivo genome editing

gene edited HSPCs go back to human

agricultural biotechnology

introduce genes to break down waste or plastics or pest resistance

Agrobacterium

gram neg, soil dwelling bacterium

known for efficient conjugation with plant cells

agrobacterium gene delivery

plasmid removed from bacterium and T-DNA is cut by a restriction enzyme

foreign DNA is cut by the same enzyme

foreign DNA is inserted into T-DNA of plasmid

Plasmid is reinserted into bacterium

bacterium is used to insert the T-DNA carrying the foreign gene into the chromosome of a plant cell

plant cells grown in culture

plant generated from a cell clone, all cells carry the foreign gene and may express it as a new trait

agrobacterium can deliver these genes

Bacillus thuringiensis toxin (undesirable to pests)

herbicide resistance

drought tolerance

promote shelf life

suppression of ripening genes

change nutritional content

human proteins (Muc-rice) makes cholera less hurtful

golden rice

has beta-carotene gene to rice

more vitamin-A

terminator gene

Monsanto’s Terminator

sold these specific seeds that have an inducible promoter that is activated by a specific chemical so the plants can start growth at any temp/environment

company sells these seeds and chemicals so can make more money

bioremediation

use of biological organisms to detoxify environmental pollutants

composting

arranging organic waste to promote microbial degredation

bioaugmentation

enhancing the performance of indigenous bacterial populations through the addition of commercially prepared bacterial strains with specific catabolic activities

compost starter pack

xenobiotics

resistant to degredation

Deinococcus radiodurans

polyextremophile

most radiation-resistant organisms known (found at Chernobyl)

can digest toluene and ionic mercury produced from highly radioactive nuclear waste

Ideonella sakaiensis

can break down plastics

Pseudomonas

strains identified with 2 plasmids that have hydrogen carbon degraddative pathways

degrade agent orange, a deadly herbicide: 2,4,5-trichlorophenoxyacetic acid

forms biofilms

Genetically engineered microbes

Ralstonia eutropha

natural inhabitant of soil that pull up heavy metals so not in water

transfected with mouse methallothionein gene = enhanced ability to sequester heavy metals

metallothionein in this GEM was expressed on the outer surface of the cells to help in sequestering of cadmium

toxic metal within the soil remains bound to microbe, so the tit is less likely to be taken up plant biomass or leaked into water supply

phytoremediation

sunflowers can suck up radioactive toxic waste

phytovolatilization: volatilization of pollutants

phytodegredations: degredation of organic compounds

phytoextraction: concentration of contaminants from soil to plant tissues

phytostabilization: reduction of bioavailability of contaminants

rhizofiltration: adsorption and absorption of pollutants from water

mycoremediation

use of fungi to clean up contaminated environments

waste water treatment

primary treatment - removal of solids

secondary treatment - removal of biological matter used microbes

tertiary treatment - disinfected chemically or physically (lagoon)

landfills

harvesting natural gas

methanogens - obligate anaerobes, oxidize decomposing organic waste in landfills, end-product of their metabolism is methane gas

biofuels

alternative energy sources using microbes

biomass → (enzymes) sugars → (microorganisms) → methane, ethanol, methyl ester compounds

biomass harvested and cut into shreds and pretreated with heat and chemicals to make cellulose accessible to enzymes

enzymes break down cellulose chains into sugars and microbes ferment sugars into ethanol which is purified through distillation and prepared for distribution

not all the same as some are not as green as others

algae oil

carbon dioxide, nutrients, and sunlight which produces oxygen

fats (oils) grow inside algae which is extracted and converted to biodiesel

microbial diversity in humans

40-100 trillion bacteria live in and on us (10x more than human cells)

over 1000 species

2-6 pounds

microbiome

collection of all microbes found in a particular region or body

mostly look the same, most uncultivable, some unidentified, some very rare

symbiotic

separate species living close together

mututalistic

both species benefit from

commensal

one species benefits and the other is unharmed

parasitic

one species harms another

bacterial benign

promotion of fat storage, promotion of angiogenesis, development and training of immune system, biosynthesis of vitamins and amino acids, metabolism of therapeutics, modification of the nervous system, breaking down food compounds, resistance to pathogens, protection against epithelial injury, modulation of bone-mass density

human microbiome project

goals: determining whether individuals share a core human microbiome

understanding whether changes in the human microbiome can be correlated with change in human health

microbiome analytical pipline

cells, isolate DNA and perform PCR, DNA sequencing and analysis, aligned rRNA gene sequences and make tree

16S rRNA sequencing analysis typically uses 97% identity to assign Operational Taxonomic Units (OTU)

early microbiome findings

sampled forearms of 6 people

found 1221 bacterial species

98 newly identified identified species

permanent residents of the skin (4 genera comprise 50% of species - staphylococcus, streptococcus, propionibacteria, corynebacteria)

microbiome development

host genetics, diet, host environment, host physiology, late life impacts, early life exposure

microbiome evolution

can be symbiont gain or loss through evolution or through the uptake of bacteria from environment

led to specialization such that microbes from one host will not colonize an unrelated host. we cannot thrive without them and they cannot grow without us

alpha diversity

measure of species diversity in a site or habitat at a local scale

diversity within a samplebe

beta diversity

measure of distance or dissimilarity between two samples at the same site on different individuals

difference between two samples

dysbiosis

microbial imbalance on or inside the body

host genetics (gene mutation), lifestyle (diet, stress), early colonization (birth in hospitals, altered exposure to microbes), medical practices (vaccination use, antibiotics, hygiene)

can lead to disease or health which are changes to immune system

dysbiosis and Clostridium difficile

~5% of people admitted to hospital get it, antibiotic exposure reduces microbiome diversity that can promote C. difficile colonization

C. diff rates higher after antibiotics, people taking laxatives, people taking proton blockers (antacids)

fecal microbiota transplantation has almost 100% cure rate

dysbiosis and yeast overgrowth

antibiotic treatment of bacteria vaginosis also kills beneficial vaginal bacteria

without the good bacteria, odor causing bacteria grows unchecked

thrush is fungal infection of the mouth

dysbiosis and necrotizing enterocolitis

~1 in 2000-4000 of all births, 70% cases occur in premature babies

affects 2-5% of premature babies

Bifidobacterium - gram pos anaerobic gut bacterium, ferments breast milk oligosaccharides in babies, works as a preventative colonization

human milk oligosaccharides (HMOs) act as bifidobacterial growth-promoting factors

microbiome change over life

internal and external environment shape our microbiome over our lifetime

irritable bowel syndrome

higher in developed countries, associated with use of antibiotics, different microbiome profiles

people with IBS have lower bacterial numbers and diversity (14 vs 800 million bacteria)

prebiotics influencing Bifidobacterium, E. coli, fecal transplants can improve symptoms

microbiome and pregnancy

altered microbiota as pregnancy goes on

microbiota from first trimester and third trimester implanted into mouse and first gives normal mice and third gives fatter mouse with insulin desensitization

protein folding

natural folding by hydrophobicity in water, structure determines function

primary is chain of amino acids

secondary has alpha helix and beta pleated sheet linked by hydrogen bonds

tertiary has attractions between the alpha/beta links

quaternary has more than one amino acid chain

unique features of prions

only known infectious pathogens that are devoid of nucleic acid, made of one protein called PRP

extremely resistant to heat, chemicals, and decomposition

prion diseases can be either infectious, genetic or sporadic

result from accumulation of PrPSc protein which is an isomer of the normal PRPC protein

prion

PROteinaceous INfectious Particle

infectious proteins of PrP that are misfolded (can’t be degraded) resulting in protein aggregation and clumping that leads to enurodegeneration of the central nervous system

scrapie

sheep disease

first reported in 18th century england and cases increased as selective breeding for wool was more common

efforts to identify - brain vacuolation, infectious: symptomatic sheep with healthy sheep, infectivity experiments failed

1939 - transmitted scrapie to two healthy sheep by intraocular inoculation of brain or spinal cord tissue but took 1-2 years to develop disease

1939 - large outbreak after sheep vaccination against looping illness which was prepared from formaldehyde-treated sheep brain extract

kuru

Fore people were starving so would eat dead tribemates in ritual for nutrients

took 9-24 months to kill them after first symptoms

slow unconventional virus, connection to scrapie

D. Carleton Gajdusek

won nobel prize for discovering how it spread by taking infected brain material and injecting it into monkeys

“some yet-overlooked, chronic, slowly progressive, microbial infection may be involved in kuru pathogenesis”

Bill Hadlow

was a veterinarian that noticed it was similar to scrapie

Tikvah Alper

noticed resistance to UV irradiation suggesting it might be devoid of nucleic acid

took sheep brain material and isolated it and radiated, saw nothing, and put it into mice and still killed

Stanley Prusiner

won the Nobel prize for discovery of prions and identified that it is caused by a protein and that it was infectious

took brain material from infected sheep and purified it, saw it was resistant to DNAses and RNAses and agents that inactivate viruses and saw that a protein fraction is the infectious agent

put it into mouse

Prp

found in every mammal (encoded in human chromosome 20)

~250 amino acids

normal protein called cellular Prp or PrpC

changes its 3-D shape to form rogue/prion/resistance/diseased Prp shape called PrpSC

model for prion disease

normal is a soluble, cellular prion protein

disease causing is protease resistant and causes other proteins to misfiled when in close proximity

prions cause this disease

Transmissible Spongiform Encephalopathy (TSE) in mammals

class of neurodegenerative diseases caused by Prp that effects central nervous system

brain vacuolation, apoptosis of neurons, astrogliosis, and accumulation of misfiled protein called PrPSc, no inflammatory response or antibody response

normal function of PrpC

expressed in variety of organs and tissues

high expression levels in the central and peripheral nervous systems

resides extracellularly in lipid rafts, where it is attached to a glycosyl phosphoinosityl (GPI) anchor

undergoes endocytosis and cleavage

knockout mice show unclear function

weird, cleaved at many places

prion animal studies in mice

Prp knockout mice show mixed and mild phenotypes

studies with rodents demonstrate that oral infection with prions can occur, but is inefficient compared to intracerebral inoculation

removing Prp make mice resistant to TSE

mouse with P102L mutation (equivalent) to a human spontaneous CJD mutations causes the mouse to develop TSE

need own form of Prp to get diseaseD

Dr. Alison krauss

uses high resolution imaging with Cryo-EM at Case Western to visualize the proteins

PrpC is monomeric with high alpha-helix

PrpSc is multimeric amyloid fibril, parallel in-register architecture and high beta-sheet

prion theory

can spread silently across a person’s brain for years without causing any symptoms

rate of conversion will gradually amplify as concentration of PrpSc form increases

prions start to kill neurons, and once symptoms strike, the person has a very rapid decline

amyloids

misfiled, insoluble, protease resistant, fibrous protein aggregates that have a distinct staining properties

cause over 20 neurodegenerative diseases but contain different protein aggregates

some proteins (amyloidogenicproteinds) have greater potential to misfold

long chains called amyloid fibers

neurodegenerative diseases are not prions but both form amyloids

forms of prion diseases

sporadic - spontaneous conversion, somatic mutation

acquired - acquired conversion

inherited - gremlin mutation, spontaneous conversion more likely

Creutzfeldt-Jakob Disease

discovered in Austria and Germany in 1920s, 85% of all human prion diseases, 1 case per million, 85% of diagnoses die within a year, mostly in people 50-70, presents as psychiatric disorder and has painful sensory symptoms, ataxia, myoclonus and dementia leading to loss of motor function and coma

causes of Creutzfeldt-Jakob disease

genetic predisposition - familial CJD (10%)

Iatrogenic (iCJD) (<5%) - from surgery, hormones derived for brain like HGH and gonadotropin, electrodes, dural grafts

variant CJD - ingestion of contaminated meat

sporadic or classical CJD

Mad cow disease

Bovine Spongiform Encephalopathy

due to industrial cannibalism and offal, peaked in the UK

possible links to spread of vCJD by consumption of contaminated meat

experimental studies show orally ingested prions is absorbed through intestine into blood, transports it to lymphoid organs and accumulates before transporting to the brain by the peripheral nerves

fatal familial insomnia

genetic disease, autosomal dominant (few cases sporadic), 40-80 families worldwide

one specific mutations in the Prp gene at codon 178, hypothalamus function is initial target, progressive untreatable insomnia, loss of circadian rhythm, endocrine disorders, motor disorders and dementia

gerstmann-straussler-scheinker syndrome

genetic-autosomal dominant, sometimes considered a subclass of CJD, 1 in 10-100 million people, avg age 35-50

survival - 5 years after first symptoms

50% of cases have mutation in Prp codon 102 - often disrupts and removes GPI anchor

new prions

variably protease-sensitive prionopathy (VPSPr) in 2006

sporadic, codon 129

why different forms of Prion diseases

different mutations within Prp will convert the normal conformation into unique structural forms of PrpSc

initiated in different locations on the brain

for familial form, different Prp mutations will induce slightly different forms of the diseases Prp

chronic wasting disease

origin uncertain, high contagious, spread direct and indirectly. feces, body fluids, aerosols

10-80% of some deer populations harbor CWD prion

no strong evidence for transmission from deer/elk to humans

prion treatment and diagnosis

universally fatal and relentlessly progressive

no treatments - some drugs use to alleviate symptoms

definitive diagnosis - autopsy and histological analysis of the brain

no premortem diagnostic test, but several tests can help build a diagnosis - clinical presentation & family history, electroencephalography, cerehrospinal fluid-based tests - Real Time-Quaking-Induced Conversion (RT-QulC) is sensitive detection of prions in spinal fluid, brain biopsy, MRI, presence PrPSc in peripheral lymphoid tissue

prions extra

Prions are winespread in fungi kingdom, but these proteins are able to switch protein shape back and forth

heterokaryon incompatibility, nitrogen catabolism, translation termination

viruses

microscopic particle that can infect the cells of an organism

word means toxin, slime, venom or poison

obligatory intracellular pathogen - viruses can only replicate themselves by infecting a host cell - acellular

20-300 nm

viruses infect animals, plants (agricultural), fungi, protists, prokaryotes, contribute to bacterial evolution

some attack other viruses too

viral therapeutics

phage therapy - viruses that kill bacteria (highly species specific)

genetic therapy, oncolytic viruses and vaccination

viral structure

capsid/capsomere/nucleocapsid - a protein shell

genetic material - RNA or DNA inside of the capsid, some have three capsid

viral structural proteins

proteins that make up the viral capsids and other structural components in the virion

viral regulatory proteins

enzymes - polymerases, helicases

transcription factors - make the host better for them

other proteins that influence host cell functions to make the environment more suitable for viral production

T-antigen in Polyomaviruses

example of regulatory protein

binds to p53 and prevents function which leads to cancer bc p53 protects DNA integrity

binds to pRB, P107, p130 which are tumor suppressor genes so it leads to over production of cells

helical viruses

ebola, rabies (lyssavirus), tobacco mosaic virus, vesicular stomatitis, respiratory syncytia virus

icosahedral

polyhedral

20 sided

polyomavirus, papillomavirus, adenovirus, poliovirus

spherical

corona virus, measles, influenza

lipid envelope, protein envelope, neuraminidase, hemagglutinin, RNA nucleoprotein

complex virus

bacteriophage

variola virus, (poxvirus familty brick shaped)

enveloped viruses

surrounded by a lipid membrane derived from the host cell

viral glycoproteins protrude out of envelope

naked or non-enveloped

viral infection strategies

acute - infections are rapid and self-limiting (colds, norovirus)

persistant - infections last for months to years, but are sometimes cleared

latent - infection is an extreme version of a persistent infection that remains dormant but can become reactivated (herpesvirus)

viral life cycle

1. attachment - a virus comes into contact with a host cell and attaches to a specific molecules on the cell called receptors

2. entry/trafficking - entry into the cell and locates to a particular intracellular location

3. replication - once inside the cell the viruses job is to make lots of copies of its genome and translation its proteins. often done at expense of the host cell

4. assembly - cellular activity of the host cell help assemble new viruses until the host cell is filled with new viruses

   1. release - the new visions can bud off of the cell or be released if the cell bursts open

goal of viral life cycle

copy its genome and translate its viral proteins

viral attachment

viral attachment proteins on viruses interact with specific molecular on the cell surface called viral receptors and co-receptors (sometimes many)

TMPRSS2 cleaves attachment for entry and attaches for ACE2

viral receptor

molecule in the cell membrane often protein that binds to a ligand to transmit a cellular signal, many receptors have sugars linked to them

normal ligands bind to the receptor leading to intracellular signals and cell events

viruses “hijack” the receptor and binds it in order to stick to surface and enter cells, may bind to same or different places than natural ligands

viral attachment with receptors

viruses have unique and specific receptors, some have co-receptors

mediates viral binding to host cells

elicits intracellular signals necessary for viral entry

directs viral trafficking

receptor presence determines virus tropism

viral attachment receptors examples

reovirus - silica acid and JAM-A

Polyomavirus - infects kidney and brain

HIV - CD4+ T-cells, individuals who are homozygous for deletions in CCR5 are resistant to HIV

Rhinovirus - ICAM which is present in nasal epithelium

tropism

preferentially targeting of a specific host species or cell type. determined by host cell factors like receptors

permissively - the ability of a virus to enter a host cell and complete an infection cycle

host range determined by specific host attachment sites and other cellular factors

usually narrow

narrow vs broad host range

receptors are most important determinant of tropism

a) host level determinants: HCV example so a change in receptor from a mutation or genetics

b) species level determinant: MERS-CoV example, seen in bats, humans, camels but not mouse ferret or guinea pig

lyssavirus spread from fox to a lot of other species

hACE2 tropism

SARS-CoV-2 only binds to human ACE2 but not mouse

sialic acid tropism

influenza uses silica acid as viral receptor

bird flu uses alpha 2,3 linked SA

human flu uses alpha 2,6 linked SA

negatively charged and linked to a monosaccharide

in lung tissue birds have alpha 2,3 linked SA in airways and humans have alpha 2,6-linked SA in upper respiratory tract

swine have both types

viral entry processes

endocytosis - attaches to membrane and then forms a membrane ring around it inside the cell

fusion - typically from enveloped viruses as the viral envelop fuses with the cell membrane and inserts genetic material

penetration - bacteriophage injects viral genome into cell

trafficking - movement of virus from surface to site of replication, microtubules often help shuttle the virus

endosomal escape

uncaring - disassembly of the virus capsid to be able to release genetic material

influenza virus changes upon exposure to low pH it finds in the endosome after entry

viral release from host

budding of enveloped viruses take some of the host cell exterior

lytic bursting of nonenveloped viruses to release newly made viruses

trafficking destinations

DNA virus in the nucleus

RNA virus in the cytoplasm

viral replication

making copies of viral genome, depending on if the virus can use host or viral enzymes to facilitate

viral genome size

vary from ~4000 base pairs to over 1 million

20nm-750nm

5 proteins to over 2500

Linnaeus taxonomy of viruses

family names end in -viridae

genus names end in -virus

species: group of viruses sharing the same genetic information and ecological niche (host)

subspecies are designated by a number