Micro 2/2 Flashcards
baltiWhat are biofilms made of?
- prokaryotes (and many microbial fungi) prefer to grow in communities / consortium of different species that stick together on organic or inorganic surfaces forming specialized structures
Where can biofilms form?
- range of surfaces
- biotic, abiotic, organic, inorganic
How are biofilms important in medicine?
- pathogenic bacteria and fungi can form biofilms on human tissue and medical equipment and implanted devices
- difficult to treat with antimicrobials
What is trapped in microbial biofilms?
- within the EPS matrix are bacterial secreted proteins and extracellular DNA fragments, lipids, soluble proteins, outermembrane vesicles
- slimy layer in which microbes grow
How can biofilms be beneficial?
- Allow microbes to work together effectively(e.g.in insect & animal intestinal systems)
- Normal microbiota biofilms of plants and animals are beneficial/essential
How can biofilms be detrimental?
- Damage equipment, degrade infrastructure (e.g. concrete, pipes)
- Colonize abiotic surface put into the body (e.g.heart valves, replacement joints, catheters)
- Biofilms of pathogenic bacteria are a huge problem in medicine (e.g. plaque biofilms & tooth decay)
- Biofilm bacteria are highly resistant to antimicrobials and killing by immune response cells and defenses
What are microbial biofilms?
- complex assemblies of microbial communities attached to surfaces and surrounded in a sticky and adhesive extracellular polysaccharide (EPS) matrix (secreted by the microbial cells)
How are biofilms formed?
- Attachment of planktonic bacteria (bind to surface)
- Attached bacteria form microcolonies (start dividing)
- EPS secretion (protects it from environmental insults)
- Biofilm elaboration and maturation (towers)
- Dissolution and dispersal (due to surr. nutrients)
Where do bacterial biofilms form?
- where nutrients are plentiful
- attach to surfaces via cell- envelope/surface appendages and proteins
What happens when nutrients become scarce for biofilms?
- individuals detach from the community to forage for new sources of nutrients
What is quorum sensing?
- process of assessing bacterial density
- mechanism for regulating, density-dependent community behaviours
What are some of the forms and functions biofilms can form for different species?
- most are a consortium of different species
- Single species biofilms can form
- formation of biofilms can be cued by different environmental signals in different species
- Biofilm formation is regulated by quorum sensing
What does quorum sensing involve?
- signalling molecules, or autoinducers, secreted into the surrounding environment
- Bacteria ‘assess’ signal concentration
- Extracellular concentration of inducer increases with population density
How does an autoinducer work in quorum sensing?
- freely diffuses in and out of bacterial cells, binds to a cytoplasmic receptor protein, a transcriptional regulator
- At a certain inducer concentration (“quorum”), the transcription regulator is activated and binds to DNA activating quorum-sensing regulated genes
- result: co-ordinated response by all cells in the community
(gene cannot bind in low cell density, can bind with lots of autoinducers)
Describe the kingdom fungi (eumycota)
- largest phyla
- basidiomycota
- ascomycota
- diverse group of heterotrophs; decomposers and saprotrophs, majority live in soil)
Distinguish between Basidiomycota and Ascomycota
B: mushroom like
A: how they carry out sexual spores (visible)
Distinguish between microfungi and macrofungi
micro: mostly invisible to naked eye (ex: yeasts, moulds)
macro: produce easily visible fruiting bodies (mushrooms, puffballs, etc)
How do saprotrophs absorb material?
- material must pass through plasma membrane
- absorptive metabolism: have to absorb food from environment
What are some distinctive traits of fungi?
- cell wall (chitin)
- ergosterol: sterol similar to animal cholesterol
- hyphae
- sexual and asexual life cycles
- micro- and macro- produce spores
What are saprotrophs?
- Principal decomposers of dead/decaying
organic matter - Essential role in environmental nutrient cycling, converting organic matter to inorganic molecules
- Cannot ingest particulate food
- Secrete enzymes for extracellular degradation and absorption of resulting nutrients (area of research)
What is a mycelium?
- Extending and branching form a mass of hyphae
- spore germinates to form hyphae: extends/branches into mycelium
- Mycelia can differentiate into spore-forming structures – fruiting bodies
- everything starts with hyphae-> mycelia; then differentiation
Describe hyphae and its alternate forms
- hyphae: extended, multinucleuate cellular filaments
- septate: separate neighbouring cells
neighbouring cells connect via pores - non-septate: one continuous hyphae (“cube” of cytoplasm)
- pseudohyphae: no connection via cytoplasm, elongated cells are independent of connection with neighbouring cells
Describe the cell wall of fungi
- made of glucans and chitin
- inner wall: conserves, made of chitin and branched B-1,3-glucan
- outer wall is variable (many yeasts, comprised of mannan and mannoproteins)
Describe the production of spores in micro and macrofungi
- Dormant structures formed to resist environmental stresses and for dissemination
- sexual reproduction and disseminating their cells
Differentiate between sexual and asexual life cycles of fungi
sexual: Produce sexual spores (meiospores such as basidiospores and ascospores) in sexual fruiting bodies
asexual: Produce asexual spores (mitospores; formed through mitosis, such as conidiospores and sporangiospores) in asexual fruiting bodies
How does asexual reproduction work?
- and + mating type; plasmogamy take place: organisms meet and fuse (cytosols and nuclei mix: dikaryotic (2 different types of nuclei, where they haven’t fused yet), form ascus; within spore-forming structure creates karyogamy
- +/- mating: haploid: dikaryotic: spore-forming dikaryotic structure: karyogamy, nuceli fuse and nucleus is diploid (2 copies of genetic material)
How does sexual reproduction work (in ascomyota, ex)
- Sexual ascospores are endospores; form within a sac-like meiocyte that form an ascus (pl. asci)
- Sexual fruiting body = ascoma
- Different kinds; shown here are the open cup-like apothecium
Describe yeasts
- Unicellular organisms, 3-10μm
- Grows via budding
- Some can form strings of connected budding cells (pseudohyphae), or hyphae
- ex: saccharomyces cerevisiae, dimorphic patogen candida albicans
What are shmoos and how do they work in yeasts sexual reproduction?
- nodule that cells grow together
- shmoos grow towards each other and fuse
- diploid gros vegatively, grows through sporulation, making spores with ascus, which can be disseminated
What are capsules in yeasts and some examples?
- pathogen: cryptococcus neoformans
What is symbiosis?
- “to live together”
- organisms adapt to the presence of others; they do not live alone
- range of positive to negative replationships; partners evolve in response
- organisms (usually 2) in association called symbiont
What are the 3 types of symbiotic interactions?
commensalism, parasitism, mutualism
What is commensalism?
One partner benefits, while the other is unaffected
What is parasitism?
intimate association where one partner benefits, while harming a specific host
What are some human pathogens?
- Emerging, globally important invasive pathogens (in immunosuppressed patients) with very high mortality rates
- candida, cryptococcus, aspergillus
What is mutualism?
Each partner gains benefits from the other (in some cases, may not grow independently)
- 2+ microbial partners
- 1+ microbial partners with plant/animal host
- removal of microbial partner leads to death or decreased growth of host
- microbial genome shows extensive degeneration (reduction) of normally essential genes for metabolism and protective structures.
- ex: lichens, gut microbiomes, mycorrhizae…
What are lichens?
multiple fungal species and algae/cyanobacteria
- cannot survive independently (reduced genomes)
- grow almost anywhere
- algae/cyanobacteria: photobiont (receives water and minerals/nutrients from fungi)
- fungi recieves carbs produced by algae
- fungi protects photobiont
- nutrient exchange @ photobiont layer
What is some general information about viruses?
- acellular entities (cannot replicate on its own)
- obligate intracellular parasites (cannot replicate by itself)
- hijacks host cellular machinery to replicate
- displays tropism (interacts with specific hosts/host ranges)
- interacts with host cell surfaces (specificity of interaction determines host range)
Compare a narrow and wide host range
narrow: cold and influenza viruses infect human respiratory epithelial cells
wide: rabies virus- dogs, foxes, raccoons, humans
What are virions?
become viruses when the host becomes infected
What is the range of impact viruses have?
innocuous to lethal
- most devastating impacts on human society
What are 2 risk group 4 agents?
risk group 4: high individual and community risk: often untreatable, readily transmittable
- filoviridae: ebola
What can bacteriophages be used to do in viruses?
- control infections as an alternative to antibiotics or disinfectants
- Viruses can be modified as delivery vehicles for gene therapy
- Bacteriophage can be used in molecular cloning, as cloning vectors
How have viruses evolutionized?
- Insertion of virus into host genome can impact gene expression at insertion site
- Excision of viral genome from host and subsequent packaging impacts gene movement (e.g., horizontal gene transfer)
- genes may be regulated or down-regulated because of viruses
- viruses can be both pos and neg
What is the general structure of viruses?
- protein coat (capsid): surrounds nucleic acid
- enclosed in a protein-containing membrane (enveloped), or not (naked or unenveloped)
- nucleic acid either RNA or DNA; encodes viral proteins
What is the classification of viruses?
complex: origin and evolutionary history largely uncharacterized
- based on: nature of genome, ALL cells/viruses need to make mRNA to make protein, viral mRNA produced from viral genome inside host
- genome can vary in size to accomodate varying lengths of genomic material
- r/s between genome and mRNA produced is central to baltimore virus classification system
What is the baltimore virus classification system?
1- double-stranded DNA (uses its own or host DNA polymerase for replication)
2- single-stranded DNA (req. DNA polymerase to generate complementary strand)
3- double-stranded RNA (req. RNA-dependent RNA polymerase to make mRNA and genomic RNA)
4- + single-stranded RNA (req. RNA-dependent RNA polymerase to make template for mRNA and genomic RNA)
5- - single-stranded RNA (req. RNA-dependent RNA polymerase to make mRNA and replicate genome)
6- retrovirus (packages its own reverse transcriptase to make dsDNA)
7- double-stranded DNA pararetrovirus (req. plant host reverse transcriptase to make DNA)
What is phage lambda?
- infects E. coli
- chloroviruses infect algae
- herpesviruses cause chickenpox, genital infections, birth defects
- poxviruses cause smallpox and monkeypox
- papillomavirus strains cause warts and tumors
What is rhinovirus?
- coronavirus; cause severe respiratory disease
- flavivirus; cause hepatitus C, Zika fever, West Nile disease, yellow fever, dengue fever
What is rabies virus?
filovirus: Ebola, causes severe hemorrhagic disease
orthomyxoviruses: cause influenza
What is human immuno-deficiency virus?
- Lentivirus: HIV,AIDS
What is viral infection and reproduction?
attachment
penetration
uncoating
release
assembly
biosynthesis
*know to spot step
What is the viral structure?
filamentous capsid, icosahedral capsid, complex viruses; bacteriophages
- phage binds to trail sheath
What is biosynthesis in reproduction?
viral RNA enters nucleus, where it is replicated by viral RNA polymerase
- released into cell
- complicated by the presence of an envelope and the nature of the genome
What occurs when the bacteriophage attaches to specific host cell receptors?
- Phage genome is injected through the cell wall and membrane and the capsid is shed
What is the bacteriophage life cycle for a tailed phage?
lytic cycle (T2, T4, ebola)
- rapid phage replication
- host cell lyses/ruptures
lysogenic cycle
- phage infects and inserts its DNA into host chromosome as a prophage
- don’t initially destroy host cell
- amplifies amount of viruses that can be produced
- activated to excise and follow lytic life cycle by certain triggers
What is amensalism?
1) One species benefits
2) The other species is harmed by the
interaction
3) The interaction is non-specific
EX: streptomyces and other soil bacteria
What are the types of symbiosis?
mutualism, synergism, commensalism, amensalism, parasitism
What is synergism?
1) Each partner benefits from the other
2) Partners can be easily (?) separated and grown independently of each other
EX: cow rumen microbiome
What is mutualism?
partner species may require each other – an example of extreme coevolution
1) Removal of one partner leads to death or reduced growth of the other
2) The genomes of each species show advanced degeneration
3) Products produced by one partner are utilized by the other – often both ways
EX: LICHENS
What is commensalism?
1) One species benefits
2) The other species is neither harmed nor benefitted by
the interaction
EX: beggiatoa and other sulfur spring microbes
What is parasitism?
1) One species benefits
2) The other species is harmed by the
interaction
3) The interaction is specific, and usually obligatory for the parasite
EX: legionella pneumophila, amebas and human lung macrophages
What is endosymbiosis?
endosymbionts: many insect species infected by intracellular bacteria
- hard to tell type of relationships: parasitic or mutualistic
What are microbial ecosystems?
- microbes usually found as microbiomes, rarely as single species ecosystems
- microbes form foundation of all Earth’s ecosystems
What is the ‘omics revolution?
- use of high-throughput methods to look at molecular signatures of microbes
- genomics, transcriptomics, metabolomics, proteomics
What are the 3 main questions microbial ecologists ask of the microbiomes (ecosystems) they study?
WHO is there?
WHAT are they doing?
HOW do they respond to different conditions
* we can use DNA, RNA, proteins, metabolites to figure this out
WHO is there? How can we determine this?
culture, DNA and RNA sequencing
What is culture?
- petri dishes with agar
- not supportive of many bacterial species
- developed by Petri and Hesse
What is the great plate count anomaly?
- Many bacterial species from the environment find lab conditions so alien that they cannot survive
- Some bacterial cells in an ecosystem may be oligotrophic , or otherwise fastidious
- Some may depend directly on other species, or be inhibited by them
- Some may be non-viable, or ‘viable but not culturable’ (VBNC)
What is culturomics (high-throughput culture)?
- Reduces labour intensity by using AI & robots
- Allows culture under hundreds of different conditions
- Allows picking of thousands of colonies into multi-well plates
What are the types of DNA sequencing?
- An ecosystem is sampled, gDNA is extracted and subjected to either:
amplicon or metagenomic sequencing - Both methods can reveal ‘alpha diversity’, or the species richness (# of species present), evenness (close together) and dominance
Describe amplicon sequencing
- A target gene is amplified, barcoded and sequenced
- Most common method amplifies regions from the 16S rRNA gene from bacteria, a taxonomic marker
- cheaper and quicker
Describe metagenomic (shotgun) sequencing
- The extracted gDNA is broken up into bits (or not, depending on sequencing method), barcoded and directly sequenced
- A powerful computer is used to pull out signature genes from the sequenced pool
- better: tells us much more about what’s in the system
Describe RNA-sequencing
- Extract the RNA (ideally, mRNA) from a community
- Transcribe to DNA (using a viral reverse transcriptase (gene being transcribed) enzyme)
- not all genes always being transcribed: will tell you what the RNA was doing at the exact time of transcription
- (or not, depending on the sequence method)
- Barcode and sequence
- Match transcripts to known genomes
- access to what is alive and actively transcribing at the moment; unalive cells do not transcribe
What are the microbes able to do/actually doing in the community?
1- predictive
2- direct (proteomics, metabolomics, metatranscriptomics)
WHAT are they doing predictively?
- powerful computer to assemble MAGs
- software to annotate genes and predict possible functions
Describe proteomics
- Extract all the proteins in the sample, sequence peptide fragments using mass-spectrometry
- Use a (powerful!) computer to match peptides to proteins, and then proteins to genes (ideally, from the metagenome)
- pros: lots of info, seeing trancribed proteins
- cons: not all of mRNA will get processed into proteins (regulation of metabolism, done outside essential dogma)
Describe the process of metabolomics
- Extract all the molecules in the sample
- Subject directly to mass-spectrometry or NMR spectroscopy
- Use a (powerful!) computer to match compound signatures from obtained spectra to standards
pros: seeing all things being produced, small molecules may not br proteins (couldn’t predict from looking at genome)
Contrast metabolomics and metabonomics
metabolomics: looking at all metabolites from given organism
metabonomics: looking at all metabolites from given ecosystem
What are metatranscriptomics?
- mRNA content reflects active transcription – what the cells are doing/making in response to their environment
What are other “omes”?
lipidome, secretome, resistome, phenome
How do microbiomes vary under different conditions?
- If you take a single sample and carry out all of the so-far mentioned ‘omics analysis on it, will you get a full picture of what is going on?
- You have taken a ‘snapshot’ of the microbiome in time
- Microbiomes are dynamic systems – they change in response to
their environment and/or what you do to them experimentally
What is multi-omics integrations?
relatively new development in field
- multiple omics studies on given sample longitudinally
- integrate metadata
- computer to combine datasets
- can also integrate host genomics, transcriptomics, metabolomics, epigenomics
What is microbiome computational biology (bioinformatics)
- develop and carry out complex computational pipelines to interpret and understand enormous amounts of data
What is humus?
- leftover of what has been all been broken down
- very N2 rich
- good at holding moisture
- will hold soil together
- product of digestion
What does a particle of soil do?
- support miniature colonies, biofilms, filaments of bacteria and fungi
- interact with each other and plant roots
- diverse ecosystem
What is Streptomyces?
a major genus of soil bacteria, notable for the diversity of antibiotics they make
What do Microbes in the rhizosphere do?
- help protect plants from pathogens
- may fix nitrogen (diazotrophs)
- they feed on nutrients provided by the plant (root secretes nutrients)
What is Ectomycorrhizae?
- colonize the rhizoplane
- form a thick, protective mantle around the root
- extend outwards to absorb nutrients
- good for agriculture: allowing the plant to extend its root to intake more nutrients
Decribe features of Endomycorrhizae
- Grow inside plant cells - Dependent on their hosts
- Lack sexual cycles
- Exist entirely underground
- Relatively small number of endomycorrhizae species – but they are extremely important to the ecosystem
- endosymbiosis (can’t live without each other very well)
What are endophytes?
- grow within plant tissues
- can be bacterial or fungal
Describe the relationship between plant roots and rhizobia
endophytic
- bacterial cells adapt to life within nodules to form a nitrogen fixing ‘organ’ for the host plant (leguminous plants)
- nodules are pink: hemoglobin, due to oxygen carriers
What are microbial hitchhikers?
- human body teems with them
- majority are bacterial, harmless
- many are beneficial to host
What protects the human body from constant attack from microbial invaders?
- non specific defenses
- adaptive and non-adaptive immune defenses
What are commensal organisms?
- Microbes normally found at various non-sterile body sites are called
- may be incorrect: commensal means they are not doing anything, yet they are
What is the human microbiota or microbiome?
- consortium of colonizing microbes
microbiota: cell consortium
microbiome: genetic potential of consortium
Why are microbe populations dynamic?
- Vary with type of tissue, condition
- Can cause a disease if they reach an abnormal location
What are ruminants
rely on microbes for digestion of cellulose
Why do some insects require microbes?
allow digestion of their dietary substrates
- termites
- demodex mites: efficient gut microbiota, once thought they have no anus (most have them on face, they have sex and poop on your face every night)
What is the human bacteria ratio?
1 : 1.3
- approximately 200 bacterial species per person
Why don’t we look like bacteria?
- Bacterial cells are far smaller than human cells
- On average 1/100 to 1/1000 of the size
- Each gram of feces contains ~ 1011 bacterial cells
- 10 trillion cells in the average bowel movement!
Which diseases is thought to emerge from imbalanced microbiota?
autism, colorectal cancer, type 1 juvenile diabetes, chronic depression, parkinson’s disease
What are the layers of the skin?
epidermis, dermis, subcutaneous tissue
Why is the skin difficult to colonize?
dry, salty, acidic, protective oils
Describe the skin
10^12 microbes in moist areas
- mostly gram positive bacteria (more resistant to salt and dryness)
Give an example of gram positive bacteria in the skin
- staphyloccocus epidermis
- cutibacterium acnes (degrades skin oil, inflames sebaceous glands; also affected by human hormonal changes)
What is a human infant’s mouth first colonized by?
- non-pathogenic Neisseria spp.
- streptococcous and lactobacillus spp.
What bacteria start growing as teeth emerge?
Prevotella and Fusobacterium spp.: between gums and teeth
*Streptococcus mutans: tooth enamel
* Many oral bacteria are strict anaerobes
What is the most common site of infection of humans?
- oral and respiratory tract
- when bacteria are on the surface, they make biofilms which restrict oxygen, beneficial for anaerobic growth
What bacteria dominate the nostrils and nasopharynx?
- bacillota and actinomycetota
- one species or even genus dominates over the other
What is the nasopharynx populated by?
staphylcoccus aureus and staph. epidermis
- some strains are better than others at keeping us healthy
What is the oropharynx?
- similar composition of microbes to saliva
- Neisseria meningitids lives as commensal in 10% all adults and normally doesn’t cause issues
Describe the lungs
- surface area 75m2
- once thought to be sterile: actually community of microbes present, mainly anaerobes
How do lungs change between healthy and diseased?
- Microbiota in diseases such as COPD, cystic fibrosis and asthma seems to be distinct for each condition
What is the microcilliatory escalator?
- constantly sweeps inhaled particles up towards throat
- whooping cough: caused by toxin from bacteria
What is the urogenital tract?
kidneys and urinary bladder: normally sterile or near-sterile
How does the vaginal microbiota composition change?
- changes with menstrual cycle
- acidic: favour lactobacillus spp.
- lactobacillus spp. presence appears to protect from STIs and improve reproductive success
- rare; having a diverse microbial community is not good, lactobacillus will present diversity
What bacteria does the urethra contain?
S. epidermidis and some members of the Enterobacteriaceae
- can cause UTIs
Describe the stomach
- very low pH (usual lethal to microbes)
- few microbes survive
What is helicobacter pylori?
- present in stomach
- survives at pH 1
- burrows into protective mucus
- can cause gastric ulcers
What happens when stomach acidity is decreased?
hypochlorhydria
- caused by malnourishment
* Also now caused deliberately e.g. through PPI use
* Can lead to intestinal disease
stomach is a barrier between mouth and intestines
What is in the lower intestine?
contains 10^9-10^11 per gm of feces
* Ratio of 1000 anaerobes to 1 facultative anaerobe
What is the colon and intestine?
- colon: where most important microbial ecosystem in the human body
intestine: - community does as much metabolic work for us as the average liver; hence recently has become to be regarded as the ‘forgotten organ’
- most human microbiome research focuses on gut microbiome
What is important about your gut microbiome?
- the species’ metabolic potentials, not the species themselves
- lots of microbes share genes
What do our gut microbes do for us?
- Regulate the immune system
- Help to extract energy from foods
- Control potential pathogens
- Make some essential metabolites, including vitamins and cofactors
- Improve intestinal function
- Remove toxins and carcinogens
- As important to us as a liver
How do the gut microbes help improve intestinal function?
- block pain receptors
- IBS: if you’re missing particular microbes that modulate receptors in the gut
How do we acquire our microbes?
- fetus is sterile
- vaginal delivery (fetus exposed to environment, baby swallowing microbes as it passes down body)
- breastfeeding as a newborn
- interaction with environment as an infant (putting things on lips; pick up microbes from environment; evolutionary theory)
How do the gut microbes help to extract energy from foods?
- colon most heavily colonized by microbes
- microbes can break down foods well to release calories
How do the gut microbes remove toxins and carcinogens?
- good at breaking down compounds
- comes from food; toxic if not broken down
- same for drugs
How do we lose our microbes?
- C-section delivery
- maternal antibiotics
- formula feeding (do not contain human oligosaccharides)
- indoor living (seasonal)
- excessive sanitation
- chemical preservation of food
- window for proper gut microbiota development is narrow ! (0-5 years old)
Describe a high gut microbial diversity of species
- Healthy ecosystem
- Balance
- Functional redundancy
- High gene count
- Resistance to damage
Describe a low gut microbial diversity of species
- Sick ecosystem
- Imbalance
- Functional disability
- Low gene count
- Susceptibility to damage
Has microbiome diversity been eroded?
- missing microbiota hypothesis: we are removing microbes faster than replaced
- Loss of microbiota generally compounds over generations, and recent changes in lifestyle have greatly exacerbated this loss
- Yanomami (remote hunter-gatherers) have a more diverse microbiome than industrial: reflective of ancestral microbiome
How has the honey bee gut environment been tested?
robogut: RoBeeGut
- reps what a bumble bee or honey bee eats
How does the human colon act as a type of bioreactor?
- grow, isolate microbes; how they behave in nature
- bioreactors can be used to emulate the human colonic environment
How is the host of the robogut comprimised?
- microbiota kept in check by physical barriers and immune system
- Accidental penetration of the barrier, or damage in immune system: microbiota behaves badly
- The microbes that breach the barriers: opportunistic pathogens or pathobionts
- patient may be repeatedly infected by normal microbiota
Describe the features of a robo-gut
- Seeded with fresh feces or defined communities and set to model the ecosystem of the colon
- Host-free system
- Can be used to ‘culture the unculturable’
- Can support whole gut microbial ecosystems for several weeks at a time
- We can model the gut microbiota under different perturbation conditions
- We can learn how to protect against the effects of these perturbations
How is the microbiota protective?
- competitive exclusion: colonization of a niche to prevent pathogen from growing there
- environment modification: lactobacillus in vagina
- host stimulation: bacteroides fragilis using host cytokine
- direct pacification: secreted factors from members of microbiome can prevent virulence gene expression
Describe the microbiome-gut-brain-axis
- brain and the gut (microbiota) signal to each other
- full of neurons
- types of microbes you have in your gut, and the metabolites they make, can affect your mood and behaviour
What are the respective effects of the brain on the microbiome and vice versa?
brain -> microbiome
motility, secretion, nutrient delivery, microbiota balance
microbiome -> brain
neurotransmitter release, stress/anxiety, mood behaviour
What happens when microbial balance is compromised?
containment breaches, niche disturbance, extinction events
less bifido bacteria (lactic acid) in hunter gatherers: do not drink milk
- beneficial microbes
- adapted to industrial lifestyle
Describe why scientists don’t like the word dysbiosis
no frame reference for a healthy microbiome, everyone’s is different
Describe the relation between obesity and “dysbiosis”
- Gut microbiota influences nutrient acquisition and regulation of energy metabolism and fat storage
- microbiome balance is different from that of healthy people for obesity
- less diverse microbiome (but differs per person)
- obesity associated with low-grade intestinal microbiome
- insight from mouse experiments (high fat diet at first, can switch to a lean microbiome and lose weight)
Describe tight junctions between epithelial cells in gut
- Potential loss of microbes increasing them
- cells that line your gut, thin layer: has to maintain barrier and produces proteins that helps epithelial cells stick together and prevent things form crossing
What are gnotobiotic animals?
- mice
- animal where the associated microbiota is known and defined: bred/raised in environment devoid of microbes
- germ free animals
- expensive to manage, valuable for microbiome research
What is the physiology of germ-free animals?
- Poorly developed immune systems
- Lower cardiac output
- Requirement for more calories to maintain body weight
- Thin, poorly developed intestinal walls, including stunted villi
- Abnormal, enlarged ceca
- Odd behaviour
- Misshapen mitochondria (crescents in germ free)
Who was David Vetter?
- boy in the bubble
- SCID: impaired immune system, lived in a sterile environment for 12 years
Why are we not always getting sick if we are surrounded by microbes?
Not all microbes are pathogens
* We have various physical and chemical barriers: mucus, skin, stomach acid
* The immune system comes into play when barriers are breached
* A highly complex system of organs, tissues, cells and cell products that work in concert to recognize and neutralize potentially pathogenic threats
What is the immune system?
- highly complex system of organs, tissues, cells and cell products that work in concert to recognize and neutralize potentially pathogenic threats
- complex collection of cells and soluble proteins is capable of responding to almost any foreign molecular structure
What are the 2 arms of the immune system?
innate and adaptive
Describe the innate (non-adaptive, non-specific) immune system
- Ancient – evolved early in invertebrates and carried over to vertebrates, including (eventually) humans
- A ‘Rapid Response system’ Includes: physical barriers, chemical and cellular responses
- ‘hardwired’ into the body
- Present at birth
Compare infection and sisease
- Contact with an infectious agent does not guarantee that a person will get sick
- If number of infecting organisms is small and/or the immune system is effective, disease will not normally follow infection
What does a pathogen need to do to cause disease?
- Breach host defenses
- Survive innate defense mechanisms
- Begin to multiply
What are some physical barriers preventing infection?
- Skin (difficult to penetrate when intact)
- Mucous membranes (selectively permeable; epithelial cells tightly connected to support strong barrier function)
- lungs (mucociliatory escalator, large microbes -100um trapped by cilia and hair in nose; sneezing to remove foreign material from respiratory tract)
What are lymphoid organs?
- physical barriers are tightly connected to lymphoid tissue
- Specialized cells monitor and sample these sites continuously, present antigens to immune cells in lymph nodes
PRIMARY (factory for lymphoid cells): bone marrow and thymus
SECONDARY (stations for antigen encounters): mechanisms to enable encounters to happen in limited areas
What is SALT and GALT?
SALT: Skin Associated Lymphoid Tissue
GALT: Gut Associated Lymphoid Tissue
What is the body’s surveillance system?
the complement system
1. cripples
2. activates
3. kills by puncturing holes
Describe the complement system
“complement”: set of proteins made by liver
- named because they “complement” antibodies in the killing of bacteria
- proteins circulate in blood and enter tissues all over the body: inactive forms, proteolytically cleaved to make them active
- ~30 soluble and membrane bound protein components of the complement system (as of now)
- C1-C9
What are the 3 complement activation pathways and how do they converge?
- The classical pathway
- The lectin pathway
- The alternative pathway: only one associate with innate immune system
- converge on lytic pathway
What are some important things to know about the alternative pathway?
- C3 splits into two smaller proteins (C3a smaller than C3b)
- C3b: opsonin (marker for other parts of immune system)
- C3a C5a: anaphylotoxins (signals) direct immune cell traffic to where it is most needed, very reactive and unstable
- Membrane Attack Complex – punches holes in target bacterial cells, killing them
What are cytokines and an example?
- molecules secreted by cells which have an effect on other cells
ex: interferon (intruder alert cytokine) - cell is infected by a virus, secretes interferons helping other cells nearby to fend off virus, to limit the spread of infection
- The neighbouring cells step up their defense systems, ex by inhibiting viral entry
What is the body’s mace (noxious, toxic spray)?
- natural antimicrobial peptides
- act as antimicrobial elements produced in close proximity to cell
- Small molecules able to lyse most microbial cells and some enveloped viruses (produced at all times, short half-life and produced in high concentration)
What are defensins?
- natural antimicrobial peptide
- affects our own host cells: producing strategically over quantitatively !
- high concentration in gut
- Higher in close proximity to the crypts of the epithelium
- Secretion is from crypts
- Keeps out even the normal microbiota
What are the body’s “attack dogs”?
cells in blood:
- red blood cells
- white blood cells **: part of immune system
- platelets
What consists of white blood cells?
- Polymorphonuclear leukocytes (PMNs,
granulocytes or polys) - Monocytes & macrophages
- Dendritic cells
- Mast cells
- Lymphocytes
- “white”: white blood cells and platelets, excreted from blood as well as plasma and red blood cells
- useful indicator of state of health
What comes from the lymphoid and myeloid stem cell (bone)?
lymphoid: natural killer cell
myeloid: mast cell, neutrophil, monocyte, macrophage, dendritic cell
What does the myeloid bone marrow stem cells differentiate into?
- phagocyte cells : cells that eat
- takes up bacterium and encases it in “prison” (phagosome) which merges with lysosome and kills bacterium, breaking it up into many small parts
Why are bacterial cells difficult to catch and how can this be made easier?
- carry a negative charge, like phagocytes do
- C3b binds non-specifically to the surface of bacterial cells and makes it easier for phagocytes to stick to and ingest them
What are NETs?
- neutrophil extracellular trap
- NETosis: unusual form of cell death by the neutrophil
- neutrophil senses invader, spews latticework of chromatin and antimicrobial compounds into vicinity (prevents pathogen spread, allowing rapid phagocytosis)
- inappropriate NETosis thought to underlie lupus
What do monocytes differentiate into and how?
- macrophages
- monocytes circulate in blood stream
- like neutrophils: attracted by chemical signals (cytokines) to needed sites
- sticky: they roll on surface towards signal
- as they travel through blood vessels (extravasation), they differentiate into macrophages (secretes more cytokines to attract other phagocytes)
Describe macrophages
- large structures that can ingest many microbes at one time
- many body organs also contain patrolling resident macrophages (kuppfer cells in liver, langerhans in skin, microglia in brain, alveolar in lung)
Why do monocytes have to differentiate into macrophages?
- macrophages secrete cytokines which would be dangerous in blood vessels
Why do SEM not have colour
- use electrons, wavelength used is less than wavelength of light
What are dendrocytes?
- dynamic cells (constantly moving)
- possess long protrusions that can squeeze through tight spaces to sample microbes
- very thin: can squeeze through tight junctions of epithelial layer in gut to directly sample gut content
What is the language of our immune system?
- cytokines, chemokines, interferons
- Close-range acting ‘hormone’ system
- Allows cells of the body to communicate with each other
- Particularly effective at signaling ‘danger’
- some cytokines are important for anti-inflammatory signals after danger has passed (Reset the homeostasis)
What are cardinal signs of acute inflammation?
1) Heat at the site (loss of extravasation)
2) Edema (swelling)
3) Redness
4) Pain
5) Altered function (cell trying to deal with an infection won’t deal with a pimple)
What is pus?
- white blood cells; dead neutrophils
What are some example of chronic inflammation?
- Mycobacterium TB (slow growing, microbes hard for macrophages to kill as their cell wall is very waxy)
- fish tank and liver granuloma
- crohn’s disease (ulcers)
What are fevers?
- can be part of an inflammatory response, and heightens the activity of components of the innate immune system
What are antigen presenting cells (APCs?
- mainly phagocytes, can also be macrophages and dendrocytes
- Process the antigens they ingest and display them on their surfaces for T cells
- Form a link between the innate and adaptive immune system
- when they digest their prey, they work to break them up into component parts, excreting waste, also taking some and posting them on their surface like a marker
Describe the microbiome in the gut
- Peyer’s patches: specialist sites within small intestine
- rich in M-cells: specialist sites of uptake of antigens in the gut for presentation to macrophages
- Help the body differentiate between friend and foe when faced with a complex microbiome
How do bacterial capsules interfere with the antigen recognition process?
- hide major components, slippery, can be decoys
- form halo around bacterial cells: keeping dye away from them
- Many pathogens are encapsulated to help them evade the innate immune system
- best way to deal with: filter through blood: spleen)
How are pathogens recognized by the immune system?
- innate immune system !
- Cells of the innate immune system have specialized sets of receptors to recognize invariant and essential microbial factors unique to the microbe: Pattern Recognition Receptors (PRRs)
= PRRs recognize MAMPs (Microbe Associated Molecular Patterns): they don’t discriminate between ‘good’ and ‘bad’ microbes
What are PRRs I?
- toll-like receptors (TLRs)
- Transmembrane receptors on some immune cells that recognize viral and bacterial products
On binding to ligand: - Stimulate cytokines to signal inflammatory response
- Induce macrophages to produce antimicrobial proteins and peptides
What are PRRs II?
- NODs and NOD-like receptors (NLRs)
- TLRs monitor the outside of a host cell, so cannot sense them
internal (cytoplasmic) NOD like receptors (NLRs) bind MAMPs and:
* Activate cytokine production
* Form a complex called an inflammasome that activates the adaptive immune response, and triggers apoptosis (‘good’ cell death!)
Where do NLRs get their name from?
NODs
- similar host defense proteins found in plants
What is good and bad cell death?
bad: necrosis
- enzymatic digestion and leakage of cellular components
good: apoptosis
- neat “suicide”
- phagocytis of apoptotic cells and fragments
What are TLRs vs NLRs?
TLR- mostly outside of cell
NLR- inside of cell
What is an inflammasome?
- a protein complex that allows rapid secretion of certain cytokines
Describe natural killer cells
“specialized hit squad”
- not phagocytic
- lymphocytic cell (distinct from T and B cells)
- large and granular
* Make up ~2% of lymphocytes in the body
* main role is in innate defense (bridge between adaptive and immune)
* Don’t attack pathogens themselves, but instead attack host cells that have become overwhelmed by pathogens
- defense against cancerous cells
Describe an infected host cell
- infected cell signals altered self response
- needs to alert body that there is a pathogen or it will hide in the body
- adaptive immune system labels infected cell with antibodies
- when it finds altered cells it kills them very quickly
What is the mechanism of action for NK cells?
- alerted by interferons ( itself) or macrophage-generated cytokines (other cells)
- infected host cell signals altered self response
- NK cell binds to infected cell (always patrolling bloodstream)
- porforin kept in membrane bound granule so it doesnt kill itself
- granzyme move through pores, induces infected cell to undergo apoptosis
Why do NK cells kill the infected cells in this way?
- induces cells to undergo a good cell death (apoptosis) to keep inflammation at a minimum
What is efferocytosis?
- dying cell neatly packages itself up for disposal (apoptotic bodies)
- small enough for neutrophils to consume through efferocytosis
- neutrophils dispose of apoptotic cell bodies along with intruder microbes
- minimum inflammation
- after apoptosis
Describe the adaptive immune response
- branch of immune sys. with memory
- develops when needed
- 2 intertwined types (cell-mediated and humoral)
- “weapons cache”, defends body against any foreign invader
Compare the two types of adaptive immune response
Cell-mediated immunity
* Teams of T-cells (T-lymphocytes) and cells from thymus work together to recognize antigens displayed on infected cells
* Target infections in the body’s cells
Humoral immunity
* Antibodies directly target microbial invaders (B-cell response)
* Target infections in the body’s fluids (humors)
What happens when the adaptive immune system is missing?
- usually lethal mutation: SCID
- boy in the bubble
Describe adaptive immunity
- Develops over ~3-4 day period following exposure to an invading microbe
- Immune system recognizes small pieces of a given antigen antigen, called antigenic determinants or epitopes (May be 2- or 3-dimensional)
- Phagocytosis produces many epitopes to show the adaptive immune system (APCs display surface of the cell with broken down microbes so they can recognize them)
How are antigens released from the cell?
- bacterial cells made of antigens (every surface/cytoplasmic structure)
- each can have 1+ epitopes
- most epitopes are immunogens (some haptens)
- each immunogen can generate immune response by itself (large enough), haptens cannot
What is a hapten?
- small molecule that is too small to elicit an immune response on its own, but when bound to a larger molecule it can act as an antigen
- ex: nickel ions, or urushiol.
- innate immune systems can only see antigens at specific size (haptens are too small)
- must be bound to larger molecule
What can the adaptive immune system recognize?
immunogenicity: effectiveness by which an antigen elicits an immune respons
worse->best antigen: nucleic acids and lipids (conserved), carbs, proteins (large 3D)
- proteins are best as they have different shapes that are 3D
How can the immune system recognize and respond to epitopes without responding to itself?
- T cells are born from hematopoietic stem cells found in the bone marrow
- As these cells mature, each will develop a unique T cell receptor (TCR); each TCR reacts to a different random epitope.
- thymus gland: where T cells learn what to do
- This organ is fully developed in utero but shrinks as you grow older
- T cell education begins before birth
What do T cells require to mature and be released into body?
- Audition for their ability to identify self Major Histocompatibility Complex peptides
1. “Positive selection” – can they understand commands? - Are tested for reactivity against self antigens in the thymus
2. “negative selection” – are they well-disciplined? - killed if they fail (98%)
How do T cells of older adults mature if “thymus college” is not maintained after puberty?
- most T cell education occurs before and just after time of birth
- reserves of educated T cells maintained by body and reproduce at balanced rate
What are two types of “effector” (mature) T-cells?
- cytotoxic T cells / CD8+ cells
- helper T cells / CD4+ cells
Describe cytotoxic T cells
‘assassins’
* Trained to seek and destroy cells presenting noxious antigens (indicating they may be infected)
- indicates they may be infected just like NK cellsi
Describe helper T cells
‘intelligence officers’
* Trained to memorize databanks of antigens and to alert B-cells if circulating antigen is detected
What are memory and regulatory T cells?
- effector T cells and TH cells differentiate into memory TH cells
- some naive T cells differentiate to regulatory T cells (do not promote an immune response but help to restore homeostasis after infection; limit collateral damage)
- lack of Treg cells = chronic inflammation
Describe memory Th cells
- Retain antigen affinity of the originally activated T cell
- used to act as later effector cells during reinfection
- Don’t have a particularly long lifecycle, but are able to clonally replicate and pass traits to the next generation (imperfect)
- Overtime, memory gets hazy
- if pathogen returns much later, there may not be a strong immune response as T cell does not recognize as strongly (reason for boosters)
What are MHC proteins?
Major Histocompatibility Complex glycoproteins
* These are the proteins onto which infected cells or APCs place
antigens for display to the immune system
* Two main types: MHCI and MHCII (so they are not attacked, immune system has to know difference between the antigens so they use MHCs)
What are MHCIs?
- display antigens on the surface of an infected cell (i.e. the infected cell signals that it is infected)
- Cytotoxic T cell assassins will promptly dispatch the infected cells
What are MHCIIs?
display antigens on the surface of APCs
* Helper T cells will use this intelligence to alert the army and take further steps to neutralize infection
Where do T cells travel to after testing?
- lymph nodes of body (armpits, neck, lower back) (garrisons for army)
- approx. 4x10^11 T cells in body
- up to 10 T cells will recognize same pattern: clonotype
What is the initiation of cell-mediated response?
- APCs that have phagocytosed a pathogen travel to the lymph nodes to display their captured antigens to T cells
- The binding of antigen-loaded MHCI or MHCII to T cell receptors (TCRs) activates the T cells, and the cell-mediated adaptive immune response begins
- Remember, activated cytotoxic T cells can directly kill an infected host cell (using methods similar to those of NK cells)
- They produce cytokines that initiate a macrophage feeding frenzy at the site of infection
What are some amazing B cell facts?
- body contains ~10 billion B cells, each recognizes a particular epitope
- Response to an infection by a microbe involves thousands of different B-cells, each recognizes a different part of the threat
- Plasma cells are short-lived (4-5 days) but can produce 2000 antibodies per second!
- Some activated B cells differentiate into long-lived memory B cells (can survive decade +, linger to speed immune response to same invader)
- Basis for immunization
What do activated helper T cells and B cells do?
- some help to activate cytotoxic T cells, but others interact with B cells
- B cells also in lymph nodes (born in bone marrow, but educated and selected there, not thymus college)
- Each B cell is programmed to recognize only 1 antigen epitope
- helper T cells: conduit b/w APCs and B cells
- B cells are agents of humoral immune response
- Free-floating antigens from the microbe bind to B cell receptors that are specific for an epitope of that antigen
- If a helper T cell presents the same antigen to a B cell as the one already on its B cell receptor, the B cell becomes activated
- Differentiates into a plasma cell, a factory for antibodies
How do we learn to live with the antigens of our own bodies/the foreign antigens of our microbiota?
- antigen dose is important
- If antigen dose is over a threshold value, then B- and T-cells become over- stimulated
- T-cells become non-functional
- B-cells do not respond to subsequent antigen exposures to make antibodies
This is partly why we don’t respond to the antigens of the microbes of a healthy microbiome - T-cells are extensively trained in the thymus and tested for lack of sensitivity to self-antigens-
What are the keys to immunological specificity?
- antibodies (immunoglobulins)
- know/understand shape
What is the typical antibody shape?
- 4 polypeptide chains
- 2 large heavy chains
- 2 smaller light chains
- Bound together by disulfide bonds
What else do the helper T cells do?
- some can help B cells respond to T-cell dependent antigens
What are the staging areas of the immune response?
- APCs engulf a pathogen and travel to the nearest lymph node
- In transit, they digest the pathogen and display its antigens on their surface
- At the lymph node garrison, the APCs activate T cells and B cells
- Plasma cells, memory cells and cytotoxic T cells leave the garrison and enter the circulation
- Cytotoxic T cells travel directly to the site of infection (fight)
- Plasma cells and memory cells travel to the bone marrow
- Plasma cells soon die off, but memory cells persist
Describe the regions of antibodies
- constant and variable
- Defined by ‘constant’ regions of conserved aa sequences * 5 heavy chain types: α, μ, γ, δ, ε
- 2 light chain types: κ, λ
- Heavy chain defines the antibody class * α=IgA,μ=IgM,γ=IgG;δ=IgD;ε=IgE
- Each class is common to a species (called an isotype)
- For example, IgE is the same for all humans but different from that of monkeys
What is an isotype?
defines the various heavy chains of a species (e.g. human)
What is an allotype?
differences in the constant region shared by some but not all members of a species
What is an idiotype?
differences in the hypervariable region within an individual
What is IgG?
- Simplest, smallest and most abundant antibody in blood and tissue fluids
- A monomer, with 4 classes (IgG1-4)
- Each class varies in aa sequence and interchain cross-linking
- Binds and opsonizes microbes (allowing phagocytes to grab them more
easily) - Binds and neutralizes viruses
- Activates the classical complement pathway
What is IgA?
- Major secreted antibody of mucosal surfaces
- Most commonly found as a dimer, linked by disulfide bonds to the J (joining)-chain protein
- Secretory piece is wrapped around both molecules during secretion
- “secretory IgA” (sIgA) is found in tears, breast milk and on mucosal surfaces
- slgA can bind 4 antigens
What is IgM?
- Can be found as monomers on the surface of B-cells (part of the B cell receptor)
- But is most commonly found as a pentamer held together by J-protein (large monomer)
- First antibody isotype detected during the course of an infection
- can bind 10 antigens
What is IgD?
- Present in trace amounts in the blood
- Exists in monomeric form on surface of B-cells
- Does not bind complement
- Plays a role in B-cell activation
- Function not well understood
- Recent work suggests that IgD enhances mucosal homeostasis and immune surveillance by arming basophils and mast cells against mucosal antigens, including microbiota members
What is IgE?
- Present in trace amounts in the blood
- Found more prominently on the surfaces of mast cells and basophils
- These cells are loaded with inflammatory mediators (histamine) (held in granules)
- When 2 molecules of IgE on mast cells or basophils are cross- linked by antigen, cells degranulate and act to quickly amplify the immune response
- Primary role of IgE is to amplify the body’s response to invaders * They help orchestrate the acute response to invasion, early during
infection
What is an allergy?
- antigens which are harmless to most people are perceived as threats
- Anti-allergen IgE triggers release of chemicals such as histamine from
mast cells, or leukotrienes from eosinophils - Leukotrienes are the major chemical mediators of asthma
- Causes itching, swelling, wheezing (if in the lungs)
- immune system not getting feedback it needs from environment
When does allergy occur?
- large amount of IgE made after first encounter, attaching to mast cells
- cross-linking occurs on second encounter, mast cells triggers release of histamine
- immune response: sneezing, runny nose, itchiness, etc
What is anaphylaxis?
- very severe allergy form
- Excess histamine triggers smooth muscle contraction
- Contraction of lung smooth muscles interferes with breathing
- Excess histamine also weakens junctions between cells lining blood vessels
- Causes them to leak
- Fluid forced from circulation into tissues
- fluid contains histamine and the reaction spreads rapidly
How does an epipen help with anaphylaxis?
- pumps epinephrine, dilating blood vessels and opens airways
- quickly increases levels of cyclic AMP, stopping mast cells from dumping histamine to stop reaction
How is complement part of the adaptive immunity?
- alternative complement pathway: (C3b binds to bacteria and initiates the formation of the Membrane Attack
Complex) - Antibodies made as part of the adaptive response activate complement through the ‘classical’ pathway
- Requires a few additional proteins, but C3 is again the major player
Describe the lectin-mediated pathway
- similar to classical
- Lectins are produced by the liver and bind sugars on bacterial cells (target for immune system)
- Allow complement proteins to bind and trigger the formation of C3 convertase, then the pathway is the same as the classical pathway (converges)
Describe the classical pathway
- C1 complex cleaves C4 and C2, fragments re-join to make C3 convertase (cleaves C3 to C3a+C3b)
- C5 made with C3b, C5 convertase cleaves C5 to C5a+C5b; C5b forms membrane attack complex
Why do we need 3 complement pathways?
- Patients who have deficiencies in complement are extremely susceptible to certain blood-borne pathogens that display ‘phase variation’
- These are ‘shapeshifting pathogens’
- virulence strategy: change their antigens regularly to elude the immune system
- New antigens are not recognized by antibodies and are thus invisible to cytotoxic T cells
How does the body fend off invaders while also hosting a vast and complex ecosystem like the gut microbiota?
- Epithelial barrier is studded with T cells that have encountered antigens
- Dendritic cells reach between epithelial cells to sample antigens from the microbiota
- Specialized cells called M cells (found in ‘Peyer’s patches) also sample antigens
- Sampled antigens are constantly presented to a rich supply of macrophages, B and T cells in the layer under the epithelial cells
What is sIgA?
- Secreted into the lumen of the gut
- Coats microbiota components considered to be threats
- Prevents bound microbes from penetrating the barrier
- May promote the colonization of certain important beneficial microbes by adhering to them
Why is the positioning and response of TLRs important?
- TLRs on the epithelial cell side facing the gut lumen (many antigens), some also on basal side of layer (fewer antigens, only from invasive pathogens)
- TLRs on basal side are much more reactive than luminal side
- beneficial microbes have evolved to dampen TLR signaling
Why do you have to rest?
- immune system can have access to all the energy that it can get hold of
- most complex biological system other than brain
What is antigenic/immunologic specificity?
- degree to which an antibody recognizes an antigen
- can distinguish between several similar-looking antigens
- earliest clues: smallopox
What is variolation for smallpox?
- pick off scabs, put them in a bowl with herbs and grind them, let it dry
- take powder and shoot up noise, rub into skin, would cause a milder form of disease (2% would die)
Describe characteristics of smallpox
- extremely painful, lasts 2 weeks
- doesn’t exist anymore (besides in 2 labs)
- risk: children and pregnant women
- killed 1/3 of infected, scarred others
- variola major:same class as monkeypox
Why do we have to get booster shots and why do we get so many colds in our lifetimes?
- cold viruses (many, approx 200 viruses) evolve very quickly
- boosters: COVID drifts antigenically, changes the proteins on its surface (similar to influenza: better)
Who was Edward Jenner?
- first to put into practice that milkmaids contracted cowpox (closely related), but causes less prominent disease
- if milkmaid came into contact, they may get small lesions, but it provided immunity for smallpox and was much less severe
- cross-protection: the major antigen cowpox and smallpox produce is the same
Where does the word vaccination come from?
cows; vacca- latin
Why is a cure not that great?
- Suffering an infection with a pathogen gives immune response to the pathogen and (usually) acquired immunity-driven protection against subsequent infection
- high morbidity and sometimes mortality for nasty pathogens
- risk of infecting others nearby (epi/pandemics)
What is immunization?
- trick the body into seeing a pathogen and raising an immune response without the risk of pathogen-mediated disease
- ex: vaccination; works because of our adaptive immune response (needs to be primed against specific antigen)
What are the 4 types of vaccination?
- killed whole organisms
- live attenuated organisms
- subunit vaccines
- nucleic acid vaccines (mRNA, viral vector)
Explain the vaccination type of killing whole organisms
- take pathogen, kill it and administer killed pathogen to patient, providing antigens that immune system needs to respond (no infection as organism is dead)
- vaccines for hep. A, polio (salK)
BENEFITS: easy to produce, many antigens are presented to the immune system for a robust response
DRAWBACKS: Complete inactivation of the organism difficult to achieve
What is the Cutter incident?
- cutter lab manufactured salk vaccine
- batches incorrectly tested/passed, contained live virus
- 250+ polio cases, many paralysis cases
- legislation introduced to stop this happening again
Explain the vaccination type of live attenuated organisms
- organisms weakened (not dead) before administration, to give the immune system the upper hand
EX: BCG, sabin polio vaccine
BENEFITS: pathogen infection process is appropriate, many antigens are presented to the immune system for a robust response
DRAWBACKS: difficult to produce; Contraindicated for those who are immunocompromised; cold chain distribution usually required
Explain the vaccination type of subunit vaccines
- selected, purified antigenic components of pathogens (have to be very aware of pathogen)
EX: toxoid vaccine, S. pneumoniae, viral capsids from human papillomavirus
BENEFITS: easy to produce; no chance of infection
DRAWBACKS: hard to find protective antigen - some subunit vaccines contain subunits of antigens from multiple pathogens
What are toxoids?
- many pathogens produce them, they give you the infection; virulence component
- protect against what is actually causing the disease
- if you change stuff on its surface, you don’t change toxin shape, but it no longer acts as a toxi
Explain the vaccination type of nucleic acid vaccines
- mRNA vaccines contain mRNA that codes for a specific antigen, that is wrapped in a lipid layer and injected
EX: covid vaccines
BENEFITS: super-easy to manufacture, and relatively quick to get to market; no chance of infection
DRAWBACKS: Cold chain distribution required; very poor public understanding/excessive fearmongering, mRNA very unstable
Why are so many vaccinations given early in childhood?
- your immune system is the most open to infection
- immunity from breast milk and placenta wanes after 6 months
Why are most vaccines given in another dose?
- better response
- first dose leads to early synthesis of IgM followed by IgG
- second shot is ‘booster’ shot; a rapid response because memory B cells formed during first response
- booster shot ensures sufficient antibodies with sufficient reactivity towards antigen will be circulating in the body, protecting against reinfection
How do pathogens change their antigens rapidly?
- vaccine primes the body against an antigen that may be lost/changed into something else within short space of time
- ex: influenza, SARS-CoV-2;
- need a slightly different vaccine to fit the new antigens, as soon as the virus mutates (which is often)
What is the waning memory?
- Memory T cells and memory B cells have long lifespans, but not indefinite
- As they replicate, they can lose specificity for their cognate antigen
- Eventually they become unprotective
Why are live attenuated vaccines superior to inactivated, subunit or mRNA vaccines?
- live pathogen activates the immune system appropriately
Compare the salk (dead; injected) and sabin (live attenuated; drops) polio vaccines
- polio transmitted through fecal-oral route
- replicates in gut mucosa, moves to regional lymph nodes + induces viremia; some replicated virus can attack nervous system and cause paralysis
- salk vaccine is injected; antibody response is protective against paralysis, does not elicit mucosal response
- a person can later get mild form of disease through fecal-oral route; they can then pass this on to unvaccinated people
- may get transient infection, body can get over it quickly but can be passed on before body clears it
Why are COVID-19 mRNA vaccines injected and why is this not preventative?
- antibody response is protective against severe disease, but does not elicit the typical lung mucosal response
- respiratory disease, but gets injected into arm (prevents more severe forms of disease)
- person can still get a mild form of disease through natural infection
- “breakthrough infection”; they can pass it on to others
- Do all people within a community need to be vaccinated in order for protection of the community?
- No–vaccinating a large part of the community effectively interrupts the transmission of the disease
What is herd immunity?
- large community is immune
- depends upon many factors including transmission rate, population density
- only works for diseases transmitted person-to-person
Why is herd immunity important?
- protects immunocompromised people who cannot tolerate, or do not respond to vaccinations
- vaccination is not an option, and they rely on the vaccination of others
What is vaccine hesitancy?
- problem at the moment (largely because of the social media megaphone, and lack of fact-checking)
- Not new – Jenner’s work promoted public hysteria in the 18th century
- People believed cowpox would turn them into cows
Why is vaccination worth it?
- alternative: serious infectious disease and the potential to cause an epidemic
- no medical procedure is without risk, but social media has blown risk way out of proportion to benefit
How did antibiotics save lives?
- would not be alive today had antibiotics not been available to save you
- Infections we consider minor today often killed their victims even just 80 or 90 years ago
Describe the history of penicillin’s discovery
- people used molds and soil microbes as home remedies for centuries prior to the discovery of penicillin
- Ernst Duchesne originally discovered the antibiotic properties of Penicillium molds (not credited)
- Fleming rediscovered it; didnt garner much interest as it was very unstable
- ‘Howard Florey and Ernst Chain are credited with purification of penicillin (kept it stable for use)
Who was Gerhard Domagk?
- German physician (1895-1964) who worked at Bayer
- daughter had serious infection after pinprick
- injected her with dye; Prontosil (was being investigated as possible antimicrobial, no activity on plates)
What are sulfa drugs?
- saved hundreds of thousands of lives
- ## prontosil and sulfanilamide
Who was Selman Waksman?
- screened 10,000 strains of soil bacteria for antimicrobial activity; discovered streptomycin
How do antibiotics exhibit selective toxicity?
- used to be innovative that a drug should be selectively toxic against a pathogen and leave host alone
- possible because there are key elements of microbial physiology that eukaryotic cells do not share
- ex: peptidoglycan, ribosomes
Why are archaea ribosomes not used in antibiotics?
- archaea have similar ribosomal structure/processes to eukaryotes
- no archaeal vaccines, we don’t necessarily know of archaeal pathogens
How are antimicrobials classified?
- according to activity
- Antifungal, antibacterial, antiprotozoan, antiviral
- “antibiotic” usually reserved for compounds that affect bacteria
Compare narrow vs broad spectrums
narrow: agents affect few target organisms
broad: agents affect a much larger number of target organisms
Compare bactericidal and bacteriostatic
bactericidal: antibiotics kill bacterium
bacteriostatic: antibiotics prevent growth of bacterium, but do not kill it
(can be narrow or broad spectrums, choice depends on infection and its site, and health state)
Why are bacteriostatic antibiotics still effective, even though they do not kill pathogen?
- prevents reproduction of microbe
- suspends growth, buys time for immune system to take over
What does antibiotic effectiveness depend on?
- The organism being treated
- The attainable tissue levels of the drug
- The route of administration (oral, topical, injected)
How can we measure the minimum inhibitory conc. (MIC) ?of an antibiotic against its target?
- Serial dilution in a 96-well plate
- E-strips (MIC strip tests)
- Kirby-Bauer disk diffusion assay
*downside of these tests: it takes time, not possible for time-sensitive situations
What are some classic targets of antibiotics?
- Cell wall synthesis
- Cell membrane integrity
- DNA synthesis
- RNA synthesis
- Protein synthesis
- Metabolism
What are some cell wall inhibitors?
- penicillins
- cephalosporins
- vancvomycin
- bacitracin
- monobactams
What are some cell membrane inhibitors?
- polymyxins
- daptomycin
- gramicidin
What is the general process of making peptidoglycan?
- Sugar molecules N-acetylmuramic acid (NAM) and N-acetyl glucosamine (NAG) are made in the cytosol
- Linked together by a transglycosylase enzyme at the cell wall
- side chains of adjacent NAM molecules are cross- linked by transpeptidase to provide rigidity to the cell wall
- each step is a separate target for different antibiotic classes
Explain steps 1-4 of making peptidoglycan
- AA add to NAM
- NAM pentapeptide transfers to bactoprenol
- NAG links to NAM
Explain steps 5-8 of making peptidoglycan
- bactoprenol flips; moving NAM-NAG to outer side of cytoplasmic membrane
- transglycosylace attaches new disaccharide unit to chain
- pentaglycine connects L-Lys on one side chain and D-Ala on other
- one phosphate on liberated bactoprenol removed, lipid moves back to cytoplasmic side of membrane
What are beta-lactam antibiotics?
- Derived from fungi, consist of a beta lactam ring structure to which a number of R groups can be added
- each R group provides different properties (stability, spectrum of activity)
- work by making the cell wall fall apart
What are penicillin binding proteins?
- transpeptidase and transglycosylase enzymes
- involved in cell wall building
How can we be resistant to beta-lactam antibiotics?
- inheritance of a gene that codes for beta-lactamase gene (can be overcome by its inhibitors; clavulanic acid)
- inheritance of a gene that codes for an altered PBP that does not bind antibiotic (mutations)
What are cephalosporins?
- type of beta-lactam antibiotic
- continuously synthetically altered to combat the development of resistance
- microbes are quick to adapt
- some only used in worst-case scenarios, to slow does resistance development
What is bacitracin?
- binds to the bactoprenol lipid carrier and inhibits transport of the peptidoglycan monomers to the growing chain
- Very toxic and can only be used topically
What is cycloserine?
- inhibits the two enzymes that make a precursor peptide of the NAM side-chain
- Useful for the treatment of tuberculosis
What is vancomycin?
- binds to the D-Ala-D-Ala terminal end of the disaccharide unit & prevents bindings of transglycosylases and transpeptidases
- Some bacteria have incorporated D-lactate into the D-Ala terminus to develop resistance
What drugs affect bacterial membrane integrity?
gramicidin, polymyxin, daptomycin
What is gramicidin?
- cyclic peptide that inserts into the bacterial membrane
- only used topically (activity towards mammalian cell membranes)
What is polymyxin?
- Binds to both outer and inner membranes of G- bacteria, disrupting the inner membrane like a detergent
- only used topically (activity towards mammalian cell membranes)
What is daptomycin?
- aggregates in G+ bacterial membranes to form channels
- Very effective against MRSA (for now)
What are metabolic inhibitors?
- sulfonamides
- trimethoprim
- metronidazole
What is a DNA replication inhibitor?
- quinolones
What are RNA polymerase inhibitors?
- rifampin
- pyronins
What drugs affect DNA synthesis and integrity?
- sulfa drugs (antimetabolites)
- quinolones
What are sulfa drugs?
- interfere with nucleic acid synthesis by preventing the synthesis of tetrahydrofolic acid (THF), an important cofactor in the synthesis of nucleic acid precursors
- selectively toxic to bacteria as bacteria are the only ones that can make it, due to their biochemical pathway
What are quinolones?
- target microbial topoisomerases, enzymes that are essential for catalyzing changes in DNA topology to allow replication and transcription
- toxic to mitochondria as they are also bacteria: they use the same mechanisms and enzymes to replicate their genomes; mitochondria replicately separately from human genome
What is metronidazole?
- pro-druge
- Activated on reduction by microbial flavodoxin or ferrodoxin, found in microaerophilic and anaerobic bacteria
- Nicks DNA at random once activated
- Not effective against aerobic bacteria
What are some examples of RNA synthesis inhibitors?
- rifampicin (used clinically) and actinomycin D
How do RNA synthesis inhibitors work?
- Binds to the exit tunnel of bacterial RNA polymerase, blocking RNA from exiting the polymerase structure
- Halts transcription
What are some examples of 50s ribosome subunit of protein synthesis inhibitors?
- chloramphenicol
- macrolides (erythromycin)
- clindamycin
- oxazolidinones (linezolid)
- streptogrammins (synercid)
What are some examples of 30s ribosome subunit of protein synthesis inhibitors?
- aminoglycosides (gentamicin)
- tetracyclines (doxycycline)
How do protein synthesis inhibitors work?
- Bind and interfere with the function of bacterial rRNA
- Bacterial ribosomes and eukaryotic ribosomes have
fundamentally different properties – bacterial ribosomes are useful antibiotic targets - ribosomes catalyze linkage of amino acids during translation, using mRNA as the code and tRNAs as the source of amino acids
- Bacterial ribosomes are made up of small (30S) and large (50S) subunits
How do we target the 30S subunit?
- Aminoglycosides: Bind 16S ribosomal RNA (part of the 30S ribosome) and causes translation misreading of mRNA (jumbled peptides)
- Tetracyclines: Bind to and distort the ribosomal A site (that accepts incoming tRNAs) (interferes with bone development in children)
How do Macrolides and lincosamides target the 50s subunit?
- inhibit translocation of the growing peptide chain
How does Chloramphenicol target the 50s subunit?
- inhibits peptidyltransferase activity
- Can depress production of blood cells in the bone marrow
How do Oxazolidinones target the 50s subunit?
- Bind to the 23S rRNA component of the 50S ribosome and prevent formation of the protein synthesis 70S initiation complex
How do Streptogramins target the 50s subunit?
- 2 types; both bind to the peptidyltransferase site
- types A and B are used together and act synergistically
How does Mupirocin target aminoacyl tRNA synthetases?
- binds to bacterial enzymes that attach amino acids to end of tRNA molecules, halting protein synthesis
- Used topically in creams to treat infections caused by G+ bacteria
- Cannot be used internally because it is rapidly degraded in the bloodstream
What is the issue with antibiotic resistance?
- many derived from nature
- we have used high concentrations of
antibiotics for long periods (exerts selective pressure on bacteria to evolve)
What is a strategy for AMR?
- keep antibiotics out of cell !
Destroy the antibiotic before it can enter the cell - Decrease membrane permeability across the outer
membrane (express narrower pores) - Pump the antibiotic out of the cell using specific
transporters
What is another strategy for AMR?
- Prevent antibiotics from binding their target!
- Modify the target so that it no longer binds the antibiotic
- Add modifying groups to the antibiotic so that the antibiotic is inactivated
What is a third strategy for AMR?
- Dislodge an antibiotic bound to its target!
- Ribosome protection or rescue (some G+ make proteins that bind to ribosomes to prevent antibiotic binding near peptidyltransferase site)
How can resistance be?
- intrinsic (natural resistance)
- genetic mutations
- through receipt of antibiotic resistance gene (carried on mobile genetic element; plasmid, transposon, integron)
Why do people living close to the soil harbour many antimicrobial resistance genes?
- many genes originate in environment
- human activity and travel amplified and disseminated them
How do antibiotics have the potential to destroy the microbiome’s ecological balance?
- Collateral damage
- A huge number of diseases are now recognized to be associated with a lack of diversity in the gut microbiome
- still should take; lifesaving drugs if used correctly!
Why are we having such a large problem with AMR?
- have not used antibiotics appropriately since their
development - used in enormous quantities in agriculture
- often prescribed inappropriately
- taken inappropriately
- bacteria can exist in biofilms where resistant strains can persist
- pharmaceutical companies have no desire to develop new antibiotics
How can we prevent inappropriate antimicrobial use?
- never use to treat viral infections
- Do not use an antibiotic to treat an infection if the patient’s microbiome has resistant strains
- know which antibiotic resistant strains are prevalent within community before prescription
- consider length needed to take antibiotic
- use narrow-spectrum antibiotics when possible
- screen infectious agents for AMR genes
- ensure those with chronic infections maintain antibiotic regiment until infection is cleared
What is the influenza virus?
- a negative strand RNA virus
What is influenza A virus?
- most common life-threatening viruses of the western world
- 10% of north america infected
- pregnant women and elderly
What is influenza B virus?
- Narrower host range than influenza A
- Can cause serious disease but mutates much more slowly
- no current circulating strands
What is influenza C virus?
- Narrower host range than influenza A
- Causes mostly mild disease and does not spread easily
What is influenza D virus?
- never been detected in humans. Associated with swine and cattle.
- Relatively rare; emerged in 2011
What are influenza pandemics?
- usually influenza A
- cyclic appearance of extremely virulent strains that cause pandemic mortality
- Spanish flu, Asian flue, Hong Kong flu
What is a recent influenza concern?
- H7N9 – causes very serious disease in a high proportion of infected people
- not currently transmissible person-person
What are some differences between flu and a cold?
- influenza: severe headache and extreme, longer duration, some GI symptoms
What is a virion structure?
- no geometrical capsid
- shell of matrix proteins (M1) that surround the 8 RNA chromosome fragments
- matrix surrounded by membrane envelope (derived from host cell during budding; originates from last person’s cells who was infected)
- Viral envelope proteins hemagglutinin (HA) and neuraminidase (NA) stud the surface of the virus
What is tamiflu?
- block activity
- does not work for COVID
- antiviral drugs are very specific for the viruses they affect
Describe the 8 negative sense RNA segments?
- Each is coated with nucleocapsid proteins (NPs)
- Each encodes 1 protein
- 2 segments undergo splicing to encode 2 further proteins
- genome segments line up precisely
Why is each segment of virion genome packaged with an RNA-dependent RNA polymerase complex?
- if you make your own viral RNA polymerase and pack it with genome, as soon as it gets released, you can get to start genome
- quickly replicating genome (race against time)
How are segments packaged precisely during viral assembly in an infected cell?
- They link to each other in order as they arrange themselves
- Each segment lines up like a bundle of sticks
- Tiny molecular extensions seem to connect these sticks
What’s the main advantage of a segmented genome?
- most dangerous aspects of influenza virus is its ability to continually change its antigenic determinants
- Segmented genomes allow for re-assortment of genetic information, generating drastically new strains more quickly than viruses with non-segmented genomes
What is an “H” bit?
- hemagglutinin (18 HA subtypes)
- genome out of cytoplasm to work effectively; due to hemagglutinin
- allows fusion of the viral membrane with the host cell membrane
How does the H bit allow fusion of the viral membrane with the host cell membrane?
- 1) HA C-terminal domain recognizes and binds to host cell sialic acid receptor
- 2) Triggers uptake of virion by endocytosis
- 3) Endocytic vesicle acidifies and produces a conformational change that exposes the N-terminal fusion peptide
- 4) Fusion of host and viral membranes can now take place (pH sensitive)
- 5) Triggers release of the genome cargo into the host cytosol
What is the avian, swine, and human flu?
- birds are the natural infleunza A virus reservoir
- pigs have both types receptors and they get infected with 2 strains simultaneously; providing an opportunity for reassortment
- emergence of a dangerous pandemic strain
- influenza A binds to sialic acid glycoprotein (found on epithelial cells lining lungs and intestines)
- host determinant: nature of the cell-surface glycoproteins on host cells that bind the HA and allow endocytosis
What happens after pigs get both receptor types?
1) Viral segments travel to nucleus and enter nuclear pores
2) Attached viral RNA polymerase synthesizes (+) strand RNA (used as mRNA or as templates for generating
progeny (-)RNA)
3) mRNA travels to cytoplasm for translation to viral proteins – these are processed by the ER/Golgi & sent to the host cell membrane
What is the N bit?
- Envelope proteins and viral genome packages travel to cell membrane for packaging into new virions
- 11 neuraminidase N variants
- Within the cell membrane, envelope proteins assemble around the genome and matrix proteins
- Virion then buds out of the host cell
- Neuraminidase cuts the virion loose from host glycoproteins to release it
to the extracellular space
What is drifting?
- ability of influenza virus (A and B) to mutate and change slightly
- RNA replication errors in HA and NA genes
What is shifting?
- big change in the structure of the ‘flu virus
- Can be caused by jumping of the virus into a new species
- caused by re-assortment of the genes from 2 different viruses mixing in a single host (usually a pig)
How does flu vaccine production occur?
- WHO provides data on current season’s circulating strains
- for northern hemisphere, meeting takes place in february (recommendations about which viruses to include are made)
- begins in early spring; 6 months to produce enough
- strain can arrive late in season, predicting can be challenging
What is the potential for a universal future flu vaccine?
- annual ’flu shots is a drag, and community uptake is never optimal enough to prevent outbreaks
- Several groups are working in developing vaccines to more
conserved regions of the influenza virus - don’t change between viral strains
- Best candidate: the stalk region of the HA
- body’s attention is diverted towards the head part of HA and
antibodies are not made to the stalk region (a viral defense tactic)
*directing the immune system to recognize the stalk region may be the key
