Spacer integrase complex for CRISPR
Cas1 and Cas2 heterohexamer. (Cas1(2) - Cas2)2 i.e., 4 Cas1 and 2 Cas2
How are newly acquired spacers arrange in the CRISPR locus?
Newly acquired spacers are ligate in between repeat domains in the CRISPR array, with the ‘leading’ (newest) spacers being transcribed into crRNA at a much higher rate. In this fashion, the cell is afforded greater protection to more recent invasions.
Protospacer-adjacent motif, recognized by CRISPR systems for the acquisition of new spacers.
Issues with using mice as model system to study Mtb.
- No sterilizing immunity
- No granuloma formation
- forms chronic, low grade infection unable to be controlled by adaptive immunity, unlike human infection (i.e. no latency)
Intracellular pathways activated by Mtb permeabilization of phagosome
cGAS-STING (IFNa/B) and AIM2-NLRP3 (Caspase1/IL-1B)
How does Mtb (and other bacteria) transmit extracellular information internally?
Serine/Threonine Protein Kinases
Proteolytic Signal Transduction (release of sigma factors that affect transcription)
How long is antibiotic treatment therapy for Mtb?
Shortest duration that will cure 95% of patients (without drug-resistance) need at least 6 months of Antibiotics. 70% can be cured with 2-3 months, but there is no way of telling who they are. Culture negativity is not indicative of cure.
Original isolation of bacteriophage
Edward Twort (1915) and Felix d’Herelle (1917)
What is the DNA/RNA bias between eukaryotic viruses versus bacteriophage?
Bacteriophage are predominantly DNA viruses, while eukaryotic viruses are predominantly RNA viruses
Why does bacteriophage nucleic acids quickly circularize (if not already circularized) upon injection/infection?
“free” dsDNA ends are highly targeted by nearly all organisms for either repair or degradation - it indicates a break in the genetic material or an infection.
Two different outcomes of successful bacteriophage infection of bacteria
Lytic (cell lyses and progeny is released)
Lysogenic (DNA is integrated into bacterial chromosome, replicated throughout daughter cells and later activated (i.e. by DNA damage sensing)).
What is bidirectional reporter?
Some bacteriophages have one promoter in their circular genome that can induce transcription to either the left or right. Often the lytic and lysogenic genes are segregated to one or the other side of this promoter, allowing for control of cycles.
Approx. length of CRISPR spacers/repeats
Luria-Delbruck fluctuation test
1969 Nobel prize in Medicine. Showed that genetic mutations arise in the absence of selection, rather than as a result of selection, using phage T1 as the selective pressure on a population of bacteria.
- Showed evidence for mobile genetic elements in a Salmonella population via phage (using a filter that bacteria cannot pass through (but phages can)).
confirmed that DNA is the source of genetic information using bacteriophages.
determined the triplet nature of the genetic code in 1961 using phage.
when non-virulent strains of bacteria become toxic due to the integration of a pro-phage-encoded toxin
Types of bacterial resistance to phage infection
Surface modification of entry peptides Superinfection exclusion Restriction Modification Abortive infection CRISPR
What bacteria does T4 phage typically target?
Bacterial cells containing an integrated prophage, which can be induced, excised from the chromosome and enter the lytic cycle
The observation that different regions (genes and gene blocks) of the phage genomes have distinct evolutionary histories, owing to horizontal gene transfer events.
Most common morphologic features of isolated phages
Tailed and dsDNA genomes.
Three absorption-blocking mechanisms against bacteriophages
the blocking of phage receptors,
the production of extracellular matrix
and the production of competitive inhibitors.
The organelle responsible for ‘secretion’ of virulence proteins by the ‘type III secretion’ mechanism.
F0F1 Proton Translocase
A large and complex enzyme
in the mitochondrial inner membrane that catalyses the synthesis of ATP, which is driven by a flow of protons
Targets of the Yop proteins injected by Yersinia through the type III secretion system.
- destruction/disruption of cytoskeleton
- blocking of the inflammatory response
- promotion of apoptosis of macrophages
differentiation of bone marrow myeloid progenitors to mature neutrophils, driven by granulocyte colony stimulating factor (G‑CSF)165, which is produced in response to interleukin‑17A (IL‑17A) synthesized by T cells.
Two methods of signaling for emergency granulopoiesis
Direct - Sensing of PAMPs by HSCs leads to autocrine signaling that stimulates their differentiation.
Indirect - Sensing of PAMPs by HSCs and mesenchymic stem cells int he periphery leads to GM-CSF production which stimulates granulopoiesis.
Three Bacterial killing mechanisms by neutrophils
Degranulation, Phagocytosis, and NETs
Three steps of neutrophil recruitment
- initiation of adherence to activated endothelial cells and rolling,
- neutrophil arrest caused by firm attachment to the endothelium,
- and finally migrating across the endothelial barrier to the infection site.
Three enzymes produced by neutrophils after successful phagocytosis of pathogens
NADPH oxidase (generates superoxides), myeloperoxidase (MPO) (converts hydrogen peroxide to HOCl), or nitric oxide (NO) synthetase (makes NO)
Three types of granules in neutrophils
and gelatinase granule
Azurophilic (primary) granule contents
a spectrum of neutrophil serine
proteases (NSPs): cathepsin G (CG), neutrophil elastase
(NE), proteinase 3 (PR3), and the recently discovered neu-
trophil serine protease-4 (NSP4)
Specific granule contents
The specific granules are smaller with 0.1 𝜇m diameter
and formed after azurophilic granules. These granules do not contain MPO and are characterized by the presence of the glycoprotein lactoferrin.
They primarily contain a wide range of antimicrobial compounds including calprotectin, lactoferrin,
neutrophil gelatinase-associated lipocalin (NGAL), hCAP-18, and lysozymes
Also known as S100A, has
been shown to inhibit microbial growth through chelation
of nutrient Mn2+ and Zn2+, resulting in reprogramming of
the bacterial transcriptome. Accounts for approximately 40-50% of the protein in neutrophil cytoplasm.
staphylococcal superantigen-like 5 (SSL5)
can block neutrophil adhesion to endothelial cells by binding to PSGL-1 and consequently blocking
its interaction with the natural ligand P-selectin
Two enzymes involved in initiation of NETosis
Two enzymes in the ROS pathway have critical roles in NETosis. ROS generated by NAPDH oxidase stimulate MPO to trigger the activation and translocation of NE from azurophilic granules to the nucleus, where NE proteolytically processes histones to disrupt chromatin packing. Subsequently, MPO binds to chromatin and synergizes with NE in decondensing chromatin independently of its enzymatic activity. NAPH oxidase activity can be redundant in response to some stimuli.
Direct cell damage is implicated in infection, sepsis, autoimmunity and diabetes. By licensing macrophages for inflammation, NETs drive atherosclerosis. The increased propensity for NETosis promotes inflammation and organ damage in cancer and ischaemia–reperfusion injury. NET formation in the circulation promotes coagulation, vascular occlusion and thrombosis. NETs in capillaries can also capture and, potentially through other mechanisms, promote tumour metastasis. Finally, although NETs can promote inflammation, an accumulation of NETs promotes the resolution of inflammation through the degradation of cytokines and chemokines.
Differences in arginine metabolism in “M1” or “M2” macrophages
Arginine is primarily processed into bactericidal NO* by iNOS in M1s, while being processed into antiinflammatory ornithine by arginase in M2 Mqs. This is largely an in vitro polarization however, and Mqs in vivo likely exhibit both.
Properties of Intestinal lamina propria macrophages
Steady-state: 1) Reduced TLRs 2) Copious IL-10 promote Tregs 3) Capture translocating commensals 4) Replenished by recruited monocytes (CCR2+LY6C+) which differentiate into Lamina propria macrophages (CX3CRhiLY6C-). 5) Clear apoptotic or senescent Epithelial cells 6) Capture invading commensal bacteria
Metabolic relationships in
which one member provides
nutrients to another
bacterial phyla in the gut
Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria
Two methods of colonization resistance conferred by the microbiota
The gut microbiome can manifest colonization resistance through a number of potential mechanisms: 1)‘passively’ by out-competing bacteria for space and trophic resources
2) actively by the generation of bacteriocidal factors
A serum protein that binds free
haemoglobin and inhibits its
A haem-scavenging protein
that is found in serum and
binds haem with high affinity
mediates energy-dependent transport
of siderophores through non-selective porins in the outer membranes of
lipocalin-2 effector mechanism
binds to and sequesters certain Fe siderophores, providing nutritional immunity.
Steps in cytokinesis/septation
(1) selection of the site where the sep tum will be formed;
(2) assembly of the Z ring, which is composed of the cytoskeletal protein FtsZ;
(3) assembly of the cell wall-synthesizing machinery (i.e., for synthesis of peptidoglycan and other cell wall constituents); and
(4) constriction of the cell and septum formation
protein controlling the elongation of rod-shaped bacteria undergoing cell division. Actin homologue.
homologue of eukaryotic intermediate filaments that localizes to one side of the bacterial cell, where it slows the insertion of new peptidoglycan units into the peptidoglycan sacculus. The resulting asymmetric cell wall growth gives rise to the inner curvature that characterizes this cell shape (comma shaped)
Enzymes used by faculative anaerobes or aerobes to catalyze destruction of super-oxide radicals and hydrogen peroxide
superoxide dismutase (SOD) and catalase
enzymes that inactivate the antibiotic by hydrolyzing a bond in the Beta-lactam ring, thus conferring penicillin resistance to the bacterium.
First virus detected and year
tobacco mosaic virus in 1892 by Ivanosky
EBV genome type
Latent membrane protein 1, expressed by EBV-infected B cells. Resembles a constitutively active CD40 receptor, and leads to proliferation of the B cell.
Latent membrane protein 2A, expressed by EBV-infected B cells. Resembles a BCR, and can lead to survival of BCR-deficient B cell lines. Includes intracellular ITAM motifs.
Genetic mutation that leads to Burkitt’s lymphoma
translocation of the MYC gene to an Ig chain region, leading to constitutive expression of the MYC protein.
How did the chamberland filter contribute to the discovery of viruses?
The chamberland filter was used to purify drinking water (to remove bacteria), and it was discovered that ‘purified’ water that passed through the filter was able to still transmit rabies. - thus rabies must be smaler than bacteria.
What are the two ‘options’ for viral capsid/nucleocapsid structure?
Capsids can be icosahedral or helical and can be ‘naked’ or enveloped with plasma membrane
How do (+)ssRNA viruses replicate?
First they need to use an RNA-dependent RNA polymerase to make a negative strand copy, off of which the same polymerase can make multiple copies of the original (+)ssRNA
RNA-dependent RNA polymerase
included in the viral capsid of ssRNA viruses in order to make the opposite ssRNA for replication.
How does VSV (chicken pox) enter latency?
Infects T cells and eventually skin, leading to lesions. Then undergoes retrograde transport to the spinal cord and exists in the nuclei of ddorsal root ganglia as dsDNA episomes until reactivation, wherein it is er-transcribed, and undergoes anterograde transport back to the skin (shingles)
What transcription factor does EBV protein LMP1 signal through
NFkB, leading to cell survival, proliferation and growth.
What cell is another reservoir for HIV other than CD4+ T cells?
Macrophages. PBL paper showed that CD8+ CTLs are less efficient at killing HIV-infected macrophages.
What is crucial for a pathogen to be successful in a (new) host?
For a pathogen to be successful, it must balance between avoiding immune responses and not killing the host before it is able to spread to other hosts - viruses both activate and inhibit the immune response.
Which cytokines primarily mediate anti-viral innate immunity?
Interferons, particularly viral RNA and DNA leading to type I (alpha and Beta) interferon.
Which cells are the main producers of interferon alph and Beta?
pDC, or plasmacytoid DCs.
Which cytoplasmic sensors detect viral ds or ssDNA?
cGAS, IFI16, DAI, AIM2,
All activate STING
Which cytoplasmic sensors detect ds or ssRNA?
Both activate MAVs
Which transcription factors are downstream from viral genetic material sensing in the cytoplasm?
IRF3, IRF7, and NFkB.
Leads to IFN production (among others)
How does type I IFN signaling transduce through the cell?
Ligation with the receptor leads to heterodimers of STAT1 and STAT2, which activate IRF9, leading to interferon stimulated gene production.
What does the Interferon-stimulated gene CH25H do?
This ISG helps to prevent viral fusion to the plasma membrane by breaking down cholesterol in the membrane, changing the lipid composition of the membrane.
What process does the ISG TRIM5a interfere with?
The uncoating of HIV, by interacting with gag proteins.
What process does the ISG RNAseL interfere with?
This gene produces an RNAse that indiscriminately degrades RNA in the cytoplasm - also targets host RNA.
What process does the ISG Viperin interfere with?
Interferes with the replication of viral genetic material by modifying nucleotides to prevent addition of new nucleotides.
Protein called tetherin that is an ISG that tethers newly produced viral capsids to the cell membrane, preventing their dissemination. HIV encodes a protein called Vpu that prevents CD137 from reaching the surface.
Promotes deamination of G -> C of viral genetic material, thus neutralizing it. HIV encodes a protein that targets this for degradation.
What cytokines other than type I interfereons are induced by viral detection?
IL-6, activation of caspase-1 (IL-1), TNF, IL-12
How do NK cells contribute to ADCC (antibody-dependent cell cytotoxicity)?
NK cells express high levels of CD16 (FcgRIII), so they can recognize IgG that is bound to host cells.
What are three ways that viruses can evade NK cell targeting?
- producing soluble proteins that block activating receptors
- Produce viral ligands for inhibitory receptors
- Mechanisms for downregulating activating receptors
What is the natural course of HIV infection in an individual in terms of levels of virus and immune cells?
The first two weeks show a rapid increase in viral load, corresponding with a decrease in CD4+ T cells (as they are targeted). As CTLs specific for HIV begin to rise, the viral load fall backdown to a viral setpoint, which persists for years and CD4 cells transiently go back up, but ultimately decline over time.
What are the categories of HIV disease progression?
- Rapid progressers often die within the first 2 years,
- Intermediate progressers initially control the. infection, but begin to develop symptoms years later
- Slow progressers find a balance between CD4+ and viral load
There are also ‘elite controllers’ that get the virus down to an undetectable level - we are not sure how, but seems to be host mediated.
Where is the most common SNP that shows up in most ‘elite controllers’ of HIV infection?
Single significant hit on that maps to the human leukocyte antigen (HLA) B locus, i.e. MHC-I (chromosome), indicating a role for CTLs. The specific SNPs mapped to the peptide binding groove.
How does epitope amino acid networking in HIV proteins confer protection?
When targeted epitopes for a particular HLA are highly networked within the folded protein, it is more difficult for HIV to change the individual amino acids without disrupting the structure of the protein. However, if the epitope is on a lower-networked ‘loop’, the amino acids are ‘more avaialble’ for mutation.
What is the function of the Nef protein in HIV?
Downregulates MHC I. More potent at downregulating HLA-A than HLA-B.
What is the balance that a virus must strike when downregulating MHC I molecules?
Downregulation of MHC I will lead to more probable evasion of killing by CD8+ CTLs, but a higher probability of being targeted by NK cells which can recognize the lack of MHC I.
Why are antibodies not effective for HIV infections?
Most of the antibody response is non-neutralizing, and the targeted spike is highly looped and thus easily mutated.
Low density of surface expression of Env Antigenetic variability Glycan sheilding Transient epitope exposure Steric occlusion Nonfunctional Env
What is unique about braodly neutralizing antibodies against HIV?
Besides being exceedingly rare, they exhibit an extraordinary amount of SHM, suggesting that they ust go through tons of rounds of GC selection and thus need lots of Tfh help
How can SARs Co-V2 still mutate when it has a proof-reading mechanism?
The proofreading mechanism is still not perfect - it still can make mistakes
Also, there is host-mediated RNA editing by APOBEC and ADAR1 that can lead to mutations
Coinfection can also lead to genetic recombination