All MM notes from

What happens in the body when you get pseudomonas

ciliary cells stop working, mucus gathers, allows formation of biofilm, this hinders PMN phagocytosis which leads to chronic inflammation

Biofilm

bacteria attach, lose flagella, and create a polysaccharide that promotes harm and inflammation

Quorum sensing

bacterial strategy to determine number of other bacteria around it

pyocyanin

produced most abundantly in low iron concentration, oxidizes NADPH to NADP, kills bacterial competition of p. aeruginosa, may also affect epithelial cells and immune function, this is also a pigmented virulence factor

Dissemination of mycobacterium

infected macrophages are carried through the lymphatic system and spread through the blood stream

What diseases does S. pyogenes cause

Scarlett fever, erysipelas and cellulitis, necrotizing fasciitis, acute rheumatic fever, acute glomerulonephritis (S EC NF ARF AG) SECARF NoFAG (yikes!!)

M-protein (in relation to S. pyogenes)

• virulence factor involved in anti-phagocytosis, adherence, internalization, and invasion/spread

• prevents C3b opsonization which would normally tag S. p for macrophage destruction

• helps with molecular mimicry

Hyaluronic acid (as relates to S. pyogenes)

• virulence factor involved in anti-phagocytosis, adherence, and invasion/spread

• HA capsule prevents receptors from binding to cell wall

• hyaluronidase breaks down host connective tissue and allows deeper spread of bacteria

Mycobacterium TB gram

gram positive but lack gram stain b/c cell wall rich in lipids

How to stain M. tb

acid fast stain

1. carbonyl fusion

2. heat

3. rinse

4. decolorize with acid

5. counterstain w/ methyl blue

Immune response to M. tb

CD4+ T cells which leads to a gamma interferon release assay

How does M. tb get into phagocyte

cofactor recognition: CR1, CR3, and CR4, as well as LAM recognition

TB virulence factors

LAM, phosphatases (PtpA, PtpB, SapM) all come in through sec and tat pathways, type VII secretion, siderophore

LAM

inhibits phagosome/lysosome fusion

PtpA

stops lysosome from docking

PtpB

delays apoptosis and prevents inflammation

SapM

prevents recruitment of membrane trafficking proteins

Rab

GTPases that make sure vesicles get to the right place (phosphatases and LAM interfere with the signaling cascade and mess up rab)

How does legionella enter a macrophage

• complement-mediated opsonization and uptake by alveolar macrophages

• host cell kinases are involved

• actin polymerization at the site of phagocytosis

The bacterium uses a Dot/Icm type IV secretion system (T4SS) to inject effectors that manipulate host membrane trafficking, forcing the macrophage to form a specialized, protective vacuole (Legionella-containing vacuole, LCV) for replication

RalF

• part of legionella

• recruits membrane fusion proteins (Arf1) and stops lysosome by secreting RalM

• prevent activation of Rab5 to prevent lysosome fusion

SidM

• part of legionella

• recruits membrane docking proteins (Rab1) and adenylates it which leads to increased recruitment of ER-derived vesicles to the LCV. Adenylation locks Rab1 in an active state

• is reversed by SidD for recycling of Rab1

AnkX

• part of the legionella system

• Fic protein that adds phosphocholine to Rab1 at golgi membrane

• further promotes recruitment of ER derived vesicles to the LCV by modifying Rab1 post-translationally

Extrinsic caspase

• part of the legionella system

• external ligands bind which triggers caspase 8 activation

• C8 cleaves Bid into its active form

• travels to mitochondria and amplifies apoptotic signal

Intrinsic caspase

• part of legionella system

• pathway triggered by internal cell stress (Baa)

• BCl-2 inhibits release of pro-apoptotic factors

• once signal is strong enough, cytochrome C is released from the mitochondria

• Cytochrome C binds with Apaf-1 to activate caspase 9

• Caspase 8 and 9 both lead to the activation of caspase 3 or the “executioner” caspase which cleaves proteins that lead to structural changes and eventual cell death

SdhA

It prevents the vacuole from breaking down, allowing the bacteria to avoid cytoplasmic degradation and preventing premature host cell death

rho/rab system

specifically host Rab GTPases and Rho GTPases are manipulated by bacterial Dot/Icm effectors to remodel the host cell membrane, allowing the pathogen to create a protected, nutrient-rich environment for replication

GEF (rho/rab)

promotes GDP leaving and GTP coming in to replace it (activation)

GAP (rho/rab)

hydrolyzes Rho GTP and brings it back to inactivated state (quality control mechanism in cells that are monopolized by bacteria)

GDI (rho/rab)

storage factor that binds to inactive GDP and keeps it soluble in cytoplasm while avoiding being prematurely activated by GEF

PKR system activation

viral RNA gets inside cell —> produces interferons —> dimerization and autophosphorylation of PKR

3 pathways of PKR

1. elf2-alpha is phosphorylated by active PKR which inhibits protein synthesis— the cell gives up its own ability to replicate to stop the virus

2. FADD activation which activates caspase 8 (apoptosis initiator), then apoptosis

3. phosphorylate Ikk-alpha beta which phosphorylates NF-kappa-B, a transcription factor that translocates from the cytoplasm to the nucleus once phosphorylated and starts expressing genes involved in inflammation which tag the infected host cell as infected

dot/icm system outcomes

1. recruitment of ER-derived vesicles: Rab1 and Arf1 are recruited which makes host send vesicles from ER to the LCV, then effectors like SidC and SdcA bind lipids to the surface to secure the vesicles

2. manipulation of apoptosis: SidF and SidA block apoptotic pathways which prevents apoptosis

3. protein synthesis and stress response: Sid1 shut down host protein synthesis, which gives bacteria resources and production of defensive proteins

4. ubiquitination

Coxiella Burnetti

• uses IV secretion system

• uses dot/icm

• survives and is stable inside lysosome and uses that space for replication

• binds to cell via repurposed integrins

• Rab7 and LAMP1 assist with lysosome fusion

• reminder that pH goes down as we get closer to the lysosome

Viruses, what are they?

infectious, obligate, intracellular molecular parasite with nucleic acid genome

Class 1 virus

dsDNA

Class II virus

ssDNA that have to become dsDNA to replicate

Class III virus

dsRNA

Class IV virus

+sense ssRNA that has to replicate into a minus sense RNA that’s used as a template.

Class V virus

-ssRNA that carry complement RNA and cannot be translated, do nothing with ribosomes, have to bring in RNA polymerase

Class VI virus

ssRNA (+ and -sense), reverse transcription

virus life cycle

attachment and entry → partial disassembly of RNA into cytoplasm → translation → polyprotein produced and cut up → genetic material now in genome → virus released

Viral assays

viral enzyme activity, serology, nucleic acid detection, centrifugation

alpha viruses

icosahedral, envelope, “perfect virus”, nearly every particle is virus causing

Viral centrifuge process

1. radiolabeled virus

2. cells washed and lysed with detergent

3. antibodies added

4. centrifuged

5. proteins eluted

6. SDS page separates them

Western blot

1. antigen samples in separation gel

2. blotting tank

3. immuno staining blot + labeled antibody

4. audioradiography

5. cleaned up antigen bands

what it does: light only emerges where enzyme is or where protein has attached to antibody, tells us the size and measure of intensity (kinda)

ELISA (serology)

sample pad with MABs, serum runs over test and control line, if the test has something to attach to, they will show up red

nucleic acid detection

shows which nucleic acids are present, yellow = equal amounts

RNA vs DNA

RNA mutates at a much higher frequency

Diversity of viral genome

ss vs ds, DNA vs RNA vs DNA with RNA segments, linear vs circular

What does not carry genes for antibiotic resistance

insertion sequence element

Why might expression of ptlA in E.coli fail to secrete PT

PT uses type IV secretion, it would need both the a and b subunits

What MHC recognizes intracellular viral antigens

MHC class 1

What T cells go along with MHC class 1

CD8

What is required for activation of T-cell

1. MHC connection

2. costimulatory signals

Main component in our saliva, sweat, or tears that constitute a chemical defense against a pathogen

lysozyme

two benefits of normal bacterial flora

1. suppress growth

2. allow uptake

3. assist immune system

4. keep digestive system working

3 mechanisms by which bacteria combat antibiotics

1. biofilm production

2. enzyme inactivation

3. eflux

If you don’t see a gram stain, what did you forget

safranin

gram negative

thin cell walls

protein has an N-terminal secretion signal containing two arginine residues, what secretion pathway does it use and is it folded or unfolded

it uses twin arginine or Tat pathways and it’s folded

What are differences between prokaryotes and eukaryotes

only eukaryotes have a nucleus, the ribosomes are larger in eukaryotes, prokaryotes divide by binary fission, which eukaryotes divide by mitosis, both can be single-celled

features of pathogenicity islands

• distinct GC content

• evidence of transposase

• flanked by direct repeats

mutant missing gene encoding usher protein, what virulence factor is it missing? and would it be a good vaccine

it’s missing FIM, and it would not be a good vaccine because it is no longer antigenic

What makes a crosslink with D-Ala of the peptide chain during peptidoglycan synthesis in gram negative bacteria

DAP (diaminopimelic acid)

What modification occurs on BvgA to activate it

phosphorylated on aspartate residue

How does BvgA regulate expression of intermediate phase genes like BipA

intermediate form of phosphorylation

How does the bordetella CyaA toxin become an active enzyme only inside eukaryotic host cell

only activated by Calmodulin in eukaryotic cell

Rig1

recognizes viral flags like non-capped RNA and long RNA strands and activates protective pathways like NF-kB

Mda5

recognizes viral RNA and promotes interferon and cytokine production

toll-like receptors

part of the innate immune system on the outside of immune cells, once detected produce pro-inflammatory cytokines

(p)ppGpp

• alarmone

• acts as secondary messenger → when its levels rise, it triggers global reprogramming of cell

• downregulates growth functions (i.e. ribosome synthesis)

• upregulates “survival functions” (i.e. amino acid biosynthesis and virulence factors)

What happens when legionella is in a stress state

1. lack of nutrients (amino acids, carbon, etc) causes a spike in (p)ppGpp levels

2. RelA and SpoT act as synthetases

3. enzymes convert GTP into (p)ppGpp

what happens when legionella is back in homeostasis (vgr)

1. (p)ppGpp levels go down

2. SpoT acts as a hydrolase and breaks (p)ppGpp back into GTP and PPi

3. CgtA interacts with SpoT to promote hydrolase activity

SpoT

synthesize ppGpp when stress levels are high

hydrolyze ppGpp into GTP and PPi when legionella is back in homeostasis

S. pyogenes virulence factors attachment

1. M-protein: binds several host proteins

2. Sfbl or Fibronectin binding protein: coasts host cell epithelia

3. HA capsule: binds CD44