How can the evolution of bacteria be studied?
16s rRNA sequencing?
Where is the Genome located in Bacteria, Archae and Eukaryotes?
Bacteria: Cytoplasm
Archaea: Cytoplasm
Eukaryotes: Nukleus
What shape has the Genom in B, A and E?
Bacteria: usually circular
Archaea: usually circular
Eukaryotes: usually linear
ist the DNA supercoiled in B, A and E?
Bacteria: yes
Archaea: mostly
Eukaryotes: yes
Are there Histones in B, A, E?
Bacteria: no
Archaea: yes
Are there Plasmids in B,A, E?
Bacteria: frequent
Archaea: frequent
Eukaryotes: rare
what is needed for a supercoiled DNA?
Topoisomerase 2
Do B, A and E have Operons?
Eukaryotes: no
Do B,A and E have mRNA modifikations?
Archaea: no
Eukaryotes: Capping and poly A Tail
Do B, A; E have Introns?
Bacteria: very rare
Archaea: very rare
Eukaryotes: most genes
What factor is necessary for the initiation of Transcribtion in bacteria?
sigma factor -> recognises promoter region
What factor is necessary for the termination of Transcribtion in bacteria?
rho factor -> releases the RNA pol
how can we interfere with Transcribtion in Bacteria?
how is sporulation activated on a gene level?
special sigma factrors in both compartments
Ribosomes, start AA, sensitive to Diphteria toxin, sensitive to Chloramphenicol?
What factors are important for translation
Initiantion F
Elongation F
Release F
Rescue Factor
can transcribed mRNA be used directly for Translation?
yes (in bacteria?)
Examples of antibiotics and toxins that inhibit translation
repeat the start and ending points in bacterial transcribtion and translation
what is the membrane build out of in B,A and E?
(Molecules, Connection, Sterols, Hopanoids)
How is the membrane permeability (for H+) in B,A and E?
What does the cell wall consist of in B, A, and E?
How do beta lactams work?
β-lactams (penicillins, cephalosporins, vancomycin): bind to or inhibit proteins involved in peptidoglycan synthesis → prevent cell wall formation
How do Amiosides work?
Aminosides: promote the misfolding of proteins that can decrease membrane stability
Why do Bacteria disrupt the eukaryotic cytoskeleton?
what general classes of adaptation of pathogen to the host defense are there?
Metabolism (Iron), Antimicrobial resistaance (efflux pumps), Biofilm formation, virulence (Exotoxin secretion)
explain transformation, transduction and conjugation
In transformation, a bacterium takes up a piece of DNA floating in its environment.
In transduction, DNA is accidentally moved from one bacterium to another by a virus.
In conjugation, DNA is transferred between bacteria through a tube between cells. (Pilus)
What surfaces do cells adhere to?
artificial surfaces (Biofilms, implants)
extracellular matrix (wounds, damaged tissues)
Endothelial cells (endocvascoular infections)
epithelial cells (nasal, skin)
What is the main sonstrain against bacteria ashesion to host surfaces?
FLOW (e.g. in Lungs, bladder, Intestine)
done by mucin secretion, ciliated cells, and smooth muscle shaping epithelium
Why does the bacteria want to attach?
What types of bacterial adhesins are there?
Supramolecular protein structures
Pili
flagella
curli fibres
Auto transporters
trimeric autotransporter adhesins (TAA)
surface adhesins
microbial surface components recognizing adhesive matrix Molecules (MSCRAMM)
Outer membrane proteins (OMP)
what do Pilis consist of?
What kinds of pili/fimbriae are there?
pilin subunits
Pilus: conjungtion or trasfer pilus
fimbriae
typ 1: adhesion via FimH
Type P: adhesion via PapG
TYpe IV pili: twithcig motility
curly: biofilm formation
How are Typ 1 and Type P Pili assembled? (in Gramm negative bacteria)
and how in Gramm positive bacteria?
What are catch bonds?
catch bonds enhance by tensile mechanical force
shown for blood proteins (selectins) and bacterial proteins (FimH and PilY)
Where are TAAs found?
found in the OM of Gramm negative bacteria
How are TAAs translocated?
What are Outer membrane proteins (in adhesion)?
Was machen die so?
cell wall anchored adhesins in gramm negative bacteria
adhesion and entry into host cell,…
an was kann das P1 OMP binden?
what kinds of adhesions are there in Gramm prositive bacteria?
anchoring in the peptidoglycan (in the cell wall)
How do the cell wall anchored adhesins in gramm positive bacteria get assembled?
What types of cell wall anchored adhesins in gramm positive bacteria are there?
How and why can bacteria (s.aureus) switch between adhesins and toxins production?
adhesions for biofilm formation and exponantial growth
toxins for infecting and spreading
-> switches stages
What non-protein adhesins do bacteria express?
What host receptors bind to WTA of s.aureus?
SREC Scavanger receptor (on nasal epithel)
how can bacteria bind to artificial implants?
via hydrophobuc interactions
or
binding to fibronectin coated plastic or metal surfaces via fibroonectin binding proteins
just a conclusion for adhesion because wtf
What factors do bacteria need to deal with in water and in infection?
what parameters can be measured by bacteria?
termperature
oxidative stress
antimicrobial peptides
low O2, high CO2
iron resitriction
explain quorum sensing
explain quorum sensing cheaters
dont produce the signal to save energy
whats the signal pathway that leads to tumbling in chemotaxis?
FliM (rotor protein) is triggered in absence of signal
example of chemotaxis in heliobacter pylori
colonizes stomach mucous membrane
-> senses urea to move toward stomach mucosa
How is Iron taken away from bacteria?
Free iron ions -> complexed by transferrin and lactoferrin
free heme -> complexed by hemopexin
free hemoglobin -> complexed by haptoglobin
how do bacteria snatch iron anyways?
siderophores bind free iron more efficiently than transferrin and lactoferrin
highly efficient heme uptake systems in s.aureus
borellia gurgendorferi uses Mn for Fe
what energy class are most bacteria?
Chemoheterorophs (chemical energy source, organic substances as Carbon and electron source)
Whats the difference between respiratory energy metabolism and fermentation?
in respiratory you have oxygen -> electrons are transferred to Oxygen products (O2, NO3-, SO42-)
in fermentation there is no oxygen -> electron tarnsfer directly back to organic products (Pyruvate, Acetyl CoA) —> NO ATP from electron transport phosphoreliation only by substrate level phosphorelation
whats the problem of fermentation for bacteria?
SCFA and other products can be sensed by scavaner receptors of leucocytes
advantage and disadvantage of reduced metabolism?
harder to detect but slower reproduction (less variants)
Which bacteria class has transport directly into a host cell?
(Gramm +/-)
only Gramm negative
post translational transport proteins
SecA=???
SecB= chaperon, prevents folding of protein and transports prottein to membrane
SecYEG=Translocon (pore)
AA- Structur of membrane proteins (IM)
Why?
Alpha helices (they saitsfy the H-Bonds)
How does the translocon know which orientation to put the protein in the membrane?
positive charges are always on the inside
(can be removed to flip the protein)
whats the advantage of beta sheets?
What AA are common in beta sheets?
they have two sides: one faces the inside (polar) one the outside (hydrophobic) of the pore.
Gly and Ala, because the have a short side chain.
what bacteria have Beta barrels?
Gramm negative (because its only in the OM)
How does the SecYEG translocon what protein needs to be inserted in the IM and what needs to be “shipped trough” to the OM?
IM= every AA must be in contact with the membrane (hydrophobic)
OM=only ever second AA must be hydrophobic (enegetically bad to insert into IM)
where can you find beta barrels in eukariots?
mitochondria and chloroplasts
Secretion path for OM proteins
Sec —> SurA (and skp?) —> BamABCDE complex
what model of membrane protein assembly and insertion for OM beta barrels is true?
BamA-hybrid barrel
How do periplasmic proteins stick to the membrane?
they get a lipid anker attached (Diacylglycerol)
How do lipoproteins get transpoorted from the outer leaflet of the IM to the inner leaflet of the OM?
How can a lipoprotein avoid this transport?
LolCDE complex (ATPase —> inside the cell, reahces through the membrane) —> LolA (Chaperone) —> LolB (implements lipoprotein in the OM)
having an Asp next to the Cys, where the lipid is attached prohibits this shuttel
Type 1 SS
Inner membrane ABC Transporter
Periplasmic adapter protein
OM pore
Type 2 SS
IM Sec translocon -> co translational transport of Pilin Subunits into the membrane -> get cut of and asseblmed in the base complex -> piston pushing mode -> secretion of folded proteins -> double walled beta barrel that is big enough
once the pili is attached to something in can reverse the ATPase direction and retrackt the pilus for movement
Or toxins are attached to the tip of the pilus
Type 3 SS
needle
ruler
chaperons -> unfoldet proteins
ATPase
self secreting
Type 4 SS
“a mess”
does a lot of things
conjungtion
DNA release
DNA uptake
Toxin delivery
effector molecule delivery
two classes A & B (one DNA + proteins, one only Proteins)
one or two step secretion process into host cell
build from outside to inside
Type 55 SS
most simple
two steps
one protein that builds its own membrane
trough IM via Sec translocon
trough OM via BamA and its own pore
part thats supposed to be secreted then gets cut from the pore
often ashesins
Type 6 SS
spring loaded spike
phage derived
to kill eukariotic or prokaryotic victims
found in almost all bacteria
Type 9 SS
largest beta barrel
transport of folded proteins across the OM
folded C-terminal secretion signal
proton motive force driven
two openings: one the side and the bottom -> Schleuse
SS in Gramm positive bacteria?
less complex
but dense cell wall (possibly ExPortals -> channels in cell wall
Type 3,4,7 also found in Gramm positive (but only IM components)
Def.
Exotoxins
Endotoxins
Exotoxins= aprotein toxin secreted by bacteria that kills or damages host cells
Endotoxins= a lipopolysaccharide in the OM of Gramm negative bacteria that becomes toxic to the host after the bacteria cell has lysed
examples of exotoxin attack points
membrane
cytoskeleton
protein synthesis
cellc cycle
signal transduction
cell-cell adhesion
vesicular trafficking
exocytosis
—> 9 classes
AB-toxins
A: active toxin
B: mediated uptake (endocytosis or pore formation)
-> gets activated once split
streptolysin O
pore forming toxin of streptococci
ologomerize and form huge openings
alpha toxin
staph aureus
forms a seven member pore
vibrio cholerae RTX toxin
Repeates in ToXin motife
secreted by T1SS
four functional entities
Cystein protease domain
actin crosslinking domain
Rho activating domain
alpha/beta hydolase (cleaves itself)
Salmonella Toxins
secreted by T3SS
guanine exchange factor
GPase activating protein
causes membrane ruffeling and invagination
reversable to keep the cell alive
2 examples of protein synthesis disrupting proteins
Shiga toxin
Diphteria toxin
cell cycle disrupting toxins
cytolethal distending toxins
DNAse triggering G2 cell cycle arrest
leads to enlarged or distended (aufblähed) cells
e.g. typoid toxin
cell cell adherance disturbing toxin
exfoliative toxins of s.aureus
acts on outside of the cells
to get deeper in the tissue
superantigens
bridege TCR and MHC calss 2 Moleküle
zytokinsturm
e.g. toxic shock syndrom toxin of s.aureus
difference microbiome and microbiota and microgenome
symbiotic
mutualism
commensal
pathogen
pathobiont
what parts of the body are steril?
inner parts of the body (Blood, tissue, cerebrospinal fluid)
eyes
respiratory trakt (mostly)
is the urinary trakt sterile?
what bacteria is predominant in the vagina?
lactobacillus (98% after puperty)
three functions of commensal bacteria in the intestine?
examples of diseases from microbiota dysbiosis
cancer
diabetes, obesity
MS, Autism, Alzheimer, Parkinson
Chrons disease, ulcerative solitis
allergies, asthma, atherosclerosis
how does staphylococcus get through the physical barrier of the skin?
exfoliatin A, B and D -> cleave cadherins (desmosome) -> deattachement of the upper epidermis
how does the mucus protect from bacteria?
mucus rinses bacteria off
steady state of producting mucus and bacteria rying to break through -> Germ free mice overproduce mucus -> colon gets huge
difference in mucus in small instestine vs large intestine
small
single, thinner layer of mucus (beacuse nutrients need to be absorbable
protection via antimicrobial modulators
large
two, thicker layer
outer layer is colonized
advantages and disadvantages of metagenomics
advantages and disadvantages of culture based characterization
what is an important substance that our gut bacteria produce for us?
Short chain fatty acids!
vitamins
how does the microbiom differ from person to person?
diversity and abundance varies widely amogst healthy subjects
but functional capacity is conserved!
how does inflammation effect our microbiom
imfalmmation kills sommensals while pathogens are resitant
-> high diversity -> health
-> low diversity -> disease
psychobiotoic
bacteria that improve xour mood
bacteria can produce neurotransmitters?
what animal classes have innate and adaptive immunity?
innate: all
adaptive: only vertebrata
host antimicorbial weapons (what types are ther?)
antimicrobial
lipids (damage bacteria membrane) -> in sebaceous glands (Talgdrüsen)
peptides (damage membrane)
enzymes (detruct cell wall and membrane)
Myeloperoxidase (production of toxic oxygen and nitrogen compounds)
terminal complement components (membrane attack complex)
4 ways bacteria can cope with antimicrobial fatty acids
what Eigenschaft do all antimicrobial peptides have?
all are cationic -> Cationic Antimicrobial peptides (CAMPs)
e.g. alpha defensins, HIstatins, beta defensins
how do CAMPs work?
what antimicorbial peptides do paneth cells produce?
alpha defensins -> to protect epithelial stem cells
how can bacteria get resistant to antimicrobial peptides?
examples of antimicrobial enzymes
lysozyme (cleaves peptidoglycan) -> s.aureus is resistnt because it has an O-Acetylation at the cleavesite
elastase (cleaves bacterial proteins)
phospholipase PLAII (cleaves bacterial phosphoolipids)
how are toxic oxygen derivates generated?
O2 —> NADPH Oxidase —> O2- —> Superoxid mutase —> H2O2 Myeloperoxidase —> HOCl or —> Fenton reaction (Fe+) —> HO
How are toxic Nitrogen derivates generated?
Arginin -> NO Synthase -> NO -> ONOO- -> NO2 + HO
how do bacteria prrotect themselves from oxidative stress?
55 signs of inflammation
redness, warmth, pain, swelling, altered function
3 pro inflammatory cytokines
producers
effects
systemic effects
TNF alpha
IL-1beta
IL-6
produced by activated macrophages, endothelial and epithelial cells
effects: secretion of defensins
endothelial pereability
activation of macrophages and lymphocytes
systemic efffects: fever
relase of acute phase proteins
schock if to much
chemokines
def.
examples
signal kaskade of TLR
same pathway as IL-1
-> MyD88 -> kinase kaskade -> NF-kB -> transkribtion
bacterial molecule with the strongest infalmmatory activity
and how does chlamydia evade that?
Lipopolsaccaride (LPS)
sensed by TLR4 -> senses lipid anchor
chlamydia can “inactivate” its LPS (only express 4-5 instead of 6 fatty acids?) so it stays unrecognized
pseudomonas also changes the number of fatt acids depending on its environment (at the cost of being more sensitive for defensins)
how can TLR9 dicriminate self from non-self DNA
depending on the methylation patterns of CpG sequences
NOD-like receptors
and bacterial modulations
what receptor causes chemotaxis? and what MAMPs are they following
GPCR
bacterial peptides (formyl peptides) or fermentation products (short chain fatty acids)
Formylated peptides because baceteria use formyl-Met as protein synthesis start
PROBLEM: Mitochondria us that as well
how does S.aureus evade Chemotaxis?
produces CHIPS (Chemotaxis inhibitor protein of S.aureus) that inhibits RPR1 (Formyl peptide receptor -> GPCR
what are the two major phagocytes (cell types)
Neutrophil granulocytes (=polymorphonuclear leukocytes =
PMN)
resident macrophages
What are the functions of Nuetrophils?
Phagocytosis
Degranulation (release toxins against bacteria)
NETs (neutrophil extracellular traps)
How ca Neutrophils kill bacteria?
What happends if the NADPHOxidase isn’t working?
Explains NETs
How can phagocytes recognise their prey
Non-opsonic phagocytosis (direct recognition)
C-type lectin receptors -> carbohydrates
Scavanger receptors -> charges molecules such as LPS, LTA, WTA
Adhesins -> sialylated glycans
Opsonic Phagocytosis (labeling, marking by others)
complement system (C4b,…)
Collectins
antibodies
what are the main steps in the complement system
activation via one of three pathways (Classic (C1 binds to Antibody), lectin (MBL binds to carbohydrates), alternative(spontaneus hydrolysis of C3 -> if bacterial surface present its not reversed?)
C3 convertase formation
C5 convertase formation
Membrane attack complex
-> MLB deficiency ist the most common inherited immune deficiency
how can mycobacteria use the Lectin pathway for their advatage?
express mannose -> MLB binds to mannose and activates complement system -> macrophages phagocyte bacteria -> bacteria inhibits phagosomal degredation -> persist in the macrophage (hides)
MLB deficiency gives resitstance against mycobacteria (e.g. tuberculosis)
review all three comlement pathywas (the initiation)
how do human cells protect themselves from activating the complement system?
And how do bacteria exploit that?
Factor H binds to sialic acid (modified sugar) to prevent C3 conversion
Neisseria incorporates sialic acid into its LPS
How does the complement system attract leucocytes?
split products =Anaphylatoxins have pro Inflammatory activity
C4a, C3a, C5a -> chemotaxis, vasodilation, histamine release,…
What parts of the complement system do what?
How does the membrane attack complex work?
How can bacteria defend themselves against phagocytosis
Type 3 effector proteins -> inhibit cytosceleon rearengement
can produce leukocidins -> e.g streptolysin I -> formes pore in membrane -> kills leucocyte
What is immunogenicity?
the effectiveness by which and antigen elicts an immune response
What are the most effective antigens?
proteins (form more diverse chemical forms ad maintain tertiary structure)
What Antibodies appear in the first response and the second?
firstt: IgM then IgG
second: mostly IgG
What abtibodies are present on naive B-cells?
monomeric IgM and IgD
When does a B cell produce IgM and when IgG, IgA,…
when they encounter the antigen on thei own there is no calss switch -> IgM
if they get help by a ThCell they can switch classes
functions of the different AB classes
what is Isotype, Allotype and Idiotype?
Differences in the constant regions may lead to:
- Isotypes: same person, different AB classes (IgE and IgM)
- Allotypes: my IgG againsts Spike protein and Annikas IgG against spike protein (same class, same antigen but still different)
- Idiotypes: my IgG against spike protein and my IgG against Influenza (same, person, same class, different antigen)
How do B-cells develop/get their AB?
What are the hypervariable regions?
What are the effects of AB binding?
What are the recombination signal sequences? (RSSs)
parts between the VDJ regions that enable the recombination of the different parts
How does the VDJ recombination take place and what enzymes are involved?
What is affinity maturation and how does it work?
How does AB class switching take place on a genetic level?
-> regulated by T-helper cells with the help of cytokines
Why do pathogens evolve so rapidly?
quick replication time
strong selective pressure
How can pathogens evade the AB recognition?
switch antigens! e.g. tryoanosoma brucei
salmonella enterica -> two versions of flagellin
neisseria -> different variations of pilin protein
how can staph aureus hide from the immune system, even once labeld by AB?
produces Protein A which binds to FC part of the AB -> immune cells cant bind the AB anymore
frequently used vaccine adjuvants
Aluminiumhydroxid
sepcific lipid/water mix
define trained immunity
-> epigenetic modifications
people with vaccine against BCG also dies less from tuberculosis
How does the tarining of the innate Immune system look like? (Epigenetik)
What are possible epigenetic reprogrammings?
explain negative and postive selection in thymocyte development
What types of T helper cells are there?
Whats TCR?
What binds to the TCR?
and what helper molecule does it need?
T cell receptor is Ig like heterodimer in membran bound form
TCR bind antigen if presented by MHC2
needs CD3 as coreceptor
What is needed to trigger an activation of B/T cells?
What do MHC I and II present?
MHC I = intracellular antigens
MHC II = extracellular antigens
How does MHC II get processes and loaded with an antigen?
How does MHC I get processes and loaded with an antigen?
BUT: Distinction between antigen processing
pathways for loading peptides on class I and
class II MHC is not absolute cross
presentation
Why do T cells need CD4/8
what do they bind to?
How does the contact between T helper cells and Antigen presenting cells look like?
How does a Tc cell kill an infected cell?
if antigen is presented -> secretes perforin -> forms pore
secretes granzymes -> enters cell trough pores -> induces apoptosis
OR binds to fas ligand -> induces caspases -> apoptosis
How do the B cells get activated?
TH0 binds MHC II on antigen-presenting cell.
If T-cell receptor binds antigen . . .
- Activation to become TH2 cell
- Proliferation of TH2 cells
B cells bind antigen with antibodies:
- Endocytoses and presents antigen on MHC II
- TH2 CD40L binds CD40
- Recognition of B cell
- CD4 binds MHC II
If T-cell receptor binds to antigen . . .
Activated TH2 activates B cell:
- Secretes Il-4, IL-6
- Stimulates B-cell proliferation, differentiation
B cells undergo class switching:
- Form plasma cells
- Secreted antibodies coat extracellular antigens.
- Prevent virus binding to target cells
Form memory B cells
- Reactivate if binds antigen again
- Fast response—no need for TH2 binding
revision humoral and cellular immune response
what determines what kind of T helper cell is going to develop?
the present cytokines
Can foreign lipids be presented as well?
yes with CD1
examples of Immune evasion
shielding or inhibition of MAMPS
Antigenic variantion
inhibition of psonation
inhibition of ROS
resistance to antimicrobial peptides
inhibition of fusion of phagosome with lysosome —> altering of membrane trafficking is common (e.g tubercolosis, salmonella
survival within phagosome
escape from phagosome
Modulation of T cell response
superantigen -> cytokin storm -> overreaction
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