Flashcards
Lecture 2_bacteriology
Cards in this deck (145)
- 1Why are commensal bacteria important?1. Development
- Induction of blood capillaries
- Infection can change gene expression ex. Bacteroids thetalotaomicron: shown with gnotobiotic mice and infected mice
2. Nutrition
- absorption of energy: allow additional breakdown (energy storage host)
3. Immunity: allergies and cancer
Allergies: correlation between early antibiotic use in children and development of asthma and allergies.
Exposure to bacterial endotoxin prevents allergy.
4. Defense: take up space which in itself is a form of defense - 2What bacteria can you find in the: a) intestine b) skin c) nasal pathway d) urogenital pathwaya) In the beginning we mostly have aerobes--> more and more anaerobes (obligate) as we get older- such as bacteroides
b) S. aureus, s.epidermis, propionobacterium (acne)
c) streptococci
d) sterile with exception of vagina which may have lactobacillus, corynebacteria, streptococcus, staphylococci, bacteroides - 3What effect does Bacteroides thetaiotaomicron have on the host?The bacteroid thetaiotaomicron, breaks down carbohydrates which the host could not break down, to simple sugars which the host and other bacteria can utilize. Experiment with mice:
Gnotobiotic mice + bacteroides--> increase of 50 percent in fat tissue despite same diet as before and an increase with 30 percent when administred LCD. - 4Explain the experiment demonstrating the correlation between certain microbiota and obesity. What is characteristic for lean vs. obese individuals?Sequencing of microbiota demonstrates that:
obese individuals: Low bacteroides and high firmicutes
Lean individuals: high bacteriodes and low firmicutes
Further proven by injecting germ free mice. - 5How do we classify eubacterial pathogens?We classify according to:
- rRNA
- morphology (biochemistry, staining)
- lifestyle: 6 different lifestyles
-pathogenesis (human disease) - 6Importance of following external structures of the bacteria: - Cell wall - capsule - outer membrane -endospores- Cell wall: prevents rupture of cell (osmotic pressure)
- Capsule: For adherence and resistance to engulfment
- Endospore: protective resting state
-Outer membrane: With LPS (endotoxin) - 7What are the differences between gram (+) and gram (-) bacteria?Gram (+)
- Thick homogenous layer: 10-100 nm
- Teichnoic acid
- Different PG composition compared to gram (-)
- 90 % CW
- Penicillin: targets interpeptide bond formation between DAP and D-ala
- Lysozyme: G-M (PG)
- Mostly cocci
Gram (-)
- Ex. E.coli
- Two heterogenous layers
-Inner and outer membrane
- periplasmic space
Endotoxin+LPS - 8Capsules- Composed of polysaccharides deposed outside the bacterium
- Protection against phagocytosis
- Adherence: ex. oral streptococci
- protection against drying
- reserves of nutrients
- Biofilms for protection against antimicrobial agents and the immune system
- May also form mucoid- gummy like appearance such as seen by bacillus anthracis - 9Endospores- Not a form of reproduction- rather a state which is induced by deleterious enviroment: vegetative cell--> endospore
- takes about 6 hours
- Show no sign of life: dormant state
- several unique layers which are not found in vegetative cells
- Highly resistant to heat, boiling, pH, antibiotics etc
- High concentration of dipocolinic acid in spores: calcium binds and stabilizes the structure
- - 10—QRT-PCR: utilizes TaqMan polymerase
- 11—Q-RT PCR: SYBR-green is a fluorescent dye which binds to double stranded DNA and can be visualized--> can visualize the amount of PCR this way. Cheaper and less precise than TaqMan.
- 12What is RT-PCR and what is it used for? What is qRT-PCR?Reverse transcriptase PCR: use cDNA (RNA stored as "DNA"- more stable than RNA) together with reverse transcriptase enzyme to give DNA.
qRT-PCR where q stands for "quantitive"- two different ways of assaying the amount of DNA synthesised:
1) TaqMan- more expensive & more precise
2) SYBR-green - 13Mechanism of gene duplication (TANDEM GENE REPLICATION): non-reciprocal recombination.
- 14TANDEM GENE REPLICATION: Rolling circle model
- 15What is the significance of gene amplification?1. Tandem gene amplification: non-reciprocal recombination and rolling-circle recombination: most common when the solution is to increase gene expression.
2. Difficult to observe as it is readily reversible and doesn't really leave any evolutionary marks.
3. Allows secondary mutations to occur. - 16What is phase variation? Give an example of phase variation.Phase variation is when the expression of a phenotype can be switched ON/OFF.
Ex. salmonella flagella (FliC)
- By moving the promoter- so that DNA is synthesised in the opposite direction and doesn't synthesise the repressor: expression of proteins neccesary for building the flagella.
- 17What is Antigenic variation? What does it have for similarities with phase variation?Antigenic variation- the expression of alternative antigens on the cell surface, can also be changed by similar mechanisms for phase variation: simply changing the direction of synthesis (inversion of DNA segments- allows synthesis of alternative Surface Layer Proteins (SLP))
- 18What are the possible advantages of phase variation? What does it have for evolutionary meaning?1. More options: variation (mixed population- aquire different genes) means higher chance of survival for the population.
2. Gene regulation may be by different triggers- enviromental changes, enviroment, immune system - 19Describe why Neisseria Gonorrhoeae is so succesful at infecting the human host despite strong antibody response?No strong immunity against Neisseria gonorrhoeae because the pathogen has antigenic variation- especially the pilus which is the target of the antibodys produced by the human host (IgA). Has several different genes for opa- pilE- pilC-pilS - can shift promoter and therefor change synthesis of which protein building blocks to use (change antigen)
- 20Neisseria gonorrhoeae: explain the cause of the antigenic variation seen in the pilus- Several different PilE, PilC, PilS and opa: move synthesis direction- can then synthesise different segments
- How? High frequency of slippage- easy to get a frame-shift: sequences consist of CTCC- in a order which readily easily give a STOP-codon or different gene segement: more specifically, antigenic variation occur when silent pilS recombine with active pilE- FURTHERMORE- by transformation (take up DNA from enviroment-HGT) can take up PilE.
- 21Give specific examples of how virulence factors can be regulated1. Phase variation: ON/OFF
2. Antigenic variation: alternative antigenes
Phase variation
a) Inverted repeats shifted
b) epigenetic control
c) mRNA
b) Epigenetic control: pap fimbriae in E. coli. There are three major components:
1. The local regulator papI
2. The global regulator Leucin Regulator Protein (LRP)
3. DNA adenine methylase (DAM)
We have the pap fimbriae sequence. There are two important promotors: The proximal promotor (site 1,2,3) and the distal promotor (Site 4,5,6).
OFF-state: LRP is bound to the proximal promotor and DAM cannot methylate the promotor and thereby activate it.
ON-state: papI binds to the distal promotor and increases the affinity of LRP to the distal promotor-which then leaves the proximal promotor, allowing DAM to bind to the proximal promotor --> transcription of fimbria sequence
Epigenetic control: One of the products of the sequence is papB- a positive regulator of papI (positive feedback for fimbria synthesis). Other protein regulators- such as N-HS which can bind to the sequence and influence the ON- and OFF state: epigenetic control. - 22Give an example of mRNA transcription regulator
- 23What is H-NS?- Regulates promotor accesability
- A small chromatin-associated-protein which acts as a repressor in non-intestinal enviroments
-Binds to curved DNA - 24On what levels can regulation occur?Following levels:
- - 25Describe in general how two-component systems work1. Stimulus--> Autophosphorylation of histidine residue on sensor kinase
2. The phosphate group is transferred from the high energy histidine residue to the lower energy arginine residue on the regulator domain on the response regulator
3. Activation of the regulatory domain and subsequent activation of transcription of certain genes - 26What are the two main types of sensor receptors and what do they respond to?1. Membrane associated sensor kinases: Enviroment
2. Intracellular receptor: intracellular responses - 27Explain how chemotaxis is controlled by a two-component system in detail1. Stimulus--> MCP--> autophosphorylation of CheA (ATP--> ADP)--> phosphorylation of CheY--> flagellar motor (rotation)
REGULATORS:
CheB: de-methylates MCP
CheR: methylates MCP
CheZ: de-phosphorylates and therefor inactivates the flagellar motor - 28What are the name of the different flagellar placements?
- 29Explain flagella motility in E.coli when: a) MCP is not bound by ligand b) MCP is bound by liganda) MCP--> autophosphorylation of CheA--> phosphorylation of Che-Y: flagellar motor protein active: TUMBLE
CheB--> demethylates MCP--> ligand binds to MCP, when ligand binds to MCP CheZ is activated and de-phosphorylates CheY --> RUNNING
b) MCP is methylated--> CheA autophosphorylates--> Che-Y is phosphorylated--> flageller motor: Tumbles,
CheR--> methylates MCP--> flagellar motor keeps being on TUMBLES - 30Describe the principle of QS in gram (-) bacteria (WITHIN SAME SPECIES)1. LuxI/LuxR system
LuxI: synthesise AI (autoinducers) which in gram (-) bacteria typically are homoserine lactones
LuxR: Response regulator- which binds to the AI (complex) and then acts on the promotor of the target sequence- inducing expression of the gene(s)
Example: Vibrio fisheri and its autoinducer N-acetyl homoserine lactone - 31Describe the principle of QS in gram (+) bacteria (WITHIN SAME SPECIES)1. AI produced by a special process involving modification and processing--> oligopeptide
2. The AI is then sensed by Two-Component System - 32Give an example of gram (-) bacteria QS which do not follow the typical LuxI-LuxR system, and explain how it differsa) Vibrio Harveyi-
LCD:
- Low levels of AIs
- LuxN, LuxPQ, CqsS act as kinases
-phosphorylates LuxO: transcription of Qrr1-5 and no bioluminescence
HCD:
- High levels of AIs
- LuxN, LuxPQ, CqsS act as phosphatases
- de-phosphorylates LuxO: no transcription of Qrr1-5 and Bioluminescence
*Similar in Vibrio Cholorae but with HapR--> Bioluminescence instead of LuxR- also no LuxN involved (but involves LuxPQ and CqsS - 33What is surprising about QS in Vibrio Cholerae?- QS promotes biofilm formation and production of virulence factors at LCD: the opposite of the usual case
Why?
2 reasons
(1) May be beneficial at early stage infection
(2) At HCD exist the host- gets out in the external enviroment where it may be beneficial to switch of these genes for virulence and biofilm formation - 34How is sporulation initiated?Sporulation is well-studied in Bacillus SubtiHow is sporulation initiatelus.
When there is limitation in nutrients the vegetative cell undergoes sporulation: Spo0A initiates sporulation- activation of Spo0A is controlled by phosphorylation--> by multiway pathways - 35What are some sources of oxidative stress for bacteria?(1) Intracellular enzyme autoxidation
(2) Enviromental redox reactions
(3) Competing microorganisms secrete hydrogen peroxide
(4) phagocytes NADPH
(5) Redox cycling antibiotics - 36How do bacteria detect reactive oxygen species?In E. coli
Detect:
Hydrogen peroxide: OxyR: thiol disulfide redox switch
Superoxide and nitric oxide: SoxR: 2Fe-2S cluster - 37Describe how OxyR is switched ON and OFF1. Hydrogen peroxide: oxidation of OxyR: disulfide bond between cys199-cys208: active
2. GSH acts as an electron donor: reduces cysteine - 38How does E.coli cope with temperature above 37 Celsius Degrees?- Sigma 32: Bind to promotor which will allow transcription of rpoH genes- proteins to deal with heat shock
- Sigma E: Deals with extreme heat
Sigma 32 is controlled by:
- mRNA
-Stability
-negatively regulated by chaperones - 39What are some major types of pili for adhesion?1. Chaperone Usher poli (type 1 pili & p-pili)
2. Type IV pili
3. Curli
4. TAA - 40General characteristics of pseudonomas- Gram (-) bacteria
- soil bacteria
-obligate aerobe--> can in anoxical environment use nitrogen
- resistant to multiple drugs - 41pseudomonas aeruginosa (oppertunistic pathogen)- what type of infections can they cause?- CF patients
- Burn wounds
- Eye contact lenses - 42Why are CF patients sensitive to Pseudomonas aeruginosa?healthy individual: Two biphasic layers of mucous: upper is vicious and the lower is thicker- which when the pathogen comes to the lower, can then be ejected to the esophagus.
In Cystic fibrosis patients there is only one mucous layer which is viscuous- the bacteria get stucks there and aren't handled properly - 43How would you describe the QS system in Pseudomonas aeruginosa?1. Primary system: Las system (LasI & LasR)
2. Secondary system: circuits AIs and induce Rhl system
3. Third system: PQS signal- balances system 1 and 2 - 44Why are pseudomonas aeruginosa so resistant to antibiotics?- Mutators: high mutation rate
- Inactivation of mismatch-repair so they have even higher mutation rate
- Strong selection in CF patients: treated heavily continously with antibiotics
- have developed drug efflux - 45What is characteristic for pseudomonas aeruginosa in the lungs of CF patients?Form biofilms of pseudomonas aeruginosa which increases mutation rate even further, protects against phagocytes, creates inflammatory response in host, adapts to the enviroment of the host and antimicrobial therapy: slow growth. Efflux pumps and chromosomal beta-lactamases
- 46How is Tuberculosis transmitted?* Person-person transmission: airborne so when you talk, laugh, cough, sneeze etc.
Virulence depends on:
- TB pathogen itself
- Exposure
-How infectious the person is
- 47What is the difference between primary- and secondary antibiotic resistance?Primary Ab resistance: The pathogen which is transmitted during infection is antibiotic resistant.
Secondary Ab resistance: Ab resistance occure during treatment, either because the patient did not listen to the orders or because doctor did not give proper regimen to the patient. - 48What therapies for tuberculosis are there?1) DOTS: Direct observed therapy short course, where health care workers monitor that patients are taking their medications. Patients are receiving a cocktail of different antimicrobial drugs. Important that the regimen is strictly followed to avoid resistance.
2) DOTS plus: montly monitoring patients for the appearance of resistance and changing regimen accordingly. - 49How do you test for latent tuberculosis (LTBI)?- Mantoux tuberculin skin test (TST)
- Inferon gamma release assays (IGRAs) - 50How does the disease go from LTBI to active TBI?1. Individual infected with tuberculosis bacili- macrophages handle these. While the immune system can handle these- the person will be asymptomatic and non-contagious. However, a few bacili will be resistant to the macrophages- and will replicate.
2. Macrophages will be recruited more and more as the bacili replicate and will form a barrier around the bacilli called tubercle- at this stage some patients may display symptoms of the disease, but can still be asymptomatic.
3. The tubercle can calcify and rupture resulting in the bacili escaping and infecting other individuals- and other body parts of the host: bones, lungs, kidneys, brain, thorax etc. - 51What's the difference between toxins and effector molecules?Toxins:
i) Secreted from the bacteria
ii) Cause covalent modifications
Effector:
i) Generally secreted into eukaryotic cells
ii) By secretion systems - 52What are the characteristics of endotoxin?Endotoxin:
1) Gram (-) bacteria outer membrane as Lipopolysachardie (LPS) and is relieved when the bacterial cell is lysed by complement, phagocytosis or autolysis.
2) Less potent than exotoxin because it doesn't act enzymatically. It is also less specific.
3) If endotoxin reaches the blood it can cause shock or even death.
4) At low concentration: fever, vasodilation, inflammation.
5) Toxicity is related to lipidcontent (lipidA) - 53How does exotoxins (LPS) cause endotoxic shock?Endotoxins (LPS) binds to LBP (lipopolysacharide binding protein) in the human serum which proceeds to bind to CD14 on the macrophage cell membrane, and thereafter acting on TLR4: activating a pro-inflammatory cascade (cytokines)--> endotoxic shock
- 54The general characteristics of exotoxinsi) More potent and specific than endotoxins
ii) Typically the genes encoding exotoxins are located on the same plasmid as the genes encoding secretion systems
iii) Exotoxins:
- SA: Superantigen
-E: Enzymatic virulence
- AB: A-B toxin *very bad
-CT: Cytolytic toxin
- Act outside the animal cell
iv) Exotoxins are secreted by both gram (-) and gram (+) bacteria - 55Give example of some exotoxins and their function1) Hyaluronidase: Acts on hyaluronic acid in tissues and facilitates bacterial spreading (ex. streptococci)
2) Streptokinase/staphylokinase: Cause blood clotting in patients, aids in bacterial spreading (ex. streptococci) - 56What type of exotoxins are there?Type 1: cell-surface active: superantigen
Type 2: membrane damaging: phospholipases, ex. hemolysin
Type 3: intracellular toxin: AB-toxin - 57How do superantigens work?Superantigens:
Bind to TCR and MHCII complex--> makes the adaptive immune system unspecific- and overloading the system with many unspecific T-cell responses which overwhelmes the system and results in fever, coma and death. - 58How do heat-stable-toxins act?Heat-stable-proteins are secreted and targets for instance cGMP and can act in temperatures as high as 100 celsius degrees.
- 59Type II toxins can be divided into two large families, which are these?I. Channel forming toxins: can be divided to two families: (i) cholesterol-dependent cytolysins and (ii) RTX toxin cytolysins: alpha-hemolysing--> cell lysis due to osmotic pressure due to disruption of ion gradient
2. Enzymatic active toxins: alpha-toxin--> lysis - 60Describe how Penton Valentine Leukocidin worksPentone Valentine Leukocidin (PVL)
- beta-channel pore-forming toxin
- increases virulence in certain strains of s.aureus
-tissue necrosis
- Assembles two components on necrosis cell forming a pore--> lysis (high concentration)- can also mediate lysis by acting on mitochondria at low concentration: the lysis of neutrophil cell releases ROS which are believed to cause tissue damage - 61—The AB toxin is a intracellular toxin. The B-paprt binds to a specific cell surface receptor and the A part is translocated. Affect: ribosome, G-protein function etc.
- 62Give an example of an AB-toxinAn AB toxin: cholera toxin--> A-subunit activates AC--> upregulates cAMP-concentration--> higher concentration of calcium--> opening of calcium-dependent chloride channels which results in efflux of chloride ions: and secretion of potassium and other ions in the intestinal lumen--> diarrhea
- 63Summerize the virulence factors of staphylococcus aureus(1) Surface proteins: protein A & Clumping factor
(2) Extracellular enzymes: coagulase, lipidase, DNase etc.
(3) Toxins: SSSS, TSST, enterotoxin, heamolysin - 64Medically important staphylococcusi) Staphylococcus epidermis: found on skin- typically not pathogenic but can be pathogenic: problems with medical implants, bacteremia, UTI.
ii) Coagulase negative staphylococcus: oppertunistic infection
iii) Staphylococcus saprophyticus: Cause of UTI in young females - 65What are some general features of gene transfer in bacteria?- Unidirectional from donor to recipient
- Can be between species
- Do not transfer an entire chromosome - 66What are the four different ways of HGT?1) Transformation: Take up DNA from the environment
2) Transduction: Virus as vector
3) Conjugation: bacterial sex involving pili
4) Transposition: transposable elements - 67TransformationUnidirectional transfer of extracellular DNA into bacteria- could be plasmid or chromosomal DNA--> requires recombination.
Factors affecting transformation:
- compotence of recipient--> E.coli is not naturally competent, in a lab have to apply methods for E.coli to perform transformation-electric shock for instance
- DNA size and state: sensitivity to nucleases - 68What are some characteristics of Pathogenecity Islands?Pathogenicity Islands:
1. Different GC content and codon usage
2. Flanked by direct repeats
3. Often has a tRNA gene
4. Commonly encode virulence factors
5. 10-200 kb long
6. Commonly present in pathogenic organisms and not in commensal
7. Located on chromosome-plasmid-bacteriophage - 69How common is the different HGT methods?Transformation: very general
Conjugation: Promiscuous
Transduction: often strain-specific - 70How is the foreign DNA: 1) Stabilized 2) Expressed1) i. Homologous recombination
ii. Site specific recombination
iiii. Plasmid self-replicating
2. i. Recognise the promotor by hosy polymerase
ii. Silence of host DNA by DNA binding protein
iii. Positive and negative regulation - 71Give example of a medical important HGT and explain further the underlying detailsA medical important HGT is transformation of resistance genes to penicilin in Neiserria and streptococcus:
1. Resistance gene is neisseria flavence penA gene--> encodes for PBP2, allowing binding to this protein instead of PBP which is the target of penicilin and disturbes the building of the peptidoglycan wall of bacteria - 72Give an example of a pathogen of medical importance- which has aquired its main pathogenecity through transduction.An example is, Vibrio Cholera encodes two important virulence factors: pili and cholera toxin.
Phage VPI and CTX
VPI phage--> pilus
CTX--> Cholera
How do cholera acts?
* ADP-ribose --> arginine residue of ADP - 73What are some characteristics of biofilms?- Microbial community + extra-polymeric molecules: same species or different species adhered to either a surface, or air-liquid interface or tissue
- Formation: Planktonic--> attachment--> microcolony--> macrocolony
- Very resistant to antibiotics
- Very resistant to phagocytosis
- Helps HGT
- Promote secretion of cytokines resulting in inflammation and tissue destruction - 74What is dental plaque?Dental plaque is a thin organic film of glycoproteins--> streptococcus bacteria can easily adhere to these and cause multi-species barrier
- 75How are kidney stones caused by pathogens?Kidney stones:
- Staphylococcus, klebsiella etc. create biofilm which concentrate the urease--> when it is converted to ammonium--> crystals - 76CF patients: the biofilm formed by Pseudomonas aeruginosa, describe it- Alginate: confer antibiotic resistance and resistance towards phagocytosis
- PSL: mannose-rich and galactose rich: crucial to hold the biofilm together
-PAL: glucose rich polysacharide: crucial for cell-cell interaction - 77Why are biofilms so tolerant to antibiotics?i) Limited bacterial diffusion
ii) Lack of growth
iii) Induction of tolerance mechanisms - 78How is antimicrobial treatment of biofilms adressed?I) Antimicrobial treatments that targets the production of the bacterial cell wall- targets more metabolically active cells found in the top layer but not the bottom layer cells which are not as metabolically active.
II) Antimicrobial treatment which targets the structure of the bacterial membrane- such as colistin- targets the lower level (deeper level) bacterial cells of the biofilm but not the top layer metabolically active cells.
Solution: Combination antimicrobial therapy- however, even this is not always succesful - 79What are some negative and positive outcomes of biofilms?negative:
- Medical implants: prostethic valves (s.epidermis, pneumonas auerguginosa etc.), urethra cathether: e.coli, s.epidermis etc.
-Piping system: biofilm formation- corrosion--> impossible to remove
- Positive outcomes:
(i) can bind to contaminated soil-particicles and relieve the contamination from the soil: help degrade soil-bound contaminations
(ii) Important in agriculture - 80Let's say you have an unpathogenic and a pathogenic strain of bacteria and the difference between these are 800 genes, how would you set out to figure out which genes are responsible for the pathogenecity of the pathogenic strain?1. Bioinformatic approach:
- sequencing: compare the evolutionary relationship
- Compare pathogenic and non-pathogenic bacteria of closely related strains
- Check for HGT: different GC content and codon usage
- Functional protein families
2. Brute force screening
- Knockouts: (i) Transposones: random and genome wide (ii) lambda-red-recombineering: specific, requires that you know which gene you want to knockout
- In vitro first followed by in vivo
3. Screenings for genes expressed in vivo
- IVET
-DFI
4. Screenings for genes required in vivo
- Signature Tagged Mutagenesis (STM) - 81What is the biologicaly active iron?Ferric iron: Fe3+
- 82Where can you find ferric iron?You can find ferric iron in:
- cytochrome
-iron-sulphur clusters
-ferritin
-oxyhemoglobin - 83What do bacteria need iron for?Bacteria need iron for:
(1) Respiration & ATP- production: requires ferric iron in the center of the cytochrome
(2) Fe-S clusters are central in redox reactions- a for nitrogenases for instance
(3) Iron is a co-factor for many metalloproteins
(4) Redox reactions - 84Explain the concept of nutritional immunity and how it worksNutritional immunity is basically when the vertebrate host restricts extracellular iron levels by a number of mechanisms, such as:
- when erythrocytes lysis- hemoglobulin is bound by haptoglobulin and hemopexin
- when erythrocytes lysis- free iron is bound by transferin (lactoferrin in milk) - 85Where in the body can you find iron?In total we have 4-5 gram of iron in our body.
* 2.5 gram: Hemoglobulin (RBCs)
* 2.0 gram: Ferritin complexes- bone marrow, spleen, kidneys: 400 mg in pregnant women or menstruating
*400 mg in myoglobulin
*3-4 in transferrin - 86Describe the Fenton reaction
- 87What is hemochromatosis and thalamassia? What does it result in, in animal models?Thalamassia: loss of iron from RBC
Hemochromatosis: absorb excessive amounts of iron in diet
--> Increased virulence - 88How much free iron do we have in the body?We have 10^.18 --> 10^-9 M depending on site
- 89How does bacteria aquire iron in the host body?1) Fur: ferric-uptake-regulator: to switch ON genes for iron uptake during low-iron conditions
2) Low iron levels is a signal for bacteria that it is in a host- will secrete toxins such as haemolysins--> lyse RBC--> heme and iron
3) Another common target is lactoferrin and transferin!
4) Inflammatory response and cytokines- can also result in an increase in extracellular iron - 90Explain siderophoresSiderophores are molecules with high affinity for Fe3+ secreted from the bacteria- which is aimed to bind to Fe3+ and therafter return to the bacterial cell and bind to the cell surface- translocated across the cell membrane with the help of ABC transporter (driven by hydrolysis of ATP)
- 91If a bacteria does not make a siderophore, how can it get iron?Can make cell surface receptors which bind to other bacterias siderophore- stealing!
- 92Summerize the different strategies which bacteria can use for uptake of iron from host1. Siderophore: secreted molecules from bacteria with high affinity for Fe3+--> bind to bacterial cell surface receptor and is translocated across the cell membrane via ABC-transporter (hydrolysis of ATP)
2. Direct binding: direct binding to transferrin and lactoferrin
3. Hemophore: can remove heme from hemopexin and hemoglobin
4. Stealth siderophore: The innate immune system has a defense against siderophore- siderocalin, however the stealth siderophore prevent siderocalin binding - 93How is bacterial iron acquisition regulated?- 19 bp palindromic consensus sequence
-Fur is a repressor: bind at high conc. iron
- 94Describe the general secretion systemThe general secretion system- translocation of unfolded protein with N-terminal, the ribosome marks the peptides that shall be translocated- the peptide can be moved to secA by the chaperones secB or not, and then is translocated across.
Examples: exotoxins - 95How does type 1 secretion system work?Type 1 secretion system is also called the general secretion system or sacA secretion system: proteins synthesised in the periplasm- transported to ABC-translocon via chaperon secB: ATP hydrolysis powers translocation of protein across.
ABC: ATP binding casette protein- utilizes ATP hydrolysis to drive translocation of substrate across the membrane
2. MFP: Membrane Fusion Protein
3. OMP: Outer membrane protein - 96What are drug efflux protein similar to?Similar to transport 1 secretion system- but uses often PMF
Present in both gram (+) and gram (-) bacteria - 97Type III secretion system characteristics- "injectisome"
- goes through the entire cell envelope and to the host cell- can therefor directly inject virulence factors into the host cell
-OBS: not all type III secretion system directly inject into host cell
- similar to flagella (homologous-common ancestry)
- 98Type IV secretion system1. Conjugation
2. Transformation
3. Effector molecules- virulence
- 99—Legionella Pneumophila- can use T4SS to inject effector molecules into the host macrophage which transforms the phagosomal compartment to a compartment that resembles more like the ER- making the macrophage to lose its lytic abilities and therefor the legionella pneumophila can live intracellularly-replicate within the macrophage.
- 100Secretion system 6 (T6SS) - Give examples of organisms that uses this - what does it have homology to?- Vibrio cholera
-Pseudomonas aeruginosa
- phage injection system - 101What can type IV pili be used for by bacteria?Type IV pili can be used for:
1. Twitching motility
2. DNA binding and uptake- related to conjugative pili
3. Adherence- signaling for formation of microvilli which is important for internalization of bacteria into host cell
4. Electrical signals- nanowires - 102Discuss two reasons why bacteria might produce toxins?i) Destroy tissue cells to get access to other tissues
ii) to release nutrients such as toxins - 103Describe in as much details as you can the structure of the Cholera toxin and its functionThe cholera toxin- secreted by for instance vibrio cholera (transduction) is an AB-toxin, with A1- and B5-subunit.
At first the AB5 subunit is endocytosed- enters the golgi and then the ER where the A-subunit is cleaved giving free A1-subunit. The A1 subunit then activates AC--> higher concentration of cAMP which results in higher calcium concentration and opening of "cystic fibrosis transmembrane receptor" a chloride ion transmembrane receptor which results in efflux of potassium, HCO-3 and chloride ions resulting in severe diarrhea - 104Name one important factor you should consider when choosing an animal model?The animal model should be representable for the specific question, human infection.
- 105What is the key to models for the evolution of virulence?i) Higher virulence in HGT in non-relatives
ii) Lower virulence in vertical transmission
WHY?
Three hypothesis:
(1) Co-incidental evolution hypothesis
(2) Short-sighted evolution hypothesis
(3) Trade-off evolution hypothesis
*Can also be that the pathogen involves within the host! - 106Explain what co-incidental evolution is and give an exampleCo-incidental evolution is when a bacteria have a trait or secrete a molecule which can be considered a virulence factor- but did not develop this for infecting the host. Accidental evolution. An example is Clostridium tetani- which secretes a neurotoxin in humans, however does not transmit from human hosts and has its natural environment in soil (soil bacteria)
- 107What is short-sighted evolution?Short-sighted evolution is when the bacteria or virus replicate on the behalf of t he host health to the extant where it serves as an actual disadvantage to the pathogen in the long run- such as by killing the host far too soon and thereby limiting the chance of transmission, a good methaphore is humans on planet earth.
Example: HIV- which can target CTXR4 co-receptors--> no more transmission, and also the final blow to the host immune system: killing the host - 108Trade-off hypothesisThe trade-off hypothesis: there is an optimal balance between transmission and pathogenecity,
cannot be too virulent then the host dies before transmission can occur etc.
Example: HIV-1 is more virulent and makes more virus particles--> more likely to be transmitted than HIV-2
- If transmission and virulence is positively correlated- it means that some level of virulence should be detected
- Natural selection: favour transmission - 109—Myxoma virus- an example of trade-off evolution
- At first killed of 99 percent: rapidly dropped
- selection for less virulent strains
- positive coupling between transmission- and virulence
- 110How can you test for trade off experimentally?- E.coli infected with phage 1
- Infection is not lethal but slows the growth of the E.coli with 30 percent
- Two main strains:
1. Vertical transmission
2. Horizontal transmission
Another dimension:
1. Vertical transmission + 1 day of HGT
2. Vertical transmission + 8 days of HGT
The experiment ran for a total of 24 days.
Then the success was evaluated by counting the number of offspring
Results:
Trade-off
1. HGT--> higher virulence
2. VT--> lower virulence
Further confirmed by the secondary dimension - 111The Basic reproduction number, R0: what does it mean?How many secondary infected cases there are generated by the primary case in a suceptible population
- Used in epidemiology- to asses the behavior of a spread of disease
R0 > 1: Infection will spread
R0<1: Infection will most likely die - 112What does the reproduction number depen on?- Susceptibility in population: transmibility
- Behavior of the infected individual: rate of contact per time unit
- incubation period
- - 113Harming the host can reduce the amount of time that a pathogen infects a host so why wouldn't low virulence evolve in all situations?The answer might be that the bacteria need to produce these certain proteins or other molecules for their own survival and for competition purposes- but also for transmission purposes: positive correlation between virulence and transmission, such as producing a symptom like coughing for spreading
- 114What does the Trade-off hypothesis suggest?The trade off hypothesis suggest that evolution is driving pathogens to evolve virulence to the degree where it gives optimal transmission rate
- 115What are some complications for the Trade-off Hypothesis?1) Within host evolution: Mostly highly virulent strains may realize the benefit of higher transmission
2) When a pathogen is introduced to a new host: may be more virulent and develop lower virulence for appropriate transmission--> an example would be ebola - 116Give example of two infections that give different symptoms in mice compared to humans- what these symptoms are1) Salmonella typhi
- typhi fever in humans
- nothin in mice
2) Salmonella typhimurium
- causes diarrhea in humans
- typhoid-like fever in mice - 117Practical considerations using animal models1) PK: Adsorption, distribution, metabolism, excretion- dose should be relative to the dose expected in human use
2) Question which is answered and requires an animal model
3) Realize the limitations of the animal model
4) Animals requires: should not include excessive number of animals- but enough for the results to be significant
5) Control: Positive & Negative control
Positive: reference compound
Negative: vechile compound (ex. water)
6) Variability in result will occur- try to control the environment as much as possible! BY:
- homogeneity of animals
-analytical methods
7) Questions must be clearly formulated and sources of bias need to be eliminated and plan statistical analysis prior the experiment
8) The infection route (inoculation)- what these have for significance for the questions asked- any potential bottleneck?
9) Anesthesia for animal- duration, post-effects, Long-term and short-term effects, should not be too long or too short etc. - 118What are some factors influencing antimicrobial activity in vivo?- Inoculation number
- Immune status (immune depression?)
- Stress
- Starvation
- Growth in vivo vs. generation time in vivo
- Development of resistance in vivo
-inactivation of compound in vivo
-PK--> Effect of drug on animal
- PD--> Effect of drug on bacteria - 119Why use mice in particular?- Low costs and easy to store
- Easy to manipulate them genetically
-surprisingly closely genetically to humans
- Gnotobiotic mice and also mice infected only with one pathogen
-Immunosuppressive mice
-Transgenic mice - 120What is a competition assay?Assert the fitness of different strains, for instance WT vs mutant and thereafter count success and make conclusions. The result is noted as Competition Index (CI)
- 121Why is adhesion important?Adhesion is important for:
- initial attachment to host
- invasion of host cells
- To resistant wash away by mucosa secretions
- Mediates signaling events
-Promotes anti-inflammatory events - 122Adhesion to what?Adhesion to:
- epithelial cells surface
- endothelial layer
-mucosa
-extracellular matrix
-blood - 123Which bacterial adherence structures are not very specific?1. Capsules
2. LPS
3. flagella
4. cellulose - 124What are the different adhesin families for gram (-) and gram (+) ?Gram (-): Type 1 pilus, type IV pilus, fimbriae, Yada TAA, Curli
Gram (+): Secretase-assembled pilus - 125Describe how type I pilus is assembledType 1 pilus is assembled by multiple proteins and components.
The rod of the adhesin is fimA, and the tip fimbriae is fimH- which is the part which contacts the host cell or other surfaces.
- 126What is the role of type 4 pilus and where can it be found?Type IV pilus play a role in:
- structural role in biofilm and motility
- DNA binding and uptake
- adhesion to host cell
- electrical communication (nanowires)
- long and thin: great tensile strength
Found in all gram (-) bacteria and some gram (+) such as streptococcus and clostrdium
- 127Describe the interactions between Neisseria Gonorrhoea and the host cell surfaceThe initial interaction of Neisseria Gonorrhoea is mediated by type IV pilus- the E-subunit.
Thereafter stronger attachment is mediated by Opa proteins- "opacity" which refers to the characteristic colour which the protein gives.
N. Gonorrhoea is so successful because of phase variation and antigenic variation (in particularly in the pili which make the initial attachment).
How does phase variation and antigenic variation occur in the pili?
Antigenic variation: PilS + PilE
Phase variation: PilC can be ON/OFF depending on frameshift mutation in poly-G tract near the end of the piliC gene - 128Describe the role of curli and its characteristicsCurli is involved in:
- Attachment to host cell
- Pro-inflammatory responses
-Biofilm formation
-bacterial aggregation
-Adhesion to surfaces
Very sticky- rather non-specific- and thin aggregative fimbriae
M.tuberculosis has similar curli - 129TAA- Adhesins - What does TAA stand for? - characteristics -role?TAA= Trimeric Autotransporter Adhesins
- Three major proteins- three independent binding sites
- Involved in adhesion to host cell and bacterial interactions
- signaling events
- 130What can bacteria use lipid rafts for?Bacteria can hijack lipid rafts- avoid meeting the intracellular degradative pathway. Can also make a vacuole which express raft-proteins on the surface and in that way avoid the immune system
- 131What are some clinically important staphylococci?- Staphylococci Aureus: major pathogen
- Staphylococci Epidermis: Oppertunistic
- Staphylocci Saprophyticus: UTI - 132What are some features of staphylocci?- Gram (+)
- Grape-like clusters
- No flagella
-Salt tolerant
- carried in nasopharynx and on skin
-No spores
-Facultative anaerobe - 133How can you distinguish between S.aureus and S.epidermis on an agarplate?S.aureus is hemolytic- fade area around it compared to S.epidermis
- 134What are some infections caused by S.aureus? (8)- osteoarticular infections
-bacteremia: blood infection
- medical devices
- infective endocarditis
- skin- and soft tissue infections
-pleumonary infections
- SSS
- TSS - 135What kind of class does TSS in S.aureus belong to?Superantigens!
Binds non-specifically to T-cells and MHC-II cells - 136What virulence factors does S.aureus have?1) Surface proteins:
surface protein A
clumping factor: hide from phagocytes
2) Extracellular enzymes: Catalase, coagulase, hyalyronidase (attack hyaluronase acid), lipase, fibrolysin,DNase
3) Toxins: TSST, SSSS toxin, Haemolysin, Leucosidins - 137What is another clinically interesting staphylococcus?Coagulase-negative staphyloccous
- frquently involved in nosocomial and oppertunistic infections - 138Which E.coli pathotype/strain can cause UTI?ExPEC
- 139Give an example of a virulence factor of pathogenic E.coliLT-toxin: Heat labile enterotoxin
Mechanism:
The A-subunit (A-B6) has ADP-ribosylation activity which covalently modifies the alpha-subunit of Gs resulting in activation of AC and an increase in cAMP resulting in an increase in calcium concentration and therefor opening of cystic fibrosis transmembrane receptor resulting in efflux of chloride ions and other ions resulting in diarrhea
ST-toxin: bind cGMP--> cAMP upregulation--> similar to LT toxin effect - 140Why are Fe-S clusters important in bacteria and overall in various organisms?I) Electron transfer
II) Redox catalyst
III) Non-redox catalyst
IV) Gene regulation: good to use for detecting iron levels in environment (bacteria) - 141What is the API test for?To identify which bacteria it is based on carbon source
- 142How can you differentiate between the different medically important staphyloccous?Staphylococcus aureus: coagulase
staphylococcus epidermis: sensitive to novobiocin
staphylococcus ssaprophyticus: resistant to novobiocin - 143What are the limitations of microarray analysis?- Random knock-outs or altered function: depending on the specificity of the transposon
- Does not tell the function of the gene
- Spatial limititation: gene could be switch on or off momentarily or on a later stage - 144What is special about the transposon used in the study?The transposon: Tn10 with IPTG promotor- adjacent to genomic region allow for transcription into adjacent genomic region- uniquely identify position (T7 promotors). Tn10 targets 8 nucleotides- important to consider
- 145What is the significance of the infection route used?The natural oral route was found to create a biological/physiological bottleneck where less than 1% of the original inoculum breached the intestinal barrier and colonize systemic tissues. Bottlenecks can lead to the stochastic reduction of the mutant library, and the IP route has been shown to have minimal impact on reproducibility of libraries. To address the possibility of a IP bottleneck, the authors compared the two passages of infection with SAM statistical analysis of microarrays.
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