week 3 Flashcards
1
Q
Hierarchy of regulatory mechanisms
A
DNA structure –presence, amplification, modification by porA/pili
Transcriptional regulation – activation,repression by sigma factor/fur
Post transcriptional regulation – translation repression, transcript stability by sRNA, riboswitch, RNAT
2
Q
Gene
A
promoter, ORF, terminator
3
Q
Operon
A
two or more genes connected all activated by a common promoter
4
Q
Regulon
A
couple of genes alle regulated by the same regulator
5
Q
Stimulon
A
genes activated by the same stimulus
6
Q
Finetuning expression of genes why
A
Essential to adapt to growh conditions
Express virulence for pathogenesis and survival in host
7
Q
function promotor, sigma, shine delgarno
A
Promotor is recognized by RNA polymerase and sigma factor, which recognizes -30 and -10
shine dalgarno in transcriptional region but not translational region AG rich is recognized by ribosome, recognizes AGG
8
Q
DNA sequence alterations;
A
Slipped strand mispairing – DNA polymerase slips forward of back, leads illegitimate base pairing of repetitive DNA during replication and to insertion or deletion. Way to alter gene expression but very random. Caused by triplets or stretches of the same nucleotides
9
Q
porA gene of n meningitidis
A
spacer becomes shorter, expression level of protein becomes less, because less binding strength RNA polymerase
10
Q
transcriptional regulation
growth conditions
A
nutrients, oxygen, iron, glucose, pH
11
Q
iron why needed in bacteria
A
needed to support growth, stimulus to produce new proteins used for survival (virulence factors). Could be potential vaccine candidates
12
Q
identify iron regulated proteins in bacteria
A
RNAseq. Grow bacteria with and without iron, isolate cDNA, how many transcripts are produced can be measured. mRNA, RNA fragments, DNA framgents, map against sequence, reading map
13
Q
transcriptional regulation
sigma factor
A
in contact with -10 and -35, equipped with RNA polymerase. Some sigma can bind specific sequences. Sigma 70 for genes that are always on and are general, sigma 32 for stress, sigmaE whe heatshocked, sigma 54 when nitrogen is present
14
Q
mode of action of sigma factor
A
recognize sequence near promotor region, mode of action on repressor or activator protein
15
Q
mode of action of repressor protein
A
steric hindrance (spacer region blocked for RNAP), blocking elongation, DNA looping
16
Q
mode of action of activator protein
A
class I activation, class II activation, conformation change
17
Q
sigma factor;
A
specific promotor sequence recognition
18
Q
fur;
A
specific transcriptional regulator stimulated by Fe
A ferritine uptake regulator. Fur box is partly overlapping with promotor. Condition that there is a lot of iron present, fur will bind to fur box. No transcription is needed. Low iron – no binding fur to furbox, no transcription.
19
Q
iron responsive regulon –
A
lactoferrin/transferrin binding protein
pili
* kapsel
* iron-acquisition proteins
* proteins complexed with iron (enzymes)
20
Q
sRNA –
A
encoded in intergenic region or anti sense strand
regulate stability and translation mRNA, multiple mRNA targets
antisense to the 5UTR of target mRNA that they regulate
environmental stress induces – induces stress response
some contain fur box
21
Q
mechanisms of riboregulation
A
translational repression – binding RBS
translation activation – remove pin like structure
mRNA degradation
mRNA stability
22
Q
four regulatory mechanisms per sRNA
A
- Coupled degradation: both sRNA and mRNA degraded
- Sequestration: both sRNA and mRNA stabilization
- Catalytic degradation: only mRNA degraded
- Activation: with Qrr degradation
23
Q
sRNA interaction with target
A
sRNA contains 4 stemloops that can disappear when bound to mRNA. When stemloop is intact, protein production is intact.
24
Q
No loop after binding sRNA
A
RNAse can bind and degrade mRNA and sRNA
25
luxMN
degraded due to loss stemloop 1
26
luxO
not degraded due to stemloop 1 still being present
27
riboswitches –
elements in 5UTR of mRNA
operates by changing structure in response to binding metabolite
translation is on when RBS is fee, but when RBS binds to anti RBS then translation is blocked. Different ligands to block translation
28
RNATs
RNA thermometers
Also conformational change but due to temperature
RBS is in stemloop, when temperature raises RBS is free, ribosome can bind
29
Q fever symptoms
* 60% asymptomatic
* 20% fever like: headache, fever, nausea, muscle pain
* 20% serious; fever, chest pain, severe head ache, diarrhoea, vomiting, atypical pneumonia (not reacting to normal AB), hepatitis, pericarditis, meningo encephalitis
* 1-3% chronic (could be in any group); endocarditis and intravasculair infections. Increased risk in pregnant, vascular disease, intravascular patches. Leads to abortion, premature birth
30
Q fever cause
Coxiella burnetii
* Spores survive up to 40 months
* In rodents, birds, farming animals, cats, dogs, ticks. Mainly sheep, goats, cattle
* Found in dung, urine, milk etc, spread through dried excreta, air borne
* Very rarely contaminated food or ticks
Treatment; acute doxy 200mg 1dd 14d, chronic; combination therapy 18m
31
Diagnosis q fever
* First 2 weeks pcr on dna Coxiella
* Week 3 and later Ab detection phase 2 IgM IgG
* Chromic; IgG phase 1
How was origin of q fever found – abortus storm by goats
32
Q fever policy of blood transfusion
* Decided that it was not a threat
* However – PCR on hottest samples, serology on half of group (follow up after second transfusion). ELISA serological plot has shown that 1/10 had antibodies in affected area
* Could be transfused, however blood was needed so PCR tests were implemented
33
Why weren’t farms shut down earlier after thousands of notifications
because of the financial impact of shutting down farms.
34
Virulence –
* competition
* invasion
* Sensing
* Attachment
* Overturning host defenses
* Interference with host processes
35
Secretion system
membrane complex
Secretion systems in gram negative cell envelope
36
class 1-6 protein complexes
based upon order of discovery. Macromolecular structures present on surface; in part adaptation from pili, flagella, conjugation system
* One step – crosses both membranes, with help of chaperones and ATP
* Two step (2 and 5) – first inner membrane via sec/tat, periplasmic chaperones, no energy source at outer membrane
37
Crystallography
refraction of beam line when x ray shines on cristal
* Purified protein in large amount
* Prepare using precipitation (trial and error)
* Obtain diffraction pattern using x ray
* Determine structure from pattern
38
Crystallography not suitable for membrane proteins why
* hydrophobic surface
* aggregation rather than crystallization
* difficult to purify in large amounts out of membrane
* need to solubilize them using detergents
39
electron microscopy
electron beams reflected by electron dense material
* biological samples need staining with heavy metals
* cyro EM – freezing at low temp. prevents radiation damage, no staining needed
* suitable for bacterial cells, sliced samples, purified complexes with lower concentrations, less pure, image averaging improves resolution and samples can be tilted which gives 3d tomography
40
type III system
‘injection needle’ makes hole in eukaryotic cell, from cytosol bacteria to cytosol eukaryotic cell
* example; yersinia outer proteins (YOPS) secreted via type 3 modulate host cell processes
* intimate attachment and anti-apoptotic and anti-inflammatory signals
41
cryo-EM tomography of type III complex
* prepare vitrified cells
* perform tomography
* image with EM
* do averaging of identified complexed
* no purification
42
secretion system VI
* two homologues of T4SS
* IM and ATP binding
* Other genes in cluster different from
43
T4SS
* Other bacteria carried similar genomic islands
* Kill other bacteria
* hcpI part between rings, very similar
44
gpVN for phage tail
* VrgG is also ring like with shaft like structure underneath
* In conclusion – functions like bacteriophage
45
Model of type VI secretion
* Build up
* Signalling
* Contraction/ejection
* Degradation
46
What gets secreted using VI
* Peptidoglycan degradation
* Nucleases
* Insertion into membrane – pore formation
47
Where can VI excretion be found
in opportunistic bacteria when microflora is present
48
What triggers VI
* Defense system – 2 component regulatory system works as kinase system
* Attack system – cell to cell contact. OmpA in outer membrane. Also periplasmic relay proteins such as TsIA, triggers assembly baseplate
49
Autotransporters
* secretion system consisting of one protein and three domains
* Two step mechanism uses sec in inner membrane, to outer membrane protein
* Folding outside is driving force of pulling protein out
50
Hemoglobin protease
plasmid encoded virulence vector encoded by E coli. Degrades hemoglobin, peritonitis
51
AFM
used to unfold and refold protein such as Hbp by lowering and lifting tip of machine.
* Release beta strand releases energy
* C terminal part is most stable part, most likely to emerge first from membrane
52
Application of autotransporters
* Vesicles (not whole bacteria) with non cleaved Hbp
* However, more complex – less proteins produced
* Hbp-spy system allows attaching proteins to outer membrane
* Hbp secretion as target for novel antibiotics
53
BamA
inhibition as target of drug inhibitors, since BamA is necessary for bacteria to survive
54
Bcg vaccine
live attenuated strain, still used today but only partly effective (only in children)
55
Treatment tuberculosis
antibiotic combination therapy
56
Prevalence tuberculosis
Sub saharan africa, south asia
57
Problem tuberculosis
* Current vaccine does not protect enough
* HIV and tuberculosis is deathly
* Antibiotic resistance
* Highly unusual and slow growing bacterium -20-24h doubling time
* No optimal infection model
* Envelope complicates analyses
58
Infection cycle tuberculosis
* Bacteria enters lungs, infects alveolar macrophages which form granulomas in response to immune response
* Bacteria inside are still alive, T cells surrounding. No symptoms, can be stable for decades and is called latent TB
* Reactivation and caseation of center; replicate and spread leads to active TB
59
Disseminated TB
* Lungs first
* Meninges
* Intestines
* Milk duct
* Bones
60
Tissue damage and death due to m tuberculosis
* Not toxins, which give little direct cell damage
* Due to granuloma and weight loss due to pro inflammatory cytokines
61
Micobacteria
different types, usually slow growth rate
Fast growth rate = 1-2 hours
62
Mycobacterial cell envelope
more than 60% lipids, not permeable leads to virulence. Also gram variable
63
Gram staining characteristics
* Gram positive had One membrane and thick peptidoglycan
* Gram negative has two membranes and thin peptidoglycan layer
* Mycobacteria – mycobacterial outer membrane, no LPS, contain arabinogalactan and peptidoglycan. So it has two membranes but still stains a bit sometimes
64
TB survival inside macrophage
phagosome doesn’t fuse with lysosome, also can escape phagosome
65
Escape of TB visualization
* Galectin 3 in macrophage labeled with GFP. Once there is damage it binds to damaged membrane
66
Esx5 in TB
is essential for growth. When outer layer of membrane is mutated, this complex is not essential anymore. Only seen in slow growing mycobacteria. Required for uptake of fatty acids
mspA found in fast growing, essential for growth. Alleviates essentiality of esx5
67
how do ATPase channels open up for transport, and how are they transported through the outer membrane?
No answer yet
68
HGT and population structure
* Population with diversity reduction – mutations that bring burden decrease in diversity. Focused towards clones that adapted best
* With horizontal transfer – adaptations to possible problems to come
* Clone dominance
69
Genome
all chromosomes and plasmids. Bacterial often circular, can be linear. Plasmids are smaller DNA elements that are circular. Megaplasmids and small chromosomose hard to distinguish. No plasmid transfer then its probably chromosome.
70
Mechanisms of HGT
* Bacterial transformation – take up DNA
* Bacterial transduction - bacteriophage
* Bacterial conjugation – bridge two bacteria
71
Transposons
* Jumping gene
* Replicating and inserting somewhere else
* Requirements – gene encoding transposase, flanked by inverted repeat sequences
72
Composite transposon
additional gene between flanking inverted repeats or insertion sequences
73
Insertion sequence
on host chromosome and plasmid, can align and cross and therefore plasmid can integrate into host genome
74
Restriction modification (RM) sequences
to neutralize incoming DNA. Incoming DNA gets digested by REase, but not when DNA is methylated, then it attacks own DNA.
75
Role of RM in recombination
before getting degraded can associate via chi sequence on DNA and integrate
* Mostly in opportunistic pathogens – if they collect all types of foreign DNA it leads to combinations that help survive. Less in obligate pathogens because they live mostly in one site
76
Pathogenicity/genomic island
* Contains genes associated with virulence, regulation and mobilituy
* Inserted in single site, often adjacent to tRNA gene
* Different GC content
* Some pathogenicity islands (PAIs) hijack a phage to spread; PICI, phage-inductible chromosomal islands
77
Applciations of bacterial genome plasticity
* Diagnostic marker
* Diagnostic antigen
* Marker for prognosis
* Vaccine candidates
* Outbreak investigation
78
MRSA test
mecA gene encoded, can be tested by PCR
79
s aureus kenmerken
* gram positive
* spherical shaped
* around 30% carries in their nose
* can infect all barrier tissues
80
health care burden s aureus
* recurrent infections, so no protective immunity
* rise in resistance
* s aureus in blood is lethal in 20%
81
s aureus vaccine development
* we don’t know the precise correlates of protection (signs that host is immune)
* insights from immune defects associated with s aureus infection (neutrophils and T cells)
82
neutrophils kenmerken
* most abundant in blood (40%)
* polymorphic nucleus
* short lived
* granules with antimicrobial peptides; hydrolases, peroxidases etc
* migration via chemotaxis
83
killing s aureus by neutrophils
* netosis
* phagocytosis
* stimulated by phagocytosis, T cells, antibodies, components
84
s aureus vaccine hurdles
* immune evasion
* translation
* strain variation
85
Immune evasion by s aureus problem for vaccine development
* Evasion of opsonization – proteins that counteract Ab binding to s aureus. Can inhibit complement by binding c3 convertase.
* Evasion of phagocytosis and killing – blocking recruitment, blocking phagocytosis, blocking NET formation, blocking formation proteases
* Targeting immune cells – toxins that form pores in immune cells. Superantigens activate more t cells (25% activation instead of 2% leading to exhaustion)
86
Translation pre clinical studies to humans problem for s aureus vaccine development
* LD50 (dose needed for 50% mortality) differs a lot per animal, unknown for human
* Not all species have same pore forming toxins
87
Merck vaccine
iron regulated surface derterminant protein B (IsdB), immunogenic antigen. Goal to produce antibodies. Seemed to work in animals, but higher mortality in humans. Pre existing immunity leads to failure of the vaccine.
88
Strain variation hurdle in s aureus problem for vaccine development
staphVAX vaccine
targets polysaccharides (CPS). Promising in mice. In humans ineffective
89
future vaccines against s aureus focus
* neutralize immune evasion (antibodies)
* models that better resemble human infections
* target conserved or essential bacterial antigens
90
vaccine strategies
microbiome function
* food digestion
* vitamin production
* colonizationresistance
* educates immune system
* but, can also contain potential pathogens
91
vaccine with toxin neutralization
prevents cytolysis of host target cells by production Ab to neutralize toxin
92
vaccine promoting recognition and killing
pore is formed leading to lysis by activating the complement system. Only works for gram negative. Phagocytosis and killing can be done in gram negative and positive by coating with antibody.
93
Capsular polysaccharides (CPS) as virulence
* Encapsulated bacteria have the ability or cause invasive disease
* Cps composition differs between species but also within species – diagnostic value
* CPS are important virulence factors by providing resistance phagocytosis and killing, resistance to serum killing
94
Serogroup
determined by polysaccharide composition. In meningitis A, B, C, W and Y most prevalent diseasae causing
95
Negative correlation antibodies and invasive disease in meningitis
* Low incidence in newborns due to high levels of antibodies
96
Plain polysaccharide vaccines
* Protection provided with several drawbacks
* Not immunogenic in young children because immune system is not developed enough
* No memory, repeated immunizations needed
* Limited class switching, only IgM, IgG
* Ineffective against carriage, only individual protectio
97
Protein conjugation vaccine
* Extract polysaccharide, chemically link it to protein
* Provides immunological memory with class switching and maturation - high IgG
* Effective in infants
* Herd immunity by reducing carriage and transmission
98
Limitations CPS vaccine
* Coverage is limited to subgroup of max 20 (while s pneumonae has 90 serotypes for example)
* Glycan antigen is not immunogenic
* Glycan structures are similar to human structures
* Non encapsulated strains not covered
99
GAS
* Pyogenes, group A
* Gram positive cocci
* Hemolytic
* Humans are only reservoir
* Strain diversity for epidemiological surveillance; variation in emm gene
100
GAS infection
* Common commensal of skin and nasopharynx
* Superficial infections, which can lead to invasive infections (flesh eating, sepsis) and post infectious/immune sequelae
101
Why is there no GAS vaccine
* Global diversity in desease causing strains, highly conserved vaccine target
* Autoreactivity of possible vaccine targets – antigens should not induce autoimmunity
* Capsule mimics host hyaluronan – autoimmunity
102
GAC modify vaccine
to keep the vaccine function but without autoimmunity
Making mutants to see if it changes the physiology or biosynthesis (reverse genetics)
Found gene deletion that creates mutant glycoprotein structure – not as virulent
No cross reactivity with heart antigen
103
FATAL FLAW SLIDE AANVULLEN
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