Progress Exam 3 (November 7 - 8 - 11) Flashcards
(116 cards)
1
Q
How many deaths by 2050 because of the rise of drug-resistant infections (to 70%)?
A
40 million deaths because of antimicrobial resistance (AMR)
2
Q
On what ten fronts can we tackle antimicrobial resistance (AMR)?
A
- Public awareness
- Antibiotics in agriculture and the environment
- Surveillance
- Human capital
- Global innovation fund
- Sanitation and hygiene
- Vaccines and alternatives
- Rapid diagnostics
- Drugs
- International coalition for action
3
Q
Tackling AMR on population level
A
National level (laboratory-based) surveillance:
* Incidence
* Outbreaks
* Vaccine effectiveness
4
Q
Tackling AMR on individual level
A
Vaccines in national immunization program
* Childhood vaccines
* Adult vaccines
5
Q
Two ways in which vaccines can exert their function (mechanisms).
A
- Toxin neutralization: binding of anti-toxins (antibodies) that catch the toxins.
- Promoting recognition and killing:
- Complement activation and lysis for Gram-negative bacteria
- Phagocytosis and killing for Gram-negative and Gram-positive bacteria.
6
Q
What are desired antigen properties (for vaccination)?
A
- Abundant
- Available
- Conserved
7
Q
Which of the desired antigen properties apply to capsular polysaccharides?
A
- Abundant
- Available
8
Q
What is the protective function of capsular polysaccharides (CPS)?
A
CPS offers protection from complement-mediated killing and phagocytosis.
They also use antigenic variation for immune escape.
9
Q
Four examples of encapsulated bacteria that cause invasive disease in humans
A
- S. pneumoniae
- Group B Streptococcus
- H. influenzae B
- N. meningitidis
Italics are in the National Immunization program.
10
Q
Characteristics - both clinical and bacterial - of N. meningitidis
A
- Gram-negative diplococcus
- Human-specific
- Natural habitat: human nasopharynx (5 - 10% of population colonized)
- ~0.01% of colonized hosts develop invasive disease
- Invasive meningococcal disease (IMD): sepsis + meningitis
11
Q
What are the most prevalent disease-causing meningococcal serogroups?
A
A, B, C, W and Y.
12
Q
What is a key observation of bactericidal antibodies and IMD cases?
A
Inverse correlation group-specific bactericidal antibodies and IMD cases.
13
Q
What kind of immune response does the plain polysaccharide vaccine (PPV) induce?
A
T cell-independent responses
14
Q
What are the restrictions of vaccines focused on the plain polysaccharide of N. meningitidis?
A
Insufficient for population-wide application:
* Not immunogenicity in young children.
* No immunological memory –> repeated immunizations required.
* Limited class switching (only IgM, IgG2) and afifnity maturation.
* Ineffective against carriage –> only individual protection.
15
Q
What kind of response does protein conjugation (PCV) elicit?
A
T cell-dependent responses
16
Q
What is the difference between conjugate (PCV) versus plain polysaccharide (PPV) vaccines?
A
- Immunological memory
- Class switching and affinity maturation –> high affinity IgG
- Effective in infants
- Herd immunity: by reducing carriage and transmission, protection is extended to non-vaccinated individuals.
17
Q
Protein conjugation is only focused on …
A
Only protects against one serogroup.
18
Q
What kind of vaccine is now used against N. meningitidis?
A
A multivalent conjugate vaccine targeting CPS ACWY-serogroups.
19
Q
What are the four limitations/challenges for CPS-based vaccines?
A
- Vaccine coverage is limited to subgroup of bacteria (there are more than 90 serotypes, but there’s currently maximum 24-valent vaccine)
- Glycan antigen is not immunogenic.
- Glycan structures are similar to human structures (mimicry). There’s a risk of inducing autoreactive antibodies.
- Disease cause by low or non-encapsulated species/strains (i.e. non-typeable strains)
20
Q
What does an infection with Strep A cause?
A
Wide range of disease manifestations. Localized (strep throat), invasive (toxic shock), post-infectious sequelae.
21
Q
Why is a Strep A vaccine feasible?
A
There’s natural exposure and infection with Strep A and that results in protective immunity.
22
Q
What is the challenge for Strep A vaccines?
A
- Unclear correlates of protection.
- Risk for induction of autoreactivity.
- Capsule mimics host hyaluronan and is therefore not immunogenic.
23
Q
What protein provides type-specific protective immunity against meningococcus?
A
M-protein
- Large antigenic variation with > 200 emm types identified.
- Large global diversity in emm types that cause disease.
- Molecular mimicry with host molecules, risk of autoimmune sequelae?
24
Q
What type of antigen would cover all Strep A emm types?
A
Conserved antigen
25
What structure is conserved across Strep A population?
Group A carbohydrates (GAC)
26
What is the immunogenic part of Group A carbohydrates (GAC)?
GlcNAc
27
What are the pros and cons of using GAC as vaccine antigen in the battle against Strep A infections?
Advantages
* Conserved epitope: universal protection
* Surface exposed
* Protective capacity as glycoconjugate vaccine in mouse models
Disadvantages
* Evidence that GAC GlcNAc side chain is associated with autoimmune sequelae.
28
What is important for full virulence of Strep A?
The GlcNAc side chain!
Glycosyltransferase Gacl is essential for GlcNAc side chain formation.
29
Is there an effect of antibodies against GAC in vitro and in vivo?
* In vitro: enhanced clearance
* In vivo: enhanced protection
30
What changes the quality of the immunological response resulting in immune memory, protection in young children and herd immunity?
Conjugation of glycans to proteins
31
Two examples of vertical gene transfer
1. Spontaneous mutation
2. Deleterious spontaneous mutation
32
What are the two possible ways of gene transfer?
* Horizontal gene transfer ('localized sex')
* Vertical gene transfer
33
What does the genome comprises of?
All chromosome + all plasmids
34
Bacterial chromosomes are often ..., but can be ...
Bacterial chromosomes are often **circular**, but can be **linear**. Combinatons occur.
35
How many plasmids does *Borrelia burgdorferi* has?
21 plasmids
36
What mechanisms do bacteria use for horizontal gene transfer?
* Bacterial transformation by releasing DNA (incl. for example an antibiotic resistance gene)
* Bacterial transduction by releasing phages.
* Bacterial conjugation by transposons that jump into plasmids and get transferred to recipient cells.
37
What are transposons?
Jumping genes with the minimal requirements ('IS elements' - inserting sequence):
* Gene encoding transposase (catalyzes transfer or copying of the element).
* Flanked by inverted-repeat sequences (target for transposase).
38
Function of transposases
Recognizing inverted repeats and excises the transposon.
39
What do composite transposons contain?
Additional genes between two inverted copies of IS elements ('mini-transposons').
40
Restriction-modification systems consist of:
* Restriction enzymes
* Modification methylase
When methylated restriction of that particular site is not possible anymore.
41
Loss of restriction modification gene complex results in
* Cell death
* Repair
* Genome rearrangements
42
Difference between incoming DNA from:
1. Closely related bacterium
2. Phage
1. Restriction of incoming DNA from a closely related bacterium (harboring similar Chi sequences) generates DNA fragments which can be utilized as substrates for homologous recombination by the RecBCD pathway.
2. In contrast, the fragments generated by the restriction of phage DNA (lacking the Chi sequence) are recognized as nonself and subjected to further degradation by the RecBCD pathway.
43
Causes of genome evolution in bacteria
* Deletion, insertion and recombination
* Horizontal gene transfer
* Core and flexible gene pool
44
Relation between genome, core genome and the accessory genome
The more genomes, the smaller the core genome and the larger the accessory genome.
45
What are pathogenicity/genomic islands?
* Large (~ 40 kb)
* Contain genes associated with virulence, regulation and mobility.
* Inserted in a single site, often adjacent to a tRNA gene.
* Has different GC content.
* At least some PAIs hijack a phage to spread; PICI (phage-inducible chromosomal islands); for example by inhibiting thte recognition of a certain protein that normally incorporates the phage cosB gene into the phage by TerS by changing it to Rpp-TerS that incorporates PICI cosB gene.
46
What are typical PAIs?
Distinct regions of DNA that are present in the genome of pathogenic bacteria but absent in nonpathogenic strains of the same or related species.
47
Core genome incorporates PAIs and then can form three different *E. coli* forms:
1. Enterohemorhagic *E. coli*
2. Enteropathogenic *E. coli*
3. Uropathogenic *E. coli*
48
What can induce for example vancomycine resistant *Enterococcus*?
The insertion of PAIs, phages, plasmids and Van resistance island into the strain genome.
49
What is *Streptococcus suis*?
The pneumococcus of the pig
50
What are the characteristics of *S. suis* and *S. pneumonia*?
* Healthy carriers
* Multiple serotypes (capsule polysaccharides)
* Serotype associated with pathogenicity
* Meningitis, sepsis, pneumonia and arthritis
* Risk of disease increases after viral infection
* Vaccines available
51
What is a sign of zoonotic *S. suis* infection?
Irreversible hearing loss (60%) combined with a low mortality meningitis.
Septicaemia (high mortality) and endocarditis, peritonitis and arthritis.
52
Core genomes
Pool of genes shared by all members of a bacterial species.
53
Accessory or dispensable genome
Pool of genes present in some but not all genomes within the same bacterial species.
54
Pangenome
Global gene repertoire of a bacterial species, comprised of core genome + accessory genome.
55
Metagenome
Global gene repertoire of mixed microbial population.
56
What may lead to increased virulence and zoonotic potential?
Loss and acquisition of genes.
**Polysaccharide capsule switch**
57
The serotypes of *S. suis*
Serotype 2, CC1, zoonotic
Serotype 2, CC20, zoonotic
Serotype 9, CC16
58
After infection with serotype ... there is a lack of protective immunity.
*S. suis* serotype 2
59
What alleles of *S. suis* yields 4 unique HsdS proteins and determines the location in the genome that is methylated?
Four alleles of SsuCC20p
60
The inverted repeats (IR) flanking ... and ... allow for recombination.
TRD1 and TRD3.
This recombination is mediated by the recombinase present within the locus.
You can do FAM assays to detect the alleles.
61
Recombination stops if you knock out this gene from the SsuCC20p
xerD
62
The 2 endemic lineages of *S. suis* in Thailand
CC233 and CC104
63
From which group did CC104 and CC233 acquire their cps2 capsule?
CC1
64
Characteristics of the polysaccharides of *S. suis*
* Repeated oligosaccharide units.
* Key virulence factor, confers protection.
* Serotype 2 (and 14) have terminal sialic acids on the side chain.
65
What are Siglecs?
Sialig immunoglobulin like lectin immune receptors exploited by bacterial pathogens.
* Sialic acids present on surface of mammalian cells.
* Siglecs interact with polysaccharides terminating with sialic acids.
* Avoid autoimmune response.
* Exploited by pathogens to evade immune evasion.
66
What do streptococci contain in their accessory genome?
Integrative and Conjugative Elements (ICE)
ICE combines features of phages, transposons and plasmids. Under certain conditions ICE excises itself from the chromosome and forms a circular intermediate. Circular intermediate replicates and is transferred through a mating pore to a recipient cell. Replicated intermediates integrate into the chromosomes of the host cells.
67
The main driver of antibiotic resistance is
Antibiotic consumption/exposure
68
What principle is important while thinking of transmission of AMR *E. coli*?
One Health
But key interventions are host and domain independent.
69
How is the colistin (polymyxin B) resistance mediated?
Mobile Colistin Resistance (MCR) - plasmid mediated colistin resistance
High prevalence of fecal colonization with mcr-1 in community in rural-suburban southern Vietnam
70
How does mcr-1 spreads?
1. Integration of mcr-1 in transposon.
2. Insertion and transposition between plasmid backgrounds.
3. Stabilisation of mcr-1 in plasmid backgrounds (insertion sequences lost over time).
4. Plasmid-mediated spread between hosts.
71
Antibiotics targeting the cell wall synthesis
Penicillins, cephalosporins, bacitracin, vancomycin
72
Antibiotics targeting the protein synthesis
Chloramphenicol, erythromycin, tetracylins, streptomycin
73
Antibiotics targeting the nucleic acid replication and transcription
Quinolones, rifampin
74
Antibiotics targeting the plasmamembrane
or
Targeting the synthesis of essential metabolites
Polymyxin B
or
Sulfonamide, trimetophrim
75
Intrinsic mechanisms of resistance
Efflux pump to pump out antibiotics that cannot bind the target protein.
76
What is the function of penicillin binding proteins (PBPs)?
They synthesize peptidoglycan.
77
AcrA an AcrB (+ TolC) are involved in
An MDR efflux pumps in Gram-negative bacteria (Resistance Nodulation Division).
The expression of Acr is controlled by TetR family repressors.
78
What is the function of TetR?
TetR represses transcription of acrAB. AraC can relieve repression by TetR and thus activate acrAB transcription. araC is repressed by multiple antibiotic resistance protein MarR.
--> Expression AcrA and AcrB low
79
How can MarR derepress?
* Antibiotic binding of MarR (unable to repress araC
* Mutations in TetR
Leading to an increased expression of the RND efflux pump AcrB and the periplasmic adaptor AcrA.
80
Two ways in which the target site can be changed
1. Target site mutation
2. Target site protection (binding something else instead)
81
MRSA express PBP2a, but how is this obtained?
By horizontal gene transfer from *S. epidermidis*. Encodes a PBP with different structure, not binding penicillins. Peptidoglycan synthesis is however not affected.
82
What target site mutation in *M. tuberculosis* causes rifampicin resistance?
Mutation in rpoB (RNA polymerase)
83
TetM target site protection in ribosome against ...
Tetracyclin
84
AMR by direct interaction with antibiotics (two options)
1. Inactivation by hydrolysis
2. Inactivation by steric hindrance (changing the surface proteins on the cells)
85
ß-lactamases
Beta-lactamases are enzymes produced by bacteria that provide multi-resistance to beta-lactam antibiotics
86
Resistance to ß-lactam antibiotics
* Efflux
* Direct interaction: ESBL, carbapenemases
* Target site change (MRSA)
87
Classes of antimicrobial peptides
* ß-stranded: alpha- and beta-defensins, protegrin
* alpha-helical: LL-37 and cecropin
88
Membrane disruption by antimicrobial peptides. Three models:
1. Barrel stave model
2. Toroidal pore model
3. Carpet model
89
90
What are thrombocidins?
Cationic AMPs from human blood platelets
91
TC84 actions
* Gradual membrane depolarization
* Membrane permeabilization
* Membrane invaginations
* Cytoplasmic content leakage, so membrane disruption
* TC84 'unmixes' membrane, fluid domains
* TC84 colocalizes with fluid domains
* TC84 colocalizes with membrane invaginations
92
Mode of action of TC-19
AMPs interact with bilayer. Fluid domains arise in the membrane --> compromised
93
Which antibiotic resistant isolates are susceptible to honey?
MRSA and ESBL
94
Predominant pathogens in catheter-related bloodstream infections
- *S. aureus*
- *S. epidermidis*
95
What is the antimicrobial peptide in honey?
Bee defensin-1
96
Where are the Type II, III and IV secretion systems derived from?
* Type II: Pili
* Type III: Flagella
* Type IV: Conjugation systems
97
Which secretion systems do one-step secretion?
Type I, III, IV and VI
* Cytoplasmic chaperones
* Targeting to/selection by secretion machine via motifs or chaperones
98
Which secretion systems do two-step secretion?
Types II and V
* Sec/Tat using a signal peptide
* Periplasmic chaperones
* No energy source at OM
99
Crystallography not suitable for
Membrane proteins:
* Hydrophobic surface: aggregation rather than crystallization
* Difficult to purify in large quantities from membranes: need to solubilize them using detergents.
100
Why use Cryo-EM?
Prevents radiation damage to samples and there's no staining needed.
101
In what species are Type III secretion systems present?
*Yersinia* species
* Intimate attachment
* Anti-apoptotic and anti-inflammatory responses
102
*V. cholera* had gene clusters with two homologues of T4SS (IM and ATP binding)
Other genes in cluster not related to T4SS.
103
*V. cholerae* kills host. Insertion of a ... attenuate virulence. This is coding for ..
Transposon.
Hcp/VgrG in Vibrio supernatant.
Type VI SS kill other bacteria
104
Type VI resembles
gpVn and Hcp1
gpVn is a protein encoded by bacteriophages --> phage tail
105
Type VI and bacteriophages
* VrgG complex resembles the tip of the bacteriophage sheath.
* Hcp rings of the sheath
* gpVn through membrane
Tail sheath homologue (VipA/VipB)
106
Type VI secretion
1. Build up
2. Signaling
3. Contraction/ejection
4. Degradation
107
Triggers for T5SS based on attack
**Cell-cell contact**
OmpA in OM
Periplasmic relay protein
* TslA
* Triggers assembly baseplate
108
Triggers for T5SS based on defense
Membrane damage triggers response
* Via 2-component regulatory system
* Phosphorylation
109
Autotransporters and the types
One protein, three major domains with a two step mechanism that uses **Sec** protein machine in IM
110
Hemoglobin Protease (Hbp)
Plasmid-encoded virulence factor in EB1 (*E. coli*)
* Degrades hemoglobin
* Peritonitis
* Model autotransporter --> secretion mechanism
111
Autotransporter secretion
Uses Sec on the IM
Bam complex on the OM (proto-barrel ß-domain)
112
Passengers that are being secreted can be used as target. How do we change them?
By insertion into passenger (f.e. sidechains). Let them carry antigen at cell surface.
PspA, Pneumolysin antigens in Hbp (*S. pneumoniae)*
113
What is a novel target for antibacterial treatments?
**BamA8**
114
Autotransporters are effective vaccine-platform used to
decorate OMVs with antigens (outer membrane vesicles)
115
Problems in combating tuberculosis
* The current vaccine (M. bovis BCG, 1921) does not protect adults efficiently
* Antibiotic resistance
* Highly unusual and slow growing bacterium
* Extremely slow-growing pathogen, doubling time 20-24 hours… Experiments at BSLIII level
* No optimal infection model (apart from monkeys) and animal experiments also lengthy
* Built-up of mycobacterial cell envelope complicates molecular and biochemical analyses
116
Take home messages tuberculosis
M. tuberculosis is currently still the deadliest bacterial pathogen
Issues with combating tuberculosis are rise of multi-drug resistance and absence of effective vaccine
Mycobacterial cell envelope has a specific and highly impermeable OM
Type VII secretion systems mediate transport of proteins across this cell envelope
ESX-1 is a crucial for intracellular survival as it mediates phagosomal escape
ESX-5 is essential as it (most-likely) mediates the insertion of nutrient transporters in the OM
The ESX-5 membrane channel spans solely the inner membrane and has unusual flexible ATPase subunits.
Current research is focusing on understanding how the channel opens and identifying the MOM channel of T7SS.
