Molecular Infection Biology Flashcards
a. Explain the molecular principles of VIDISCA for virus discovery and mention what the biggest contaminant is and why
VIDISCA begins with a treatment to selectively enrich for viral nucleic acid, which includes a centrifugation step to remove residual cells and mitochondria. In addition, a DNase treatment is to remove interfering chromosomal DNA and mitochondrial DNA from degraded cells, whereas RNases in the sample will degrade RNA. RNA is reverse transcribed into cDNA, and second-strand synthesis is performed to make dsDNA (from a viral RNA or DNA genome). The dsDNA is digested with frequently cutting restriction enzymes. The target is subsequently PCR-amplified with primers that anneal to the anchor sequences, followed by a round of selective amplification with primers that are extended with one nucleotide (G, A, T, or C). Problem: ribosomes; contain RNA and are size of viral particle.
b. A new virus has been discovered using VIDISCA, what set of rules do investigators use to determine if the virus really causes a disease, also mention the rules themselves.
Koch’s postulates:
- The pathogen must be present in every case of disease
- The pathogen must be isolated from the host with the disease and grown in pure culture
- The specific disease must be reproduced when a pure culture of the pathogen is inoculated into a healthy susceptible host/animal
- The pathogen must be isolated again from these ill indivuals/animals
c. Explain how you can link a new virus to a disease, when the virus cannot be cultured.
In this case, you can only check postulate 1 and include careful controls; check its prevalence in patient groups and healthy controls and determine whether there is an association with symptoms (first Koch’s postulate), you can transplant infected tissue to animals and see if it causes a similar infection.
- In 2004 seminal paper of S. Hedrick (The acquired immune system: a vantage from beneath) mentioned: “The acquired immunity is not a prerequisite for survival in the face of infections in complex animals. It may be an incremental advantage.”
a. Explain what he means with this comment and what arguments are there to demonstrate this point
It links to the red queen hypothesis, each evolutionary novelty in host pathogen interactions gives temporary advantage that is lost once the other side (pathogen) adapts to this. Animals without an adaptive immune system live a long life, can be as large and can be as intelligent/complex.
- In 2004 seminal paper of S. Hedrick (The acquired immune system: a vantage from beneath) mentioned: “The acquired immunity is not a prerequisite for survival in the face of infections in complex animals. It may be an incremental advantage.”
b. Discuss whether all pathogens have to develop mechanisms to interact with the acquired (adaptive) immune system
No, not all pathogens, so called hit-and-run pathogens only have to cope with the innate immune system. Also, pathogens that only infect hosts without an innate immune system do not need this.
- In 2004 seminal paper of S. Hedrick (The acquired immune system: a vantage from beneath) mentioned: “The acquired immunity is not a prerequisite for survival in the face of infections in complex animals. It may be an incremental advantage.”
c. Describe how you would identify virulence factors of a pathogen that are required to escape eradication by the acquired immune system.
To do this, you will have to describe a system where you make sure that the adaptive immune system is on AND you have to exclude factors required for the innate immune system. For instance, perform selection procedure with TraDIS in a naïve animal to find the innate immune factors and then repeat this experiment with an immunized animal. The difference between the two are factors that could play a role in the adaptive immune response.
- Two-component signal transduction plays an important role in the regulation of Type III Secretion in Salmonella enterica
a. Describe how the two-component signal transduction works in general and which 3 proteins are involved in this process.
Two-component systems are a (histidyl-aspartyl) phosphorelay system, inner membrane component a signal which results in autophosphorylation, this results in phosphorylation of the regulator , which gets activated and then binds the promotor (operator). Sensor kinase, response regulator, and phosphatase that inactivates the regulator
- Two-component signal transduction plays an important role in the regulation of Type III Secretion in Salmonella enterica
b. Describe and explain the difference in regulation between Spi1 and Spi2 systems of Salmonella enterica.
Spi1 is required in the lumen of the gut to induce inflammation and invasion of epithelial cells and therefore activated in the lumen, downregulated in tissue. Spi3 is required inside the host cell for survival and making the SCV on therefore induced once inside host cells. Both systems are regulated via various regulators, including two-competent systems (network) and response to different stimuli Spi1 anaerobic growth, Spi2 osmolarity, antimicrobial peptides.
- Two-component signal transduction plays an important role in the regulation of Type III Secretion in Salmonella enterica
c. Explain how you can identify whether a newly identified regulator, BlfR, is affecting in vivo regulation of Spi1 and/or Spi2. Include controls in your answer
If it is one regulator, like in this example, BifR, you should make a knockout mutant, and do not forget complement version. Measure of Spi1, Spi2 induction by using a fusion of the promotors to GFP or other indicator inside your mutant (also in WT and complemented strain as control). Wrong answer: IVET, DFI (not genome wide and therefore no answer for BlfR.
- Genome variation is an important aspect of microbial evolution
a. Explain the terms Pangenome and Synteny
Pangenome: All genes from all strains of the same species
Synteny: Comparison based on gene order on the chromosome
- Genome variation is an important aspect of microbial evolution
b. One source of genome variation in bacteria is the presence of filamentous phages. Describe how filamentous phages can contribute to virulence and give an example.
Filamentous phages can play a role in transduction (HGT by phages), but they can also carry directly virulence factors, for instance Vibrio cholera filamentous phage that carries both genes of cholera toxin
- Genome variation is an important aspect of microbial evolution
c. Describe the consequence for a viral pathogen if there is no gene transfer and name an example
Viral pathogens hardly show gene transfer, they then normally evolve by point mutations, example: any virus apart from influenza A (Corona)
- For in vivo experiments, most researchers use the mouse as a model host organism. However, for Mycobacterium tuberculosis and Staphylococcus aureus the mouse does not seem to be a good model
a. Clearly describe what the problem is to study tuberculosis in a mouse model and whether (and how) you could circumvent this (and still use mice)
Problems: no well organized granuloma (stratification), also, no hypoxia and necrosis and high bacterial load numbers, so therefore some virulence factors and treatment are completely different.
No good solution for this yet, but people try different mouse lines and mice with immune system replacement
- For in vivo experiments, most researchers use the mouse as a model host organism. However, for Mycobacterium tuberculosis and Staphylococcus aureus the mouse does not seem to be a good model
b. Clearly describe what the problem is to study Staphylococcus aureus in a mouse model and whether (and how) you could circumvent this (and still use mice)
S. aureus of humans is not pathogenic for mice because many virulence factors are human specific.
Solutions: humanized mice that produce some human genes for the S. aureus virulence factors, such as C5aR
- For Neisseria meningitides vaccines against Group B strains, they sometimes use polyvalent OMV based vaccines
a. Explain what is meant with polyvalent and with OMV in this respect and what role is of each in the immunization process
Polyvalent: different version of the same protein(s), for instance outer membrane proteins, different versions are required to cover a broad range of strains
OMV: Outer Membrane Vesicle, presents the protein in its native conformation and provides adjuvants properties
- For Neisseria meningitides vaccines against Group B strains, they sometimes use polyvalent OMV based vaccines
b. Explain why researchers are tinkering with the lipid A biosynthesis pathway
Lipid A is a strong inducer of the innate immune response, but induction can be too strong, therefore, researchers make mutations that result in altered versions of lipid A with reduced (optimal) induction responses
- For Neisseria meningitides vaccines against Group B strains, they sometimes use polyvalent OMV based vaccines
c. Discuss whether the same approach (polyvalent OMV-based vaccine) can be used to create a vaccine for Neisseria gonorrhoea.
Questionable, NG is more variable in surface antigens and it is not yet known what type of immunity will work against NG. Probably not only antibody response. (Not a robust animal model that mimics human disease).
- The Red Queen Hypothesis escribes the evolution of virulence factors as a part of an ongoing battle between the host and its pathogens:
a. Enterobactin is a strong siderophore of E. coli and binds iron with a very high affinity to transport iron back to the bacterial cells. Virulent E. coli strains produce, in addition to this strong siderophore, the much weaker siderophore aerobactin. Explain why the additional but much weaker siderophore aerobactin enables pathogenic E. coli strains to gain efficiently iron compared to a non-pathogenic strain.
The strong siderophore enterobactin is bound an inactivated by the host by the innate immune … lipocalin. However, aerobactin is not bound by lipocalin and can therefore provide iron even though the binding affinity is lower.
b. The formylated-peptide receptor (FPR1/FPR2) are receptors of the innate immune system to activate neutrophils to migate to the site of infection. These receptors recognize the presence of bacteria. Explain what substrate is recognized by this receptor and what underlying mechanism makes this receptor specific to bacteria.
Bacteria start protein synthesis always with a formyl-methionine residue instead of a normal methione residue. When bacterial proteins are degraded the fragments containing the N-terminus will have a formyl-met and can be recognized by the FPRs. Eukaryotic proteins start just with methionine and are therefore not alarming the immune system.
c. Discuss whether the S. aureus inhibitor of FPR1 receptor (CHIPS) can be identified using a TraDIS approach.
Migration of the neutrophils will be blocked by an extracellular protein. If one mutant does not make this protein it will not affect the overall effect of the migration of neutrophils and the survival of this one mutant as compared to the others, so the answer must be no.
- Many microbes use glycolysis to generate ATP. Respiration and oxidative phosphorylation can generate even more ATP
a. Glycolysis is considered a good target pathway for drugs against the infectious form of Trypanosoma brucei. Recently, studies have shown that fat tissue is a niche of T. brucei in the mammalian host. Explain how T. brucei was found to adapt to this niche and how affect the potential of glycolysis as a target pathway against T. brucei?
The trypanosomes start to express enzymes in β ixidation. Fatty acids can be an alternative ATP source. This makes glycolysis redundant for ATP generation and hence a less relevant target pathway.
b. Nitric oxide produced by neutrophils is a respiratory poison to S. aureus. S. aureus will respond by expression of the enzyme lactate dehydrogenase. How would this benefit S. aureus?
S. aureus stops using the respiratory enzymes. But electrons need a terminal electron acceptor. With lactate dehydrogenase, pyruvate can be the terminal electron acceptor.
- Salmonella typhimurium and Listeria monocytogenes. Both regulate actin polymerization t enter the cell
a. Indicate for each organism how the entry system is called.
Salmonella: trigger.
Listeria: zipper
- Salmonella typhimurium and Listeria monocytogenes. Both regulate actin polymerization t enter the cell
b. Describe for each organism one bacterial protein/host protein interaction and how this interaction initiates actin polymerization.
Salmonella prevents fusion with lysosomes. Listeria escapes the phagosome.
- Salmonella typhimurium and Listeria monocytogenes. Both regulate actin polymerization t enter the cell
Once inside the cell, both organisms avoid death by lysosomes.
c. Indicate for each organism how they avoid death by lysosomes.
d. Describe for each organism one bacterial protein that is essential for this process, and how it orchestrates this on a molecular level.
Salmonella: SopD2 acts as Rab7 effector blocking lysosome maturation. SifA sequesters Rab9 blocking cathespin delivery/lysosome maturation. SopB, Rab5 retention.
Listeria: LLO, destabilizing membrane by pores. PLCa or PLCb, lipases that destroy the phagosomal membrane.
Legionella pneumophilla is a pathogen that can cause lung infections. Once inside the host Legionella pneumophilla modulates membrane trafficking and fusion by secreting effector molecules into the host cell. One of them, called DrrA regulates small GTPases.
e. Explain the GTPase cycle. Indicate when it is one, and when it is off and which three roles DrrA could have to interfere with this cycle.
Small GTPases hydrolyze GTP to GDP, whoch can be stimulates by GAPs (GTPase Activating Proteins). The GDP can be exchanged for a GTP stimulated by a GEF (Guanine Exchange Factor). When bound to GTP they are ON and able to bind to an effector, when bound to GDP they are off. The 3 possible roles of DrrA are: GEF, GAP or effector.
- Genome variation is an important aspect of microbial evolution.
a. Explain the terms ‘genetic headroom’ and ‘core-genome’.
Genetic headroom: the amount of dispensable information in the genome, i.e. the fraction that is not part of the core-genome. Determines genetic flexibility, evolutionary potential.
b. There are different methods to predict which genes have been acquired by horizontal gene transfer. One of these methods is by examining codon usage. Describe what this method is based on and how it works,
How to determine HGT: codon usage. There are multiple codons for arginine, for example. A species could favour one combination over the other. GC content (take into account amino acid composition). Gene order/synteny. However, we see an underestimation, because the differences are very small due to homology.
