L1. Introduction Flashcards
How many pathogenic bacterial species are described?
Less than 100 pathogenic bacterial species are described.
What percentage of bacterial diseases are caused by a small number of species?
1% of bacterial diseases are caused by several thousands of species, even though most infections are made up by the 100 described pathogenic species.
Does bacterial interaction occur in isolation?
No, bacterial interaction does not occur in a vacuum but only when interacting with a host (Nothing in this module happens in a vacuum, only when interacting with a host).
What happens during a real-life infection in terms of microbial presence?
During a real-life infection, there are lots of bacteria, fungi, archaea, etc., present in the environment, but the focus is on the one causing the disease; however, the others impact the infection massively.
How are non-pathogenic members of the normal flora described?
Non-pathogenic members of the normal flora are described as “commensals.”
Define commensalism.
Commensalism is an association between two organisms in which one benefits and the other derives neither benefit nor harm.
What is mutualism in the context of microbiota and an example?
Mutualism is a symbiotic relationship where both organisms benefit, such as the gut microbiome providing vitamin B12 and contributing to the calorie content of food.
How do gut microbiota benefit the host’s epithelial cells?
All energy supplied to epithelial cells comes from short-chained fatty acids produced by gut microbiota.
Describe a parasitic relationship in the context of microbiota.
A parasitic relationship causes slight harm to the host by using resources, but it is not too detrimental.
What can commensal organisms do under the right circumstances?
Commensal organisms can sometimes cause disease under the right circumstances, becoming opportunistic pathogens.
What is the focus of the module in terms of bacteria?
The focus is on bacteria that can become opportunistic pathogens.
What are the two possible outcomes of a pathogen infecting a host?
Asymptomatic carriage or disease development.
What is asymptomatic carriage?
When no symptoms of disease are shown, but the individual can still pass on the pathogen or even develop the disease later.
Give an example of an asymptomatic carrier.
Typhoid Mary.
Why don’t pathogens evolve to become less pathogenic?
It’s not in the pathogen’s interest to kill the host, but it makes no difference as once it can transmit and spread quickly, there is no selective pressure against death.
What happens if a pathogen isn’t good at transmitting?
Pathogenicity may decrease over time.
Why do some pathogens benefit from the host’s death?
Some anaerobes, like Clostridium tetanus, can’t live in the presence of oxygen and have evolved to kill the host, making the host an anaerobic fermenter (food source).
What are the two classes of virulence factors?
Factors for colonizing the host and factors that damage the host.
Name some virulence factors used for colonizing the host.
Adhesions, invasions, nutrient acquisition (e.g., Fe scavenging), motility, and chemotaxis.
Name some virulence factors that damage the host.
Exotoxins, endotoxins, proteases, DNase, lipase, and hemolysins.
How do host defenses push pathogens to become more asymptomatic or lead to recovery from disease?
Through physical barriers, innate immunity, and adaptive immunity.
What are the components of innate immunity that help fight pathogens?
Complement secretion, macrophages, and antimicrobial peptides.
What role do physical barriers play in host defense?
Skin and gut epithelium prevent bacteria from accessing more nutrients and usually need to be damaged for pathogens to enter.
What is the role of adaptive immunity in host defense?
B cells and T cells respond to pathogens, especially if the pathogen survives the initial innate immune response.
What is the importance of complement in innate immunity?
It involves the secretion of complement proteins that form a cascade to attack and lyse pathogens.
How do antimicrobial peptides contribute to host defense?
They lyse bacterial membranes, as are cationic peptides that insert and disrupt membranes, helping to kill pathogens.
What are Koch’s Postulates?
> In 1876 Robert Koch put forward 4 criteria that must be met in order to identify the etiological agent of a disease
- The microorganism must be found in abundance in all organisms suffering from the disease but not in healthy organisms.
- The microorganism must be isolated from a diseased organism and grown in pure culture.
- The cultured microorganism should cause disease when introduced into a healthy organism.
- The microorganism must be re-isolated from the diseased experimental host and identified as being identical to the original causative agent.
What are the molecular Kock’s postulates and what are they for?
> These are how to distinguish what a virulence factor is and what it does:
- The phenotype or property under investigation should be associated with pathogenic members of a genus or pathogenic strains of a species.
>Some commensals have strains which are pathogenic, so the genes studied are specific to the pathogen not the whole genus. - Specific inactivation of the gene(s) associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence.
>If you knockout a gene with a virulence trait, the bacteria pathogen becomes less pathogenic. - Reversion or allelic replacement of the mutated gene should lead to restoration of pathogenicity.
>Now referred to as complementation, if you want to know what a gene does- disrupt or remove gene, study change in phenotype, then restore genotype back, gives confidence that gene is for a phenotype.
What is one method used to study pathogenesis?
Genetic manipulation, which involves some readout of virulence using animal or surrogate models to assess virulence.
What does reductionist microbiology focus on in the study of pathogenesis?
Identifying virulence factors such as toxins, degradative enzymes, adhesins, invasins, chemotaxis and motility systems, nutrient acquisition, etc.
Why is reductionist microbiology often used in studying pathogenesis?
Because it is easier to work with pure cultures or single genes, whereas studying a pathogen in a mixed population is not easy.
What challenge arises from the fact that many virulence factors are also produced by non-pathogenic organisms?
It makes it difficult to identify a true virulence factor.
How does the presence of virulence factors in both pathogens and commensals complicate the identification of true virulence factors?
It raises the question of whether a factor is required for disease if both pathogens and commensals produce it.
Are all virulence factors clearly identifiable as essential for disease?
No, some virulence factors are clearly required for disease, while others are not as clear.
What organism releases the tetanus toxin?
The gram-positive spore-forming anaerobe Clostridium tetani, related to Clostridiodes difficile.
How does the tetanus toxin cause death?
The clostridial neurotoxin induces rigid paralysis by acting at the neuromuscular junction, preventing muscles from relaxing. This can lead to suffocation and broken bones due to muscle spasms.
What is the potency of the tetanus toxin compared to other toxins?
It is the 2nd most potent toxin known with an LD50 of 2.5 ng/kg. The most potent toxin is botulinum toxin with an LD50 of 1 ng/kg.
What role does OmpA play in E. coli virulence?
OmpA is a dominant outer membrane protein essential for evasion of macrophage killing and invasion of the blood-brain barrier, causing meningitis in neonates.
Why is OmpA considered a virulence factor despite being present in all E. coli strains?
Although OmpA is present in all E. coli strains and contributes to virulence, it is necessary but not sufficient alone to make the bacteria virulent.
What does reverse genetics seek to do?
Reverse genetics seeks to assign a function to a particular gene or sequence.
How does reverse genetics typically start?
It starts with a hypothesis for what a gene does or what species it is found in.
What method is used in reverse genetics for a specific gene?
Directed mutagenesis.
How does reverse genetics use hypothesis-driven analysis?
The hypothesis directs experimental analysis, such as using readout for toxin production if thinking a gene produces a toxin.
What is the goal of forward genetics?
Forward genetics seeks to identify the genetic basis of a particular phenotype.
Does forward genetics require prior knowledge of a gene?
No, it does not require prior knowledge of a gene, only a known phenotype to study.
What method is used in forward genetics?
Random mutagenesis.
How is an experimental approach designed in forward genetics?
It is designed to screen for phenotype, such as making thousands of mutants and assessing which ones can’t adhere.
What is often used to back up findings in reverse genetics?
Another sort of data, such as biochemical assays to demonstrate enzyme activity or protein-protein interactions.
What is a limitation of genetic tools in molecular microbiology?
Not all tools are available in all organisms and some organisms are intractable.
What postulates do reverse genetics often satisfy?
Molecular Koch’s Postulates.
What are the steps in reverse genetics?
- Start with gene.
- Knock it out.
- Test phenotype.
- Complement knockout (put gene back).
- Test phenotype and see if it’s restored.
What types of mutations can be used in more subtle genetics instead of knockout?
- Point mutations.
- Loss of function mutations.
- Gain of function mutations.
- Dominant negative mutations.
What does a dominant negative mutation do?
Expression of a mutated allele suppresses the phenotype of a wild-type allele.
What are the two main options for making a gene knock out?
Insertional and deletion.
What is an insertional knock out?
Adding something to a gene to disrupt it, typically an antibiotic resistance gene.
What are the pros and cons of an insertional knock out?
Pros: Quicker and easier.
Cons: Can have downstream effects, especially if the gene is in an operon, potentially disrupting the expression of other genes (polar effects).
What is a deletion knock out?
Deleting some or all of the upper reading frame or a whole chunk of the gene.
Why can a deletion knock out be more effective than an insertional knock out?
It can precisely remove the gene without affecting the rest of the operon.
What are some techniques used for making knock outs?
Lambda Red (phage), Group II introns (TargeTron), homologous recombination, phage transduction, and CRISPR.
Why are natural systems often better at molecular biology than we are?
Because these systems have evolved to perform these tasks efficiently, which we can exploit for genetic manipulation.
What are the two main options for complementing a gene (putting it back in)?
> Using a self-replicating plasmid.
> Knocking it into the chromosome.
What are the pros and cons of using a self-replicating plasmid for gene complementation?
> Pros: Easiest to do, involves one cloning step then transformation into the mutant.
> Cons: Plasmids are often present in multiple copies, which can lead to overexpression and potential survival issues.
What are the pros and cons of knocking a gene into the chromosome for complementation?
> Pros: Effective, single copy, usually inserted into known spots in the genome.
> Cons: Harder to do, requires cloning into a recombinant vector and insertion into the chromosome.
Why does the location of insertion in the genome matter for gene expression?
Genes located near the origin of replication tend to be expressed at higher levels because they are present in higher copy numbers as DNA is constantly being replicated.
What do we need for complementing a gene using a plasmid?
Selectable markers, origins of replication, transformation/conjugation methods, and promoters.
What do we need for complementing a gene using insertion?
A suitable insertion site, homologous recombination, and counter selection.
Why are specific insertion sites important for gene complementation?
Because the location shouldn’t have a negative impact on the cell
Why is counter selection important in the process of gene complementation?
It helps select against bacteria that have the delivery plasmid, ensuring the plasmid is unstable or conditional.
Ensuring Plasmid Instability or Conditionality:
Plasmid Stability: In gene complementation experiments, the delivery plasmid (carrying the gene of interest) should be unstable or conditional. This means the plasmid should not be permanently maintained in the bacterial population under non-selective conditions.
Conditional Maintenance: Counter selection ensures that the plasmid can only be maintained under specific conditions. For example, if the plasmid carries an antibiotic resistance gene, bacteria will maintain the plasmid only when the antibiotic is present. When the antibiotic is removed, the plasmid is lost over generations.
Selecting Against Bacteria with the Delivery Plasmid:
Eliminating Delivery Plasmid: After the gene of interest is integrated into the host genome or the desired genetic modification is achieved, the presence of the delivery plasmid is no longer needed. In fact, it might be undesirable as it can interfere with further genetic analysis or experiments.
Counter Selective Markers: Counter selection involves using markers that are negatively selected under certain conditions. For example, a plasmid might carry a gene that confers sensitivity to a specific compound (like sucrose or certain antibiotics) when expressed. When the bacteria are exposed to this compound, only those that have lost the plasmid will survive.
Achieving Clean Genetic Backgrounds:
Removing Extraneous Genetic Material: To ensure that the observed phenotypes are solely due to the chromosomal integration or the desired genetic changes, counter selection helps remove the extraneous plasmid DNA.
Precision in Genetic Experiments: By selecting against bacteria with the delivery plasmid, researchers can ensure that the genetic background of the bacteria is as clean as possible, leading to more accurate and interpretable experimental results.
Practical Example:
In a typical gene complementation experiment:
Introduction of Plasmid: A plasmid carrying the gene of interest and a counter-selectable marker (like a gene conferring sensitivity to a specific compound) is introduced into the bacteria.
Selection for Plasmid: Initially, bacteria are grown under conditions that select for the presence of the plasmid (e.g., antibiotic selection).
Inducing Integration: Conditions are changed to induce integration of the gene of interest into the bacterial chromosome or to achieve the desired genetic modification.
Counter Selection: The bacteria are then grown under conditions that select against the presence of the plasmid (e.g., presence of the compound to which the counter-selectable marker confers sensitivity).
Isolating Desired Clones: Only bacteria that have lost the plasmid (and ideally have the gene of interest integrated into the genome) will survive, ensuring a clean genetic background.
What is the starting point in forward genetics?
Start with a wanted phenotype but no idea of the genetic bases of this phenotype.
What is the first step in forward genetics to study a wanted phenotype?
Randomly mutagenize bacteria and study the genes of those that don’t have the phenotype.
Give an example of a forward genetics approach for a toxin.
Testing for a toxin that kills mice. If no obvious toxin genes are found, incubate bacteria with cultured human cells. If the bacteria produce toxins, they kill human cells. Identify human cultured cells sensitive to the toxin and use them as a screen. Look for bacterial mutants that don’t kill the cells (don’t produce the toxin) to see which gene was mutated.
What is needed for an efficient forward genetics screen?
A high-frequency screen with 100s-1000s of mutants.
What are the requirements for a phenotypic screen in forward genetics?
The screen needs to be relatively straightforward and scalable.
What is a challenge in identifying the responsible mutation in forward genetics and how has this been made easier?
Random mutagenesis makes it challenging because the gene causing the phenotype is unknown. Whole genome sequencing has made identifying the responsible mutation easier.
What is the overall process of forward genetics?
Start with a phenotype, devise a screen, make a lot of mutants, and test the mutants to identify those that do not exhibit the phenotype, then determine which gene was mutated.
How was random mutagenesis originally done?
Using chemical mutagens or radiation which damage DNA. Most organisms die, some survive by repairing DNA, but the DNA repair process is error-prone, causing random mutations.
What is now largely used instead of chemical mutagens for random mutagenesis?
Transposons, which are genetic parasites that randomly insert into the genome.
What is the advantage of using transposons for random mutagenesis?
They can be used 10-20,000 times and screened to have one mutant in every single gene in a genome.
What are the 3 different systems used for screening mutants?
- Early systems: 10-20,000 screened individually.
- Signature tagged: 10-20,000 screened in pools of 96.
- TraDIS/TnSeq: 50,000+ screened in a single pool. (JUST KNOW THIS ONE)
What is TraDIS?
Transposon Directed Insertion site Sequencing, a method to screen very large numbers of mutants simultaneously.
Describe the process of TraDIS in 7 steps.
- Library generation: Each bacterium contains one random transposon mutation, creating a pool of different mutants.
- Transposon mutant pool: If transposon goes into a crucial gene, the bacteria die immediately, so 10% of the genome cannot be mutated.
- Genomic DNA from the mutant pool: Extract genomic DNA from surviving 90% of bacteria with transposons in different places.
- Fragments are sequenced using Illumina: PCR specific amplification with primers inside transposons and Illumina adaptors on DNA.
- Reads are mapped to a reference: Sequence DNA to identify insertion sites of every transposon, mapping all mutants across the genome.
- Essential genes don’t have insertions: Genes with no insertions are essential since mutants died.
- Add selection pressures: Determine which mutants die with specific pressures to identify essential genes for survival.
What does TraDIS reveal about essential genes?
Essential genes don’t have insertions because mutants with these insertions die and are not present in the library.
How does TraDIS help understand gene function under different conditions?
By applying selective pressures and comparing mutants present with and without the pressure, it identifies genes necessary for survival under those conditions.
Give an example of using TraDIS to study conditional essentiality.
Growing mutants in bile salts to see which genes help survive. Mutants that die in bile salts have lost their mutation for survival, indicating genes necessary for bile salts resistance.
How was TraDIS used in C. difficile and what did it identify?
> Made 77,000 transposon mutants with an insertion every 54bp.
> Identified 404 essential genes out of approximately 4,000.
> Applied selection pressure by sporulation, identifying 798 sporulation genes required for survival.
Why are rodents the most commonly used animal models?
> Inbred lines reduce variation, making them very homogenous genetically.
> They are small, cheap, and reproduce quickly.
> Many available tools (reagents), such as antibodies recognizing mouse proteins and mutant lines.
> Commonly used rodents include mice, rats, rabbits, and hamsters.
What are 3 limitations of using rodents as animal models?
- License restrictions (tightly regulated in the UK; more relaxed in the US).
- Ethical issues.
- Mice aren’t humans, leading to limitations in applicability.
Why are fish and non-vertebrate models becoming more popular as models?
> Many tools are in development, particularly for zebrafish.
> Light touch regulation, with fewer ethical concerns for embryos.
> Large group sizes can be used for statistical robustness due to their small size.
> Can visualize fluorescence of macrophages to watch immune defense.
What are 3 disadvantages of using fish and non-vertebrate models?
- They are genetically and evolutionarily further away from humans.
- They have more primitive immune systems, with only innate immunity and no adaptive immunity in embryos.
- They are good for studying macrophages, etc.
What are 3 advantages of using cultured cell lines?
- Can use human cells.
- Normally immortalized, making them easy to culture and scalable.
- No current ethical issues, although there may be concerns about their origin.
What are 5 disadvantages of using cultured cell lines?
- Usually derived from cancer cells, which have many genetic changes, including alterations in surface protein expression.
- Not genetically identical to the tissues they originated from, especially surface proteins, as these are mutated to become motile.
- No disease model as don’t have symptoms like whole human, so only specific aspects of virulence can be studied (e.g., adhesion, invasion, intoxication).
- Cannot test for symptoms like diarrhea but can study secretions from cells that would cause this.
- Often behave quite differently from the parental cell type/tissue.
