Micro Exam 2

Question 1

How did the Fluctuation test demonstrate that mutations are spontaneous?

Answer 1

Luria and Delbruck set up cultures with a small number of bacteria in each allowing them to evolve possible genes that resist viral infection and then plated them with a bacteriophage virus. The number of plates containing resistant bacteria greatly varied between cultures indicating mutations are spontaneous. A “jackpot” culture containing lots of resistant bacteria may have picked up a resistant mutation early and other cultures may not have picked up any resistant mutations and resulted in no resistant bacteria.

Question 2

What would the results of the Fluctuation test be if mutations were directed?

Answer 2

If mutations were directed in response to environmental conditions, the number of resistant bacteria in each plate would be around the same in each culture.

Question 3

What are the 5 stages of a bacteriophage life cycle?

Answer 3

• attachment to cell
• entry of phage DNA and degradation of host DNA
• synthesis of viral genomes and proteins
• assembly of phage
• release of assembled phages resulting in cell lysing

Question 4

What are suppressor tRNA’s? What are the results of a cell using a suppressor tRNA

Answer 4

A suppressor tRNA has an anticodon that recognizes a stop codon on an mRNA strand but it has an amino acid attached to it. This means translation is not stopped in the presence of a stop codon and a peptide chain is extended, which also means a peptide chain is not terminated in the presence of a nonsense mutation.

Other kinds of suppressor tRNA’s encode one amino acid instead of another leading to missense mutations. Generally cells containing suppressor tRNA’s are not as healthy as wild type.

Question 5

What are conditional mutations?

Answer 5

Mutations that render an essential protein more sensitive to changes in temperature, pH, salt, and other environmental conditions. These are generally missense mutations that prevent a protein from retaining the correct tertiary or quaternary structure under more restrictive conditions.

Question 6

What are revertant mutations

Answer 6

Mutations that restore a wild type phenotype through base changes that restore it to its original genotype or second mutations that restore normal gene function

Question 7

What are the steps for determining how a cellular process works using microbial genetics?

Answer 7

1. Identify the question you want to answer
2. Develop a strategy for identifying a mutation in the process of interest, commonly screening
3. Increase your probability of success by adding a selection step. This can occur by introducing a physical/chemical mutagen or a transposon insertion that causes a mutation of interest. This can also include using particular selection media or for example using antibiotics to select for bacteria with antibiotic resistance genes
4. Identify a mutation of interest and clone the DNA to obtain a sequence and identify genes of interest
5. Make a null mutation in the identified gene sequence and repeat strategy for identifying mutation

Question 8

How was microbial genetics used to identify the genes required for B. subtilis sporulation?

Answer 8

A screening process was used to identify whether a given mutation prevented sporulation since colonies unable to sporulate were shown to turn white after 48 hours of growing on a sporulation medium (DSM), and wild type colonies were shown to turn brown.

Question 9

What kinds of methods are generally used to increase the probability of identifying mutant genes (without the use of transposons)?

Answer 9

• Selection steps that select for more mutations scientists are looking for, such as selecting against cells that make amino acids with penicillin when looking at amino acid biosynthesis
• Using a chemical or physical mutagen in order to increase the mutation rate and lower the number of colonies needed to screen for. Mutagens damage DNA and induce the SOS repair system which often introduces more errors

Question 10

How does transposon mutagenesis help increase the probability of identifying important genes?

Answer 10

Transposons are introduced in plasmids that can’t replicate and integrate in different positions in the genome. Then they are grown in a medium with more permissive conditions and replicated, and bacteria are then selected for a transposon marker, which requires more restrictive conditions. Then to determine genes that allow for growth in iron conditions for example, they are grown in low and high iron mediums and colonies that are grown in low iron mediums but not in high iron mediums are targeted.

TnSeq can be used to determine the relative abundance of mutants without screening for specific colonies. Used to determine conditions where if genes are affected in mice correlations of mutants

Question 11

What are some of the drawbacks of transposon mutagensis?

Answer 11

More biased than other mutagens since they can only be inserted in specific regions of a genome

Can also have polar effects on downstream genes

Only allows for the identification of loss of function mutations

Question 12

How is a gene disrupted by a transposon identified and cloned?

Answer 12

Transposon can be flanked by primers and a sequence can be obtained from cloning the disrupted genome. These sequences can be used to determine candidate genes that may have been interrupted by the transposon but first cloning of transposon insertions needs to occur. Then a null mutation can be made in specific genes of interest to test if loss of function occurs.

Question 13

What is commonly used to make a null mutation in an identified gene?

Answer 13

A nonreplicating plasmid inserts within a targeted gene disrupting function and causing it to become null either via single or double crossover disruption.

Question 14

What are the similarities and differences between conjugation, transduction, and transformation?

Answer 14

They all serve as ways bacteria exchange DNA
- Conjugation occurs when bacteria exchange DNA directly
- Transduction occurs when DNA exchanged is mediated via a virus
- Transformation occurs when bacterial DNA is uptaken from its environment

Question 15

What is the role of the F plasmid in conjugation?

Answer 15

The proteins required for pilus formation are encoded on the F plasmid.

1) A conjugation bridge is formed between organisms from the pilus retracting

2) The F plasmid is nicked in one strand by traI (encoded in the tra operon of the F plasmid)

3) one strand of the F+ cell is transferred to the F- cell and the F plasmid is simultaneously replicated in the F+ cell.

4) The synthesis of a complementary strand then begins in the recipient cell

5) cells separate after the completion of DNA transfer and synthesis of the F plasmid.

Question 16

Are Hfr strains able to initiate conjugation even though the F plasmid is integrated into the bacterial chromosome?

Answer 16

Yes, the F plasmid is able to be nicked and transferred to the F- bacteria even when it is a part of the bacterial genome.

Question 17

How are Hfr genomes used to map mutations of interest?

Answer 17

They do this via interrupted mating

Use appropriate Hfr strains selected from F+ and F- crosses and perform interrupted mating

Frequency of genetic markers among recombinants can be measured over time

Hfr and F- cells are mixed and the transfer of F plasmid can be mapped by looking at which genes show up first based on gene expression

You can see what genes are transferred from donor to recipient over time

Depending on time point of when a gene is replicated in recipient you can map it on the chromosome

Question 18

What are the steps of generalized transduction using a bacteriophage?

Answer 18

• Phage attaches to bacteria
• Phage DNA is replicated within bacterium and bacterial DNA is cut into small pieces
• When phages assemble within bacteria, some bacterial DNA may be packaged in phage heads and these phages are then released
• Phage containing bacterial DNA may attach and replicate its DNA in a bacterial cell
• This injected DNA is then incorporated into a bacterial chromosome
• This led to toxin genes being transferred from Shigella into E. Coli to make a more toxic E. Coli strain

Question 19

What occurs during artificial transformation?

Answer 19

Artificial transformation occurs when double-stranded DNA is forced through a cytoplasmic membrane and linear fragments are integrated through a homologous recombination or replication of a plasmid. Cells can be made to be competent, where they are able to take up DNA and integrate it into their genome, either by chemical treatment or electroporation.

Question 20

How do naturally competent bacteria mediate the uptake and incorporation of exogenous DNA?

Answer 20

Naturally competent bacteria have a competence regulon, a set of genes whose products mediate the uptake/incorporation of DNA (PilA, ComEA, ComEC, RecA)

• PilA binds to dsDNA (exogenous DNA) and retracts it by depolymerizng the pilus, where the polymer is converted into smaller subunits
• ComEA binds to dsDNA in the periplasm and pulls it through the pilus
• The dsDNA unwinds and one strand enters the cytoplasm through the ComEC channel and the other strand is degraded
• RecA recruits ssDNA to the homologous site for integration with the genome

Question 21

What are 4 types of naturally competent bacteria?

Answer 21

• Streptococcus pneumoniae
• Haemophilus influenzae
• Bacillus subtilis
• Vibrio cholerae

Question 22

What are different examples of ways gene transfer occurs between cells?

Answer 22

• Specialized and generalized transducing phages (transduction)
• DNA transfer via nanotubes (conjugation)
• DNA transfer via vesicles (transformation)
• Transfer of DNA plasmids via T4SS membranes (conjugation)
• DNA secretion and uptake (transformation)

Question 23

Genomic islands

Answer 23

Clusters of genes within a bacteria genome that appear to have been acquired via horizontal gene transfer

Question 24

What are 4 examples of genomic islands?

Answer 24

• Pathogenicity island in E. Coli containing acquired genes that allow for virulence
• Metabolic island in Salmonella senftenberg containing acquired genes that allow for sucrose uptake
• Symbiosis island in Mesorhizobium loti containing acquired genes that allow for symbiosis
• Resistance island in S. aureus containing genes that allow for Methicillin resistance

Question 25

What are the steps of the environmental regulatory system?

Answer 25

• Sensor kinase detects an environmental signal outside the cell
• The signal triggers or prevents autophosphorylation of a conserved histidine group
• When autophosphorylation is triggered, a phosphate is transferred from the sensor kinase to a response regulator in the cytoplasm
• The response regulator binds to DNA and either stimulates or represses target genes
• A phosphatase removes the phosphate and downregulates the system, but the response regulator may be phosphorylated again

Question 26

What are the functions and characteristics of the sensor histidine protein kinase?

Answer 26

• A phospho-group from the histidine residue is transmitted to an aspartate residue on a response regulator
• It consists of a conserved transmitter domain (the histidine residue) and variable response domain (the histidine protein kinase that detects environmental signal)
• Sensors usually form dimers and autokinase activity (autophosphorylation-dependent protein kinase) is present. When autophosphorylation is activated, ATP binds to one subunit and phosphorylates the other, a process known as transphosphorylation
• It is membrane associated or cytoplasmic

Question 27

What are the functions and characteristics of the response regulator?

Answer 27

• Regulators consist of at least two domains, a conserved receiver domain containing the aspartate residue, where the phosphate group is transferred, and a variable regulator
• Different regulators have different half lives when they are phosphorylated. The length of the half life determines the duration of the response to changing environmental conditions
• Many response regulators dimerize, combine with another molecule, after phosphorylation and bind to DNA
• CheY and CheB are two response regulators involved in chemotaxis. They modulate the activity of other proteins without binding to DNA

Question 28

How does the EnvZ/OmpR system of E. Coli operate?

Answer 28

• Signal binding causes the activation of the autokinase domain of the sensor histidine kinase EnvZ resulting in ATP hydrolysis and the phosphorylation of a histidine on the phosphotransferase on the regulator domain of response regulator OmpR
• The phosphoryl group from phosphotransfer domain of EnvZ is transferred to the aspartyl group on OmpR

Question 29

What is the purpose of having regulatory systems with multiple components?

Answer 29

It allows for the integration of various signals since proteins can fuse into multidomain proteins in response to different signals.

Question 30

What is chemotaxis and what is its use?

Answer 30

It refers to the gradient sensing mechanism that allows prokaryotes to compare concentrations of a chemical outside of its membrane over a short time interval using the flagellar motor. This makes it possible for prokaryotes to make spatial comparisons over many cell lengths rather than a single cell length. It allows bacteria to quickly move towards attractants and from repellants.

Question 31

How does a flagellar motor operate?

Answer 31

H+ moves from a high to low concentration gradient activating a mot protein and propelling the movement of the Fli proteins (motor switch) which moves the MS ring and then further moves the L and P rings and then the hook outside the outer membrane.

Question 32

How does bacterial movement differ in the presence of an attractant or repellant compared to when there is an absence of a chemical gradient?

Answer 32

In the absence of a chemical gradient bacteria move randomly where they “run” smoothly and then “tumble.” When a chemical gradient is present, bacteria bias this random motion to include longer runs and fewer tumbles up the concentration gradient of an attractant or down the concentration gradient of a repellant. This directed motion is known as chemotaxis.

Question 33

How is chemotaxis modulated in E. Coli?

Answer 33

A signal transduction system that include MCP in the outer membrane, a sensor histidine kinase, and a response regulator are responsible for modulating chemotaxis in E. coli by alternating the duration of period of smooth swimming (counterclockwise rotation) and tumbling (clockwise rotation) of the flagellar motor to include more smooth swimming and less tumbling either towards an attractant or away from a repellant.

Question 34

MCP

Answer 34

Methyl accepting chemotaxis proteins that sense attractants and repellants and trigger a chemotaxis response in E. Coli and eventually affect flagellar motion. Located in the bacterial “nose.” Tar is one example that senses aspartate and maltose as well as the repellants cobalt and nickel. Attractants and repellants generally bind to different sites.

Question 35

What is the function of CheA and what will occur if a mutation is present in the presence of an attractant and repellant?

Answer 35

Function: Sensor histidine kinase. When bound to an activated MCP a conformational change occurs that changes the rate of autophosphorylation with the help of CheW. Rate of autophosphorylation decreases in the presence of an attractant and increases in the presence of a repellant

Mutation: In the presence of an attractant, autophosphorylation will not slow meaning cheY will remain phosphorylated. Since phosphorylated CheY interacts with FliM and induces clockwise rotation of flagellar motor, motor will remain clockwise resulting in more tumbling and less smooth swimming.

In the presence of a repellant, autophosphorylation will not increase so cheY phosphorylation will not increase. This would mean counter-clockwise rotation and smooth swimming would continue even in the presence of a repellant.

Question 36

What is the function of CheY and what will occur if a mutation is present?

Answer 36

Function: When CheA is phosphorylated, phosphate is transferred to the aspartate residue on the response regulator CheY. Phosphorylated CheY then interacts with FliM, which is a component of the flagellar motor that changes the direction of rotation from counterclockwise to clockwise resulting in tumbling.

Mutation: When a mutation occurs, CheY is unable to be phosphorylated and/or unable to interact with FliM and induce clockwise, tumbling rotation so this results in more smooth swimming and ccw rotation

Question 37

When occurs in the flagellar motion signal transduction pathway in E. Coli when an attractant is present?

Answer 37

• Attractant is bound to MCP (methyl accepting chemotaxis proteins) which affects thr pathway
• CheA decreases its rate of phosphorylation when attractant binds to an MCP, which means CheA is more likely to remain unphosphorylated
• Since CheA is not phosphorylated, CheY remains unphosphorylated and does not interact with FliM
• Disruption of CheY and FliM interaction switches the flagellar motor to counterclockwise rotation which means smooth swimming occurs

Question 38

What is the function of CheW and what will occur if a mutation is present?

Answer 38

Function: Helps slow the rate of autophosphorylation to CheA when an attractant binds to MCP. Keeps CheA dephosphorylated

Mutation: If a mutation is present CheA is less likely to remain dephosphorylated in the presence of an attractant, so this means the phosphorylation of CheY is more likely to occur resulting in more clockwise rotation and tumbling

Question 39

What is the function of CheZ and what will occur if a mutation is present?

Answer 39

Function: Keeps CheY dephosphorylated in the presence of low levels of phosphorylated CheA

Mutation: If a mutation is present, CheY will be more likely to be phosphorylated resulting in more cheY FliM interaction and therefore more clockwise rotation and tumbling

Question 40

What occurs in the flagellar motion signal transduction pathway when a repellant is added?

Answer 40

• The binding of a repellant to MCP leads to an increase in CheA autophosphorylation
• Phosphorylated CheA transfers its phosphate to CheY to stimulate its interaction with FliM
• Phosphorylated CheY interacts with FliM and the flagellar motor switches to clockwise rotation and more tumbling

Question 41

How does adaption affect the flagellar motion of cells?

Answer 41

It allows for the resetting of the signaling state of the MCPs. This means in the presence of high but unchanging concentration gradients of attractant, the presence of attractants eventually doesn’t trigger the same flagellar motor pathway and cells are able to randomly move rather than maintain their biased random walk until they reach the highest concentration of attractant.

Question 42

What role does Quorum sensing play in sporulation?

Answer 42

Rap phosphatases block the transmission of phosphate to Spo0A by dephosphorylating Spo0F. When there is a high cell density, cells secrete and respond to small peptides via ABC transporter Spo0K, and since peptides inactivate the Rap phosphatases, this can cause Spo0A to therefore be phosphorylated and activated. This means sigH gene can be further transcribed and sigma H can be activated since sigma H is partially activated by the transcription of the sigH gene that is dependent on the phosphorylation of Spo0A.

Question 43

What role do Spo0A and Sigma H play in sporulation?

Answer 43

They drive the expression of genes that are responsible for the switch from medial to polar septation.

Question 44

What is the role of Sigma E and Sigma F in sporulation?

Answer 44

They are required for the engulfment of the cell that would form a spore.

Question 45

What is the role of Sigma G in sporulation?

Answer 45

Sigma G is responsible for activation of sigma K in the forespore. It sends a signal across the forespore membrane to activate Sigma K in the mother cell.

Engulfment is coupled with activation in the forespore

Question 46

What is the role of Sigma K in sporulation?

Answer 46

Induces the expression of genes that allow for a coat formation

Question 47

How is the spore coat formed in an endospore and what is its role?

Answer 47

Gene expression in the mother cell leads to the formation of a spore coat. This coats the spore DNA with proteins that protect the spore from solar radiation and also helps dehydrate the spore allowing it to be resistant to high temperatures. After the coat is formed the mother cell lyses releasing the endospore.

Question 48

What is a transporter and what are three types?

Answer 48

A transporter is a membrane spanning protein that typically contains 12 alpha-helices that form a channel through the membrane.

Three types are uniporters, antiporters, and symporters

Question 49

What kind of transporter is LacY and what role does it play in gene regulation?

Answer 49

LacY is a symporter that takes in lactose with H+ and depletes the proton gradient. It is coded by a gene on the lac operon.

The movement of lactose into the cell then induces the lac operon where small amounts of lactose are converted to allolactose by LacZ, which is also coded by a gene on the lac operon.

Question 50

How is the lac operon repressed?

Answer 50

The repressor LacI binds to lacO and the bound protein overlaps the lacZYA promoter creating a DNA loop and preventing transcription. This process is known as repression.

Question 51

How does repression induction occur in the lac operon?

Answer 51

The inducer is allolactose, made from lactose using lacZ, which reduces the lacI affinity for lacO and allows transcription to occur. This process is known as repression induction.

Question 52

How is the lac operon activated via catabolite activation?

Answer 52

Uniporter IIB phosphorylates glucose as it enters the cell rather than IIA as part of the phosphotransferase system and this means unphosphorylated IIA accumulates and inhibits LacY. When glucose is absent phosphorylated IIA accumulates and LacY is free to transport lactose leading to the increased formation of allolactose and increased lac operon expression. This is known as catabolite repression. cAMP which is a lac operon activator can also accumulate and induce expression, this is activation. The entire system is catabolite repression/activation.

Question 53

What type of transport is the phototransferase system (PTS) and what are the key steps of the phototransferase system?

Answer 53

PTS is a uniporter that allows glucose to enter the cell after several conditions are met. First, phosphoenolpyruvate supplies a high energy phosphate bond to Enzyme I. HPr is then used to help transfer the bond to Enzyme II and as glucose moves across the membrane through enzyme II-c it is phosphorylated into glucose-6-phosphate.

Question 54

How does catabolite repression occur in xylose regulon expression in B subtilis?

Answer 54

HPr is produced when glucose is present and HPr-P (produced via the phototransferase system) binds to and activates CcpA which represses the xylose operon in B. subtilis. This is known as catabolite repression.

Question 55

What are different characteristics and types of ABC transporters?

Answer 55

They generally have high affinity substrate binding. In gram-positive bacteria the substrate binding protein is anchored in the plasma membrane and in gram negative bacteria the substrate binding protein is floating in the periplasmic space.

It is required for cell-cell signaling. Maltose binding protein for example part of the maltose transporter allows it to be easily bound and transported at a high efficiency making it ideal for protein purification use.

Question 56

What are the different secretion routes and what proteins are they correlated with.

Answer 56

Sec, SRP, and Tat pathways go towards and through the inner membrane into the periplasm from the cytoplasm.

Types II and V secretion systems go towards and across the outer membrane from the cytoplasm and stops in the periplasm, but the type II protein has a component through the inner membrane. Proteins moving through Tat and Sec pathways can go through Type II proteins to get from periplasm to outside the cell through outer membrane.

Type I secretion system goes from cytoplasm to outside the cell

Type III, IV, and VI secretion systems to from cytoplasm to outside the cell but sometimes they attach to another host cell

Question 57

How does the Sec/SRP Pathway move proteins from the cytoplasm to the periplasm?

Answer 57

SecB attaches to the protein and SecA is recruited to push the protein through SecYEG into the periplasm. To recruit a protein into the inner membrane, SRP recruits a protein and FtsY helps move it towards SecYEG which then inserts it.

Question 58

How does the Tat pathway help move proteins from the cytoplasm to the periplasm?

Answer 58

TatB and TatC bind to a protein and guide it towards TatA, which it moves through to enter the periplasm.

Question 59

How does the Type I Secretion pathway transport proteins across the bacterial envelope? How does this mechanism occur in the secretion of Hemolysin A in E. Coli.

Answer 59

ATP-binding cassette (ABC) containing transporters are used.

Type I secretion is used to secrete Hemolysin A (HlyA) from E. Coli to a host cell membrane. First HlyA interacts with a HlyB ATP binding cassette and a HlyD membrane fusion protein and a proton motive force is used to attach HlyA. It is then transported across the inner and outer membrane, and this process requires ATP hydrolysis and/or binding. TolC, an outer membrane protein so it is usually found in gram-negative bacteria since gram-positive don’t have an outer membrane

HlyA is a hemolytic protein with a calcium binding repeated segment, and this is thought to interact with the host cell membrane and trigger integration. Integration in HlyA creates pores in the plasma membrane and allows cytoplasmic contents to leak out killing the cell.

Question 60

What is required for Type II secretion?

Answer 60

An N terminal signal sequence is required and a signal peptidase that is located in the periplasm of Gram-negative cells process the signal sequence once the peptide is across an inner membrane.

Since the periplasm is an oxidizing environment and the cytoplasm is a reducing environment, proteins that are secreted in this way assume their proper tertiary and quaternary structure in the periplasm using chaperones and specialized proteins that reshuffle disulfide bonds.

Question 61

How does Type II Secretion usually occur in E. Coli?

Answer 61

Generally after emerging from the ribosome, a preprotein is bound by SecB protein which prevents complete tertiary/quaternary folding. This substrate is transferred to SecA and translocated through SecYEG into the periplasm using ATP. This preprotein is then processed in order to refold into its proper tertiary and quaternary structure. To rearrange disulfide bonds specifically, electrons flow from thioredoxin to DsbC via DsbD, a cytoplasmic membrane protein.

Substrate is usually recognized by type II machinery after it is fully formed. So instead after it moves to the periplasm via SecYEG, the outer membrane secretin GspD is recruited and serves as a translocator for PulA to move it outside the cell.

Question 62

What is required for Type III Secretion and how does it usually occur?

Answer 62

Yersinia is the type III secretion machinery that is made up of plasma membrane proteins and the outer membrane secretin SctC. When type III secretion is needed, SctF is transported by this machinery and assembles into a needle-like structure on the bacterial surface on top of the Yersinia complex. This is thought to be somewhat related to flagellar assembly and it is possible SctJ is a rough equivalent of flagellar MS rings.

Question 63

What proteins are involved in Type III secretion?

Answer 63

YopE is an effector protein that kills eukaryotic cells.

Ysc is the protein complex that forms the type III secretion system.

YopN is the “cork” that blocks the secretion system until a bacterium encounters a eukaryotic cell

YopB/D proteins on the host cell translocate YopE and other effector proteins into the host cell. These effector proteins be used to disable macrophages and other immune cells.

Question 64

How was it determined what is necessary and sufficient for YopE secretion???

Answer 64

Hybrid protein analysis was used to determine that the first 15 amino acids of YopE/N are sufficient for secretion since none of the frame shifts after 15 amino acids affected secretion. They were able to conclude that one of the signals for Yop secretion is dependent on mRNA sequence/secondary structure. Deletion analysis was also used to conclude that SycE binding to residues 15-100 of YopE is also needed for secretion.

Question 65

What is Type IV secretion generally used for?

Answer 65

The machinery used is similar to conjugation so it can generally transport both DNA and proteins. It is the mechanism of Crown Gall Disease where a virus transfers T-DNA and proteins that cause a nick in host DNA and allow viral DNA to be inserted.

Bordetella pertussis also uses Type IV secretion to secrete a Pertussis toxin across an inner and outer membrane into an extracellular environment which then binds to a mammalian cell membrane, so Type IV secretion doesn’t always involve DNA transfer.

It is also the mechanism by which Legionella survives in phagosomes and prevents phagosome-lysosome fusion.

Question 66

What typically occurs during adherence?

Answer 66

It usually involves an interaction between a molecule on the bacterial cell surface and a receptor or other cell surface protein on the host cell, and it is specific for both the host and the tissue.

There is generally a type I Pili that binds to glycoproteins and mediate this attachment. Sometimes adherence induces the formation of “pedestals” by intestinal epithelial cells.

Question 67

What typically occurs during invasion?

Answer 67

Pathogens have to get past the cell epithelium, and this often occurs through breaks/lesions in either the epithelium or the mucosal membrane. There are intracellular and extracellular pathogens, and invasion by intracellular pathogens usually requires that they enter host cells. Legionella for example is usually engulfed by macrophages.

Question 68

What typically occurs during colonization?

Answer 68

Inoculum is rarely enough to cause disease, so bacteria must be able to grow inside the host. This means colonization is dependent on the availability of nutrients and growth factors, and is generally limited to specific tissues in which the organism is well adapted. Brucella abortus for example only grows well in the placenta of infected cattle since elevated concentrations of erythritol promote its growth. HIV also only grows well in CD4 T cells.

Colonization is assisted by pathogen specific factors such as siderophores that can help the bacterium strip iron from iron binding proteins that are derived from its host such as transferrin. Most infections are localized but some can enter the blood stream via lymph vessels/nodes causing a systemic infection and bacteremia, the shedding of bacteria in the blood, can even develop.

Question 69

How does botox function as a virulence factor and what effects can this have?

Answer 69

Botox is an endotoxin that causes flaccid paralysis, and it can lead to respiratory paralysis and cardiac failure/death in more severe cases. It is secreted from Clostridium botulinum and is typically due to eating uncooked/processed foods.

But it is also used in plastic surgery since it can eliminate wrinkles using this mechanism of making cells flaccid and it can be used to treat migraine headaches.

Question 70

What are three nonspecific host defenses?

Answer 70

Skin

Mucosal lay

The complement system (direct pathogen killing, opsonization, continued inflammation)

Phagocytes

Question 71

Why do the skin an nails serve as protective features that prevent microbial entry?

Answer 71

Dead cells on the skin suface provide a dry environment that is not idea for microbial growth so viruses cannot replicate here. Cells are pushed towards the surface of the skin and these cells produce keratin which is difficult for microbes to digest. Nails are also keratinized.

SALT (skin associated lymphoid tissue) is just below the surface of the skin and it is the specific defense system connected to the skin. This specific response is initiated by Langerhans cells which are phagocytic cells that engulf and destroy microbes. Langerhans then signal other immune cells. This means the skin has to be breached, and an insect, cut, or burn can do this.

Question 72

What is the role of the Mucosal membrane in protecting against pathogens?

Answer 72

It is inside the body but generally in areas that are constantly exposed to material from external environment (intestinal tract, respiratory tract, vaginal tract, bladder). The lining of these areas consist of a few layers of mucosal cells, but barriers have to be thin and exposed to body fluids since fluids or gases have to pass through them.

Mucin provides the primary barrier for mucosal membranes and consist of a mixture of proteins and polysaccharides. This traps the microbes and prevents them from reaching epithelial cells (internal and external). It can also cause lubrication to prevent mechanical damage. It also contains antimicrobial compounds such as lactoferrin, lysozyme, and defensins, which are small proteins that make pores in microbial cell membranes. Mucus is constantly being shed and replaced in order to expel microbes trapped, and the underlying epithelial cells are also shed.

It is associated with specific host defense MALT (mucosa-associated lymphoid tissue) containing phagocytic cells underlying it that engulf invades and signal the specific immune system.

Question 73

What is the role of Natural Killer cells in protecting against pathogens?

Answer 73

NK cells target intracellular pathogens. They are part of the adaptive immune system and target specific antigens. They attach to infected cells and kill them by releasing granules that contain molecules that are toxic to human cells. This process is similar to cytotoxic T cells, but they are less specific, and they recognize cancer cells in labs.

Question 74

What is the role of neutrophils in protecting against pathogens? How do they attack?

Answer 74

Neutrophils target extracellular microbes and are known as polymorphonuclear leukocytes and are commonly produced in the bone marrow. Even though they are short lived, they circulate in the blood stream constantly. Polymorphonuclear refers to the multilobed structure of the nucleus. They attack as the microbe is engulfed and enclosed in a phagosome.

The interior then acidifies in response to the active transport of protons into it by a membrane ATPase. The neutrophil also containss granules called lysosomes that contain substances that are toxic to the microbe such as myeloperoxidase. When a phagosome and lysosome fuse, myeloperoxidase is activated and HOCl from hydrogen peroxide is produced along with chloride anions. Peroxide and superoxides are also produced from the respiratory burst and contained in a lysosome. These radicals and enzymes must be contained in the lysosome since they are also toxic to bacterial cells.

Question 75

How is the complement system activated and what are three ways it can protect against pathogens?

Answer 75

It is partly activated by molecules such as lipopolysaccharide (LPS on the gram-negative surface) and lipoteichoic acid (LTA on gram-positive surface) signatures on the bacterial cell surface

Inflammation: promotes the continued and amplified response to infection

Opsonization: coats the surface of pathogens that allow phagocytes to target it

Direct pathogen killing: directly forms pores in the pathogen membrane

Question 76

What are different ways collateral damage via the immune system can occur?

Answer 76

MAC indiscriminately and can cause collateral damage to “bystander” red blood cells

Toxic contents from neutrophils can sometimes leak out into surrounding tissue

Neutrophils can damage blood vessel walls as they squeeze out

Redness/swelling comes from leakage of blood into tissue

Pain occurs from damage to nerve endings by toxic substances released from neutrophils

Pus and dead tissue are also possibly the result of neutrophil attack

Question 77

What role do cytokines play in the immune response?

Answer 77

They are small proteins that are secreted and produced by many cell types especially the endothelial cells line the blood vessels/macrophages

Cytokine-producing cells have a receptor on surface which binds to bacterial factors (LPS, LTA, peptidoglycan)

Binding to LPS stimulates the cell to release cytokines which further stimulate neutrophils to kill invaders

Question 78

What is septic shock?

Answer 78

May develop if inflammatory response is triggered all over the body. When this occurs, neutrophils attach to blood vessels throughout the body and exit the blood stream meaning blood pressure drops and fluid loss occurs. Neutrophils may attack blood vessels exacerbating this, and this starves internal organs leading to death/disability.

Question 79

What are bacterial countermeasures against neutrophils, phagosomes, and MACs?

Answer 79

A microbe could contain a capsule and resist infection

A microbe could kill a neutrophil

A microbe could grow inside a phagocyte/phagosome freeing bacteria in the cytoplasm

Bacteria could produce a protease that degrades C5a to stop neutrophils from following it

Antiphagocytic capsule on bacterial cell surface can prevent uptake or mimic human cell surface with sialic acid to prevent neutrophil engulfment

Bacteria could prevent phagosome/lysosome fusion

Cell wall of bacterium could protect it from toxic chemicals released from phagosome

LPS can be modified and make it resistant to MAC formation

Question 80

What can antibodies do?

Answer 80

Opsinization can occur where antibodies can tag foreign pathogens for elimination

Antibodies can neutralize a toxin and prevent it from binding to a cell

Antibodies can activate the complement system

Antibodies can help trap microbes in mucin

Question 81

What are the 5 Immunoglobin classes and their functions/characteristics

Answer 81

IgM - Pentamer with 10 antigen binding sites, main antibody of primary responses, best at fixing complement sites, monomer form is a B cell receptor

IgG - Main antibody of secondary responses and crosses placenta, makes up 80% of antibody serum, main blood antibody of secondary responses and binds to phagocytes, neutralizes toxins and opsonization

IgA - Dimer that is secreted into mucus, tears, saliva, and colostrum

IgE - Antibody of allergy and antiparasitic activity, binds to mast cells and basophils

IgD - Serves as a B cell receptor

Question 82

B-cells

Answer 82

Antibody producing lymphocytes that mature in the bone-marrow. They are responsible for antigen interaction, antibody production, and immune memory and contain Ig molecules on their surface. Surface Igs recognize an antigen in the native conformation of a pathogen and it is then activated.

They divide into memory cells, which can proliferate and secrete specific antibodies if exposed to the same antigen, and plasma cells, which are antibody producing cells that only live for a few days and produce a large amount of specific antibodies in response to an antigen.

Question 83

T-cells

Answer 83

Lymphocytes that mature in the thymus and contain antigen specific T cells receptors on their surface and interact specifically with an antigen. Antigen is processed and presented to the TCR as an APC.

CD8 are cytotoxic T-cells that kill antigen bearing cells through an interaction between a cell surface antigen on a target cell and TCR, usually involves the release of granules

CD4 cells are helping cells. In response to an antigen Th1 recruit nonspecific effector cells like phagocytes to a specific cell and Th2 cells stimulate B lymphocytes to produce antibodies which then convert memory cells into plasma cells which then more quickly secrete antibodies.

Question 84

APCs (antigen presenting cells)

Answer 84

Amateurs are infected cells that are made up of foreign proteins which are made inside the cell from viral DNA to protect against viruses

Professionals are phagocytic cells that ingest foreign particles and present proteolyzed components of an antigen

Question 85

What is a TCR and how does it contrast to antibodies?

Answer 85

TCR is expressed in the surface of all T cells and consists of a constant region and variable region that can bind to specific antigens when presented on the surface of an MHC. It can recognize millions of different antigens.

In contrast to antibodies, TCRs recognize an antigen through the MHC (major histocompatibility complex) which recognize a wide variety of antigens relative to Igs and TCRs. Class I MHCs are on the surface of amatuer APCs (infected cells) and class II are on the surface of professional APCs like macrophages.

Question 86

How do cytotoxic T cells target host or cancer cells?

Answer 86

An antigen is made in a host cell and bound to the cell surface by an MHC class I after going through the endoplasmic reticulum. This MHC Class I presenting antigen then interacts with the TCR on the surface of cytotoxic T cells, and T cells then activate to kill the cell, granules are usually released.

Question 87

How do MHC Class II APCs and helper T cells target extracellular pathogens via Humoral Immunity?

Answer 87

Antigens are processed from a pathogen by an APC (usually a phagocyte) after being ingested, processed, and then displayed as an MHC to form an MHC II complex.

T helper cells then recognize this antigen the TCR binds to the MHC I. Then a superantigen binds which allows T cells to reactivate and systemic inflammation and/or shock can occur.

Question 88

Interleukins

Answer 88

Cytokines that mediate the interactions between leukocytes, usually Th2 helper cells, macrophages containing MHC I complexes, and B cells.

IL-1 are secreted by macrophages and TH cells are the main target

IL-2 is produced by an activated TH cell in response to IL-1. It binds to the IL-2R surface of a TH cell and it induces the cell to divide and make clonal copies, this is autocrine signaling

ILL-4 is produced by an activated TH cell in response to IL-1 and IL-2 so it binds to IL-4R on B cells. B cells are stimulated to proliferate and differentiate into antibody producing plasma cells

Question 89

Primary response vs secondary response and direct B cell stimulation??

Answer 89

After an antigen is introduced, there is a latent period before an antibody appears in the blood and this is followed by a gradual antibody increase and slow fall.

When second exposure to an antigen occurs, several days/weeks later an antibody titer rises to 10-100 times the primary response.

Question 90

How does T cell tolerance develop???

Answer 90

Positive selection occurs as immature T cells leave the bone marrow and travel to the thymus. T cells interact with the MHC via TCRs and those that don’t die in a process called apoptosis.

Negative selection occurs as T cells that recognize MHC are bound to antigens presented by thymus cells that bind tightly to thymus tissue and then die. If T cells don’t bind tightly, they migrate to the spleen and lymph and then encounter foreign antigens

Question 91

How is adaptive immunity acquired?

Answer 91

Natural active immunity where an infection renders an individual immune.

Artificial active immunity (immunization) where an injection of an antigen results in antibody formation.

Artificial passive immunity where the injection of antiserum from an individual who has developed active immunity can lead to immunity

Natural passive immunity where newborns retain maternal IgGs in blood stream for months after birth.

Question 92

How can adaptive immunity lead to complications?

Answer 92

They could miss an infectious agent, misidentify an external compound as an infectious agent (allergies), or misidentify a bodypart as an infectious agent (autoimmune)

Question 93

How is Wolbachia transmitted and why did releasing Wolbachia infected mosquitos in Indonesia reduce dengue infection rates?

Answer 93

Wolbachia is an intracellular parasite and it has colonized over half of the world’s insect species. It is transmitted from mother to offspring through the germ line.

Wolbachia infected mosquitos experience virus inhibition which prevented dengue spread.

Question 94

What are 4 kinds of reproductive manipulation?

Answer 94

Feminization where genetic males develop as females, possibly due to insertion into a chromosome or the evolution of a new sex chromosome

Parthenogenesis where unfertilized eggs are developed

Male killing where the male embryos of infected females experience embryonic lethality

Cytoplasmic incompatibility where an infected male and uninfected female cannot produce offspring

Question 95

How do squid acquire Vibrio fischeri? How is host filtering used in this process?

Answer 95

Squid acquire Vibrio from environment through horizontal transmission where Vibrio colonize crypts inside a light organ and the squid then eject the Vibrio bacteria from crypts and reacquire the bacteria throughout the day.

Squid secretes chitobiose which is a chemoattractant in the mucus at the entrance of the light organ attracting bacteria. The squid excludes all bacteria other than V. fischeri using its innate immune system and other factors. Specifically cilated and mucus-producing endothelial cells prevent bacterial adhesion
Innate immune cells secrete antibacterial factors that prevent invasion/colonization of bacteria attracted to it. The presence of bacteria can initiate cellular changes in the host, such as luminescence in squid due to V. fischeri and the development of a light organ.

Question 96

Are there any symbiotic elements in the relationship between the Wolbachia parasite and insects?

Answer 96

Wolbachia can prevent viral infection in insects. Specialized transporters allow Wolbachia to obtain nutrients from their host, genes for biosynthesis of purines/pyrimidines are conserved in Wolbachia, and nucleotide metabolism gene expression is changed in Wolbachia-infected insects so that when nutrients are limited, Wolbachia is beneficial

Question 97

What factors control what kinds of microbes are colonized and what persists?

Answer 97

Dispersal - the movement of microbes between environments determines which microbes are available, and sometimes host interactions (such as squid and light organs) contribute to this

Selection - Host filtering can exclude or recruit microbes through the immune system and select based on changes to the host environment

Diversification - Once microbes colonize an organism, they can evolve into new species

Ecological Drift - Random processes may cause some microbial populations to vanish

Priority effects - The first colonized microbe influences subsequent colonization and the resulting microbial community

Question 98

What is phylosymbiosis and what can lead to it?

Answer 98

Phylosymbiosis is the microbial community relationships that recapitulate or are analogous to the phylogeny of their hosts. This can occur due to ecoevolutionary processes such as microbial dispersal, microbial selection and host filtering, microbial diversification, ecological drift, and priority effects

Question 99

What are different ways Vibrio luminescence in squid is regulated?

Answer 99

Vibrio MAMPs drive expression of the cry light receptor genes in the squid which is a clock gene. This means expression of light receptor clock genes are expressed more frequently at night and luminescence therefore increases at night.

Quorum sensing can also regulate bioluminescence through the Luxl protein mechanism. Luxl protein initially synthesizes an acyl homoserine lactone autoinducer (AI). The AI then diffuses and accumulates, and at a threshold concentration, AI can diffuse into the cell and bind to LuxR, which activates lux transcription. This means lux transcription tends to occur when there is a greater biomass.

Question 100

How do Vibrio contribute to the development of its host?

Answer 100

The presence of Vibrio is necessary for light organ development since Vibrio secretes TCT (tracheal cytotoxin) which triggers tissue reorganization and can lead to light organ development. This was proven since HPLC used a similar molecule? and the addition of TCT to a developing squid resulted in the complete development of a light organ.

Question 101

What are Genus Rhizobium and what role does it play in agriculture?

Answer 101

They associate with legumes and are able to fix nitrogen from the air, so this means farmers generally rotate crops with legumes in order to maintain the nutrients in the soil.

They are gram-negative, associate with the roots (sometimes stems) in a species specific manner, enter the root via root hairs, invade plant cells and differentiate into semi-dormant nitrogen fixing state (bacteroids). This leads to the formation of nodules (groups of plant cells with high numbers of bacteroids)

Question 102

How does nodule formation occur?

Answer 102

1) Rhizobium recognizes and attaches to root hairs, and this is mediated by Rhicadhesin which is a calcium binding protein in the rhizosphere

2) The excretion of rod factors in the bacteria cause root hair curling

3) Rhizobia penetrate the root hair and multiply using an infection thread.

4) Bacteria in this infection thread grow towards the root cell and the invaded plant cells are then stimulated to divide

5) Bacteroids form within plant cells

6) There is then continued plant and bacterial cell division and reproduction of nodules as the root continues to grow

Question 103

What plasmid is used in nod formation and what products are formed/activated?

Answer 103

A region of the sym plasmid in Rhizobium bacteria are key in nodulation, and the sym plasmid also contains genes that restrict infection to a specific host plant.

nodD is an activator for the transcription of nod genes which mediate the interaction of bacteria with plant root hairs. They do this by bending DNA at the promoter and enhance RNAP binding and transcription.

nodA, nodB, and nodC produce nod factors, that are chitin like molecules that induce root hair curling and nodule formation. Exogenous nod factors can also induce nodule formation even in the absence of bacteria?? (are these on the plant?)

Question 104

How is nodD induced?

Answer 104

It is activated by inducers, one of which are plant flavonoids. Flavonoids are organic molecules that are secreted in large quantities by the roots of leguminous plants. They are involved in growth regulation and they attract pollinating animals along with other activities that promote growth/reproduction.

Question 105

Why are nodules red in color?

Answer 105

Rhizobium can only fix N2 under microaerophilic conditions (low O2). The bacteria needs some O2 for nitrogen fixation, but the nitrogenase is inactivated by high levels of O2. Therefore the plant produced binding protein leghemoglobin keeps O2 levels low and allows Rhizobium to fix nitrogen. Leghemoglobin is an iron containing protein, and it gives the nodules their red color.

Question 106

What do plant cells provide Rhizobium bacteria in a nodule and what do bacteria provide?

Answer 106

Plant cells provide carbohydrates/organic acids which are used to create a proton gradient and produce ATP via the citric acid cycle and NADH. They also provide O2 which acts as the terminal acceptor in a proton gradient and produces more ATP.

Bacterial cells use NADH and ATP to fix N2 into NH3 which can be used by the cell to produce amino acids and ureides. This gives plants a significant growth advantage in nutrient poor soils

Question 107

Xenorhabdus nematophilia

Answer 107

Gram-negative bacteria that has a mutualistic relationship with S. carpocapsae which is a nematode. X. nematophilia lives in a specialized vesicle in nematode intestines.

With the nematode, X. nematophilia infects and kills a wide variety of insect larvae. It needs the nematode for delivery, but the bacteria is the main killer and secretes toxins that kill larvae within 48 hours of infection.

Question 108

How is X. nematophilia both a symbiont and pathogen?

Answer 108

It is a symbiont since it kills insects and this allows nemotade growth and development where insects are used as food. Bacteria then colonize nematodes as they grow and reproduce and colonize larvae leading to more colonized nematodes.

X. nematophilia is also a pathogen since when the colonized nematodes migrate to insect hosts, bacteria is released into the haemolymph of insects (usually insect larvae) which then kills the insect host. The bacteria then recolonizes the nematodes as they develop from the insect cadaver and process continues.

Question 109

What did an in vitro colonization assay reveal about the role Sigma-S plays in its mutualistic relationship with the nematode S. carpocapsae and parasitic relationship with insects such as the larvae of M. sexta?

Answer 109

rpoS was looked at since it encodes the transcription factor Sigma-S. This regulon has been implicated in the stress response, survival, and host interactions which makes it a logical candidate.

rpoS mutants were not able to colonize their nematode host, but they were able to kill insect larvae which indicates that Sigma-S is required for the mutalistic relationship between X. nematophilia and their nematode hosts but not for pathogenesis.

Question 110

Vibrio cholera

Answer 110

Caused by a gram-negative “comma shaped” bacterium. It is commonly acquired by drinking water that is contaminated with human feces or can sometimes occur from eating food washed in contaminated water.

In the small intestine, V. cholerae attaches to the mucosal surface and produces a cholera exotoxin that decreases the net flow of sodium into tissues and water and chloride ions out resulting in electrolyte imbalance and diarrhea.

A person with cholera can lose up to 20 L of water a day and this initially leads to a loss of skin plasticity/sunken eyes and later leads to the collapse of the circulatory system. Death toll is an estimated of 95,000.

Question 111

What was John Snow’s theory regarding cholera and how did he prove this?

Answer 111

He conducted an early epidemiology study in London and identified contaminated pump water as the source of a cholera outbreak. People at the time believed cholera was spread by miasmas in foul smelling air, but he conducted extensive interviews with families of victims and identified an infected pump on Broad street since most cases were near there rather than nearby pumps. Epidemic came to a halt when city officials removed the pump.

Question 112

What virulence factors are associated with cholera?

Answer 112

Flagella - V. cholerae have a single polar flagellum that enables bacteria to move through mucosal layers in the small intestine and adhere to the surfaces of epithelial cells

Cholera Toxin (CT) - an exotoxin with an A subunit and 5 B subunits. The B portion binds to a target host cell and the A portion enters the cell.

TCP pili - genes for TCP pili are co-regulated with the cholera toxin genes and they allow V. cholerae to attach to host cells. They can also act as a receptor for CTX phages. They are controlled by ToxR, ToxS, and ToxT

Question 113

How is cholera converted into a deadly pathogen?

Answer 113

CTX bacteriophage enters an uninfected cholera bacterium by pili formed from expression of TCP pili genes, and these genes are acquired from a TCP bacteriophage. When CTX phage is inside the bacterium, the cholera toxin gene is activated by toxT, the same gene that turns on the TCP pili gene.

Question 114

How can cholera infection be treated? What can be done to avoid cholera infection?

Answer 114

Immediate replacement of fluid/salts lost through diarrhea

Oral rehydration solution (prepack with sugar/salts mixed with water and drunk in large amounts)

IV fluid replacement

Antibiotics (not as important as rehydration)

Prompt medical attention

Vaccines (one variant contains killed V. cholerae 01 cells and CT B subunit, other variant contains killed V. cholerae 01 and V. cholerae 0139 cells

“Boil it, cook it, peel, it, or forget it”

Question 115

How are S. Aureus infections transmitted? What are some virulence factors associated with it?

Answer 115

It is a foodborne illness with humans being a major carrier but can be spread through domesticated animals, dry environmental surfaces, skin-to-skin contact, contact with objects by infected persons.

Can cause soft tissue infections, impetigo (skin infection), osteomyelitis (direct infection of bone associated with open/penetrating fractures), or TSS (involves a superantigen)

Some virulence factors are surface proteins that promote colonization of host tissues, invasins that promote bacterial spread in tissues, capsules that inhibit the engulfment of phagocytes, and exotoxins that damage the membrane of cells in host tissues

Question 116

What are nonsocomial infections, and how do they relate to S. Aureus?

Answer 116

S. aureus is accounted for in ~13% of all nosocomial infections between 1990 and 1996. It is the most common cause of surgical wound infections and MRSA is becoming a problems since it is so common in hospitals.

Question 117

How did multi-drug resistant S. aureus develop (MRSA)? What is the last line of defense against MRSA and what are the pathways when it is effective or not effective?

Answer 117

Resistance to methicillin and beta-lactam antibiotics is due to the presence of a second copy of a cell wall synthesis gene (pbpB-2) altering its affinity for antimicrobial compounds.

PBP binds to peptide side chains and forms a cross-link between two peptide side chains to form a cell well and it disassociates once the cross-link has been formed, but when penicillin is added to the system, it enters the active site of PBP and reacts with a serine group. The beta-lactam ring of penicillin is irreversible opened and permanently blocks the active site preventing it from activating and forming the cross-link.

However, PBP2a does not bind Penicillin and remains active in the presence of penicillin.

Question 118

Where is Streptococcus pyogenes found and what kinds of pathologies/diseases can it cause?

Answer 118

It is responsible for strep throat (~50% of sore throats), scarlet fever (via erythrogenic toxin Spe), rheumatic fever (by provoking an autoimmune response), “flesh eating bacteria” (exotoxin Spe is used to necrotize subcutaneous tissue)

Question 119

CMT

Answer 119

Cytolysin-Mediated Translocation that is SLO dependent and allows a supernatant pathogen to enter the cell.

Question 120

M-preotin

Answer 120

“Fuzzy coat” component of S. pyogenes cell surface protein that helps a bacterium evade phagocytic cells. It acts by binding to factor H of the complement system and prevents opsonization. Antibodies can be effective but there are 80 serotypes of M protein.

Question 121

Superantigens

Answer 121

Group of toxins that bind to the MHC and “break” the specificity of the adaptive response so it can lead to inflammation and septic shock due to simultaneous activation of T cells

TSS toxin (superantigen) enters bloodstream to cause TSS

S. pyogenes bacteria growing in an infected wound can enter bloodstream and produce Spe and other exotoxins leading to TSLS (Toxic shock like syndrome)

Question 122

What is necrotizing fasciitis? How does it happen?

Answer 122

Caused by S. pyogenes being made into flesh eating bacteria. But MRSA has developed flesh eating capabilities and 14 out of 843 patients in a study acquired MRSA infections with flesh eating capabilities.

Question 123

What is one organic location and one inorganic location where biofilms form?

Answer 123

Bacterial populations are usually embedded in a biofilm, or a polysaccharide matrix attached to a solid surface such as a rusty automobile or teeth.

Question 124

What problems can biofouling cause and how can we get around these problems?

Answer 124

It is typically initiated by the formation of bacterial biofilms and can form on pipes and cause blockage/corrosion. Antifouling paints are usually used to prevent this.

Question 125

What are the stages of biofilm development?

Answer 125

Reversible attachment, irreversible attachment (where microcolonies tend to form), stage 1 maturation, stage 2 maturation, dispersion

Question 126

How can confocal microscopy be used to study biofilms?

Answer 126

They can be used to visualize 3D structures using point illumination to acquire images and generate 3D reconstructions of solid objects such as biofilms. This means out of focus images are not used.

Question 127

How can flow cell reactors be used to study biofilms?

Answer 127

Media can be continuously pumped into a slide to study how biofilms react in different conditions.

Question 128

How can microtiter dishes be used to generate a colorimetric assay and study biofilms?

Answer 128

Bacteria were grown in polystyrene microtiter dishes, washed out, adherent bacteria that are part of a biofilm were stained with Crystal Violet, and bacterial density was determined.

Question 129

How were the genes used to identify biofilm formation identified?

Answer 129

A screen was used to identify the factors required for pseudomonas biofilms which is a leading cause of death for CF patients. Variation of this screen was used to identify factors required for biofilm-dependent antibiotic resistance.

Genes encoding Type IV pili required for adherence to surfaces and twitching motility along with a gene encoding fliP (gene involved in flagella biosynthesis). Further studies also identified flagella and twiching motility as preludes to biofilm attachement/formation.

Question 130

Why are biofilms bad when pathogens form them? What are four ways biofilms can make antibiotics less effective? How does antibiotic resistance contribute to this?

Answer 130

1) Metabolic activity of cells in biofilm is reduced due to the presence of substrate concentration gradients and better microenvironments. Reduced microbial activity means less susceptibility to antimicrobials

2) Bacteria within the inner layers of a biofilm may be able to carry out protective stress responses at the expense of other cells since they have more time

3) Multicellularity in biofilm results in better bacterial defenses due to higher “persister cells.” It is also conducive to horizontal gene transfer since there is increased genetic competence

4) Outer layers of a biofilm can absorb damage from antimicrobials

Question 131

How does B. subtilis regulate biofilm formation?

Answer 131

Environmental sensing through a two component system

Activation of genes required for surface attachment of a cell such as pili and adhesins and downregulation of genes that induce flagellar motion

Quorum sensing and the induction of cell density response factors leads to the production of factors required for extracellular matrix production/biofilm ultrastructure

Outer membrane proteins allow the architecture for biofilms to be secreted

Question 132

How does Pseudomona aeruginosa regulate biofilm formation?

Answer 132

Flagella are required to bring bacteria in proximity with a surface and LPS mediates early interactions with other bacterial surfaces using outer membrane proteins. Type IV pilus-mediated twiching motility is required for cells to agregate and changes in gene regulation include upregulation of biosynthesis genes that allow for alignment and downregulation of flagellar synthesis.

Question 133

How does E. Coli regulate biofilm formation?

Answer 133

Flagellum-mediated swimming is required for approaching and moving across the surface. Organism-surface interactions require type I pili and outer membrane protein Ag43 and EPS (colanic acid) is required for development of normal E. Coli biofilm architecture.

Question 134

How does Vibrio cholerae regulate biofilm formation?

Answer 134

Vps is formed by B. cholera to form a mature biofilm.

Question 135

Are phagocytes attracted to or repelled by biofilms? Why are they less effective against biofilms than other planktonic bacteria?

Answer 135

Phagocytes are attracted to the biofilms and phagocytosis is blocked but phagocytic enzymes are released. Phagocytic enzymes damage tissue around biofilm and as bacteria are released from biofilm, dissemination can lead to infection in neighboring tissue.

Question 136

Enterecoccus faecalis

Answer 136

Commensal organism in human gut and grows at a wide range of temperature, pH, salt concentration. It quickly adapts to survive in the presence of detergents and can serve as a reservoir for antibiotic resistance.

Enterococcus bloodstream infections from catheters produced more biofilm than non-catheter related infections and 100% of E. faecalis formed biofilms.

Question 137

Cystic fibrosis

Answer 137

Biofilm formation in Pseudomonas is one of the most common infections in patients. Recurring infection and inflammation damages the lungs and causes respiratory failure.

Question 138

Otis media

Answer 138

Middle ear infection caused by bacteria/viruses. Usually repeated because of biofilms. Haemophilius influenzae biofilm on middle-ear mucosa was visualized. The biofilm was formed within 24 hours of infection and only began to dissipate after 21 days.

Question 139

What are two ways biofilms are treated and controlled?

Answer 139

Prevent adherence and microcolony formation.

Promote detachment and formation of planktonic cells.

Question 140

What role does lactoferrin play in biofilm formation and maintenance? What happens in environments that lack free iron?

Answer 140

Dr. Pradeep Singh determined that Lactoferrin, compound of innate immune system, sequesters iron which is an essential nutrient for biofilms and bacteria remained in planktonic form when lactoferrin/iron was not present.

Question 141

What did Kolodkin-Gal et al. demonstrate?

Answer 141

They identified norspermidine as a substance produced by B. subtilis that promoted biofilm dispersal. It works in conjunction with D-amino acids to inhibit formation and was shown to prevent biofilm formation in B. subtilis, E. coli, and S. aureus

It inhibits planktonic growth but does not break down biofilms.

Question 142

EPEC

Answer 142

Eneteropathogenic E. Coli. Strain of E. Coli that causes watery/bloody diarrhea by attaching to epithelial cells, up to 50% mortality.

Typically have type I Pili that binds to a host cell glycoprotein and mediates attachment. Adherence induces formation of “pedestals” when Tir is injected by T3SS. Tir translocates to the host membrane and bacterial adhesion binds the loop region that projects in into the extracellular space allowing bacteria to be firmly anchored. Then N and C termini of Tir invokes actin phosphorylation EPEC and actin pedestal formation is mediated by Tyr474 phosphorylation preventing phagocytosis.

Question 143

What is shigella and how does shigella infectivity occur?

Answer 143

Requires a set of factors (Ipa, Spa, Mxi, and vir genes) to enter non phagocytic epthelial cell. VirR oligomerizes and prevents expression of genes at 30 deg C but at 37 deg C virR can no longer disrupt its ability to inhibit gene expression of targets, so this signals invasion can occur.

Question 144

What is needed for shigella infection?

Answer 144

37 deg temperature is needed and in the absence of VirR, VirF can bind to virB promoter to induce VirB expression which can then activate ipa, mxi, and spa promoters. spa and mxi operon activation leads to the assembly of a Mxi-Spa Type III secretion apparatus where products of ipa operon are secreted into host cell.

Question 145

What does the ipa operon code for and what do IpaA, IpaB, IpaC, and IpaD do?

Answer 145

It codes for actin motility and allows Shigella to use actin motility to escape a macrophage when encased in a lysosome. IpaB and IpaC are integrated into the host membrane and IpaD guides this. IpaC signals the induction of lamellipodia and ipaA in conjunction with vinculin promotes lamellipodia formation (actin) near the lysosome containing Shigella. VirA then stimulates the membrane allowing Shigella to escape from a lysosome.

Question 146

What happens when “innocent” organisms get inside a eukaryotic host cell?

Answer 146

They are enclosed in the phagosome which is a compartment created when host cell membrane is pinched off. Active protein transports acidifies interior of phagosome and lysosome, another compartment, contains substances that are toxic to microbes such as proteases, defensins, myeloperoxidase. These are activated upon fusion and these radicals/enzymes kill bacterial cells.

Question 147

How is phagosome-lysosome fusion stimulated?

Answer 147

Can be stimulated by Th1 cells

Question 148

How do some organisms survive in the host cell?

Answer 148

Myobacterium spp and Ehrlichia spp block the maturation of phagosomes early on so pH cannot lower and it cannot progress into late endosomal stage

Salmonella allows slightly more maturation and Coxiella, however, is delievered to phagolysosome

Listeria and Rickettsia escape into cytoplasm and Legionella and Brucella assocate with membranes of endoplasmic reticulum

Chlamydia can induce inclusion bodies that associate with mitochondria

Question 149

Listeria

Answer 149

Has adherens that hold adjacent cells together through E-cadherin

Intracellular E-cadherin recruits a and B catenin which bridges cytoskeleton and E-cadherin. This forms actin cables at cell-cell junctions

Question 150

What is actin and how does actin based motility impact listeria survival in a host?

Answer 150

Listeria can mimic host proteins required for actin polymerization (VASP and ActA) so Actin assembly in regions proximal to Listeria can occur. actA mutants are not motile. Listeria can co-opt host cell machinery to move and then escape the cell, it can almost look like a comet tail.

Question 151

What are the characteristics of M. tuberculosis and how does it spread?

Answer 151

Spread by droplet nuceli and expelled when an infectious person secretes droplets so close contacts are at risk of infection. Transmission of active Tb occurs from person with infections.

It is a nonmotile rod-shaped bacterium that is fairly large. It is an obligate aerobe, slow grower, and acid fast (greasy cell wall that forms a lipid shell and resists antibiotics, resistant to killing by acidic compounds, and resistance to lysis) so neither gram positive or gram negative

Question 152

How does Tb bypass lung’s defenses and what secretion systems does it use?

Answer 152

It is able to bypass being killed by macrophages by surviving in macrophages after being consumed using an early endosome which is dynamic, accessible, etc and does not fuse with late endosomes and lysosomes that generally contain toxins. It is then able to multiply within macrophages, burst, and then spread TB and immune response in lungs forms granuloma which causes symptoms

It uses a conventional sec secretion apparatus and recent research as identified a new secretion system Esx, the type VII secretion system T7SS which prevents phagosome lysosome fusion.

Question 153

What is the Tb disease progression?

Answer 153

Bacteria is generally inhaled into lungs and ingested by alveolar macrophages. TB multiplies within unactivated macrophages until they burst and spread. Lymphocytes then begin to infiltrate T cells forming granuloma. Granuloma then becomes surrounded by fibrin which calcifies into a tubercle.

Question 154

What are characteristics of legionella pneumophilia? Where is it found? How does it enter humans? What cells does it infect in a human host?

Answer 154

Acute pneumonia. Fever, malaise, myalgia, headache, etc in the first 24-48 hours then patient becomes ill. It is aerobic, motile, cell wall has fatty acid composition, and there are fastidious growth requirements with high levels of cysteine and iron. It is commonly found in contaminated water sources and cooling towers for air conditioning systems (shower, water fountain, vegetable misters in stores)

Question 155

What are the symptoms and epidemiology behind Legionnaires’ disease? Where do we commonly see infections?

Answer 155

Generally seen in outbreaks with 10% being fatal and can cause up to 15% of community-acquired pneumonia. 300-400 cases originally reported but retrospective study estimated 8000-18000 cases per year.

Question 156

What toxins are exported by L. pneumophila? What are the importance of dot/icm genes in treating Legionnaires’ disease? What secretion system is this associated with?

Answer 156

icm (intracellular multiplication) and dot (defect in organelle trafficking) genes were shown to be important and resemble a plasmid transfer system where there is adherence, pilus retraction, membrane fusion, DNA processing, ssDNA transfer, complementary DNA strand synthesis, cell separation

It is a T4SS Type IV secretion system that secretes over 300 toxins. This is needed since it has to manipulate the host and keep it alive for 24 hours. This secretion system also allows Legionella to survive within the phagosomes of macrophages.

Question 157

What is cryo-electron microscopy and what can we use it for?

Answer 157

Form of TEM where samples are studied at cryogenic temperatures. It allows for the observation of unstained and unfixed specimen in native environments.

Question 158

What are three types of exotoxins?

Answer 158

Membrane disrupting exotoxins that insert pores and other proteins into the host cell membrane

Protein-synthesis disrupting that inhibit protein synthesis

Signal transduction disrupting that alter cell signaling

Question 159

How does the cholera toxin enter the cell and what is the mechanism? What effects do we see?

Answer 159

Entry is dependent on monosialosyl (GM1) ganglioside receptors on surface of intestinal mucosal cells. Cells normally have the diasialosyl form on their surface, but bacterium produces neuraminidase during colonization which degrades gangliosides to monosialosyl form and allows it to serve as a cholera toxin receptor.

There is an A and B subunit. The B subunit binds to the monosialosyl receptor and allows the A subunit to ente. After the A subunit enters, it is cleaved at a disulfide bond into two peptides, A1 and A2. A1 catalyzes the hydrolysis of NAD and the transfer of ADP-ribose group to G proteins, blocking GTPase activity. This causes the rapid elevation of cAMP levels and cAMP-dependent protein kinase A is activated. cAMP-dependent protein kinase A activates the proteins involved in the secretion of chloride ions, bicarbonate, and water resulting in more sodium, chloride, and water exiting the cell.

The only way to control water flow into and out of tissues is to control ion balance, and by blocking G protein function, the cholera toxin alters the activity of sodium and chloride transporters producing an imbalance that leads to a net loss of water and ions from blood and tissues.

Question 160

How is the cholera regulatory circuit initiated?

Answer 160

Genes encoding for CT and TCP (toxin co-regulated pili) production are controlled by a pathway that consists of ToxT, TcpPH, ToxRS, and AphAB transcriptional regulatory proteins.

ToxRS and TcpPH are membrane associated transcriptional regulators that activate the transcription of the ToxT gene which then directly activates genes encoding for CT and TCP.

AphAB is a cytoplasmic protein that functions as an activator for tcpPH gene expression. Mutation in the efflux system reduced tcpPH and toxT transcription which resulted in significantly reduced amounts of CT and TCP while also blocking the colonization of the small intestine.

Question 161

What are virulence factors of S. pyogenes?

Answer 161

Adhesins (M proteins that bind other proteins, F1 and F2 that bind fibronectin, Epa that binds collagen)

Extracellular enzymes (Streptolysin O (SLO) and SLS that lyse red blood cells, S. pyogenes NAD-Glycohydrolase)

Superantigens and antiphagocytic factors

Also Lipotechoic acid can activate cytokine production and may act synergistically with superantigens to lead to septic shock

Question 162

What are some of the advantages and drawbacks of transposon mutagensis?

Answer 162

Advantages

Effective, higher mutation frequency and lower chance of killing
organism, ease of cloning

Able to induce single hit mutations, incorporate selectable markers, Recover
genes after mutagenesis

Drawbacks

More biased than other mutagens since they can only be inserted in specific regions of a genome

Can also have polar effects on downstream genes

Only allows for the identification of loss of function mutations

TnSeq can be used to determine the relative abundance of mutants without screening for specific colonies. Used to determine conditions where if genes are affected in mice correlations of mutants