Midterm Exam 3 FA24 Study Guide PDF

**CH8: MICROBIAL METABOLISM** - Know the different methods for energy production in living cells: **[Substrate Level Phosphorylation ]** *Substrate Level Phosphorylation* is one of two mechanisms for producing ATP. In substrate-level phosphorylation, a phosphate group is removed from an organi...

**CH8: MICROBIAL METABOLISM** - Know the different methods for energy production in living cells: **[Substrate Level Phosphorylation ]** *Substrate Level Phosphorylation* is one of two mechanisms for producing ATP. In substrate-level phosphorylation, a phosphate group is removed from an organic molecule and is directly transferred to an available ADP molecule, producing ATP. - During glycolysis, high-energy phosphate groups from the intermediate molecules are added to ADP to make ATP. Overall, during this process of glycolysis, the net gain from the breakdown of a single glucose molecule is: - Two ATP molecules, - Two NADH molecules, and - Two pyruvate molecules **[Photophosphorylation ]** *Photophosphorylation* is the process by which plants use light energy from the sun to convert ADP into ATP during photosynthesis. Essentially, it's the synthesis of ATP using light energy absorbed by chlorophyll molecules within the plant cell. -  Involves the movement of electrons through an electron transport chain, which generates a proton gradient across a membrane, ultimately driving ATP synthesis through ATP synthase. **[Oxidative Phosphorylation]** *Oxidative Phosphorylation* is a cellular process that generates ATP by using an electron transport chain to transfer electrons from NADH and FADH~2~ to oxygen, with the energy released being used to pump protons across a membrane, creating a gradient that then drives ATP synthesis. - Primary method for generating ATP in most cells, providing energy for cellular functions. - Ion pumps are used to pump H^+^ out of the bacterial cytoplasm into the extracellular space. - H^+^ flow back down the electrochemical gradient into the bacterial cytoplasm through ATP synthase, providing the energy for ATP production. - Know the difference in respiration and fermentation. *Bacteria:* enzyme breaks reactive oxygen species (ROS): superoxide, dismutase, peroxidase, and catalase lack a sufficient amount of enzyme, inorganic/final electron acceptor, electron transport system. (inorganic final electron acceptor could be environmentally dependent) - During carbohydrate catabolism where Photophosphorylation or Oxidative Phosphorylation take place (during glycolysis, citric acid cycle?) During carbohydrate catabolism, **oxidate phosphorylation** takes place primarily during the citric acid cycle, as this is where the majority of electron carriers (NADH and FADH~2~) are produced, which are then used in the electron transport chain to generate ATP through oxidative phosphorylation. **Photophosphorylation** is specific to photosynthesis and does not occur during carbohydrate catabolism. - What are different glycolysis pathways/alternate glycolysis pathways in bacteria? In which glycolysis a maximum ATP is produced, which glycolysis no ATP is produced? Which glycolysis helps in making pentose sugar/ components for nucleic acid. **[Glycolysis]** *Glycolysis* is the most common pathway for the catabolism of glucose; it produces energy, reduced electron carriers, and precursor molecules for cellular metabolism. - Does not use O~2~ itself, however, it can be coupled with additional metabolic processes that are either aerobic or anaerobic. - Takes place in the cytoplasm of prokaryotic and eukaryotic cells. - Begins with a single six-carbon glucose molecule and ends with two molecules of a three-carbon sugar called pyruvate. [Types of Glycolysis Pathways:] **Embden-Meyerhof-Parnas (EMP) Pathway** EMP is a type of glycolysis found in animals and is the most common in microbes. Glycolysis using the EMP Pathway consists of two distinct phases: 1. Energy Investment Phase: - Uses energy from two ATP molecules to modify a glucose molecule so that the six-carbon molecule can be split evenly into two phosphorylated three-carbon molecules called glyceraldehyde 3-phosphate (G3P). 2. Energy Payoff Phase: - Extracts energy by oxidizing G3P to pyruvate, producing four ATP and reducing two molecules of NAD^+^ to two molecules of NADG, using electrons that originated from glucose. 4 ATP produced, 2 ATP used, net gain = 2 ATP with 2 NADH **Substrate-Level Phosphorylation** [Types of Alternate Glycolysis Pathways:] **Entner-Doudoroff (ED) Pathway** Although some bacteria, including the opportunistic gram-negative pathogen *Pseudomonas aeruginosa,* contain only the ED pathway for glycolysis, other bacteria, like *E.coli,* have the ability to use the ED pathway of the EMP pathway. ED converts glucose to ethanol via pyruvate (1ATP, 1 NADPH, 1 NADH). **Pentose Phosphate Pathway (PPP)** Also called the **phosphogluconate pathway** or the **hexose monophosphate shunt.** The intermediates from the PPP are used for the biosynthesis of nucleotides and amino acids. This glycolytic pathway may be favored when the cell has need for nucleic acid and/or protein synthesis. PPP generates NADPH and five-carbon sugars as well as ribose 5-phosphate. - How much ATP is produced in EMP pathways? How much net ATP gain in EMP pathway from one molecule of glucose? In EMP Pathways, 4 ATP are produces, 2 ATP are used and the net gain are 2 ATP with 2 NADH. - How many pyruvates are made from one molecule of glucose? - Know the glycolysis, Breakdown of pyruvate, Citric acid cycle and Oxidative phosphorylation take place in bacteria vs eukaryotic cells. 1. Glycolysis (glucose converted to pyruvate): in cytoplasm. 2. Breakdown of Pyruvate (Pyruvic Acid is converted to Acetyl coenzyme A) occur at mitochondria / prokaryotes -- cytoplasm. 3. Citric Acid Cycle (Chemical Reaction which utilize Acetyl coenzyme A and produce ATP or reducing agent like NADH) occur at mitochondria / prokaryotes -- cytoplasm. 4. Oxidative Phosphorylation (occur at mitochondria, generate ATP from reducing agent: chemiosmosis / prokaryotes -- cell membrane. **Transition Reaction, Coenzyme A, and the Krebs Cycle** For pyruvate to enter the next oxidative pathway, it must first be decarboxylated by the enzyme complex pyruvate dehydrogenase to a two-carbon acetyl group in the **transition state,** also called the **bridge reaction.** - Electrons are also transferred to NAD^+^ to form NADH. - Occurs in the mitochondrial matrix of eukaryotes; in prokaryotes, it occurs in the cytoplasm because prokaryotes lack membrane-enclosed organelles. To proceed to the next phase, the comparatively tiny two-carbon acetyl must be attached to a very large carrier compound called **coenzyme A (CoA).** **[Citric Acid Cycle]**  Also called the ***Citric Acid Cycle**,* or the ***tricarboxylic acid cycle (TCA)*** because citric acid has three carboxyl groups in its structure. It transfers remaining electrons from the acetyl group produced during the transition reaction to electron carrier molecules, thus reducing them. It is a closed loop: the last part of the pathway regenerates the compound used in the first step; for each molecule of glucose that enters cellular respiration because there are two pyruvates. - Occurs in the cytoplasm of prokaryotes along with glycolysis and the transition reaction. - Takes place in the mitochondrial matrix of eukaryotic cells where the transition reaction also occurs. Generates two CO~2~ molecules, three NADH, one FADH, and one ATP or GTP. - Who is the final electron acceptor in respiration or in fermentation, what happens if pyruvate is the final electron acceptor in glucose catabolism? If pyruvate is the final electron acceptor, it indicates a fermentation process occurring, where pyruvate is used to regenerate NAD^+^ from NADH, allowing glycolysis to continue in the absence of oxygen and producing fermentation products like lactate or ethanol. - What is siderophore, why it is important in bacteria? Siderophore is a small molecule produced by bacteria that acts as a high-affinity iron chelator, essentially allowing bacteria so "scavenge" iron from their environment by binding to it very tightly, making iron readily available for the bacterial cell to utilize. This is crucial for bacterial survival as iron is often limited in the environment, and siderophores play a key role in bacterial pathogenesis by enabling pathogens to acquire iron from their host tissues. Iron and bacteria: enzyme co-factor DNA polymerases and ribonucleotide reductases, energy production (Cytochromes -- prosthetic group), Fenton reaction, peptidoglycan synthesis. - Does bacteria make more ATP from one molecule of glucose than eukaryotic cells? If yes, then why? Yes, bacteria generally produce slightly more ATP per glucose molecule than eukaryotic cells because in bacteria, the entire process of cellular respiration happens in the cytoplasm, whereas in eukaryotes, some energy is used to transport molecules into the mitochondria, resulting in a slightly lower net ATP yield for eukaryotes. Typically, bacteria produce 38 ATP per glucose while eukaryotes produce 36 ATP per glucose. - Know about photosynthesis in bacteria and its difference from eukaryotic cells. **[Photosynthesis]** *Photosynthesis* is the biochemical process by which phototrophic organisms convert solar energy (sunlight) into chemical energy. [Two Sequential Stages:] 1. **Light-dependent reaction**: energy from sunlight is absorbed by pigment molecules in photosynthetic membranes and converted by pigment molecules in photosynthetic membranes and converted into stored chemical energy. - Produce ATP, O~2~ and either NADPH or NADH to temporarily store energy. - These energy carriers are used in the light-independent reactions to drive the energetically unfavorable process of. "fixing" inorganic CO~2~ in an organic form, sugar. - Take place in thylakoid membranes. 2. **Light-independent reactions (Calvin Cycle)**: chemical energy produced by the light-dependent reactions is used to drive the assembly of sugar energy molecules using CO~2~. - However, these reactions are still light-dependent because the products of the light-dependent reactions are necessary for driving them are short-lived. - Chemical energy -- Glucose Formation - Occurs in stroma. - Uses ATP and NADPH to incorporate CO~2~ into carbohydrate. - Used by plants and photoautotrophic bacteria, and enzyme RuBisCO. **[Photosynthetic Structures in Eukaryotes and Prokaryotes]** [Eukaryotes ] In all phototrophic eukaryotes, photosynthesis takes place inside a **chloroplast,** an organelle that arose in eukaryotes by endosymbiosis of photosynthetic bacterium. - Chloroplasts are enclosed by a double membrane with inner and outer layers. - Within the chloroplast is a third membrane that forms stacked, disc-shaped photosynthetic structures called **thylakoids.** - A stack of thylakoids is called a **granum**, and the space surrounding the granum within the chloroplast is called **stroma.** [Prokaryotes]  Photosynthetic membranes in prokaryotes are not organized into distinct membrane-enclosed organelles; rather, they are infolded regions of the plasma membrane. - E.g., In cyanobacteria, these infolded regions are also referred to as thylakoids. - Embedded within the thylakoid membranes or other photosynthetic bacterial membranes are **photosynthetic pigment** molecules organized into one or more photosystems, where light is converted into chemical energy. - Photosynthetic pigments within the membranes are organized into photosystems, each of which is composed of a **light-harvesting** (antennae) complex and **reaction center**. - Light-harvesting complex consists of multiple proteins and associated pigments that each may absorb light energy, thus, become excited. - The reaction center contains a pigment molecule that can undergo oxidation upon excitation, giving up an electron. - Light energy is converted into an excited electron in this step. \*LH pigments absorb light energy, converting it into chemical energy. This energy is passed from one LH pigment to another until it reaches a RC pigment, exciting an electron. This high-energy electron is lost from the RC pigment and passed through an ETS, ultimately producing NADH or NADPH and ATP. A reduced molecule (H~2~A) donates an electron, replacing electrons to the electron-deficient RC pigment.\* **[Oxygenic and Anoxygenic Photosynthesis]** [Oxygenic Photosynthesis] In *Oxygenic Photosynthesis,* H~2~O is split and supplies the electron to the reaction center. O~2~ is generated as a byproduct and is released. - Eukaryotes and cyanobacteria carry out oxygenic photosynthesis, producing O~2~.  [Anoxygenic Photosynthesis] When reduced compounds serve as the electron donor, O~2~ is not generated. - Hydrogen sulfide (H~2~S) or thiosulfate (S~2~O^2-^~3~) can serve as the electron donor, generating elemental sulfur and sulfate (SO^2-^~4~) ions as a result. **CH6: ACELLULAR PATHOGENS** - **Know about the virus and its structure (envelop, matrix, capsid, genome)** **[Characteristics of Viruses]** - **Infectious, acellular pathogens.** - **Obligate intracellular parasites with host and cell-type specificity.** - **DNA or RNA genome (never both).** - **Genome is surrounded by a protein capsid, and, in some cases, a phospholipid membrane studded with viral glycoproteins.** - **Lack genes for many products needed for successful reproduction, requiring exploitation of host-cell genomes to reproduce.** **[Viral Structures]** **Virion is the complete, infective form of a virus outside a host cell, with a core of RNA or DNA and a capsid.** **In general, virions (viral particles) are small and cannot be observed using a regular light microscope.** **[Size ]** **The size of a virion can range from 20nm (for small viruses) up to 900nm (for typical, large viruses).** - ***Pandoravirus salinus* and *Pithovirus sibericum* (1500nm) -- sizes approaching that of a bacterial cell.** - **The size of a virus is small relative to the size of most bacterial and eukaryotic cells and their organelles.** - **Adaptation that allows viruses to infect these larger cells.** [Capsid] Viruses consist of a nucleic acid (either DNA or RNA, never both) surrounded by a protein coat called a **capsid**. - Interiors is not filled with cytosol, instead it contains the bare necessities in terms of genome and enzymes needed to direct the synthesis of new virions. - Is composed of protein subunits called **capsomeres** made of one or more different types of capsomere proteins that interlock to form the closely packed capsid. [Nucleic Acid] [General Composition] 1. **Naked Viruses or Nonenveloped Viruses:** viruses formed from only a nucleic acid and capsid. 2. **Viral Envelope or Enveloped Viruses:** viruses formed with a nucleic-acid packaged capsid surrounded by a lipid layer. - Viral envelope is a small portion of phospholipid membrane obtained as the virion buds from a host cell. - May either be intracellular or cytoplasmic in origin. - Made up of carbohydrates and lipids; exclusively obtained from cells during maturation. Extending outward and away from the capsid on some naked viruses and enveloped viruses are protein structures called **spikes.** - At the tips of these spikes are structures that allow the virus to attach and enter a cell - E.g., influenza virus hemagglutinin spikes (H) or enzymes like the neuraminidase (N) influenza spikes that allow the virus to detach from the cells surface during the release of new virions. - **What is tropism and why it is important for virus infection?** **Many viruses are host specific, meaning they only infect a certain type of host; and most viruses only infect certain types of cells within tissues. This specificity is called a tissue tropism.** - **E.g., Poliovirus exhibits tropism for the tissues of the brain and spinal cord. Influenza virus has a primary tropism for the respiratory tract.** By infecting a virus's tropism, researchers can predict which organs or tissues will be affected during an infection, helping to identify potential disease complications. - **Which was the 1^st^ human virus discovered?** **[Two Critical Experiments of Early Virology]** 1. **By Adolf Mayer in 1886: infectious or contagious.** 2. **By Dimitri Ivanovski in 1892 and Martinus Beijerincle in 1898:** - **Filterable agent, smaller than bacteria.** - **Virus: liquid toxin or poison.** - **Ivanovski is credited as the original discover of viruses and founder of virology field.** - **How we classify the virus based on their shape (e.g. Helical, Polyhedral. Complex): Know more about it.** **[Shapes ]** **Viruses vary in the shape of their capsids, which can be either helical, polyhedral, or complex.** **[Helical]** **A *helical* capsid forms the shape of tobacco mosaic virus (TMV), a naked helical, and Ebola virus (an enveloped helical virus).** - **The capsid is cylindrical, or rod shaped, with the genome fitting just inside the length of the capsid.** **[Polyhedral ]** ***Polyhedral* capsids form the shapes of poliovirus and rhinovirus and consists of a nucleic acid surrounded by a polyhedral (many-sided) capsid in the form of an icosahedron.** - **An icosahedral capsid is a three-dimensional, 20-sided structure with 12 vertices.** - **Resemble a soccer ball.** - **E.g., Adenovirus** **[Complex ]** **Both helical and polyhedral viruses can have envelopes. Viral shapes seen in certain types of bacteriophages, such as T4 phage, and poxviruses (e.g., vaccinia virus) have many features of both polyhedral and helical viruses so they are described as *complex viral shape.*** - **In bacteriophage complex form, the genome is located within the polyhedral head and the sheath connects the head to the tail fibers and tail pins that help the virus attach to receptors on the host cell's surface.** - **Poxviruses that have complex shapes are often brick shaped, with intricate surface characteristics not seen in other categories of capsid.** - **What is structural or nonstructural proteins in a virus?** - **From where does a virus take its Envelope, does the virus take its spike from the same place?** **Envelopes are acquired as nucleocapsids bud through cellular membranes. These portions of cellular membranes are modified by viruses and contain the viral glycoproteins (not cellular proteins). These glycoproteins appear as spikes on the surface of the virus particles.** **[Functions of Spikes (Glycoproteins)]** - **Binding sites for cell surface receptors.** - **Major antigenic determinants.** - **Mediates virus fusion with cellular membranes.** - **Why virus need to be assembled/capsid? Where does virus assembly take place?** **A virus needs to be assembled into a capsid to protect its genetic material (DNA or RNA) while traveling outside of a host cell, allowing it to effectively deliver the genome to a new host cell by maintaining its structural integrity.** - **Protection Barrier: nucleic acids (DNA or RNA) are very sensitive to the nucleases or mechanical shearing and chemical modifications such as by UV from the sunlight.** - **Essential for Viral Infectivity: in order for viruses to reproduce themselves in the cells, they need proteins on the surface of the virus to bind to the cell receptors to initiate infection, in some cases deliver the genome into the cells.** - **Acts as a protective shell around the viral nucleic acid, shielding it from degradation by enzymes in the host or environment.** **This assembly typically takes place within the cytoplasm of the infected host cell, though some viruses assemble in the nucleus depending on their replication cycle and genome type.** **Cytoplasm: most RNA viruses assemble in the cytoplasm of the host cell.** **Nucleus: DNA viruses that replicate in the nucleus often assemble there as well.** **How to Assemble:** - **Several RNA viruses undergo *self-assembly* as a helical nucleocapsid.** - **TMV: Heinz Fraenkel-Conrat and R.C. Williams in 1957.** - **The symmetry structure is held together by protein-protein, protein-nucleic acid, and protein-lipid interactions (hydrophobic and electrostatic).** - **Because of symmetry, the structure is in free-energy state and stable and therefore the favored structure of the components.** - **Virus starts to assemble spontaneously when there are accumulated viral components.** - **Which virus has two capsid layers? Which virus is diploid?** **Reoviridae has two capsid layers.** - **What do you understand with (+) or (-) sense RNA virus or (+) or (-) sense viral RNA?** - **Which one is "**bullet" **shaped virus which one is brick shaped virus?** **Bullet-shaped -- Rhabdovirus** **Brick-shaped -- Poxvirus** **Know about following virus if they are DNA or RNA virus/ enveloped or non-enveloped virus?** **[Hepatitis B virus]** - **DNA (double-stranded) Virus** - **Enveloped** - **Reverse transcriptase** **[Herpes virus]** - **DNA (double stranded) Virus** - **Enveloped** - ***Varicella-zoster virus, cytomegalovirus, Epstein-Barr virus*** **[Pox virus - Poxviridae]** - **DNA (double stranded) Virus** - **Enveloped** - **Smallpox (variola), monkeypox** **[Flavivirus (Zika Virus)]** - **RNA (single-stranded, positive sense RNA)** - **Enveloped** - **HCV, Zika, other viral hemorrhagic fevers** **[Parvovirus]** - **DNA (single-stranded) Virus** - **Non-enveloped** - **Gastroenteritis** **[Coronavirus]** - **RNA (single-stranded) Virus** - **Enveloped** **[Orthomyxovirus/Influenza-Flu]** - **RNA (single-stranded, negative sense RNA) Virus** - **Enveloped** **[Retro Virus- HIV]** - **RNA (retrovirus) Virus** - **Enveloped** - **Retroviridae** - **Know about virus replication cycle/ steps, you may have a picture for virus replication, and you need to identify which type of virus it is such as RNA virus (positive or negative sense) etc.** **A virus replication cycle typically involves the following:** 1. **Attachment to a host cell,** 2. **Entry (penetration),** 3. **Uncoating,** 4. **Replication of the viral genome,** 5. **Assembly of new viral particles, and** 6. **Release from the host cell.** **The nature of the genome determines how the genome is replicated and expressed as viral proteins.** **If the genome is ssDNA, host enzymes will be used to synthesize a second strand that is complementary to the genome strand, thus producing dsDNA. The dsDNA can now be replicated, transcribed, and translated similar to host DNA.** **There are three types of RNA genome:** 1. **dsRNA** 2. **Positive (+) Single-Strand RNA (+ssRNA)** - **If a virus has a +ssRNA genome, it can be translated directly to make viral proteins.** 3. **Negative (-) Single-Strand RNA (-ssRNA)** - **If a virus contains a -ssRNA genome, the host ribosomes cannot translate it until the -ssRNA is replicated into +ssRNA by viral RNA-dependent RNA polymerase (RdRp).** **RdRp is an important enzyme for the replication of dsRNA viruses because it uses the negative strand of the double-stranded genome as a template to create +ssRNA.** RNA viruses contain +ssRNA that can be directly read by the ribosome to synthesize viral proteins. Viruses containing -ssRNA must first use the -ssRNA as a template for the synthesis of +ssRNA before viral proteins can be synthesized. - **Know exceptions: Almost all RNA virus replicates in cytoplasm (Except Influenza virus, retrovirus). Almost all DNA virus replicated in nucleus/ use nucleus (expect Pox virus and other?)** **[Retrovirus Replication]** **ssRNA viruses carry a special enzyme called *reverse transcriptase* within the capsid that synthesizes a complementary ssDNA copy using the +ssRNA genome as a template.** **ssDNA is made into dsDNA, which can integrate into the host chromosome and become a permanent part of the host.** **The integrated viral genome is called a *provirus.*** - HIV, an enveloped, icosahedral retrovirus, attaches to a cell surface receptor of an immune cell and fuses with the cell membrane. - Viral contents are released into the cell, where viral enzymes convert the single-stranded RNA genome into DNA and incorporate it into the host genome. **[Influenza Virus]** *Influenza Virus* is one of the few RNA viruses that replicates in the nucleus of cells. In influenza virus infection, viral glycoproteins attach the virus to a host epithelial cell. As a result, the virus is engulfed. Viral RNA and viral proteins are made and assembled into new virions that are released by budding. - Contains eight RNA segments (-ss) and an outer lipid bilayer. - **Know big picture of Baltimore virus classification.** **The Baltimore Virus Classification is an alternative to ICTV nomenclature. It classifies viruses according to their genomes (DNA or RNA, singe versus double stranded, and mode of replication). This system thus creates seven groups of viruses that have common genetics and biology.** **Categories may include naked or enveloped structure, single-stranded (ss) or double-stranded (ds) DNA or RNA genomes, segmented or non-segmented genomes, and positive-strand (+) or negative-strand (-) RNA.** **[Class I: ds DNA Viruses]** - **Same mechanism of protein synthesis as eukaryotic cells.** - **Use DNA dependent RNA polymerase (cellular) to make mRNA from viral DNA (transcription).** - **Translation (make proteins from mRNAs): using cellular machinery (tRNA, rRNA).** **[Class II: ss DNA Viruses]** - **Use ds DNA as intermediate to synthesize mRNA and ssDNA progeny.** **[Class III: ds RNA Viruses with Segmented Genome]** - **Virus encodes RNA dependent RNA polymerase (RdRp) to make mRNA from (-) RNA.** - **Each segment is transcribed individually to produce mRNA.** **[Class IV: ss (+) RNA Viruses]** - **Genomic or sub-genomic RNA as mRNA.** - **RdRp makes sub-genomic mRNAs and replicates genome.** - **Genomic RNA is infectious.** **[Class V: ss (-) RNA Viruses]** - **RdRp makes mRNA or sub-genomic mRNAs and replicates genome.** - **Genomic RNA is not infectious.** **[Class VI: ss (+) RNA Virus with DNA Intermediate (Retrovirus)]** - **Virus encodes reverse transcriptase to make DNA from RNA.** - **Then DNA to mRNA to protein using cellular polymerase.** **[Class VII: ds DNA virus (Pox Virus)]** - **Virus encodes DNA dependent RNA polymerase to make early mRNA.** **\*RdRp lacks proof reading mechanism, therefore, high mutation rates during virus replication accumulate. Quansispecies RNA viruses: population mixed and keep changing.\*** **\*Viral DNA Polymerase: much more stable, still higher than the cellular DNA polymerase. RT: unique, profile the mRNA population, cellular or viral by using RT-PCT.\*** - **Know about virus shift and drift.** **[Virus Shift]** ***Antigenic shift* is a major change in spike proteins due to gene reassortment. This reassortment for antigenic shift occurs typically when two different influenza viruses infect the same host.** **[Virus Drift]** ***Antigenic drift* is the result of point mutations causing slight changes in the spike proteins hemagglutinin (H) and neuraminidase (N).** - **Why is pig known as a mixing vessel for the influenza virus?** **Pigs are considered "mixing vessels" for the influenza virus because they can be infected by both avian and human influenza viruses simultaneously, allowing for the genetic material from these different strains to mix and create new, potentially pandemic influenza viruses (genetic assortment). They act as a host where different influenza viruses can swap genes, generating novel strains with the potential to jump species and infect humans.** - **Why mainly influenza A cause pandemic among other influenza virus (A, B, C, and D).** **Influenza A viruses are the only influenza viruses known to cause pandemics because they can undergo genetic changes that allow them to spread easily between people and infect people who have little or no immunity.** - **Pigs can act as "mixing vessel".** - **When an animal influenza virus undergoes genetic changes that allow it to infect humans.** - **when the surface of the influenza A virus combines in new ways, creating a new strain that can infect people.** **Influenza B viruses are less common than Influenza A viruses and mutate more slowly, so pandemics generally do not occur with Influenza B. Influenza C viruses cause mild illness and are not thought to cause human epidemics. Influenza D viruses primarily affect cattle and are not known to infect people.** - **Know Viral Growth Curve, eclipse period, PFU, CFU** **[Viral Growth Curve]** In a one-step multiplication curve, the host cells lyse, releasing many viral particles to the medium, which leads to a very steep rise in ***viral titer*** (the number of virions per unit volume). If no viable host cell remains, the viral particles begin to degrade during the decline of the culture. **[Eclipse Period]** **In the *eclipse phase,* viruses bind and penetrate the cells with no virions detected in the medium.** - **Know about the Lysogenic Cycle, Lytic Cycle, Prophase, and phase conversion.** **[Lytic Cycle]** **During the *lytic cycle* of virulent phage, the bacteriophage takes over the cell, reproduces new phages. And destroys the cell.** **[Lysogenic Cycle]** **In a *lysogenic cycle,* the phage genome enters the cell through attachment and penetration. Instead of killing the host, the phage genome integrates into the bacterial chromosome and becomes part of the host.** **[Prophase]** ***Prophase* is the integrated phage genome. Bacterial host with a prophage is called a lysogen.** **[Phase Conversion]** **It is typical of temperate phages to be latent or inactive within the cell. As the bacterium replicates its chromosome, it also replicates the phage's DNA and passes it on to new daughter cells during reproduction. The presence of the phage may alter the phenotype of the bacterium since it can bring in extra genes. This change is host phenotype is called *lysogenic conversion* or *phage conversion.*** - **Know the difference between transfection, transduction, conjugation and transformation.** **[Transfection]** ***Transfection* is a gene transfer technique that introduces nucleic acids (DNA or RNA) into eukaryotic cells using nonviral methods (chemical, biological, or physical).** **[Transfection Types:]** 1. **Transient Transfection: the introduced nucleic acid is only expressed for a limited period of time and does not replicate.** 2. **Stable Transfection: the introduced nucleic acid integrates into the genome of the recipient and replicates when the host genome replicates.** **[Transduction]** ***Transduction* occurs when a bacteriophage transfers bacterial DNA from one bacterium to another during sequential infections.** **[Two types of Transductions: ]** 1. **Generalized Transduction: occurs when a random piece of bacterial chromosomal DNA is transferred by the phage during the lytic cycle.** 2. **Specialized Transduction: occurs at the end of the lysogenic cycle, when the prophage is excised, and the bacteriophage enters the lytic cycle.** \*An integrated phage excises, bringing with it a piece of the DNA adjacent to its insertion point. On reinfection of a new bacterium, the phage DNA integrates along with the genetic material acquired from the previous host.\* **[Conjugation ]** *Conjugation* refers to the process where one bacterial cell transfers genetic material to another bacterial cell through direct physical contact, essentially acting as a "donor" cell transferring DNA to a "recipient" cell, often facilitated by a pilus. - It is a form of horizontal gene transfer between bacteria where the cells temporarily join together to exchange genetic information. **[Transformation ]** *Transformation* refers to the process where a cell takes up and incorporates exogenous DNA (foreign genetic material) from its surroundings, resulting in a stable genetic change within the cell, often occurring when the cell is in a state called "competence" where it is receptive to DNA uptake. - **What is a big difference in RNA vs DNA virus replication (immediate early, early and late proteins?)** **The major difference lies in where and how the "immediate early" proteins are produced. With most RNA viruses, the viral genome itself can directly act as mRNA, allowing for immediate translation of proteins upon entry into the host cell, while DNA viruses typically require an initial transcription step to produce mRNA for the "immediate early" proteins, which then regulate the expression of subsequent early and late genes.** **RNA viruses often have a quicker "early" phase due to their ability to directly translate their genome, while DNA viruses have a more defined "immediate early" phase with dedicated regulatory proteins.** - **Where you can grow the virus (know the advantages and disadvantages for these techniques/host)** **[Isolation of Viruses]** **Viruses require a living host cell for replication. Infected host cells (eukaryotic or prokaryotic) can be cultured and grown, and then the growth medium can be harvested as a source of virus. Virions in the liquid medium can be separated from the host cells by either centrifugation or filtration. Filters can physically remove anything present in the solution that is larger than the virions; the viruses can then be collected in the filtrate.** **[Cultivation of Viruses]** **Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro (outside a living organism in cells in an artificial environment). Flat horizontal cell culture flasks are a common vessel used for in vitro work. Bacteriophages can be grown in the presence of a dense layer of bacteria (bacterial lawn) grown in 0.7% soft agar in a Petri dish. As the phage kills the bacteria, many plaques are observed among the cloudy bacterial lawn.** - **each plaque corresponds to a single virus; can be expressed as plaque-forming units (PFU).** **[Animal Viruses]** **Animal viruses require cells within a host animal or tissue-culture cells derived from an animal.** **In Vivo:** - **Host sources can be a developing embryo in an embryonated bird's egg or a whole animal.** - **The embryo or host animal serves as an incubator for viral replication.** - **Many have a tissue tropism, therefore, must be introduced into a specific site for growth.** - **Viral infection may damage tissue membranes, producing lesions (pox); disrupt embryonic development; or cause the death of the embryo.** **In Vitro:** - **A cell culture is freshly prepared from animal organs or tissues.** **[Serological Tests]** **A serological test is used to detect the presence of certain types of viruses in patient serum.** - **Western blotting, ELISA, IFA** **[Nucleic Acid Amplification Test]** **NAAT are used to detect unique nucleic acid sequences of viruses in patient samples.** - **PCR is used to detect the presence of viral DNA in a patient's tissue of body fluid.** - **RT-PCR is used to detect the presence of RNA virus.** - **What is Cytopathic Effects and how you will identify that?** **Cytopathic effects (CPEs) are distinct observable cell abnormalities due to viral infections. CPEs can include loss of adherence to the surface of the container, changes in cell shape from flat to round, shrinkage of the nucleus, vacuoles in cytoplasm, fusion of** **cytoplasmic membranes and the formation of the multinucleated syncytia, inclusion bodies in the nucleus or cytoplasm, and complete cell lysis.** **Further pathological changes include viral disruption of the host genome and altering normal cells into transformed cells, which are the types of cells associated with carcinomas and sarcomas.** - **Observable cell abnormalities.** - **Know about/difference in virus, virion, Viroids, Virusoids and Prions.** **[Viroid]** ***Viroids* consist only of a short strand of circular RNA capable of self-replication. They do not have a protein coat to protect their genetic information.** - **1^st^ viroid discovered was found to cause potato tuber spindle disease, which causes slower sprouting and various deformities in potato plants.** - **Takes control of the host machinery to replicate their RNA genome.** - **Can result in devastating losses of commercially important agricultural food crops grown in fields and orchards.** - **Other viroids that cause diseases in plants:** - **Tomato planta macho viroid (TPMVd) infects tomato plants, which causes loss of chlorophyll, disfigured and brittle leaves, and very small tomatoes.** - **Avocado sunblotch viroid (ASBVd) results in lower yields and poorer-quality fruit.** - **Smallest viroid discovered.** - **Peach latent mosaic viroid (PLMVd) can cause necrosis of flower buds and branches, and wounding of ripened fruit, which leads to fungal and bacterial growth in the fruit.** **In general, viroids can be dispersed mechanically during crop maintenance or harvesting, vegetative reproduction, and possible via seeds and insects, resulting in a severe drop in food availability and devastating economic consequences.** **[Virusoids]** **A second type of pathogenic RNA that can infect commercially important agricultural crops are the *virusoids,* which are subviral particles best described as non-self-replicating ssRNAs. They require that the cell also be infected with a specific "helper" virus. They are small genomes, only 220 to 388 subunits long. A virusoid genome does not code for any proteins, but instead serves only to replicate virusoid RNA.** **Helper Virus:** - **Once it enters the host cell, the virusoids are released and can be found free in plant cell cytoplasm, where they possess ribozyme activity.** - **Undergoes typical viral replication independent of the activity of the virusoid.** **[Prions ]** **A *prion (proteinaceous infectious particles)* is a misfolded rogue form of a normal protein (PrPc) found in the cell. This rogue protein (PrPsc), which may be caused by a genetic mutation or occur spontaneously, can be infectious, stimulating other endogenous normal proteins to become misfolded, forming plaques.** - **PrPc: normal cellular prion protein, produces disease on the cell surface.** - **PrPsc: scrapie protein, produces disease in brain cells, forming plaques.** **[Transmissible Spongiform Encephalopathy (TSE)]** ***TSE* is a rare degenerative disorder that affects the brain and nervous system.** - **The accumulation of rogue proteins causes the brain tissue to become sponge-like, killing brain cells and forming holes in the tissue, leading to brain damage, loss of motor coordination, and dementia.** - There is no cure; infected individuals are mentally impaired and become unable to move or speak. TSE in humans: - **Fatal Familial Insomnia** - **Gerstmann-Straussler-Scheinker disease** - **Creutzfeldt-Jakob disease** **TSE in animals:** - **Mad Cow Disease** - **Scrapie (in sheep and goats)** **Prions are extremely difficult to destroy because they are resistant to heat, chemicals, and radiation. Even standard sterilization procedures do not ensure the destruction of these particles.** - **How Prion disease is transmitted. Know more about it from the PowerPoint or textbook.** **DISEASE** **MECHANISM(S) OF TRANSMISSION** -------------------------------------------- --------------------------------------------------------------------------------------------------------------------------- **Sporadic CJD (sCJD)** **Not known; possibly by alteration of normal prior protein (PrP) to rogue form due to somatic mutation.** **Variant CJD (vCJD)** **Eating contaminated cattle products and by secondary bloodborne transmission.** **Familial CJD (fCJD)** **Mutation in germline Prp gene** **Latrogenic CJD (iCJD)** **Contaminated neurosurgical instruments, corneal graft, gonadotrophic hormone, and, secondarily, by blood transfusion.** **Kuru** **Eating infected meat through ritualistic cannibalism.** **Gerstmann-Straussler-Scheinker Disease** **Mutation in germline PrP gene.** **Fatal Familial Insomnia (FFI)** **Mutation in germline PrP gene.** - **To know steps in virus replication** **Be prepare for figure type question in where you need to identify virus (+ sense RNA, - sense RNA, retrovirus, DNA VIRUS)** **CH15: MICROBIAL MECHANISMS OF PATHOGENICITY** - **Know Acute, chronic, latent diseases and asymptomatic carrier.** **[Acute Disease]** **For an *acute disease,* pathologic changes occur over a relatively short time (e.g., hours, days, or a few weeks) and involve a rapid onset of disease conditions.** **[Chronic Disease]** **For a *chronic disease,* pathologic changes can occur over longer time spans (e.g., months, years, or a lifetime).** **[Latent Disease]** **In *latent diseases,* the causal pathogen goes dormant for extended periods of time with no active replication.** - **Herpes virus** **[Asymptomatic or Subclinical]** **They do not present any noticeable signs or symptoms.** - **Know about pathogen-associated molecular pattern, pathogen recognition receptor, where they are present, and what is their function, what happen when they interacts?** - **What is cytokine, what is its role in the immune system?** **A *cytokine* is a chemical messenger. The excessive production of cytokines (cytokine storm) elicits a strong immune and inflammatory response that can cause life-threatening high fevers, low blood pressure, multi-organ failure, shock, and death.** - **Which cell produces antibodies?** - **What is superantigen?** ***Superantigens* are exotoxins that trigger an excessive, nonspecific stimulation of immune cells to secrete cytokines.** **The prototype superantigen is the toxic shock syndrome toxin of *S. aureus*.** - **Most TSS cases are associated with vaginal colonization by toxin-producing S. aureus in menstruating females.** - **However, colonization of other body sites can also occur.** - **Some strains of *Streptococcus pyogenes* also produce superantigens; they are referred to as the streptococcal mitogenic exotoxins and the streptococcal pyrogenic toxins.** **Process:** 1. **Toxins bind directly to MHC II on macrophages (without being processed) and form a crosslink with T cell receptors.** 2. **Crosslinking causes stimulation of up to 1 in 5 T cells in the body (normal antigens cause stimulation 1 in 10,000).** 3. **Excessive IL-2 production results from the massive stimulation of T helper cells.** 4. **Stimulation of other cytokines by IL-2 lead to shock.** - **Difference between endotoxin and exotoxin** **[Endotoxin]** ***Endotoxins* are the lipid portions of lipopolysaccharides (LPSs) that are part of the outer membrane of the cell wall of gram-negative bacteria. Endotoxins are liberated when the bacteria die, and the cell wall breaks apart.** **[Exotoxin]** ***Exotoxins* are protein produced inside pathogenic bacteria. Most commonly gram-positive bacteria, as part of their growth and metabolism. The exotoxins are then secreted or released into the surrounding medium following lysis.** **Three Roles:** 1. **Intracellular Targeting:** 2. **Membrane Disrupting:** 3. **Superantigens:** - **Which bacteria produce endotoxin/exotoxin? Know the difference between endotoxin and exotoxin?** **Characteristic** **Endotoxin** **Exotoxin** -------------------- ----------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------- **Source** **Gram-negative bacteria** **Gram-positive bacteria (primarily) and gram-negative bacteria** **Composition** **Lipid A component of lipopolysaccharide** **Protein** **Effect on Host** **General systematic symptoms of inflammation and fever** **Specific damage to cells dependent upon receptor-mediated targeting of cells and specific mechanisms of action** **Heat Stability** **Heat stable** **Most are heat labile, but some are heat stable** **LD~50~** **High** **Low**