Virology: Infections and Host Response

Viruses & Infections

• Cell tropism determines the specificity of a virus for certain cell types which is affected by specific surface proteins and host cell receptors
• For a virus to infect a cell it requires compatible receptors for attachment, and the intracellular machinery for replication
• HIV targets CD4+ T cells due to the presence of CD4 receptors

Direct/Indirect Effects of Viral Infections

• Direct effects of viral infections include cell lysis, cell fusion, and altered cell function
• Indirect effects of viral infections result from the immune response, like inflammation and tissue damage
• Some viruses cause cellular transformation that can lead to cancer

Host System and Viral Disease Resolution

• The immune system resolves viral infections, with the innate immune system as the first line of defense
• The adaptive immune system offers specific responses to clear the virus via T cells and antibodies

Cultivation and Enumeration of Viruses

• Cultivating viruses requires inoculating an appropriate living host
• Host cells are required because viruses need living cells to replicate
• Enumeration methods include plaque assays, endpoint dilution assays, and direct particle counting
• Accurate virus growth requires proper sterile techniques and controlled environments

Viroids and Satellites

• Viroids consist of only RNA, while satellites consist of only nucleic acids
• Viroids are small, circular RNA molecules that infect plants, lacking protein-coding genes; they rely on host enzymes for replication
• Satellites are subviral agents that require a helper virus for replication and encode their own proteins

Prions: Infection and Damage

• Prions are proteins only
• They are misfolded proteins that cause neurodegenerative diseases by inducing normal proteins to adopt an abnormal conformation
• Prions lack nucleic acids and spread through direct contact with infected tissue
• Cellular damage occurs as prions build up in the brain, causing nerve cell death and sponge-like holes, which leads to memory loss, movement problems, and death
• Mad cow disease and Creutzfeldt-Jakob disease (CJD) are examples of prion diseases

Bacterial Cell Cycle

• The bacterial cell cycle involves binary fission; where a single cell grows, replicates its chromosome, and divides into identical daughter cells
• The cycle has 3 main stages: growth, DNA replication, and cytokinesis
• Eukaryotic cell cycle includes interphase (G1, S, G2,) and mitosis which has more complex processes

The Role of FtsZ and Mre B

• FtsZ is a protein that forms a Z ring at the division site, guiding septum formation during cytokinesis and it is essential for bacterial cell division
  • Septation is the formation of a cross wall between two daughter cells
    • Steps include selection of site for septum formation - assembly of Z ring (composed of FstZ) - assembly of cell wall synthesizing machinery - constriction of cell and septum formation
• Protein FtsZ: tubulin homologous, found in most bacteria and archaea and it assists in building new cell wall
  • The MinCDE system in E. coli ensures the Z ring forms only in the middle
• MreB is an actin-like protein that helps maintain rod-shaped bacteria cell shape by directing cell wall synthesis and chromosome movement; it determines the location for cell wall elongation before cell divides

Specific Gene Products in Chromosome Partitioning and Cell Wall Biosynthesis

• ParA and ParB proteins involved in chromosome partitioning, helping segregate replicated chromosomes
  • ParA polymerizes to form filaments that pull one copy of the DNA to a cell pole; ParB binds DNA at paS site near origin of replication to stabilize the DNA
• Penicillin-Binding Proteins (PBPs): enzymes that link peptidoglycan strand, helping with cell wall remodeling during growth
• Autolysins: special PBPs that break down small portions of the cell wall to allow new peptidoglycan insertion

The Role of Bactoprenol

• Bactoprenol: is a lipid carrier that transports peptidoglycan precursors across the cytoplasmic membrane, which helps deliver building blocks to the growing cell wall during bacterial cell division
• Bactoprenol: essential for bacterial growth and maintaining cell shape

Cardinal Conditions

• Cardinal conditions are the minimum, optimum, and maximum physical conditions (temp, pH, osmolarity) required for microbial growth
  • Minimum is the lowest condition where growth is possible
  • Optimal is the condition where growth occurs at the fastest rate
  • Maximum is the highest condition where growth can still occur before the environment becomes lethal
• Ex: thermophiles thrive in high heat

Oxygen Requirements

• Aerobe: grows in presence of atmospheric oxygen (O2), which is 20% O2
  • Obligate aerobes require oxygen to grow
• Anaerobe: grows in the absence of O2
  • Facultative anaerobes grow with or without oxygen but prefer oxygen to grow better
  • Obligate anaerobes cannot grow in presence of oxygen and are killed in its presence
  • Aerotolerant anaerobes grow without O2
  • Microaerophiles require low oxygen levels (2-10% O2)

Types of Culture Media

• Defined/synthetic media: each ingredient can be defined with a chemical formula
• Complex media: contains some ingredients of unknown chemical compositions (yeast)
• Selective media: supports growth of specific microbes while inhibiting others
  • MacConkey agar selects for gram- bacteria
• Differential media: distinguishes between species by visible changes
  • Blood agar distinguishes between hemolytic vs nonhemolytic bacteria
  • MacConkey agar distinguishes between lactose fermenters vs nonfermenters

Bacterial Growth Curve Phases

• Lag phase: length can vary, cells adjust to the environment, there is no division and cells are synthesizing new components
• Log (exponential phase): rapid cell division and growth, population is most uniform in chemical and physical properties during this phase
• Stationary phase: growth rate equals death rate due to nutrient depletion

Stationary Phase Reasons

• Nutrient limitation, limited oxygen, toxic waste accumulation, and critical population density are all reasons for the stationary phase
• Death phase: cells die as resources are exhausted, and the number of viable cells declines exponentially
• Long-term stationary phase: a few genetically adapted survivors persist, leading to natural selection and evolution over time

Enzymes Needed for Toxic Oxygen Control

• Oxygen is reduced to reactive oxygen species (ROS): superoxide radical, hydrogen peroxide, hydroxyl radical
• Aerobes produce protective enzymes: SOD, catalase, and peroxidase
  • Superoxide dismutase (SOD): converts superoxide radicals to oxygen and hydroxide peroxide
  • Catalase: breaks hydrogen peroxide into water and oxygen
  • Peroxidase: converts hydrogen peroxide into water without releasing oxygen

Modifications for Hot and Cold Temperatures

• Psychrophiles: thrive in cold (0-20°C) with unsaturated fatty acids in membranes for flexibility
• Mesophiles: prefer moderate temp (20-45°C) including most human pathogens
• Thermophiles: adapt to hot environments (45-85° C) using heat-stable proteins and saturated fatty acids
• Hyperthermophiles: live in extreme heat (85-113° C) with specialized DNA protecting proteins and stable membranes
• Halophiles: grow optimally in the presence of salts

Biofilm Characteristics

• Biofilms are structured communities of bacteria embedded in a self-produced matrix (EPS – extracellular polymeric substance)
• Stages of biofilm formation: initial attachment to surface→ maturation as cells produce EPS → dispersion when some bacteria leave to colonize new areas
• Biofilms offer protection from antibiotics, environmental stress, and the immune system
• They develop through attachment, growth, and dispersal, common on medical devices and natural surfaces
• Growth refers to the population growth rather than individual cells

pH and Temperature

• pH: acidophiles, neutrophiles, and alkaliphiles (most bacteria & protist are neutrophiles, most fungi= acidic) archaea are acidophiles
• Temperature: microbes cannot regulate their internal temp, enzymes have optimal temp at which they function optimally, high temp may inhibit enzyme functioning & be lethal

Pressure and Radiation

• Pressure: microbes on land and water = 1 atm
  • Barotolerant: affected by increased pressure but not nontolerant
  • Barophilic: require or grow more rapidly in presence of high pressure and change membrane fatty acids to adapt to high pressure
• Radiation: mutations result in death and DNA damage can be repaired by repair mechanisms
  • Visible light: at high intensities generates singlet oxygen, a powerful oxidizing agent

Therapeutic Dose

• The therapeutic dose is the minimum concentration of a drug needed to effectively treat an infection without causing harm to the patient
• Balance between drug efficiency and safety (drug level requires for clinical treatment)
• Therapeutic index is calculated as toxic dose divided by therapeutic dose
• Toxic dose is the drug level that becomes too toxic for the patient

Kirby Bauer Test

• It assesses the sensitivity of bacteria to antimicrobial agents. Antibiotic-impregnated paper disks are placed on an agar plate with bacteria in question
• As antibiotics diffuse, they create a zone of inhibition where bacterial growth is prevented and the size of zones are measured to determine whether bacteria are susceptible, intermediate, or resistant to the antibiotic
• The Kirby-Bauer test measures bacterial susceptibility to antibiotics and antibiotic impregnates discs are placed on agar plate with bacteria
• The size of the clear zone around each disc indicates the drug's effectiveness
• It is a standardized method for disk diffusion and it uses tablets relating zone diameter with microbial resistance to determine sensitivity/resistance
• Table values plotted are used to determine if the effective concentration of the drug in the body can be reached

Types of Drug Resistance

• Intrinsic resistance is natural resistance due to structural features (ex: gram-neg bacteria outer membrane)
  • Mycoplasma resistance to B-lactam antibiotics and other cell wall inhibitors occurs because these bacteria lack a cell wall
• Acquired resistance is resistance gained through mutations or horizontal gene transfer, which occurs when there is a change in the genome of a bacterium that converts it from one susceptible to resistant
• Adaptive resistance is temporary resistance due to environmental stress, which disappears when conditions change; bacteria lack mechanisms for antibiotic resistance and ignore the presence of antibiotics usually because they are embedded in biofilms that antibiotics cannot effectively penetrate or are growing too slowly to be inhibited
• Drug-tolerant bacteria persisters lack mechanisms for antibiotic resistance and ignore the presence of antibiotics because they are embedded in biofilms that antibiotics cannot effectively penetrate or are growing too slowly to be inhibited

Mechanisms of Drug Resistance

• Mechanisms of drug resistance include modifying the target of antibiotics, drug inactivation, minimizing the concentration of antibiotics in the cell, bypassing the biochemical reaction inhibited by the agent to increase the production of the target metabolite
• To overcome drug resistance, give the drug in an appropriate concentration to destroy microbes, give two or more drugs simultaneously, and use drugs only when necessary

Sterilants, Disinfectants, Sanitizers, and Antiseptics

• Sterilant: kills all microbes, including spores (autoclaving, ethylene oxide, hydrogen peroxide vapor)
• Disinfectants: kill most pathogens on an inanimate surface, but not spores (bleach, phenol)
  • Phenolics: used in labs and hospitals disinfectants, act by denaturing proteins and disrupting cell membranes
  • Alcohols: widely used, most common are ethanol and isopropanol, inactivate some viruses, denature proteins and possibly dissolve membrane lipids
  • Halogens-Iodine/chlorine:
    • Iodine: skin antiseptic, oxidizes cell constituents and iodinates proteins, and at high concentrations may kill endospores
    • Chlorine: disinfection of water, used in dairy and food industries, effective household disinfectant, destroys vegetative bacteria and fungi, and chlorine gas is sporicidal
• Sanitizers: reduce microbial numbers to safe levels (hand sanitizer alcohol based)
• Antiseptics: kill or inhibit microbes on living tissue (iodine)

Bacteriostatic, Bactericidal, and Bacteriolytic

• Bacteriostatic: inhibits bacterial growth without killing (tetracycline)
• Bactericidal: kills bacteria without lysing them (penicillin)
• Bacteriolytic: kills bacteria by lysing (bursting) their cells (lysozyme)

Other Ways to Control Microorganisms

• Physical: heat (autoclaving), radiation (UV, gamma), filtration
• Mechanical: hand washing, air filters
• Biological: using bacteriophages to target bacteria

Antimicrobial Drugs

• Antimicrobial drugs can be grouped based on their general targets (cell types or cell targets)
  • Cell wall synthesis inhibitors: (e.g., penicillin) most are 6-aminopenicillanic acid derivatives that differ in the side chain attached to amino acids
    • A crucial feature is the B-lactam ring, which is essential for bioactivity and many penicillin-resistant organisms produce B-lactamase, which hydrolyzes a bond in this ring and Penicillin targets peptidoglycan formation
      • Penicillin blocks the enzyme that catalyzes transpeptidation (formation of cross-links in peptidoglycan), which prevents the synthesis of a complete cell wall leading to lysis of the cell and it acts only on growing bacteria synthesizing new peptidoglycan
      • Naturally occurring penicillin's (penicillin V and G) are narrow spectrum
      • Semisynthetic penicillin's are broader spectrum, their bulkier side chains making them more difficult for B-lactamase enzymes to degrade
      • Aminopenicillins have broader coverage that includes gram-neg bacteria
      • Cephalosporins are similar to penicillin and are used by most patients allergic to penicillin with four categories based on spectrum of activity
      • Vancomycin is glycopeptide antibiotics, which inhibit cell wall synthesis and treats antibiotic-resistant staphylococcal infections
• Protein synthesis inhibitors affect bacterial ribosomes that translate mRNA into proteins
  • Tetracyclines have a four-ring structure to which side chains are attached and they are broad spectrum & bacteriostatic
    • They target the 30S subunit of the ribosome, inhibiting protein synthesis
  • Macrolides such as erythromycin are composed of 12-22 carbon lactone rings
    • They bind to the 50S ribosomal subunit to inhibit bacterial protein elongation
  • Lincosamines are produced by streptomyces bacteria and exert antibiotic activity against anaerobic microbes

Chloramphenicol and Oxazolidinones

• Chloramphenicol binds to the 40S ribosomal subunit and inhibits peptidyl transferase, stopping bacterial protein production
  • It is effective against gram-positive and gram-negative bacteria, anaerobes, and some intracellular pathogens
• Oxazolidinones bind to the 50S subunit, targeting rRNA and preventing the formation of the initiation complex
  • They are active against gram-positive bacteria and MRSA

Nucleic Acid Synthesis Inhibitors

• Most commonly used antibacterial drugs inhibit DNA polymerase and topoisomerases and RNA polymerase (rifamycin)
• They are not as selectively toxic as other antibiotics because bacteria and eukaryotes do not differ in the way they synthesize nucleic acids
  • Fluoroquinolones are synthetic drugs containing a four-quinoline ring and they act by inhibiting bacterial DNA gyrase and topoisomerase II, treating a wide variety of infections

Metabolic Pathway Disruptors

• Metabolic pathway disruptors (sulfonamides) act as antimetabolites
• They antagonize or block the functioning of metabolic pathways by competitively inhibiting the use of metabolites by key enzymes, they are structural analogs that are similar and compete with natural metabolic intermediates to block normal cellular metabolism
  • Sulfonamide or sulfa drugs: block folic acid synthesis
  • Trimethoprim: synthetic antibiotic that interferes with folic acid production and combined with sulfa drugs to increase efficacy
• Cell membrane disruptors (polymyxins) damage bacterial membranes

Specific Drug Targets

• Penicillin/cephalosporins: inhibit peptidoglycan synthesis
• Rifampin: blocks RNA polymerase, preventing transcription
• Tetracycline: inhibits ribosomal function (protein synthesis)
• Sulfonamides: block folic acid synthesis (metabolic pathway)

Characteristics of a Toxic Drug

• Drugs become toxic due to a lack of target specificity, accumulation in tissues, disruption of host cell functions, or causing allergic reactions

Action of Tamiflu

• Tamiflu inhibits the influenza virus neuraminidase enzyme, preventing viral release from infected cells and limiting viral spread

HIV Cocktail of Chemotherapy

• Reverse transcriptase inhibitors block viral RNA to DNA conversion
• Protease inhibitors prevent viral protein maturation
• Integrase inhibitors block viral DNA integration into the host genome
• Entry/fusion inhibitors prevent HIV from binding to and entering cells
• Anti-HIV drugs:
  • Nucleoside reverse transcriptase inhibitors (NRTIs) block reverse transcriptase enzyme, and they target and intersperse with steps in viral replicative processes
  • Protease inhibitors (PIs) block the activity of HIV protease needed for the production of all viral proteins

Bacterial Resistance

• Enzymatic inactivation: bacteria produce enzymes that breakdown or modify antibiotics
• Alteration of target sites: bacteria change the structure of the antibiotics target, making the drug ineffective

Antimicrobials to Control or Inhibit Fungus, Viruses, and Parasites

• Antifungals:
  • Azoles inhibit ergosterol synthesis, disrupting fungal membranes
  • Polyenes bind to ergosterol, causing cell lysis
• Antivirals: target viral replication
  • Acyclovir inhibits viral DNA polymerase and zanamivir blocks influenza neuraminidase
  • Acyclovir and vidarabine: used to treat herpes infections and shingles
  • Ganciclovir: used to treat systemic cytomegalovirus illness
  • Foscarnet: used in cases of acyclovir or ganciclovir resistance which treats illness caused by herpes
  • Cidofovir: inhibits viral DNA polymerase
  • Anti-HIV drugs: nucleoside reverse transcriptase inhibitors (NRTIs) target and interfere with critical steps in viral replicative processes and protease inhibitors (PIs) block the activity of the HIV protease needed for the production of all viral proteins
• Nonnucleoside reverse transcriptase inhibitors (NNRTIs) prevent HIV DNA synthesis by selectively binding to and inhibiting the viral reverse transcriptase enzyme
• Integrase inhibitors prevent the incorporation of the HIV genome into the hosts chromosomes
• Fusion inhibitors prevent HIV entry into cells
• Antiparasitic:
  • Chloroquine inhibits heme detoxification (malaria)
  • Metronidazole disrupts DNA in anaerobic protozoa

Metabolism

• Metabolism obeys the laws of thermodynamics
  • Energy cells obtain from their environment is most often conserved as ATP
  • Oxidation-reduction reactions play a role in energy conservation
  • Chemical reactions that occur in cells are organized into pathways
  • Each reaction of a pathway is catalyzed by an enzyme or ribozyme
  • The functioning of the biochemical pathway is regulated
• Regulation of metabolism relies on 3 important mechanisms
• Metabolism is the total of all chemical reactions in the cell and is divided into :
  • Catabolism: breaks down molecules to release energy (breaking glucose down for ATP) and generate precursors for biosynthesis
  • Anabolism: builds complex molecules using energy (making proteins or DNA)
• Organisms are grouped by energy classes including energy source, electron source, and carbon source

Grouping by Energy Source

• Phototrophs use light for energy
• Chemotrophs use chemicals for energy

Grouping by Electron Source

• Lithotrophs use inorganic compounds
• Organotrophs use organic compounds

Grouping by Carbon Source

• Autotrophs use CO2 as a carbon source
• Heterotrophs use organic molecules for carbon

Redox Tower

• The redox tower shows how easily molecules donate or accept electrons
• The top of the tower has strong electron donors (glucose)
• The bottom of the tower has strong electron acceptors (oxygen)
• A greater difference between the E0 of the donor and the E0 of the acceptor means a more negative AG
  • Equilibrium constant for oxidation-reduction measures the tendency of the reducing agent to lose electrons, it also means that a more negative EO means better electron donor
• When electrons move down the tower during cellular processes, energy is released and powers ATP production

Energy-Rich Compounds

• Energy-rich compounds store energy in their chemical bonds
• ATP is the main energy carrier, a currency for reducing power to supply electrons for chemical reactions
• Phosphoenolpyruvate (PEP) is a high-energy intermediate
• Acetyl-CoA important in energy and biosynthesis
• When these bonds break, energy is released to drive cell processes

Fermentation, Aerobic Respiration, and Anaerobic Respiration

• Fermentation does not use an electron transport chain, generates a protomotive motive force, and ATP is synthesized only by substrate-level phosphorylation
• Aerobic respiration catabolizes an organic energy source using CO2
  • Glycolytic pathways like glycolysis and the TCA cycle use oxygen as the final electron acceptor
  • Produces ATP indirectly via the activity of the electron transport chain and high-energy electron carriers
• Anaerobic respiration has inorganic nitrate or sulfate(NO3, SO4 Fe3+, SeO42-) electron acceptor

Electron Carriers

• Electron transfer electrons through ETS to generate a proton gradient for ATP production
  • FAD: flavin adenine dinucleotide
  • FMN: flavin mononucleotide, riboflavin phosphate
  • Coenzyme Q: aka ubiquinone
  • Cytochromes: use iron to transfer electrons (part of heme)
  • Nonheme iron-sulfur proteins: use iron to transport electrons (iron is not part of a heme group)

1. NADH & FADH2: brings electrons from earlier pathways (glycolysis, Krebs cycle)
2. Flavoproteins: accept the first electrons in the ETC
3. Quinones: small, mobile carrier that pass electrons between complexes
4. Cytochromes: proteins with iron that pass electrons near end of chain
5. Terminal electron acceptor: oxygen (aerobic) or another molecules (anaerobic)

Proton Motive Force

• PMF is the energy from protons (H+) being pumped across the membrane during ETC
  • What: proton gradient across the cell membrane in bacteria
  • How: as electrons move through the ETC, protons are pumped outside the cell
  • Why: the gradient drives ATP synthesis through ATP synthase and can also power flagella
• Diffusion of proton back across membrane (down gradient) drives formation of ATP
• ATP synthase:

Energy Conservation

• Energy conservation means capturing energy from catabolic reactions and using it to make ATP
• Microbes use:
  • Substrate-level phosphorylation is direct ATP production during glycolysis
  • Oxidative phosphorylation is ATP from ETC and PMF
  • Only 4 ATP molecules are synthesized directly from oxidation of glucose to CO2; most ATP is made when NADH and FADH2 are oxidized in the ETC

Sugars and Polysaccharides

• Prokaryotes make sugars through gluconeogenesis (reverse of glycolysis)
  • Precursors come from glycolysis and the Krebs cycle
• Polysaccharides are made by linking sugars

Pentose Phosphate Pathway

• The PP helps breakdown glucose as an alternative way:
  • Produces NADPH for biosynthesis
  • Provides ribose-5-phosphate to make DNA and RNA
  • Supplies intermediates for amino acid production
• The pentose phosphate pathway is also called the hexose monophosphate pathway
• It operate at the same time as the glycolytic pathway or enter-duodoroff pathway, aerobically or anaerobically
• It is an amphibolic pathway: glucose-6-P + 12NADP+ + 7H2O → 6CO2 + 12NADPH + 12H+ Pi

Amino Acids and Metabolic Processes

• Amino acids are made from metabolic intermediates: Glycolysis provides precursors for serine and alanine Krebs cycle provides a precursor for glutamate and aspartate Called the citric acid cycle it is common in aerobic bacteria, archaea, free-living protists, and fungi and it has a major role as a source of precursors for metabolites to use in biosynthesis
• The pentose phosphate pathway provides precursors for aromatic amino acids

Nucleic Acid Base Precursors

• Microbes make DNA and RNA bases from: P-PP provides ribose-5-phosphate (sugar part)
• Amino acids supply nitrogen and carbon atoms

Fatty Acids

• Precursors are acetyl-CoA from Krebs used to build fatty acids
• Entry point: fatty acids are broken down by beta oxidation, producing acetyl-coA which enters the Krebs cycle for energy
• Triglycerides are common energy sources hydrolyzed to glycerol and fatty acids by lipases, glycerol degraded via the glycolytic pathway, fatty acids often oxidized via B-oxidation pathway

Chemotrophs vs Autotrophs

• Chemotrophs: energy source is chemicals and carbon source uses organic compounds
  • An example is E.coli
• Autotrophs: energy source is light or chemicals, carbon source is CO2( inorganic)
  • An example is cyanobacteria

Enzymes

• Apoenzyme: protein component oof an enzyme
• Cofactor: nonprotein component of an enzyme
  • Prosthetic groups- firmly attaches, coenzyme-loose attached
• Holoenzyme : apoenzyme + cofactor

Enzyme Reactions and Activity

Enzyme reactions entail A+B → C +D : transition state, activation energy, where enzymes speed up reaction by lowering Ea, by increasing concentrations of substrate at the active site of enzyme which results in induced fit Enzyme activity is the impact it has on substrate concentration, pH, temperature Ribozymes splice pre-rRNA, mitochondrial rRNA, and mRNA. Catalyze peptide bond formation and self-replicate