CMMB 343 - Lab Exam 2 - Presentations

what are antibiotics?

super drugs that inhibit the growth and multiplication of pathogenic microbes, high efficacy when first introduced or prescribed, however a small number of bacteria attain resistance and survive 

what are beta-lactam antibiotics?

large class of antibiotics, lyse bacteria by inhibiting synthesis of peptidoglycan walls, bind irreversibly to enzymes responsible for cross-linking in peptidoglycans, has structural similarity to D-Ala-D-Ala dipeptide (target substrate of enzymes)

explain the antibiotic resistance pathway

misuse/overuse antibiotic → spread of resistant genes via lateral/horizontal gene transfer → development of multi-drug resistant pathogens → new methods are needed to combat this (where siderophore-antibiotic conjugates come in)

what are beta-lactamase enzymes?

threatens the efficacy of beta-lactam antibiotics, deactivates beta-lactams before it can bind to target bacteria 

what are siderophores?

chelating compounds synthesized by microorganisms which sequester iron from the surrounding environment, a method for selective antibiotic delivery (siderophore-antibiotic conjugates)

so bacteria can sequester iron - crucial for transportation of oxygen and a co-factor for enzymes involved in DNA synthesis/repair and antioxidant defense mechanisms

what is the trojan-horse approach?

• combines a siderophore with an antibiotic component (sideromycins or siderophore-antibiotic conjugate) → bacterium recognizes the iron-siderophore complex → complex is actively transported into the cell → the antibiotic is released and results in cell lysis 

  • Linkage between antibiotic and siderophore cleavable (reversibly linked)

  • Bond breaks in response to chemical or biological signal that indicates arrival at target 

  • Enter through outer membrane transporter and bind to siderophore binding protein (SBP)

what are advantages of siderophore delivery?

• Siderophore delivered actively through membrane, drugs are undetected

• Delivered in more potent doses than would be via diffusion

• Selects for non-virulent bacterial strains that lack siderophore receptors, limits development of new virulent strains 

what are disadvantages of siderophore delivery?

• Difficult to design a cleavable linkage

• May be toxic to organs 

• Inner membrane of G -ve poorly characterized, restricts antibiotics 

what are lentiviruses?

subgroup of retroviruses, can infect both dividing and non-dividing cells, has a ssRNA genome with enzymatic proteins, structural proteins, and supplementary genes

what is the life cycle of a lentivirus?

1. Viral entry, envelope targets specific cell type, membrane fusion

2. Uncoating, capsid degrades, RNA and proteins released into host cytoplasm

3. Reverse transcription (viral RNA → dsDNA)

4. Nuclear import and integration, proviral DNA imported into nucleus and integrated into host genome (via viral integrase)

5. Replication, transcription, and translation using the host cells machinery

what are lentiviral vectors?

genetically modified lentivirus, designed to deliver genes safely into cells → transgenes

• Can be integrated into many cell types 

• Can be used on its own, can’t target specific genome regions without Cas9

• Integrated via the PIC and integrase protein, often need promoters to drive transgene production

why are lentiviruses suitable for gene editing?

• Relatively simple

• Lentiviruses produced as replication-defective particles (virus gene inactive after reverse transcription)

• Spliced transgene cassette to be independently expressed 

what’s the mechanism of lentiviral gene editing?

• Identify and insert transgene into LV, insert Cas9 gene and sgRNA into LV plasmids

• Insert LV package into cells 

• Transduction - RNA reverse transcribed, Cas9 enzymes produced by cell, transgene integrated into genome

what’s the significance of plasmids in lentiviral gene editing?

viral vectors could recombine into a functional lentivirus → LV kept as 3 separate plasmids then combined for use in packaging cells 

what’s the role of promoters in lentiviral gene editing?

inserted genes need appropriate promoters, change is permanent, need compatible promoters for long term

compare lentiviral and non-viral vector therapies

• Non-viral 

  • Improved safety due to inability to replicate or infect

  • Carry larger inserts, easier to engineer

  • Low transfection efficiency, lack of targeting

  • Cannot effectively deliver CRISPR/Cas9

• Lentiviral 

  • Integrate into genome

  • Best suited for dividing cells

  • Commonly used in ex vivo

  • Long-term function, genetic material is copied into new cells 

what is the microbiome?

include bacteria, fungi, archaea, and viruses, includes skin, GI tract, respiratory tracts, diversity and structure influenced by age, geography, medications, genetic factors, disease states, and diet and lifestyle

what are the common bacteria in the GI tract?

bacteroidetes, firmicutes, proteobacteria, tenericutes 

what is cancer?

disease caused by unchecked proliferation of cells and evolve under natural selection

how do gut microbes influence cancer therapy through immune system modulation?

Microbes interact with immune cells 

what is eubiosis?

diverse, symbiotic community that supports immune function

what is dysbiosis?

microbial imbalance, promotes inflammation and weakens immune regulation

how do gut microbes influence cancer therapy via microbial metabolic signaling?

• Dysbiosis linked to decreased synthesis of short-chain fatty acids (produced through bacterial fermentation of fiber)

• Metabolites important to maintain epithelial barrier, control inflammatory responses, and supporting metabolic regulation in peripheral tissues

how do gut microbes influence cancer therapy via drug metabolism and biotransformation?

• Pharmacomicrobiomics: microbiota-drug interactions

  • Gut microbiome can alter metabolism, efficacy and toxicity of chemotherapeutic agents

what are immune checkpoint inhibitors?

class of cancer immunotherapy that targets immune regulator pathways (PD-1, PD-L1) that act as brakes on the T-cells (prevents excessive immune response under normal conditions)

• Can block inhibitory interactions

• Restore T cell activation 

• Results in anti-tumor immune responses - cytotoxic T-cells can recognize and destroy cancer cells

• anti-PD-1 (block PD1 or PD-L1)

how do microbes in the gut impact cancer therapy?

in the gut, activate T-cells 

Can provide ICI efficacy, increase infiltration of killer T-cells 

how do antibiotics impact cancer therapy success?

can reduce therapy success, reduce key immune-signaling microbes, impairing T-cell activation and reducing effectiveness of ICIs

how is chemotherapy linked to gut microbiota?

• Drugs used in chemo can be chemically modified by enzymes produced by bacteria (activation or inactivation)

  • E.g. irinotecan 

• Dysbiosis can be caused by chemo drugs killing bacteria → can cause inflammatory response, inhibition of cell differentiation 

• Proteobacteria - able to reactivate irinotecan back into its toxic state, killing cells in the intestine 

what are probiotics?

live microorganisms that provide health benefits to a host, promote beneficial bacteria, reduce pathogens, compete with pathogens, can produce biofilms to increase resistance to pathogens 

what are prebiotics?

dietary ingredients that help the growth and functioning of probiotics 

what’s a fecal microbiota transplantation?

stool transplant to restore a functional microbial community, risks with immunocompromised patients (increase vulnerbaility to infection or sepsis, bacterial translocation)

what is a CRISPR-Cas system?

adaptive immune mechanisms by which bacteria and archaea defend against foreign nucleic acids and viral threats, use complimentary crRNA to guide proteins with nuclease capabilities towards a foreign RNA with the purpose of cleaving it out and degrading it 

• Multiple types of CRISPR systems, all cleave/alter RNA to defend against viral threats 

what do Cas1 and Cas2 do?

recognize a fragment (protospacer) of bacterial DNA which is then inserted into the host’s CRISPR array (spacer) → CRISPR array is transcribed into crRNA and processed by Cas proteins → cell can now identify that section of the bacteriophage DNA

what are protospacer-adjacent motifs (PAMs)?

signify that a sequence is foreign rather than self, located downstream of the viral protospacer

what are the classes of CRISPR-Cas systems?

• Two major classes

  • Class 1: multi-protein effector complexes

    • Types I, III, IV

  • Class 2: single effector protein

    • Types II, V, VI

• Type I - multi-protein complex, targets DNA

• Type II - Cas9, targets DNA

• Type III - targets DNA and RNA

• Type V - Cas12, targets DNA

• Type VI - Cas13, targets RNA 

what are gut microbiota?

community of bacteria, archaea, eukarya, and viruses in the GI tract, evolved with humans to support immune function, metabolism, and defense against pathogens, includes mouth to anus 

what are the most common bacteria in the gut microbiome?

• Firmicutes (G+) and bacteroidetes (G-) make up 90% of phyla in the gut

• Others include actinobacteria, proteobacteria, fusobacteria, and verrucomicrobia

what kind of bacteria is mostly in the large intestine?

Mostly obligate anaerobic bacteria, low oxygen content and neutral to slightly acidic pH in large intestine

what kind of bacteria is mostly in the small intestine?

Small intestine has more aerobes and facultative anaerobes (more oxygen content)

what are bacteriophages?

viruses that infect and replicate only in bacteria, very small, composed of head/capsid, tail and tail fibers 

• Caudovirales most common group 

• Phages mostly found in mucus, can translocate to intestines and act as modulators of the gut microbiota

• Act as predators, control bacterial density and maintain balance 

• Can suppress potentially pathogenic or overgrowing bacteria 

• 90% of gut viruses 

what is the lytic cycle?

phage immediately replicates within host, leading to cell lysis and release of new virions

what is the lysogenic cycle?

phage genome integrates into or coexists with the bacterial chromosome as a prophage, remaining inactive until triggered to enter the lytic phase 

what are pros of bacteriophages in the gut?

• Highly specific bacterial attackers, target specific taxa (harmful bacteria), maintain beneficial microbes

• Can contribute to HGT for evolution and adaptability 

• Genes that enhance bacterial fitness, stress tolerance, and metabolic potential 

• Interact with host immune system and enhance immune responses (e.g. cancer immunotherapy)

• Actually pretty safe - minimally allergenic, little to no side effects, non-toxic to mammalian cells

what are cons of bacteriophages in the gut?

• Endotoxin release - rapid destruction of bacteria can release endotoxins

• Temperate phages can transfer viral genetic material to bacteria instead of killing them, gives them ability to produce harmful toxins, and antibiotic resistance 

• Can trigger inflammatory immune responses (colitis)

• Environmental sensitivity 

• Narrow host range

• Bacterial resistance mechanisms 

how can phage therapy help bacterial infections?

• Phage therapy better than antibiotics perchance

• Can target and lyse pathogenic bacteria

• Phages can be destroyed by harsh pH of GI tract 

• Phage-based in situ gene editing - adjust genome of gut bacteria

• Diagnostics - bacteriophages as biomarkers for cancers, can predict colorectal cancer and adenoma, also pancreatic cancer

• Fecal viral transplantation - can take healthy stool samples from one individual and transplant healthy gut viral populations into the colon of another and repopulate the gut, proliferation of beneficial species

what are biofilms?

free floating cells trapped in a polysaccharide matrix, composed of a single microorganism or a mixture (bacteria, fungi, archaea, etc.)

• Almost all bacteria can form biofilms

• Microbial growth and colonization leads to development

• Cell dispersal: microbes in mature biofilms can disperse and colonize new sites

how do biofilms form? (5 steps)

1. Bacteria cling to surfaces via flagella, pili, or physical force

2. Interactions with surface become irreversible and agglomeration begins

3. Microcolonies form and cells begin to multiply and divide

4. Cell maturation and stabilization of biofilm 

5. Bacteria disperse and spread infection

why are biofilms harmful?

• Biofilms can form on many surfaces, also antibiotic resistant

• Can grow under anaerobic conditions 

• Hypermutable - fast evolution 

• Can be harmful to humans, can’t remove with chemicals 

• 70% of bacterial infections are associated with biofilms which can lead to infectious diseases 

what is cystic fibrosis?

inherited disease that causes thick and sticky mucous secretions

what bacteria is most common in CF?

Pseudomonas aeruginosa: most common pathogen involved in CF, produces alginate

what is alginate?

mucoid exopolysaccharide involved in biofilms, formation induced by inflammatory response, causes chronic infections 

what are potential therapies for CF?

• DNAse 1 to destroy extracellular DNA of P. aeruginosa biofilms 

• Peptide antibiotics 

• Anti-inflammatory therapy before biofilm is established

what is dental plaque?

microbial biofilm of over 1000 bacterial species, survive through organic and inorganic materials found in saliva, gingival fluid, and bacterial products, exopolysaccharide protects cells against dehydration and harmful agents 

Microorganisms are capable of synthesizing and degrading their own exopolysaccharides in oral biofilms

how do dental biofilms form?

• Early plaque = mostly G+ (aerobic, oxygen rich)

• Biofilm matures 

  • Cells proliferate, EPS production

  • More cellular O2 consumption → less O2 available → anaerobic

• Existing plaque allows more aggressive G- bacteria to adhere (anaerobic, nutrient rich)

why is dental plaque bad?

• Leads to gingival inflammation 

• Bone and soft tissue destruction 

• Can gain access to circulatory system → CVD, Alzheimer’s, pancreatic cancer

• Brush teeth, use mouthrinses (antiseptic), scaling

how are biofilms related to prosthetic joint infections?

PJIs are highly susceptible to biofilms, commonly ESKAPE bacteria 

Economic burden, diagnostic hurdles, surgical complexity, immune system evasion