Antifungal agents and others

what are the three types of antibacterials?

small molecules, antibacterial peptides and bacteriophages

describe what antimicrobial peptides are like

-              Antimicrobial peptides have the same structure as proteins (primary, secondary, tertiary and quaternary)

-              The primary sequence may be shorter

-              They can be alpha helical or beta pleated in structure or may have a mix of both (differences in secondary and tertiary structure)

-              They have different sources eg different organisms such as animals or bacteria or fungi etc or can be synthetic

describe the mechanism of action of antimicrobial peptides in the cell membrane

Electrostatic interactions - AMP’s are cationic and amphipathic so can bind to the negatively charged bacterial membrane

Insertion into membranes - after binding, AMP’s can insert themselves into the membrane and adopt many conformations eg alpha helix or beta pleats. Amphipathic nature means AMP’s can interact with hydrophilic and hydrophobic regions

Pore formation and membrane disruption

what is the barrel - stave model

AMPs form a barrel-like structure, embedding themselves into the membrane, creating channels

what is the carpet model?

AMPs coat the membrane like a carpet, disrupting it as their concentration increases

what is the toroidal model?

• AMPs bend the membrane lipids, creating a toroidal pore 

• These processes lead to membrane depolarization, leakage of cytoplasmic contents, and eventually bacterial lysis (death of bacterial cell)

Describe the mechanism of action of AMP’s intracellular

-              The antimicrobial peptide is capable of entering the cytoplasm

-              Here, the peptide can target the bacterial DNA (inhibition of DNA replication)

-              The peptide can also target the ribosome to block translation

Describe what bacteriophages are like

-              Have high host specificity and can commonly only infect only one bacteria species

-              They have both lytic and non-lytic phages, where lytic phages cause lysis of the bacteria

-              Bacteriophages are sensitive to heat

-              They come in many shapes and sizes including filamentous, icosahedral with tail and without tail

-              They are involved in transduction (upon infection, they can remove some of the host cells DNA and transfer it to a new host)

-              They have 2 ways of replication – lytic and lysogenic

They are used in both diagnosis and treatment

Describe the structure of a bacteriophage

o   Tadpole shaped

o   Capsid head – hexagonal in shape and is composed of coat protein and encapsulates the phage DNA

o   Genome DNA – double stranded DNA that codes for enzymes and proteins necessary to replicate more viruses

o   Tail sheath – hollow core covered in contractile sheath

§  When infecting bacteria, DNA travels from the head to the bacteria via the sheath

o   Tail fibre – helps anchor the phage on the membrane

o   Spike – a protein complex that penetrates into the host cell membrane during infection and facilitates the injection of the bacteriophage DNA into the host cell

Describe the injection machinery of a bacteriophage

o   There is a conformational change on the baseplate upon binding to the cell membrane

o   The sheath contracts from the extended state to the contracted state

o   During sheath contraction, the rigid tail tube pierces the host cell’s outer membrane

o   The bacteriophage DNA then begins to enter the host cell

Describe the lytic replication pathway of bacteriophages

-              The virus material is injected into the bacterial cell

-              DNA replication of the viral DNA occurs using proteins from the virus or using the host cell’s proteins

-              The products formed are used to assemble the bacteriophage

-              They break out of the host to infect other bacterial cells

-              Important to note that the viral DNA does not become integrated in the host’s DNA

Describe the lysoghenic pathway for bacteriophages

• Adsorption 

  • Phage attaches to bacterial cell surface  

• Penetration  

  • Phage injects genetic material (DNA or RNA) into host cell 

• Integration 

  • Phage DNA integrates into host chromosome forming a prophage 

• Replication 

  • Prophage replicates and as host divides it is passed on to daughter cells - each daughter cell contains the viral DNA integrated into its genome 

• Induction 

  • When daughter cell is induced (triggered by something e.g. UV radiation, stress) the viral DNA is excised (cut out) of the host DNA  

• Synthesis 

  • Phage DNA and proteins are synthesised using host cell machinery  

• Assembly 

  • Phage DNA and proteins assemble to form new viruses - these are called infective virions  

• Release 

  • Host cell undergoes lysis and virions are released  

why may bacteriophages be eliminated quickly from the body?

can be recognised as non-self

How can recognition of bacteriophages as foreign be overcome?

-              by administering the bacteriophage directly to the site of infection so it can eliminate the infection before It is eliminated by the body

-              The bacteriophages may cause an inflammatory response as they are detected by the macrophages but they themselves do not cause disease

-              A challenge may occur if the infection is an area where it is hard to administrate the bacteriophage

Describe how bacteriophages can be used clinically?

-              The bacteria that is infecting the patient can be sampled and screened against the bacteriophages to identify which would the most effective to use in the patient

-              The bacteriophage species would then be isolated and then tested on animal models and then tested on the patient

What are the limitations of bacteriophage therapy

-              Expensive to develop as it is personalised medicine

-              Bacteria can gain resistance

-              A bank of bacteriophages needs to be made to screen the bacteria to find a suitable species

-              May be difficult to find a phage that works against a specific bacterium

-              Inflammation may occur

-              Difficulty in administering if the infection is in a hard to reach area

Briefly, what is the life cycle of a fungus?

Infection and initial colonisation

Sexual reproduction

Spread and new infections

Describe what happens during infection and initial colonisation

• Infection: 

  • Fungal infections begin when spores (the reproductive units of fungi) land on a suitable host. These spores are typically carried by the air or other means 

  • When the spores find the right environment (moisture, temperature, nutrients), they germinate and begin to grow, forming hyphae (thread-like structures that make up the fungus) 

• Hyphal Growth: 

  • The hyphae invade the tissues of the host, causing damage and spreading 

  • As they grow, they release enzymes that break down the host tissue to access nutrients in the host  

  • The fungus may form a mycelium, which is a dense network of hyphae that colonizes the tissue even more  

• Asexual Reproduction: 

  • Most fungal infections rely on asexual reproduction for rapid spread 

  • In this phase, the fungus produces conidia (or spores) asexually 

  • Conidia are released into the environment and can infect new hosts or spread within the same host 

    • This process allows the fungus to reproduce quickly and spread over a wide area 

Describe what happens during sexual reproduction of a fungus

• Environmental Trigger: 

  • Sexual reproduction happens when conditions become less favourable for asexual reproduction, or when the fungus has to ensure genetic diversity 

• Fusion of Hyphae (Plasmogamy): 

  • In many fungi, two genetically distinct hyphae of different mating types come into contact. These hyphae fuse their cytoplasm = plasmogamy 

  • This step results in a dikaryotic (two nuclei) cell, where each cell contains two separate nuclei, one from each mating type 

• Karyogamy (Fusion of Nuclei): 

  • After plasmogamy, the two nuclei fuse during karyogamy, forming a diploid (two sets of chromosomes) zygote 

  • This diploid cell is short lived and undergoes meiosis to produce genetically diverse haploid spores 

• Formation and Release of Sexual Spores: 

  • The sexual spores (like ascospores in Ascomycota or basidiospores in Basidiomycota) are produced in specialized structures (like asci or basidia) 

    • These spores are released into the environment and can then infect new hosts or form new colonies 

Describe what happens during the spread and for new infections for fungi

• Asexual Spores: 

  • These spores are the primary method for rapid infection and can spread quickly, leading to acute infections 

• Sexual Spores: 

  • Sexual spores are important for the fungus’s ability to adapt to changing environments and ensure survival in diverse conditions 

what is the difference between a bacterial and fungal cell wall

fungal - chitin

bacteria - peptidoglycan

Describe the fungal infection mechanism

-              Route of invasion of the fungal pathogen

-              Pathogen recognition and response via the host’s immune response

o   Use of pathogen molecular pattern molecules (PAMPs)

-              Morphological modulation of fungal cells for immune invasion

Pulmonary transmission and pathogenesis of invasive fungal infections

how can pulmonary transmission of fungal infections occur?

o   Inhalation of spores or conidia

o   Entry into the alveoli

o   Eliciting the first line defence

o   Depletion of phagocytic cells, leading to disease progression as pulmonary nodules and pneumonia

o   Macrophages phagocytose the fungal cells or encapsulate them and form granuloma

o   Fungal cells parasitize the macrophages, leading to vomocytosis of intact fungi and their circulation in the bloodstream

o   These can then cross the BBB to cause systemic infection

Describe what imidiazoles are like and how they work

o   Inhibit sterol 14alpha-demethylase, preventing the conversion of lanosterol to ergosterol, which is critical for fungal cell membrane stability and fluidity

o   Broad spectrum and effective against Candida and other pathogens

o   Has multiple administration routes

o   May also induce oxidative stress to enhance the antifungal effects

describe what polyenes are like and how they work

-              eg nystatin and amphotericin B

o   These bind to ergosterol in the fungal cell membrane, forming pores, leading to leakage of cell content and eventually cell death

o   These are highly effective and used for serious fungal infections

o   Amphotericin B triggers oxidative stress, amplifying the antifungal activity

describe what echinocandins are like and how they work

-              eg caspofungin, micafungin, anidulafungin

o   Inhibit 1,3-beta-d-glucan synthase, preventing the synthesis of 1,3-beta-d-glucan, which is a component of the fungal cell wall

o   Effective against Candida and Aspergillus

describe what allylamines are like and how they work

-              eg terbinafine and naftifine

o   Thee block squalene epoxidase, which is an enzyme required for the conversion of squalene lanosterol in the ergosterol biosynthesis pathway

o   Effective for superficial infections

describe what pyrimidine analogues are like and how they work

-               eg 5-Flurorcytosine

o   Enters the fungal cell walls via cytosine permeases, which is then converted into 5-fluoracil

o   This interferes with RNA and DNA synthesis, impairing protein production

o   Effective for infections caused by Candida and Cryptococcus

what factors contribute to fungal resistance

o   Fungal adaptation eg phenotypic plasticity, genetic mutations, chromosomal changes, sexual reproduction and horizontal gene transfer

How do fungal biofilms contribute to resistance

-              Candida forms biofilms on medical equipment

o   This means that fungal cells can be protected with an extracellular polymeric matrix, physically blocking the antifungal drugs from reaching the fungal cells

o   Biofilms also contain persister cells – dormant of metabolically inactive cells, allowing them to survive when most fungal cells die, making them resistant

o   Fungi grows slow so, drugs that target actively dividing cells is likely to be ineffective

which antifungals are more prone to resistance?

-              Azoles are more prone to resistance whilst polyenes have lower rates of resistance

what is the resistance mechanism for azaleas?

§  Overexpression of ABS (ATP binding cassette) and MFS efflux pumps, allowing fungal cells to remove antifungals quicker

§  Point mutation of ERG11 or CYP51 changes the shape of the enzyme so, drug can no longer bind effectively

§  Mitochondrial dysfunction and stress signalling increases survival so, the mitochondria can alter energy production and activated pathways can counteract the effect of the drug

what is the resistance mechanism for polyenes?

§  Loss of ERG3 gene and enzyme function, reducing ergosterol synthesis, so drug is less able to bind

what is the resistance mechanism for echinocandins?

§  Mutation in FKS gene changes the chape of synthase enzyme decreasing drug binding affinity

what is the resistance mechanism for pyrimidine analogues?

§  Mutations in cytosine permease (Fcy2) protein, preventing drug entry into the fungal cell

§  Mutations in cytosine deaminase (Fcy1) protein, preventing the activation of the drug so it no longer has efficacy

what is the resistance mechanisms for allylamines?

§  Mutations in the ERG1 gene, reducing drug binding and activity

what are anti fungal peptides?

-              These are small and naturally occurring proteins, made by higher organisms as a defence mechanism against fungal pathogens

-              These are part of the immune system

-              Examples include thaumatin, thionins, plant defensins

what is the mechanism of action of anti fungal peptides?

-              Mechanisms of action are not fuly understood

-              May disrupt fungal cell membrane, forming pores, which leads to cell lysis

-              May interfere with DNA, RNA etc, stopping growth and replication

-              May bind to fungal surface receptors and becomes internalised, acting directly on the cell

-              May trigger different pathways in the cell, causing apoptosis

-              Can bind to cell wall components such as glucans, weakening it

what is specific translation using exogenous mRNA?

-              introduction of synthetic mRNA into the body

how does specific translation using exogenous mRNA work?

o   The synthetic mRNA codes for a protein of interest eg a virus protein or human protein

o   This synthetic mRNA is delivered into the patient’s cells, where it is then translated to form the protein

o   Eg mRNA vaccine for COVID-19 – codes for the spike protein

what is post-transcriptional modulation?

-              regulates gene expression by affection RNA molecules

how does post-transcriptional modulation work?

o   Modification of the mRNA or degradation to prevent the expression of a protein

o   Short interfering RNA (siRNA) – short double stranded RNA, which can bind to a specific mRNA sequence

§  Once bound, these activate a cellular process that leads to the degradation of the mRNA, so the protein is not produced

§  Eg can be used in some diseases to silence the faulty protein or gene

o   MicroRNA (miRNA) – small naturally occurring RNA’s that regulate gene expression by binding to mRNA to prevent gene translation or to trigger degradation

§  Synthetic miRNA can be used to restore activity of specific miRNA that aren’t present in specific diseases

§  Eg synthetic miRNA can be used to correct improper gene regulation in CVD or cancer

o   Antisense oligonucleotides (ASO) – short RNA or DNA, which are designed to bind to complementary regions of mRNA

§  These block the ability of mRNA to be translated into a protein, promoting the degradation of the mRNA

§  Eg in spinal muscular atrophy (SMA), ASO’s are used to restore the production of a missing or defensive protein

what is gene activation and editing?

targets the DNA, modifying gene expression

how does gene activation and editing work?

o   Short acting RNA (saRNA) – small RNA molecules that can activate specific genes, by interacting with DNA or the machinery involved in gene transcription to increase expression

§  Useful in diseases where a specific protein is not produced enough eg CF

o   CRISPR-Cas9 – gene editing technology that uses RNA to direct a Cas9 protein to a specific location on the DNA

§  Acts as molecular scissors to cut DNA

§  Allows the disabling of a gene, correction of a mutation or insertion of new genetic material

§  Eg can be used to edit out defective genes that cause diseases like sickle cell anaemia, allowing for the genetic mutation to be corrected

what is protein inhibition or degradation using RNA aptamers?

-              small single stranded RNA molecules that can bind to specific proteins and either block their activity or mark them for destruction

how does protein inhibition or degradation using RNA aptamers work?

o   Aptamers are designed to fold into 3D shapes that allow them to bind tightly to a particular protein or target molecule

o   Once bound, the RNA aptamer can inhibit the protein’s function or mark the protein for destruction

o   Eg research to bind to cancer proteins to stop their activity and prevent further cell growth

what are traditional vaccines like?

o   Contain weakened or inactivated forms of pathogens or pathogen components

o   Production is slower and more complex, requiring virus growth or protein extraction

o   Takes years to develop, especially for new pathogens

o   More expensive due to complex production processes

what are mRNA vaccines like?

o   Contain synthetic mRNA that instructs cells to produce a therapeutic protein

o   Production is faster and simpler, with no need to grow the virus

o   Can be developed in weeks once the virus sequence is known

o   More cost-effective due to streamlined production - less complex and involved less steps

what are the advantages mRNA vaccines?

o   Faster Development: Can be quickly adapted for new viruses or variants by changing the mRNA sequence

o   Lower Cost: Simplified production process reduces costs

o   Platform Flexibility: Once the mRNA platform is established, it can be easily adjusted for different pathogens

o    Streamlined Clinical Trials: Less testing required for each new variant, reducing time and costs

o   Simpler Components: No need for complex virus culturing or protein purification

o   Adaptability: New variants can be quickly targeted by modifying the mRNA sequence

what are the disadvantages of mRNA vaccines?

o   Storage and Stability: Require very low temperatures, making storage and transportation challenging.

o   Immune Response Variability: Effectiveness may vary between individuals.

o   Side Effects: Short-term side effects are common, and there are rare instances of more serious side effects (e.g., myocarditis).

o   Limited Long-Term Data: New technology, so long-term safety and effectiveness are still being studied

o   Need to update the vaccine frequently every (6 months) due to the evolution of viruses and new strains developing - as one vaccine

targets one strain

describe the structure of mRNA vaccines

o   5’ cap – protects mRNA from degradation and helps to initiate translation by enabling ribosome recognition

o   Facilitates mRNA transport from the nucleus to the cytoplasm

what is the coding sequence in a mRNA vaccine?

-              carries the genetic instructions to make the viral protein

o   Consists of codons that encode amino acids to form the protein

what the untranslated regions in a mRNA vaccine?

-              Noncoding regions at the 5’ and 3’ ends

o   5’ UTR – influences how efficiently the ribosome will initiate translation

o   3’ UTR – stabilises the mRNA and contributes to the regulation of mRNA degradation

what is the poly A tail in an mRNA vaccine?

-              a string of adenosine nucleotides added to the 3’ prime end of mRNA

o   Protects mRNA from degradation, aids stability and promotes translation

what is not part of mRNA but is essential for mRNA vaccines?

-              Lipid nanoparticles (delivery system) – not part of mRNA but is crucial for delivery

o   Encapsulates the mRNA to protect it and allows it to enter cells by fusing with the cell membrane

describe the mechanism of action of mRNA vaccines

o   fter injection, the mRNA vaccine is taken up by antigen presenting cells (APC’s) eg dendritic cells

o   Once it enters the cell, it escapes the endosome and enters the cytosol

o   The mRNA is translated by the ribosomes into a viral protein

o   The translated protein is processed inside of the cell, where it is broken down into smaller fragments by the proteasome

o   These protein fragments are then displayed on the surface of APC’s using MHC class I proteins, presenting the fragments to CD8 T-cells

o   These cytotoxic T-cells recognise the antigens presented by MHC class I so, become activated

o   The activated CD8 cells kill the infected cells by releasing cytolytic molecules, such as perforin and granzyme

o   Some of the translated viral protein is secreted from the APCs and can be taken up by other immune cells

o   The secreted antigen is degraded within endosomes and are presented on the cell surface by MHC class II proteins to CD4 cells (helper T-cells)

o   Activated CD4 cells further the immune response by helping B-cells to produce neutralising antibodies to target and neutralise circulating pathogens

o   Helper T-cells activate phagocytes through the release of inflammatory cytokines, enhancing the clearance of pathogens from the body

describe the manufacturing process of mRNA vaccines

o   Target sequence identification – identify the gene encoding the pathogen, isolate it and clone into a plasmic

o   Transformation and fermentation – plasmid containing target DNA is introduced into bacterial cells for rapid growth so many plasmids can be isolated

o   Restriction enzymes – these cut DNA of the plasmid at a specific sequence, linearising the plasmid for easier transcription

o   In-vitro transcription – the linearised plasmid is used as a template for in-vitro transcription where RNA polymerase reads the DNA and synthesises mRNA (mirrors the code of the protein)

o   Lipid nanoparticle formation – encapsulates the mRNA for delivery into human cells

o   Production – finalisation of the final formulation, performing quality checks and packaging

what are nano cages?

-              hollow self-assembly structures, which are composed of molecules like proteins

o   Can be applied to drug delivery, vaccine development and diagnostics

o   Nanocages can encapsulate therapeutic agents or display antigens on their surface

o   Ferritin nanocages – protein that naturally forms a hollow nanocage for storing iron

§  Can be used to encapsulate chemotherapeutics, protecting from degradation and allowing for targeted release

§  These can also carry imagining agents for diagnostic purposes

§  Can be used for antigen presentation so, can be used to stabilise vaccine antigens and enhance the immune response

what are mosaic vaccines?

o   use of nanocages to display antigens from multiple virus strains at the same time so, can be used against multiple strains of a virus

§  This is good for vaccines with high mutation rates such as COVID-19 or influenza

§  Stimulates a bigger and cross-reactive immune response

How does CRISPR Cas 9 work?

-              Bacteriophages attack bacteria by injecting their DNA

-              Bacteria fights back by cutting pieces of the bacteriophage DNA and storing it in their own DNA as a memory

-              This region is called the CRISPR array

-              Next time the bacteriophage attcks, the bacteria uses the stored DNA to create a guide RNA

-              This finds the matching part of the viral DNA

-              The guide RNA directs Cas9 (a nuclease) to cut the viral DNA at the site matching the guide RNA

-              Cas9 then cuts the bacteriophage DNA, stopping it from spreading

-              This technology can be used in other therapeutics (ie creating a guide RNA for Cas9 to target specific diseased DNA)

give some examples where CRISP Cas 9 is used?

-              Eg blood disorders – sickle cell anaemia and thalassemia by correcting the faulty blood cell genes

-              Eg HIV – used to cut out the HIV viral DNA, preventing replication and editing the CCR5 gene

o   CCR5 receptor that is used by HIV to enter cells

o   Editing this gene prevents further infection of other cells

-              EG A;S research – fixing of SOD1 gene that causes ALS

what are the challenges of CRISP Cas 9?

o   Ethical implications – editing genes in embryos can lead to permanent changes that can be passed down to future generations

o   Cutting DNA at the wrong place can cause unintended mutations

Also difficult to get CRISPR components into cells