fungal biotechnology Flashcards
Sir Alexander Fleming
o In 1928, working with staphylococci (a bacterium that can cause sepsis)
o Noticed clear zones with no bacterial growth around a mould growing on the plate
o Discovered moult was effective against bacteria causing scarlet fever, meningitis, diptheria
o Published results but was largely ignored
Sir Alexander Fleming
o In 1928, working with staphylococci (a bacterium that can cause sepsis)
o Noticed clear zones with no bacterial growth around a mould growing on the plate
o Discovered moult was effective against bacteria causing scarlet fever, meningitis, diphtheria
o Published results but was largely ignored
Howard Florey & Sir Ernst Boris Chain
o Running a research programme on antibacterial substances
o In 1939, used penicillin culture in mice following reading Fleming’s paper
o Showed it had both antibacterial properties and lack of mammalian toxicity
o Norman Heatley (recruited by Florey) devised methods for mass culture in bed pans, assay, and extraction using solvents
Early use of penicillin
Albert Alexander
▪ Sratched by rose thorn
▪ Got sepsis, caused by pathogenic bacteria
▪ Abscesses over head and shoulders, eye infected and removed, lungs infected. Near death.
▪ Given penicillin, fever broke within a day
▪ Not enough penicillin to continue treatment (despite best efforts – extraction from urine!!). Died.
▪ Still showed value of penicillin
WW2
▪ Death rates from infection near 0, contrast to 15% in WW1
▪ Government subsidised factories willing to make penicillin for war effort
Later developments with penicillin
Drug development & Gene research
▪ Transition from using bed pans to fermenters
▪ Unusually for eukaryotic genes, found clustered together – more similar to bacterial operon!
▪ Genes from different penicillin species have v high homology
▪ Also high homology to to β-lactam antibiotics produced by Streptomycetes bacteria
→ suggesting common ancestry and bacterial to fungal lateral gene transfer ~370mya
▪ Modern drug development based mainly on duplication of gene clusters, causing ~15,000x increase in yield per g of culture in 70yrs. Colocalisation means they are easy to replicate.
How to antibiotics work?
• There are multiple targeted sites of action in bacteria
o Penicillin targets bacterial cell wall
o Inhibition of 30S/50S bacterial ribosomal subunits, interfering in protein synthesis
- Ribosomes are different to ours so can be targeted :)
o Cytoplasmic membrane structure
Why do antibiotics stop working?
Bacteria have their own mechanisms to become immune to antibiotic o Prevent drug uptake o Drug modification o Drug inactivation o Alteration of drug target
Antimicrobial resistance
o Naturally occurring isolates with resistance will occur
- Overuse of antibiotics selects for resistance
- Conjugation transfers plasmids at high frequency (lateral gene transfer)
- Many antibiotic resistance genes are plasmid encoded
Eg. MRSA (methicillin-resistant Staphlylococcus aureus)
o Increasingly pathogens are becoming resistant to multiple drugs
Eg. Multi-drug-resistant Mycobacterium tuberculosis (MDR-TB)
- We thought we could get rid of TB, now nearly untreatable
Solutions to antibiotic resistance
- give 2
- New antibiotics
o However, these take 10-25 yrs to develop, cost millions of dollars
o Not attractive to drug companies anymore due to the speed at which they become completely obsolete - Abstinence
o Only use when absolutely necessary
o Removes the selection pressure for resistant bacteria, bacteria lose plasmids
Characteristics of a fungus suitable for industrial use
▪ Able to grow and form product in large-scale culture
▪ Spores easily to inoculate and germinate in large fermenters
▪ Rapid growth to produce desired product
▪ Grows in inexpensive nutrient eg. Waste starch
▪ Not pathogenic
▪ Able to GM
Uses of fungi in industry
o Food
- Eating whole (mushrooms)
- Brewing, baking, cheese making
- Quorn mycoprotein
- For primary metabolites eg. Organic acids
o Useful products
- Antibiotics
- Alkaloids and gibberellins
- For secondary metabolites eg. Enzymes, β-lactam antibiotics
o As hosts for secretion eg. Of mammal proteins
o Bioremediation & waste treatment
o Biological control
We must be wary of alfatoxins (eg. Secondary metabolite produced by Aspergillus flavus)
Name 2 fungal species used in industry and what they’re used for
Aspergillus niger (ascomycete)
- Produces citric acid
- Used in food and drink flavouring (eg. jam)
- Fungus is deprived of iron, tricking it into producing excess citric acid as a siderophore to try and acquire iron from the environment in chelate form
Aspergillus niger (ascomycete)
Aspergillus niger (ascomycete)
- Produces citric acid
- Used in food and drink flavouring (eg. jam)
- Fungus is deprived of iron, tricking it into producing excess citric acid as a siderophore to try and acquire iron from the environment in chelate form
Puccina graminis
Basidiomycete
• Causes wheat stem rust
• Serious pathogen, causing up to 100% yield loss
• Has caused great historical famines
Two host plants
- Barberry leaf
- Wheat
Infection event – biotrophy in leaves
- When spore lands on suitable surface (eg. Barberry leaf)
- It germinates, forms germ tube that moves along leaf and forms appressorium that grows a pointed hyphae through the epidermis and between cells in the cortex
- This is done using turgor pressure in the infection peg. Sometimes enzymes are also employed
- Mycelium propagates in leaf mesophyll, some hyphae enter cells and form haustoria which absorb nutrients
Can plants resist fungal diseases?
How?
Localised apoptosis
• Localised, plant-induced cell death deprives biotrophic pathogen of food
• Often only a single cell dies, so no observable symptoms
• White spotting on leaves shows where appressorium formed and started to penetrate a plant cell, which underwent apoptosis! – hypersensitive response
Who’s Harold H Flor? What did he do?
Harold H Flor: originator of the gene-for-gene concept
o Explained genetic interactions between flax and a fungus
o Put into practice in his breeding program to develop rust-resistant flax
Flor’s experiment
Flor’s experiment: Gene for gene interaction between host and parasite
▪ Crossed flax plants together and pathogen lines together to generate new genotypes of both species
▪ Challenged flax plants with pathogens and identified those which were resistant, identified resistance alleles
▪ For every resistance allele found in the flax, he found a corresponding allele in the pathogen
▪ He called these corresponding pathogen genes ‘avirulence genes’
How does gene-for-gene work?
▪ Both a functional plant resistance gene (R) and a functional avirulence gene (avr) are needed for the plant to be resistant to infection
The Elicitor-Receptor model of how it works
Pathogen avr gene produces elicitor (extracellular gene product)
Plant R gene produces receptor that recognises gene product and binds to it
The plant knows the pathogen is attacking and induces hypersensitive response!! Apoptosis and spotting!!
There are 3 ways the pathogen can attack and 1 way the plant can resist
• If no receptor, no detection
• If no elicitor gene product, nothing to detect!
What makes a plant susceptible or resistant?
Both a functional plant resistance gene and a functional avirulence gene (avr) are needed for the plant to be resistant to infection
▪ Eg. Plants containing copy of dominant resistance allele infected with pathogen containing copy of dominant avirulence allele were resistant
▪ If either plant/pathogen doesn’t have a dominant copy, it is susceptible to disease
Many wheat stem outbreaks INITIALLY solved by…
Sr31 - Wheat resistance gene
• Problem of wheat stem outbreaks solved by the introduction of wheat resistance genes eg. Sr31
• Effectiveness of Sr31 so great that wheat stem rust declined to almost insignificant levels by the 1990s
Why did wheat become susceptible to wheat stem rust again?
Susceptible again! – Ug99 strain of P. graminis
- Ug99 - Ug(anda) 19(99) – lineage of wheat stem rust present in Africa and the middle east that had lost the avirulence gene made an elicitor that was recognised by Sr31 receptor!!!!
- Wheat is susceptible again
- Virulent against almost all resistance genes, including Sr31
- This is a nightmare. Can cause major crop losses.
- Likely to spread to almost worldwide - both urediniospores and aeciospores are designed to be wind dispersed
FINALLY how was wheat stem rust solved (after second bout of outbreaks)
Sr35 & Sr33 – confer immunity to Ug99!!
• Both advances restore gene-for-gene resistance against Ug99
• Durability remains to be seen
• Global initiative – more than 60 varieties of wheat resistant to stem rust Ug99 have now been released in at-risk countries
How is wheat stem rust an example of
Constant evolutionary arms race between plant and pathogen
- Plants race to develop disease resistance via evolving resistance genes to improve their fitness
- Pathogens race to develop infection of plants via evolving virulence (vir) genes and evading detection by changing avirulence genes to improve their fitness
