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Pathogenetics

©2006 Lee Bardwell

Bio 97

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Pathogenetics lecture outline - 1

• What are bacteria?• What are viruses?• Antibiotic resistance is a big

problem– What is an antibiotic?– Why don't antibiotics kill us?– Why don't antibiotics kill viruses?

• How do bacteria become resistant to antibiotic (biochemical mechanism)?

©2005 Lee Bardwell

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Genetics of pathogens - 2

• How do bacteria become resistant to antibiotic (genetic mechanism)?– What's a plasmid?– What's a transposable element?– How do these things move from cell

to cell?

• What are other ways for DNA to move from cell-to-cell?– Transformation– Viral Transduction

©2005 Lee Bardwell

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Bacterial life cycle

s

Circularchrm

Bacteria • Prokaryotic• Unicellular• Haploid

Question

Does the statement “loss-of-function alleles are often recessive” make

sense when applied to bacteria?

©2005 Lee Bardwell

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Viruses and Phages• Non-cellular micro-organism• Consist minimally of DNA or RNA

genome and some protein• Infect host cells• Can replicate only within host cells• No intrinsic metabolism- relies on

host for energy, precursor molecules

• No ribosomes - relies on host for protein synthesis

©2005 Lee Bardwell

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Antibiotic resistance is a big problem in bacteria

• Most bacteria isolated from clininal infection are resistant to multiple antibiotics

• Some are resistant to all antibiotics in routine use

• Some of what used to be the best antibiotics (very effective, few side effects) are now virtually useless

©2005 Lee Bardwell

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What is an antibiotic ?

• A substance that kills or halts the growth of a micro-organism (typically a bacterium)

• Usually made by other micro-organisms (fungi, other bacteria)

• Examples• Pencillin• Streptomycin• Chloramphenicol

©2005 Lee Bardwell

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Why don’t antibiotics kill us ?

• Bacteria and humans share many core processes Bacteria polymerases resemble human

polymerases Bacterial ribosomes resemble human ribosomes,

etc.

• But there are some things that are completely different bacteria have a peptidoglycan cell wall and we

don’t

• The best (least toxic to us) antibiotics target these “completely different” structures

©2005 Lee Bardwell

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Inhibition of bacterial cell-wall synthesis by certain* antibiotics

*penicillin, vancomycin, others

QuickTime™ and aGIF decompressor

are needed to see this picture.

How do antibiotics kill bacteria ?

NOTE: movies won’t work on downloads/pdfs

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No need to memorize

BONUSMATERIAL !

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No need to memorize

BONUSMATERIAL !

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Inhibition of bacterial protein synthesis by macrolide* antibiotics

*erythromycin, azithromycin, others

QuickTime™ and aGIF decompressor

are needed to see this picture.

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Why don’t antibiotics

kill viruses ?

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How do bacteria become resistant ?

They can acquire certain _____ that encode ______ that function to neutralizethe antibiotic

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DNA can move between bacteria

• Many DNA sequences in bacteria are mobile – they can be transferred between individuals and even between species• Plasmids• Transposable Elements

• Antibiotic resistance genes are often contained in these mobile elements

• Other means of moving DNA between cells• Transformation• Viral Transduction

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PlasmidsPlasmids are circular DNA

molecules that replicate independently of the bacterial

chromosome

• They often carry antibiotic resistance genes

• They are used in genetic engineering as gene

transfer vectors

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F factor Plasmid

• The F (fertility) factor plasmid is a low-copy-number plasmid ~100 kb in length, and is present in 1–2 copies per cell

• It replicates once per cell cycle and segregates to both daughter cells in cell division

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F factor and Conjugation• Conjugation is a process in which

DNA is transferred from bacterial donor cell to a recipient cell by cell-to-cell contact

• The transfer is mediated by a tube-like structure called a pilus, formed between the cells, through which the plasmid DNA passes

• The ~20 proteins that make up the pilus are encoded by the F-factor plasmid

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Mobility of smaller plasmids

• Many small plasmids don’t have the genes necessary for pillus formation

• They can recombine with F and tag along for the ride.

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Transposable Elements

• Transposable elements are DNA sequences that can jump from one position to another within a chrm, or from one DNA molecule to another

• Bacterial TE’s often contain antibiotic resistance genes

• They can jump into plasmids, and move with ‘em• The smallest and simplest are 1–3 kb in length

and encode the transposase protein required for transposition and one or more additional proteins that regulate the rate of transposition

• TE’s are also found in eukaryotes, including humans

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Bacterial Transformation

• The process of genetic alteration by pure DNA is transformation

• Recipient cells acquire genes from DNA outside the cell

• DNA is taken up by cell and often recombines with genes on bacterial chromosome

• Bacterial transformation showed that DNA is the genetic material (Avery, MacLeod, McCarty 1944)

• Transformation may alter phenotype of recipient cells

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Transduction

• In the process of transduction, bacterial DNA is transferred from one bacterial cell to another by a phage

• The transferred DNA may be integrated into the host chrm by recombination

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Phage Life Cycle

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Fig. 7.16

In the process of transduction, bacterial DNA is transferred from one bacterial cell to another by a phage

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The transferred DNA may be integrated into the host chrm by recombination

Fig. 7.16

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Brief review of diploid genetics

©2005 Lee Bardwell

Bio 97

31Dominance, Recessiveness

– Heterozygous genotype --> normal phenotype

– The mutant allele is ___________

©2001 Lee Bardwell

32Dominance, Recessiveness

– Heterozygous genotype --> disease phenotype

– The mutant allele is ___________

©2001 Lee Bardwell

33Mutant (disease causing) alleles

• Loss-of-function mutations --> allele encodes a protein that...– Is not made, or has a reduced function, or is non-

functional• (e.g Hemophilia) Usually recessive

• Gain-of-function mutations --> allele encodes a protein that...– has a new, disease-causing function

• (e.g Huntington’s disease --> mutant protein forms toxic aggregates) Usually dominant

©2005 Lee Bardwell

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L.O.F. alleles • In many cases, having only half

the normal amount of a given protein is okay– Heterozygous genotype -->

normal phenotype– The mutant (loss-of-function)

allele is recessive

©2001 Lee Bardwell

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L.O.F. alleles II• In some cases, having only half

the normal amount of a given protein is NOT okay– Heterozygous genotype -->

disease phenotype– Loss-of-function allele is

dominant(= haploinsufficiency)

©2001 Lee Bardwell

HUNTINGTON’S DISEASE

PHENOTYPE • PROGRESSIVE INVOLUTARY MOVEMENTS INCLUDING CHOREA (GREEK=DANCE),

COGNITIVE DEFICITS, AND PSYCHIATRIC DISORDERS

GENETICS• FREQUENCY = 1/10,000 (European origin) • AUTOSOMAL DOMINANT - COMPLETE PENETRANCE

• LATE ONSET: 35-50 years of age 10-20 YEAR COURSE

• ANTICIPATION -TRINUCLEOTIDE REPEAT EXPANSION (pp. 427-8)

NO EFFECTIVE TREATMENT OR CURE

Disease of the Day

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HD is characterized by neuronal degeneration

MATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQQQPPP

PPPPPPPPQLPQPPPQAQPLLPQPQPPPPPPPPPPGPAVAEEPLHRP

KKELSATKKDRVNHCLTICENIVAQSVRNSPEFQK… (3142 total aa’s)

HUNTINGTIN protein

350 kD protein

10-11 kb transcript

ubiquitously expressed

function unknown

correlation between repeatsize and age of onset

Gene cloned by Huntington’s Disease

Collaborative ResearchGroup 1993

The repeated trinucleotide is AGCwhich encodes Q (glutamine)

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Most accepted current theory:

The extended glutamine tracts are thought to promote the formation of toxic aggregates, leading to cell death

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Sex Selection

©2005 Lee Bardwell

Using genetic techniquesTo pick the sex of your kid

Ethical Issue

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Sex Selection

• Sperm sorting – (X is heavier) 75% success if boy is desired, 90% if girl is

desired

• Preimplantation Genetic Diagnosis– In Vitro Fertilization– Determine sex of 4-8 cell embryos– Only implant the ones you want

• Chorionic Villus Sampling - sample the placenta

– Done at 8-9 weeks of gestation

• Amniocentesis - sample the amniotic fluid

– Done at 4-5 months©2005 Lee Bardwell

Ethical Issue

Using genetic techniquesTo pick the sex of your kid

How ?

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Sex Selection

• To prevent the birth of sons to women carriers of X-linked recessive diseases

• For family “balancing” • Because of economic and cultural

pressures that lead to sons being more desirable

©2005 Lee Bardwell

Ethical Issue

Picking the sex of your kid

Why ?

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Sex Selection

©2005 Lee Bardwell

Ethical Issue

Picking the sex of your kid

How do you feel about it ?