Antibiotics (Cao)

Bacteria

• large domain

• prokaryotic microorganisms

• spheres, rods, spirals

Prokaryotes

• organisms made of cells that lack nucleus/membrane bound organelles

• genetic material (DNA) is not bound within nucleus

• DNA is single loop (less structured)

Infection

• invasion of germs (bacteria, virus)

  • from coughing, sneezing

• makes you sick

• immune system must work to fight it off

Good bacteria function

• keep balance

• healthy body has 85% good bacteria

• probiotics, gut microbiome

Bad bacteria function

• cause disease

Gram stain technique

1. Apply crystal violet (primary dye)

   1. both cell walls stained

2. Gram’s iodine (mordant)

   1. gram positive cells trap dye

3. Alcohol (decolorizer)

   1. Gram positive cell retains dye (gram negative loses)

4. Safranin (red dye counterstain)

   1. Gram positive cell remains violet

   2. Gram negative cell stains red

Gram positive bacteria

• cell wall: thick layer of murein

Gram negative bacteria

• cell wall: thin layer of murein

• cell wall surrounded by second outer lipid bilayer

• lipopolysaccharide (LPS) in outer membrane

Murein structure

• Peptide side chains

• peptide cross bridge

• Carbohydrate backbone

  • N-acetylglucosamine (NAG)

  • N-acetylmuramic acid (NAM)

Central dogma

• 2 step process

• information in genes flows into proteins

• transcription → translation

• DNA → RNA → protein

Differences in human and bacteria central dogma

• Topoisomerase (DNA replication)

• RNA polymerase (RNA transcription)

• Ribosomes (protein translation)

Major drug treatments focus on interruption to bacterial

• DNA replication

• transcription

• translation

History of DNA discovery

• Frederick Griffith: factor in diseased bacteria can transform harmless bacteria into deadly bacteria (1928)

• Rosalind Franklin: X ray photo of DNA (1952)

• Watson and Crick: described DNA molecule from Franklin’s X ray (1953)

• Crick, Watson, and Maurice Wilkins: Novel Prize

DNA nucleotide building block

• Phosphate group

  • on C5 of sugar

• Sugar (deoxyribose)

• Nitrogenous base

  • on C1 of sugar

Difference between ribose and deoxyribose

• ribose has OH on 2C

• Deoxyribose has H on 2C (missing an oxygen)

Watson/Crick Complementary Rule

• A-T

  • 2 H bonds

• C-G

  • 3 H bonds

• DNA made of 2 long strands of nucleotides arranged in specific way

• specific pairing between nitrogenous bases

DNA

• deoxyribonucleic acid

• molecule that contains genetic code of all living organisms

• in each cell in organism and tells cells what proteins to make

• a cell’s proteins determine it’s function

DNA structure

• polymerized deoxyribonucleotide

• phosphodiester bonds between nucleotides

  • link 3-OH to 5-OH

• Hydrogen bonding between DNA strands

• bacterial: circular

• eukaryotic: linear

For replication to begin, DNA double helix must

• unwind

• separate DNA

Topoisomerase functions

• remove excess DNA supercoils during replication

• separate intertwined progeny DNA by breaking

• rotate and religate DNA strands

Type I topoisomerse

• form and reseal single-stranded breaks in DNA

• decrease positive supercoiling

Type II topoisomerase

• nuclease and ligase operations on both strands of DNA

• remove excess DNA to permit segregation of DNA to daughter cells

DNA replication

• semiconservative model (Watson and Crick)

• 2 strands separate, and each functions as template for synthesis of new complementary strand

DNA polymerase

• adds nucleotides to existing nucleic acid strand

• needs primer

• only synthesuze from 5→3

Why is replication necessary?

so both new cells will have correct DNA

When does replication occur

• during interphase

• S phase

Describe how replication works

• enzymes unzip DNA

• complementary nucleotides join each original strand

RNA polymerase

• Bacterial

  • 5 subunits form holoenzyme

  • 2a, 1B, 1B’, 1o

• RNA polymerase differs between bacteria and humans and can serve as selective target for anti-bacterial drug action

• Eukaryotes express 3 different nuclear RNA polymerases

Stages of Transcription

1. initiation

   1. RNA polymerase holoenzyme recognizes promoter on DNA

   2. holoenzyme separates strands of DNA

   3. exposes start site for transcription and begins synthesis of new RNA strand

2. elongation

   1. extend RNA strand in 5’→3’ direction

   2. blocked by rifampin

3. termination

   1. RNA polymerase reaches termination sequence

   2. DNA, RNA pol, and new RNA separate

Rifampin

• blocks transcription elongation

• complex with Beta subunit of RNA polymerase

Ribosomes

• number and composition of rRNA differ between bacterial and human ribosomes

• bacterial ribosomes: selective targets

• 30S subunit: 16S

• 50S subunit: 23S and 5S

• rRNA, not protein components of ribosomes, are responsible for ribosome’s key activities

Translation

• cell uses information from messenger RNA to produce proteins

Stages of Translation

• Initiation

  • mRNA joins with 30S subunit of bacterial ribosome

  • tRNA linked to formylated methionine (fMet)

  • 50S subunit joins with 30S subunit to form the complete 70S ribosome

  • fMet-rRNA now occupies P site of 70S ribosome

• Elongation

  • peptidyl transferase: enzyme whose activity derives from 23S rRNA of the 50S subunit

  • translocation

• Termination

  • A site meets stop codon

Bacterial 70S ribosome

• 30S subunit and 50S subunit

• rRNAs are responsible for activities

Drugs that target 70S ribosome (Target translation)

• aminoglycosides

• spectinomycin

• tetracyclin

• macrolides

• chloramphenicol

• lincosamides

• streptogramins

• oxazolidinones

• pleuromutilins

Drugs targets on bacteria

• bacteria structure: peptidoglycan (cell wall)

• DNA replication: DNA helicase, DNA polymerase, Topoisomerase (I, II, and IV), DNA ligase, primase

• Transcription: RNA polymerase (initiation, elongation, termination)

• Translation: initiation, elongation, termination

Why is transcription necessary

transcription makes mRNA to carry the code for proteins

Describe transcription

RNA polymerase binds to DNA, separates the strands, then uses one strand as a template to assemble mRNA

Why is translation necessary

translation assures that the right amino acids are joined together by peptides to form the correct protein

Describe translation

the cell uses information from mRNA to produce proteins

Main differences between DNA and RNA

• DNA: deoxyribose

• RNA: ribose

• DNA: 2 strands

• RNA: 1 strand

• DNA: thymine

• RNA: uracil

Synthesis of bacterial cell wall

1. in cytoplasmic phase of murein monomers synthesis

2. polymerization stage

3. cell wall biosynthesis

   1. adjacent glycopeptide polymers cross-linked in reaction catalyed by bacterial transpeptidases

Drugs that target murein synthesis

• Fosfomycin, Fosmidomycin

• Cycloserine

Drugs that target Muerin polymerization

• Beta-lactams

• Penicillins

• Cephalosporins

• Monobactams

• Carbapenems

• Vancomycin

• Telcoplanin

• Bacitracin

Transpeptidase action

• catalyze transpeptidation

• reaction only occurs in bacterial cells

• inhibited by penicillin

Bacterial pathogenicity

• ability of bacteria to cause disease

• expressed in terms of virulence

• helped by fimbriae, flagella, toxins, invasion, bacteriophages, plasmids, capsules

  • invasion ex: tuberculosis

Salmonella typhimurium

• ability to destroy intestinal cells

• cause severe diarrhea

Plasmids

• responsible for surface antigens on bacteria

• give bacteria multiple drug resistance

• infection difficult to treat

Capsulated bacteria

Klebsiella pneumoniae

haemophilus influenzae

Drugs that target Murein

• Vancomycin, telavancin, and teicoplanin

  • bind D-Ala-d-Ala terminus of bactoprenol-conjugated murein monomer unit

• Beta lactam antibiotics (penicillins, cephalosporins, monobactams, carbapenems)

  • inhibit transpeptidase

  • stops cross-linking of adhacent peptidoglycan polymers

Beta lactam antibiotics

• penicillins, cephalosporins, monobactams, carbapenems

• differ from one another in their backbone structures

• individual drugs within subclasses differ in R groups