Bacteriology

• • Eukaryote vs Prokaryote
  • P = smaller, no organelles, cell wall, smaller ribosomes, no nucleus - free flowing DNA with no histones
  • E = larger, more organised with organelles, nucleus, larger ribosomes, chromosomes, no cell wall
• Bacteria Requirements - General

  • Correct Temperature
  • Correct pH
  • Carbon source
  • Ion availability such as magnesium, iron, potassium, cofactors
  • Oxygen
• Bacteria Requirements - Temperature

  • Psychrophiles = under 15
  • Mesophiles = 25 - 40
  • Thermophiles = 50 - 60
• Bacteria Requirements - pH

  • Most require neutral 6.5-7
  • Acidophiles = less than 5.4
  • Neutrophiles = 5.4 - 8.5
  • Alkaliphiles = 7 - 12 or higher
• Bacterial Envelope

  Where the host interacts and recognises bacteria
• What affects if the host becomes sick

  • Ability of the pathogen to colonise and damage host
  • Immune status of host
  • Exposure to pathogen doesn't mean illness will occur
• Key abilities of bacterial pathogens

  • Transmit between hosts
  • Colonise hosts
  • Cause host damage
• Factors involved in effective host colonisation

  • Adhere to host cells and resist removal
  • Invade host cells
  • Compete for iron and nutrients
  • Evade the immune system
• Promote attachment and resist removal

  • Fimbriae - gram -
  • Pilli - gram -
  • Adhesins - gram+
  • Motility - resist removal
• Invasion of host cells

  • Invasins - promote cell entry via phagocytosis, engulf bacteria to allow invasion. Can evade immune system in a host cell and feed off its nutrients
  • Extracellular proteases - punch holes in cells allowing entry
  • Type 3 secretion system - Injectosome on bacteria pierces through host cell, Effector molecules interfere with cytoskeleton, Encourage phagocytosis, Eventual cell spread, Can also code for cell lysis to release bacteria from cell
• Compete for nutrients

  • Carbon
  • Nitrogen
  • Iron
• Iron competition

  • Host requires iron for:
  • Transferrin - serum
  • Haemoglobin - red blood cells,
  • Lactoferrin - tears, sweat, saliva, mucus
  • Bacteria also requires iron for their function so bacteria will want to steal
  • Bacterial cells have siderophores which are iron collating agents (bind iron very strongly)
  • Bacteria releases siderophores when the bacteria is intracellular in a host cell
  • Siderophore gets sent out to scavenge iron ions
  • Siderophore can rip iron out of glycoproteins such as haemoglobin or transferrin and take it into the bacteria cell
• Evading the immune system- coating and epitopes

  • s.aureus forms an enzyme called coagulase
  • Fibrinogen to fibrin which = clotting
  • Fibrin coats surface of bacteria allowing phagocytosis evasion - phagocytes cant recognise as foreign due to coat
  • Coagulase coating only seen with pathogenic s.aureus
  • Some bacteria can change their epitope to avoid recognition by the immune system (s. aurues has protein A which affects antibody binding) - Makes the antibody bind in the wrong direction/orientation
• Bacterial defence against lysozymes (tears)

  Gram+ evolved N terminal deacetylation to protect against lysozyme action
• Bacterial Toxins

  Benefit bacteria as they kill neutrophils + macrophages, therefore protecting bacteria from phagocytes that could kill them, Toxins that kill human cells will release iron/carbon sources - bacteria thrives
• Toxin Types

  • Exotoxin = secreted proteins and peptides
  • Endotoxin = components of the cell wall e.g. lipids
• Botulinum (botox)

  Botulism - food borne disease, causes paralysis, if injected and not treated causes respiratory collapse, Found in spores in food
• Endotoxins

  • LPS - found on gram-, Chain structure with a polysaccharide O chain at the top, outer and inner core and lipid A which helps anchor it onto the cell surface
  • O chain at the top of LPS is toxic - Activates host complement serological specificity/host defence systems, Manipulates cytokine production to cause damage
• Exotoxins

  • Type 1 - do not enter host cells (superantigens)
  • Type 2 - disrupt eukaryotic cell membranes e.g. phospholipases
  • Type 3 - A-B Toxins, act intracellularly. B part binds to Eukaryotic cell via receptor, A part enters the cytoplasm
• Type 1 (Superantigen)

  • Bypass normal antigen presentation by binding to MHC Class II. When the MHC goes to present the epitope to the T cell, it binds to non specific regions of the T cell antigen receptor. This creates an exaggerated (above antigen specific) reaction resulting in massive cytokine release
  • Usual T cell activation = 1 in 1000 cells become active and start secreting cytokines
  • Sag activation = 1 in 5 cells
  • Excess interlukin2 and tnf A production leads to tissue damage and cell lysis which helps the nutrients get out and help the bacteria grow and spread
• Type 2 (Pore forming toxins)

  • Disrupt membranes - make holes via osmotic pressure of host cytoplasm causing cell lysis
  • When they find a receptor they change conformation (helix) and start drilling into the membrane
  • Membrane insertion often aided by acidic pH
  • Makes cell burst as pressure drops
• Type 3 (A-B toxins)

  • B is binding domain, A is enzymatic domain
  • B will bind to receptor causing the cell to phagocytose allowing the bacteria to enter the cell in an endosome
  • A will facilitate breaking out of the endosome and modify its target to cause damage
  • Examples: Cholera enterotoxin, Diphtheria toxin, Shiga toxin, Tetanus toxin
• Beneficial uses of bacterial toxins

  • Vaccines - diphtheria and tetanus – target toxins
  • Agriculture – insecticide
  • Cosmetic – botox
  • Immunotoxins – can be used to target cancer cells and viral infections
  • Bacterial toxins are potential therapeutic targets
• Prokaryote genetics

  • Single circular genome
  • Extra-chromosomal plasmids
  • Cytoplasmic "free" location
  • Haploid
  • Coupled transcription and translation - translation starts when mRNA is still being synthesized
  • Bacteria contain mostly coding DNA (codes for proteins)
• • Promoter regions are non-coding regions in DNA required for gene transcription
• • The 5′ untranslated region (also known as 5′ UTR, leader sequence, transcript leader, or leader RNA) is the region of a messenger RNA (mRNA) that is directly upstream from the initiation codon (helps transcribe genes)
• • The 5′UTR sequence has a critical role in the recruitment of ribosomes to mRNA as well as in many processes related to the mechanisms regulating translation
• • Non coding regions are usually promoters which helps regulate how the gene works
• • Promoters contain conserved sequences that are recognised by RNA polymerase
  • -35 is upstream from the start codon
  • -10 is upstream from the start codon
  • represent the number of base pairs upstream from each other
  • Then a Transcription Start Point (TSP) - either CAT or CGT start codon
  • Can be a variable length away from the Ribosome Binding Site (RBS) - AGGAGG
  • After RBS (usually 4-7 bp away) is the start codon
• before start codon is promoter region
• • RNA polymerase holoenzyme sits at bacterial promoter sites
  • RNA Polymerase core enzyme sits at promotor region and sigma factor comes in and binds the different sections of the promoter (-35, -10, etc)
  • Allows core enzymes to be orientated properly for the Transcription start point (TSP)

  
• • Holoenzyme = a core enzyme + sigma factor
• • A sigma factor is a protein needed for initiation of transcription in bacteria. It is a bacterial transcription initiation factor that enables specific binding of RNA polymerase to gene promoters
• without the appropriate sigma factor, RNA polymerase enzymes will not work.
• transcriptional regulators

  transcription repressors

  transcription activators
• Transcriptional Repressors

  regulatory protein binds at promoter region to block transcription by blocking RNA polymerase
• why repress transcription?

  may need to turn off a gene (e.g. a gene which is needed for avoiding digestion when intracellular in a macrophage will not be needed when bacteria is not in a macrophage)

  wasting energy

  creating proteins that arent needed

  may be detrimental for the cell
• transcriptional repression is AKA as negative control
• transcriptional activation is AKA as positive control
• Transcription activators

  genes may be dormant and will require activation

  regulatory protein binds upstream of the DNA/gene (even before the promoter region)

  will attract ribosomes to start the transcription process

  regulatory protein binds to RNA Polymerase