Bacterial Toxin Types and Mechanisms

• Lecture topic: Secretion and Efflux
• Date: Feb. 6, 2025

Lecture Learning Outcomes

• To describe how AB toxins, pore-forming toxins, and superantigens lead to damage in host tissues.
• To describe how proteins are translocated through the cytoplasmic membrane by the Sec system, Tat system, and ABC transporters.
• To describe how proteins are targeted to the outer membrane.
• To describe how bacteria secrete substances using secretion systems, efflux pumps, and extracellular vesicles.
• To describe how gram-positives attach proteins to their surfaces

Exotoxins

• Exotoxins are proteins secreted by pathogens that disrupt host cells.
• They contribute to many bacterial infections, such as pertussis and cholera.
• They are categorized by mechanism and structure into AB toxins, pore-forming toxins, and superantigens.

AB Toxins

• AB toxins interfere with processes inside host cells.
• They are comprised of active (A) and binding (B) components that form a complex.
• The B component binds to a receptor on the host cell, triggering endocytosis and helping the A component enter.
• The A component is an enzyme and causes a toxic effect.

Diphtheria Toxin (AB Toxin)

• Diphtheria is caused by Corynebacterium diphtheriae.
• Diphtheria toxin damages cardiac and nervous tissue.
• The B component activates endocytosis.
• An A component is released from the vacuole and modifies elongation factor, blocking translation.
• One toxin molecule can modify all EF-2 in the cell.

Shiga Toxin (Stx)

• Disease is caused by enterohemorrhagic E. coli.
• This causes severe inflammation and gastrointestinal bleeding.
• The toxin damages vascular endothelium.
• Stx carries a prophage where it encodes Shiga toxins.
• It is considered an AB toxin:
  • The A component removes a nucleobase from the ribosomal RNA(rRNA).
  • Disables ribosomes, therefore inhibiting protein synthesis.

Pore-Forming Toxins

• They insert into host cell membranes creating channels.
• This disrupts ion gradients, causes cytoplasmic contents to leak out.
• This causes Water to enter, leading to swelling and cell lysis.
• They lyse host cells to release nutrients.
• Hemolysins lyse red blood cells which releases heme/iron which is scavenged by the bacteria.
• Facilitates bacteria to escape from phagosomes.
• Some bacterial examples include Listeria monocytogenes,
  • an intracellular pathogen
  • replicates in immune cells after escape.
  • it spreads through blood, which often causes bacterial meningitis

Superantigens

• Macrophages and dendritic cells (DCs) present antigens to T-cells.
• Superantigens bind these cells together regardless of antigen presence, triggering pro-inflammatory cytokines overproduction.
• This leads to fever, low blood pressure, and organ failure.
• An example includes Staphylococcus aureus TSS toxin, which causes toxic shock syndrome

Protein Translocation and Secretion

• Approximately 1 in 3 bacterial proteins leave the cytoplasm.
• Each type can be targeted to every part of the cells, and it has distinct transport strategy
• Translocation is transport across the membrane, including insertion into the membrane.
• Secretion is the release into the external environment which requires translocation through one or two membranes

Crossing the Cytoplasmic Membrane

• Proteins are produced in the cytoplasm and may have folding assistance with chaperones
• Translocated/secreted proteins often have signal peptides. These have a specific amino avid at N-terminus to be recognized by transport systems
• Proteins translocated to the periplasm utilize Sec/Tat systems and ABC exporters.

Sec System: Post-Translational Translocation:

• The unfolded proteins are translocated into the periplasm utilizing this system.
• Proteins are stablized by chaperones prior to the translocation in the periplasm.
• The secA binds to peptide signal and escorts it to the SecYEG
• SecA drives translocation to the secYEG with the use of ATP.
• Post-translational translocation occurs once translation is complete

Sec System: Periplasmic Proteins

• Signal peptidase (SPase) recognizes and cuts the signal peptide; which occurs during translocation
• The translocated protein folds in the periplasm with assistance from periplasmic chaperones.
• Periplasm must be in an oxidizing environment to promote disulfide bond formation.
• Periplasmic proteins ensure correct disulfide formation, e.g, protein disulfide isomerase

Tat System

• Not all proteins will be folded in the cell, and may require co-insertion for example:
  • Iron-sulfur clusters need >10 proteins.
• Cytoplasmic chaperones help with assembly into complexes, and tat system translates into periplasm
• Tat= twin arginine translocase
• The Tat system uses a different signal peptide than the Sec system

Additional information on the Tat System

• Protein folds in cytoplasm and is transported through the periplasm
• Is targeted to Tat ABC with a signal peptide
• No targeting faction is needed, unlike Sec system which uses SecA
• The proton motive force acts as the energy component
• SP is then cleaved off

ABC Exporters

• ATP-binding cassette (ABC) transporters: utilize primary active transport.
  • They import many substances.
• ATP-dependent
• Others as exporters:
  • Waste, antibiotics and translocation proteins
  • it can recognize the peptide signal, and cleaves it during export

Sec System and Membrane Proteins

• Sec system can also insert proteins into cytoplasmic membrane
• The Peptide signal is translated 1st, very hydro-phobic for membrane, and is recognized by the peptide signal with:
  • Halts to the translation
  • Escorts ribosomes to the SecYEG
• The translation then continuous, and the protein is inserted with the combination of translocation.

Gram-Negative BAM Complex

• ẞ-Barrel proteins targeted to outer membrane:
  • Outer membrane proteins (OMPs)
• Unfolded protein translocated with the Sec system.
• Is stabilized with Periplasmic Chaperones (SurA and SKP)
• Is then delivered to the ẞ Barrel assembly machinery(BAM) which then can insert the proteins into the membrane.

Gram-Negative Secretion Systems

• Secretion systems transport proteins out of the cell to be:
  • • Bound to the cell surface
  • Released into the external environment
  • Injected into other cells

Type V Secretion System (T5SS)

• Tat or Sec translocates proteins to Peliplasm.
• Proteins are then translated to theẞ Barrrel in the outer membrane, and then is secreted outside the cell.

Type I Secretion System (T1SS)

• Is made of of Three components:
  • ABC transporter
  • Membrane fusion
  • ẞ – barrel proteins
• ABC Transporters:
  • Power transport
  • Determines specificity

Type III Secretion Systems (T3SSs)

• The T3ss are injected into eukaryotic cells which are then labeled effector proteins
• Effector travels from bacterial cytoplasm to the hosts
• Effectors then manipulate host cell structure to facilitate colonization.

T3ss Structure and Assembly

• Made up of >20 proteins
• Related to flagella
  • Basal Body, needle/filaments
  • Export Functions
• During Assembly:
  • Sub units travel through hollow central channels.
• After assembly the plug blocks travel.

T3SS Effectors

• Once T3SS binds to the host cell the channels open
• Chaperones deliver unfolded effectors
• Effector travels through T3SS, and folds the the specific targets
  • Transport powered by ATP, proton motive force
• A single T3SS, can secrete many different targets.
  • This is only the recognition to the conserved N-terminal section signal.

T3SS Effector Proteins

• Effectors target host cytoskeleton, and signal transduction
  • Help bacteria attach, and invade.
• Salmonella:, a intracellular pathogen that causes : -Gastroenteritis -Systemic illnesses
• Salmonella T3SS effectors

Efflux pumps

• Transport molecules to the periplasm
• Several Classes:
  • ABC transporters -RND tripartite system -Resembles T1SS

Efflux pumps and Antibiotic Resistance

• Major contributor to multidrug resistance
• Single pumps can pump out different antibiotics
• Mutations can increase the # of pumps produced.

Extracellular Vesicles (EVs)

• Bacteria export substances in extracellular vesicles
• They are spherical buds released from cell surface of :
  • Gram-positives and gram negatives:
    • Gram negatives: outer membrane vesicles (OMVs)
• Contain protein and DNA
• contribute to horizontal gene transfer, virulence and antibiotic resistance.

Extracellular Vesicles (EVs) and Virulence

• EVs deliver cargo between the cells. This can be used by cells such as :
  • Enter-toxogenic E. coli (ETEC) - Can also contribute to travelers diarrhea - EV's can release to the intestines which can causes water toxins and diarrhea/

EVs and Antibiotic Resistance

• Can protect against antibiotics
• EX: EVs can contain beta-lactamases
• Beta lactams enter EV through porins, and the degrade the B-lactems

Gram-Positive Bacteria and Secretion

• Protein secretion simpler than Gram negatives, it does not have Elaborate secretion
• The do however utilize Sec and Tat System
• Some secrete proteins get attached to cell surface - Sortase attaches proteins that have cell wall sorting signal

Gram-Positive Surface Proteins

• Surface proteins ofen contribute to virulence.
• they are have a high attachment rate to the host cells
• Nutrient acquisition in host ex:
• The immune Evasion:
  • Protease = degrade bodies
  • protein A: preventing opsonization

This lesson covers bacterial toxins, including AB toxins, pore-forming toxins, and superantigens, detailing their mechanisms of action and the types of cells they disrupt. It explains how proteins are translocated across membranes and the roles of different toxin components in host cell damage. It also highlights specific examples like diphtheria and Shiga toxins.