Lecture_SecretionSystems_PvU_2025

Protein Machines in the Gram-negative Cell Envelope

  • Classification: Secretion systems classified I-VI (currently at XI) based on their order of discovery.

  • Adapted Structures: Evolved from macromolecular structures on bacterial surfaces:

    • Pili (II)

    • Flagella (III)

    • Conjugation systems (IV)

Secretion Mechanisms

  • Two-step secretion (T2SS and autotransporters - T5SS):

    • Involves:

      • Cytoplasmic membrane: Sec/Tat pathway utilizing a signal peptide

      • Periplasmic chaperones

      • T5SS transports directly through autotransporter, T2SS through chaperone

  • Energy Requirement: No energy used at the OM; energy derived from protein folding.

One-step Secretion:

  • Involves: T1SS, T3SS, T4SS

    • Cytoplasmic chaperones for targeting to secretion machinery

      • Motifs that recognize machinery/chaperones in cytosol that guide substrate

    • ATP hydrolysis drives the process.

Structural Biology Techniques for Investigating Secretion Mechanisms

  • Techniques Discussed:

    • X-ray crystallography

    • Electron microscopy (EM)

  • Examination of different secretion systems:

    • Type I Secretion System

    • Type III Secretion System (injection needle)

    • Type VI Secretion Systems (T6SS) - identified through bioinformatics and structural biology tools.

X-ray Crystallography

  • Classical technique for determining protein structure:

    • Requires purification of proteins in large quantities.

    • Crystallization through trial and error methods.

    • Obtaining diffraction patterns to determine structures.

Limitations of Crystallography

  • Challenges with membrane proteins:

    • Hydrophobic surfaces,

    • Low solubility in crystallization solutions.

    • Issues with purification and high aggregation tendency.

Electron Microscopy

  • Overview: Detects electron-dense materials;

  • Needs heavy metal staining for biological samples.

  • Introduction of cryo-EM to avoid radiation damage and reduce staining needs, suitable for various samples.

  • 3D Tomography: Allows for detailed visualization of structures.

Cryo-EM and Single Particle Analysis

  • Preparation of purified samples on grids, followed by imaging.

  • Grouping structures and fitting protein structures effectively.

Advances in Cryo-EM Techniques

  • Enhanced electron detectors allow for better resolutions and techniques like plunging samples in liquid ethane for preservation.

  • Tomography: Achieves high-resolution structures in native conditions,

  • Used for Cryo-EM imaging of Type III complexes.

Type I Secretion System (T1SS)

  • Key Components:

    • Transporters: ABC transporter facilitates translocation via trimeric b-barrels in OM.

    • Adaptor Protein: Provides a link between the inner membrane complex and the OM channel.

  • Functionality: Secretes various proteins including toxins and enzymes.

Multidrug Efflux Pump in E.coli

  • Role: TolC used as the OMP channel, specific transporters based on systems.

  • Substances: Pumps out antibiotics and harmful chemicals, effectively countering threats.

Type III Secretion in Yersinia Species

  • Linked to various diseases:

    • Y. pestis: Bubonic plague

    • Y. enterocolitica: Enteric diseases, diarrhea

    • Y. pseudotuberculosis: Enteric diseases

  • Mechanism: Type III secretion system facilitates interaction with host cells, promoting pathogen survival.

Type III and Type VI Secretion Systems

  • Type III: Yersinia outer proteins modulate host processes, secreted via T3SS, inducing apoptosis and anti-inflammatory responses.

  • Type VI Secretion: Identified in V. cholerae, essential for inter-species competition. Type VI is an inverted bacteriophage injection machine

Molecular Ruler for Needle Length

  • YscP Function: Regulates length based on protein repeat changes; correlated with virulence.

  • Importance: Correct needle length required for optimal interaction with host cells and evasion of immune responses.

Summary of Secretion Systems

  • Type I: Solved structures of trimeric OMPs and mechanisms unveiled through crystallography and cryo-EM.

  • Type III: Needle length precision ensures effective interaction mechanisms.

  • Type VI: Derived from bacteriophage systems, reflecting complexity in bacterium-host interactions and competition for environmental niches.