Microbiology: Quorum Sensing and Virulence Gene Regulation

Virulence Gene Regulation

Quorum Sensing

• A process of cell-to-cell communication that allows bacteria to coordinate gene expression in response to population density
• Involves the production and detection of signaling molecules (e.g. autoinducers)
• Regulates virulence factors, such as biofilm formation, toxin production, and adherence
• Examples: LuxI/LuxR in Vibrio harveyi, LasI/LasR in Pseudomonas aeruginosa

Two-Component Systems

• A signaling pathway that allows bacteria to respond to environmental stimuli
• Consists of a sensor histidine kinase and a response regulator protein
• Phosphorylation of the response regulator activates or represses transcription of target genes
• Regulates virulence factors, such as adhesion, invasion, and toxin production
• Examples: PhoP/PhoQ in Salmonella, BvgS/BvgA in Bordetella pertussis

Sigma Factors

• A subunit of RNA polymerase that recognizes specific promoter sequences
• Regulates transcription of virulence genes in response to environmental signals
• Examples: σS (RpoS) in E. coli and Salmonella, σB in Bacillus subtilis
• Alternative sigma factors can redirect transcriptional machinery to express virulence genes

Horizontal Gene Transfer

• The transfer of genetic material between bacteria other than by vertical inheritance
• Mechanisms: transformation, conjugation, transduction
• Allows for the acquisition of new virulence factors and increased pathogenicity
• Examples: Streptococcus pneumoniae acquiring antibiotic resistance genes, E. coli acquiring virulence plasmids

Regulation of Virulence Factors

• Virulence factors are often regulated by multiple mechanisms, including quorum sensing, two-component systems, and sigma factors
• Regulators can act at the transcriptional, translational, or post-translational level
• Examples: ToxR in Vibrio cholerae regulates cholera toxin production, PrfA in Listeria monocytogenes regulates listeriolysin O production
• Regulation of virulence factors can be influenced by environmental cues, such as temperature, pH, and nutrient availability

Virulence Gene Regulation

Quorum Sensing

• Bacteria communicate through signaling molecules (autoinducers) to coordinate gene expression based on population density
• Regulates virulence factors, including biofilm formation, toxin production, and adherence
• Examples: LuxI/LuxR system in Vibrio harveyi and LasI/LasR system in Pseudomonas aeruginosa

Two-Component Systems

• Sensor histidine kinase responds to environmental stimuli, phosphorylating the response regulator protein
• Phosphorylated response regulator activates or represses transcription of target genes
• Regulates virulence factors, including adhesion, invasion, and toxin production
• Examples: PhoP/PhoQ system in Salmonella and BvgS/BvgA system in Bordetella pertussis

Sigma Factors

• RNA polymerase subunit recognizes specific promoter sequences to regulate transcription of virulence genes
• Responds to environmental signals, redirecting transcriptional machinery to express virulence genes
• Examples: σS (RpoS) in E.coli and Salmonella and σB in Bacillus subtilis

Horizontal Gene Transfer

• Transfer of genetic material between bacteria occurs through transformation, conjugation, and transduction
• Allows for acquisition of new virulence factors and increased pathogenicity
• Examples: Streptococcus pneumoniae acquiring antibiotic resistance genes and E.coli acquiring virulence plasmids

Regulation of Virulence Factors

• Virulence factors are regulated by multiple mechanisms, including quorum sensing, two-component systems, and sigma factors
• Regulation occurs at transcriptional, translational, or post-translational levels
• Examples: ToxR regulates cholera toxin production in Vibrio cholerae and PrfA regulates listeriolysin O production in Listeria monocytogenes
• Environmental cues, such as temperature, pH, and nutrient availability, influence virulence factor regulation

Secretion Systems

Type I Secretion System

• Known as ATP-binding cassette (ABC) transporter, found in both Gram-negative and Gram-positive bacteria
• Transports small molecules, including toxins, antibiotics, and signaling molecules
• Consists of three components: inner membrane transporter, periplasmic adapter protein, and outer membrane porin
• ATP hydrolysis drives the transport process

Type II Secretion System

• Also known as the general secretory pathway (GSP), found in Gram-negative bacteria
• Involved in the transport of proteins, such as enzymes, toxins, and adhesins
• Composed of 12-14 proteins that form a channel across both inner and outer membranes
• Requires the Sec machinery for protein translocation across the inner membrane

Type III Secretion System

• Found in Gram-negative bacteria, involved in direct transport of proteins into host cells
• Transports proteins, including toxins and effector proteins
• Composed of 20-30 proteins that form a needle-like structure
• Uses type III secretion ATPase to provide energy for secretion

Type IV Secretion System

• Found in both Gram-negative and Gram-positive bacteria
• Involved in the transport of DNA, proteins, and other molecules
• Composed of 10-12 proteins that form a channel across both inner and outer membranes
• Uses type IV secretion ATPase to provide energy for secretion

Type VI Secretion System

• Found in Gram-negative bacteria, involved in the transport of proteins into target cells
• Transports proteins, including toxins and effector proteins
• Composed of 12-15 proteins that form a dynamic structure
• Uses type VI secretion ATPase to provide energy for secretion
• Also plays a role in interbacterial competition and killing of neighboring bacteria

Outer Membrane Vesicles (OMVs)

Biogenesis

• Formed through outer membrane budding and fission, regulated by peptidoglycan layer and outer membrane curvature
• Induced by environmental stress, bacterial growth phase, and cellular responses to antibiotics
• Highly regulated process involving multiple protein components and cellular pathways

Immunomodulation

• Play a crucial role in modulating host immune response, stimulating or suppressing immune cell activation
• Induce production of pro-inflammatory cytokines and modulate Th1/Th2 immune response
• Interact with immune system through recognition by pattern recognition receptors, inducing type I interferon responses
• Modulate antigen presentation and processing

Gram-negative Bacteria

• Produce OMVs for virulence factor delivery, quorum sensing, and horizontal gene transfer
• Contain toxins, enzymes, and other virulence factors, modulating host immune response
• Play a role in biofilm formation and dispersal

Pathogenic Bacteria

Biochemical Tests

• Identify and characterize bacteria based on metabolic properties
• Catalase test: detects enzyme catalase, breaking down hydrogen peroxide
• Coagulase test: detects enzyme coagulase, causing blood to clot
• Oxidase test: detects enzyme oxidase, oxidizing certain compounds
• Urease test: detects enzyme urease, breaking down urea
• Helps identify bacteria and determine pathogenic potential

Microscopy Techniques

• Visualize and characterize bacteria
• Bright-field microscopy: uses visible light to illuminate the sample
• Phase-contrast microscopy: visualizes live cells
• Fluorescence microscopy: uses fluorescent dyes to stain specific structures or molecules
• Scanning electron microscopy (SEM): produces high-resolution image
• Helps identify bacterial morphology and structure

Molecular Diagnostics

• Detect and identify bacteria using molecular biology techniques
• Polymerase chain reaction (PCR): amplifies specific DNA sequences
• Real-time PCR: monitors amplification process in real-time
• DNA-DNA hybridization: detects specific DNA sequences
• 16S rRNA gene sequencing: identifies bacteria based on 16S ribosomal RNA gene sequence
• Provides rapid and sensitive detection of pathogenic bacteria

Selective Media

Baird Parker Agar

• Isolates and identifies Staphylococcus aureus
• Contains lithium chloride, inhibiting growth of most bacteria except S. aureus
• Contains tellurite, reduced to tellurium, producing a black colony

EMB Agar

• Isolates and identifies Escherichia coli and other Enterobacteriaceae
• Contains eosin and methylene blue, inhibiting growth of most bacteria except E. coli and other Enterobacteriaceae
• Contains lactose, fermented by E. coli and other Enterobacteriaceae, producing a pink or purple colony

CRISPR System

Overview

• A type of RNA-guided DNA endonuclease that provides bacteria with immunity against bacteriophages.
• Consists of two main components: guide RNA (gRNA) and Cas9 enzyme.

Guide RNA (gRNA)

• A small RNA molecule that recognizes a specific DNA sequence.
• Guides the Cas9 enzyme to the target location.

Cas9 Enzyme

• An endonuclease that cleaves double-stranded DNA.
• Guided by the gRNA to recognize and cleave specific DNA sequences.
• Highly specific and can be programmed to target specific sequences.
• Responsible for cutting the DNA, which is then repaired by the bacterium's own repair machinery.

Bacterial Immunity against Bacteriophages

• The CRISPR system targets and degrades the viral DNA to provide immunity.
• Based on the bacterium's ability to remember and recognize previous infections.
• Bacterium captures a small piece of viral DNA and stores it in its own genome.
• This piece of DNA is used to create a gRNA that targets the viral DNA.
• When the bacterium encounters the same bacteriophage again, the CRISPR system is activated, and the Cas9 enzyme cuts the viral DNA.
• Provides long-term immunity against the specific bacteriophage.

Bacterial Adherence and Colonization

Biofilm Formation

• Bacteria form complex communities on surfaces, protected by a self-produced matrix of polysaccharides, proteins, and DNA
• Biofilms provide protection from environmental stresses, antimicrobials, and host immune responses
• Characteristics of biofilms:
  • Irreversible attachment
  • Microcolony formation
  • Matrix production
  • Resistance to antimicrobials and host defenses

Adhesin Proteins

• Bacterial surface proteins mediating adherence to host cells and surfaces
• Types of adhesin proteins:
  • Fimbrial adhesins (pili)
  • Afimbrial adhesins (non-pilus)
  • MSCRAMMs (microbial surface components recognizing adhesive matrix molecules)
• Functions of adhesin proteins:
  • Recognition of host receptors
  • Binding to extracellular matrix components
  • Facilitating biofilm formation

Host-Microbe Interactions

• Bacterial adherence triggers host responses:
  • Inflammation
  • Immune cell activation
  • Production of antimicrobial peptides and cytokines
• Host receptors involved:
  • Integrins
  • Cadherins
  • Proteoglycans

Surface Receptors

• Bacterial surface structures involved in adhesion:
  • Fimbriae (pili)
  • Curli
  • Autotransporters
  • MSCRAMMs
• Recognize host receptors, facilitating adherence and colonization

Quorum Sensing

• Bacterial communication system regulating gene expression in response to population density
• Regulates virulence factor production, including adhesins and biofilm formation
• Autoinducer molecules involved:
  • Autoinducer-1 (AI-1)
  • Autoinducer-2 (AI-2)

Adhesion Mechanisms

• Fimbral adhesion:
  • Involves pili (fimbriae)
  • Mediates adherence to host cells and surfaces
• Afimbral adhesion:
  • Lacking pili
  • Mediates adherence through afimbrial adhesins

Type 5 Secretion Adhesion

• Autotransporter system secreting adhesins across the outer membrane
• Involved in adherence to host cells and surfaces
• Examples:
  • E.coli AIDA-I autotransporter

MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules)

• Microbial surface proteins recognizing and binding to host extracellular matrix components
• Examples:
  • Fibronectin-binding proteins
  • Collagen-binding proteins

E.coli Adhesins

• Examples:
  • Type 1 fimbriae (FimH)
  • P-fimbriae (PapG)
  • AIDA-I autotransporter
  • MSCRAMMs (e.g., FdeC)

Bacterial Invasion

Intracellular Survival

• Bacteria can evade the immune system by surviving and replicating within host cells
• Inhibition of phagosome-lysosome fusion prevents the host's immune system from eliminating bacteria
• Modulation of autophagy helps bacteria to survive within host cells
• Production of antioxidants counteracts reactive oxygen species (ROS) to maintain bacterial survival

Immune Evasion

• Bacteria evade the host's immune response by producing immunosuppressive factors
• Inhibition of cytokine production reduces the host's immune response
• Disguise as host cells or apoptotic cells helps bacteria to evade the immune system
• Exploitation of immune cells for replication and dissemination enables bacterial spread

Virulence Factors

• Adhesins facilitate bacterial attachment to host cells
• Toxins contribute to bacterial pathogenesis
• Invasins enable bacterial invasion of host cells
• Immune modulators alter the host's immune response
• Nutrient acquisition systems allow bacteria to obtain necessary nutrients

Adhesion

• Bacteria bind to host cells through specific adhesins
• Adhesins recognize specific receptors on host cells
• Examples of adhesins include pili, fimbriae, and outer membrane proteins

Zipper Mechanism

• The zipper mechanism is a type of bacterial invasion
• Invasins bind to host cells and induce uptake
• The zipper mechanism involves a tight interaction between bacterial invasins and host cell receptors
• Examples of bacteria using the zipper mechanism include Listeria monocytogenes and Shigella flexneri

Trigger Mechanism

• The trigger mechanism is another type of bacterial invasion
• Toxins induce host cell membrane ruffling and uptake
• The trigger mechanism involves a signaling cascade triggered by toxin interaction with host cell receptors
• Salmonella is an example of a bacterium that uses the trigger mechanism

Listeria monocytogenes

• Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis
• It uses the zipper mechanism to invade host cells
• LLO is a virulence factor produced by Listeria monocytogenes
• Listeria monocytogenes can cross the blood-brain barrier and placenta

Shigella flexneri

• Shigella flexneri is a Gram-negative bacterium that causes shigellosis
• It uses the zipper mechanism to invade host cells
• IpaB is a virulence factor produced by Shigella flexneri, which induces host cell uptake
• Shigella flexneri can cause severe diarrhea and dysentery

Salmonella

• Salmonella is a Gram-negative bacterium that causes salmonellosis
• It uses the trigger mechanism to invade host cells
• SipA is a virulence factor produced by Salmonella, which induces host cell membrane ruffling
• Salmonella can cause food poisoning and typhoid fever

Cellular Invasion

• Intracellular pathogens invade host cells to replicate and evade the host's immune system, using mechanisms such as adhesins, invasins, and direct penetration
• Adhesins bind to host cell receptors, while invasins trigger host cell signaling pathways to internalize the pathogen
• Direct penetration involves forceful entry into the host cell

Cytoskeleton Manipulation

• Intracellular pathogens manipulate the host cell's cytoskeleton to facilitate their own replication and survival
• Actin cytoskeleton rearrangement creates a replicative niche for the pathogen, while microtubule manipulation disrupts host cell trafficking and signaling

Host-cell Signaling

• Intracellular pathogens manipulate host cell signaling pathways to create a favorable environment for their own replication and survival
• Mechanisms include suppression of pro-inflammatory signaling, activation of anti-inflammatory signaling, and modulation of cell death pathways

Immune Evasion

• Intracellular pathogens evade the host's immune system to avoid detection and elimination
• Mechanisms include antigen presentation avoidance, cytokine modulation, and immune cell suppression

Coxiella burnetii

• Forms a replicative vacuole that fuses with lysosomes, allowing the pathogen to survive and replicate in the harsh environment
• Manipulates host cell signaling to create a favorable environment for replication

Salmonella

• Uses type III secretion system to inject effector proteins into host cells, triggering actin cytoskeleton rearrangement and invasion
• Manipulates host cell signaling to evade immune detection and facilitate replication

Mycobacteria

• Has a thick cell wall that resists host cell defenses
• Manipulates host cell signaling to suppress pro-inflammatory responses and facilitate replication

Chlamydia

• Forms a replicative inclusion that recruits host cell organelles and nutrients
• Manipulates host cell signaling to evade immune detection and facilitate replication

Legionella Life Cycle

• Ingestion by host cell is followed by formation of a replicative vacuole, replication and survival in the vacuole, and finally lysis of the host cell and release of progeny
• Manipulates host cell signaling to facilitate replication and survival

Metal Ion Acquisition

• Microbial growth and survival depend on metal ion uptake, which involves transporting metal ions across the cell membrane through various mechanisms.

Mechanisms of Metal Ion Uptake

• Passive diffusion: metal ions move across the membrane without energy input
• Active transport: energy is used to transport metal ions against their concentration gradient
• Chelation-mediated transport: metal ions are bound to chelating agents, facilitating transport across the membrane

Types of Transporters

• ABC (ATP-Binding Cassette) transporters: use ATP to transport metal ions across the membrane
• Major Facilitator Superfamily (MFS) transporters: use proton motive force to transport metal ions
• P-type ATPases: use ATP to transport metal ions across the membrane
• Cation diffusion facilitators (CDFs): transport metal ions out of the cell

Ion Binding Proteins

• Ferritin: binds to iron, facilitating its uptake
• Metallothionein: binds to zinc, copper, and other metal ions, facilitating their uptake
• CopZ: binds to copper, facilitating its uptake

Siderophores

• Low-molecular-weight compounds produced by microorganisms to acquire iron
• Bind to ferric iron, forming a complex that can be transported into the cell
• Examples: enterobactin (E. coli), pyoverdine (Pseudomonas aeruginosa)

Metallophores

• Compounds that bind to metal ions, facilitating their uptake
• Examples: ferrichrome (binds to iron), coprogen (binds to copper)
• Can be produced by microorganisms or derived from environmental sources

Therapeutic Targetting of Metal Acquisition

• Inhibiting metal ion acquisition as a strategy to combat microbial infections
• Examples: inhibitors of siderophore production, inhibitors of metal ion transporters, chelators that bind to metal ions, making them unavailable to microorganisms

Bacterial Immune Evasion

Antigenic Variation

• Bacteria alter their surface antigens to evade the host's immune system
• Antigens are proteins or carbohydrates on the bacterial surface that stimulate an immune response
• Variation in antigens prevents recognition by the host's immune system, allowing bacteria to evade immune responses
• Examples include Neisseria gonorrhoeae (gonorrhea) and Borrelia burgdorferi (Lyme disease)

Immune Suppression

• Bacteria produce immunosuppressive factors, such as cytokines and chemokines, to suppress the host's immune response
• Bacteria inhibit immune cell activation and proliferation
• Bacteria induce regulatory T cells that suppress immune responses
• Bacterial toxins inhibit immune cell function
• Examples include Mycobacterium tuberculosis and Streptococcus pneumoniae

Complement Evasion

• Bacteria produce proteins that inhibit complement activation
• Bacteria express surface molecules that bind to and inactivate complement proteins
• Bacteria release soluble factors that inhibit complement activation
• The complement system is a group of proteins that work together to help eliminate pathogens from the body

Evading Complement

• Bacteria use capsular polysaccharides to inhibit complement activation (e.g., Streptococcus pneumoniae)
• Bacteria express surface proteins that bind to and inactivate complement proteins (e.g., Neisseria meningitidis)
• Bacteria release soluble factors that inhibit complement activation (e.g., Staphylococcus aureus)

Evading Immune Cells

• Bacteria inhibit phagocytosis by immune cells (e.g., Mycobacterium tuberculosis)
• Bacteria produce toxins that inhibit immune cell function (e.g., Staphylococcus aureus)
• Bacteria express surface molecules that inhibit immune cell activation (e.g., Streptococcus pneumoniae)

Genetic Exchange in Gonorrhoea

• Neisseria gonorrhoeae (gonorrhea) uses horizontal gene transfer to exchange genetic material with other bacteria
• This process allows for the rapid spread of antibiotic resistance and antigenic variation

Phase Variation in Salmonella

• Salmonella enterica uses phase variation to evade the host's immune system
• Phase variation involves the on/off switching of gene expression, allowing for changes in surface antigens
• This process allows Salmonella to evade recognition by the host's immune system and adapt to changing environments