Understanding Bacterial Virulence Factors

Medical Microbiology Course

• Introduction to Microorganisms
• Normal flora of Human Body
• Virulence Factors
• Gram Positive Cocci of Medical Importance
• Gram Positive Bacilli of Medical Importance
• Gram Negative Cocci
• Gram Negative Bacilli
• Acid Fast Bacteria
• Biochemical Identification of Microorganisms

Table of Content:

• Comparison of Gram negative and Gram positive cell wall
• Pathogenesis of Infection
• Koch’s Postulates
• Types of Infections
• Transmission Cycle
• Bacterial Cell & Its Virulence Factors
• Pathogenicity islands
• Regulation of Virulence Factors

Comparison of Gram negative and Gram positive cell wall

Peptidoglycan Layer

• Gram-positive: Thick peptidoglycan layer (20–80 nm) forming the outermost structure.
• Gram-negative: Thin peptidoglycan layer (2–7 nm) located between the inner and outer membranes.

Outer Membrane

• Gram-positive: Absent.
• Gram-negative: Present; contains lipopolysaccharides (LPS), porins, and proteins.

Teichoic Acids

• Gram-positive: Present; includes wall teichoic acid and lipoteichoic acid, which help in adherence and rigidity.
• Gram-negative: Absent.

Lipopolysaccharide (LPS)

• Gram-positive: Absent.
• Gram-negative: Present in the outer membrane; acts as an endotoxin and elicits immune responses.

Periplasmic Space

• Gram-positive: Usually absent or very narrow.
• Gram-negative: Well-defined periplasmic space containing enzymes and transport proteins.

Staining Reaction

• Gram-positive: Retains crystal violet stain, appears purple.
• Gram-negative: Loses crystal violet stain during decolorization, counterstained by safranin, appears pink/red.

Cell Wall Rigidity and Resistance

• Gram-positive: More resistant to physical disruption due to thick peptidoglycan.
• Gram-negative: More resistant to certain antibiotics and detergents due to the outer membrane.

Antibiotic Sensitivity

• Gram-positive: More sensitive to β-lactam antibiotics (e.g., penicillin).
• Gram-negative: Often more resistant due to the protective outer membrane.

Examples

• Gram-positive: Staphylococcus aureus, Streptococcus pyogenes.
• Gram-negative: Escherichia coli, Salmonella typhi.

Pathogenesis of Infection

The pathogenesis of bacterial infection includes:

Initiation of the Infectious Process:

• The first step in bacterial pathogenesis involves the entry of bacteria into the host's body through portals such as the respiratory tract, gastrointestinal tract, urogenital tract, skin abrasions, or mucous membranes.
• This stage includes the survival of bacteria against harsh environmental conditions (e.g., stomach acid, enzymes, bile) and their ability to reach a favorable site in the host.
• The bacteria must overcome natural physical barriers (like epithelial cells), mechanical clearance (like cilia and mucus), and initial immune defenses (like phagocytes and antimicrobial peptides).
• Success at this stage is essential for colonization and further progression of infection.

Mechanisms Leading to Development of Signs and Symptoms of Disease

• Once bacteria establish themselves in the host, they begin to multiply and interact with host tissues.
• They may release toxins, damage host cells, and disrupt normal tissue function, which triggers immune responses.
• The host’s immune response (such as inflammation, fever, pain, or swelling) contributes significantly to the symptoms experienced during infection.
• Depending on the type of pathogen and the affected tissue, the clinical manifestations can vary—from localized infections (e.g., boils) to systemic diseases (e.g., sepsis or meningitis).

Characteristics of bacteria that are pathogens include:

Transmissibility

• Ability to spread from one host to another via air, water, direct contact, or vectors.
• Some bacteria form spores or biofilms to enhance survival and transmission.

Adherence to Host Cells

• Bacteria attach to host tissues using structures like pili, fimbriae, or adhesins.
• This prevents their removal and allows them to colonize surfaces like mucosal membranes.

Invasion of Host Cells and Tissues

• Some pathogens penetrate epithelial barriers and invade deeper tissues or even host cells.
• Invasion helps bacteria spread within the host and avoid immune detection.

Toxigenicity

• Pathogens may produce exotoxins (secreted toxins) or endotoxins (like LPS in Gram-negative bacteria).
• These toxins can damage cells, disrupt body functions, and trigger severe immune reactions.

Evasion of the Host Immune System

• Pathogens avoid immune responses using capsules, antigenic variation, immune-modulating enzymes, or by hiding inside host cells.
• These strategies help them persist and cause prolonged or chronic infections.

Koch’s Postulates

Developed by Robert Koch in the late 19th century, Koch’s postulates are a set of criteria used to establish a causal relationship between a microorganism and a specific disease.

The Four Classical Postulates

1. The microorganism must be found in all organisms suffering from the disease, but not in healthy individuals.
2. The microorganism must be isolated from the diseased organism and grown in pure culture.
3. The cultured microorganism should cause disease when introduced into a healthy, susceptible host.
4. The microorganism must be re-isolated from the experimentally infected host and identified as identical to the original causative agent.

Types of Infections

Infections can be classified based on several criteria, including:

Based on Causative Agents

Viral Infections

• Caused by viruses such as influenza virus, HIV, or coronavirus.
• Often lead to conditions like the flu, COVID-19, hepatitis, or cold sores.

Bacterial Infections

• Result from bacterial pathogens like Staphylococcus aureus, E. coli, or Mycobacterium tuberculosis.
• Examples include strep throat, UTIs, and tuberculosis.

Fungal Infections

• Caused by fungi such as Candida or Aspergillus.
• Common conditions include athlete’s foot, candidiasis, and ringworm.

Based on Organ or System Involved

Skin Infections

• Infections affecting the skin, such as cellulitis, boils, or fungal dermatitis.

Respiratory Infections

• Affect the lungs and airways, including pneumonia, bronchitis, and tuberculosis.

Gastrointestinal Infections

• Involve the digestive tract, caused by pathogens like Salmonella or Rotavirus.
• Symptoms often include diarrhea, vomiting, and abdominal pain.

Based on Route/Pathogenesis of Infection

Local Infection

• Confined to a single area or tissue, such as an abscess or skin wound.

Systemic Infection

• Spreads throughout the body via the bloodstream, like sepsis or HIV.

Primary Infection

• The initial infection caused by a pathogen in a previously healthy host.

Secondary Infection

• Occurs when a second pathogen infects the host during or after treatment for a primary infection (e.g., fungal infection after antibiotics).

Transmission Cycle

The transmission cycle describes how an infectious agent moves from one host to another, including the points of entry and exit, possible vectors, and routes of transmission. Understanding this cycle is crucial for breaking the chain of infection.

Portal of Entry

• The site where pathogens enter the host's body.
• Common portals include:
• Respiratory tract (e.g., through inhalation of droplets – flu, COVID-19)
• Gastrointestinal tract (e.g., ingestion of contaminated food/water – cholera, typhoid)
• Urogenital tract (e.g., during sexual contact – HIV, gonorrhea)
• Broken skin or mucous membranes (e.g., cuts, wounds – tetanus, rabies)
• Placental transmission (e.g., from mother to fetus – syphilis, Zika)

Portal of Exit

• The route through which the pathogen leaves the host to infect others.
• Major exit points include:
• Respiratory secretions (coughing, sneezing – TB, influenza)
• Feces or urine (e.g., typhoid, hepatitis A)
• Blood (e.g., via needles or transfusion – HIV, hepatitis B/C)
• Saliva, semen, or vaginal secretions (e.g., STDs)
• Skin lesions or pus (e.g., in skin infections like impetigo)

Intermediate Host

• A host that temporarily harbors the pathogen and may be essential for part of its life cycle.
• Often not the final host, but necessary for development or transmission.
• Example: Snails in the life cycle of Schistosoma parasites.
• Pigs or dogs in some parasitic tapeworm infections.

Vector

• A living organism that transmits pathogens from one host to another, often without getting infected itself.
• Can be mechanical (physically carries pathogen) or biological (pathogen develops within vector).
• Examples:
• Mosquitoes (malaria, dengue, Zika)
• Ticks (Lyme disease, Rocky Mountain spotted fever)
• Fleas (plague)

Route of Transmission

• The path the pathogen follows from the reservoir or source to the new host.
• Main types include:
• Direct contact (e.g., kissing, touching – herpes, scabies)
• Indirect contact (e.g., contaminated surfaces – norovirus)
• Droplet transmission (e.g., sneezing, coughing – flu, COVID-19)
• Airborne transmission (e.g., TB, measles)
• Vector-borne transmission (e.g., malaria, leishmaniasis)
• Fecal-oral route (e.g., cholera, hepatitis A)
• Vertical transmission (from mother to child – HIV, Zika)

Bacterial Cell & Its Virulence Factors

Pathogenic bacteria possess various virulence factors that enable them to infect hosts, evade the immune system, and cause disease. These factors are often encoded on genetic elements such as plasmids or pathogenicity islands.

Virulence Factors

• Traits that enhance a bacterium's ability to cause disease in a host.
• Include structural components, secreted products, and genetic traits.
• Help in colonization, immune evasion, nutrient acquisition, and tissue damage.

Adherence Factors

• Enable bacteria to attach to host tissues, a critical first step in colonization.
• Use surface structures like:
• Fimbriae or Pili – hair-like appendages for specific binding to receptors.
• Adhesins – molecules on the bacterial surface that bind to host cells.

Pili

• Thin, filamentous structures on the bacterial surface.
• Many bacteria possess pili—thin, hair-like structures that project from their surface and play a key role in attaching to host cell surfaces.
• For instance, certain strains of E. coli produce type 1 pili, which enable them to bind specifically to receptors on epithelial cells.
• Strains of E. coli responsible for diarrheal diseases use these pili to adhere tightly to the lining of the intestinal epithelium, facilitating colonization and infection.
• Functions:
• Adhesion to host cells or surfaces.
• In some bacteria, pili are involved in DNA transfer (conjugation).
• Help in biofilm formation, increasing resistance to host defenses and antibiotics.

Fimbriae

• Fimbriae are hair-like surface structures that contribute to the adherence and virulence of Streptococcus pyogenes (Group A streptococci).
• These fimbriae are associated with lipoteichoic acid, protein F, and M protein, all of which play important roles in the infection process.
• Lipoteichoic acid and protein F facilitate the bacterium’s attachment to buccal epithelial cells in the oral cavity.
• This adherence is mediated by fibronectin, a host cell receptor protein that binds to these bacterial components.
• The M protein serves as a key antiphagocytic factor, helping the bacteria evade immune system attack.
• M protein is considered a major virulence determinant, as it prevents engulfment by phagocytic cells, promoting bacterial survival.

Antiphagocytic Factors

Polysaccharide Capsules

• Found in bacteria like Streptococcus pneumoniae and Neisseria meningitidis.
• These capsules form a protective outer layer that inhibits recognition and ingestion by phagocytic cells such as macrophages and neutrophils.
• The capsule helps the bacteria evade the host immune system, contributing to increased virulence.

M Protein

• A surface protein produced by Group A streptococci (Streptococcus pyogenes).
• It prevents phagocytosis by interfering with opsonization, a process where antibodies and complement mark pathogens for destruction.
• M protein is considered a major virulence factor and is also involved in host cell adhesion.

Adhesins

Yersinia enterocolitica

• Adheres to the host cell membrane, triggering the extrusion of protoplasmic projections.
• The bacteria are then engulfed by the host cell, leading to the formation of a vacuole.
• Once inside, the vacuolar membrane dissolves, releasing the bacteria into the cytoplasm.

Listeria monocytogenes

• The adhesion and invasion of macrophages is mediated by a specific protein called internalin.
• This protein promotes the bacteria’s entry into host cells by interacting with receptors on macrophages.

Neisseria gonorrhoeae

• Uses pili as the primary adhesins to attach to host cells.
• Opacity-associated proteins (Opa) serve as secondary adhesins, further facilitating attachment and invasion.
• Without adhesion, bacteria are likely to be cleared from the body by host defenses.

Toxins

• Poisonous substances produced by bacteria to damage host cells.
• Two main types:

Exotoxins 

• secreted proteins (e.g., botulinum toxin, diphtheria toxin).
• Produced and secreted by bacteria into the surrounding environment.
• Protein in nature, often heat-labile (can be denatured by heat).
• Highly potent and can cause significant damage to host cells, tissues, or organs.
• Can be specific in their target (e.g., neurotoxins affecting the nervous system, enterotoxins affecting the gastrointestinal tract).
• Typically produced by Gram-positive and Gram-negative bacteria.
• Can be used as vaccines (e.g., tetanus toxoid, diphtheria toxoid).
• Exotoxins are often composed of two subunits:
• B subunit: Responsible for binding the toxin to host cell receptors and facilitating entry into the cell.
• A subunit: Carries the enzymatic or toxic activity that disrupts host cell function.
• Examples of Important Exotoxins:

Diphtheria Toxin

• Produced by Corynebacterium diphtheriae strains carrying a temperate bacteriophage.
• The phage encodes the toxin gene, enabling the bacteria to cause diphtheria.

Tetanospasmin (Tetanus Toxin)

• Released by Clostridium tetani.
• Interferes with neurotransmitter release, causing spastic paralysis by affecting motor neurons.

Botulinum Toxin

• Produced by Clostridium botulinum and absorbed from the intestinal tract.
• Binds to presynaptic membranes of motor neurons, blocking acetylcholine release, leading to flaccid paralysis.

Lecithinase (Alpha Toxin)

• Secreted by Clostridium perfringens.
• Destroys cell membranes by hydrolyzing lecithin into phosphorylcholine and diglyceride.

Toxic Shock Syndrome Toxin (TSST-1)

• Secreted by Staphylococcus aureus.
• A superantigen that causes massive immune system activation, leading to fever, rash, hypotension, and shock.

Exotoxin A

• Produced by Streptococcus pyogenes and other streptococcal strains.
• Contributes to tissue destruction and immune-mediated damage.

Cholera Toxin

• Secreted by Vibrio cholerae.
• Stimulates cAMP production in intestinal cells, leading to watery diarrhea (rice-water stools).

Endotoxins

• Endotoxins are part of the outer membrane of Gram-negative bacteria and consist of lipopolysaccharide (LPS).
• The toxic component is Lipid A, which is released when bacteria die and disintegrate.
• Regardless of bacterial species, the pathophysiologic effects of endotoxins are similar and often severe.
• Common Effects of Endotoxins:
• Fever (due to cytokine release like IL-1 and TNF-alpha)
• Leukopenia (reduction in white blood cells)
• Hypoglycemia (low blood sugar levels)
• Hypotension (low blood pressure)
• Septic shock with organ dysfunction (affecting brain, heart, and kidneys)
• Intravascular coagulation (may lead to Disseminated Intravascular Coagulation – DIC)
• Death can occur in severe cases due to multi-organ failure

Enzymes

• Aid in invasion and tissue destruction.
• Examples include:
• Hyaluronidase – breaks down connective tissue.
• Collagenase – degrades collagen in host tissues.
• Coagulase – causes clotting to wall off infection.
• Hemolysins – lyse red blood cells to release nutrients.
• Staphylokinase – digests blood clots.
• DNase – digests DNA.
• Lipases – digest oils; enhances colonization on skin.
• Penicillinase – inactivates penicillin.
• Cytolysins — they dissolve red blood cells (hemolysins) or kill tissue cells or leukocytes (leukocidins).
• IgA Proteases-inactive primary antibody

Genetic elements

• Carry genes responsible for virulence and antibiotic resistance.
• Include:
• Plasmids – extra-chromosomal DNA carrying resistance or toxin genes.
• Transposons – mobile genetic elements that can move between DNA regions.
• Pathogenicity islands – clusters of virulence genes acquired through horizontal gene transfer.
• These elements enhance bacterial adaptability and pathogenic potential.

Pathogenicity islands

• Pathogenicity islands (PAIs) are large clusters of genes located on the bacterial chromosome that contribute to a bacterium’s ability to cause disease.
• These DNA segments are typically 10 to 200 kilobases in size and are organized in a way that allows coordinated expression of virulence factors.
• PAIs contain one or more virulence genes, which can include those responsible for toxin production, adhesion, or invasion.
• They are present in pathogenic strains of bacteria but are absent in non-pathogenic members of the same species, highlighting their role in disease development.
• PAIs often have a distinct guanine plus cytosine (G+C) content, which differs from the rest of the bacterial genome, suggesting they were acquired through horizontal gene transfer.
• These regions are frequently associated with mobile genetic elements such as transposons or plasmids, making them genetically unstable and capable of mobility within or between genomes.

Regulation of Virulence Factors

• The diphtheria toxin gene in Corynebacterium diphtheriae is carried by temperate bacteriophages, and only strains lysogenized by these phages can produce the toxin.
• Toxin production in C. diphtheriae is enhanced in low-iron environments, indicating that iron availability plays a regulatory role in virulence factor expression.
• In Bordetella pertussis, the expression of virulence genes increases at 37°C, which is human body temperature, suggesting temperature-dependent regulation.
• The same virulence genes in B. pertussis are suppressed when the bacteria are grown at lower temperatures or in the presence of high levels of magnesium sulfate or nicotinic acid.
• In Vibrio cholerae, regulation of virulence factors, including cholera toxin, is influenced by multiple environmental signals such as temperature and pH.
• Expression of cholera toxin is higher at pH 6.0 than at pH 8.5, and greater at 30°C than at 37°C, showing how environmental conditions can modulate virulence.