Bacterial virulence factors

Question 1

What are the two types of pathogen? Give an example of each.

Answer 1

1. Primary pathogen: cause disease in an immunocompetent host, e.g. Mycobacterium tuberculosis, influenza virus
2. Opportunistic pathogen: cause disease in an immunocompromised host, and are sometimes members of the normal flora, e.g. Candida albicans (normal), Pseudomonas aeruginosa​ (environmental)

Question 2

How can virulence be compared (measured) between two microbes?

Answer 2

Different doses (number of cells) of innoculum are transfered into a mouse model and the rate of death is compared between the two, microbes/strains that cause death at a lower dose are more virulent.

Question 3

What are the different stages of pathogenesis by a microbe? [7]

Answer 3

1. Exposure
2. Adherence: of the pathogen to the host, e.g. epithelial surface, skin etc.
3. Invasion: microbe employes a strategy to invade the tissue.
4. Colonisation and growth: production of virulence factors.
5. Toxicity: systemic (e.g. tetanus toxin) or local effects.
6. Invasiveness: further growth at original site and distant sites, e.g. septicaemia, meningits.
7. Tissue damage, disease

Question 4

What is a bacterial virulence factor? Give examples.

Answer 4

A virulence factor is anything that contributes to pathogenicity. It can be a single virulence factor, or a wide variety, and can be transient.

• Endotoxin in LPS layer
• Enterotoxin
• Type 1 fimbriae
• Cytotoxin
• Vi capsule antigen
• Flagellum
• O antigen
• Anti phagocytic proteins

Question 5

Define exotoxin. What are the different types? [5]

Answer 5

A toxin (secreted enzymes and waste products) released by a living bacterial cell into its surroundings. These include:

• Cytolytic exotoxins: destroy red blood cells or nucleated cells, including haemolysins and leukocydins in Streptococcus pyogenes.
• Invasive enzymes: important for pathogenesis, including hyalurinidase (S. aureus) and collagenase (C. perfringens).
• Break down clots: to release bacteria, including streptokinase and staphylokinase.
• Form clots: protects bacteria from phagocytic cells, including coagulase (S. aureus).
• A-B exotoxins: classic toxins containing two components, e.g. tetanus toxin.

Question 6

Define endotoxin. What are the different types? [2]

Answer 6

A toxin present inside a bacterial cell (cell wall components, usually in gram -ve cells) that is released when it disintegrates.

• Lipopolysaccharide (LPS): long sugar molecule.
• Lipooligosaccharide (OPS): shorter fat-sugar molecule.

Question 7

Outline adherence of a microbe to a host surface.

Answer 7

Pathogens have specific mechanisms for adhering to host cells involving specific ligand-receptor interactions. Often there is tissue tropism in infection, which is usually the result of this specific pathogen ligand-host-cell-receptor interaction. There are various molecules and specialised organelle-like structures on the surface of bacteria involved in adherence.

Question 8

What are the major adherence factors? [4] Give examples.

Answer 8

• Glycocalyx/capsule/slime layer: a capsule around a bacterium that is a slime layer:
  
  • Streptococcus mutans- glycocalyx promotes adherence to brush border of intestinal vili.
  • Pathogenic E. coli- dextran glycocalyx promotes adhesion to tooth surfaces.
• Pili and fimbriae: spike-like protrusions important for adherence in some bacteria:
  
  • Neisseria gonorrhoea- pili facilitate binding to the urogenital epithelium.
  • Salmonella spp.- type 1 fimbriae facilitate binding to the epithelium of the small intestine.
  • Pathogenic E. coli- colonisation factor antigens (CFAs) - fimbriae - facilitate binding to small intestine epithelium.
• Adherence proteins: specific proteins on the outer memebrane of gram -ve and +ve cells that are prone to antigenic variability:
  
  • Streptococcus pyogenes- M protein on the cell surface binds to receptors on respiratory mucosa.
  • Neisseria gonorrhoea- Opa protein binds to receptors on genital tract epithelium.
• Lipoteichoic acid: an important component of gram +ve bacteria that is an endotoxin (similar to LPS), which is important for adherence to cell surfaces:
  
  • Streptococcus pyogenes- facilitates binding to respiratory epithelium (along with M protein).
• Biofilms: a thin but robust layer of mucilage adhering to a solid surface and containing a community of bacteria and other microorganisms. Allows for extracellular DNA, protein, and polysaccharide exchange.
  
  • Streptococcus mutans- dextrans in the oral cavity.
  • Neisseria gonorrhoea
  • Pseudomonas aeruginosa in the lungs of cystic fibrosis.

Question 9

Outline invasion of a microbe into host cells and tissues.

Answer 9

Most organisms need to invade to cause disease, but there are some exceptions to this such as Clostridium tetani which produces tetanus toxin, that has systemic effects from a localised infection. Breakage of the skin/membrane is often needed to penetrate the epithelium, and sometimes colonisation of the surface occurs when the normal flora has been altered or eliminated, e.g. in immunocompromised individuals. Once through the epithelium, colonisation is local initially and may then spread around the body via the bloodstream (septicaemia).

Question 10

Outline colonisation and growth of a microbe in a host.

Answer 10

After invasion, colonisation and growth occur, which depends upon a number of factors:

• Right ecological niche/environment for the pathogen to grow, i.e. temperature, nutrients, pH.

There is multiplication and localisation, which may form a local infection such as a boil or skin infection. Alternatively there may be a spread of infection to other sites, leading to a generalised systemic infection involving bacteraemia, sepcticaemia etc.

Question 11

Define toxicity and invasiveness of a microbe in a host. Give an example of each.

Answer 11

Toxicity- the capacity of an organism to cause disease by means of toxins produced by the organism, e.g. tetanus (Clostridium tetani), diptheria (Corynebacterium diptheriae).

Invasiveness- the ability of an organism to multiply to high levels in tissues and cause disease and is not dependent on the toxins, i.e. dependent on other virulence factors such as invasive enzymes, clot forming, e.g. Streptococcus pneumoniae has a capsule that inhibits phagocytosis.

Some microbes use a mixture of both of these strategies, e.g. Clostridium perfringens produces a toxin and invasive enzymes.

Question 12

List the virulence factors associated with colonisation and invasion [3]. Give examples.

Answer 12

Enzymes that help the microbe colonise and invade tissues. Various extracellular enzymes may contribute to microbial virulence:

• Hyaluronidase (spreading factor): breaks down hyaluronic acid (intracellular cement), e.g. Streptococcus spp, Staph. aureus, certain Clostridium spp.
• Collagenase: breaks down the collagen network supporting tissues, e.g. certain Clostridium spp.
• Proteases, nucleases, lipases: depolymerise host proteins, nucleic acids and lipids.

Question 13

Outline the different types of bacterial toxins.

Answer 13

• Exotoxins are released from living bacterial cells, which may travel from the focus of infection to other sites in the body.
  
  • Cytolytic: actively lyse cells.
  • A-B toxins
  • Superantigen toxins: are potent and able to have an exaggerated immune response with systemic effects.
• Endotoxins are part of the cell wall and are released upon death.

Question 14

Outline the action of cytolytic toxins.

Answer 14

Cytolytic toxins are exotoxins that act directly on the cell membranes by creating holes in the membrane, e.g. haemolysins, leukocidins, phospholipases, lecithinases, pore-forming toxins.

Blood agar can be used as a dianostic medium for the production of haemolysins by a microbe, which form a ‘halo’, e.g. Streptococcus.

Question 15

List some examples of A-B toxins and their diseases [4].

Answer 15

• Diptheria (Corynebacterium diptheriae): toxin inhibits protein synthesis in heart muscle and other cells.
• Tetanus (Clostridium tetani): toxin affects neuromuscular junctions leading to a constant release of acetlycholine, which leads to irreversible contraction of muscles and spastic paralysis.
• Botulism (Clostridium botulinum): toxin affects neuromuscular junctions, preventing the release of acetylcholine leading to a lack of stimulus to muscles, causing flaccid paralysis.
• Cholera (Vibrio cholerae): toxin activates adenyl cyclase in intestinal cells, leading to the disruption of soidium ion influx and loss of water to the lumen, and diarrhoea.

Question 16

Outline superantigen toxins. Give an example.

Answer 16

Superantigen toxins have similarities to a number of different immune molecules, and are able to stimulate large numbers of lymphocytes, creating a cytokine storm (massive cytokine release). This causes fever, systemic toxicity, immune suppression and shock, e.g. toxic shock syndrome toxin (TSST) from Staphylococcus aureus.

Question 17

How can tetanus be prevented and treated?

Answer 17

Prevention: tetanus toxoid vaccine targets the A-B toxin and is an antibody vaccination that neutralises the toxin, effective for 8-10 years and requires boosters to remain effective. DPT is given to children, immunising against diptheria, pertussis and tetanus.

Treatment: physicians quickly response to possible exposure to tetanus toxin by giving passive immunisation (purified antibodies raised in another organism) at the site of infection. The physician will need to know:

• Whether the patient knows their immunisation history and if they have had a shot in past 8 years.

Question 18

Outline the action of cholera toxin.

Answer 18

The cholera pathology is caused by the Vibrio cholerae enterotoxin, which an exotoxin that affects the intestinal tract. The bacterium adheres to the endothelial cells (via fimbriae) and secretes an A-B enterotoxin. The toxin has two distinct parts: B attaches to the membrane GM1 ganglioside, and A enters the cell, dramatically increasing cAMP production. This causes a high concentration of ions to be pumped into the lumen, causing water to flow out of the tissues in an attempt to create an equilibrium in solute concentration. The quantity of water loss from the body can be huge.

Question 19

Outline LPS action.

Answer 19

Lipid A (endotoxin), the hydrophobic anchor of lipopolysaccharide (LPS), is a glucosamine-based phospholipid that makes up the outer monolayer of the outer membranes of most Gram-negative bacteria.

In macrophages, lipid A activation of TLR4 triggers the biosynthesis of diverse mediators of inflammation, such as TNF-α and IL1-β, and activates the production of co-stimulatory molecules required for the adaptive immune response.

Lipid A stimulates an overreaction of the body’s phagocytic cells in which normal body tissue is destroyed and inflammation is excessive.

Question 20

What are the main differences between gram negative and gram positive envelopes?

Answer 20

• Gram positive cells have a thicker cell wall (20-30 nm) than gram negative cells (8-12 nm).
• Gram positive cells have a thick layer of peptidoglycan, whereas gram negative cells have a thin layer.
• GN cells have a high LPS content, whereas GP cells have very few.
• GP cells contain techoic and lipotechoic acid, GN cells do not.

Question 21

List the gram negative membrane components [3].

Answer 21

1. Inner (cytoplasmic) membrane: active transport, respiratory chain components, energy transducing systems, H⁺/ATP proton pumps, biosynthetic enzymes for membrane phospholipids, PG, LPS, capsule.
2. Periplasm: peptidoglycan, degradative enzymes, B lactams, binding proteins, signalling molecules.
3. Outer membrane: porins, transporters, B lactams, specific uptakes of B12, iron, and maltose.

Question 22

The peptidoglycan network of the bacterial cell wall comprises ______ chains of β-1,4-linked N-acetylmuramic acid (______) and N-acetylglucosamine (______), cross-linked by short peptide stems of l- and d-amino acids anchored to the lactyl moiety of ______.

During growth and maturation, the PGN is degraded by dedicated hydrolase enzymes causing PGN fragments (____________) to be shed from the cell wall into the environment, a process termed PGN turnover. These fragments, released from the PGN layer, can stimulate host ______ immune responses through Nod1 and Nod2. Human Nod1 (hNod1) is strongly activated by mesoDAP-containing tripeptides (______), found predominantly in Gram-________ bacteria, whereas the minimal activating structure is the dipeptide γ-d-Glu-mesoDAP (iE-DAP).

The mature PG architecture is also marked by a high degree of direct peptide cross-links. Overall, 70 to 80% of the peptides are cross-linked, in two kinds of linkages. One type is between the _-___ at position 4 of one peptide and the meso-DAP at position 3 of an adjacent peptide. These “_-_” linkages are catalyzed by typical d, d-transpeptidases, also known as penicillin-binding proteins (PBPs), which can be inhibited by various classes of beta-lactam antibiotics. The other type of linkage is between two DAP residues, also known as a “_-_” linkage, that are catalyzed by the concerted activity of d,d-carboxypeptidases and l, d-transpeptidases, the latter of which have been found to be resistant to most beta-lactam antibiotics, except for the carbapenem class

The polymerization of the sugar backbone and cross-linking of the peptides of the PG are carried out by a variety of _________________, _______________, and _________________.

Answer 22

The peptidoglycan network of the bacterial cell wall comprises glycan chains of β-1,4-linked N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc), cross-linked by short peptide stems of l- and d-amino acids anchored to the lactyl moiety of MurNAc.

During growth and maturation, the PGN is degraded by dedicated hydrolase enzymes causing PGN fragments (muropeptides) to be shed from the cell wall into the environment, a process termed PGN turnover. These fragments, released from the PGN layer, can stimulate host innate immune responses through Nod1 and Nod2. Human Nod1 (hNod1) is strongly activated by mesoDAP-containing tripeptides (TriDAP), found predominantly in Gram-negative bacteria.

The mature PG architecture is also marked by a high degree of direct peptide cross-links. Overall, 70 to 80% of the peptides are cross-linked, in two kinds of linkages. One type is between the d-Ala at position 4 of one peptide and the meso-DAP at position 3 of an adjacent peptide. These “4-3” linkages are catalyzed by typical d, d-transpeptidases, also known as penicillin-binding proteins (PBPs), which can be inhibited by various classes of beta-lactam antibiotics. The other type of linkage is between two DAP residues, also known as a “3-3” linkage, that are catalyzed by the concerted activity of d,d-carboxypeptidases and l, d-transpeptidases, the latter of which have been found to be mostly resistant to most beta-lactam antibiotics.

The polymerization of the sugar backbone and cross-linking of the peptides of the PG are carried out by a variety of transglycosylases, transpeptidases, and carboxypeptidases.

Question 23

Gram-________bacteria have two distinct membranes: an inner membrane and an outer membrane. A prominent constituent of the outer leaflet of the outer membrane is __________________ (LPS). The LPS components of many bacteria are toxic.

The LPS molecule can be divided into three parts: lipid _, core _______________ and _-_______ repeats.

Lipid _ represents the hydrophobic component of LPS which locates in the outer leaflet of the outer membrane, while core _______________ and _-_______ repeats are displayed on the surface of the bacterial cells. Lipid _ is known to be responsible for the toxic effects of infections with Gram-________ bacteria. The detailed structure of LPS varies from one bacterium to another, and this variation could affect the _________ of the bacterium. The biosynthetic pathway of LPS has been well characterized in E. coli. The biosynthetic pathway and export mechanisms of LPS are common to most Gram-________ bacteria, but some bacterial pathogens can further modify the basic structure of their LPS.

Answer 23

Gram-negative bacteria have two distinct membranes: an inner membrane and an outer membrane. A prominent constituent of the outer leaflet of the outer membrane is lipopolysaccharide (LPS). The LPS components of many bacteria are toxic.

The LPS molecule can be divided into three parts: lipid A, core polysaccharides and O-antigen repeats.

Lipid A represents the hydrophobic component of LPS which locates in the outer leaflet of the outer membrane, while core polysaccharides and O-antigen repeats are displayed on the surface of the bacterial cells. Lipid A is known to be responsible for the toxic effects of infections with Gram-negative bacteria. The detailed structure of LPS varies from one bacterium to another, and this variation could affect the virulence of the bacterium. The biosynthetic pathway of LPS has been well characterized in E. coli. The biosynthetic pathway and export mechanisms of LPS are common to most Gram-negative bacteria, but some bacterial pathogens can further modify the basic structure of their LPS.

Question 24

Outline what LPS is and its structure.

Answer 24

Lipopolysaccharide (LPS) is a large and variable complex glycolipid, and is an integral structural component of the outer membrane of Gram-negative bacteria. Localized in the outer leaflet of the outer membrane, LPS molecules maintain the barrier function of the outer membrane and mediate several interactions between the bacterium and its surrounding environment.

It is composed of three domains:

1. a hydrophobic domain termed lipid A (or endotoxin), which is embedded in the outer membrane
2. a relatively conserved non-repeating core oligosaccharide
3. a variable outermost polysaccharide (or O-antigen).

The three LPS domains differ in structure, and therefore confer different biological properties: The lipid A domain interacts with immune receptors and confers the LPS molecule with a range of immunological and potential endotoxic properties; the core oligosaccharide influences permeation properties of the outer membrane; and the O-antigen contributes to the antigenicity and serospecificity of the molecule

Question 25

What can be used to target **peptidoglycan**?

Answer 25

Penicillin, cephalosporins, and vancomycin.

Question 26

Outline **teichoic** and **lipoteichoic acids**.

Answer 26

**Techoic acids** are negatively charged polyglycerol or polyribotol phosphate polymers that are covalently linked to PG in gram-positive cells. **Lipotechoic acid** is a lipophilic glycolipid anchored in the cytoplasmic membrane. Most **Gram-positive bacteria** have **teichoic acids** (TAs) or related glycopolymers that play crucial roles in bacterial **survival** under disadvantageous conditions and in other basic cellular processes. TAs are usually constitutively produced and either connected to peptidoglycan (wall teichoic acids, WTA) or to the cytoplasmic membrane (**lipoteichoic acids**, LTA). The actual functions of TAs and the reasons why most Gram-positive bacteria produce both LTA and WTA at the same time remain incompletely understood. WTA has been shown to be dispensable for viability of *S. aureus* and *Bacillus subtilis* under laboratory conditions but to play important roles during **colonization** and infection *in vivo.*

Question 27

What is the function of the **bacterial capsule**?

Answer 27

The bacterial capsule is the outermost layer (up to 10 um) of some **gram-positive** and **negative** bacteria. It is usually viscous **polysaccharide** and is not essential for viability. Sometimes it is less discrete, i.e. slime capsules, and helps shield the bacteria from **complement** and **phagocytosis**.

Question 28

Outline the **structure** and **function** of **flagella**.

Answer 28

A **flagellum** consists of **three** parts: the basal body, the hook, and the filament. The basal body consists of a reversible rotary motor and a protein export apparatus that translocates the flagellar components and some bacterial virulence proteins, while the hook and the filament function as a universal joint and a propeller, respectively. * Flagellin is the major structural protein of the flagellar filaments; it comprises four linearly connected domains, two filament core domains (D0, D1) and two hypervariable-region domains (D2, D3). The N- and C-terminal chains of flagellin form packed α-helical structures, which constitute the D0 and D1 domains positioned in the filament core. The variable region of flagellin is exposed as a folded β-sheet structure (D2 and D3 domains) on the filament outer surface Flagella are essential structures in the pathogenic bacteria, by providing **motility**. However, flagella are involved in bacterial pathogenicity not just by conferring motility but also through other pathways: * promoting **adherence** by acting as bacterial **adhesins** and by providing force-generating motility. * promoting bacterial **biofilm formation** supporting pathogen survival in vivo. * translocating virulence proteins into host cells via special **type III secretion systems.** * triggering host pro-inflammatory responses through the Toll-like receptor 5 (**TLR5**) signaling pathway.

Question 29

What is the function of **pili**?

Answer 29

* Conjugation: *E.coli* F pilus * DNA uptake (*Neisseria*) * Adhesion * Phage receptors * Early steps in biofilm formation * Resist phagocytosis * Antigenic variation (*Neisseria gonorrhoea*)

Question 30

The process of transferring the genes from alien genomes into the host genomes is known as __________ gene transfer, which is in contrast to lateral gene transfer, where gene transfers occur within the host genomes. When comparing the genomic region of pathogenicity islands and the remaining parts of the host genome, we can usually find that PAIs have their own genomic characteristics such as containing ________ genes, containing _________ genes, and having their own sequence \_\_\_\_\_\_\_\_\_. In general, each genome has its unique genome signature, which can be measured by \_+\_ content, dinucleotide frequencies (or other k-mer frequencies), and codon usage. The \_+\_ (%) contents (i.e., the percentage of _______ and _________ bases) in PAIs are often different from that of the host organisms. Another important property of PAIs is that they contain \_\_\_\_\_\_\_\_\_-associated genes. Depending on the environment that bacteria live in, the proteins encoding virulence-associated genes in PAIs can include the following categories: (a) \_\_\_\_\_\_\_\_\_, which are cell-surface components that facilitate bacteria adhere to eukaryotic cells; (b) Type ___ and __ secretion systems, which have needle-like structures that detect the presence of host organisms, and secrete effector proteins into the host cell; (c) \_\_\_\_\_\_\_\_, which facilitate bacteria to invade eukaryotic epithelial cells; (d) \_\_\_\_\_\_, which can be exotoxins, proteases, lipases, and enterotoxins; and (e) Iron uptake systems.

Answer 30

The process of transferring the genes from alien genomes into the host genomes is known as **horizontal** gene transfer, which is in contrast to lateral gene transfer, where gene transfers occur within the host genomes. When comparing the genomic region of pathogenicity islands and the remaining parts of the host genome, we can usually find that PAIs have their own genomic characteristics such as containing **mobility** genes, **virulence** genes, and having their own sequence **signature**. In general, each genome has its unique genome signature, which can be measured by **G+C** content, dinucleotide frequencies (or other k-mer frequencies), and codon usage. The **G+C** (%) contents (i.e., the percentage of **guanine** and **cytosine** bases) in PAIs are often different from that of the host organisms. Another important property of PAIs is that they contain **virulence**-associated genes. Depending on the environment that bacteria live in, the proteins encoding virulence-associated genes in PAIs can include the following categories: (a) **Adhesins**, which are cell-surface components that facilitate bacteria adhere to eukaryotic cells; (b) Type **III** and **IV** secretion systems, which have needle-like structures that detect the presence of host organisms, and secrete effector proteins into the host cell; (c) **Invasins**, which facilitate bacteria to invade eukaryotic epithelial cells; (d) **Toxins**, which can be exotoxins, proteases, lipases, and enterotoxins; and (e) Iron uptake systems.

Question 31

What is a **pathogenicity island**? List some features.

Answer 31

Pathogenicity islands are **segments** of the bacterial chromosome harboring large clusters of **virulence genes.** They are *present* in **pathogenic** strains but *absent* or sporadically distributed in non-pathogenic strains/species. They typically have a **G+C** **content different** from that of the rest of the chromosome/ They are often associated with tRNA genes and/or **mobile genetic elements** at their boundaries. * PAI carry one or more **virulence genes**; genomic elements with characteristics similar to PAI but lacking virulence genes are referred to as genomic or metabolic islands. * PAI are present in the genomes of a **pathogenic bacterium** but absent from the genomes of a nonpathogenic representative of the same species or a closely related species. * PAI occupy relatively **large genomic regions**. The majority of PAI are in the range of 10 to 200 kb. * PAI often differ from the core genome in their **base composition** and also show a different **codon usage**. The base composition is expressed as percentage of guanine and cytosine (**G+C**) bases, and the average G+C content of bacterial DNA can range from 25 to 75%. * PAI are frequently located adjacent to **tRNA** genes. Certain bacteriophages use tRNA genes as specific insertion points in the host genome. tRNA genes may represent specific anchor points for the integration of foreign DNA. * PAI are frequently associated with **mobile genetic elements**. They are often flanked by direct repeats (DR). DR are defined as DNA sequences of 16 to 20 bp (up to 130 bp) with a perfect or nearly perfect sequence repetition. * PAI often are **unstable** and delete with distinct frequencies. Virulence functions encoded by certain PAI are lost with a frequency that is higher than the normal rate of mutation.

Question 32

The physical process of **transferring DNA** from one bacterial cell to another can be accomplished by generalized transduction, conjugation or transformation. **Outline** these three processes.

Answer 32

1. Transduction simply describes the transfer of DNA from one cell to another via bacteriophage (phage), and typically refers to the transfer of the phage's own DNA from one cell to another and its integration into the bacterial chromosome at a specific site. **Generalized transduction** is a variation of the transduction process, where the newly forming phage particles package bacterial chromosomal or plasmid DNA instead of phage DNA. It is not known if this is accidental or programmed, and why only some bacteriophage do this but not others. 2. **Conjugation** involves the transfer of DNA from one cell to another directly through a connecting tube (**pilus**) or pore. The *tra* genes encode the necessary proteins and show similarity to type IV secretion systems and are carried exclusively on mobile genetic elements. 3. **Transformation** is the uptake of free DNA from the environment by a bacterium, usually via bacterially encoded **competence proteins** that form a pore-like structure in the bacterial cell membrane. This mechanism is extremely efficient in a range of bacteria, such as *Streptococcus pneumonia.*