Question

4. In this chapter, we have studied a number of toxins that are responsible for many of the disease symptoms associated with potent pathogens. For each of the following toxins, describe the mechanism of action on host cells in terms of the structure of the toxin, how it gains access to its target, what it does to its target, and the outcome.

A. DT

B. Shiga toxin

C. E. coli heat-stable toxin

D. Cholera toxin

E. Superantigen

F. YopE

G. Listeriolysin O

Answer 1

A. DT

Mechanism in which the bacterial DT invades the host cell membrane. The toxin enters through the membrane by endocytosis with the aid of fragment B. Fragment A is then actively exposed to the cytoplasm. In acidic endosomes, its translocation domain inserts into endosomal membranes and facilitates the transport of the catalytic domain (DTA) from endosomal lumen into the host cell cytosol and DTA ADP-ribosylates elongation factor 2 inhibits protein synthesis and leads to cell death

B. Shiga toxin The toxin has two subunits and is one of the AB5 toxins. The B subunit is a pentamer that binds to specific glycolipids on the host cell, specifically globotriaosylceramide (Gb3). Where as the A subunit is internalised and cleaved into two parts. The A1 component then binds to the ribosome, disrupting protein synthesis. Stx-2 has been found to be about 400 times more toxic than Stx-1.

subunit B to Gb3 causes induction of narrow tubular membrane invaginations, which drives formation of inward membrane tubules for the bacterial uptake into the cell.

These tubules are essential for uptake into the host cell. The Shiga toxin is transferred to the cytosol via Golgi network and ER. From the Golgi the toxin is trafficked to the ER then it  act to inhibit protein synthesis within target cells by a mechanism. After entering a cell via a macropinosome, the protein (A subunit) cleaves a specific adenine nucleobase from the 28S RNA of the 60S subunit of the ribosome, thereby halting protein synthesis.

As they mainly act on the lining of the blood vessels, the vascular endothelium, a breakdown of the lining and hemorrhage eventually occurs. The first response is commonly a bloody diarrhea. This is because Shiga toxin is usually taken in with contaminated food or water.

C. E. coli heat-stable toxin

Heat-stable enterotoxins (STs) producing Enterotoxigenic Escherichia coli  strains are ranked eighth among enteropathogens leading to diarrhea with mortality , accounting for 3.2% total diarrhea with mortality among all age groups,

common cause of acute diarrheal disease in both humans and farm animals, spread via fecal–oral transmission among hosts and several virulence factors, such as adhesins and enterotoxins, play an important role in its pathogenesis.

Heat-stable enterotoxins produced by ETEC are secreted peptides that can be divided in two types, STa and STb,  the STa enterotoxin is more relevant in diarrhea induction in humans, Enterotoxin STa is methanol soluble and protease resistant, while STb is methanol insoluble and protease sensitive. According to the host species, STa is further classified into two subtypes, known as STp and STh,

The release of STs into the small intestine aids binding to target receptors in the brush border membrane of the small intestinal epithelial cells, that activates intracellular signaling cascades, resulting in a disruption of the electrolyte homeostasis and finally leading to fluid secretion.

Heat-stable enterotoxin STa binds to the guanylate cyclase C receptor and activates its intracellular catalytic domain, further causes the hydrolysis of guanosine triphosphate (GTP) and accumulation of intracellular cyclic GMP (cGMP) levels. These increased cGMP levels activate cGMP-dependent protein kinase II (PKGII).

In addition, cGMP was shown to inhibit phosphodiesterase 3 (PDE3), leading to the activation of cAMP-dependent protein kinase A (PKA). Activated PKGII and PKA phosphorylate and open the cystic fibrosis transmembrane conductance regulator Cl− channel, inducing Cl− and HCO3− release into the intestinal lumen. Protein kinase A also phosphorylates the sodium/hydrogen exchanger 3 inhibits the Na+ reabsorption

D. Cholera toxin

The cholera toxin is an oligomeric complex made up of six protein subunits: a single copy of the A subunit enzymatic,  and five copies of the B subunit.

The B subunit ring of the cholera toxin binds to GM1 gangliosides on the surface of target cells. The B subunit can also bind to cells lacking GM1. The toxin then mostly binds to other types of glycans,

Once bound, the entire toxin complex is endocytosed by the cell and the cholera toxin A1 chain is released by the reduction of a disulfide bridge. The endosome is moved to the Golgi apparatus, where the A1 protein is recognized by the endoplasmic reticulum chaperone, protein disulfide isomerase. The A1 chain is then unfolded and delivered to the membrane, where Ero1 triggers the release of the A1 protein by oxidation of protein disulfide isomerase complex. As it moves from the ER into the cytoplasm by the Sec61 channel, it refolds and avoids deactivation.

A change in the shape of CTA1 exposes its active site and enables its catalytic activity. The CTA1 fragment catalyses ADP-ribosylation of the Gs alpha subunit (Gαs) proteins using NAD. The ADP-ribosylation causes the Gαs subunit to lose its catalytic activity of GTP hydrolysis into GDP + Pi, thus maintains Gαs in its activated state.

This leads to increased adenylate cyclase activity, which increases the intracellular concentration of 3',5'-cyclic AMP (cAMP) to more than 100-fold over normal and over-activates cytosolic PKA and further activates phosphorylate the cystic fibrosis transmembrane conductance regulator  chloride channel proteins, leads to ATP-mediated efflux of chloride ions and secretion of H2O, Na+, K+, and HCO3− into the intestinal lumen.

In addition, the entry of Na+ and consequently the entry of water into enterocytes are diminished. The combined effects result in rapid fluid loss from the intestine, up to 2 liters per hour, leading to severe dehydration and other factors associated with cholera, including a rice-water stool.

E. Superantigen

Superantigens bind first to the MHC class II and then coordinate to the variable alpha- or beta chain of T-cell Receptors  Binding to the α-chain puts the SAg in the appropriate position to coordinate to the TCR.  SAgs attach to the polymorphic MHC class II β-chain in an interaction mediated by a zinc ion coordination complex between three SAg residues and a highly conserved region of the HLA-DR β chain and activate a large proportion of the T-cell population.

Group I SAgs contact the Vβ at the CDR2 and framework region of the molecule. SAgs of Group II interact with the Vβ region using conformation dependent mechanism. These interactions are for the most part independent of specific Vβ amino acid side-chains. Group IV SAgs engages all three CDR loops of certain Vβ forms.

The interaction takes place in a cleft between the small and large domains of the SAg and allows the SAg to act as a wedge between the TCR and MHC. This displaces the antigenic peptide away from the TCR and circumvents the normal mechanism for T-cell activation.

The SAg cross-links the MHC and the TCR inducing a signaling pathway that results in the proliferation of the cell and production of cytokines.

the T-cells are stimulated and produce excess amounts of cytokine resulting in cytokine-mediated suppression of T-cells and deletion of the activated cells as the body returns to homeostasis. The toxic effects of the microbe and SAg also damage tissue and organ systems, a condition known as toxic shock syndrome. At initial stages it causes allergies and inflammation.

F. YopE

YopE has 4 alpha helices arranged in a left handed Four-helical up-and-down bundle. This bundle acts as the GAP domain, because arginine from an alpha helix is inserted into a GTP-ase which catalyses GTP hydrolysis through stabilisation of the transition state

It functions as a Rho GTPase-activating protein (GAP). YopE acts as both a virulence factor and a protective antigen. In order to evade detection by the host, YopE uses a number of different eukaryotic signalling pathways to counteract innate and adaptive immune responses of the host. YopE targets the small GTPases: RhoA, Rac1, and Rac2. YopE GAP activity inhibits two common methods of host immunity - phagocytosis and reactive oxygen species generation. Additionally, it is thought that YopE targets the following immune cells, mostly B lymphocytes, macrophages, dendritic cells and neutrophils.

It utilizes other invasive factors to aid its entry into host cells. requires invasive factors to enter into host cells, and during the later stages, it must produce antiphagocytic factors to escape uptake by host cells.

Adhesion of Psa to the surface of host cells facilitates the delivery of Yops into host cells, which further blocks the phagocytic ability of the host cells.

G. Listeriolysin O

Listeriolysin O (LLO) is a hemolysin produced by the bacterium Listeria monocytogenes, the pathogen responsible for causing listeriosis.

Listeriolysin O is a non-enzymatic, cytolytic, thiol-activated, cholesterol-dependent, pore-forming toxin protein; hence, it is activated by reducing agents and inhibited by oxidizing agents.

When gets activated within the acidic phagosomes ( pH ~ 5.9) of cells that have phagocytosed L. monocytogenes. After LLO lyses the phagosome, the bacterium escapes into the cytosol, where it can grow intracellularly. Upon release from the phagosome, the toxin has reduced activity in the more basic cytosol.

LLO permits L. monocytogenes to escape from phagosomes into the cytosol without damaging the plasma membrane of the infected cell. This allows the bacteria to live intracellularly, where they are protected from extracellular immune system factors such as the complement system and antibodies.

Also causes dephosphorylation of histone H3 and deacetylation of histone H4 during the early phases of infection, The alterations of the histones cause the down regulation of genes encoding proteins involved in the inflammatory response. So LLO may be important in subverting the host immune response to L. monocytogenes. Further protein degradation occurs