Salmonella toxin with their toxic effects

Salmonella toxins, produced by the bacterium Salmonella, play a crucial role in the pathogenesis of infections caused by this pathogen. These toxins primarily include enterotoxins, which disrupt the normal function of the intestines by increasing fluid secretion and causing diarrhea, and cytotoxins, which damage host cells and contribute to inflammation. The resulting symptoms can range from mild gastroenteritis, characterized by abdominal cramps and diarrhea, to severe systemic infections, especially in cases caused by strains like Salmonella Typhi. The toxins not only cause direct harm to the gastrointestinal tract but can also lead to more serious health complications if the bacteria enter the bloodstream. Effective management and prevention of Salmonella infections involve good food handling practices and, in some cases, vaccination

1. Characterization of Toxin Produced by Salmonella spp. Dr. Rony Ibne Masud MS in Microbiology Department of Microbiology and Hygiene Bangladesh Agricultural University

2. Introduction Rod-shaped, gram-negative bacteria Family Enterobacteriaceae Non-spore-forming, Predominantly motile enterobacteria Cell diameters 0.7 and 1.5 μm , Lengths 2 to 5 μm Peritrichous flagella Chemotrophs, obtain their energy from oxidation and reduction reactions Facultative anaerobes, capable of generating adenosine triphosphate with oxygen

3. Classification:

4. Toxin produced by salmonella spp Salmonella toxin Endotoxin Exotoxin Cytotoxin Cell wall Lipopolysaccharide A toxin is a naturally occurring organic poison produced by metabolic activities of living cells or organisms. They occur especially as proteins, often conjugated Salmolysin Typhoid toxin Enterotoxin Cytolethal distending toxin ADP Ribosylating toxin

5. Toxin produced by different salmonella spp Toxin name Types Species Typhoid toxin Exotoxin Salmonella typhi Salmonella paratyphi Cytolethal distending toxin Exotoxin Salmonella typhi Salmonella paratyphi Enterotoxin Exotoxin Salmonella enteritidis Salmonella typhimarium Hemolysin Exotoxin Salmonella spp. ADP Ribosylating toxin Exotoxin Salmonella enterica sub spp Lipopolysacchride Endotoxin Salmonella spp.

6. Typhoid toxin Typhoid toxin belongs to the family of bacterial AB toxins consisting of an “A” enzymatic subunit and a “B” receptor binding subunit Length of approximately 90 Å and a width of approximately 60 Å, Demonstrates an unprecedented A 2 B 5 organization with two covalently linked A subunits, CdtB and PltA , non-covalently associated with a pentameric B subunit PltB The evolution of uniquely positioned Cys residues in both CdtB and PltA has resulted in the adaptation of CdtB for its tethering to the PltA-PltB complex

7. MOA of Typhoid toxin Secretion: Typhoid toxin is secreted by Salmonella Typhi bacteria after they invade the host's intestinal epithelial cells. It is a protein toxin consisting of two subunits: the enzymatic subunit, called the A subunit, and the binding subunit, called the B subunit Cellular binding and internalization: The B subunit of typhoid toxin binds to specific receptors on the surface of host cells, facilitating its internalization into the target cells. Intracellular trafficking: Once inside the host cell, the toxin undergoes intracellular trafficking, ultimately reaching the endoplasmic reticulum (ER), a cellular organelle involved in protein synthesis and processing

8. Cont. A subunit translocation: The A subunit of typhoid toxin is transported from the ER into the cytoplasm of the host cell. This transport involves the enzymatic activity of the A subunit itself, which acts as an ADP- Ribosyltransferase ADP-ribosylation: In the cytoplasm, the A subunit of typhoid toxin ADP- Ribosylates specific target proteins. ADP-ribosylation is a post-translational modification that involves adding ADP-ribose groups to target proteins, thereby disrupting their normal function. Typhoid toxin primarily targets and modifies host proteins involved in cellular signaling pathways that are crucial for immune responses.

9. Toxic effects Inhibition of protein synthesis: D isrupt the host cell's protein synthesis machinery, primarily by targeting the ribosome. This inhibition of protein synthesis can lead to cell damage and dysfunction. Disruption of host cell signaling: The toxin ADP- ribosylates specific host proteins involved in cellular signaling pathways. By modifying these proteins, typhoid toxin interferes with normal cellular processes, including signaling cascades that regulate inflammation and immune responses Immune suppression: inhibits the production of proinflammatory cytokines, such as interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α). This suppression of the host immune response impairs the body's ability to recognize and respond infection Apoptosis: Typhoid toxin can induce apoptosis, which is programmed cell death, in certain host cell types. This can contribute to tissue damage and the spread of infection.

10. Cont. Disruption of the intestinal barrier: A ffect the integrity of the intestinal epithelial barrier, making it more permeable. This can lead to the translocation of bacteria and toxins from the gut into the bloodstream, potentially causing systemic effects Vascular leakage: The toxin can cause increased vascular permeability, leading to fluid leakage from blood vessels into tissues. This can contribute to symptoms such as fever, diarrhea, and dehydration seen in typhoid fever. Systemic effects: Typhoid toxin's actions on host cells and immune responses can lead to systemic effects, including fever, malaise, abdominal pain, and other symptoms characteristic of typhoid fever Inhibition of neutrophil function: Neutrophils are a type of immune cell that plays a crucial role in the defense against bacterial infections. Typhoid toxin impairs the function of neutrophils, reducing their ability to phagocytose (engulf) and kill bacteria

11. Cytolethal distending toxin CDT is a protein toxin composed of CdtA , CdtB , and CdtC . CdtB is the catalytic subunit responsible for the toxicity The molecular weight of CdtB , the catalytic subunit, is approximately 28-30 kDa . CdtB is an endonuclease and is responsible for the DNA-damaging activity of CDT. CdtA and CdtC are believed to play roles in binding to host cells and facilitating the delivery of CdtB into the host cell. CDT exerts its toxic effects by causing DNA damage within host cells. DNA damage leads to cell cycle arrest, primarily in the G2 phase, which results in cell death.

12. Mechanism of action Delivery Endonuclease activity Transfer DNA damage Cell cycle arrest Cellular distention Cell death

13. Toxic effects

14. Enterotoxin Small protein and its structure consists of a single polypeptide chain. It has a three-dimensional structure stabilized by disulfide bonds The molecular weight of Salmonella enterotoxin varies depending on the specific strain but is typically in the range of 17-21 kDa . Heat-stable Primarily exerts its toxic effects by binding to receptors on the surface of intestinal epithelial cells in the host's small intestine.

15. Mechanism of action Binding Ion transport alteration cGMP elevation Fluid secretion Cell cycle arrest Severe diarrhea

16. Toxic effects Diarrhea Dehydration Electrolyte imbalance Abdominal cramp Nausea Vomiting Malabsorption Inflammatory response

17. Endotoxin Molecular Weight: LPS molecules can be quite large, often exceeding 10,000 Daltons Structure : composed of three central regions: the lipid A, the core oligosaccharide, and the O-antigen polysaccharide. Amphiphilic Nature : LPS molecules are amphiphilic, meaning they have both hydrophilic (water-attracting) and hydrophobic (water-repellent) regions Pyrogenicity : a potent pyrogen that can induce fever when introduced into the bloodstream. It activates immune cells, such as macrophages, to release pro-inflammatory cytokines, leading to fever Immunogenicity : LPS molecules can be recognized by the host immune system, leading to the production of antibodies against Salmonella LPS Heat Stability : LPS molecules are heat-stable, meaning they can withstand exposure to high temperatures Solubility : The solubility of LPS in water can vary depending on the specific structure of the molecule and the presence of divalent cations. In the absence of divalent cations, LPS can form aggregates and be less soluble

18. Structure of endotoxin

19. Mechanism of action Recognition Endonuclease activity Binding to TLR-4 Activation of NF- κ B: Cytokine Production Activation Immune Cells Complement Activation

20. Toxic effects Systemic Inflammation Septic Shock Vascular Changes Multiple Organ Dysfunction Syndrome Disseminated Intravascular Coagulation Endotoxemic Shock Organ-Specific Effects Neurological Effects Renal Dysfunction

21. Isolation and Purification of Endotoxin Ammonium sulfate precipitation method of toxin purification

22. Hot phenol water method Purification of bacterial isolates LPS extraction and purification Detection of impuritues Bacterial suspension centrifuged: 10000*g- 5 min Pellets were washed twice with PBS Resuspended at 10 ml PBS and sonicated for 10 minutes Treatment with proteinase k, DNase, RNase

23. Cont. Incubate overnight at 37 Suspension cooled on ice and transferred to polypropylene tube Hot(65-70) phenol addition(90%) in same volume and shaking Centrifuged at 8500*g for 5 minutes Supernatants were collected Reextracted by 3000 micro ml distilled water Stored -20 C overnight

24. Cont. Again centrifuged at 2000*g for 15 minutes Extensive dialysis with double distilled water to eliminate phenols Pellets were resuspended in 1 ml distilled water Finally LPS producted were lyophilized and stored at 4 C

25. Cont. Confirmation is done by Silver comassic blue and ethidium bromide stain High-performance liquid chromatography Limulus amebocyte lysate assay Rabbit pyrogen test

26. Westphal method of purification Lyophilized salmonella cell stabilized in buffer solution Enzyme inactivation at 75C for 10 minutes Treated with Bovine serum albumin Treated with10 micro ml trypsin for 90 minutes at 37C Enzyme inactivation repeated Centrifuge at 30000*g for 1 hour Pellets were incubated 100 ml acetone at 37C

27. Cont. Centrifuge at 1500 *g for 15 Minutes Cell suspension centrifuge at 1500 *g for 20 Minutes Extracted with chloroform-methanol at 20 C overnight Suspension preheated with distilled water Extracted equal volume of phenol water for 20 minutes centrifuge at 1500 *g for 20 Minutes The phenol phase was reextracted with water, dialyzed against distilled water for three days and freeze-dried

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