Bacterial Pathogenesis

Mechanism of Bacterial Pathogenesis and associated definitions

1. SAEEDA RIAZ M.Sc. MLT UHS, Lahore. Bacterial Pathogenesis(1)

2. KEY POINTS: Koch’s Postulates General Terms Used In Pathogenesis Virulence Factors and its examples Super Antigen Infection and its types Mechanism Of Pathogenesis Transmission Adherence Invasion and inflammation Toxin Production Immuno -pathogenesis

3. KOCH'S POSTULATES (MODIFIED) 1. The organism must always be found in humans with the infectious disease but not found in healthy ones. 2. The organism must be isolated from humans with the infectious disease and grown in pure culture. 3. The organism isolated in pure culture must initiate disease when re-inoculated into susceptible animals. 4. The organism should be re-isolated from the experimentally infected animals. Postulates 3. and 4. are extremely important in definite proof of the role of agent in human disease. However, this depends on the ability to develop animal models that resemble the human disease. In many cases such models do not exist.

4. General Terms Used In Pathogenesis: A pathogen is a microorganism that is able to cause disease in a plant, animal or insect. Pathogenicity is the ability to produce disease in a host organism. Microbes express their pathogenicity by means of their virulence. OR Pathogenesis is a multi-factorial process which depends on the immune status of the host, the nature of the species or strain (virulence factors) and the number of organisms in the initial exposure. virulence , a term which refers to the degree of pathogenicity of the microbe. Hence, the determinants of virulence of a pathogen are any of its genetic or biochemical or structural features that enable it to produce disease in a host .

5. Oppotunistic Pathogen: those that take advantage of certain situations usually do not cause disease in a healthy host, one with a healthy immune system . A compromised immune system, however, presents an "opportunity" for the pathogen to infect . Parasites: An organism that grows, feeds, and is sheltered on or in a different organism while contributing nothing to the survival of its host . Obligate Intra Celullar Parasite: cannot reproduce outside their host cell, meaning that the parasite's reproduction is entirely reliant on intracellular resources.e.g . Viruses and some bacteria i.e chlamydia and rickettesia .

6. Virulence factors : These are molecules expressed and secreted by pathogens (bacteria, viruses, fungi and protozoa) that enable them to achieve the following : colonization of a niche in the host (this includes adhesion to cells) Immuno evasion , evasion of the host's immune response Immunosuppression, inhibition of the host's immune response entry into and exit out of cells (if the pathogen is an intracellular one ) obtain nutrition from the host. Pathogens possess a wide array of virulence factors. Some are intrinsic to the bacteria (e.g. capsules and endotoxin) whereas others are obtained from plasmids (e.g. some toxins).

7. Examples Of Virulance Factors: Examples of virulence factors for Staphylococcus aureus are hyaluronidase , protease, coagulase, lipases, deoxyribonucleases and enterotoxins. Some examples of virulence factors for Streptococcus pyogenes are: M protein, lipoteichoic acid, hyaluronic acid capsule , invasins such as streptokinase , hyaluronidase , and streptolysins , and exotoxins adhesion factors , extracellular enzymes, toxins and antiphagocytic factors.

8. Super antigen: Superantigens ( SAgs ) are a class of antigens which cause non-specific activation of T-cells resulting in oligoclonal T cell activation and massive cytokine release. produced by pathogenic microbes (including viruses, mycoplasma, and bacteria ) 1. as a defense mechanism against the immune system. 2. Compared to a normal antigen-induced T-cell response where . 001-.0001% of the body’s T-cells are activated, capable of activating up to 20% of the body’s T-cells. Anti-CD3 and Anti-CD28 Antibodies have also shown to be highly potent superantigens (and can activate up to 100% of T cells).

9. 1. Bind to TCR and activate T cells 2. Autoimmune-like responses 3. S. aureus =>Toxic shock syndrome toxin S. pyogenes => Erythrogenic toxin A orC

10. Colonization Colonization occurs whenever any one or more species populate an area. derived from the Latin colere , "to inhabit, cultivate, originally related to human. Infection: Infection: The growth of a parasitic organism within the body.e.g . Bacteria A person with an infection has another organism (a "germ") growing within him, drawing its nourishment from the person.

11. Types Of Infections: Communicable Infection: An infection that can be transmitted from one individual to another either directly by contact or indirectly by fomites and vectors . Asymptomatic Infection: a disease is considered asymptomatic if a patient is a carrier for a disease or infection but experiences no symptoms . A condition might be asymptomatic if it fails to show the noticeable symptoms with which it is usually associated also called subclinical infections . The term clinically silent is also used.

12. S ymptomatic Infection: a disease is considered symptomatic if a patient is a carrier for a disease or infection and express symptoms . e.g. fever. P andemic infection: pandemic is an epidemic of infectious disease that is spreading through human populations across a large region; for instance multiple continents, or even worldwide. e.g. HIV E pidemic infection: an epidemic occurs when new cases of a certain disease, in a given human population, and during a given period. e .g. dengue in punjab . Endemic infection: Endemic infection Prevalent in or restricted to a particular region, community, or group of people . e.g. cholera

13. Infection Results In : 1.carrier state: A carrier state occurs when someone has been exposed to a pathogen (such as TB). A person may live as a carrier of a pathogen and would never know it unless tested antibodies against the pathogen.it lives in the host's body and can be passed on to others, without ever actually causing measurable harm to the host. 2.latent state: In a latent state, the virus is simply "at rest". person infected with the pathogen will show signs of the disease, at some time in the individual's life. common in many diseases, complimented by the "lytic" or active state of the pathogen/ disease.

14. Mechanism Of Bacterial Pathogenesis: Two broad qualities of pathogenic bacteria underlie the means by which they cause disease 1. Invasiveness: It is the ability to invade tissues. encompasses mechanisms for colonization (adherence and initial multiplication ) production of extracellular substances which facilitate invasion ( invasins ).

15. 2. Toxigenesis : It is the ability to produce toxins. Bacteria may produce two types of toxins called exotoxins and endotoxins . Exotoxins: These are released from bacterial cells and may act at tissue sites removed from the site of bacterial growth . Endotoxins: These are cell-associated substance. released from growing bacterial cells and cells that are lysed as a result of effective host defense (e.g. lysozyme) or the activities of certain antibiotics (e.g. penicillins and cephalosporins ).

16. Determinants Of Pathogenesis: 1.Transmission. 2.Adhesion. 3.Invasion and Inflammation. 4.Toxin Production. 5.Immunopathogenesis.

17. 1.TRANSMISSION Specific bacterial species (or strains within a species) initiate infection after being transmitted by different routes to specific sites in the human body. Four major routes of transmission: 1.Skin i.e. through cuts or wounds. 2.Gastro intestinal tract i.e. by ingestion of food or water. 3.Respiratory tract i.e.in airborne droplets. 4.Genital Tract i.e. through sexual contact. 1.TRANSMISSION:

18. Disease can be directly transmitted in two ways: Horizontal disease transmission – from one individual to another in the same generation (peers in the same age group ). Horizontal transmission can occur by either direct contact (licking, touching, biting ) indirect contact air – cough or sneeze (vectors or fomites that allow the transmission of disease without physical contact ). Vertical disease transmission – passing a disease causing agent vertically from parent to offspring , such as perinatal transmission .

19. Different Mode Of Pathogen Transmission

20. TRANSMISSION THROUGH AEROSOLS:

21. Adherence: The process or condition of attachment. Mechanisms of Adherence to Cell or Tissue Surfaces The mechanisms for adherence may involve two steps: 1. nonspecific adherence : reversible attachment of the bacterium to the eukaryotic surface (sometimes called "docking") 2. specific adherence : irreversible permanent attachment of the microorganism to the surface (sometimes called "anchoring").

22. Possible interactions and forces involved are: 1 . hydrophobic interactions 2. electrostatic attractions 3. atomic and molecular vibrations resulting from fluctuating dipoles of similar frequencies 4. Brownian movement 5. recruitment and trapping by biofilm polymers interacting with the bacterial glycocalyx (capsule )

23. Specific adherence involves complementary chemical interactions between the host cell or tissue surface and the bacterial surface. In the language of medical microbiologist, a bacterial " adhesin " attaches covalently to a host "receptor" so that the bacterium "docks" itself on the host surface. The adhesins of bacterial cells are chemical components of capsules, cell walls, pili or fimbriae. The host receptors are usually glycoproteins located on the cell membrane or tissue surface.

24. 2. INVASION and Inflammation: The invasion of a host by a pathogen may be aided by the production of bacterial extracellular substances which act against the host by breaking down primary or secondary defenses of the body. Medical microbiologists refer to these substances as invasins . Most invasins are proteins (enzymes) that act locally to damage host cells and/or have the immediate effect of facilitating the growth and spread of the pathogen . The damage to the host as a result of this invasive activity may become part of the pathology of an infectious disease.

25. Inflammation: A basic way in which the body reacts to infection, irritation or other injury, the key feature being redness, warmth, swelling and pain. a type of nonspecific immune response . 1.Pyogenic inflammation: Capable of generating pus . Neutrophils are predominant cells. Streptococcus , Staphocococcus , and bowel bacteria are the primary pyogenic organisms . 2.Granulomatous inflammation: usually chronic, marked by granuloma formation . Macrophages and T-cells are predominant. Most important is Mycobacterium Tuberculosis

26. The extracellular proteins produced by bacteria which promote their invasion are not clearly distinguished from some extracellular protein toxins ("exotoxins") which also damage the host. usually act at a short range (in the immediate vicinity of bacterial growth) and may not actually kill cells as part of their range of activity exotoxins are often cytotoxic and may act at remote sites (removed from the site of bacterial growth). Also, exotoxins typically are more specific and more potent in their activity than invasins. Even so, some classic exotoxins (e.g. diphtheria toxin, anthrax toxin) may play some role in colonization or invasion in the early stages of an infection, and some invasins (e.g. staphylococcal leukocidin) have a relatively specific cytopathic effect.

27. Bacterial Invasins : Spreading Factors: " Spreading Factors" is a descriptive term for a family of bacterial enzymes that affect the physical properties of tissue matrices and intercellular spaces, thereby promoting the spread of the pathogen. Hyaluronidase . is the original spreading factor. It is produced by streptococci. staphylococci, and clostridia. The enzyme attacks the interstitial cement ("ground substance") of connective tissue by depolymerizing hyaluronic acid.e.g . strept pyogenes . Collagenase: is produced by Clostridium histolyticum and Clostridium perfringens . It breaks down collagen, the framework of muscles, which facilitates gas gangrene due to these organisms.e.g . strept pyogenes .

28. Neuraminidase: is produced by intestinal pathogens such as Vibrio cholerae and Shigella dysenteriae . It degrades neuraminic acid (also called sialic acid), an intercellular cement of the epithelial cells of the intestinal mucosa. Streptokinase and staphylokinase : are produced by streptococci and staphylococci, respectively. Kinase enzymes convert inactive plasminogen to plasmin which digests fibrin and prevents clotting of the blood. The relative absence of fibrin in spreading bacterial lesions allows more rapid diffusion of the infectious bacteria.

29. Enzymes. that Cause Hemolysis and/or Leucolysis Phospholipases . produced by Clostridium perferingens (i.e., alpha toxin), hydrolyze phospholipids in cell membranes by removal of polar head groups. Lecithinases . also produced by Clostridium perferingens , destroy lecithin (phosphatidylcholine) in cell membranes . Hemolysins . notably produced by staphylococci (i.e., alpha toxin), streptococci (i.e., streptolysin ) and various clostridia, may be channel-forming proteins or phospholipases or lecithinases that destroy red blood cells and other cells (i.e., phagocytes) by lysis. Coagulase. Coagulase, formed by Staphylococcus aureus , is a cell-associated and diffusible enzyme that converts fibrinogen to fibrin which causes clotting.

30. Invasin Bacteria Involved Activity Hyaluronidase Streptococci, staphylococci and clostridia Degrades hyaluronic of connective tissue Collagenase Clostridium species Dissolves collagen framework of muscles Neuraminidase Vibrio cholerae and   Shigella dysenteriae Degrades neuraminic acid of intestinal mucosa Coagulase Staphylococcus aureus Converts fibrinogen to fibrin which causes clotting Some Extra Cellular Bacterial Proteins That Act As Invasins:

31. Kinases Staphylococci and streptococci Converts plasminogen to plasmin which digests fibrin Leukocidin Staphylococcus aureus Disrupts neutrophil membranes and causes discharge of lysosomal granules Streptolysin Streptococcus pyogenes Repels phagocytes and disrupts phagocyte membrane and causes discharge of lysosomal granules Hemolysins Streptococci, staphylococci and clostridia  Phospholipases or lecithinases that destroy red blood cells (and other cells) by lysis Lecithinases Clostridium perfringens Destroy lecithin in cell membranes

32. Phospholipases  Clostridium perfringens Phospholipases Clostridium perfringens Destroy phospholipids in cell membrane. Anthrax EF  Bacillus anthracis Anthrax EF Bacillus anthracis One component (EF) is an adenylate cyclase which causes increased levels of intracellular cyclic AMP Pertussis AC Bordetella pertussis Pertussis AC Bordetella pertussis One toxin component is an adenylate cyclase that acts locally producing an increase in intracellular cyclic AMP .

33. EXOTOXINS: Polypeptide in nature. Secreted by both gram positive and gram negative species. Induce high titer antibodies called antitoxins. Most toxic substance known. Toxoid used as vaccine. Can be divided into three categories: Cytotoxins Neurotoxins Enterotoxins

34. Exotoxins: Many bacteria secrete proteins (exotoxins) that modify, by enzymatic action, or otherwise destroy certain cellular structures . Effects of exotoxins are usually seen acutely, since they are sufficiently potent that serious effects (e.g. death) often result. Examples of this are botulism, anthrax, cholera and diphtheria. Classes of exotoxins include: Toxins that act on the extracellular matrix of connective tissue. e.g . Clostridium perfringens collagenase, Staphylococcus aureus hyaluronidase .

35. Toxins that have a cell binding "B" component and an active "A" enzymatic component (A-B type toxins) These include: a) Those with ADP-ribosylating activity e.g. cholera toxin, E. coli heat labile toxin, Pseudomonas aeruginosa and diphtheria toxins. b) Those with a lytic activity on 28S rRNA e.g. shiga and shiga-like (vero) toxins. c) Those with a partially characterized site of action e.g. botulinum toxin, tetanus toxin and anthrax lethal toxin. Membrane Damaging Toxins e.g. Staphylococcus aureus delta toxin

36. Toxins which act extracellularly . These include proteases, collagenases and hyaluronidases . For example, Clostridium perfringens produces a potent collagenase, whilst Staphylococcus aureus produces a hyaluronidase . Damage to the connective tissue matrix (by hyaluronidase and collagenase) can "loosen up" the tissue fibers allowing the organism to spread through the tissues more readily. Also included in this group is the exfoliatin of Staphylococcus aureus which causes separation of the layers within the epidermis and is the causative agent of scalded skin syndrome in the newborn. A - B Toxins . Such toxins consist of two components. One binds to cell surfaces and the other passes into the cell membrane or cytoplasm where it acts. The classical toxins demonstrated to act in this fashion are those of cholera and diphtheria.

37. ( i ) ADP- ribosylating exotoxins Diphtheria toxin (produced by Corynebacterium diphtheriae ). The toxin is synthesized as one polypeptide chain and readily nicked into two chains held together by a disulfide bond. B binds to cells and A has the enzymatic activity. A is endocytosed and from the endosome passes into the cytosol. Diphtheria toxin ADP- ribosylates elongation factor (EF2) in ribosomes, thus inhibiting protein synthesis. Pseudomonas exotoxin A has an similar mode of action to diphtheria toxin. Cholera toxin has several subunits which form a ring with one A subunit inserted in the center. B binds to gangliosides on the cell surface and appear to provide a channel through which A penetrates. after internalization ADP- ribosylates a cell membrane regulator complex (using NADH as a substrate), in turn causing increase activation of adenylate cyclase . Activation of adenylate cyclase causes an increase in cyclic AMP production with resulting decrease in electrolytes

38. (iii) Partially characterized site of action Botulinum neurotoxins, tetanospasmin and the lethal toxin of B. anthracis appear to be A-B type exotoxins. Botulinum toxin acts by causing inhibition of release of acetylcholine at the neuromuscular junction. Tetanus toxin is taken up at neuromuscular junctions and transported in axons to synapses. Botulinal exotoxin, produced by Clostridium botulinum .This causes a flaccid paralysis , a weakening of the involved muscles. Death is usually from respiratory failure. While two exotoxins of C. botulinum catalyze ADP- ribosylation of host cell proteins, the botulinal toxin that affects neurons does not. Since the botulinal toxin is able to cause a weakening of muscles, it is now being used therapeutically to treat certain neurologic disorders such as dystonia and achalasia that result in abnormal sustained muscle contractions, as well as a treatment to remove facial lines.

39. Tetanus Toxins: Tetanus exotoxin ( tetanospasmin ), produced by Clostridium tetani . This is a neurotoxin that binds to inhibitory interneurons of the spinal cord and blocks their release of inhibitor molecules. The toxin, by blocking the release of inhibitors, keeps the involved muscles in a state of contraction and leads to spastic paralysis , a condition where opposing flexor and extensor muscles simultaneously contract. Death is usually from respiratory failure.

40. Membrane Damaging Toxins: These toxins enzymatically digest the phospholipid (or protein) components of membranes or behave as detergents. In each case holes are punched in the cell membrane and the cytoplasmic contents can leach out. The phospholipase ("toxin") of C. perfringens is an example of a membrane damaging toxin. It destroys blood vessels . This also helps create an anaerobic environment which is important in the growth of this strict anaerobe

41. Endotoxins Endotoxins are toxic components of the bacterial cell. Lipopolysaccharide in nature . peptidoglycan displays many endotoxin-like properties. Endotoxins are "non-specific" inciters of inflammation. For example, cells of the immune system and elsewhere are stimulated to release cytokines (including interleukin 1 and tumor necrosis factor ). LIPID A: causes septic shock involves hypotension (due to tissue pooling of fluids), disseminated intravascular coagulation and fever and is often fatal from massive system failure. This includes lack of effective oxygenation of sensitive tissues such as the brain. There is no effective therapy to reverse the toxic activity of lipid A or peptidoglycan in patients.

42. Endotoxins also activate the alternate complement pathway. cytokines production results in attraction of polymorphonuclear cells into affected tissues . PG and LPS and certain other cell wall components (e.g. pneumococcal teichoic acid) are also activators of the alternate complement cascade Endotoxins are also potent polyclonal B cell activators and adjuvants (for both antibodies and cell mediated immunity); this plays a role in the development of a suitable chronic immune response in handling the microbes if they are not eliminated acutely. In a "primary" infection during the acute phase "non-antigen specific" immunity will be of utmost importance in eradicating the infection. important in chronic infections such as tuberculosis, leprosy, Lyme disease and syphilis

43. Basic structure of endotoxin (lipopolysaccharide) from Gram-negative bacteria

44. Immunopathology: can occur in acute and chronic infections. Over stimulation of cytokine production and complement activation by endotoxins can cause tissue injury in the absence of an immune response . Continuously generated antigens released from persisting viable microbes will subsequently elicit humoral antibodies and cell mediated immunity resulting in chronic immunopathology . Certain poorly degradable antigens ( e.g pneumococcal polysaccharide and group A streptococcal cell walls) can maintain immunopathology