Microbial Pathogenesis
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1 Microbial Pathogenesis September 15, 2008 by David E. Briles Whether a pathogen is able to cause symptoms (disease) is dependent on the interaction of the pathogen with the host. This interaction is called Microbial Pathogenesis. 1
2 Microbial Pathogenesis Innate immunity & antigen-specific adaptive immunity Protect the host from pathogens and tumors Microbial virulence mechanisms (mediated by virulence factors) Enable the pathogen to combat host immunity Understanding Microbial Pathogenesis leads to: New anti-microbial drugs (antibiotics) New and improved vaccines Use of cytokines to stimulate innate immunity Better supportive therapy New preventive measures 2
3 Antibiotics Originally from natural products (selected in nature from the competition between microbes). Antibiotics block essential microbial-specific functions. (DNA replication, protein synthesis, cell wall synthesis etc.) Antibiotic resistance is overcome by mutations and gene exchange leading to new variants of the original bacterial genes. Future antibiotics will exploit new bacterial targets. In vivo studies are conducted by Administering of drugs, cytokines, antibodies or gene product that target specific bacterial or host processes. Genetic knock-out mutants in, the host (effector molecules and receptors) the pathogen (virulence factors) 3
4 Adapted to Man No Classification of Microbes (with respect to human health) Type Properties Does not colonize or infect man Free living or in other hosts No Yes Yes Opportunistic Normal flora Disease Producing, Maintained by a. colonization b. infection Causes disease in immune compromised a. infants b. elderly c. sick individuals d. immune deficient e. AIDS patients Commensal or symbiotic -- cause no symptoms Can cause clinical symptoms & sometimes death a. occasionally cause disease b. always cause disease Reside in: Normal Flora gut, especially the large intestine oral cavity upper airways genital tract urinary tract skin 4
5 Normal Flora Cause no clinical symptoms In some cases they are beneficial: produce some vitamins (especially vitamine K) exclude pathogens by occupying available niches Attributes of a Pathogenic Microbe Acquisition by a host Exploitation of a niche environment Usually this niche is in sterile tissue Partially evade host defenses Multiplication in the host Produce disease symptoms Transmission to others 5
6 The evolutionary success of a pathogen is measured by its ability to infect or colonize additional hosts, not in its ability to kill. All pathogens have developed ways to be transferred to others, and most have evolved to keep the host alive for a long time to maximize their ability to pass to other hosts. 6
7 Some pathogens such as HIV, which causes AIDS, do not have to be passed efficiently. They have figured out how to remain in the host a long time to maximize there chance to be passed to others before the original host dies. Pathogens such as Vibrio cholera (or Anthrax) don t mind killing their host because in doing so they produce enough bacteria in a watery diarrhea (or spores on the savanna) to guaranty transmission to a new host. 7
8 From the Perspective of the Host: Host Defense Mechanisms Mechanical barriers: skin epithelial cells mucus cilia stomach acid, etc. Phagocytes: macrophage kuffer cell neutrophil Complement C-reactive protein Major host defenses Antibody IgA, IgM, IgG, IgE T cells CD4 CD8 NK cells Cytokines Interferon!, ", # TNF-!, " IL-6 etc. 8
9 Relative Concentration of Selected Immune Mediators Mediator 2!g/ml Specific Antibody 2ng/ml Specific Antibody Conc./ml blood molecules molecules PMN 5 x 106 Mononuclear cells 1 x 106 CD4 T cells (antigen specific) CD8 T cells (antigen specific) 2 x 106 ( ) 2 x 106 ( ) Phagocytes (PMN & Macrophages) Ingest and destroy microbes Find micobes by: Chance encounters Chemotaxis bacterial breakdown products (N-formyl methionine) complement fragments (C5a) leukotriene from stimulated lymphocytes Recognize microbes by molecules on their surface Deposited complement (ic3b) Antibody Fc Repeating structures on microbe surfaces (manosebinding lectin) 9
10 Protective Action of Antibody Alone blocks adherence blocks toxin activity blocks enzymatic activity In conjunction with fixed complement aggregates and opsonizes toxins aggregates and opsonizes microbes blocks enzymatic activity lyses host cells bearing bacterial or viral antigens (containing bacteria or viruses) lyses gram negative bacteria Complement role in opsonophagocytosis C3 is Activated Through the alternative pathway (factor B) by many microbial surfaces. (Extracellular pathogens generally inhibit this pathway). Through the classical pathway (C1q) by IgG and IgM antibody bound to pathogen surfaces. Through the manose pathway, where manose is exposed on the bacterial surface. C3 activation and surface deposition Mediates chemotaxis (C5a) opsonization (ic3b) lysis (membrane attack complex (C5b, C6, C7, C8, C9). 10
11 NK cells Lyse host cells identified by: IgG (ADCC) -- kills cells infected with virus and bacteria tumor antigens -- tumor surveillance Significant producer of!-interferon Cell mediated immunity -- intracellular bacteria and viruses Interferons Interfere with viral replication and enhance cell-mediated immunity (by activating monocytes and lymphocytes) 11
12 ! interferon -- produced by immune T cells " & # interferons -- produced by infected cells Helper (CD4) T cells: A. Activate (help) B cells to make antibody B. Mediate cell mediated immunity 1. Enhance production of cytotoxic T cells 2. Activate macrophages to kill intracellular bacteria. 12
13 Cell Mediated Immunity, augments macrophage function. destroys intracellular pathogens and the host tissues containing them. Inflammation, enhances microbial killing at the expense of host tissue. 13
14 Inflammation is mediated by, microbial products (LPS, cell wall, some toxins) antibody and complement cell mediated immunity Two types of pathogenic bacteria Extracellular -- replicate outside of cells and must therefore avoid being killed by phagocytes or complement. Intracellular -- replicate inside cells and must therefore avoid being killed by phagocytosis and the antibacterial properties of lysozomes. 14
15 Extracellular Bacteria Host Resistance mediated by: Antibody Complement opsonization of all bacteria lysis of gram negative only Phagocytes Intracellular Bacteria Resistance mediated by: TH1 cells Macrophages CD8 T cells cells NK cells (probably) 15
16 Viruses Resistance mediated by: antibody - to block extracellular spread interferon - to block intracellular growth NK and CD8 cells - to kill virus producing cells From the Perspective of the Pathogen 16
17 The properties that allow pathogens to survive, spread, and cause disease are called Virulence Factors Virulence Properties Exploiting a particular niche Adherence to host tissues Escape from host immunity Protects it self from Ab and C' attack Invasion into cells & avoidance of degredation in lysozomes Interference with host and/or adaptive immunity Live in protected tissue sites Means of acquiring nutrients Enzymes, siderophores, and transport machinery Mechanism for transmission to the next host 17
18 Streptococcus pneumoniae Streptococcus mutans Helicobacter pylori Listeria and Salmonella Salmonella (typhoid) E. coli Group B streptococci Mycobacterium tuberculosis HIV Finding a Niche Asymptomatic colonization of upper airways. Infects cavities (lung, ear, spinal fluid, eye, etc.) Adheres to glucan layer it applies to teeth Lives in mucus lining of the stomach Survive in phagocytes Survive in bile and infect liver and spleen Colonize gut, adhere to surface of bladder and ureter Asymptomatic colonization of gut, but can infect and kill newborn infants Grows slowly to minimize elicitation of host immunity Grows in and kills CD4 T-cells, thus eliminating host anti-viral response Adherence by many pathogens is important because, 1. It prevents them from being cleared by mucus flow. 2. It is necessary to subsequent to tissue and cellular invasion. 3. Presence on the mucosa is frequently critical for transmission. 18
19 Critical adhesion targets of some pathogens Pathogen Influenzae virus Mycoplasma pneumoniae Streptococcus mutans Neisseria gonorrhoeae Escherichia coli Malaria (Plasmodium vivax) Giardia lamblia Host Tissue Respiratory epithelium Respiratory epithelium Tooth enamel Uretheral epithelium Intestinal epithelium urinary tract epithelium erythrocytes Duodenum & jejunum Attachment site on host cell Neuraminic acid Neuraminic acid Bacterial glucan Carbohydrate on host cells D-mannose Duffy blood group antigen Mannose-6-phosphate Spread of Pathogens Pathogens Mechanism Salmonella Cholera Streptococcus mutans Rhinoviruses Syphilis, Chlamydia, Gonococci, HIV Yersinia pestis, Lime disease, St. Louis encephalitis virus & West Nile virus Rabies Spread in feces, survives in water Induces tremendous volumes of diarrhea, Survives in water Saliva Sneezing dripping noses Sex Fleas & Rats Ticks & Deer Mosquitoes Animal bites 19
20 Bacterial toxins Exotoxins -- secreted molecules that can kill, damage, or alter host cells Endotoxins -- Lipopolysaccharides (LPS) from the outer membrane of gram negative bacteria. Some Toxins that Contribute to Pathogenesis Type of Toxin Name of Toxin Function A-B (B subunit targets cells, A subunit enters and kills) A-B (B subunit targets cells, A subunit enters and acts) Membrane disrupting Diphtheria toxin Cholera toxin Listeriolysin Pneumolysin ADP-ribosylation of host elongation factor 2. Stops protein synthesis ADP-ribosylation of host regulatory protein. Leads to massive fluid loss. Escape from phagosomes Non-protective inflammation Superantigen toxic shock toxin Non-protective inflammation Bacterial products Cell wall + others Inflammation and necrosis 20
21 The Host Controls Extracellular Bacterial Growth by Limiting Availability of Iron Host Sequesters iron via, 1. Transferrin -- transports and sequesters iron for host use 2. Lactoferrin -- sequesters iron from pathogens and host 3. Fever -- increases the requirement of bacteria for iron Bacteria get iron via Siderophores (secreted molecules that bind iron ions) Transport of siderophore+fe +++ Cytoplasmic degredation siderophore-fe +++ to release Fe +++ Transport of iron-binding transferrin, lactoferrin or heme. Growing intracellularly (where iron is more readily available) 21
22 Evasion of Phagocytosis Structure Mechanism Pathogens Polysaccharide anti-complementary Streptococcus pneumoniae capsule antigenically unique Neisseria meningitidis Haemophilus influenzae M-protein anti-complementary Streptococcus pyogenes antigenically unique PspA & PspC anti-complementary Streptococcus pneumoniae Antigenic variation Pilin PII LOS create new antigens Neisseria gonorrhoeae Pathogens Evasion of Intracellular Killing Mycobacterium tuberculosis Mechanism Glycolipid cell wall resists nonactivated macrophages. Slow growth fails to elicit strong Cell Mediated Immunity. Listeria monocytogenes Listeriolysin - escape to cytoplasm Actin tails - drive it into adjacent cells Rickettsiae Escape to cytoplasm Salmonella Prevents lysosome-phagosome fusion 22
23 Regulation of Virulence Factors in Response to Host Conditions Temperature Carbon source Concentration of Iron, Manganese, or Calcium Osmolarity ph Stress Vaccines Elicitation of Protective Immune Response by Host. - Antibody - Cell Mediated Immunity 23
24 Vaccine Approaches Antibody Immunize with toxins (cholera, diphtheria, tetanus etc.) Immunize with adhesions (flu) Immunize with unique surface components (pneumococcus) Cell Mediated Immunity Intracellular bacteria and viruses Polysaccharides are poorly immunogenic in the children less than two years of age. 24
25 Protein-Polysaccharide Conjugates To elicit a T-cell help dependent antipolysaccharide response Boostable response Higher titer response To permit immunization of young children To better immunize the elderly Polysaccharide-protein conjugate vaccines are available for group B Haemophilus influenzae Streptococcus pneumoniae (7 capsular types) Groups A and C Neisseria meningitidis. 25
26 Inclusion of cytokines and immune modulators with antigen To enhance immune responses To focus immune responses on a. humoral b. cell mediated To focus immune responses on a. TH1 b. TH2 Mucosal Immunization elicits systemic and mucosal immunity avoids giving a shot adjuvants for human use are still needed Mucosal Immunity can block colonization and transmission (herd immunity) prevent invasion block local action of toxins 26
27 Thanks for your attention If you have questions, or Learn and understand concepts Elements of Microbial Pathogenesis 1. Permissive Host / Virulent Pathogen 2. Pathogen Evades Host Defenses 3. Pathogen Multiplies and Spreads to others 4. Disease/Symptoms/Inflammation 27
28 Identify roles of immunity mediated by Complement and T-cells for two disease models in mice Salmonella infection (mouse typhoid) Pneumococcal infection of the blood Eliminate complement C3 with Cobra Venom factor No Effect Increased susceptibility Eliminate T-cells with mutation Increased susceptibility No effect Pathogenesis involves complex in vivo events Results obtained in vitro (outside the body) must be verified by in vivo studies (inside the body) 28
29 Enterobactin (an example of a virulence factor) Enterobactin is an iron chelating siderophore released by enteric bacteria to bind iron 3+ and deliver it to the pathogen. Mutants unable to make enterobactin do not grow in the gut. Repair of mutation, or feeding enterobactin will permit growth. 29
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