260 summer 2016 pathogenesis

Clicker questions about viral replication Which viruses can use the host’s replication enzymes to make new genomes Which viruses have a genome that can be directly used as an mRNA Which viruses require a RNA-dep RNA pol Which viruses need to carry the RNA-dep RNA pol in the capsid Reverse transcriptase uses a ____ template to make ____ A = DNA viruses B = all RNA viruses C = all RNA viruses except not retroviruses D=retroviruses only A = DNA  DNA B = DNA  RNA C = RNA  DNA C = RNA  RNA A = dsDNA virus B = dsRNA virus C = “+”sense RNA D = “-”sense RNA E= retrovirus

About PCR Compare replication to PCR 1. list the things you need (reagents, enzymes) for replication and in what order 2. list the things you put into your PCR tube (reagents, enzymes). 3. compare – for the things in replication that are missing in the PCR tube, what did you add to fill that missing role?

The study of disease Pathogenesis – the study of disease progression Next class – epidemiology we’ll study disease again but from a public health perspective

Pathogenesis (chapter 16) Principles of disease aka The Chapter of Many Definitions

Why do care? What specific agent causes disease How does the agent cause disease in the body How infectious agents are spread

A brief history Diseases and plagues attributed to supernatural forces, incurable and terrifying Observation: disease seems to be communicable 1600s there are microbes 1800s Microbes cause disease Robert Koch (German physician 1843-1910) – systematic proof of causative agent

Koch’s Postulates The same pathogen must be present in every case of the disease The pathogen must be isolated from the diseased host and grown in pure culture The pathogen from the pure culture must cause the disease when it is inoculated into a healthy, susceptible lab animal The pathogen must be isolated from the inoculated animal and must be shown to be the original organism

Koch’s Postulates Obtain pure culture of the organism Pure culture used to infect healthy lab animal causes the disease Same organism is present in every case of the disease Figure 14.3

Koch’s Postulates Organism cultured from infected animal is identical to original infection Figure 14.3

Koch’s Postulates Koch's postulates can be used to prove the cause of an infectious disease.. But can Koch’s postulates be used to identify the agent/microbe responsible for all diseases? Some pathogens can cause several disease conditions Some pathogens cause disease only in humans Some pathogens are not easily cultivated

Pathology, Infection, and Disease Pathology: The study of disease Etiology: The study of the cause of a disease Pathogenesis: The development of disease Infection: Colonization of the body by pathogens Disease: An abnormal state in which the body is not functioning normally

Normal Microbiota and the Host Transient microbiota may be present for days, weeks, or months then disappear Normal microbiota permanently colonize the host Symbiosis is the relationship between normal microbiota and the host

Representative Normal Microbiota Figure 14.1

Is this bad news?

Symbiosis In commensalism, one organism benefits, and the other is unaffected In mutualism, both organisms benefit In parasitism, one organism benefits at the expense of the other Some normal microbiota are opportunistic pathogens such as?

Normal Microbiota on the Human Body Table 14.1

What is the role of normal microbiota?

Normal Microbiota and the Host Microbial antagonism is a competition between microbes. Normal microbiota protect the host by Occupying niches that pathogens might occupy Producing waste eg. acids Producing bacteriocins Probiotics: Live microbes applied to or ingested into the body, intended to exert a beneficial effect

Effect of antibiotics Why are you at risk of a yeast infection or bacterial GI illness after taking antibiotics?

Principles of disease Symptom: A subjective change in body function that is felt by a patient as a result of disease Sign: An objective change in a body that can be measured or observed as a result of disease Syndrome: A specific group of signs and symptoms that accompany a disease

For example You catch a respiratory virus You feel tired, have a headache, your sinuses itch, you have a sore throat The doctor finds swollen lymph nodes, notes an elevated body temperature and views redness in your throat and nasal passages What are the symptoms, signs How are the signs and symptoms related to the concept of a syndrome

The Stages of a Disease Figure 14.5

Development of disease Incubation period – from transmission to first symptoms Prodromal period – early mild symptoms Period of illness – period of severe symptoms Period of decline – number of pathogens declines (may overlap with severe symptoms) Period of convalescence (numbers continue to drop; mild or no symptoms)

For example 10/8 – your partner sneezes on you 10/10 – sore throat 10/12 – liquid goo phase, sneezing, fever 10/17 – back to salsa dancing! Give the dates of the incubation period, prodromal period and period of illness

Severity or Duration of a Disease Acute disease: Symptoms develop rapidly Chronic disease: Disease develops slowly Subacute disease: Symptoms in between acute and chronic Subclinical disease: infection without noticeable symptoms Latent disease: Disease with a period of no symptoms when the causative agent is inactive

Order of disease Primary infection: Acute infection that causes the initial illness Secondary infection: Opportunistic infection after a primary (predisposing) infection

For example 1. a few weeks after the salsa dancing success, you find you have a bacterial sinus infection 2. a patient with HIV develops thrush

Types of infection Local infection: Pathogens are limited to a small area of the body Systemic infection: Microbe or toxins spread throughout the body by the blood/lymph Focal infection: Infection that began as a local infection spreads via the blood/lymph

Spread of disease – inside the body Bacteremia: Bacteria present in the blood Toxemia: Toxins present in the blood Viremia: Viruses present in the blood Septicemia: “blood poisoning” - systemic infection arising from multiplication of pathogens in the blood

Question What’s the difference between bacteremia and bacterial septicemia What’s the difference between bacteremia and toxemia

Spread of disease – in the population Communicable disease: A disease that is spread from one host to another Contagious disease: A disease that is easily spread from one host to another Noncommunicable disease: A disease that is not transmitted from one host to another – SUCH AS??

For example Determine whether these are communicable or not and if so, are they contagious HIV Influenza Botulism

Mechanisms of Pathogenicity Pathogenicity: Ability to cause disease Are all microbes pathogens? Virulence: The extent of pathogenicity ID50: Dose that causes infection in 50% of population LD50: Dose of toxin that is lethal for 50% of test population

Questions For a given microbe, is the ID50 the same in every situation? Which is less likely to cause disease – a microbe with a higher ID50or lower ID50?

Toxins Which organism has the most potent toxin?? Toxin LD50 Botulinum 0.03 ng/kg Shiga toxin 250 ng/kg Staphylococcal enterotoxin 1350 ng/kg Which organism has the most potent toxin??

reservoirs What do you think? Human Animal (zoonotic) Environment Carrier – human without symptoms

For example What is the reservoir for HIV Rabies Mycobacterium tuberculosis Clostridium botulinum

Modes of transmission Direct contact transmission – touch Indirect contact transmission - fomite Vehicle transmission – a vector brings the pathogen Mechanical vector (rides the roof) Biological vector (rides inside)

question What is the reservoir for malaria What kind of transmission is it? Direct contact, indirect contact, zoonotic, mechanical vector, biological vector More than one right answer

Portals of Entry Preferred route of entry Some have multiple routes (e.g. Yersina pestis causes plague; B. anthracis causes anthrax) Other routes may not cause disease (e.g. Salmonella typhi on skin; Streptococci pneumoniae that are ingested)

Portals of Entry How do microbes get into your body? Mucous membranes GI tract, respiratory tract most common Genitourinary tract, conjunctiva (membrane of eyes) Skin Major barrier to entry, mostly inaccessible to pathogens Some openings: hair follicles, sweat gland ducts Hookwork larvae can bore through skin; some fungi grow on keratin Parenteral route Penetration of skin /mucous membranes (punctures, cuts, bites, etc.)

Bacillus anthracis Portal of Entry ID50 Skin 10–50 endospores Inhalation 10,000–20,000 endospores Ingestion 250,000–1,000,000 endospores Which portal is most likely to cause infection??

So for example Plasmodium (malaria) – what route? C botulinum (botulism) – what route? Influenzavirus – what route? HIV (AIDS) – what route? Bacillus anthracis (anthrax) – what route? Why are these routes different?

biofilms ~65% of infections Dental plaque Implants Contact lenses

Adherence

Alveolar macrophage attacking E. coli; RBC at top left capsules Can prevent phagocytosis – make cells “invisible” Alveolar macrophage attacking E. coli; RBC at top left

Alveolar macrophage attacking E. coli; RBC at top left Capsules Capsules prevent phagocytosis Streptococcus pneumoniae; Haemophilus influenzae; Bacillus anthracis Often avirulent without capsule since phagocytosed (eg S. pneumoniae) Note - many nonpathogenic bacteria also produce capsules (capsulation alone is not sufficient to designate a pathogen) Alveolar macrophage attacking E. coli; RBC at top left

Cell Wall Components M protein resists phagocytosis Surface of fimbriae Streptococcus pyogenes (strep throat, scarlet fever, TSS) Opa protein inhibits T helper cells fimbriae Neisseria gonorrhoeae Mycolic acid resists digestion Waxy lipid where? Mycobacterium tuberculosis

Hijacking host Cytoskeleton Cytoskeleton filament - actin Microbes (e.g. Salmonella; E. coli) produce invasins that rearrange actin filaments of cytoskeleton  membrane ruffling, penetration of cell Some species use actin to propel themselves through and between cells (e.g. Shigella; Listeria; Ricksettia) BEGIN HERE

Membrane Ruffling Invasins: alters host actin to enter host cell eg.Salmonella Figure 15.2

Comet tails Some bacteria organize actin behind them for propulsion eg.Listeria, Shigella, Ricksettia

Damage to Host Cells Consumption of host’s nutrients Direct damage in vicinity of invasion TOXINS  blood/lymph  distant sites

Consumption of host nutrients Lack of iron limits bacterial growth Secrete siderophores to capture Take up from blood; some lyse cells to release

Human Herpes Virus 6 budding off Direct damage Disrupt host cell function Release wastes Rupture  release more viruses/bacteria Human Herpes Virus 6 budding off

Toxins Toxin: Substance that contributes to pathogenicity Toxigenicity: Ability to produce a toxin Toxemia: Presence of toxin in the host's blood Toxoid: Inactivated toxin used in a vaccine Antitoxin: Antibodies against a specific toxin

All about toxins May be transported by blood or lymph what is the important implication? May  fever, diarrhea, shock, cardiovascular and nervous system disruption ~220 known types; ~40% damage cell membranes Two general types: Exotoxins Endotoxins What do you think the difference is?

Exotoxins and Endotoxins Lipid A of lipopolysaccharides (LPS) of outer membrane of Gram negatives Proteins produced by bacteria and secreted or released following lysis Figure 15.4

Exotoxins Specific for a structure or function in host cell Figure 15.4a

Naming exotoxins By type of cell affected By associated disease (e.g., neurotoxins, cardiotoxins, hepatotoxins, leukotoxins, enterotoxins, cytotoxins) What does each affect? By associated disease (e.g., diphtheria toxin, tetanus toxin) What disease is associated with each toxin? By bacterium producing (e.g., botulinum toxin, Vibrio enterotoxin What organism produces each of these toxins?

What are exotoxins, really? Proteins, usually enzymes  even small amounts harmful 1 mg botulinum exotoxin can kill 1 million guinea pigs Genes usually carried on ? Soluble in host fluids  transported widely plasmids, phage – WHY?? START HERE

What are exotoxins, really? Proteins, usually enzymes  even small amounts harmful 1 mg botulinum exotoxin can kill 1 million guinea pigs Genes usually carried on ? Soluble in host fluids  transported widely Mode of action– 3 main modes Inhibit metabolism (usually protein synthesis) Destroy parts of host cells overactivate immune system plasmids, phage START HERE

What are exotoxins, really? Proteins, usually enzymes  even small amounts harmful 1 mg botulinum exotoxin can kill 1 million guinea pigs Genes usually carried on ? Soluble in host fluids  transported widely Mode of action Inhibit metabolism (usually protein synthesis) Destroy parts of host cells overactivate immune system Types A-B membrane-disrupting superantigen plasmids, phage START HERE

Exotoxin type: A-B Exotoxin eg. diptheria, botulinum, tetanus, vibrio (cholera) Two parts – A and B, proteins B (binding) – binds host receptor A-B enter A and B separate A – enzyme, usually inhibits protein synthesis Figure 15.5

Membrane-Disrupting exotoxin Lyse host’s cells in one of two ways Making protein channels in the plasma membrane Leukocidins Hemolysins Streptolysins (b-hemolysis Streptococcus) Disrupting phospholipid bilayer Phospholipases remove polar head –gas gangrene (C. perfringens) Why is this bad?

Exotoxin type: Superantigens Evoke intense immune response Proliferation T cells (lymphocytes)  excess cytokines Symptoms: fever, nausea, vomiting, diarrhea, shock, death Eg. Staphylococcus (food poisoning, toxic shock syndrome)

Last words about exotoxins Exotoxins are disease specific Due to exotoxin, not bacterial infection foodborne intoxication botulism, staphylococcal food poisoning Fighting exotoxins Body makes antibodies (antitoxins)  provide immunity Inactivate (heat, HCHO, iodine)  toxoids Toxoids used as vaccine to stimulate antibody production tetanus, diphtheria START HERE

Endotoxins – part of the cell Part of outer membrane of Gram-negatives Lipid portion of LPS, called lipid A Death and lysis of bacteria; antibiotics can worsen Stimulate macrophages -release high level cytokines Fever, nausea, vomiting, diarrhea, sometimes shock, death (similar to but not as potent as superantigen exotoxins…) Remember this?

Endotoxins and the Pyrogenic Response Figure 15.6

Damage to Host Cells

Exotoxin Source Gram? Relation to microbe ?made/part of Chemistry What kind of molecule? Neutralized by antitoxin? Y/N? LD50 Need a little or a lot? Figure 15.4a

Exotoxin Source Mostly Gram + Relation to microbe Made by growing cell Chemistry Protein Neutralized by antitoxin? Yes LD50 Small Figure 15.4a

Endotoxins Source Gram? Relation to Microbe Made/part? Chemistry Molecule? Neutralized by Antitoxin? Y/N? LD50 How much you need? Figure 15.4b

Endotoxins Source Gram Relation to Microbe Outer membrane Chemistry Lipid A Neutralized by Antitoxin? No LD50 Relatively large Figure 15.4b

Horizontal transmission Where do bacteria get their toxin genes?

Horizontal transmission Transformation Conjugation Transduction

A word about plasmids Circular small DNA containing 4-5 genes What did we just transform into E. coli in lab? What genes were on that plasmid? DIAGRAM WHITE BOARD Many antibiotic resistance genes are transmitted between species by sharing plasmids Transformation and conjugation

Plasmids can code for Antibiotic resistance genes Metabolic enzymes An example from lab? Metabolic enzymes Examples from this class? AND toxins Tetanus neurotoxin Staphylococcal enterotoxin Dextransucrase (Streptococcus mutans  tooth decay) Adhesins and coagulase (Staphylococcus aureus) Fimbria (Enteropathogenic E. coli)

Other ways to get toxins: transduction Lysogenic conversion: prophage in chromosome Shiga toxin of E. coli O157 Virbrio enterotoxin of Vibrio cholerae (cholera) Botulinum neurotoxin Capsule of Streptococcus pneumoniae (virulence factor) Diphtheria toxin

Exotoxins due to Lysogenic Conversion Corynebacterium diphtheriae A-B toxin Streptococcus pyogenes Membrane-disrupting erythrogenic toxin Clostridium botulinum A-B toxin; neurotoxin C. tetani Vibrio cholerae A-B toxin; enterotoxin Staphylococcus aureus Superantigen

Pathogenicity of Viruses Gain entry via attachment sites for cell receptors Resulting damage termed cytopathic effects Production of virus particles (inclusion bodies) Inhibition of biosynthesis (DNA, RNA, protein synthesis) Host cell lysis Inclusion body in brain tissue of rabies victim

Syncytium (giant cell) caused by measles virus Cytopathic effects Cause lysosomes to release enzymes Cause cells to fuse into syncytium Changes in host cell function Production of interferon Chromosomal damage Loss of contact inhibition Syncytium (giant cell) caused by measles virus

Pathogenic Properties of Fungi May cause disease, but usually lack toxins Fungal waste products may cause symptoms Chronic infections provoke an allergic response Proteases Candida Capsule prevents phagocytosis Cryptococcus (meningitis)

Exceptions - Fungal toxins Ergot toxin - hallucinations Claviceps purpurea Aflatoxin (peanut butter) Aspergillus Mycotoxins (mushrooms) Neurotoxins: Phalloidin, amanitin Amanita phalloides

Pathogenic Properties of Protozoa Presence of protozoa Protozoan waste products may cause symptoms Avoid host defenses by Growing in phagocytes Antigenic variation

Plasmodium What disease? reproduction in host cells

Giardia Giardia lamblia (diarrhea) – digest cells and tissue fluids

Trypanosomes African trypanosomiasis – sleeping sickness Chagas disease (US) reproduction inside host

Helminths Use host tissue and resources Interfere with host function Waste can cause symptoms

Algae A few species produce toxins Paralytic shellfish poisoning – “red tide” Dinoflagellates Saxitoxin

Portals of Exit Why do we care how the pathogen gets out?

Portals of Exit Important in spread of disease (epidemiology) Respiratory tract Coughing and sneezing Gastrointestinal tract Feces and saliva; “oral-fecal” Genitourinary tract STDs, urine Skin Wound infections; drainage Blood vectors important Biting arthropods and needles or syringes

Mechanisms of Pathogenicity Figure 15.9