Molecular Biology of THE Molecular Biology of THE CELLCELL……Chapter 25Chapter 25

Pathogens, Infection, and Innate ImmunityPathogens, Infection, and Innate Immunity

Shainn-Wei Wang, Ph.D.Shainn-Wei Wang, Ph.D.Institute of Molecular MedicineInstitute of Molecular Medicine

N.C.K.U. College of MedicineN.C.K.U. College of Medicine

Infectious Diseases- 1/3 deaths in the world- Iceberg concept of infection - Epidemic/Endemic/Pandemic- Acute vs. Chronic- persistent vs. latent- Ancient diseases: TB, Malaria- New infectious diseases: SARS, HIV

General Introduction

Iceberg concept of infection …disease is a kinetic process (v.) not a state (n.)

Host responseCell Response

Lysis of cell Death

Clinical Disease

SubclinicalDisease

DiscernableEffect

Below VisualChange

Infections

Incomplete or Invalid infection ?

Abolished ?

Asymptomatic

Invalid exposure

Pathogen effect

Cell dysfunction

transformation

Classical & SevereSymptom

Moderate or mildillness

Hostpopulation

Pathogendependent

Host dependent

Pandemic…Pandemic… an epidemic that affects a wide geographic areaan epidemic that affects a wide geographic area

Garber D. A., et al., Lancet Infec. Dis., 2004

Global distribution of HIV-1 subtypes and recombinants

Acute- Having a - Having a shortshort and relatively and relatively severe severe coursecourse, usually , usually

refers to primary infectionrefers to primary infection

Chronic- Persisting over a - Persisting over a longlong periodperiod ofof timetime after primary infection after primary infection

Persistent- Continuing to Continuing to existexist in spite of in spite of interferenceinterference or or treatmenttreatment, ,

tending to tending to recurrecur.. Latent

- A type of infection that lays - A type of infection that lays dormantdormant in a host but can still in a host but can still

be passed on to others.be passed on to others.

SSPE:Subacute-sclerosing panencephalitis

(Tolerant: high level; active replication; Non-lytic)

(Breakthrough, low level; Active replication, lytic)

(recurrent activation, lytic)

PathogenesisPathogenesis - Direct cell damage

- Release of exotoxins

endotoxins

irritant enzymes/proteins

- Induction of host immune response

Host Defense Innate immune responses

- act immediately after an infection- do not depend on the host’s prior exposure

Adaptive immune responses

- operate later in an infection- highly specific for the pathogen that induce

them

Innate and Adaptive Immunities

Innate Immunity…nonspecific host defense

1. Tissue barriers Skin : skin associated lymphoid tissue (SALT) underlies epidermis, contains

Langerhans cells, phagocytes that destroy invading microbes. Mucosal surfaces: all inner channels (respiratory, GI tract, vagina, bladder)

are lined with mucus-producing cells = mucosal cells. Mucus secretion is slimy polysaccharide, traps microbes and prevents access to epithelial cell surfaces. Mucus contains protective enzymes such as lysozyme (attacks peptidoglycan cell walls), lactoferrin (iron-binding protein), defensins (proteins that attack cell membranes). Mucosal membranes also have phagocytes, called mucosal-associated lymphoid tissue (MALT).* Respiratory tract * Intestinal tract * Genitourinary tract

2. Chemical barriers Include a variety of enzymes; lysozyme, fibronectin, hormones

(such as corticosteroids), etc.

3. Phagocytes

Major components of immune system Certain white blood cells (granulocytes (a.k.a. neutrophils, macrophages,

others) are highly mobile, carry out phagocytic activity* Phagocytosis begins with engulfment of particulate matter (can be

bacteria, clumps of virions, cell debris, etc.) into phagosome.

* Phagosome fuses with lysosomes: phagolysosomeTwo kinds of lysosomes:

- acid hydrolases, lysozyme, neutral proteases, myeloperoxidase; - lactoferrin, lysozyme, phospholipase A.

(These enzymes can degrade biomolecules; but many pathogenic bacteria have walls that are resistant to lysozyme)

* White blood cells are chemically attracted to foci of disease or tissue damage by process of chemotaxis "Respiratory burst" : Dramatic increase in oxygen uptake during phagocytosis, .

Produces superoxide (O2-), hydrogen peroxide (H2O2).

4. Inflammation

A nonspecific reaction to tissue damage; Triggered by several chemicals called inflammation mediators, which include: complement, prostaglandins, and cytokines. These attract phagocytes to the locus of inflammation. Effects include:

* vasodilation (opening junctions between capillary cells, allowing fluid and WBCs to leave blood and enter surrounding tissues)* redness (from heightened blood flow) * pain (from prostaglandins released by tissues binding to nerve receptors) * heat (produced by pyrogens liberated at site of inflammation)* Others: fibrin clotting, platelet aggregation, chemotactic signaling to

attract WBCs, activation of complement factor C3.

Allergy: Inflammation mediators also act on mast cells, found throughout connective tissue and mucous membranes. These release further chemicals: * Histamine binds to capillary receptors and causes dilation. * Heparin binds to a clot-inhibiting protein (AT3) and reduces the chance that leakage from blood vessels will produce a clot.

Septic Shock: Inflammation gone wild. If significant numbers of microbes get into body, can trigger a widespread systemic inflammatory response, leading to rapid swelling, loss of fluid, loss of blood pressure.

5. Complement and Opsonization

Complement = complex of > 20 proteins present in normal serum. Heat labile, destroyed @ 56 deg. C. Named C1-C9, also Factor B, D, H, I etc. Complement is used up (fixed) in antibody-antigen reactions, as a result of series of reactions called classical complement cascade. Alternatively, complement C3 can interact directly with certain chemicals (teichoic acids, LPS) found in bacterial cell walls, activate alternate pathway.

Classical cascade reaction: When Ab-Ag complex forms, base of Ab (constant region) changes shape. This activates Complement C1, which acquires esterase activity. Activated C1 not activates C2 and C4; generates a new activity which activates C3, etc. After several more steps, activate C8 and C9 membrane attack complex, creates pores in membrane of target cell lysis.

Opsonization = enhanced phagocytic activity. Stimulated by complement bound to Ab-Ag targets. Especially important in binding to capsules, triggering effective phagocytic uptake of capsulated bacteria.

Infection--- Signs and SymptonInfection--- Signs and Sympton

Caused by pathogens- lesions

- toxicity

- any selective advantage for the pathogen Caused by host’s responses

- swelling & redness

- production of pus

- fever

Introduction to pathogenIntroduction to pathogen

Normal Flora

- human (1013 cells) vs. microbes (1014) Pathogenic invaders

- opportunistic (immuno-compromise or injury)

- virulence factor:

colonization / multiplication /

nutrient competition / exit and spread

Evolved specific mechanism for host interaction…natural selection

- disease…selective advantage? Intracellular life (all viruses)

- poses problems for host to attack - must leave cells for survival- can’t survive outside at all (through cell contact or animal bites)

Extracellular life - deal with host defensive mechanisms- greater opportunity for growth, spread, colonization

Faculative intracellular life - grow either inside or outside of host cells depending on

circumstance- Shigella; Salmonella

Numbers of bacteria that colonize different parts of the body(per gram of homogenized tissue or fluid or per square centimeter of skin surface)

Appl. Environ. Microbiol., 31:310,1976

Scanning electron micrograph of a cross-section of rat colonic mucosa

Bacteria (B) immediately adjacent to colonized intestinal tissue (T)

Clinical conditions that may be caused by members of the normal flora

Mechanisms by which the normal flora competes with invading pathogens

Virulence Factors

Virulence Factors are specific adaptations that allow pathogen to:

Attach selectively to host tissues Gain access to nutrients by invading or

destroying host tissues Avoid host defenses

Examples of virulence factors:

1. Specific attachment & entry factors Pathogen must be able to bind to some receptor molecule on cell surfaces. These receptors

typically have necessary functions for cell. Most diseases are tissue specific, because only certain tissues have receptor molecule

needed. Ex: HIV binds to cells that have CD4 receptor Fimbriae or pili are used by some bacteria to attach selectively to certain tissues. Ex:

Neisseria gonorrhaea binds to genital epithelium by fimbriae.

2. Invasive enzymes Many bacteria have specific enzymes that allow cells to penetrate tissues Example 1: collagenase produced by Clostridium perfringens. Enzyme degrades collagen,

the primary structural fiber of connective tissue (25% of body's protein), allows penetration deeper into tissues. Fulminating gangrene: Strictly anaerobic process, only occurs when tissue is damaged so blood can't supply oxygen (e.g. serious wounds, frostbite).

Example 2: hemolysin enzymes produced by Streptococcus pyogenes dissolves cell membranes of tissues, produces typical symptoms of "strep throat".

Pathogen are phylogenically diverse

Bacteria: free living cells- relying on the host primarily for nutrition

Viruses: host dependent- - DNA vs. RNA / Envelop vs. non-envelop

Eukaryotic organisms- fungi / protozoa

Prion: protein (mad cow disease)- self imprinting / neurodegenerative

Parasitism at many levels

Pathogens in many forms

Poliovirus: poliomyelitis

Vibrio cholerae:diarrheal cholera

Toxoplasma gondii: Muscle & Brain

Ascaris nematodes: intestine

Phylogenetic diversity of pathogens

… based on 16S rRNA sequences

Note: 16S rRNA is highly conserved among all organisms due to the antiquity of the protein synthesizing process

Bacterial Pathogens Classified by shape

- rods, spheres, spirals, cell surface characters-Thiomargarita namibiensis (1mm);mycoplasma (0.2mm)

Surface appendages- swim or adherence

1-5 x 106 bp- yeast (12 x 106 bp); human (3000 X 106 bp)

Only a small minority are dedicated pathogens - obligate pathogens: replicate only in host - facultative pathogens: replicate in both the environment and the host - opportunistic pathogen: normally benign, but have latent ability to cause disease---replicate in injured or immunocompromised host)

Bacteria are classified into three different shapes

Bacteria are also classified into Gram-positive or Gram-negative

E. coli; SalmonellaStreptocci; Staphylococi

Cell surface projections are important for bacterial behavior

Flagellum:for movement

Straigh pilus:also calledFimbriae…for Adherence & genetic exchange)

Genetic differences between pathogens and non-pathogens…virulence

• Virulence genes: genes that contribute to the ability of an organism to cause disease• Virulence factors: the proteins encoded by virulence genes• Virulence genes are frequently clustered together, either in groups on the bacterial chromosome called pathogenicity islands or on extrachromosomal virulence plasmids

-bloody diarrhea; human - food poisoning; vertebrate

BACTERIAL INDUCED INJURY

Adhesins / receptors- Capsular material

- Proteins (Protein F, Protein M, Tir receptor)

- Lipotechoic acids

- Fimbriae & Pili Endotoxins

- Gram negative LPS cell wall components (Lipid A

and Core Sugars) Exotoxins

- Can be produced by either Gram Positive or

Gram Negative Organisms

Genetic organization of Vibrio cholerae

• Two plasmids system• Two replication origins

CTX : integrated bacteriophage genome carrying genes for cholera toxinVPI: for intestinal colonizationIntegron Island: gene structure facilitating foreign gene insertion (antibiotic resistance)ctxA and ctxB : genes for two kinds of subunit of cholera toxin (one A , five B)ace and zot: genes for other virulence factorsRS1 and RS2: for chromosomal insertion of the bacteriophage genome

Massive watery diarrhea caused bycholera exotoxin (ADP-ribosyltransferase)

Cholera toxin activates the adenylate cyclase enzyme, leading to increased levels of intracellular cAMP, and the secretion of H20, Na+, K+, Cl-, and HCO3

- into the lumen of the small intestine

Toxin contains 5 binding (B) subunits, an active (A1) subunit, and a bridging piece (A2) that links A1 to the 5 B subunits.

The bacterium produces an invasin, neuraminidase, during colonization.

Neuraminidase degrades gangliosides to the monosialosyl form, which is the specific receptor (GM1 ganglioside) for the toxin.

Once it has entered the cell, the A1 subunit interacts with ADP ribosylation

factor (ARF) to enzymatically transfers ADP ribose (ADPR) from NAD to a protein (GS) that regulates the adenylate cyclase system.

GS is a heterotrimeric stimulatory G-protein involved in G-protein signaling and is comprised of 3 subunits: alpha, beta, and gamma. if alpha is bound to GDP, then GS is inactive; When alpha is bound to GTP, GS is active.

Adenylate cyclase (AC) is activated normally by a regulatory protein (GS) and GTP; however activation is normally brief because another regulatory protein (Gi), hydrolyzes GTP.

ADPR-GS-GTP binds and activates adenyl cyclase (secondary messenger).

Activated adenyl cyclase manufactures cyclic-AMP (cAMP) using ATP as substrate.

Alpha subunit is GTPase and thus, eventually hydrolyzes bound GTP to GDP. However, the linking of ADPR to GS renders the Alpha unit incapable of continuing the hydrolysis process

Protease activation:Nicked toxin

Diphtheria toxin receptor: HB-EGF/CD-9 - HB-EGF: Heprin-binding epidermal growth factor-liked growth factor - CD-9: cluster of differentiation 9

A subunit has ADP-ribosyltransferase activity.

The substrate of the reaction is human elongation factor 2 (EF2, aminoacyl transferase), an essential part of the protein synthetic machinery.

ADP-ribosyl transferases promote the breakdown of nicotinamide adenine dinucleotide (NAD) into nicotinamide and adenine diphosphate ribose (ADPR)

EF2 + NAD ----> ADPR-EF2 + nicotinamide + H+

covalent binding of the ADPR inactivates the bound protein

Receptor mediated endocytosis of DT

Activation by ADP-ribose

Anthrax toxin

Acute infectious disease of herbivores (occasionally humans) Direct contact with spores of Gram+ Bacillus anthracis Survive in soil and highly resistant to adverse environments Secrete two toxins: lethal toxin & edema toxin Each toxin alone is sufficient to cause signs of infection Each toxin is made of two subunits (A + B) B- surface binding; A-effecter unit Lethal toxin A subunit is a zinc protease for members of MAP

kinase Kinase family … causing septic shock and death edema A subunit is a adenylyl cyclase ATP cAMP (accumulation) ion imbalance

(accumulation of extracellular fluid)

Extracellularly-Acting Toxins…for reference

Non-Membrane Damaging 1. Hyaluronidase - This is also called the spreading factor because it catalyzes the breakdown of hyaluronic acid, the substance that cements the human cells together. This allows the bacterial cells to spread through tissue causing a condition known as cellulitis. 2. Coagulase- This enzyme catalyzes the conversion of fibrinogen to fibrin with resultant clot formation. 3. Fibrinolysin - This catalyzes the conversion of plasminogen to the fibrinolytic enzyme plasmin. Thus it acts opposite of coagulase. In Staphylococcus aureus, the gene for fibrinolysin is on a bacteriophage and is expressed during lysogeny. 4. Lipase - Production of excessive amounts of lipase allow bacteria to penetrate fatty tissue with the consequent formation of abscesses. 5. IgA protease - Many bacteria which colonize the mucous membranes produce an IgA protease which degrades secretory IgA. 6. Collagenase- This enzyme catalyzes the degradation of collagen, a scleroprotein found in tendons, nails and hair.

Membrane Damaging 1. Hemolysins - There are many different types of hemolysins but, in each case, the end result is lysis of the red blood cell with resultant anemia. 2. Leukocidins- Again, there are many different types of leukocidins, and some are specific for only one type of leukocyte. However, the end result in lysis of leukocytes with resultant leukopenia. 3. Phospholipase- This enzyme attacks any cell with phospholipid in its membrane. The result is widespread cell lysis. Lecithinase - (phospholipase C) is an enzyme which breaks down the lecithin in the human cell plasma membrane, resulting in cell lysis. It is especially active on red blood cells. It is also called a toxin.

Intracellular-Acting toxin (ADP-ribosyl transferases)… for reference These promote the breakdown of nicotinamide adenine dinucleotide (NAD) into nicotinamide and adenine diphosphate ribose (ADPR) and the covalent binding of the ADPR to various proteins, thus inactivating the bound protein. 1. Diphtheria toxin. Corynebacterium diphtheriae cells lysogenized with the -phage produce a diphtheria toxin, composed of a B subunit which mediates binding to a specific human cell surface receptor and an A subunit which possesses enzymatic (ADP-ribosyltransferase) activity. The substrate of the reaction is human elongation factor 2 (EF2), an essential part of the protein synthetic machinery. The result of this reaction is inhibition of protein synthesis and cell death. 2. Pseudomonas aeruginosa exotoxin A. This works in the same manner as diphtheria toxin, i.e., it catalyzes the ADP-ribosylation of EF-2. Human epithelial cell death occurs. 3. Pseudomonas aeruginosa exoenzyme S. This is an ADP-ribosyl transferase whose substrate is unknown. However EF-2 is not the substrate. It also causes human epithelial cell death. 4. Cholera toxin. Vibrio cholerae growing in the intestine secretes an exotoxin composed of 5 B subunits, an A subunit and an A2 subunit. On exposure to small bowel epithelial cells, each B subunit binds to a receptor on the gut epithelium. Following binding the A and A2 moieties migrate through the epithelial cell membrane. The A subunit is an ADP-ribosyl transferase that catalyzes the transfer of ADPR from NAD to a guanosine triphosphate (GTP)-binding protein that regulates adenylate cyclase activity. The ADP-ribosylation of GTP binding protein inhibits the GTP turnoff reaction and causes a sustained increase in adenylate cyclase activity which results in excess secretion of isotonic fluid into the intestine with resulting diarrhea. 5. Labile toxin (LT) of Escherichia coli. This toxin is identical to that of cholera toxin. The ability to produce it is mediated by a plasmid. 6. Bordetella pertussis toxin. During an episode of whooping cough, the B.  pertussis cell produces an exotoxin composed of an A portion and 4 B portions. The A subunit is an ADP-ribosyl transferase which elevates cAMP but in a way different from cholera toxin. It ribosylates a 41,000 MW membrane protein which specifically binds guanine nucleotide (the G1 protein).

Non-Ribosylating Toxins … for reference

1. Shiga toxin. Species of Shigella carry the gene for shiga toxin on the chromosome. This toxin has an A subunit and 5 B subunits. The A subunit can be divided into A1 and A2 subunits. The A1 moiety binds to the 60S human ribosome which inhibits protein synthesis. The toxin has a multiplicity of effects; it is neurotoxic, cytotoxic and enterotoxic. 2. Anthrax toxin. Bacillus anthracis produces an exotoxin composed of three distinct proteins: protective antigen, edema factor and lethal factor. The protective antigen is the binding protein, the edema factor is an adenyl cyclase  and the lethal factor has an unknown function but is thought to be enzymatic. Dermal necrosis is the result of the toxin action. 3. Tetanus toxin. Clostridium tetani produces an endopeptidase that cleaves synaptobrevins: this interferes with vesicle formation at the myoneural junction and the neural-neural junction in the spinal cord.  The result is muscle spasm.  The tetanus toxin serologically cross-reacts with the botulinu4. Botulinum toxin. Clostridium botulinum produces an endopeptidase that blocks the release of acetylcholine at the myoneural junction.  Muscle paralysis is the result.  the botulinum toxin, like tetanus toxin, cleaves synaptobrevin thus interfering with vesicle formation.  This toxin is used clinically in the treatment of dystonias. 5. Pyocyanin. Pseudomonas aeruginosa produces this non-enzyme protein which binds to the flavoproteins of the cytochrome system. It interferes with terminal electron transport causing an energy deficit and cell death. 6.  Adenylate cyclase. Bordetella pertussis produces a calmodulin-independent adenylate cyclase which inhibits and/or  kills white blood cells. 7.  NAD glycohydrolase. Shigella flexneri, upon being phagocytized, produces a NAD glycohydrolase which rapidly depletes the phagocyte of NAD, thus blocking cellular metabolism and bacterial cell killing.

Type III secretion system deliver virulence Factors into the cytoplasm of host cells

(plague bacillus Yersinia pestis)

Antibiotic Targets

Not effective against • intracellular virus---you have to kill your own cells• fungi and protozoan parasites

- eukaryotes- form switching- narrow specificity

Dimorphism in the pathogenic fungus Histoplasma capsulatum

Filamentous:Low temperatureIn the soil

Circular buds:In lung of a mammal(histoplasmosis)

Mold form

Yeast form

The complex life cycle of malaria

• Two hosts• Zygote• gut lining• liver & blood

Three forms of Plasmodium falciparum in RBC

Note: Africa-natural selection: malaria parasites grow poorly in RBC fromeither homozygous sickle cell patients or heterozygous health carriers.

Tricks to avoid host defenses.

1. Capsules: extracellular polysaccharide; prevent quick disposal by WBCs.

2. "Nasty Enzymes"

Leukocidins: secreted chemicals that specifically kill WBCs. Ex: Staphylococcus aureus: pus at infected site is caused by dead WBCs.

Coagulase: enzyme that coagulates blood. EX: Staph. aureus typically remains localized, "walled off" from defenses, produces many

nasty types of localized infections. WBCs, other body defenses can't reach site of infection. 3. Siderophores = iron-binding factors to compete with the host for iron. * Most bacteria require iron to synthesize cytochromes. Iron in human body is tightly bound, either in hemoglobin (blood

cells), on transferrin proteins (serum and lymph), or lactoferrin (milk, tears, saliva, mucus, etc.). Note: Streptococci do not require iron (fermentative growth)

* Ex: Enterochelin produced by enteric bacteria (E. coli, Salmonella). Mutants that cannot synthesize enterochelin lose virulence.

4. Endotoxins

* Endotoxins are integral parts of Gram-negative outer membrane (= LPS, lipolysaccharide). Unlike Exotoxins, they are typically heat resistant, active only in sizable amounts, and remain bound to cells.

* Mechanism of action is very diverse; LPS turn on every defense at our disposal, including fever, decrease in iron, inflammation, blood clotting, reduced sugar in blood, etc. Most important clinical problems are fever and shock.

* Gram-negative bacteria (e.g. E. coli, Pseudomonas) enter body via clinical procedure cause sudden decrease in blood pressure (hypotension) = "septic shock". Can be lethal.

5. Exotoxins Most exotoxins are secreted proteins, often damaging tissues at some distance. Very potent, small amounts are

very toxic. Often coded by plasmid DNA (ex. E. coli) or lysogenic phage DNA (ex. botulism, diphtheria) Almost always inactivated by heat. Most are good antigens when inactive, can make toxoids (antigens without

poison activity) = strong immune response. * Ex. 1: Diphtheria toxin. From Corynebacterium diphtheriae. Enters cell, inactivates elongation factor needed for

protein synthesis. Result = cell gradually loses ability to make proteins (same toxin molecule keeps inactivating more and more factors), shuts down.

* Ex. 2: Botulin toxin, a neurotoxin (attacks nervous system). From Clostridium botulinum, anerobic soil bacterium, endospore former. Most potent toxin known --- 1 gram could kill 10 million people. Toxin interferes with synaptic transmission at nerve-muscle junctions flaccid paralysis. Occurs most typically in home canned foods that are not cooked long enough to kill endospores.

* Ex. 3: Tetanus toxin, another neurotoxin. FromClostridium tetanus, anerobic soil bacterium, endospore former. Blocks synaptic transmission to inhibitory neurons needed to relax one muscle when the other in paired muscle contracts (e.g. biceps must relax when triceps contracts, vice versa), leads to rigid paralysis. Common from deep wounds, pulled teeth. (But in 20% of cases, no history of injury!). Kills about 1 million infants/year by infecting umbilical stump. Treatment: antitoxin. Prevention: toxoid immunization (lasts 5-10 yrs).

* Ex. 4: Cholera toxin = an enterotoxin (attacks enteric tract). From Vibrio cholerae. Binds to receptors on intestinal cells, chemically alters molecule involved in c-AMP production, leaves cAMP stuck in the "on" position. Causes massive outflow of water (chasing outflow of Na+/Cl-). Similar mode of action for other enterotoxin. Epidemic in S. America currently. Pathogen is free-living in fresh water, only causes infection in humans. Can be spread by drinking water, food (shellfish common). Untreated, mortality is ~50%. With fluid replacement, <1%. Prevention: clean drinking water.

VIRAL INDUCED INJURY Direct attachment and killingDirect attachment and killing Inhibit host cell synthesis (RNA, DNA, proteins)Inhibit host cell synthesis (RNA, DNA, proteins) Intracellular replication and lysisIntracellular replication and lysis Surface targets for host immune systemSurface targets for host immune system Local direct damage leading to secondary bacterial Local direct damage leading to secondary bacterial

infectionsinfections Latent injuryLatent injury

- reactivation of viral infection at a later time

- induction of transformation and proliferation with resulting neoplasia (EBV, HPV, HBV, HTLV-1)

General host responses to viral invasion

A simple viral life cycle

• DNA vs. RNA• Single stranded vs. double stranded• Nonenveloped (protein coat) vs. Enveloped • cytolytic vs. non-cytolytic (budding)• Acute vs. Chronic• Non-oncogenic vs. oncogenic• wide variety of shapes and sizes• poxviruses: 0.45 mm; 270 Kb; dsDNA• parvoviruses: 0.02mm; 5 Kb; ssDNA• capsid vs nuceocapsid• encode at least three essential products: - for genome replication - for structural assembly - for needs of spread • vaccination : effective way for protection - smallpox (gone) - poliomyelitis (almost eradicated)

Examples of viral morphology

Schematic drawing of several types of viral genomes

Semliki forest viruses

Acquisition of a Viral envelope

A map of the HIV genome

• gag, pol, & env are common to all retroviruses• encode 6 accessory proteins from genes of rev, tat, vif, nef, vpu, & vpr for modification of gene regulation, protein trafficking, and cell cycle progression

Eradication of poliomyelitis through vaccination

(Salk vaccine)

(Sabin vaccine)

Neural degeneration in a prion infection

Bovine spongiform encephalopathy (BSE)

• Prions replicated in the host by copying an aberrant protein structure• Indistinguishable from the normal forms in sequence and in post- translational modification, but differ in folding• Misfolded prion tends to aggregate and to catalyze normal protein to adopt its misfolded structure.

Cell biology of infection Protective BarriersProtective Barriers

- epithelial / Normal flora competition / mucosal

ClearanceClearance- mucus / cilia / micturition / peristalsis

InvasionInvasion - wound / biting insects / endocytosis / phagocytosis / receptor - alter host metabolism, membrane traffic mechanism, and etc.

SpreadSpread - transmission (insect/zoonotic/feces/air/surface binding) and colonization

Drug resistanceDrug resistance - Antibiotics / mutations

Pathogen Infection & Spread…human

The spread of plague… behavior related

Cohesive aggregates of Bacterium Yersinia pestis (bubonic plaque)• painful symptom/fever/headache• enlarged lymphonode• pneumonia/sepsis

Digestive tract of a flea

Starvation effect for repeated attempts of bit

Another example:tsete fly and protozoan Trypanosoma brucei (sleeping sickness)

Adhesins : uropathogenic E.coli attachto bladder epithelial cells

Note: 1. Helicobacter pylori (stomach ulcer/cancer) expresses at least five types of adhesins

2. Bordetella pertusis (whooping cough) expresses at least four types of adhesins

Interaction of enteropathogenic E. Coli (EPEC) with host cells

Green: TirRed: actinBlue: cell

(adhesin)

Pedestal protrusion and adhesion for entering cells

Cell surface receptors Function related

- E. coli & bacteriophage: maltose transport receptor Surface abundant

- Influenza: sialic acid containing oligosaccharides Cell specific

- Rabies: nerve growth factor receptor (NGFR) nicotinic acetylcholine receptor (NACR) cell-cell adhesion molecules (N-CAM)

Viral specific- Hepatitis: 4/6 viruses used different receptors

Non-protein mediated- Herpes simplex virus; heparan sulfate proteoglycans

Primary and secondary receptors mediated- HIV CD4 and CXCR4/CCR5

Receptor and coreceptor for HIV

M tropic T tropic

Endocytic Recycling Frederic R. Maxfield & Timothy E. McGraw, Nature MCB Reviews, 2004

Endocytic membrane traffic:1. Functions of normal cellular process in endocytic recycling

membrane componentsreceptor-associated ligandsother soluble molecules

2. Intracellular targeting of therapeutic agents3. Intracellular fates of pathogens and toxins

Main pathways:Degradation in lysosomesMembrane/protein recyclingTranscytosis (polarized cells: epithelial cells/IgA

1. Clathrin coated pit formation (endosome)re-use or degradation

2. Non-clathrin coated pit formation (endosome)can later fuse with #1 pathway

3. Membrane rufflinglarge endocytic compartment (Macropinosome)Infrequent, stimulated by growth factors

4. Caveolae formationSmall (55-60nm)membrane invaginationoften coated with protein caveolincan pinch-off to form vesicles (infrequent)

5. Phagocytosis (most cells are capable of /low levels)can uptake large particles (300nm)actin dependent

6. Stem (surface-connected tubules entering M) formationLarge diameter (2m) tubules bud into the cytoplasm

Mechanisms for Internalization molecules

Models of Internalization

Caveolaeformation

Polymerized actin

Phagocytosis

Membrane ruffling

Clathrin Coated-pitformation

Non-clathrin coated-pit formation

Stemformation

phagosome Macropinosome

Endosome

Endocytic recycling pathway

Multivesicular Bodies (MVB) Biogenesis

lysosome

Late endosome

ECV/MVB

Early endosome

Golgi

Recycling Endosome

Plasma membrane

Endocytosed tracers that are destined to be degraded reach MVBs afterbeing segregated from recycling receptors, but before they reach lysosomes.

Examples of Viral Entry

Enveloped viruses: fusion

Non-Envelopedviruses: pore orendosome disruption

Virus entering:• Membrane fusion• Pore formation• Membrane disruption

The entry strategy used by the influenza virus

• HA mediated fusion• Acidification of endosome• HA fusion mediated RNA release

Uptake of Legionella pnemophila by a human Mononuclear phagocyte

• Gram negative• causing legionnaire’s disease• Pneumonia/ flu-like respiratory illness• bacteria (disease) spread only through tiny water droplet (aerosal)• not harmful if it present in drinking water

Membrane ruffling

(marcropinosomes formation)

Phagocytosis mechanisms used by bacteria

The life cycle of the intracellular parasite Toxoplasma gondii …energy required entry

Conoid:Microtubule-ribbed structure

Lipid vacuolar synthesis

Infect exculsively in cats; others: meat source

Invasion of Trypanosoma cruzi…endosomal escape by porin

(pore forming enzyme)

Transsialidase: sialic acid coating

The choices faced by an intracellular pathogen

Choice 1• All viruses• Trypanosoma. cruzi• Listeria monocytogenes• Shigella flexneri

Choice 2• Plasmodium falciparum• Mycobacterium tuberculosis• Samonella enterica• Legionella pneumophila• Chlamydia trachomatis

Choice 3• Coxiella burnetii• Leishmania

Selective destruction of phagosomal membraneBy Listeria monocytogenes

(Endosome escape by hemolysin/pH mediated mechanism)

Modifications of intracellular membraneTrafficking by bacterial pathogens

Envelope acquisition of Herpes virus

Envelope acquisition of Vaccinia virus

• Poliovirus RNA replication using ER associated polymerase• Protease 3A cleaves the TATA binding factor TFIID• ER is swallen and traffic from the ER to the Golgi is inhibited

Intracellular membrane alterations induced by a poliovirus protein-3A/TFIID interaction

Cos 7 cell

Some viruses use IRES for translation initiation

HostSome Viruses

Exploit the cytoskeleton for intracellular movement

Listeria monocytogenes• Opportunistic parasite• Saprophyte (0-45°C)• Gram positive• Rod shape• Food poisoning

Microfilaments• contractile filament of F-actin• for structure & movement

The actin based movement of Listeria monocytogenes

Red: bacteriaGreen: actin tailYellow:overlaped region

Molecular mechanisms for actin nucleation by various pathogens

ARP: Actin Related ProteinWIP: WASp-interacting proteinWASp: Wiskott Aldrich Syndrom protein

(G-)(G+)

Wolbachia associates with microtubules…segregation into two daughter cells

Red: wolbachia• symbionts of insects & invertebrates• reside in cytosol

Green: Microtubules ofmitotic spindles inDrosophila embrio

Trafficking of Herpes virus in an axon

GFP tagged Herpes

Antigenic variation in trypanosomes

• parasitic protozoan• infect human and domestic animal• sleeping sickness and leishmaniasis• lives in M•VSG: variant specific glycoprotein• 1000 distinct VSG genes• express one VSG at a time• changed repeatedly by gene rearrangement• escape of Ab-mediated clearance

Diversification of HIV-1, HIV-2, and related strains of SIV

• AIDS• HIV-1 is divided into M & O group• M is responsible for the global AIDS• M can be subdevided into A through G• B is dominant in US and Europe• B, C, E predominate in Asia• All found in Africa• SIVcpz & SIVmnd are close related

with HIV-1

Based on gag gene

Drug Resistance Bacteria

- misuse of antibiotics (persistent and chronic use)- food additive- normal floral- genetics

Viruses- genetics (reverse transcriptase / natural selection)

Strategies- producing enzyme that destroys the drug- alter the sensitivity of drug target- prevent acess to the drug target

Innate Immunity Skin and epithelial surfaceSkin and epithelial surface

- Tight junctions / Mucus layer (mucins; defensins) / Cilia- Tight junctions / Mucus layer (mucins; defensins) / Cilia Alerting signals from recognitionAlerting signals from recognition

- pathogen associated immunostimulants (Adenovirus:Raf/MAP-IL8)- pathogen associated immunostimulants (Adenovirus:Raf/MAP-IL8)

- inflamation and phagocytosis- inflamation and phagocytosis Pattern Recognition ReceptorsPattern Recognition Receptors

- soluble (components of the complement system)- soluble (components of the complement system)- membrane bound (Toll like receptor)- membrane bound (Toll like receptor)

Complement activationComplement activation- made by liver, circulating blood, and extracellular fluid- amplify and complement of the action of Ab- are also pattern recognition receptors- three distinc pathways: classical, lectin, and alternative

Phagocytosis/Cytokines/chemokinesPhagocytosis/Cytokines/chemokines- Neutrophil/Macrophage/NK/INF- Neutrophil/Macrophage/NK/INF

Epithelial defenses against microbial invasion

Intestinal lumen Crypt

Prokaryotic:Prokaryotic: - formylated methionine-peptide- formylated methionine-peptide Bacteria: Bacteria: - peptidoglycan; flagella; LPS(G-); teichoic acids(G+) - peptidoglycan; flagella; LPS(G-); teichoic acids(G+) Fungus:Fungus: - zymosan; glucan; chitin- zymosan; glucan; chitin ProtozoaProtozoa - glycosylphosphatidylinositol (plasmodium parasites)- glycosylphosphatidylinositol (plasmodium parasites) Viruses & bacteriaViruses & bacteria - DNA (CpG motif: purin-purin-CpG-pyrimidine-pyrimidine)- DNA (CpG motif: purin-purin-CpG-pyrimidine-pyrimidine) - ds RNA- ds RNA

Pattern Recognition

Pattern Recognition Receptors…Toll like proteins

Found initially in Drosophila/flyFound initially in Drosophila/fly

- A large extracellular domain (leucine-rich repeats)- A large extracellular domain (leucine-rich repeats)

- for pattern formation (dorsal-ventral polarity)- for pattern formation (dorsal-ventral polarity)

- for resistance of fungal/ bacterial infections (NF-kB mediated defensins)- for resistance of fungal/ bacterial infections (NF-kB mediated defensins)

At lease 10 TLRs found in humanAt lease 10 TLRs found in human- most of them use the NF-kB signaling pathway- most of them use the NF-kB signaling pathway

- innate/inflammation initiation and adaptive immunity induction- innate/inflammation initiation and adaptive immunity induction

- abundant in macrophage, neutrophils, and epithelial cells of lung and gut- abundant in macrophage, neutrophils, and epithelial cells of lung and gut

- involved in innate immunity in muticellular organisms- involved in innate immunity in muticellular organisms

- TLR and defensins seem to be evolutionarily ancient- TLR and defensins seem to be evolutionarily ancient

Toll-like Receptors

TLR signaling pathway…

LPS = Lipid A+ core polysaccharides + Polysaccharide side chains (O-Ag)

- Lipid A: unusual fatty acid (hydroxy-mysteric acids)-Core: unusual sugar (KDO: keto- deoxyoctulonate and heptulose (ketoheptose)). Contains two Glucosamine sugar derivatives each containing 3 Fatty acids with phosphate or pyrophosphate attached- Side chain: varies; refers to O antigen,- Act as endotoxin and can bind to CD14 on Macrophage for pro- inflammatory mediated responses

6 fatty acid tails

3 phosphates

2 glucosamin sugars

Branch sugars(3-deoxy-D-manno-octulosonic acid)

Structure of Lipopolysaccharide

The activation of a Macrophage by LPSCD14: GPI anchored receptorMyD88: myeloid differentiation

factor 88IRAK: IL-1 receptor-associated kinaseTRAF6: TNF receptor-associated

factor 6TAK1: MAP kinase kinase kinase

(mitogen-activated protein kinase kinase transforming growth factor (TGF)-beta-activated kinase 1 (TAK1))

MAPK: p38 mitogen-activated protein kinase

ERK: Extracellular signal-regulated kinase

JNK: c-Jun N-terminal kinasesIKK: IkB kinaseIkB: inhibitor kBNF-kB: nuclear factor-kappa Bc-fos: cellular DNA-binding proteins encoded by the c-fos genes

to promote immune and inflammatory responses

> 20 interacting soluble proteins in blood and > 20 interacting soluble proteins in blood and extracellular fluid; activated by infectionextracellular fluid; activated by infection

Made mainly by the liverMade mainly by the liver Ability to amplify and complement the action of AbsAbility to amplify and complement the action of Abs Some components of complement are also pattern Some components of complement are also pattern

recognition receptorsrecognition receptors Early complement componentsEarly complement components

ClassicalClassical pathway; pathway; LectinLectin pathway; pathway; AlternativeAlternative pathway pathway C3 is the pivotal component of complementsC3 is the pivotal component of complements

- C3b-large: coating microbes and induction of phagocytosis- C3b-large: coating microbes and induction of phagocytosis- C3a-small (C4 and C5 too): recruitment of inflammatory cells- C3a-small (C4 and C5 too): recruitment of inflammatory cells

The early components and C3 are all proenzymes and activate The early components and C3 are all proenzymes and activate an amplifying proteolytic cascadean amplifying proteolytic cascade

Complement activation for phagocytosis or lysis

Activated by IgG or IgM

Pattern Recognition:binds to mannoseand fucose

C3 spontaneouslyactivates at low levels

Late components:Membrane attack Complexes for pore formation

Early components:proteolytic cascade

The Principle Stages in complement activation

Assembly of the late complement componentsTo form a membrane attack complex

C3b mediatedcleavage of C5

EM of negative stained complement lesions

Plasma membrane of Red blood cell

en face side view

Phagocytic cells Macrophage and NeutrophilMacrophage and Neutrophil

Cell surface receptors to recognize and engulf pathogens- Pattern recognition receptors- Fc receptor- C3b receptor

Phagocytosis Actin polymerization Phagosome

- Acidificaton - NADPH oxidase complex- Toxic oxygen derived componds: superoxide, hydrochlorite,

hydrogen peroxide, hydroxyl radicals, and Nitric oxide)- Respiratory burst:oxygen consumption

lysosome (lysozymes; hydrolases; defensin) Direct killing (macrophage + neutrophil + eosinophils)

- defensin; lysozymes; toxic products of the respiratory burst

Strategies to avoid phagocytosis Avoid recognition by complements or receptors

- G+ bacteria: polysaccharide coat (capsule) Prevent maturation of phagosome

- Mycobacterium tuberculosis Secret enzymes to detoxify the products of the respiratory burst Other phagosome escape

Recruit additional phagocytic cells Proinflammation responses (IL1, IL6, TNF-, TGF-)

- Stimulate endothelial cells to produce proteins for blood clotting(inhibit pathogen spreading but also can cause sepsis)

- Inappropriate or overzealous inflammatory responses (asthma) Inflammatory responses

- Lipid (postaglandins) and protein (cytokines) signaling molecules- Some cytokines trigger fever- Some cytokines are chemoattractants (chemokines)- Adhesion proteins to facilitate attachment & extravasion of white blood cells, including neutrophils, lymphocytes, monocytes, and dendritic cells.

Phagocytosis

5 red blood cells& a macrophage

Eosinophils attacking a schistosome larva

Inflammation of the airways in chronic asthma restricts breathing

Responses of virus infected cells Effects of the ds RNA

RNA interference (RNAi) INF- & INF-

- autocrine and paracrine fashion- Jak/STAT signaling for suicide - ribonuclease activation - protein kinase activation to inactivate eIF-2

(protein synthesis initiation factor)- induce innate and adaptive responses - MHCI upregulation in uninfected cells

- enhance NK cells activity to kill virus-infected cells (low MHC) MHC I down regulation (avoid T cell attack)

Adenovirus / HIV: block MHCI transcription Herpes simplex virus / cytomegalovirus: block ER peptide translocators

Apoptosis death receptor / intracellular proteolytic cascade

A natural killer cell attacking a cancer cell

• NK cells do not express Surface marker of either T or B cell lineage

• Posses Fc receptor for IgG For ADCC (Antibody Dependent Cell-mediated Cytotoxicity)

• Also use perforin to kill target

Human Cytomegalovirus (HCMV) Evasion

Dislocation of MHC class I/II heavy chain from the ER

US3 physically associates with class I

MHC, retaining them in the ER

Virus Strategies to Evade the Host Virus Strategies to Evade the Host Immune ResponsesImmune Responses

Avoidance (hide from host immunity)Avoidance (hide from host immunity) Infection of immune privileged sitesInfection of immune privileged sites Down regulation of viral gene expressionDown regulation of viral gene expression Mutation to evade Ab or T cell mediated responses (HIV)Mutation to evade Ab or T cell mediated responses (HIV) Wholesale replacement of viral proteins (influenza)Wholesale replacement of viral proteins (influenza)

Inhibition (actively disrupt host immunity)Inhibition (actively disrupt host immunity) Thymic infection to induce tolerance (LCMV)Thymic infection to induce tolerance (LCMV) APC destruction / Inhibition of Ag presentationAPC destruction / Inhibition of Ag presentation Effecter interference Effecter interference (NK; cytokines; complements...)(NK; cytokines; complements...) Delay of target cell deathDelay of target cell death Infection of T or B lymphocytesInfection of T or B lymphocytes

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