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What is a pathogen?
interpreted relative to outcome to host
microbe host damage (disease)
pathogenesis:
Types of microbes causing infectious disease
Type Branch Cellular? Genome Nuclear Example
(Kingdom) membrane?
Metazoan Eucarya yes DNA yes Ascarisparasites (Animalia) lumbricoides
Protozoan Eucarya yes DNA yes Plasmodiumparasites (Protista) falciparum
Fungi / Eucarya yes DNA yes Candidayeasts (Fungi) albicans
Bacteria Eubacteria yes DNA no Streptococcus (not Archaea) pyogenes
Viruses no DNA/RNA no Herpes simplex
Prions no no genes no BSE (Mad Cow Disease)
Robert A. Koch 1843-1910
Bacteria must be associated with the lesion
Bacteria must be isolated in pure culture
Isolated bacterium must produce disease when inoculated into human or animal
Bacteria must be re-isolated from intentionally infected animal or human
Koch’s Postulates - 1882Confirming Germ theory of disease
not all bacteria can be cultivated on artificial medium
no animal models for some diseases
Nucleic acid sequence of pathogen should be found in association with disease or diseased organ
Nucleic acid sequence should be absent from healthy individual
Resolution of disease should result in decrease in pathogen associated nucleic acid sequences
Presence of pathogenic nucleic acid in a healthy individual should predict development of disease
Nature of the microorganism inferred from nucleotide sequence should be consistent with biological characteristics of organism
Sequence based findings should be reproducible
Molecular Koch’s Postulates
How to distinguish infection from colonization ~
colonization of an infectious agent no disease = asymptomatic carrier
colonization disease = infection
vs.
colonization by normal flora disease ≠ infection
Asymptomatic colonization by Asymptomatic colonization by pathogenic bacteriapathogenic bacteria
Group B streptococci .… >24% of females - vaginal (neonatal septicemia / pneumonia / meningitis)
Streptococcus pyogenes …. 20-30% - nasopharynx (strep throat / rheumatic fever / “flesh-eating disease”/ scarlet fever)
Streptococcus pneumoniae .... 20-50% - nasopharynx (pneumonia / septicemia / meningitis / ear infections)
Staphylococcus aureus …. >40% - anterior nares (hospital infections, septicemia, pneumonia)
Disease
function of susceptibilityof host
relates to mechanism ofbacterial pathogenesis
immune competent/compromisedimmunizations
agetrauma
geneticsantimicrobial therapy
I. secretion of factors (toxins)
II. direct host cell manipulation
Host bacterial defenses
Natural barriersnormal flora - affected by diet / antibiotics (competitive exclusion)
pH - acidic / alkaline (inhibits growth)
mucin - viscous glycoproteins (barrier / bacterial trapping)
defensins - antibacterial peptides (damage bacterial membranes)
collectins - lectins (bacterial aggregation) (PRR)
epithelial shedding - (removes attached bacteria)
Innate immune responsePRR - (pattern recognition receptor) macrophages / PMNs / dendritic cells / NK cells / mast cells /basophils / eosinophils
Acquired immune responseB-cells / antibody production - extracellular bacteria
T- cells / cytotoxity response - intracellular bacteria
Emergence of infectious disease ~
BoundaryBarrier
time
habitat / niche(reservoir)
homeostasis
habitat changeadapt by regulation
mutation gene transfer
emerginginfectiousdisease
re-establishbalance
most successful bacteria achieve
balance with environment (host)
Two basic types of pathogens
overt (exogenous) pathogen - aggressive pathogen, requires acquired immunity for host defense
opportunistic (endogenous) pathogen - becomes a pathogen when host is compromised
• damage to epithelium• introduction of bacteria to sites where they are not normal flora - linked to presence of foreign body, catheters, prosthesis, biofilms• disruption of normal flora by antibiotics • suppression of immune system by drugs• impairment of host defenses due to infection
Types of diseases caused by bacteria
Localized infection:
Disseminated infection:
Intoxication:
e.g. Clostridium tetani - spores enter / germinate in deep wound - toxin produced - toxin disseminates
e.g. Staphylococcus aureus - wound infection / abcess - organism invades bloodstream - organism invades heart, kidney, lungs, brain bones
e.g. Clostridium botulinum - spores in contaminated food germinate, multiply, produce toxin - toxin ingested - botulism
What is the difference between virulence and
pathogenicity?
virulence = pathogenicity
(ability to cause disease)
LD50 vs. ID50
LD50 - number of bacteria needed to kill 50% of animals
ID50 - number of bacteria necessary to infect 50% of the animals exposed to the bacterium
(Salyers and Whitt, 2002)
higher virulence lower virulence
Measurement of virulence
What is a virulence factor?
AnyAny microbial product or strategy
that contributes to disease ~
Infectious process ~
colonizationgrowth
establish disease
Virulence factors of bacteria
I. adherence / colonization:
pilimotility / chemotaxis (flagella)outer membrane proteins
II. infectious process:exotoxins / endotoxinstype III / type IV secretion processesintracellular growth (invasion)
III. protection against host defense:capsule / cell wall / outer membrane antigenic variationbiofilm formation
pilus-pili-fimbraie
Scanning electron micrograph showing microcolonies of EPEC displaying the characteristic localized adherence pattern of adherence to HEp-2 cells.
Function:- in attachment- movement on host cell surface
- immunodominant protein neutralized by host antibodies vaccine candidate
flagellum / flagella
Function:helical filament rotates - used for- swimming- chemotaxis
- filaments rotate together either clockwise or counterclockwise
ccw rotation = smooth swimming cw rotation = a “tumble”
- flagellin - PAMP
Smooth swimming Tumble
Macnab, Ornston, 1977Kahn 1978)
flagella movement:
(Copyright: ASM Hoch and Silhavy p. 91,)
counter-clockwise rotation clockwise rotation
no attractant attractant
- bacteria move by a biased random walk
chemotaxis:(movement in response to an environmental stimuli)
outer membrane proteins (OMPs)
Function:- adhesin
- invasion
(Prasadarao et al, Infect. Immun. 1999)
E. coli invasion
E. coli - OmpA facilitated binding / invasion
Antibody - against OmpA blocks invasion
II. Infectious process
Model infectious agents will be used to examine mechanisms of bacterial pathogenesis
exotoxins / endotoxinstype III / type IV secretion systemintracellular growth
Bacterial model systems ~
• mechanism of toxicity • protein design (structure vs. function) • vaccine production• tool to manipulate host cell function• toxin re-design for therapeutics• biological warfare
Bacterial toxins:
diphtheria toxin - cholera toxin
Clostridium neurotoxins
S SA-subunit B-subunit
L enzyme activity / receptor binding / internalization intracellular trafficking
Bacterial model systems ~Intracellular survival (invasion):
Shigella flexneri Listeria moncytogenes Legionella pneumophilia macropinocytosis/memb ruffling zipper-type coiling phagocytosis
(from P. Cossart, Cellular Microbiology, 2000)
Mycobacterium tuberculosisYersinia spp.
Listeria monocytogenes
Bacterial model systems ~Bacterial secretion:
(Kubori, 1998, Blocker, 2001, Plano, 2001)
• type III secretory process• direct manipulation of host cell signaling proteins• Pseudomonas aeruginosa• Salmonella spp.
Bacterial model systems ~Emerging diseases:
Comparison of pathogens
• EPEC• ETEC• EAggEC• EHEC - (OH157:H7)• EIEC• Salmonella• Shigella
Escherichia coli
III. Bacterial protection against host defense
antigenic variation
capsule / cell wall / outer membrane
biofilm formation
capsule /cell wall /outer membrane
The surface of Bacillus anthracis. The bacterial membrane is evident as the innermost layer surrounding the cytoplasm. P denotes the peptidoglycan cell wall. S refers to the S-layer which consists of two proteins including the major antigen. C denotes the poly-D-glutamic acid capsule that is exterior to and completely covers the S-layer proteins. (From Mesnage, et al. Journal of Bacteriology, 180:52-58, 1998)
capsule(highly hydrated gelatinous matrix - composed of
carbohydrates and proteins)
Streptococcus pneumoniae -Capsule is composed of polysaccharide. It is the most important determinant of virulence ~ allows the bacteria to escape phagocytes in the lung. India ink stain. (K. Todar)
Bacillus anthracis -Capsule is composed of poly-D-glutamic acid. It is anti-phagocytic and protects the bacteria from complement- mediated lysis in serum or blood. Fluorescent- tagged antibody. (CDC)
Streptococcus pyogenes - Capsule is composed of hyaluronic acid, the same polymer found in human connective tissue. This is an antigenic disguise that prevents bacterial recognition by the immune system. Transmission electron micrograph. (M. Fazio, V. Fischetti, Rockefeller University)
Function:- resistance to phagocytosis, desiccation, antibiotics, detergents - highly antigenic (K antigen of bacteria) used in vaccines- attachment / sequestration of nutrients- loss of capsule can result in loss of virulence
biofilm(living capsule - 3D-bacterial community growing on a surface)
Examples of biofilms:dental plaquegrowth on cathetersgrowth in heart tissuechronic cystic fibrosis lung infection
natural biofilm - slimy (green) mass attached to rocks in mountain streams
Function:- resistance to antibodies, phagocytosis, desiccation,antibiotics, detergents - attachment / sequestration of nutrients- formation of inter-dependent microbial communities
(www.math.utah.edu/.../ quorum_talk.html)
Bacterial biofilm - magnified 7,000x
Concepts
• terms ~ pathogenesis /virulence, colonization, infection, disease
• understanding of a pathogen ~ origin of emerging infectious disease
• types of diseases caused by bacteria
• definition of virulence factor ~ types of virulence factors those that affect: I. adherence / colonization II. infectious process III. protection against host defense