1. Pathogenic bacteria & virulence factors

2. What is a pathogen? pathogenesis:
interpreted relative to outcome to host
microbe host damage (disease)
pathogenesis:
Goal - not a comprehensive microbial pathogenesis course -
use model bacterial systems to explore mechanisms of microbial pathogenesis

3. Types of microbes causing infectious disease
Type Branch Cellular? Genome Nuclear Example
(Kingdom) membrane?
Metazoan Eucarya yes DNA yes Ascaris
parasites (Animalia) lumbricoides
Protozoan Eucarya yes DNA yes Plasmodium
parasites (Protista) falciparum
Fungi / Eucarya yes DNA yes Candida
yeasts (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)

4. How do you identify a pathogen?

5. Confirming Germ theory of disease
Robert A. Koch
Koch’s Postulates
Confirming Germ theory of disease
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
not all bacteria can be cultivated on
artificial medium
Caveats
2. Cultivation difficulties
3. Potential problem in developing appropriate animal model system
no animal models for some diseases

6. Molecular Koch’s Postulates
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

7. How to distinguish infection from colonization ~
colonization disease = infection
vs.
colonization by normal flora disease ≠ infection
Diagnosticallysometimes difficult to distinguish between normal flora and pathogen
colonization of an infectious agent no disease =
asymptomatic carrier

8. Asymptomatic colonization by pathogenic bacteria
Group B streptococci .… >24% of females - vaginal
(neonatal septicemia / pneumonia / meningitis)
Streptococcus pyogenes … % - nasopharynx
(strep throat / rheumatic fever / “flesh-eating disease”/ scarlet fever)
Streptococcus pneumoniae % - nasopharynx
(pneumonia / septicemia / meningitis / ear infections)
Staphylococcus aureus …. >40% - anterior nares
(hospital infections, septicemia, pneumonia)

9. Disease function of susceptibility of host relates to mechanism of
bacterial pathogenesis
immune competent/compromised
immunizations
age
trauma
genetics
antimicrobial therapy
I. secretion of factors (toxins)
II. direct host cell manipulation

10. Host bacterial defenses
Natural barriers
normal 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 response
PRR - (pattern recognition receptor)
macrophages / PMNs / dendritic cells / NK cells / mast cells /
basophils / eosinophils
Acquired immune response
B-cells / antibody production - extracellular bacteria
T- cells / cytotoxity response - intracellular bacteria

11. Emergence of infectious disease ~
Boundary
Barrier
habitat / niche
(reservoir)
homeostasis
emerging
infectious
disease
re-establish
balance
time
habitat change
adapt by regulation mutation
gene transfer
most successful bacteria achieve balance with environment (host)

12. 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

13. Types of diseases caused by bacteria
Localized infection:
e.g. Clostridium tetani
- spores enter / germinate in deep wound
- toxin produced
- toxin disseminates
Disseminated infection:
e.g. Staphylococcus aureus
- wound infection / abcess
- organism invades bloodstream
- organism invades heart, kidney, lungs, brain bones
Intoxication:
e.g. Clostridium botulinum
- spores in contaminated food germinate,
multiply, produce toxin
- toxin ingested - botulism

14. What is the difference between virulence and pathogenicity?
virulence = pathogenicity
(ability to cause disease)

15. Measurement of virulence
(Salyers and Whitt, 2002)
higher virulence lower virulence
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

16. What is a virulence factor?
Any microbial product or strategy
that contributes to disease ~
Infectious process ~
colonization
growth
establish disease

17. Virulence factors of bacteria
I. adherence / colonization:
pili
motility / chemotaxis (flagella)
outer membrane proteins
II. infectious process:
exotoxins / endotoxins
type III / type IV secretion processes
intracellular growth (invasion)
III. protection against host defense:
capsule / cell wall / outer membrane
antigenic variation
biofilm formation

18. I. Adherence / colonization:
pili
motility / chemotaxis (flagella)
outer membrane proteins

19. pilus-pili-fimbraie Function: - in attachment
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

20. 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

21. flagella movement: Smooth swimming Tumble
Macnab, Ornston, 1977
Kahn 1978)
Smooth swimming Tumble
(Copyright: ASM Hoch and Silhavy p. 91,)
counter-clockwise rotation clockwise rotation

22. chemotaxis: (movement in response to an environmental stimuli)
no attractant
attractant
- bacteria move by a biased random walk

23. outer membrane proteins
(Prasadarao et al, Infect. Immun. 1999)
E. coli invasion
E. coli - OmpA facilitated
binding / invasion
Antibody - against OmpA
blocks invasion
outer membrane proteins
(OMPs)
Function:
- adhesin
- invasion

24. II. Infectious process exotoxins / endotoxins
type III / type IV secretion system
intracellular growth
Model infectious agents will be used to examine mechanisms of bacterial pathogenesis

25. Bacterial model systems ~
Bacterial toxins:
diphtheria toxin - cholera toxin
Clostridium neurotoxins
S S
A-subunit
B-subunit
L enzyme activity / receptor binding / internalization
intracellular trafficking
mechanism of toxicity
protein design (structure vs. function)
vaccine production
tool to manipulate host cell function
toxin re-design for therapeutics
biological warfare

26. 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 tuberculosis
Yersinia spp.
Listeria monocytogenes

27. 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.

28. Bacterial model systems ~
Emerging diseases:
Escherichia coli
Comparison of pathogens
EPEC
ETEC
EAggEC
EHEC - (OH157:H7)
EIEC
Salmonella
Shigella
DAEC - diffusely adhering E. coli

29. III. Bacterial protection against host defense
antigenic variation
capsule / cell wall / outer membrane
biofilm formation

30. Neisseria gonorrhoeae adaptation - antigenic variation of pili

31. 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)

32. capsule
(highly hydrated gelatinous matrix - composed of carbohydrates and proteins)
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
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)

33. (living capsule - 3D-bacterial community growing on a surface)
biofilm
(living capsule - 3D-bacterial community growing on a surface)
Function:
- resistance to antibodies, phagocytosis,
desiccation,antibiotics, detergents
- attachment / sequestration of nutrients
- formation of inter-dependent microbial
communities
( quorum_talk.html)
Bacterial biofilm - magnified 7,000x
Examples of biofilms:
dental plaque
growth on catheters
growth in heart tissue
chronic cystic fibrosis lung infection
natural biofilm - slimy (green) mass attached to rocks in mountain streams

34. 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