Pathogenic bacteria &virulence factors

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)

How do you identify a pathogen?

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

I. Adherence / colonization:

pilimotility / chemotaxis (flagella)outer membrane proteins

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

Neisseria gonorrhoeae adaptation - antigenic variation of pili

Antigenic variation

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