Download MOLB – 2220 Pathogenic Microbiology

MOLB – 2220
Pathogenic Microbiology
Lecture #3
Mechanisms of Bacterial
Virulence – An Overview
Introduction - History
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1800’s to date - Physiology of bacteria under study
1890 – Exotoxin of Clostridium tetani discovered
1903 – Toxic substance from Shigella identified
1931 – polysaccharide capsule associated with
virulence of Streptococcus
• 1941 – One gene-one enzyme concept
• 1944 – “Transforming” principal
The “transforming principal” is DNA!
-from Lewin, Genes III, Wiley & Sons publishers, 1987.
History – cont.
• 1953- Watson/Crick; Hershey/Chase
experiment
• 1954 – “Toxic” factor of anthrax identified.
• 1956 – Key virulence protein of Y. pestis
(plague) discovered.
• 1958 – DNA replication; concept of
“operon”
Birth of Molecular Pathogenesis
Key Nobel Prizes:
• 1958 – Beadle and Tatum: “for their discovery that
genes act by regulating definite chemical events”.
…and Lederberg: "for his discoveries concerning
genetic recombination and the organization of the
genetic material of bacteria"
• 1962 – Watson, Crick, and Wilkins: "for their
discoveries concerning the molecular structure of
nucleic acids and its significance for information
transfer in living material"
• 1969 – Hershey, Chase, Luria, and Delbruck: “for their
discoveries concerning the replication mechanism
and the genetic structure of viruses”
Koch’s “Molecular” Postulates
1. A specific gene should be consistently
associated with the virulence phenotype.
2. When the gene is inactivated, the bacterium
should become avirulent.
3. If the wild type gene is reintroduced, the
bacterium should regain virulence.
4. If genetic manipulation is not possible, then
induction of antibodies specific for the gene
product should neutralize pathogenicity.
- From Falkow, 1988. Rev. Infect. Dis. Vol. 10, suppl 2:S274-276
General Properties of Pathogenicity
• Complex – multiple virulence factors
– Different sets of virulence factors cause different diseases
– Although, change of a single factor may change disease
• Example: EPEC versus EHEC infection
• Acquisition of large “blocks” of genetic material
– Not slow evolution (mutation of existing genes)
– Mobile DNA: mainly plasmids
• Example: Antibiotic resistance markers
– Pathogenicity “Islands”
• Examples: E.coli haemolysin; E. coli P-pili
Characteristics of Virulence Factors
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Virulence “factor” = virulence “determinant”
Gene and/or gene product
Stringently or “loosely” regulated in the bacterium
Expression of multiple virulence factors confer the
disease producing properties on the pathogen.
• Required for virulence, …or merely enhance it
• Proteins or non-proteins
• Defined or undefined biologic function with defined or
undefined mechanisms of action.
– Single or multiple functions (effects)
Common Themes in Bacterial
Pathogenesis
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2.
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4.
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8.
Toxins
Adherence
Invasion
Intracellular lifestyle
Host Immune Modulation
Iron Acquisition
Secretion Mechanisms
Gene Regulation
Bacterial Toxins
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Direct enzymatic mechanism which effects target cells.
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Facilitate spread through tissues
Damage cell membranes
Immunomodulatory
Inhibit protein synthesis
Inhibit release of neurotransmitters
Single cause for disease or just a contributor
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Anthrax vs Shigellosis
Categories:
1.
2.
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4.
5.
Sub-unit
Proteolytic
Pore-forming
Cytoskeletol modulators (CM toxins)
Pyrogenic
Sub-unit (A-B) Toxins
• A sub-unit
– enzymatic (toxic) activity
– Protein synthesis inhibitors
• Examples: E. coli LT; cholera toxin; Shiga toxin
– Adenylate cyclases
• Example: anthrax edema factor
• B sub-unit
– Mediates binding to host cell receptor.
• Host gangliosides (glycolipids) or glycoproteins
– Facilitates translocation of A sub-unit.
Proteolytic Toxins
• Zinc metalloendoproteases
– Neuropathologic effects
• Inhibit release of neurotransmitters
• Delivery-dependent disease presentations
– Oral, example: Botulinum toxin- causes flaccid paralysis
» Cleaves synaptobrevin to inhibit release of
acetylcholine in peripheral nerves
– Wound, example: Tetanus toxin- spastic paralysis
» Cleaves synaptobrevin to inhibit release of
acetylcholine in the CNS.
Proteolytic Toxins – cont.
• Immunoglobulin A (IgA) proteases
– Made by pathogens that colonize mucosal
surfaces
• Examples: Haemophilus, Neisseria, Serratia,
Helicobacter
– Unlike some other toxins, does not require
elaborate secretion system - “autosecreted”
by the bacterium
– Specifically cleaves secretory IgA1
Pore-forming Toxins
• RTX family
– Produced by many Gram negative pathogens
• Example: E. coli haemolysin
– Induce cytolytic effects by insertion into the
target cell membrane
• Sulfhydryl-activated family
– Produced by Gram positive pathogens
• Example: L. monocytogenes Listeriolysin O
• Mediates escape from macrophage vacuole
• Activity triggered by low pH
CM Toxins
• Alter host cell actin filament polymerization state.
– Anti-phagocytic and/or Necrotic effects
• Yersinia Outer Proteins (YOPs)
• E. coli cytotoxic necrotizing factor I (CNF1)
– Enhance cell-cell interactions (example: EPEC)
0.2 µM
Pyrogenic Exotoxins
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“Superantigens”
Potent activators of T-cells
Suppress B-cell responses
Enhance susceptibility to LPS
Stimulate cytokine production
Examples:
– Staphylococcus enterotoxin B (SEB)
– S. aureus toxic shock syndrome toxin
How
superantigen
toxins work…
Adherence
• Requisite to colonization and/or invasion
• Mediated by “adhesins”
– Bind host cell receptors and/or extracellular matrices
– Surface macromolecular structures (polymers)
• Fimbrial: P (Pap) pili; Type IV pili; somatic pili
• Fibrillar: Yersinia YadA and pH6 Ag; E. coli “curli”
– Other afimbrial adhesins
• B. pertusis and Haemophilus FHA
– High molecular weight secreted monomer
“Classic” vs “Curli”
A few words about Flagella
• Large filamentous polymeric structures
• Required for motility
– Tactic response: nutritional and/or physical
– Single or multiple “strands”
– Bundled or dispersed
• Virulence factor in SOME pathogens
– Example: V. cholerae
• Flagellar “motor” assembly may be more
relevant to virulence.
– Evolution of the Type-III protein secretion system
(TTSS)
Flagellar Arangement and
Motor Apparatus
monotrichous
lopho-
peri-
-from Brock, Biology of Microorganisms, 3rd Ed., Prentice Hall Inc., 1979.
Invasion
• Many bacterial pathogens can escape from the
vacuole of a phagocyte.
• …and/or, gain entry into non-phagocytic host cells.
• “Invasins”
Two types:
– Disrupt cytoskeletol architecture
• Host receptor-mediated
• Similar to CM toxins
– Examples: Yersinia Inv; Shigella Ipa
– Direct membrane digestion
• Phospholipases; example: Rickettsia
Intracellular Lifestyle Survival/Growth of pathogen within
phagocytes and/or non-phagocytic cells
• Modification of the phagocytic vacuole
– Inhibition of lysosomal fusion, eg. Brucella
– Induction of “coiling” phagocytosis
• Examples: Legionella; Borrelia
– “Transcytosis”; example: Salmonella
• Movement through target cell and release
• Escape from the vacuole
– Lysis of the vacuolar membrane
• Examples: Yersinia – YopB/D; Shigella - IpaB; Listeria – listeriolysin O
– Movement of bacteria in cytoplasm via host actin filaments
• Examples: Shigella – IscA; Listeria - ActA
Coiling Phagocytosis
-from, Ritteg, et al. 1998. Infect. Immun. 66:627.
Intracellular Virulence Factors
• O2 radical neutralizers; example: Brucella
• Resistance to bactericidal cationic
peptides; example: Legionella
• Proteases which degrade lysosomal
proteins; example: Salmonella
• Inhibition of vacuole acidification
– Example: Mycobacterium
“Alternative Lifestyle”
• Resistance to phagocytosis
– Capsule
• Polysaccharide; example: S. pneumoniae capsule
• Protein; example: Y. pestis F1 antigen
– Fimbriae (pili), example: Group A Streptococcus
– Other surface molecules
• Example: Streptococcal M protein
– Induction of apoptosis
• Programmed cell death
• Contact-dependent secreted virulence factors
– Examples: Yersinia YopJ; Shigella IpaB (note, multi-functional)
Immune Modulation
• Degradation of immune components
– IgA proteases; examples: H. influenza, Clostridium
– Complement proteases; example: S. pneumoniae
– Cytokine proteases; example: Y. pestis
• Downregulation and/or upregulation of cytokines
– Induces “wrong” immune response
– Examples: Brucella; Burkholderia
– Numerous virulence factors – mostly uncharacterized
Immune Avoidance
• Masking of the bacterial surface
– Example: Staph Protein A – binds immunoglobulin
• Antigenic variation
– pili, flagella, LPS, capsule, secreted enzymes,
outer membrane proteins
• Gonococcal pilus
– Rearrangements of over 50 copies of the pilin sub-unit
gene
• Salmonella LPS O side-chain
– Over 60 different serotypes
Iron Acquisition
• Essential to survival (in and out of the host)
• Free iron severely limited within the host
• Iron acquisition systems
– Membrane-bound receptors
• Interact with host iron-binding proteins, such as human
lactoferrin and transferrin
– Secreted (siderophores)
• “Steal” iron from host
• Moves into the pathogen by a specialized transport system.
Bacterial Protein Secretion
“What’s In and What’s Out!”
• Virulence factors must traverse two
membranes in Gram negative pathogens.
– IM: General Secretory Pathway (GSP)
– OM: Protein secretion (transport) mechanisms
More Complex
Simple
Autotransporting
Single Accessory Pathway
Chaparone/Usher
Type I
Type II
Type III
Type IV
Protein Secretion Systems
The “Simplest”
IM
Protein Secretion Systems – Types I and II
Protein Secretion Systems – Types III and IV
Virulence Gene Regulation
“What’s Hot and What’s Not!”
• Requirements for survival outside the host versus
inside the host
could be quite different.
• Most virulence genes are tightly regulated by a
number of environmental cues.
• …but, some are more “loosely” regulated than others.
– Y. pestis F1 capsule
Temperature
– Y. pestis plasminogen activator (Pla)
??
• Regulation is usually tiered.
– non-sequence-specific and sequence-specific regulators
• Conditions affecting expression:
– Temperature, pH, osmolarity, O2 tension, metabolites, [ions],
cell density, others (?)
Summary
• Bacterial pathogenicity is complex- many
virulence factors.
• Expression of virulence factors are
regulated in the host environment.
• Virulence is characterized by common
mechanisms (themes).
• Bacterial pathogens continue to evolve…