微生物学美国IndianaUniversityPurdueUniversity授课03

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(see Nester 10.13—Colony Hybridization)
– Bordetella pertussis
• Intracranial • Interperitoneal • Respiratory aspiration
BIOL 533
3
Lecture Three
Choosing an Animal Model
• Ideally, want model to:
BIOL 533
10
Lecture Three
Immunological
• Determine whether Ab to bacterial product are protective in infected animals
• Possible problem:
– Ab to bacterial surface molecules might prevent infection by opsonizing or enhancing complement action rather than inactivating virulence factor
– S. typhimurium is pathogen and normal E. coli is not; therefore, the differing
sequences may be virulence genes
BIOL 533
20
Lecture Three
Wild Type
• Experimental technique:
– Measurements on isolated molecules may not accurately reflect function in intact bacterium
• Prove function in vivo; have to take either genetic
or immunological approach
BIOL 533
19
Lecture Three
Wild Type
• Example: E. coli and S. typhimurium
– Chromosomal maps very similar
– S. typhimurium has DNA sequences that E. coli does not
-OR-
may give different symptoms
• Given disease may have a number of animal models, none of which fully satisfies characteristics of disease
BIOL 533
2
Lecture Three
– Difficult to determine specific function of virulence genes
– Example: loss of ability to invade kidney cell
• Loss of regulatory protein needed for activation?
– Use same route as human disease – Display same symptoms – Display same virulence
• Alternative: cell culture, organ culture
BIOL 533
4
Lecture Three
Cell Culture/Organ Culture
M. smegnatis is faster-growing; methods may be applicable to M. tuberculosis
BIOL 533
7
Lecture Three
Studying Pathogenic Organisms
• Approaches for identifying virulence factor and proving its importance in causing disease:
BIOL 533
15
Lecture Three
Genetic
• Strengths of genetic approach:
– Starts with function of known importance
– Isolating mutants with this function affected can lead to discovering new virulence factors that previously had no assay
BIOL 533
6
Lecture Three
Studying Pathogenic Organisms
• Look at other, similar members of the same genus; for example:
– M. smegnatis vs. – M. tuberculosis
BIOL 533
13
Lecture Three
Genetic
• Test mutants for changes in virulence
-OR-
• Introduce cloned genes back into avirulent mutants; is virulence restored?
• Use one organism’s DNA as a probe and hybridize with DNA from related organism
– If pathogenic strain contains genetic material that is absent from non-pathogenic strain, that material may encode genes that confer pathogenicity
Choosing an Animal Model
• One model may show certain aspects of disease, but not another
• Different models may rely on different routes of introducing pathogen; e.g.:
• Difficult
• Cell-lines often tumor lines that are genetically
and physiologically different (immortal—many mutations)
• Removed from effects of other organs, hormones
– Biochemical – Genetic – Immunological
• Best to combine approaches
BIOL 533
8
Lecture Three
Biochemical/Immunological
• Purify molecule and study in vitro
BIOL 533
11
Lecture Three
Immunological
• Used to ascertain that putative virulence factor is being produced in animal during infection
BIOL 533
12
Lecture Three
• Yields detailed information about
– Cofactors – General physical properties
BIOL 533
9
Lecture Three
Biochemical/Immunological
• Two limitations:
– Molecule must be assayable; most applicable if know product and function
-OR-
• Identify potential virulence genes by regulation; are they co-regulated?
BIOL 533
14
Lecture Three
Genetic
• In Vivo Experimental Technique (IVET)
Identify in vivo-induced (ivi) genes that are
highly expressed in animal tissues, but not in laboratory media
• Limitation of techniques (see slide 14):
– Each requires some understanding of lab conditions to get virulence gene expression
• Limitations of genetic approach:
– Variety and interest of mutant from a given selection or screening depends on cleverness and specificity of the procedure
• Look at phylogeny to find closely related organisms; for example:
– S. typhimurium vs. – S. typhi
One may respond more easily than the other to variety of genetic techniques
• Loss of invasin structural gene?
• Loss of genes needed for processing, localizing? • Function having some indirect effect?
BIOL 533
ห้องสมุดไป่ตู้
17
Lecture Three
Genetic
• Cells grown in artificial media differ from in vivo
• Cell lines may not express same Ag on surface as when in animal
BIOL 533
5
Lecture Three
Studying Pathogenic Organisms
BIOL 533
18
Lecture Three
Wild Type
• Sequence wild-type or mutated gene:
– Sometimes find unexpected relationships
– Useful only if match known gene sequence
Medical Microbiology
Strategies for Studying Microbial Pathogenesis
BIOL 533 Lecture 3
BIOL 533
1
Lecture Three
Choosing an Animal Model
• Pathogen may not affect animal at all
– Also, connection between genes and some aspect of virulence is established from the beginning
BIOL 533
16
Lecture Three
Genetic
• Limitations of genetic approach:
Genetic
• Sequence wild-type gene and compare to others
– Sequence identity and similarity infers function
• Hybridize to related species and make mutations in gene that encodes virulence factor
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