strategies for studying microbial pathogenesis
TRANSCRIPT
BIOL 533 1 Lecture Three
Strategies for Studying Microbial Pathogenesis
BIOL 533Lecture 3
Medical Microbiology
BIOL 533 2 Lecture Three
Choosing an Animal Model
• Pathogen may not affect animal at all -OR-
may give different symptoms
• Given disease may have a number of animal models, none of which fully satisfies characteristics of disease
BIOL 533 3 Lecture Three
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.:– Bordetella pertussis
• Intracranial• Interperitoneal• Respiratory aspiration
BIOL 533 4 Lecture Three
Choosing an Animal Model
• Ideally, want model to:– Use same route as human disease– Display same symptoms– Display same virulence
• Alternative: cell culture, organ culture
BIOL 533 5 Lecture Three
Cell Culture/Organ Culture
• Difficult• Cell-lines often tumor lines that are genetically
and physiologically different (immortal—many mutations)
• Removed from effects of other organs, hormones
• Cells grown in artificial media differ from in vivo• Cell lines may not express same Ag on surface
as when in animal
BIOL 533 6 Lecture Three
Studying Pathogenic Organisms
• 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
BIOL 533 7 Lecture Three
Studying Pathogenic Organisms
• Look at other, similar members of the same genus; for example:– M. smegnatis vs.– M. tuberculosis
M. smegnatis is faster-growing; methods may be applicable to M. tuberculosis
BIOL 533 8 Lecture Three
Studying Pathogenic Organisms
• Approaches for identifying virulence factor and proving its importance in causing disease:– Biochemical– Genetic– Immunological
• Best to combine approaches
BIOL 533 9 Lecture Three
Biochemical/Immunological
• Purify molecule and study in vitro
• Yields detailed information about– Cofactors – General physical properties
BIOL 533 10 Lecture Three
Biochemical/Immunological
• Two limitations:– Molecule must be assayable; most
applicable if know product and function
– 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 11 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
BIOL 533 12 Lecture Three
Immunological
• Used to ascertain that putative virulence factor is being produced in animal during infection
BIOL 533 13 Lecture Three
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
BIOL 533 14 Lecture Three
Genetic
• Test mutants for changes in virulence-OR-
• Introduce cloned genes back into avirulent mutants; is virulence restored?-OR-
• Identify potential virulence genes by regulation; are they co-regulated?
BIOL 533 15 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
BIOL 533 16 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
– Also, connection between genes and some aspect of virulence is established from the beginning
BIOL 533 17 Lecture Three
Genetic
• Limitations of genetic approach:– Difficult to determine specific function of
virulence genes
– Example: loss of ability to invade kidney cell• Loss of regulatory protein needed for activation?• Loss of invasin structural gene?• Loss of genes needed for processing, localizing?• Function having some indirect effect?
BIOL 533 18 Lecture Three
Genetic
• Limitations of genetic approach:– Variety and interest of mutant from a
given selection or screening depends on cleverness and specificity of the procedure
BIOL 533 19 Lecture Three
Wild Type
• Sequence wild-type or mutated gene:– Sometimes find unexpected relationships– Useful only if match known gene sequence
• 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
BIOL 533 20 Lecture Three
Wild Type
• Example: E. coli and S. typhimurium– Chromosomal maps very similar– S. typhimurium has DNA sequences that
E. coli does not– S. typhimurium is pathogen and normal
E. coli is not; therefore, the differing sequences may be virulence genes
BIOL 533 21 Lecture Three
Wild Type
• Experimental technique:(see Nester 10.13—Colony Hybridization)
Recombinant plasmids containing S. typhimurium-specific sequences identified on filter blots as not hybridizing to probe made from entire E. coli chromosome
BIOL 533 22 Lecture Three
Wild Type
• Results:
– 6.4 kb region maps to minute 60 on chromosome, and deletions abolish ability of S. typhimurium to enter epithelial cells
– Similar analyses revealed other genes
BIOL 533 23 Lecture Three
Mutant
• Cloned genes introduced into avirulent mutants or E. coli
• Works for E. coli only if foreign gene can be expressed in E. coli ; most cannot:– May not have accessory genes needed
(e.g., capsule)– May not have necessary regulatory
sequences
BIOL 533 24 Lecture Three
Mutant
• Example:– Ordinary E. coli strains don’t adhere to
or invade tissue culture monolayers– Potential adhesins and invasins can be
identified by screening for clones containing DNA sequences that enable E. coli to adhere or invade monolayers
BIOL 533 25 Lecture Three
Mutant
• Limitations:– Standard cloning techniques isolate only
small portions of genome (<30 kb)– Approach works best if only one or a few
genes are required for trait to be expressed
– Gene must be expressed in E. coli– Approach most successful when foreign
organism is closely related to E. coli
BIOL 533 26 Lecture Three
Mutants
• Construct and test mutants for changes in virulence– Common method for obtaining mutants
is to mutagenize with transposons– Screen for loss of virulence
BIOL 533 27 Lecture Three
Mutant
• Advantages:– Every selected colony has selectable
phenotype– Most disrupt a gene– Transposon serves as marker to locate
gene; useful for cloning– Can be used to detect genes not
expressed in E. coli or not closely linked to other virulence genes
BIOL 533 28 Lecture Three
Mutant
• Limitations:– Carrying transcriptional terminators
• If transposon inserts into first gene in operon, eliminates transcription for that gene and other genes as well; therefore, insertions are polar
• Avirulent phenotype could be due to loss of expression of downstream gene
– Will not work with essential genes, because organism will not survive to form colony
BIOL 533 29 Lecture Three
Mutant
• Groisman and Heffron– Pilot study– Screened 400 random transposon
mutants for virulence in mice– Results:
• 2% of mutations increased IP 50% lethal dose (LD50) by 10,000
• 6% increased oral LD50
BIOL 533 30 Lecture Three
Mutant
• If S. typhimurium has 3,000 genes, results of this pilot study would suggest that 60 to 180 genes play a role in pathogenesis.
• Further examination—must consider:– Definition of virulence gene– Defects found among avirulent mutants
BIOL 533 31 Lecture Three
Mutants
• Further examination:– Difficult to identify mutants with weak
effect on LD50
– Not ideal, because Salmonella pathogenesis varies in severity
– Many different properties affect infection process
BIOL 533 32 Lecture Three
Mutants
• Certain virulence factors decrease LD50 <100-fold in mice while others, like motility, may not affect LD50 but are important in other models
BIOL 533 33 Lecture Three
Mutant
• Therefore, using one infection model and specific definition of virulence, study probably underestimated number of virulence genes
• However, may also have overestimated if you eliminate housekeeping genes, such as recA, that have other functions
BIOL 533 34 Lecture Three
Mutant
• Can make a case that housekeeping genes contribute, as do other genes concerned with bacterial physiology
BIOL 533 35 Lecture Three
Mutant
• Identifying virulence genes by regulation– Virulence genes are frequently in
operons and regulons controled by same proteins
– If one gene found, other genes may also be found
– Approach uses transcriptional fusions
BIOL 533 36 Lecture Three
Mutant
• Introduction by plasmid (suicide vector)– Common way to introduce transposon
into chromosome– Also could be done with intact or
inactivated cloned gene
BIOL 533 37 Lecture Three
Lecture Three
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