Lecture Three BIOL 533 1
Strategies for Studying
Microbial Pathogenesis
BIOL 533
Lecture 3
Medical Microbiology
Lecture Three BIOL 533 2
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
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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
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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
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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
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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
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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
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Studying Pathogenic Organisms
? Approaches for identifying virulence factor
and proving its importance in causing
disease,
– Biochemical
– Genetic
– Immunological
? Best to combine approaches
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Biochemical/Immunological
? Purify molecule and study in vitro
? Yields detailed information about
– Cofactors
– General physical properties
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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
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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
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Immunological
? Used to ascertain that putative virulence
factor is being produced in animal during
infection
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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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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?
Lecture Three BIOL 533 15
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
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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
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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?
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Genetic
? Limitations of genetic approach,
– Variety and interest of mutant from a given
selection or screening depends on cleverness
and specificity of the procedure
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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
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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
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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
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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
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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
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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
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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
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Mutants
? Construct and test mutants for changes in
virulence
– Common method for obtaining mutants is to
mutagenize with transposons
– Screen for loss of virulence
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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
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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
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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
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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
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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
Lecture Three BIOL 533 32
Mutants
? Certain virulence factors decrease LD50
<100-fold in mice while others,like
motility,may not affect LD50 but are
important in other models
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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
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Mutant
? Can make a case that housekeeping
genes contribute,as do other genes
concerned with bacterial physiology
Lecture Three BIOL 533 35
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
Lecture Three BIOL 533 36
Mutant
? Introduction by plasmid (suicide vector)
– Common way to introduce transposon into
chromosome
– Also could be done with intact or inactivated
cloned gene
Lecture Three BIOL 533 37
Lecture Three
? Questions?
? Comments?
? Assignments..,