Strategies for Studying Microbial Pathogenesis

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Strategies for Studying Microbial Pathogenesis
Medical Microbiology

• BIOL 533
• Lecture 3

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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 (immortalmany
  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?

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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 organisms 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
  10.13Colony 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 dont 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 (lt30 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 examinationmust 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

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Mutants

• Certain virulence factors decrease LD50 lt100-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

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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

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Mutant

• Introduction by plasmid (suicide vector)
• Common way to introduce transposon into
  chromosome
• Also could be done with intact or inactivated
  cloned gene

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Lecture Three

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• Assignments...