strategies for studying microbial pathogenesis

BIOL 533 1 Lecture Three

Strategies for Studying Microbial Pathogenesis

BIOL 533Lecture 3

Medical Microbiology


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



• 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



• Ideally, want model to:– Use same route as human disease– Display same symptoms– Display same virulence

• Alternative: cell culture, organ culture


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


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



• 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



• Approaches for identifying virulence factor and proving its importance in causing disease:– Biochemical– Genetic– Immunological

• Best to combine approaches


Biochemical/Immunological

• Purify molecule and study in vitro

• Yields detailed information about– Cofactors – General physical properties


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


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


Immunological

• Used to ascertain that putative virulence factor is being produced in animal during infection


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


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?


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


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


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?


Genetic

• Limitations of genetic approach:– Variety and interest of mutant from a

given selection or screening depends on cleverness and specificity of the procedure


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


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


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


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


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


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


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


Mutants

• Construct and test mutants for changes in virulence– Common method for obtaining mutants

is to mutagenize with transposons– Screen for loss of virulence


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


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


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


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


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


Mutants

• Certain virulence factors decrease LD50 <100-fold in mice while others, like motility, may not affect LD50 but are important in other models


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


Mutant

• Can make a case that housekeeping genes contribute, as do other genes concerned with bacterial physiology


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


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

• Questions?

• Comments?

• Assignments...

strategies for studying microbial pathogenesis

Technology

potential virulence

genetic material

loss of genes

virulence gene expression

virulence alternative

aspect of virulence

inactivating virulence

putative virulence factor