Lecture 4 BIOL 533 1
Strategies for Studying
Microbial Pathogenesis
BIOL 533
Lecture 4
Medical Microbiology
Lecture 4 BIOL 533 2
Reporter Gene Fusions
? Operon fusion,transcriptional
– Promoter from foreign gene
– Translational sequence from reporter gene
Lecture 4 BIOL 533 3
Reporter Gene Fusions
? Gene fusion,translational
– Promoter and translational signal from
foreign gene
? Initiation signal/+NH2 coding sequence
– Translational signal also from reporter gene
? Initiation sequence and part of NH2 sequence of
reporter gene missing,but COOH region still
present
– Two coding sequences must be in frame
Lecture 4 BIOL 533 4
Technique
? Use transposon vector containing lac
fusion
– Carries promoterless lacZ gene and selectable
antibiotic marker
– Selection for antibiotic marker generates set
of random insertions in chromosome
– Colonies carrying transposon screened for
expression under conditions tested
Lecture 4 BIOL 533 5
Foreign Gene Expression
? Assay for production of βgal
– Higher temperature
– +— iron
– etc,
? Locate unknown gene
Lecture 4 BIOL 533 6
lacZ Fusion
? Used to find regulatory genes that control
expression of virulence gene
– lacZ fusion strain is mutagenized (chemical or
another transposon)
– Resulting colonies screened for aberrant
regulation
Lecture 4 BIOL 533 7
lacZ Fusion
? Aberrant regulation,
– Colonies are not Lac+ in presence of inducing
conditions
? Loss of activator
– Colonies are Lac+ under both inducing and
non-inducing conditions
? Loss of repressor
Lecture 4 BIOL 533 8
lacZ Fusion
? Gene tagged by transposon can be cloned
– Segment of DNA adjacent to transposon,
? Can be used as hybridization probe to clone wild-
type locus
? Can also be cloned by complementation
? Cloned DNA can be sequenced
Lecture 4 BIOL 533 9
phoA Gene Fusions
? Virulence genes are small subset of total
genes,so random mutagenesis will affect
many more non-virulence genes
? Way to optimize number of virulence
gene mutations takes advantage of fact
that most virulence genes are on bacterial
surface or excreted into medium
Lecture 4 BIOL 533 10
phoA Gene Fusions
? Translational protein fusion
? Alkaline phosphatase is expressed only in
periplasm
? Foreign gene product has to be expressed
outside of cytoplasm
? In vector,signal sequence mutations
block export of alkaline phosphatase and
render it inactive
Lecture 4 BIOL 533 11
phoA Gene Fusions
? Export and activity restored by fusing in frame a
restriction fragment to truncated phoA gene;
i.e.,lacking signal sequences
– No AUG start codon
– No ribosome binding site
? Restriction fragment has portions of NH2-
terminal genes encoding signal sequences of
heterologous proteins,such as OmpF or LamB
Lecture 4 BIOL 533 12
TnphoA Transposon
? Created TnphoA that randomly inserts
phoA into target genes
? Hybrid proteins display alkaline
phosphatase activity only if target gene
encodes a periplasmic outer membrane or
extracellular protein
Lecture 4 BIOL 533 13
TnphoA Transposon
? Use TnphoA,
– Study topology of inner membrane proteins
? βgal fusions expressed only in cytoplasm
– Identify genes that encode export proteins
? Exported proteins represent major class of
virulence factors
? Number of additional delivery systems
? Remember analysis of Groisman and Heffron
Lecture 4 BIOL 533 14
Analysis of Virulence
? Study TnphoA mutants of S,typhimurium
identified as Pho+ on L-agar plates
– Tested for virulence by oral infection of
BALB/C mice
– Found 15/150 Pho+ mutants were avirulent
? 9/15 were defective in LPS biosynthesis
? None had insertions in virulence plasmid
Lecture 4 BIOL 533 15
Analysis of Virulence
? Conclusion,same caveats as apply to
normal transposon mutagenesis,
– But use of TnphoA limits identification of
random insertions to those affecting cell
envelope factors
Lecture 4 BIOL 533 16
Analysis of Virulence
? Disadvantage,
– Limits selection to genes expressed in
S,typhimurium on L-agar
– Genes expressed at low levels or repressed
on L-agar plates would be missed
Lecture 4 BIOL 533 17
Analysis of Virulence
? Expression problem may explain why 9/15
Pho+ avirulent mutant strains were LPS—
rather than being defective in other
virulence genes
Lecture 4 BIOL 533 18
Analysis of Virulence
? TnphoA narrows,but doesn’t solve all
detection problems
– While disrupting gene,the TnphoA mutations
produce alkaline phosphatase hybrid proteins
– Could be phenotype not due to loss of gene,
but presence of deleterious hybrid protein;
– Therefore,to be sure mutation in virulence
gene,must conduct other genetic analyses
Lecture 4 BIOL 533 19
In Vivo Expression Technology
Mahan et,al.,1993,Science 259,686-688
? Based on gene that,when mutated,
causes organism to be attenuated
? Used Salmonella typhimurium
? ΔpurA — lacZY both lack promoter
(in suicide plasmid)
Lecture 4 BIOL 533 20
In Vivo Expression Technology
? Sau3A digested chromosomal DNA
?electroporated Salmonella PurA— strain
? Injected into mice twice
? After each 3 days
?harvest spleen
? Plate on artificial media to detect Lac—
strains
Lecture 4 BIOL 533 21
In Vivo Expression Technology
? Based solely on fact that ivi genes are
highly expressed in animal tissues,but
are not expressed in lab media
? Selection strategy,
– Bacterial strain with mutation that attenuated
growth in vivo
– Complemented by operon fusions to same
attenuated gene
Lecture 4 BIOL 533 22
In Vivo Expression Technology
? Criteria for IVET system,
– Fusions in single chromosomal copy
? No plasmid complications
– Fusions constructed without disruption of
gene
? Loss of gene could be lethal to bacterium
– Monitored both in animal and on lab media
Lecture 4 BIOL 533 23
pIVET Vector
? pIVET1 plasmid
– Suicide vector
? Replication dependent on Pi replication protein
Salmonella does not produce
– Promoterless purA gene and lacZ operon
? Can monitor level or transcription of entire operon
– Unique cloning site 5’ to purA gene
? Clone random fragments of DNA
– bla gene allows plasmid to be selected
Lecture 4 BIOL 533 24
pIVET Vector
? Construction of clones
– Clone Sau3A fragments into vector
– Put fragment pool into PurA deletion strain of
Salmonella (—Pi replication protein)
– Selection for ApR plasmid,integrates at
homologous Salmonella gene
? No natural lacZ gene
? Deleted purA gene
Lecture 4 BIOL 533 25
pIVET Vector
? Clones of interest
– Product of plasmid integration generates a
duplication
? One promoter drives purA lacZ while other drives
wild-type copy of virulence gene
? Integrate at unknown gene because purA and lacZ
are from E,coli; not enough homology to
Salmonella
Lecture 4 BIOL 533 26
Properties of Clones
? To survive and propagate in host,
– purA lacZ fusion has to be expressed at
sufficient level to overcome deletion of purA
– If gene of interest codes for virulence factor,
then expression also required for infection
– Monitor expression in animal and lab by
measuring βgal from strains that survive
three days and are recovered from spleen
Lecture 4 BIOL 533 27
Analysis of Virulence
– To identify genes code for virulence,
junctions of fusions cloned and sequenced
– Prepared inactivated genes
? Any effect on pathogenesis?
– Transposon insertions in 3/5 genes
– BALB/C mice infected orally have increased
LD50 (200 to 20,000 fold)
? Therefore,IVET selects for virulence
– Also,mice immunized with 1 mutant were
protected when challenged with wild-type
Lecture 4 BIOL 533 28
Identity of Genes
? Results,
– Most genes found so far are housekeeping
genes,perhaps because of screening
conditions
? Screened only for expression of genes on aerobic
media,but most body parts are anaerobic
– Crude approach; at best,identifies a set of
genes that are expressed at some point of
infection
Lecture 4 BIOL 533 29
Identity of Genes
? pheST himA operon encodes 2 su
phenylalanyl t-RNA synthetase and 1 su
host integration factor (IHF)
? Antisense Rfb O-antigen synthesis
– O-antigen synthesis mutants attenuated-
orally,but virulent IP route
– Mutations that increase LD50 do affect
virulence
Lecture 4 BIOL 533 30
Pros and Cons
? Detailed analysis of genes and gene
products expressed in animal necessary to
determine where and when during
infection the gene was expressed
? More serious problem—focuses on
transcriptional regulation
Lecture 4 BIOL 533 31
Pros and Cons
? Genes that are regulated or activated
post-transcriptionally will be missed
? Also,many genes expressed on lab media
may be important in vivo
? If some aspect of in vitro growth
condition is similar or identical to in vivo
condition (e.g.,37° C),certain genes
may be discarded
Lecture 4 BIOL 533 32
Research Strategy
? To get maximal variability in genes
selected,use different,
– Reporter genes
– Infection models
– in vitro growth conditions
? Likely that infection by different routes or
hosts characterized by unique H-P
interactions lead to different genes
Lecture 4 BIOL 533 33
Analysis of Virulence
Heithoff et,al.,1999 (Mahan’s group)
? Previously found over 100 different genes
– 25% unidentified
– All affect virulence
? Looked at regulation of genes in intestine,
liver,and spleen,plus cultured murine
macrophage
Lecture 4 BIOL 533 34
Analysis of Virulence
? Regulation in response to Mg+2,pH,and
Fe proceeded same as in vitro at
approximately the same levels
? Not true in spleen; has to be different
signals
? Think that response to Mg+2 and pH
correlates with assuming macrophage
existence
Lecture 4 BIOL 533 35
Uses
? Expected to provide Ag for vaccine
– Genes expressed only in vivo
? Assist in construction live-attenuated
vaccines (inactivated genes)
? Also can establish in vivo regulated
expression of heterologous Ag in live
vaccines
Lecture 4 BIOL 533 36
Uses
? Identification of ivi genes allows detection
of changes in metabolism,gene
regulation,and cell surface properties
during growth in animal tissues
? Allows new antimicrobial targets to be
identified
Lecture 4 BIOL 533 37
Lecture 4
? Questions?
? Comments?
? Assignments..,
Strategies for Studying
Microbial Pathogenesis
BIOL 533
Lecture 4
Medical Microbiology
Lecture 4 BIOL 533 2
Reporter Gene Fusions
? Operon fusion,transcriptional
– Promoter from foreign gene
– Translational sequence from reporter gene
Lecture 4 BIOL 533 3
Reporter Gene Fusions
? Gene fusion,translational
– Promoter and translational signal from
foreign gene
? Initiation signal/+NH2 coding sequence
– Translational signal also from reporter gene
? Initiation sequence and part of NH2 sequence of
reporter gene missing,but COOH region still
present
– Two coding sequences must be in frame
Lecture 4 BIOL 533 4
Technique
? Use transposon vector containing lac
fusion
– Carries promoterless lacZ gene and selectable
antibiotic marker
– Selection for antibiotic marker generates set
of random insertions in chromosome
– Colonies carrying transposon screened for
expression under conditions tested
Lecture 4 BIOL 533 5
Foreign Gene Expression
? Assay for production of βgal
– Higher temperature
– +— iron
– etc,
? Locate unknown gene
Lecture 4 BIOL 533 6
lacZ Fusion
? Used to find regulatory genes that control
expression of virulence gene
– lacZ fusion strain is mutagenized (chemical or
another transposon)
– Resulting colonies screened for aberrant
regulation
Lecture 4 BIOL 533 7
lacZ Fusion
? Aberrant regulation,
– Colonies are not Lac+ in presence of inducing
conditions
? Loss of activator
– Colonies are Lac+ under both inducing and
non-inducing conditions
? Loss of repressor
Lecture 4 BIOL 533 8
lacZ Fusion
? Gene tagged by transposon can be cloned
– Segment of DNA adjacent to transposon,
? Can be used as hybridization probe to clone wild-
type locus
? Can also be cloned by complementation
? Cloned DNA can be sequenced
Lecture 4 BIOL 533 9
phoA Gene Fusions
? Virulence genes are small subset of total
genes,so random mutagenesis will affect
many more non-virulence genes
? Way to optimize number of virulence
gene mutations takes advantage of fact
that most virulence genes are on bacterial
surface or excreted into medium
Lecture 4 BIOL 533 10
phoA Gene Fusions
? Translational protein fusion
? Alkaline phosphatase is expressed only in
periplasm
? Foreign gene product has to be expressed
outside of cytoplasm
? In vector,signal sequence mutations
block export of alkaline phosphatase and
render it inactive
Lecture 4 BIOL 533 11
phoA Gene Fusions
? Export and activity restored by fusing in frame a
restriction fragment to truncated phoA gene;
i.e.,lacking signal sequences
– No AUG start codon
– No ribosome binding site
? Restriction fragment has portions of NH2-
terminal genes encoding signal sequences of
heterologous proteins,such as OmpF or LamB
Lecture 4 BIOL 533 12
TnphoA Transposon
? Created TnphoA that randomly inserts
phoA into target genes
? Hybrid proteins display alkaline
phosphatase activity only if target gene
encodes a periplasmic outer membrane or
extracellular protein
Lecture 4 BIOL 533 13
TnphoA Transposon
? Use TnphoA,
– Study topology of inner membrane proteins
? βgal fusions expressed only in cytoplasm
– Identify genes that encode export proteins
? Exported proteins represent major class of
virulence factors
? Number of additional delivery systems
? Remember analysis of Groisman and Heffron
Lecture 4 BIOL 533 14
Analysis of Virulence
? Study TnphoA mutants of S,typhimurium
identified as Pho+ on L-agar plates
– Tested for virulence by oral infection of
BALB/C mice
– Found 15/150 Pho+ mutants were avirulent
? 9/15 were defective in LPS biosynthesis
? None had insertions in virulence plasmid
Lecture 4 BIOL 533 15
Analysis of Virulence
? Conclusion,same caveats as apply to
normal transposon mutagenesis,
– But use of TnphoA limits identification of
random insertions to those affecting cell
envelope factors
Lecture 4 BIOL 533 16
Analysis of Virulence
? Disadvantage,
– Limits selection to genes expressed in
S,typhimurium on L-agar
– Genes expressed at low levels or repressed
on L-agar plates would be missed
Lecture 4 BIOL 533 17
Analysis of Virulence
? Expression problem may explain why 9/15
Pho+ avirulent mutant strains were LPS—
rather than being defective in other
virulence genes
Lecture 4 BIOL 533 18
Analysis of Virulence
? TnphoA narrows,but doesn’t solve all
detection problems
– While disrupting gene,the TnphoA mutations
produce alkaline phosphatase hybrid proteins
– Could be phenotype not due to loss of gene,
but presence of deleterious hybrid protein;
– Therefore,to be sure mutation in virulence
gene,must conduct other genetic analyses
Lecture 4 BIOL 533 19
In Vivo Expression Technology
Mahan et,al.,1993,Science 259,686-688
? Based on gene that,when mutated,
causes organism to be attenuated
? Used Salmonella typhimurium
? ΔpurA — lacZY both lack promoter
(in suicide plasmid)
Lecture 4 BIOL 533 20
In Vivo Expression Technology
? Sau3A digested chromosomal DNA
?electroporated Salmonella PurA— strain
? Injected into mice twice
? After each 3 days
?harvest spleen
? Plate on artificial media to detect Lac—
strains
Lecture 4 BIOL 533 21
In Vivo Expression Technology
? Based solely on fact that ivi genes are
highly expressed in animal tissues,but
are not expressed in lab media
? Selection strategy,
– Bacterial strain with mutation that attenuated
growth in vivo
– Complemented by operon fusions to same
attenuated gene
Lecture 4 BIOL 533 22
In Vivo Expression Technology
? Criteria for IVET system,
– Fusions in single chromosomal copy
? No plasmid complications
– Fusions constructed without disruption of
gene
? Loss of gene could be lethal to bacterium
– Monitored both in animal and on lab media
Lecture 4 BIOL 533 23
pIVET Vector
? pIVET1 plasmid
– Suicide vector
? Replication dependent on Pi replication protein
Salmonella does not produce
– Promoterless purA gene and lacZ operon
? Can monitor level or transcription of entire operon
– Unique cloning site 5’ to purA gene
? Clone random fragments of DNA
– bla gene allows plasmid to be selected
Lecture 4 BIOL 533 24
pIVET Vector
? Construction of clones
– Clone Sau3A fragments into vector
– Put fragment pool into PurA deletion strain of
Salmonella (—Pi replication protein)
– Selection for ApR plasmid,integrates at
homologous Salmonella gene
? No natural lacZ gene
? Deleted purA gene
Lecture 4 BIOL 533 25
pIVET Vector
? Clones of interest
– Product of plasmid integration generates a
duplication
? One promoter drives purA lacZ while other drives
wild-type copy of virulence gene
? Integrate at unknown gene because purA and lacZ
are from E,coli; not enough homology to
Salmonella
Lecture 4 BIOL 533 26
Properties of Clones
? To survive and propagate in host,
– purA lacZ fusion has to be expressed at
sufficient level to overcome deletion of purA
– If gene of interest codes for virulence factor,
then expression also required for infection
– Monitor expression in animal and lab by
measuring βgal from strains that survive
three days and are recovered from spleen
Lecture 4 BIOL 533 27
Analysis of Virulence
– To identify genes code for virulence,
junctions of fusions cloned and sequenced
– Prepared inactivated genes
? Any effect on pathogenesis?
– Transposon insertions in 3/5 genes
– BALB/C mice infected orally have increased
LD50 (200 to 20,000 fold)
? Therefore,IVET selects for virulence
– Also,mice immunized with 1 mutant were
protected when challenged with wild-type
Lecture 4 BIOL 533 28
Identity of Genes
? Results,
– Most genes found so far are housekeeping
genes,perhaps because of screening
conditions
? Screened only for expression of genes on aerobic
media,but most body parts are anaerobic
– Crude approach; at best,identifies a set of
genes that are expressed at some point of
infection
Lecture 4 BIOL 533 29
Identity of Genes
? pheST himA operon encodes 2 su
phenylalanyl t-RNA synthetase and 1 su
host integration factor (IHF)
? Antisense Rfb O-antigen synthesis
– O-antigen synthesis mutants attenuated-
orally,but virulent IP route
– Mutations that increase LD50 do affect
virulence
Lecture 4 BIOL 533 30
Pros and Cons
? Detailed analysis of genes and gene
products expressed in animal necessary to
determine where and when during
infection the gene was expressed
? More serious problem—focuses on
transcriptional regulation
Lecture 4 BIOL 533 31
Pros and Cons
? Genes that are regulated or activated
post-transcriptionally will be missed
? Also,many genes expressed on lab media
may be important in vivo
? If some aspect of in vitro growth
condition is similar or identical to in vivo
condition (e.g.,37° C),certain genes
may be discarded
Lecture 4 BIOL 533 32
Research Strategy
? To get maximal variability in genes
selected,use different,
– Reporter genes
– Infection models
– in vitro growth conditions
? Likely that infection by different routes or
hosts characterized by unique H-P
interactions lead to different genes
Lecture 4 BIOL 533 33
Analysis of Virulence
Heithoff et,al.,1999 (Mahan’s group)
? Previously found over 100 different genes
– 25% unidentified
– All affect virulence
? Looked at regulation of genes in intestine,
liver,and spleen,plus cultured murine
macrophage
Lecture 4 BIOL 533 34
Analysis of Virulence
? Regulation in response to Mg+2,pH,and
Fe proceeded same as in vitro at
approximately the same levels
? Not true in spleen; has to be different
signals
? Think that response to Mg+2 and pH
correlates with assuming macrophage
existence
Lecture 4 BIOL 533 35
Uses
? Expected to provide Ag for vaccine
– Genes expressed only in vivo
? Assist in construction live-attenuated
vaccines (inactivated genes)
? Also can establish in vivo regulated
expression of heterologous Ag in live
vaccines
Lecture 4 BIOL 533 36
Uses
? Identification of ivi genes allows detection
of changes in metabolism,gene
regulation,and cell surface properties
during growth in animal tissues
? Allows new antimicrobial targets to be
identified
Lecture 4 BIOL 533 37
Lecture 4
? Questions?
? Comments?
? Assignments..,
