Transcript Regulation of Salmonella Enterica by: Laurel Kovach

Regulation of Salmonella Enterica
by: Laurel Kovach
Fahlen, T., Wilson, R., Boddicker, J., and, Jones, B. 2001. Hha is a negative
modulator of transcription of hilA, the Salmonella enterica serovar
Typhimurium invasion gene transcriptional activator. J. Bacteriol.
183:6620-6629.
Questions for Experimenting
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What genes negatively regulate the hilA gene?
How do different growth conditions affect Salmonella?
What will decrease the expression of the hilA gene?
How will the mutation of different genes affect the growth
of Salmonella?
What are the similarities between Salmonella and E. coli.
What hemolytic characteristics do the two bacteria
posses?
What is the effect of overexpression of the hha gene on
other invasive genes.
Background
Salmonella is known to cause
gastroenteritis and enteric fever.
 It can be caused by the ingestion of any
contaminated meat, vegetable, or liquid.
 There are many forms of Salmonella,
some of which are multidrug resistant.
 It is important to find other ways to control
Salmonella.
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Pathway of Salmonella enterica
Small intestine
 Specialized M cells of the Peyers Patches
 Regional nodes
 Liver
 Spleen
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How does Salmonella gain access
to the small intestine?
The most critical step for Salmonella is the
ability to invade the small intestine.
 First, the cellular membrane rearranges to
form actin ruffles.
 The actin ruffles phagocytize the bacteria.
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Were does the bacteria go?
Salmonella enterica localizes at the 40 kb
region of the chromosome.
 This is located at the 63rd centromere.
 This area is termed Salmonella
pathogenicity island 1 (SPI-1).
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Conditions that repress Salmonella
invasion
High oxygen
 Low osmolarity
 Low pH
 Stationary phase growth
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Rationale
The factors that regulate the hilA gene are
the same as those that regulate the
invasive phenotype of Salmonella.
 Thus, if you can regulate the hilA gene,
then you can regulate the invasive
phenotype of Salmonella.
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Explanation
Repressing the hilA gene will also repress
the invasive phenotype of Salmonella.
 On the other hand, overexpression of the
hilA gene causes a hyperinvasive
phenotype.
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Purpose and reasoning of the
experiment
hupB and ams genes were found to be
negative for hilA expression.
 Another search was performed to find
other factors that negatively regulate the
hilA gene.
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hilA Expression Under Different
Growth Conditions
- Low oxygen/high osmolarity
- increased growth from 2.5 to 3.5 fold
- High oxygen/low osomolarity
- repressed growth
- Therefore osmolarity has more of an effect
on the expression of the hilA gene
What will Decrease the Expression of the
hilA Gene
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The strain BJ70 was transformed with a
plasmid gene bank of the serovar
Thyphimurium SL1344 chromsome and
plated ampicilin- resistant transformants
on MacConkey lactose agar.
What is the point?
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The purpose was to have a plasmid strain
that would definitely repress hilA
expression.
Repressing hilA Expression
White transformants that were able to
repress the expression of the hilA gene
were transformed back into strain BJ70.
 Of these, one isolate was selected due to
the possession of a plasmid that was able
to repress hilA expression 75% more
effective than growth conditions.
 This plasmid is known as pTF120.
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pTF120
The cloned gene was sequenced and
used to search databases at the National
Center for Biotechnology Information.
 The intention was to identify the cloned
DNA fragments.
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What is the importance of the
sequencing?
The sequencing identified pTF120 to have
a similar genetic makeup to that of E. coli.
 This enables the scientists to further
experiment interchanging different material
between the bacteria.
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Interesting Find
The database identified pTF120 to have a
very similar sequencing to the hha gene of
E. coli.
 The hha gene of E. coli is known to
repress hemolysin production.
 A functional homolog of the hha gene of
Yersinia enterocolitica, known as ymoA,
also shows the ability to regulate virulence
genes.
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Can the hha gene of Salmonella function to
repress the hemolysin activity of E. coli
Hemolytic activity is detected by
inoculating a blood agar with E. coli.
 When E. coli was mixed with the
Salmonella hha gene, the zone of clearing
was completely eliminated.
 This is an indication that the Salmonella
hha gene functions to stop hemolysis.
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More Cloning is Conducted to see
if hha can Repress hilA Expression
hha from the serovar Typhimurium was
amplified and cloned into pGEM-T.
 This created pTF137.
 The pTF137 was introduced to strain
BJ70.
 Overexpression of pTF137 reduced hilA
expression by more than sevenfold.
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Rationale for Further
Experimenation
Experimentation on overexpression of the
hha gene was done to find its effect on
other invasive genes.
 The following genes were traced:
- invF
- prgH
- sipC
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Experiment
Plasmid TF137 were fused separately with
each of the invasive genes.
 Not only did pTF137 decrease repress
hilA, but it also decreased the expression
of the three invasive genes.
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Salmonella hha null mutant gene TF79
TF79 was created by replacing the
majority of the hha gene in BJ70 with a
kanamycin cassette.
 It was found that the hilA gene was still
being controlled by environmental signals.
 Further experimentation was necessary.
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The role of hha
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Expression of hha affects the expression
of hilA.
Experimentation of hilA in TF79
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The first expriment involving low
oxygen/high osmolarity and vice versa
was repeated for the hilA gene in TF79.
hha on the expression of hilA
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Expression of hilA
after mutation of hha
Comparitive analysis
between wild type
strain and hha
mutated
Results
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Wild type
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Oxygen limiting100mu, low hilA
expression
High oxygen- 5 mu,
very low expression
Low osmolarity- 15
mu, very low hilA
expression
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Hha mutated
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Oxygen limiting- 300
mu, very high hilA
expression
High oxygen- 15 muvery low expression
Low osmolarity- 100
mu, great variation
from wild type
Effect of hha mutation on
invasiveness
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Regulatory role of hha on invasiveness of
Salmonella enterica
Assessment of affecting factors
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Three factors had to be assessed prior to
experimentation:
The invasiveness of of SL1344 with plasmid
GEM-T
 The hha mutant TF80 with plasmid GEM-T
 TF80 with plasmid TF137
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Results
Invasive phenotype of TF80 was higher
than SL1344
 Lower invasion with the overexpression of
Hha from plasmid TF137 in strain TF80
 Repressed invasion of SL1344 with
plasmid TF137
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Comparative analysis of invasion between
wild type strain and Hha mutated strain
Results
Under repressing conditions
-Hha mutated gene- showed increase in
invasiveness
 High oxygen
-invasion was sixfold higher
 Low osmolarity
-invasion was twelve-fold higher
 High oxygen and low osmolarity
-invasion was sixfold higher
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Conclusion
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What is Salmonella’s relationship with the hilA gene?
By repressing the hilA gene, the invasive phenotype of Salmonella
can also be repressed.
Many things affect the repression of the hilA gene:
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Level of oxygen
Level of osmolarity
Genetic engineering