Regulation of Salmonella Enterica by: Laurel Kovach

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Regulation of Salmonella Entericaby 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.
• 1836620-6629.

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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

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What will Decrease the Expression of the hilA Gene

• 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.

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What is the point?

• The purpose was to have a plasmid strain that
  would definitely repress hilA expression.

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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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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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Experimentation of hilA in TF79

• The first expriment involving low oxygen/high
  osmolarity and vice versa was repeated for the
  hilA gene in TF79.
• A wild-type strain and an hha mutant strain were
  each grown under the two conditions.

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Results

• hilA expression is reduced in both wild-type and
  hha mutant stains under high oxygen/high
  osmolarity.
• Under low osmolarity/low oxygen hilA expression
  was increased sixfold.

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Conclusion

• To regulate the invasive phenotype of Salmonella
  enterica one must first understand its
  relationship with the hilA gene. By repressing
  the hilA gene, the invasive phenotype of
  Salmonella can also be repressed. Many things
  contribute to the repression of the hilA gene.
  These include level of oxygen and osmolarity,
  and genetic engineering. With further research
  it is hopeful that we will be able to rid of
  pathogenic Salmonella including those that are
  multidrug resistant.