Regulation of Regulation of Salmonella Salmonella Enterica Enterica by: Laurel Kovach by: Laurel Kovach Fahlen, T., Wilson, R., Boddicker, J., and, Jones, B. Fahlen, T., Wilson, R., Boddicker, J., and, Jones, B. 2001. Hha is a negative 2001. Hha is a negative modulator of transcription of modulator of transcription of hilA, the hilA, the Salmonella enterica Salmonella enterica serovar serovar Typhimurium invasion gene transcriptional Typhimurium invasion gene transcriptional activator. activator. J. Bacteriol. J. Bacteriol. 183:6620-6629. 183:6620-6629.
Transcript
Regulation of Regulation of Salmonella EntericaSalmonella Entericaby: Laurel Kovachby: Laurel Kovach
Fahlen, T., Wilson, R., Boddicker, J., and, Jones, B. 2001. Hha is a negative Fahlen, T., Wilson, R., Boddicker, J., and, Jones, B. 2001. Hha is a negative modulator of transcription of hilA, the modulator of transcription of hilA, the Salmonella enterica Salmonella enterica serovar serovar
Questions for ExperimentingQuestions for Experimenting
What genes negatively regulate the hilA gene?What genes negatively regulate the hilA gene? How do different growth conditions affect How do different growth conditions affect SalmonellaSalmonella?? What will decrease the expression of the hilA gene?What will decrease the expression of the hilA gene? How will the mutation of different genes affect the growth How will the mutation of different genes affect the growth
of of SalmonellaSalmonella?? What are the similarities between Salmonella and E. coli.What are the similarities between Salmonella and E. coli. What hemolytic characteristics do the two bacteria What hemolytic characteristics do the two bacteria
posses?posses? What is the effect of overexpression of the hha gene on What is the effect of overexpression of the hha gene on
other invasive genes.other invasive genes.
BackgroundBackground
Salmonella is known to cause Salmonella is known to cause gastroenteritis and enteric fever.gastroenteritis and enteric fever.
It can be caused by the ingestion of any It can be caused by the ingestion of any contaminated meat, vegetable, or liquid. contaminated meat, vegetable, or liquid.
There are many forms of Salmonella, There are many forms of Salmonella, some of which are multidrug resistant.some of which are multidrug resistant.
It is important to find other ways to control It is important to find other ways to control Salmonella.Salmonella.
Pathway of Pathway of Salmonella entericaSalmonella enterica
Small intestineSmall intestineSpecialized M cells of the Peyers PatchesSpecialized M cells of the Peyers PatchesRegional nodesRegional nodesLiverLiverSpleenSpleen
How does How does SalmonellaSalmonella gain access gain access to the small intestine?to the small intestine?
The most critical step for The most critical step for SalmonellaSalmonella is the is the ability to invade the small intestine.ability to invade the small intestine.
First, the cellular membrane rearranges to First, the cellular membrane rearranges to form actin ruffles.form actin ruffles.
The actin ruffles phagocytize the bacteria.The actin ruffles phagocytize the bacteria.
Were does the bacteria go?Were does the bacteria go?
Salmonella entericaSalmonella enterica localizes at the 40 kb localizes at the 40 kb region of the chromosome.region of the chromosome.
This is located at the 63This is located at the 63rdrd centromere. centromere.This area is termed This area is termed SalmonellaSalmonella
pathogenicity island 1 (SPI-1).pathogenicity island 1 (SPI-1).
Conditions that repress Conditions that repress SalmonellaSalmonella invasioninvasion
The factors that regulate the hilA gene are The factors that regulate the hilA gene are the same as those that regulate the the same as those that regulate the invasive phenotype of invasive phenotype of Salmonella.Salmonella.
Thus, if you can regulate the hilA gene, Thus, if you can regulate the hilA gene, then you can regulate the invasive then you can regulate the invasive phenotype of phenotype of SalmonellaSalmonella..
ExplanationExplanation
Repressing the hilA gene will also repress Repressing the hilA gene will also repress the invasive phenotype of the invasive phenotype of SalmonellaSalmonella..
On the other hand, overexpression of the On the other hand, overexpression of the hilA gene causes a hyperinvasive hilA gene causes a hyperinvasive phenotype.phenotype.
Purpose and reasoning of the Purpose and reasoning of the experimentexperiment
hupB and ams genes were found to be hupB and ams genes were found to be negative for hilA expression.negative for hilA expression.
Another search was performed to find Another search was performed to find other factors that negatively regulate the other factors that negatively regulate the hilA gene.hilA gene.
hilA Expression Under Different hilA Expression Under Different Growth ConditionsGrowth Conditions
- increased growth from 2.5 to 3.5 - increased growth from 2.5 to 3.5 foldfold
- High oxygen/low osomolarityHigh oxygen/low osomolarity
- repressed growth- repressed growth
- Therefore osmolarity has more of an effect - Therefore osmolarity has more of an effect on the expression of the hilA geneon the expression of the hilA gene
What will Decrease the Expression of the What will Decrease the Expression of the hilA GenehilA Gene
The strain BJ70 was transformed with a The strain BJ70 was transformed with a plasmid gene bank of the serovar plasmid gene bank of the serovar Thyphimurium SL1344 chromsome and Thyphimurium SL1344 chromsome and plated ampicilin- resistant transformants plated ampicilin- resistant transformants on MacConkey lactose agar.on MacConkey lactose agar.
What is the point?What is the point?
The purpose was to have a plasmid strain The purpose was to have a plasmid strain that would definitely repress hilA that would definitely repress hilA expression.expression.
White transformants that were able to White transformants that were able to repress the expression of the hilA gene repress the expression of the hilA gene were transformed back into strain BJ70.were transformed back into strain BJ70.
Of these, one isolate was selected due to Of these, one isolate was selected due to the possession of a plasmid that was able the possession of a plasmid that was able to repress hilA expression 75% more to repress hilA expression 75% more effective than growth conditions.effective than growth conditions.
This plasmid is known as pTF120.This plasmid is known as pTF120.
pTF120pTF120
The cloned gene was sequenced and The cloned gene was sequenced and used to search databases at the National used to search databases at the National Center for Biotechnology Information.Center for Biotechnology Information.
The intention was to identify the cloned The intention was to identify the cloned DNA fragments.DNA fragments.
What is the importance of the What is the importance of the sequencing?sequencing?
The sequencing identified pTF120 to have The sequencing identified pTF120 to have a similar genetic makeup to that of E. coli.a similar genetic makeup to that of E. coli.
This enables the scientists to further This enables the scientists to further experiment interchanging different material experiment interchanging different material between the bacteria.between the bacteria.
Interesting FindInteresting Find
The database identified pTF120 to have a The database identified pTF120 to have a very similar sequencing to the hha gene of very similar sequencing to the hha gene of E. coli.E. coli.
The hha gene of E. coli is known to The hha gene of E. coli is known to repress hemolysin production.repress hemolysin production.
A functional homolog of the hha gene of A functional homolog of the hha gene of Yersinia enterocolitica, known as ymoA, Yersinia enterocolitica, known as ymoA, also shows the ability to regulate virulence also shows the ability to regulate virulence genes.genes.
Can the hha gene of Can the hha gene of SalmonellaSalmonella function to function to repress the hemolysin activity of repress the hemolysin activity of E. coliE. coli
Hemolytic activity is detected by Hemolytic activity is detected by inoculating a blood agar with inoculating a blood agar with E. coliE. coli. .
When When E. coliE. coli was mixed with the was mixed with the SalmonellaSalmonella hha gene, the zone of clearing hha gene, the zone of clearing was completely eliminated. was completely eliminated.
This is an indication that the This is an indication that the SalmonellaSalmonella hha gene functions to stop hemolysis.hha gene functions to stop hemolysis.
More Cloning is Conducted to see if More Cloning is Conducted to see if hha can Repress hilA Expressionhha can Repress hilA Expression
hha from the serovar Typhimurium was hha from the serovar Typhimurium was amplified and cloned into pGEM-T.amplified and cloned into pGEM-T.
This created pTF137.This created pTF137.The pTF137 was introduced to strain The pTF137 was introduced to strain
BJ70.BJ70.Overexpression of pTF137 reduced hilA Overexpression of pTF137 reduced hilA
expression by more than sevenfold.expression by more than sevenfold.
Rationale for Further Rationale for Further ExperimenationExperimenation
Experimentation on overexpression of the Experimentation on overexpression of the hha gene was done to find its effect on hha gene was done to find its effect on other invasive genes. other invasive genes.
The following genes were traced:The following genes were traced:
- invF- invF
- prgH- prgH
- sipC- sipC
ExperimentExperiment
Plasmid TF137 were fused separately with Plasmid TF137 were fused separately with each of the invasive genes. each of the invasive genes.
Not only did pTF137 decrease repress Not only did pTF137 decrease repress hilA, but it also decreased the expression hilA, but it also decreased the expression of the three invasive genes.of the three invasive genes.
TF79 was created by replacing the TF79 was created by replacing the majority of the hha gene in BJ70 with a majority of the hha gene in BJ70 with a kanamycin cassette.kanamycin cassette.
It was found that the hilA gene was still It was found that the hilA gene was still being controlled by environmental signals.being controlled by environmental signals.
Further experimentation was necessary.Further experimentation was necessary.
The role of hhaThe role of hha
Expression of hha affects the expression Expression of hha affects the expression of hilA. of hilA.
Experimentation of hilA in TF79Experimentation of hilA in TF79
The first expriment involving low The first expriment involving low oxygen/high osmolarity and vice versa oxygen/high osmolarity and vice versa was repeated for the hilA gene in TF79.was repeated for the hilA gene in TF79.
hha on the expression of hilAhha on the expression of hilA
Expression of hilA Expression of hilA after mutation of hhaafter mutation of hha
Comparitive analysis Comparitive analysis between wild type between wild type strain and hha strain and hha mutatedmutated
ResultsResults
Wild typeWild type Oxygen limiting- Oxygen limiting-
mu, very high hilA mu, very high hilA expressionexpression
High oxygen- 15 mu- High oxygen- 15 mu- very low expressionvery low expression
Low osmolarity- 100 Low osmolarity- 100 mu, great variation mu, great variation from wild typefrom wild type
Effect of hha mutation on Effect of hha mutation on invasivenessinvasiveness
Regulatory role of hha on invasiveness of Regulatory role of hha on invasiveness of Salmonella entericaSalmonella enterica
Assessment of affecting factorsAssessment of affecting factors
Three factors had to be assessed prior to Three factors had to be assessed prior to experimentation:experimentation:The invasiveness of of SL1344 with plasmid The invasiveness of of SL1344 with plasmid
GEM-TGEM-TThe hha mutant TF80 with plasmid GEM-TThe hha mutant TF80 with plasmid GEM-TTF80 with plasmid TF137TF80 with plasmid TF137
ResultsResults
Invasive phenotype of TF80 was higher Invasive phenotype of TF80 was higher than SL1344than SL1344
Lower invasion with the overexpression of Lower invasion with the overexpression of Hha from plasmid TF137 in strain TF80Hha from plasmid TF137 in strain TF80
Repressed invasion of SL1344 with Repressed invasion of SL1344 with plasmid TF137plasmid TF137
Comparative analysis of invasion between Comparative analysis of invasion between wild type strain and Hha mutated strainwild type strain and Hha mutated strain
ResultsResults
Under repressing conditions Under repressing conditions -Hha mutated gene- showed increase in -Hha mutated gene- showed increase in invasiveness invasiveness
High oxygen High oxygen -invasion was sixfold higher -invasion was sixfold higher
Low osmolarity Low osmolarity -invasion was twelve-fold higher -invasion was twelve-fold higher
High oxygen and low osmolarity High oxygen and low osmolarity -invasion was sixfold higher -invasion was sixfold higher
ConclusionConclusion What is Salmonella’s relationship with the hilA gene?What is Salmonella’s relationship with the hilA gene? By repressing the hilA gene, the invasive phenotype of By repressing the hilA gene, the invasive phenotype of SalmonellaSalmonella
can also be repressed.can also be repressed. Many things affect the repression of the hilA gene:Many things affect the repression of the hilA gene:
Level of oxygen Level of oxygen Level of osmolarityLevel of osmolarity Genetic engineeringGenetic engineering
