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Proteomic analysis of Salmonella enterica serovar Enteritidis following propionate adaptation.

Calhoun LN, Liyanage R, Lay JO, Kwon YM - BMC Microbiol. (2010)

Bottom Line: However, we found the acid resistance to be fully restorable after genetic complementation.This work reveals a significant difference in the proteomes of PA adapted and unadapted S.Enteritidis and affirms the contribution of Dps and CpxR in PA induced acid resistance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell and Molecular Biology Program, Department of Poultry Science, University of Arkansas, 1260 W, Maple Avenue, Fayetteville, AR 72701, USA.

ABSTRACT

Background: Salmonella Enteritidis is a highly prevalent and persistent foodborne pathogen and is therefore a leading cause of nontyphoidal gastrointestinal disease worldwide. A variety of stresses are endured throughout its infection cycle, including high concentrations of propionate (PA) within food processing systems and within the gut of infected hosts. Prolonged PA exposure experienced in such milieus may have a drastic effect on the proteome of Salmonella Enteritidis subjected to this stress.

Results: In this study, we used 2 D gel electrophoresis to examine the proteomes of PA adapted and unadapted S. Enteritidis and have identified five proteins that are upregulated in PA adapted cultures using standard peptide mass fingerprinting by MALDI-TOF-MS and sequencing by MALDI LIFT-TOF/TOF tandem mass spectrometry. Of these five, two significant stress-related proteins (Dps and CpxR) were shown (via qRT-PCR analysis) to be upregulated at the transcriptional level as well. Unlike the wild type when adapted to PA (which demonstrates significant acid resistance), PA adapted S. Enteritidis ∆dps and S. Enteritidis ∆cpxR were at a clear disadvantage when challenged to a highly acidic environment. However, we found the acid resistance to be fully restorable after genetic complementation.

Conclusions: This work reveals a significant difference in the proteomes of PA adapted and unadapted S. Enteritidis and affirms the contribution of Dps and CpxR in PA induced acid resistance.

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Related in: MedlinePlus

qRT-PCR monitoring the expression of selected genes from PA adapted and unadapted cultures. The level of expression of each target gene in the PA adapted culture was compared to the level of gene expression of the identical target in the unadapted culture. The expression of each gene in unadapted cultures was taken to be the basal level of expression for that particular gene to which the expression in PA adapted cultures was compared, therefore allowing quantification of the relative changes in gene expression of selected targets. The relative quantification (RQ) of each target gene was subsequently calculated from the qRT-PCR data using the comparative CT (ΔΔCT) method. All data obtained from qRT-PCR experiments were normalized using 16 s rRNA. Presented data is the average of five independent trials. Standard error is represented by error bars. Genes with expression that is significantly different from the unadapted condition are indicated with an asterisk.
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Figure 3: qRT-PCR monitoring the expression of selected genes from PA adapted and unadapted cultures. The level of expression of each target gene in the PA adapted culture was compared to the level of gene expression of the identical target in the unadapted culture. The expression of each gene in unadapted cultures was taken to be the basal level of expression for that particular gene to which the expression in PA adapted cultures was compared, therefore allowing quantification of the relative changes in gene expression of selected targets. The relative quantification (RQ) of each target gene was subsequently calculated from the qRT-PCR data using the comparative CT (ΔΔCT) method. All data obtained from qRT-PCR experiments were normalized using 16 s rRNA. Presented data is the average of five independent trials. Standard error is represented by error bars. Genes with expression that is significantly different from the unadapted condition are indicated with an asterisk.

Mentions: Quantitative real-time PCR was performed to determine if the proteins upregulated in PA cultures (Dps, CpxR, SodA, RplE, and RplF) were overexpressed at the transcriptional level as well. A relative quantification experiment was performed; therefore, the level of expression of each target in the PA adapted culture was compared to the level of gene expression of the identical target gene in the unadapted culture. The expression of each gene in unadapted cultures was taken to be the basal level of expression for that particular gene (for the growth conditions used in this study) to which the expression in PA adapted cultures was compared. This method allowed the changes in gene expression of our selected targets to be carefully quantified. The relative quantification of each target gene was calculated from the data obtained using the comparative CT (ΔΔCT) method. Interestingly, qRT-PCR results did not fully coincided with all of the previously obtained proteomic results from 2 D electrophoresis (Figure 3). When compared to unadapted cultures, only two of the five targets overexpressed at the proteomic level (Dps and CpxR) showed increased expression at the transcriptional level (p < 0.05). cpxR showed a 20.8% increase in expression in PA adapted cultures, while dps from PA adapted cultures showed a 50.7% increase in expression over that from unadapted cultures. Expression of rplE and rplF in PA adapted cultures was only 82.1% and 99.5% respectively, of those from unadapted cultures. This difference in gene expression was not statistically significant (p > 0.05). Finally, sodA showed a significant decrease in expression after exposure to PA (p < 0.01). Its expression in PA adapted cultures was only 52.2% of that in unadapted cultures. Our combined results from 2D-gel and qRT-PCR experiments suggested that overexpression of cpxR and dps occurs at the transcriptional level, while rplE, rplF, and sodA were most likely upregulated post-translationally by an unidentified regulatory mechanism.


Proteomic analysis of Salmonella enterica serovar Enteritidis following propionate adaptation.

Calhoun LN, Liyanage R, Lay JO, Kwon YM - BMC Microbiol. (2010)

qRT-PCR monitoring the expression of selected genes from PA adapted and unadapted cultures. The level of expression of each target gene in the PA adapted culture was compared to the level of gene expression of the identical target in the unadapted culture. The expression of each gene in unadapted cultures was taken to be the basal level of expression for that particular gene to which the expression in PA adapted cultures was compared, therefore allowing quantification of the relative changes in gene expression of selected targets. The relative quantification (RQ) of each target gene was subsequently calculated from the qRT-PCR data using the comparative CT (ΔΔCT) method. All data obtained from qRT-PCR experiments were normalized using 16 s rRNA. Presented data is the average of five independent trials. Standard error is represented by error bars. Genes with expression that is significantly different from the unadapted condition are indicated with an asterisk.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2957393&req=5

Figure 3: qRT-PCR monitoring the expression of selected genes from PA adapted and unadapted cultures. The level of expression of each target gene in the PA adapted culture was compared to the level of gene expression of the identical target in the unadapted culture. The expression of each gene in unadapted cultures was taken to be the basal level of expression for that particular gene to which the expression in PA adapted cultures was compared, therefore allowing quantification of the relative changes in gene expression of selected targets. The relative quantification (RQ) of each target gene was subsequently calculated from the qRT-PCR data using the comparative CT (ΔΔCT) method. All data obtained from qRT-PCR experiments were normalized using 16 s rRNA. Presented data is the average of five independent trials. Standard error is represented by error bars. Genes with expression that is significantly different from the unadapted condition are indicated with an asterisk.
Mentions: Quantitative real-time PCR was performed to determine if the proteins upregulated in PA cultures (Dps, CpxR, SodA, RplE, and RplF) were overexpressed at the transcriptional level as well. A relative quantification experiment was performed; therefore, the level of expression of each target in the PA adapted culture was compared to the level of gene expression of the identical target gene in the unadapted culture. The expression of each gene in unadapted cultures was taken to be the basal level of expression for that particular gene (for the growth conditions used in this study) to which the expression in PA adapted cultures was compared. This method allowed the changes in gene expression of our selected targets to be carefully quantified. The relative quantification of each target gene was calculated from the data obtained using the comparative CT (ΔΔCT) method. Interestingly, qRT-PCR results did not fully coincided with all of the previously obtained proteomic results from 2 D electrophoresis (Figure 3). When compared to unadapted cultures, only two of the five targets overexpressed at the proteomic level (Dps and CpxR) showed increased expression at the transcriptional level (p < 0.05). cpxR showed a 20.8% increase in expression in PA adapted cultures, while dps from PA adapted cultures showed a 50.7% increase in expression over that from unadapted cultures. Expression of rplE and rplF in PA adapted cultures was only 82.1% and 99.5% respectively, of those from unadapted cultures. This difference in gene expression was not statistically significant (p > 0.05). Finally, sodA showed a significant decrease in expression after exposure to PA (p < 0.01). Its expression in PA adapted cultures was only 52.2% of that in unadapted cultures. Our combined results from 2D-gel and qRT-PCR experiments suggested that overexpression of cpxR and dps occurs at the transcriptional level, while rplE, rplF, and sodA were most likely upregulated post-translationally by an unidentified regulatory mechanism.

Bottom Line: However, we found the acid resistance to be fully restorable after genetic complementation.This work reveals a significant difference in the proteomes of PA adapted and unadapted S.Enteritidis and affirms the contribution of Dps and CpxR in PA induced acid resistance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell and Molecular Biology Program, Department of Poultry Science, University of Arkansas, 1260 W, Maple Avenue, Fayetteville, AR 72701, USA.

ABSTRACT

Background: Salmonella Enteritidis is a highly prevalent and persistent foodborne pathogen and is therefore a leading cause of nontyphoidal gastrointestinal disease worldwide. A variety of stresses are endured throughout its infection cycle, including high concentrations of propionate (PA) within food processing systems and within the gut of infected hosts. Prolonged PA exposure experienced in such milieus may have a drastic effect on the proteome of Salmonella Enteritidis subjected to this stress.

Results: In this study, we used 2 D gel electrophoresis to examine the proteomes of PA adapted and unadapted S. Enteritidis and have identified five proteins that are upregulated in PA adapted cultures using standard peptide mass fingerprinting by MALDI-TOF-MS and sequencing by MALDI LIFT-TOF/TOF tandem mass spectrometry. Of these five, two significant stress-related proteins (Dps and CpxR) were shown (via qRT-PCR analysis) to be upregulated at the transcriptional level as well. Unlike the wild type when adapted to PA (which demonstrates significant acid resistance), PA adapted S. Enteritidis ∆dps and S. Enteritidis ∆cpxR were at a clear disadvantage when challenged to a highly acidic environment. However, we found the acid resistance to be fully restorable after genetic complementation.

Conclusions: This work reveals a significant difference in the proteomes of PA adapted and unadapted S. Enteritidis and affirms the contribution of Dps and CpxR in PA induced acid resistance.

Show MeSH
Related in: MedlinePlus