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Differential gene expression and adherence of Escherichia coli O157:H7 in vitro and in ligated pig intestines.

Yin X, Zhu J, Feng Y, Chambers JR, Gong J, Gyles CL - PLoS ONE (2011)

Bottom Line: It was found that decreased adherence in vitro by bacteria grown in MB was mainly due to lactose, possibly implicating the involvement of carbon catabolite repression (CCR).When bacteria were grown in MB and Brain Heart Infusion with NaHCO(3) (BHIN) plus lactose, pH was reduced to 5.5-5.9 and there was a significant decrease in expression of the locus of enterocyte effacement (LEE) genes eae, tir, espD, grlA/R and ler, and an increase in cya (cAMP), and two negative regulators of the LEE, gadE and hfq.Presence of lactose resulted in decreased expression of LEE genes and the failure of EHEC O157:H7 to adhere to epithelial cells in vitro but this repression was overcome in vivo.

View Article: PubMed Central - PubMed

Affiliation: Guelph Food Research Center, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada.

ABSTRACT

Background: Escherichia coli O157:H7 strain 86-24 grown in MacConkey broth (MB) shows almost no adherence to cultured epithelial cells but adheres well in pig ligated intestines. This study investigated the mechanisms associated with the difference between in-vitro and in-vivo adherence of the MB culture.

Methodology/principal findings: It was found that decreased adherence in vitro by bacteria grown in MB was mainly due to lactose, possibly implicating the involvement of carbon catabolite repression (CCR). Expression of selected virulence-related genes associated with adherence and CCR was then examined by quantitative PCR. When bacteria were grown in MB and Brain Heart Infusion with NaHCO(3) (BHIN) plus lactose, pH was reduced to 5.5-5.9 and there was a significant decrease in expression of the locus of enterocyte effacement (LEE) genes eae, tir, espD, grlA/R and ler, and an increase in cya (cAMP), and two negative regulators of the LEE, gadE and hfq. Putative virulence genes stcE, hlyA, ent and nleA were also decreased in vitro. Reversal of these changes was noted for bacteria recovered from the intestine, where transcripts for qseF and fis and putative virulence factors AidA(15), TerC and Ent/EspL2 were significantly increased, and transcripts for AIDA(48), Iha, UreC, Efa1A, Efa1B, ToxB, EhxA, StcE, NleA and NleB were expressed at high levels.

Conclusions/significance: Presence of lactose resulted in decreased expression of LEE genes and the failure of EHEC O157:H7 to adhere to epithelial cells in vitro but this repression was overcome in vivo. CCR and/or acidic pH may have played a role in repression of the LEE genes. Bacterial pathogens need to integrate their nutritional metabolism with expression of virulence genes but little is known of how this is done in E. coli O157:H7. This study indicates one aspect of the subject that should be investigated further.

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Transcriptional profiles of acid-resistance regulatory and quorum sensing genes.RNAs were isolated from EHEC O157:H7 strain 86-24 grown in BHIN, BHIN+bs, BHIN+lac, BHIN+bs+lac and MB at 37°C overnight statically, and the bacteria recovered from loops inoculated with bacteria grown in BHIN (BHIN-Loop) and MB (MB-Loop) at 37°C overnight statically as well as the control loops inoculated with EMEM (Control-Loop). Data are expressed as the means ± SD for RNA extracted in 4–6 biological replicates. Relative fold expression (RFE) represents the changes in transcription compared to the bacteria grown in BHIN (value of 1.0). The levels of 16SrRNA transcripts were used to normalize the Ct values. * indicates p<0.05 as compared to BHIN; † indicates p<0.05 as compared to BHIN-Loop.
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pone-0017424-g004: Transcriptional profiles of acid-resistance regulatory and quorum sensing genes.RNAs were isolated from EHEC O157:H7 strain 86-24 grown in BHIN, BHIN+bs, BHIN+lac, BHIN+bs+lac and MB at 37°C overnight statically, and the bacteria recovered from loops inoculated with bacteria grown in BHIN (BHIN-Loop) and MB (MB-Loop) at 37°C overnight statically as well as the control loops inoculated with EMEM (Control-Loop). Data are expressed as the means ± SD for RNA extracted in 4–6 biological replicates. Relative fold expression (RFE) represents the changes in transcription compared to the bacteria grown in BHIN (value of 1.0). The levels of 16SrRNA transcripts were used to normalize the Ct values. * indicates p<0.05 as compared to BHIN; † indicates p<0.05 as compared to BHIN-Loop.

Mentions: Since growth in the presence of lactose reduced the pH, genes encoding the glutamate-dependent acid resistance (GDAR) system were examined. The key regulator of this system is GadE, which is also a negative regulator of the LEE. gadE was increased >3.8-fold by growth in the presence of lactose, bile salts and MB (Fig. 4A). gadX, gadW, ydeO, evgA and mnmE, the upstream regulators of gadE, were assessed. The data showed that gadX, ydeO and evgA were not changed by addition of lactose; on the other hand, addition of lactose decreased gadW expression (Fig. 4A). Bile salts stimulated the expression of gadXW and evgA significantly (Fig. 4A). mnmE was increased by MB, possibly via bile salts and/or mild low pH (Fig. 4A). rpoS, encoding general stress response factor sigma S, was slightly increased by lactose, and sharply increased by MB (5.2 fold) (Fig. 3B).


Differential gene expression and adherence of Escherichia coli O157:H7 in vitro and in ligated pig intestines.

Yin X, Zhu J, Feng Y, Chambers JR, Gong J, Gyles CL - PLoS ONE (2011)

Transcriptional profiles of acid-resistance regulatory and quorum sensing genes.RNAs were isolated from EHEC O157:H7 strain 86-24 grown in BHIN, BHIN+bs, BHIN+lac, BHIN+bs+lac and MB at 37°C overnight statically, and the bacteria recovered from loops inoculated with bacteria grown in BHIN (BHIN-Loop) and MB (MB-Loop) at 37°C overnight statically as well as the control loops inoculated with EMEM (Control-Loop). Data are expressed as the means ± SD for RNA extracted in 4–6 biological replicates. Relative fold expression (RFE) represents the changes in transcription compared to the bacteria grown in BHIN (value of 1.0). The levels of 16SrRNA transcripts were used to normalize the Ct values. * indicates p<0.05 as compared to BHIN; † indicates p<0.05 as compared to BHIN-Loop.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046156&req=5

pone-0017424-g004: Transcriptional profiles of acid-resistance regulatory and quorum sensing genes.RNAs were isolated from EHEC O157:H7 strain 86-24 grown in BHIN, BHIN+bs, BHIN+lac, BHIN+bs+lac and MB at 37°C overnight statically, and the bacteria recovered from loops inoculated with bacteria grown in BHIN (BHIN-Loop) and MB (MB-Loop) at 37°C overnight statically as well as the control loops inoculated with EMEM (Control-Loop). Data are expressed as the means ± SD for RNA extracted in 4–6 biological replicates. Relative fold expression (RFE) represents the changes in transcription compared to the bacteria grown in BHIN (value of 1.0). The levels of 16SrRNA transcripts were used to normalize the Ct values. * indicates p<0.05 as compared to BHIN; † indicates p<0.05 as compared to BHIN-Loop.
Mentions: Since growth in the presence of lactose reduced the pH, genes encoding the glutamate-dependent acid resistance (GDAR) system were examined. The key regulator of this system is GadE, which is also a negative regulator of the LEE. gadE was increased >3.8-fold by growth in the presence of lactose, bile salts and MB (Fig. 4A). gadX, gadW, ydeO, evgA and mnmE, the upstream regulators of gadE, were assessed. The data showed that gadX, ydeO and evgA were not changed by addition of lactose; on the other hand, addition of lactose decreased gadW expression (Fig. 4A). Bile salts stimulated the expression of gadXW and evgA significantly (Fig. 4A). mnmE was increased by MB, possibly via bile salts and/or mild low pH (Fig. 4A). rpoS, encoding general stress response factor sigma S, was slightly increased by lactose, and sharply increased by MB (5.2 fold) (Fig. 3B).

Bottom Line: It was found that decreased adherence in vitro by bacteria grown in MB was mainly due to lactose, possibly implicating the involvement of carbon catabolite repression (CCR).When bacteria were grown in MB and Brain Heart Infusion with NaHCO(3) (BHIN) plus lactose, pH was reduced to 5.5-5.9 and there was a significant decrease in expression of the locus of enterocyte effacement (LEE) genes eae, tir, espD, grlA/R and ler, and an increase in cya (cAMP), and two negative regulators of the LEE, gadE and hfq.Presence of lactose resulted in decreased expression of LEE genes and the failure of EHEC O157:H7 to adhere to epithelial cells in vitro but this repression was overcome in vivo.

View Article: PubMed Central - PubMed

Affiliation: Guelph Food Research Center, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada.

ABSTRACT

Background: Escherichia coli O157:H7 strain 86-24 grown in MacConkey broth (MB) shows almost no adherence to cultured epithelial cells but adheres well in pig ligated intestines. This study investigated the mechanisms associated with the difference between in-vitro and in-vivo adherence of the MB culture.

Methodology/principal findings: It was found that decreased adherence in vitro by bacteria grown in MB was mainly due to lactose, possibly implicating the involvement of carbon catabolite repression (CCR). Expression of selected virulence-related genes associated with adherence and CCR was then examined by quantitative PCR. When bacteria were grown in MB and Brain Heart Infusion with NaHCO(3) (BHIN) plus lactose, pH was reduced to 5.5-5.9 and there was a significant decrease in expression of the locus of enterocyte effacement (LEE) genes eae, tir, espD, grlA/R and ler, and an increase in cya (cAMP), and two negative regulators of the LEE, gadE and hfq. Putative virulence genes stcE, hlyA, ent and nleA were also decreased in vitro. Reversal of these changes was noted for bacteria recovered from the intestine, where transcripts for qseF and fis and putative virulence factors AidA(15), TerC and Ent/EspL2 were significantly increased, and transcripts for AIDA(48), Iha, UreC, Efa1A, Efa1B, ToxB, EhxA, StcE, NleA and NleB were expressed at high levels.

Conclusions/significance: Presence of lactose resulted in decreased expression of LEE genes and the failure of EHEC O157:H7 to adhere to epithelial cells in vitro but this repression was overcome in vivo. CCR and/or acidic pH may have played a role in repression of the LEE genes. Bacterial pathogens need to integrate their nutritional metabolism with expression of virulence genes but little is known of how this is done in E. coli O157:H7. This study indicates one aspect of the subject that should be investigated further.

Show MeSH
Related in: MedlinePlus