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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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Illustration of adherence of EHEC O157:H7 strain 86-24 in vitro in the presence and absence of lactose.Bacteria were grown in BHIN, BHIN+lac, and MB to IPEC-J2 cells. Adherence assays were performed for 6 h as described in Materials and Methods. The bacteria grown in MB caused almost no adherence; there was a sharp decrease in adherence when grown with lactose.
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pone-0017424-g002: Illustration of adherence of EHEC O157:H7 strain 86-24 in vitro in the presence and absence of lactose.Bacteria were grown in BHIN, BHIN+lac, and MB to IPEC-J2 cells. Adherence assays were performed for 6 h as described in Materials and Methods. The bacteria grown in MB caused almost no adherence; there was a sharp decrease in adherence when grown with lactose.

Mentions: In previous studies, there was very little adherence of bacteria cultured in MB to IPEC-J2 and HEp-2 cells [32]. The unique MB components, lactose and bile salts, were therefore examined for their effects on adherence. Addition of lactose to BHIN caused a marked reduction in total adherence and in large clusters on IPEC-J2 cells. Addition of bile salts to BHIN resulted in a slight reduction in adherence to these cells. When bile salts and lactose were both added, there was a further decrease in adherence (Figs. 1 and 2). To prove whether the effects by MB were due to other components in stead of lactose and bile salts, MB minus lactose and/or bile salts was examined in bacterial adherence to IPEC-J2 cells. Consistent with the effects by addition of lactose and bile salts to BHIN, this study showed that bacteria grown in MB minus bile salts (containing lactose) increased bacterial adherence, while MB minus lactose (containing bile salts) increased bacterial adherence more significantly, and MB minus both lactose and bile salts restored the adherence levels to those caused by bacteria grown in BHIN (Fig. 1). Similar effects were observed with HEp-2 cells (data not shown).


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)

Illustration of adherence of EHEC O157:H7 strain 86-24 in vitro in the presence and absence of lactose.Bacteria were grown in BHIN, BHIN+lac, and MB to IPEC-J2 cells. Adherence assays were performed for 6 h as described in Materials and Methods. The bacteria grown in MB caused almost no adherence; there was a sharp decrease in adherence when grown with lactose.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017424-g002: Illustration of adherence of EHEC O157:H7 strain 86-24 in vitro in the presence and absence of lactose.Bacteria were grown in BHIN, BHIN+lac, and MB to IPEC-J2 cells. Adherence assays were performed for 6 h as described in Materials and Methods. The bacteria grown in MB caused almost no adherence; there was a sharp decrease in adherence when grown with lactose.
Mentions: In previous studies, there was very little adherence of bacteria cultured in MB to IPEC-J2 and HEp-2 cells [32]. The unique MB components, lactose and bile salts, were therefore examined for their effects on adherence. Addition of lactose to BHIN caused a marked reduction in total adherence and in large clusters on IPEC-J2 cells. Addition of bile salts to BHIN resulted in a slight reduction in adherence to these cells. When bile salts and lactose were both added, there was a further decrease in adherence (Figs. 1 and 2). To prove whether the effects by MB were due to other components in stead of lactose and bile salts, MB minus lactose and/or bile salts was examined in bacterial adherence to IPEC-J2 cells. Consistent with the effects by addition of lactose and bile salts to BHIN, this study showed that bacteria grown in MB minus bile salts (containing lactose) increased bacterial adherence, while MB minus lactose (containing bile salts) increased bacterial adherence more significantly, and MB minus both lactose and bile salts restored the adherence levels to those caused by bacteria grown in BHIN (Fig. 1). Similar effects were observed with HEp-2 cells (data not shown).

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