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Catabolite and Oxygen Regulation of Enterohemorrhagic Escherichia coli Virulence

View Article: PubMed Central - PubMed

ABSTRACT

The biogeography of the gut is diverse in its longitudinal axis, as well as within specific microenvironments. Differential oxygenation and nutrient composition drive the membership of microbial communities in these habitats. Moreover, enteric pathogens can orchestrate further modifications to gain a competitive advantage toward host colonization. These pathogens are versatile and adept when exploiting the human colon. They expertly navigate complex environmental cues and interkingdom signaling to colonize and infect their hosts. Here we demonstrate how enterohemorrhagic Escherichia coli (EHEC) uses three sugar-sensing transcription factors, Cra, KdpE, and FusR, to exquisitely regulate the expression of virulence factors associated with its type III secretion system (T3SS) when exposed to various oxygen concentrations. We also explored the effect of mucin-derived nonpreferred carbon sources on EHEC growth and expression of virulence genes. Taken together, the results show that EHEC represses the expression of its T3SS when oxygen is absent, mimicking the largely anaerobic lumen, and activates its T3SS when oxygen is available through Cra. In addition, when EHEC senses mucin-derived sugars heavily present in the O-linked and N-linked glycans of the large intestine, virulence gene expression is initiated. Sugars derived from pectin, a complex plant polysaccharide digested in the large intestine, also increased virulence gene expression. Not only does EHEC sense host- and microbiota-derived interkingdom signals, it also uses oxygen availability and mucin-derived sugars liberated by the microbiota to stimulate expression of the T3SS. This precision in gene regulation allows EHEC to be an efficient pathogen with an extremely low infectious dose.

No MeSH data available.


Related in: MedlinePlus

AE lesion formation in deletion strains. (A) All DNA is stained red (bacteria and HeLa cell nuclei). AE lesions are green (actin) cups beneath red bacteria. The number of quantified HeLa cells is indicated. (B) Quantification of the average number of bacteria attached per HeLa cell. (C) Quantification of the percentage of attached bacteria to form an AE lesion. The standard deviation is indicated. P values were calculated with Student’s t test. *, P ≤ 0.05; **, P ≤ 0.005.
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fig1: AE lesion formation in deletion strains. (A) All DNA is stained red (bacteria and HeLa cell nuclei). AE lesions are green (actin) cups beneath red bacteria. The number of quantified HeLa cells is indicated. (B) Quantification of the average number of bacteria attached per HeLa cell. (C) Quantification of the percentage of attached bacteria to form an AE lesion. The standard deviation is indicated. P values were calculated with Student’s t test. *, P ≤ 0.05; **, P ≤ 0.005.

Mentions: Because Cra, KdpE, and FusR affect LEE gene expression, which is necessary for AE lesion formation, we next assessed the roles of these transcription factors in the regulation of AE lesion formation. To form an AE lesion, EHEC uses specialized effectors to intimately attach to mammalian cells and reorganize actin to cup the bacteria, forming a pedestal-like structure. To assay whether deleting cra, kdpE, or fusR either alone or in combination affects EHEC AE lesion formation, HeLa cells were infected and assessed for the amount of AE lesions formed and the amount of bacteria attached to each lesion. Compared to WT bacteria (41.99 ± 5.46), the ΔfusR (56.74 ± 7.11) and ΔfusR ΔkdpE (61.52 ± 13.07) strains had significantly more bacteria attached to the HeLa cells, with 73.68, 70.67, and 77.44% of the attached bacteria able to form AE lesions, respectively (Fig. 1A to C). Any strain with cra deleted had significantly fewer bacteria attached to HeLa cells or associated with AE lesions. While the ΔkdpE strain had more attached bacteria (61.56 ± 16.74), with 69.86% forming pedestals compared to the WT, these data were not significant. Overall, these data indicate that Cra is a strong activator of AE lesion formation in EHEC; however, deleting fusR either alone or in combination with kdpE shows that these regulators repress or alter the kinetics of AE lesion formation prior to EHEC attachment to the HeLa cell.


Catabolite and Oxygen Regulation of Enterohemorrhagic Escherichia coli Virulence
AE lesion formation in deletion strains. (A) All DNA is stained red (bacteria and HeLa cell nuclei). AE lesions are green (actin) cups beneath red bacteria. The number of quantified HeLa cells is indicated. (B) Quantification of the average number of bacteria attached per HeLa cell. (C) Quantification of the percentage of attached bacteria to form an AE lesion. The standard deviation is indicated. P values were calculated with Student’s t test. *, P ≤ 0.05; **, P ≤ 0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120142&req=5

fig1: AE lesion formation in deletion strains. (A) All DNA is stained red (bacteria and HeLa cell nuclei). AE lesions are green (actin) cups beneath red bacteria. The number of quantified HeLa cells is indicated. (B) Quantification of the average number of bacteria attached per HeLa cell. (C) Quantification of the percentage of attached bacteria to form an AE lesion. The standard deviation is indicated. P values were calculated with Student’s t test. *, P ≤ 0.05; **, P ≤ 0.005.
Mentions: Because Cra, KdpE, and FusR affect LEE gene expression, which is necessary for AE lesion formation, we next assessed the roles of these transcription factors in the regulation of AE lesion formation. To form an AE lesion, EHEC uses specialized effectors to intimately attach to mammalian cells and reorganize actin to cup the bacteria, forming a pedestal-like structure. To assay whether deleting cra, kdpE, or fusR either alone or in combination affects EHEC AE lesion formation, HeLa cells were infected and assessed for the amount of AE lesions formed and the amount of bacteria attached to each lesion. Compared to WT bacteria (41.99 ± 5.46), the ΔfusR (56.74 ± 7.11) and ΔfusR ΔkdpE (61.52 ± 13.07) strains had significantly more bacteria attached to the HeLa cells, with 73.68, 70.67, and 77.44% of the attached bacteria able to form AE lesions, respectively (Fig. 1A to C). Any strain with cra deleted had significantly fewer bacteria attached to HeLa cells or associated with AE lesions. While the ΔkdpE strain had more attached bacteria (61.56 ± 16.74), with 69.86% forming pedestals compared to the WT, these data were not significant. Overall, these data indicate that Cra is a strong activator of AE lesion formation in EHEC; however, deleting fusR either alone or in combination with kdpE shows that these regulators repress or alter the kinetics of AE lesion formation prior to EHEC attachment to the HeLa cell.

View Article: PubMed Central - PubMed

ABSTRACT

The biogeography of the gut is diverse in its longitudinal axis, as well as within specific microenvironments. Differential oxygenation and nutrient composition drive the membership of microbial communities in these habitats. Moreover, enteric pathogens can orchestrate further modifications to gain a competitive advantage toward host colonization. These pathogens are versatile and adept when exploiting the human colon. They expertly navigate complex environmental cues and interkingdom signaling to colonize and infect their hosts. Here we demonstrate how enterohemorrhagic Escherichia coli (EHEC) uses three sugar-sensing transcription factors, Cra, KdpE, and FusR, to exquisitely regulate the expression of virulence factors associated with its type III secretion system (T3SS) when exposed to various oxygen concentrations. We also explored the effect of mucin-derived nonpreferred carbon sources on EHEC growth and expression of virulence genes. Taken together, the results show that EHEC represses the expression of its T3SS when oxygen is absent, mimicking the largely anaerobic lumen, and activates its T3SS when oxygen is available through Cra. In addition, when EHEC senses mucin-derived sugars heavily present in the O-linked and N-linked glycans of the large intestine, virulence gene expression is initiated. Sugars derived from pectin, a complex plant polysaccharide digested in the large intestine, also increased virulence gene expression. Not only does EHEC sense host- and microbiota-derived interkingdom signals, it also uses oxygen availability and mucin-derived sugars liberated by the microbiota to stimulate expression of the T3SS. This precision in gene regulation allows EHEC to be an efficient pathogen with an extremely low infectious dose.

No MeSH data available.


Related in: MedlinePlus