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Collagen-like proteins in pathogenic E. coli strains.

Ghosh N, McKillop TJ, Jowitt TA, Howard M, Davies H, Holmes DF, Roberts IS, Bella J - PLoS ONE (2012)

Bottom Line: Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk.This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins.Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.

View Article: PubMed Central - PubMed

Affiliation: Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, United Kingdom.

ABSTRACT
The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.

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Far-UV CD analysis of the Col–PfC fragment after purification from rEPclA by SEC.(A) CD spectra at 4°C, 55°C and 4°C after immediately cooling back the sample (see text). The vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with a protein concentration of 0.2 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell. (B) Thermal denaturation of the Col–PfC fragment, monitored by CD at 220 nm as a function of increasing temperature between 4°C and 60°C, with a protein concentration of 0.2 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4, and a heating rate of 0.33°C/min.
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pone-0037872-g004: Far-UV CD analysis of the Col–PfC fragment after purification from rEPclA by SEC.(A) CD spectra at 4°C, 55°C and 4°C after immediately cooling back the sample (see text). The vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with a protein concentration of 0.2 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell. (B) Thermal denaturation of the Col–PfC fragment, monitored by CD at 220 nm as a function of increasing temperature between 4°C and 60°C, with a protein concentration of 0.2 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4, and a heating rate of 0.33°C/min.

Mentions: The secondary structures of rEPclA and the Col–PfC fragment were analysed by circular dichroism (CD). Interpretation of the results is easier if the Col–PfC CD data is considered first. A sample of Col–PfC was purified from a preparation of full-length rEPclA by nickel-affinity and size exclusion chromatographies. Its CD spectrum was measured at different temperatures between 195 and 260 nm. The concentration of the Col–PfC sample was calculated as 0.2 mg/ml from its UV absorption at 280 nm and an estimated molar extinction coefficient ε = 11000 M-1 cm-1. The CD spectrum of Col–PfC at 4°C (Figure 4A) shows the characteristic features of triple helical collagen: a small maximum of positive ellipticity at 220 nm and a deep minimum of negative ellipticity at around 199 nm [43]. Both these features are associated with the polyproline II conformation [44] characteristic of the collagen triple helix. The Col–PfC fragment includes mainly the collagen domain (Col) of EPclA and the C-terminal PfC domain, and thus its CD spectrum suggests that the Col domain adopts indeed a collagen-like, triple helical structure. The triple helical features disappeared from the CD spectrum upon increase of temperature, as shown by the CD curve at 55°C (Figure 4A). Interestingly, immediate cooling of the same sample back to 4°C recovered completely the triple helical structure, with a CD spectrum practically indistinguishable from the initial one (Figure 4A).


Collagen-like proteins in pathogenic E. coli strains.

Ghosh N, McKillop TJ, Jowitt TA, Howard M, Davies H, Holmes DF, Roberts IS, Bella J - PLoS ONE (2012)

Far-UV CD analysis of the Col–PfC fragment after purification from rEPclA by SEC.(A) CD spectra at 4°C, 55°C and 4°C after immediately cooling back the sample (see text). The vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with a protein concentration of 0.2 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell. (B) Thermal denaturation of the Col–PfC fragment, monitored by CD at 220 nm as a function of increasing temperature between 4°C and 60°C, with a protein concentration of 0.2 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4, and a heating rate of 0.33°C/min.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368898&req=5

pone-0037872-g004: Far-UV CD analysis of the Col–PfC fragment after purification from rEPclA by SEC.(A) CD spectra at 4°C, 55°C and 4°C after immediately cooling back the sample (see text). The vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with a protein concentration of 0.2 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell. (B) Thermal denaturation of the Col–PfC fragment, monitored by CD at 220 nm as a function of increasing temperature between 4°C and 60°C, with a protein concentration of 0.2 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4, and a heating rate of 0.33°C/min.
Mentions: The secondary structures of rEPclA and the Col–PfC fragment were analysed by circular dichroism (CD). Interpretation of the results is easier if the Col–PfC CD data is considered first. A sample of Col–PfC was purified from a preparation of full-length rEPclA by nickel-affinity and size exclusion chromatographies. Its CD spectrum was measured at different temperatures between 195 and 260 nm. The concentration of the Col–PfC sample was calculated as 0.2 mg/ml from its UV absorption at 280 nm and an estimated molar extinction coefficient ε = 11000 M-1 cm-1. The CD spectrum of Col–PfC at 4°C (Figure 4A) shows the characteristic features of triple helical collagen: a small maximum of positive ellipticity at 220 nm and a deep minimum of negative ellipticity at around 199 nm [43]. Both these features are associated with the polyproline II conformation [44] characteristic of the collagen triple helix. The Col–PfC fragment includes mainly the collagen domain (Col) of EPclA and the C-terminal PfC domain, and thus its CD spectrum suggests that the Col domain adopts indeed a collagen-like, triple helical structure. The triple helical features disappeared from the CD spectrum upon increase of temperature, as shown by the CD curve at 55°C (Figure 4A). Interestingly, immediate cooling of the same sample back to 4°C recovered completely the triple helical structure, with a CD spectrum practically indistinguishable from the initial one (Figure 4A).

Bottom Line: Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk.This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins.Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.

View Article: PubMed Central - PubMed

Affiliation: Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, United Kingdom.

ABSTRACT
The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.

Show MeSH
Related in: MedlinePlus