Crystal structure of enterotoxigenic <i><scp>E</scp>scherichia coli</i> colonization factor <scp>CS</scp>6 reveals a novel type of functional assembly

Roy, Saumendra Prasad; Rahman, Mohammad Mubinur; Yu, Xiao Di; Tuittila, Minna; Knight, Stefan D.; Zavialov, Anton V.

doi:10.1111/mmi.12044

Cited by 28 publications

(34 citation statements)

References 48 publications

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“…The stoichiometry of oligomers was not disrupted even in a less polar solvent, like 50% ethylene glycol. We did not find either homooligomers or hetero-oligomers in unequal stoichiometry, as documented by Roy et al (2012). This hetero-oligomeric assembly as a function of increasing protein concentration…”

Section: Discussionmentioning

confidence: 61%

“…The structural analysis showed that both the subunits of CS6 are mostly rich in b-sheets (Roy et al, 2012). We found that the CS6 protomers interact with each other through these b-sheets during oligomerization, by noncovalent interactions.…”

Section: Discussionmentioning

confidence: 92%

“…Here, we clearly demonstrated that CS6 oligomers were spherical in shape in solution instead of elongated, narrow rod-like structures. However, Roy et al (2012) suggested that CS6 forms linear, highly flexible, mini fibres, although they did not show the existence of these structures on the ETEC surface. Another possibility is that the CS6 might exist in pre-fimbriae form on the bacterial surface.…”

Section: Discussionmentioning

confidence: 99%

“…A recent crystallographic study showed that the CssA and CssB subunits are assembled alternately in linear fibres and form hetero-polyadhesin, which supports multivalent attachment to different host receptors. The formation of CS6 oligomers was also reported (Roy et al, 2012). To date, CS6 could not be detected on the bacterial surface with distinct morphology (Knutton et al, 1989;Nishikawa et al, 1998;Lüdi et al, 2006).…”

Section: Introductionmentioning

confidence: 91%

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Characterization of oligomeric assembly of colonization factor CS6 from enterotoxigenic Escherichia coli

et al. 2016

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The widely distributed colonization factor (CF) CS6 of enterotoxigenic Escherichia coli (ETEC) has gained importance over the years in terms of its structure and function. CS6 is an afimbrial assembly in contrast to the other ETEC CFs, which are mostly fimbrial. A recent study predicted a linear fibre model for recombinant chimeric CS6 and formation of oligomers in solution. In this study, we characterized the oligomeric assembly of CS6, purified from a clinical ETEC isolate and identified its existence in the WT strain. We found that purified CS6 forms a continuous array of higher order oligomers composed of two tightly associated subunits, CssA and CssB in an equal (1:1) stoichiometry. This oligomerization occurs by formation of (CssA-CssB) n complex where 'n' increases with the concentration. The diameter of CS6 oligomers also proportionally increases with concentration. More significantly, we showed CS6 oligomers to be spherical in shape instead of being linear fibres as predicted earlier and this was further confirmed by electron microscopy. We also showed CS6 assembled on the bacterial surface in the form of an oligomeric complex. This process depends on the expression of properly folded CssA and CssB together, guided by the chaperone CssC and usher CssD. In conclusion, our results provide evidence for the existence of concentration-dependent, spherical oligomers of CS6 comprising both the structural subunits in equal stoichiometry and the CS6 oligomeric complex on the ETEC surface.

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Section: Discussionmentioning

confidence: 61%

Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 91%

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Characterization of oligomeric assembly of colonization factor CS6 from enterotoxigenic Escherichia coli

et al. 2016

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“…However, the N termini of both subunits showed the presence of a donor strand (G d ) motif characterized by alternating hydrophobic and hydrophilic residues (10). The C termini of CssA and CssB showed the presence of a chaperone protein recognition motif, as defined for P pilus structural subunits.…”

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confidence: 99%

Functional Role of N- and C-Terminal Amino Acids in the Structural Subunits of Colonization Factor CS6 Expressed by Enterotoxigenic Escherichia coli

et al. 2016

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CS6 is a common colonization factor expressed by enterotoxigenic Escherichia coli. It is a two-subunit protein consisting of CssA and CssB in an equal stoichiometry, assembled via the chaperone-usher pathway into an afimbrial, oligomeric assembly on the bacterial cell surface. A recent structural study has predicted the involvement of the N-and C-terminal regions of the CS6 subunits in its assembly. Here, we identified the functionally important residues in the N-and C-terminal regions of the CssA and CssB subunits during CS6 assembly by alanine scanning mutagenesis. Bacteria expressing mutant proteins were tested for binding with Caco-2 cells, and the results were analyzed with respect to the surface expression of mutant CS6. In this assay, many mutant proteins were not expressed on the surface while some showed reduced expression. It appeared that some, but not all, of the residues in both the N and C termini of CssA and CssB played an important role in the intermolecular interactions between these two structural subunits, as well as chaperone protein CssC. Our results demonstrated that T20, K25, F27, S36, Y143, and V147 were important for the stability of CssA, probably through interaction of CssC. We also found that I22, V29, and I33 of CssA and G154, Y156, L160, V162, F164, and Y165 of CssB were responsible for CssA-CssB intermolecular interactions. In addition, some of the hydrophobic residues in the C terminus of CssA and the N terminus of CssB were involved in the stabilization of higher-order complex formation. Overall, the results presented here might help in understanding the pathway used to assemble CS6 and predict its structure. IMPORTANCEUnlike most other colonization factors, CS6 is nonfimbrial, and in a sense, its subunit composition and assembly are also unique. Here we report that both the N-and C-terminal amino acid residues of CssA and CssB play a critical role in the intermolecular interactions between them and assembly proteins. We found mainly that alternate hydrophobic residues present in these motifs are essential for the interaction between the structural subunits, as well as the chaperone and usher assembly proteins. Our results indicate the involvement of the side chains of identified amino acids in CS6 assembly. This study adds a step toward understanding the interactions between structural subunits of CS6 and assembly proteins during CS6 biogenesis. Enterotoxigenic Escherichia coli (ETEC) infection is the most common cause of infantile diarrhea, which accounts for approximately 210 million episodes and 380,000 deaths annually (1). Plasmid-encoded colonization factors (CFs) and heat-labile and heat-stable (LT and ST, respectively) enterotoxins are the major determinants of ETEC virulence. Colonization is the prerequisite step in initiating the virulence mediated by the interaction of CFs with their cognate receptors on the intestinal epithelium. So far, 25 different CFs have been identified and subdivided on the basis of antigenicity, molecular weight, and structural morpholo...

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Crystal structure and analysis of HdaB: The enteroaggregative Escherichia coli AAF/IV pilus tip protein

2016

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Enteroaggregative Escherichia coli is the primary cause of pediatric diarrhea in developing countries. They utilize aggregative adherence fimbriae (AAFs) to promote initial adherence to the host intestinal mucosa, promote the formation of biofilms, and mediate host invasion. Five AAFs have been identified to date and AAF/IV is amongst the most prevalent found in clinical isolates. Here we present the X‐ray crystal structure of the AAF/IV tip protein HdaB at 2.0 Å resolution. It shares high structural homology with members of the Afa/Dr superfamily of fimbriae, which are involved in host invasion. We highlight surface exposed residues that share sequence homology and propose that these may function in invasion and also non‐conserved regions that could mediate HdaB specific adhesive functions.

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Crystal structure of enterotoxigenic Escherichia coli colonization factor CS6 reveals a novel type of functional assembly

Cited by 28 publications

References 48 publications

Characterization of oligomeric assembly of colonization factor CS6 from enterotoxigenic Escherichia coli

Characterization of oligomeric assembly of colonization factor CS6 from enterotoxigenic Escherichia coli

Functional Role of N- and C-Terminal Amino Acids in the Structural Subunits of Colonization Factor CS6 Expressed by Enterotoxigenic Escherichia coli

Crystal structure and analysis of HdaB: The enteroaggregative Escherichia coli AAF/IV pilus tip protein

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