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

Anusuya Debnath, Subrata Sabui, Takeaki Wajima, Takashi Hamabata, Rajat Banerjee, Nabendu Sekhar Chatterjee

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Abstract

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. IMPORTANCE: Unlike 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.

Original languageEnglish
Pages (from-to)1429-1441
Number of pages13
JournalJournal of Bacteriology
Volume198
Issue number9
DOIs
Publication statusPublished - May 1 2016
Externally publishedYes

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Enterotoxigenic Escherichia coli
Amino Acids
Mutant Proteins
Proteins
Caco-2 Cells
Protein Subunits
Mutagenesis
Alanine
Bacteria

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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Functional Role of N-and C-Terminal Amino Acids in the Structural Subunits of Colonization Factor CS6 Expressed by Enterotoxigenic Escherichia coli. / Debnath, Anusuya; Sabui, Subrata; Wajima, Takeaki; Hamabata, Takashi; Banerjee, Rajat; Chatterjee, Nabendu Sekhar.

In: Journal of Bacteriology, Vol. 198, No. 9, 01.05.2016, p. 1429-1441.

Research output: Contribution to journalArticle

Debnath, Anusuya ; Sabui, Subrata ; Wajima, Takeaki ; Hamabata, Takashi ; Banerjee, Rajat ; Chatterjee, Nabendu Sekhar. / Functional Role of N-and C-Terminal Amino Acids in the Structural Subunits of Colonization Factor CS6 Expressed by Enterotoxigenic Escherichia coli. In: Journal of Bacteriology. 2016 ; Vol. 198, No. 9. pp. 1429-1441.
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N2 - 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. IMPORTANCE: Unlike 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.

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