title: Functional Role of N- and C-Terminal Amino Acids in the Structural Subunits of Colonization Factor CS6 Expressed by Enterotoxigenic Escherichia coli.
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.
  
  
  OBJECTIVE
  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.
authors.affiliation: National Institute of Cholera and Enteric Diseases, Kolkata, India.
authors.forename: Anusuya
authors.initials: A
authors.lastname: Debnath
authors.affiliation: University of California-Irvine, VAMCLB-151, Long Beach, California, USA.
authors.forename: Subrata
authors.initials: S
authors.lastname: Sabui
authors.affiliation: Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
authors.forename: Takeaki
authors.initials: T
authors.lastname: Wajima
authors.affiliation: National Center for Global Health and Medicine, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Hamabata
authors.affiliation: Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata, India.
authors.forename: Rajat
authors.initials: R
authors.lastname: Banerjee
authors.affiliation: National Institute of Cholera and Enteric Diseases, Kolkata, India nschatterjee@rediffmail.com.
authors.forename: Nabendu Sekhar
authors.initials: NS
authors.lastname: Chatterjee
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D001422:Bacterial Adhesion; D018938:Caco-2 Cells; D054307:Enterotoxigenic Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D009154:Mutation
chemical_list: D000596:Amino Acids; D000942:Antigens, Bacterial; C098406:CS6 antigen, E coli; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones