Assembly of CS1 pili: the role of specific residues of the major pilin, CooA

Abstract

CS1 pili are important virulence factors of enterotoxigenic Escherichia coli strains associated with human diarrheal disease. They are the prototype for a family of pili that share extensive sequence similarity among their structural and assembly proteins. Only four linked genes, cooB, cooA, cooC, and cooD, are required to produce CS1 pili in E. coli K-12. To identify amino acids important for the function of the major pilin CooA, we used alanine substitution mutagenesis targeting conserved residues in the N and C termini of the protein. To test function, we examined cooA mutants for the ability to agglutinate bovine erythrocytes. Each hemagglutination-negative (HA(-)) cooA mutant was examined to identify its assembly pathway defect. CooA has been shown to be degraded in the absence of CooB (K. Voegele, H. Sakellaris, and J. R. Scott, Proc. Natl. Acad. Sci. USA 94:13257-13261, 1997). We found several HA(-) cooA mutants that produced no detectable CooA, suggesting that recognition by CooB is mediated by residues in both the N and C termini of CooA. In addition, we found that alanine substitution for some of the conserved residues in the C-terminal motif "AGxYxG(x(6))T," which is found in all subunits of this pilus family, had no effect on pilus formation. However, alanine substitution for some of the alternating hydrophobic residues within this motif prevented CooA from interacting with CooD, which serves as both the tip adhesin and nucleation protein for pilus formation. Thus, it appears that some, but not all, of the residues in both the N and C termini of CooA play a critical role in the intermolecular interactions of the major pilin with the other structural and assembly proteins. We anticipate that the results obtained here for CS1 pili in enterotoxigenic E. coli will help develop an understanding of the pilus assembly pathway used by CS1 family members in several important human pathogens.

FIG. 1.

Model of CS1 pilus assembly at the outer membrane protein CooC. Letters specify the periplasmic chaperone CooB (B), the nucleation protein/tip adhesin CooD (D), the outer membrane protein CooC (C), and the major pilin subunit CooA (A). Pilus assembly is initiated when a periplasmic CooB-CooD complex associates with the outer membrane protein CooC. The pilus structure grows as CooA subunits, in association with the CooB chaperone, are added to CooD. With the addition of each subunit, CooB is displaced and recycled to the periplasm.

FIG. 2.

Alignment of amino acid sequences of CS1 family major pilin proteins. CLUSTAL W (52) was used to align the following: CooA, enterotoxigenic Escherichia coli CS1 pili (National Center for Biotechnology Information reference number AAA23596); CotA, enterotoxigenic Escherichia coli CS2 pili (CAA87761); CfaB, enterotoxigenic Escherichia coli CFA/I pili (P02971); CblA, Burkholderia cepacia cable type II pili (AAM56038); and TcfB, Salmonella enterica serovar Typhi Tcf pili. Identical residues are indicated by black boxes, and similar residues are indicated by gray boxes. The arrow below the alignment indicates the predicted cleavage site of the signal peptide. Asterisks below the sequence indicate conserved hydrophobic residues. Carets indicate the conserved residues comprising the C-terminal motif (AG-x-Y-x-G-x₆-T). Residues that were mutated in CooA are indicated above the sequence alignment.

FIG. 3.

Western blot of CooA in the periplasm of N-terminal cooA mutants. Periplasmic extracts were prepared from strains expressing wild-type CooA (MC4100/pEU605) (WT); negative control (MC4100) (NEG); and mutant CooA proteins produced by MC4100/pEU605 derivatives (I5A-K3A) with mutations as indicated above each lane. Mutants with substitutions at I5A-D21A were HA⁻. The mutant with the K3A substitution was HA⁺. A 20-μl aliquot of each periplasmic extract at an OD₆₀₀ of 30 was loaded onto the gel. Proteins were separated by SDS-PAGE using 12% NuPage Novex Bis-Tris polyacrylamide precast gels (Invitrogen). CooA was detected by immunoblot using CooA antiserum.

FIG. 4.

Western blot of column fractions after affinity purification to examine CooA-CooB interaction. Periplasmic extracts were recovered from the wild-type (WT) strain MC4100/pEU8102/pFDX500 and an isogenic strain carrying a pEU8102 derivative encoding CooA with an L31A substitution in the mature protein. To purify CooA-CooB complexes, periplasmic extracts were applied to a Strep-Tactin affinity column (see Materials and Methods). (A) An aliquot of each fraction (the input sample [I], 5%; column flowthrough [FT], 5%; wash fractions [W1 to W5], 10%; and eluate [E], 25%) was analyzed for the presence of CooA by immunoblot analysis with CooA antiserum. (B) CooB was detected by immunoblot analysis with Strep-Tactin alkaline phosphatase conjugate (IBA).

FIG. 5.

Western blot of column fractions after affinity purification to examine CooA-CooD interaction. Periplasmic extracts were recovered from the wild-type (WT) strain MC4100/pEU8105/pEU605 and isogenic strains carrying pEU605 derivatives with the CooA mutations indicated. Amino acid positions are numbered from the beginning of the mature protein (minus the signal sequence). To purify CooA-CooD complexes, 500 μl of periplasmic extracts, prepared from an equal amount of cells, was applied to a Strep-Tactin affinity column (see Materials and Methods). (A) An aliquot of each fraction (the input sample [I], 5%; column flowthrough [FT], 5%; wash fractions [W1 to W5], 10%; and eluate [E], 25%) was analyzed for the presence of CooA by immunoblot analysis with CooA antiserum. (B) CooD was detected by immunoblot analysis with Strep-Tactin alkaline phosphatase conjugate (IBA).

FIG. 6.

Western blot of CooA multimers in the periplasms of HA⁻ cooA mutants. Periplasmic extracts were prepared from strains expressing the following: lane 1, wild-type (WT) CooA from MC4100/pEU605; and lane 2, negative control (NEG) from MC4100. Lanes 3 to 5 contain extracts of MC4100/pEU605 derivatives expressing CooA with the following mutations: lane 3, I5A; lane 4, V7A; and lane 5, A9S. Amino acid positions are numbered from the beginning of the mature protein (minus the signal sequence). A 12-μl aliquot of each periplasmic extract at an OD₆₀₀ of 120 was loaded onto the gel. Proteins were separated by native gel electrophoresis using 4 to 20% Novex Tris-glycine polyacrylamide precast gels (Invitrogen). CooA oligomers were detected by immunoblot with CooA antiserum.

FIG. 7.

Summary of phenotypes for N-terminal and C-terminal cooA mutants. Amino acid positions are numbered from the beginning of the mature protein (minus the signal sequence). Residues in boldface type were replaced by alanines, with the exception of alanine residues, which were replaced with serines. Boxed residues are amino acids in CooA that, when mutated, resulted in an HA⁻ phenotype. Green-boxed residues are amino acids in CooA that, when mutated, reduced the amount of protein in the periplasm to undetectable levels. The pink-boxed residue, when mutated, eliminates interaction of CooA with the CooB chaperone but does not affect the interaction of CooA with the CooD minor pilin. Blue-boxed residues are amino acids involved in interaction of CooA with the CooD minor pilin. The orange-boxed residue, when mutated, had no effect on the interaction of CooA with the CooB chaperone but reduced the amount of CooA protein in the periplasm to undetectable levels when CooD was also expressed. Yellow-boxed residues, when replaced by alanine, had no effect on the interaction of CooA with either the CooB chaperone or the CooD minor pilin.