Identification and characterization of assembly proteins of CS5 pili from enterotoxigenic Escherichia coli

Abstract

<jats:title>ABSTRACT</jats:title> <jats:p> This study investigated the role of three genes comprising part of the operon which encodes CS5 pili from enterotoxigenic <jats:italic>Escherichia coli</jats:italic> . In-frame gene deletions were constructed, and the effects on biogenesis of the pili were examined. A deletion in <jats:italic>csfB</jats:italic> abolished CsfA major subunit accumulation in the periplasm, which could be restored by <jats:italic>trans</jats:italic> -complementation with a complete copy of the <jats:italic>csfB</jats:italic> gene. Localization studies using an antibody against CsfB showed that this protein was periplasmically located, and thus CsfB is likely to function as the specific chaperone for CsfA. An in-frame deletion mutation in the <jats:italic>csfE</jats:italic> gene resulted in pili approximately three times longer than those of the wild-type strain, thereby indicating a role for CsfE in pilus length regulation. Localization studies using an antibody generated against CsfE showed low-level CsfE accumulation in the outer membranes. Modulation of <jats:italic>csfE</jats:italic> expression in <jats:italic>trans</jats:italic> did not reduce the mean length of the pilus below that of the wild type, which indicated that CsfE is not rate-limiting for termination of pilus assembly. Interestingly, a deletion in the <jats:italic>csfF</jats:italic> gene also resulted in an elongated pilus morphology identical to that of the <jats:italic>csfE</jats:italic> deletion strain. However, unlike CsfE, CsfF was shown to be rate-limiting for termination of assembly, since overexpression of CsfF in a <jats:italic>csfF</jats:italic> deletion strain resulted in a significant decrease in the mean length of the pilus compared to that of the wild type. When the same construct was introduced into the wild-type strain, pilus expression was abolished. Since CsfF bears significant homology to the proposed CsfB chaperone, CsfF was predicted to act as the specific chaperone for CsfE. A double deletion in the <jats:italic>csfB</jats:italic> and <jats:italic>csfF</jats:italic> genes was shown to abolish the periplasmic accumulation of both CsfA and CsfD pilins, which could be restored individually only when the strain was <jats:italic>trans</jats:italic> -complemented with a wild-type copy of <jats:italic>csfB</jats:italic> or <jats:italic>csfF</jats:italic> , respectively. Therefore, CsfF may chaperone not only CsfE but also CsfD. A model for CS5 biogenesis is also proposed based on these and previous observations. </jats:p>