Eikenella corrodens phase variation involves a posttranslational event in pilus formation

Abstract

The human pathogen Eikenella corrodens synthesizes type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying the molecular basis of this phase variation in the clinical isolate E. corrodens VA1. A genomic fragment encoding the major type IV pilin was cloned from the S-phase variant of strain VA1. Sequence analysis of the fragment revealed four tandemly arranged potential open reading frames (ORFs), designated pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin. The protein predicted by pilB shares sequence identity with the Dichelobacter nodosus FimB fimbrial assembly protein. The protein predicted by hagA resembles a hemagglutinin. The region containing these four ORFs was designated the pilA locus. DNA hybridization and sequence analysis showed that the pilA locus of an L-phase variant of strain VA1 was identical to that of the S-phase variant. An abundant pilA1 transcript initiating upstream of pilA1 and terminating at a predicted hairpin structure between pilA1 and pilA2 was detected by several assays, as was a less abundant read-through transcript encompassing pilA1, pilA2, and pilB. Transcription from the pilA locus was nearly indistinguishable between S- and L-phase variants. Electron microscopy and immunochemical analysis showed that S-phase variants synthesize, export, and assemble pilin into pili. In contrast, L-phase variants synthesize pilin but do not export and assemble it into pili. These data suggest that a posttranslational event, possibly involving an alteration in pilin export and assembly, is responsible for phase variation in E. corrodens.

FIG. 1

Physical map of the pilA locus for E. corrodens VA1-S1. Shaded boxes indicate size and orientation of ORFs as determined by sequence analysis. Flanking and internal restriction sites are shown for enzymes used in cloning and generation of probes. Labeled horizontal bars below map identify regions that correspond to probes used in DNA and RNA hybridization analyses. B, BglII; E, EcoRI; P, PvuII.

FIG. 2

DNA hybridization analysis of pilA locus structure for S- and L-phase variants of E. corrodens VA1. Total DNA (4 μg per lane) was isolated from strain VA1-S1 (lanes 1) or VA1-L2 (lanes 2), digested with PvuII and BglII (A), PvuII (B), or PvuII and DraI (C), and subjected to blot hybridization against a DNA probe for the pilA locus of strain VA1-S1. The positions of DNA molecular size standards are shown at the left.

FIG. 3

Expression of pilA genes in E. corrodens VA1-S1 and VA1-L2. Samples of total RNA (5 μg per lane) from strain VA1-S1 (lanes 1) or VA1-L2 (lanes 2) were subjected to blot hybridization against RNA probes specific for transcripts from pilA1 (A) or pilA2 (B). The positions of RNA molecular size standards are shown at the left.

FIG. 4

Nucleotide sequence of the E. corrodens VA1-S1 pilA1 promoter region. The putative pilA1 transcription initiation site (+1) determined by primer extension analysis is boxed, as are potential ς⁷⁰ promoter sequences (−35 and −10). Potential ς⁵⁴ promoter sequences (−24 and −12) are shaded. A putative ribosome binding site (RBS) is underlined. Numbers to the right correspond to the sequence deposited in the GenBank database (accession no. AF079304).

FIG. 5

Localization of PilA1 in S- and L-phase variants of E. corrodens VA1. Surface (lanes 1 and 2), soluble (lanes 3 and 4), and insoluble (lanes 5 and 6) protein fractions from strains VA1-S1 (lanes 1, 3, and 5) and VA1-L2 (lanes 2, 4, and 6) were subjected to immunoblot analysis with a polyclonal antiserum specific for PilA1. The arrow marks the position of mature pilin. The positions of protein molecular weight standards are shown at the left.

FIG. 6

Differential piliation of S- and L-phase variants of E. corrodens VA1. (A) Immunogold electron micrograph of a strain VA1-S3 cell; (B) equivalent micrograph of a strain VA1-L2 cell. Bars = 200 nm.