Demonstration of Type IV pilus expression and a twitching phenotype by Haemophilus influenzae

Abstract

Haemophilus influenzae is considered a nonmotile organism that expresses neither flagella nor type IV pili, although H. influenzae strain Rd possesses a cryptic pilus locus. We demonstrate here that the homologous gene cluster pilABCD in an otitis media isolate of nontypeable H. influenzae strain 86-028NP encodes a surface appendage that is highly similar, structurally and functionally, to the well-characterized subgroup of bacterial pili known as type IV pili. This gene cluster includes a gene (pilA) that likely encodes the major subunit of the heretofore uncharacterized H. influenzae-expressed type IV pilus, a gene with homology to a type IV prepilin peptidase (pilD) as well as two additional uncharacterized genes (pilB and pilC). A second gene cluster (comABCDEF) was also identified by homology to other pil or type II secretion system genes. When grown in chemically defined medium at an alkaline pH, strain 86-028NP produces approximately 7-nm-diameter structures that are near polar in location. Importantly, these organisms exhibit twitching motility. A mutation in the pilA gene abolishes both expression of the pilus structure and the twitching phenotype, whereas a mutant lacking ComE, a Pseudomonas PilQ homologue, produced large appendages that appeared to be membrane bound and terminated in a slightly bulbous tip. These latter structures often showed a regular pattern of areas of constriction and expansion. The recognition that H. influenzae possesses a mechanism for twitching motility will likely profoundly influence our understanding of H. influenzae-induced diseases of the respiratory tract and their sequelae.

FIG. 1.

Open reading frame maps of the pil and com gene clusters from H. influenzae strain 86-028NP.

FIG. 2.

Southern blot demonstrating that a single pilA gene was contained within the genomes of H. influenzae strain Rd, the clinical CF isolate, and 13 of 13 low-passage clinical NTHI isolates recovered from patients with chronic otitis media. The chromosomal DNA digests were from the following strains: lane 1, 86-028NP; lane 2, 1728MEE; lane 3, 1729MEE; lane 4, 1714MEE; lane 5, 214NP; lane 6, 1236MEE; lane 7, 165NP; lane 8, 1060MEE; lane 9, 1128MEE; lane 10, 10548MEE; lane 11, 3224A; lane 12, 3185A; lane 13, 1885MEE; lane 14, 27W116791N1.

FIG. 3.

ClustalW alignment of the pilA gene products from 11 NTHI isolates and strain Rd. Arrowheads indicate the locations of four conserved cysteine residues. Braces denote two regions of sequence diversity wherein there appear to be two major variants among the 12 isolates analyzed.

FIG. 4.

Transmission electron micrograph composite of negatively stained preparations demonstrating Tfp produced by strain 86-028NP and derivatives. (A) Parent strain grown under inducing conditions. The inset shows an image at a reduced magnification of the parent strain grown under inducing conditions. (B) Free Tfp bundles. (C) The pilA mutant of strain 86-028NP grown under inducing conditions. (D) The comE mutant of strain 86-028NP expressing very large, predominantly polar appendages that appeared to be membrane bound and to contain a fibrous material. The inset shows a regularly repeating pattern of areas of constriction and expansion often noted in appendages expressed by the comE mutant when grown under inducing conditions. (E) Higher magnification of structures similar to those described above (D), demonstrating the characteristic slightly bulbous tip.

FIG. 5.

Transmission electron micrograph composite of negatively stained whole bacteria. (A) Complemented pilA mutant expressing pilus-like structures (magnification, ×∼72,000). (B) Aggregate of non-cell-associated pilin fibers present in the complemented pilA mutant preparation that appear highly similar to those shown in Fig. 4B as expressed by the parental isolate. (C) Complemented comE mutant expressing pilus-like structures (magnification, ×∼72,000). (D) Complemented comE mutant expressing near-polar pili (bracket) (magnification, ×∼72,000).

FIG. 6.

Light microscopy composite of the outermost leading edge of bacterial growth from a point inoculation site. All images were captured with a ×40 objective on a Zeiss Axioskop 40. Scale bar, 10 μm. (A to E) Growth after 7 h of incubation. (F to J) Growth after 24 h of incubation. A and F, NTHI strain 86-028NP; B and G, pilA mutant; C and H, comE mutant; D and I, pilA mutant complemented with pilA; E and J, comE mutant complemented with comEF. All strains were grown on chemically defined medium (1% agar, 3-mm thickness, pH 9.0). Note the formation of rafting growth (A and F), typical of twitching motility. This rafting phenotype is absent from the pilA mutant (B and G), whereas the comE mutant (C and H) demonstrates an intermediate growth phenotype suggesting inhibited motility. The complemented pilA mutant demonstrated a restored rafting phenotype (D and I), as did the complemented comE mutant (E and J).