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.

Nontypeable Haemophilus influenzae releases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery

“…Here, by fluorescence imaging and electron microscopy, we revealed that DNA and DNABII proteins were released from NTHI at a single subpolar location on the cell. The location of this release is consistent with the location of Tfp expression through the ComE pore (44). Unlike the less well-understood mechanism(s) by which NTHI releases DNA into the extracellular milieu, uptake of DNA from the environment is wellstudied.…”

“…4C. The NTHI Tfp is an adhesive protein that is extruded through the outer-membrane pore formed by ComE (10,44,45). In addition to its role in adherence and twitching motility, expression of the ComE pore and Tfp is also required for the uptake of DNA from the extracellular environment (44,45).…”

“…NTHI expresses Tfp from a single subpolar location along the long axis of the bacterial cell. These Tfp are essential for twitching motility, adherence to respiratory tract epithelial cells, colonization of the mammalian respiratory tract, and competence (10,44,45,50,51). The Tfp machinery in NTHI is encoded by two operons, pilABCD and comABCDEF.…”

The OxyR Regulon in Nontypeable Haemophilus influenzae

“…The oxyR gene, its 122-bp upstream intergenic region, and the first 23 bp of the 5Ј end of the downstream gene were amplified by PCR, TA cloned into pGEM-T Easy (Promega Co., Madison, WI), and then moved into pSPEC1, a derivative of the Escherichia coli-Haemophilus shuttle vector pGZRS-39A that contains a spectinomycin resistance gene (6). The pSPEC1-oxyR construct was transformed into the 86-028NP oxyR::Tn5 mutant strain, and transformants were selected on chocolate agar containing 200 g spectinomycin/ml.…”

Self-organization of bacterial biofilms is facilitated by extracellular DNA

“…Interestingly, interstitial biofilms of nontypeable Haemophilus influenzae and Acinetobacter baumanii that also actively expand via twitching motility (32,33) are not associated with the formation of intricate network patterns of trails despite the presence of eDNA in these biofilms (Fig. S9).…”