Expression of type IV pili by Moraxella catarrhalis is essential for natural competence and is affected by iron limitation

Abstract

Type IV pili, filamentous surface appendages primarily composed of a single protein subunit termed pilin, play a crucial role in the initiation of disease by a wide range of pathogenic bacteria. Although previous electron microscopic studies suggested that pili might be present on the surface of Moraxella catarrhalis isolates, detailed molecular and phenotypic analyses of these structures have not been reported to date. We identified and cloned the M. catarrhalis genes encoding PilA, the major pilin subunit, PilQ, the outer membrane secretin through which the pilus filament is extruded, and PilT, the NTPase that mediates pilin disassembly and retraction. To initiate investigation of the role of this surface organelle in pathogenesis, isogenic pilA, pilT, and pilQ mutants were constructed in M. catarrhalis strain 7169. Comparative analyses of the wild-type 7169 strain and three isogenic pil mutants demonstrated that M. catarrhalis expresses type IV pili that are essential for natural genetic transformation. Our studies suggest type IV pilus production by M. catarrhalis is constitutive and ubiquitous, although pilin expression was demonstrated to be iron responsive and Fur regulated. These data indicate that additional studies aimed at elucidating the prevalence and role of type IV pili in the pathogenesis and host response to M. catarrhalis infections are warranted.

FIG. 1.

Genetic organization of the M. catarrhalis pil biosynthesis-related homologues. Based on homologies to other systems, nine M. catarrhalis putative coding sequences with homology to genes involved in the biogenesis and assembly of type IV pili were identified (depicted in black). Thick arrows represent the size and orientation of the putative open reading frames identified. The pilA, pilT, and pilQ homologues were selected for further analysis.

FIG. 2.

To investigate the localization of PilA in the wild-type and mutant strains, whole bacterial lysates (WCL; odd lanes) or sheared surface components (SS; even lanes) were analyzed by SDS-PAGE and immunoblotting. Comparative analysis of the MAb 4G9-probed immunoblot indicated that whereas the wild-type 7169 contained pilin subunits both within the cell and on the surface (lanes 1 and 2), pilAK4 exhibited the expected PilA-null phenotype (lanes 3 and 4), pilTK3 appeared to have slightly more pilin in the SS (lanes 5 and 6), and pilQK6 contained pilin subunits within the cell but not on the cell surface (lanes 7 and 8). Molecular size standards are shown in kilodaltons.

FIG. 3.

(A) The contribution of M. catarrhalis pili to DNA competence was investigated in a quantitative transformation assay (a representative set of plates is shown). (B) These data indicate that the pil mutants are defective in DNA uptake compared to the wild-type 7169.

FIG. 4.

Piliation phenotypes as assessed by EM studies of the wild-type and pilA,-Q, and -T mutant strains. (A) EM analysis revealed short, peritrichous pili on the wild-type 7169. In contrast, whereas both the PilA-deficient (B) and PilQ-deficient (C) strains lacked detectable pili on the surface, the PilT-null mutant (D) appeared hyperpiliated with longer, tangled pili.

FIG. 5.

RT-PCR analysis of pilin expression by M. catarrhalis 7169 at various time points during broth-based growth (lane 2, 1.5 h; lane 3, 3.0 h; lane 4, 5.0 h, lane 5, 7.5 h) as well as from plate-grown organisms (lane 6). pilA transcript was detected during all phases of growth (amplicons denoted by arrowhead). rpoD served as the internal control (amplicons denoted by asterisk). One representative set of controls is depicted, including an amplification reaction that included RNA but lacked RT activation (lane 7), 7169 chromosomal DNA (lane 8), and a no-template control (lane 9). Samples were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. Molecular size standards (lanes 1 and 10) are depicted in 100-bp increments.

FIG. 6.

Analysis of iron-regulated expression of M. catarrhalis pilin. (A) Comparison of wild-type 7169 (lane 1) and the isogenic Fur mutant 7169fur1(lane 2) pilA transcripts by agarose gel analysis of RT-PCR amplicons. Control reactions, lacking RT activation, are depicted in lanes 3 and 4 for RNA isolated from 7169 and 7169fur1, respectively. (B) MAb 4G9 was used in immunoblot analysis of surface pilin expression by comparing the sheared surface components from 7169 (lane 1) and 7169fur1 (lane 2). (C) Representative depiction of the regulation of pilA expression by Fur, as demonstrated by the FURTA-positive phenotype of H1717 cells transformed with pPILAk4 (right panel) compared to the FURTA-negative phenotype of the H1717 cells transformed with the control plasmid paphA-3 (left panel). Red colonies appear dark gray and white colonies appear pale gray, as imaged by the monochromatic AlphaImager 2200 documentation and analysis system (Imgen Technologies, Alexandria, Va.).

FIG. 7.

RT-PCR analysis of pilA and pilQ expression by a panel of M. catarrhalis clinical isolates. RT-PCR amplicons, corresponding to pilA (bottom band, arrow) and pilQ (top band, asterisk) mRNA transcripts, were analyzed by agarose gel electrophoresis and ethidium bromide staining. Even lanes (2 to 26) represent reactions in the presence of RT, and odd lanes (3 to 27) represent control reactions lacking RT activation to verify RNA purity and absence of DNA contamination. Total RNA was isolated and analyzed from the following M. catarrhalis clinical isolates: lanes 2 and 3, 43617 (American Type Culture Collection); lanes 4 and 5, 7169 (Buffalo, N.Y.); lanes 6 and 7, KSA (Buffalo, N.Y.); lanes 8 and 9, BC40 (Buffalo, N.Y.); lanes 10 and 11, sk633 (Buffalo, N.Y.); lanes 12 and 13, O35E (Houston, Tex.); lanes 14 and 15, Tal1 (Philadelphia, Pa.); lanes 16 and 17, Af218 (England); lanes 18 and 19, 27335 (France); lanes 20 and 21, 27479 (Japan); lanes 22 and 23, 210044 (Angola); lanes 24 and 25, 27325 (Germany); lanes 26 and 27, 27512 (Belgium). Controls are depicted in lane 28, no-template control reaction, and lane 29, 7169 chromosomal DNA. Molecular size standards (lanes 1 and 30) are depicted in 100-bp increments.