Genetic analysis of Moraxella bovis pilus expression.

Abstract

Moraxella bovis is a member of the Moraxellaceae family and is the causative agent of infectious bovine keratoconjunctivitis. Filamentous appendages known as pili are suspected virulence factors. The pili of M. bovis belong to the type 4 family of fimbriae, which includes the pili of Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, and Dichelobacter nodosus among others. The pilin gene region of strain Epp63 has been cloned and sequenced. This region contains an invertable DNA segment about two Kb in size determining which of two types of pilin, Q or I, will be produced. The gene encoding the putative invertase, piv, has also been identified. To study factors involved in the inversion event, we evaluated the effects of Fis, HU, and IHF, three proteins known to be involved in other inversion systems. Loss of the Fis protein did not affect the frequency of inversion of the pilin gene region present on a plasmid vector in E. coli, while loss of HU decreases the rate of inversion. The involvement of IHF is still unclear. Certain aspects of colonial morphology have been used previously as indicators of piliation status in M. bovis. Spreading and corroding (SC) colonies were correlated with moderate to heavy piliation, while nonspreading and noncorroding colonies (N) did not usually exhibit pili. We isolated several morphological variants of the N colony type in order to study pilus expression and phase variation. These variants were evaluated for colony morphology, pilin production via immunoblot, autoagglutination, competence for transformation, and pilus expression as seen by electron microscopy. Several N colony types did produce pili as detectable by EM, while others produced pilin but did not express pili. Some of the pilus producing variants did not autoagglutinate. The DNA sequence of 23 variants was identified and revealed no changes in the structural gene.