Pseudomonas aeruginosa is an opportunistic pathogen of animals and humans infecting immunocompromised hosts. Pathogenesis by this bacterium involves the production of a number of extracellular virulence determinants including type IV fimbriae (or pili), which are flexible, filamentous surface appendages produced at the poles of the bacterial cell. Type IV fimbriae are produced by a number of pathogenic bacterial species. These structures mediate attachment to the host epithelial tissue and a form of surface translocation termed twitching motility. They also appear to act as receptors for certain bacteriophages.
The biogenesis and function of type IV fimbriae in P aeruginosa is dependent on at least 35 genes, which are located in several clusters on the chromosome. Our laboratory, using TnJ-B21 transposon mutagenesis and observing any subsequent alteration in twitching motility, have determined the identity and location of a number of these genes. These include genes encoding the fimbrial subunit (PilA), a leader peptidase (PilD), ancillary proteins with pre-pilin like leader sequences (PilE, PilV, PilW, PilX, FimT, FimU), inner and outer membrane proteins (PilC, PilQ), nucleotide binding proteins (PilB, PilT, PilU), other proteins whose functions are not clear (PilM-P, PilF, PilYI, PilY2, PilZ), a sigma factor (RpoN), 2 two-component sensorregulator pairs (PilS/PilR and FimS/AlgR), and a complex chemosensory signal transduction system (PilG-L, ChpA-B).
The initial objective of this thesis was to further characterise a group of five remaining Tn5-B21 mutants. Four of these mutants lacked twitching motility and the fifth exhibited impaired motility. Southern analysis was used to map the mutants to the same Kpnl restriction fragment at approximately 40' on the P. aeruginosa genome. Cloning and sequencing studies showed that all five transposon insertions occurred within the same 2.8 kb open reading frame, which was termed fimV. The product of this gene has a putative peptidoglycan-binding domain, predicted transmembrane domains, a highly acidic C-terminus, and an anomalous electrophoretic migration, indicating unusual primary or secondary structure.
The P aeruginosa genome also possesses a paralog of fimV. Homologs of fimV were also found in the sequenced genomes of other type IV fimbriated bacteria but not in those of bacteria which lack type IV fimbriae or homologs of the type IV fimbrial genes. Wild-type twitching motility was restored to fimV mutants by complementation with wild-type fimV in a dosage dependent manner. Overexpression of fimV resulted in an unusual phenotype where the cells were massively elongated and migrated in large convoys at the periphery of the colony. From this data it is suggested that FimV may be involved in remodelling of the peptidoglycan layer to enable assembly of the type IV fimbrial structure and machinery.
In light of the unusual phenotypes observed during the fimV investigation, a detailed examination of twitching motility in Pseudomonas aeruginosa was carried out. The optimal conditions in vitro (at the smooth surface formed between semi-solid growth media and plastic or glass surfaces) were determined and twitching motility was found to be extremely rapid, leading to an overall radial rate of colony expansion of 0-6 mm/hour or greater. Staining the twitching zones revealed the presence of concentric rings in which there is a high density of microcolonies, which may reflect periods of expansion and cell division.
Video microscopic analysis showed that twitching motility involves the initial formation of large projections or rafts of aggregated cells, which move away from the colony edge. Behind the rafts, individual cells move rapidly up and down trails which thin and branch out, ultimately forming a fine lattice-like network of cells. The bacteria in the lattice network then appear to settle and divide to fill out the colonised space. These observations redefine twitching motility as a rapid, highly organised mechanism of bacterial translocation by which P aeruginosa can disperse itself over large areas to colonise new territories.