Surface plasmon resonance shows that type IV pili are important in surface attachment by Pseudomonas aeruginosa

Abstract

Type IV pili have been shown to play a role in the early stages of bacterial biofilm formation, but not in initial bacterial attachment. Here, using the surface analytical technique, surface plasmon resonance (SPR), we follow the attachment of the bacterium Pseudomonas aeruginosa in real time. In contrast to previous studies, we show that type IV pili mutants are defective in attachment. Both mutants lacking pili (pilA), and those possessing an overabundance of pili (pilT), showed reduced SPR measured attachment compared with the wild-type PAO1 strain. Both pil mutants also showed reduced pathogenicity in a model insect host, as measured by percentage mortality after 24h. SPR revealed differences in the kinetics of attachment between pilA and pilT, differences obscured by endpoint assays using crystal violet stain. These results highlight the power of SPR in monitoring bacterial attachment in real time and also demonstrate an additional role for type IV pili beyond bacterial aggregation and micro-colony formation.

Figure 1

(a) Schematic showing the SPR setup used to measure bacterial attachment to gold. In this diagram, P polarized laser light is totally internally reflected, forming an energy wave at the surface. The angle θ of incident light which forms maximum excitation is sensitive to changes in the dielectric properties of thin films forming on the upper-side of the gold film. (b) Attachment of wild-type P. aeruginosa compared with pilA and pilT mutants, as measured by SPR. The rapid initial attachment of bacteria is reflected by the rapid increase in the angular minimum and is followed by a steady decline to an equilibrium (see text). Note that both pilA and pilT mutants show altered binding, but that the pattern of attachment over time differs between the two mutants.

Figure 2

Light microscopy of bacteria attached to the gold SPR substrate. Bacteria were visualized under brightfield microscopy after being taken directly from the SPR chamber and stained with crystal violet. Note the qualitative difference between the colonies formed by the wild-type (a) and mutant (b,c) bacteria. Scale bar 100 μm.

Figure 3

Endpoint assay of biofilm formation by the wild-type P. aeruginosa strain (PAO-1) and the two pil mutants (pilA and pilT) using crystal violet stain. Note that the final apparent density of the biofilm is greater in the wild-type strain than in the two mutants.

Figure 4

Percentage mortality of wax moth larvae injected with the wild-type (PAO-1) and mutant (pilA and pilT) strains of P. aeruginosa. Note the relative order of virulence: PAO-1>pilT>pilA (see text, §3).