The chaperone/usher pathways of Pseudomonas aeruginosa: identification of fimbrial gene clusters (cup) and their involvement in biofilm formation

Abstract

Pseudomonas aeruginosa, an important opportunistic human pathogen, persists in certain tissues in the form of specialized bacterial communities, referred to as biofilm. The biofilm is formed through series of interactions between cells and adherence to surfaces, resulting in an organized structure. By screening a library of Tn5 insertions in a nonpiliated P. aeruginosa strain, we identified genes involved in early stages of biofilm formation. One class of mutations identified in this study mapped in a cluster of genes specifying the components of a chaperone/usher pathway that is involved in assembly of fimbrial subunits in other microorganisms. These genes, not previously described in P. aeruginosa, were named cupA1-A5. Additional chaperone/usher systems (CupB and CupC) have been also identified in the genome of P. aeruginosa PAO1; however, they do not appear to play a role in adhesion under the conditions where the CupA system is expressed and functions in surface adherence. The identification of these putative adhesins on the cell surface of P. aeruginosa suggests that this organism possess a wide range of factors that function in biofilm formation. These structures appear to be differentially regulated and may function at distinct stages of biofilm formation, or in specific environments colonized by this organism.

Figure 1

Biofilm formation of wild-type PAK strain, PAK-NP (pilA), and PAK-NF (fliC). (A) Bars represent the measure of OD₆₀₀ as a result of four distinct experiments. The level of attachment of the mutant strains is indicated as a percentage of attachment of the wild-type PAK strain. (B) Visualization of biofilm formation in wells of a microtiter dish after crystal violet staining.

Figure 2

Flagellar motility. Motility of strains PAK, PAK-NP, and PAK-NF are compared with motility of PAKΔpilA Tn5 mutant derivatives. Strains R15, U60, and P48 appeared to be mutants affected in flagellum biogenesis (see Table 2), whereas IC41, O51, Q77, L91, II40, and N66 are affected in genes unrelated to flagellar biogenesis that were called lad. N66 (also named PAN66) is affected in a gene that we renamed cupA2. In the case of strain P48, for which slight motility is observed, the mutated gene corresponds to motY, which has a role in the function of the flagellum but not its assembly.

Figure 3

Microscopic analysis of biofilm formation. Bacteria grown in M63 medium, supplemented with glucose, MgSO₄, and Casamino acids, were allowed to attach to glass cover slides. The development of biofilm on a solid surface was visualized by phase-contrast microscopy. (A) PAKΔpilA. (B) PAN66 (ladN66/cupA2).

Figure 4

Organization of the cup gene clusters. The three cup gene clusters have been represented to scale. Genes encoding fimbrial subunits are represented as black arrows, genes encoding periplasmic chaperones are represented as gray arrows, and genes encoding ushers are represented with striped arrows. The cupB5 gene, encoding a protein similar to Fha, is represented in white. The numbers indicated above each gene correspond to the genome annotation (http://www.pseudomonas.com).

Figure 5

Quantification of biofilm formation of the PAN66 and PAN67 strains. Bars represent the measure of OD₆₀₀ as a result of four distinct experiments. The level of attachment of the mutant strains is indicated as a percentage of attachment of the wild-type PAK strain.