Biofilm-associated proteins: news from Acinetobacter

Abstract

Background: A giant protein called BAP (biofilm-associated protein) plays a role in biofilm formation and adhesion to host cells in A. baumannii. Most of the protein is made by arrays of 80-110 aa modules featuring immunoglobulin-like (Ig-like) motifs.

Results: The survey of 541 A. baumannii sequenced strains belonging to 108 STs (sequence types) revealed that BAP is highly polymorphic, distinguishable in three main types for changes both in the repetitive and the COOH region. Analyzing the different STs, we found that 29 % feature type-1, 40 % type-2 BAP, 11 % type-3 BAP, 20 % lack BAP. The type-3 variant is restricted to A. baumannii, type-1 and type-2 BAP have been identified also in other species of the Acinetobacter calcoaceticus-baumannii (ACB) complex. A. calcoaceticus and A. pittii also encode BAP-like proteins in which Ig-like repeats are replaced by long tracts of alternating serine and aspartic acid residues. We have identified in species of the ACB complex two additional proteins, BLP1 and BLP2 (BAP-like proteins 1 and 2) which feature Ig-like repeats, share with BAP a sequence motif at the NH2 terminus, and are similarly expressed in stationary growth phase. The knock-out of either BLP1 or BLP2 genes of the A. baumannii ST1 AYE strain severely affected biofilm formation, as measured by comparing biofilm biomass and thickness, and adherence to epithelial cells. BLP1 is missing in the majority of type-3 BAP strains. BLP2 is largely conserved, but is frequently missing in BAP-negative cells.

Conclusions: Multiple proteins sharing Ig-like repeats seem to be involved in biofilm formation. The uneven distribution of the different BAP types, BLP1, and BLP2 is highly indicative that alternative protein complexes involved in biofilm formation are assembled in different A. baumannii strains.

Fig. 1

Organization of BAPs coding sequences in wholly sequenced A. baumannii genomes. The organization of coding sequences homologous to BAP [15] in wholly sequenced A. baumannii genomes deposited at the KEGG (Kyoto Encyclopedia of Genes and Genomes) site is depicted. Boxes denote NH2 and COOH regions, ovals modules of the repetitive region. Modules within small ORFs spanning the repetitive region in different clones have not been reported for clarity. Numbers in parenthesis denote ORFs, numbers to the right of each protein the strain ST

Fig. 2

Repeat modules in type-1, type-2 and type-3 BAPs. Bars denote undefined repeat regions in Z-plus type-1 and type-2 BAPs. AFDL, AEPM and AFDA clones potentially express a complete type-3 BAP

Fig. 3

Alpha and beta BAPs. Changes in the organization of the 8 BAP types found in the Acinetobacter genus are highlighted. Bau, A. baumannnii; SDF, A. baumannnii SDF; Hae, A. haemolyticus; Pit, A. pittii; Nos, A. nosocomialis; Bay, A. baylyi, Rad, A. radioresistens, Cal, A. calcoaceticus. Cal1 and Cal2 refer to the wholly sequenced A. calcoaceticus PHE-A2 [41] strain and the draft genomes deposited at the NCBI, respectively. Sequences conserved at the end of all BAP types are in orange. For sake of simplicity difference in the COOH regions among BAPs are not highlighted. E, G, F and B modules are depicted as in Fig. 1

Fig. 4

Conserved motifs in BAP, BLP1 and BLP2. Sequence motifs conserved at the termini of the three proteins are highlighted. RTX refers to the 1450 aa RTX toxin (ORF1891) encoded by the A. baumannii ACICU strain

Fig. 5

A. baumannii BLP1 and BLP2 proteins. The modular organization of the two proteins is diagrammed. The 297 aa “unique region” between P and Q repeats in BLP1 is depicted by a bar

Fig. 6

CLSM analysis of biofilms formed by the A. baumannii strains ACICU (a), 4190 (b), and ATCC17978 (c). In each panel, to the left is shown the orthogonal view of Z-stacks, to the right the three-dimensional spatial distribution of the biofilm. Arrows denote biofilm heights. Bright and dark areas show cell clusters and voids in the biofilm

Fig. 7

Biofilms CLSM analysis. Biofilms formed by the AYE (a), the AYE-ΔBLP1 (b) and AYE-ΔBLP2 (c) strains. Orthogonal view of Z-stacks, three-dimensional spatial distribution of the biofilm and arrows are as in Fig. 6

Fig. 8

Quantitative analysis of biofilm formation. The thickness (white bars) and the biomass (grey bars) of biofilms formed by the A. baumannii AYE, AYE-ΔBLP1 and AYE-ΔBLP2 strains are shown

Fig. 9

Bacterial adherence of A. baumannii AYE, AYE-ΔBLP1 and AYE-ΔBLP2 strains to A549 bronchial epithelial cells. Cell surface-associated bacteria after 60 min incubation at 37 °C. Asterisks denote statistically significant (p <0.001) differences in the degree of cell adhesion of AYE and derivative clones