Identification and Characterization of an Acinetobacter baumannii Biofilm-Associated Protein

Abstract

We have identified a homologue to the staphylococcal biofilm-associated protein (Bap) in a bloodstream isolate of Acinetobacter baumannii. The fully sequenced open reading frame is 25,863 bp and encodes a protein with a predicted molecular mass of 854 kDa. Analysis of the nucleotide sequence reveals a repetitive structure consistent with bacterial cell surface adhesins. Bap-specific monoclonal antibody (MAb) 6E3 was generated to an epitope conserved among 41% of A. baumannii strains isolated during a recent outbreak in the U.S. military health care system. Flow cytometry confirms that the MAb 6E3 epitope is surface exposed. Random transposon mutagenesis was used to generate A. baumannii bap1302::EZ-Tn5, a mutant negative for surface reactivity to MAb 6E3 in which the transposon disrupts the coding sequence of bap. Time course confocal laser scanning microscopy and three-dimensional image analysis of actively growing biofilms demonstrates that this mutant is unable to sustain biofilm thickness and volume, suggesting a role for Bap in supporting the development of the mature biofilm structure. This is the first identification of a specific cell surface protein directly involved in biofilm formation by A. baumannii and suggests that Bap is involved in intercellular adhesion within the mature biofilm.

Insights into Acinetobacter baumannii pathogenicity

“…Loehfelm et al (6) identified a staphylococcal homologue of biofilm-associated protein (Bap), and after generating a transposon-mediated bap mutant in a contemporary A. baumannii clinical strain (307-0294), it was shown that the mutant produced \50% of the biofilm thickness produced by the parent strain. Bap is a surface exposed protein and, rather than being involved in bacterial cell attachment, appears to be important in maintaining mature biofilm architecture (6). Interestingly, antibodies directed to Bap bound to 93% of the four most common strain types from a clinical outbreak, as determined by MLST, whereas they bound to only 28% of the 35 less common strain types (6).…”

“…Bap is a surface exposed protein and, rather than being involved in bacterial cell attachment, appears to be important in maintaining mature biofilm architecture (6). Interestingly, antibodies directed to Bap bound to 93% of the four most common strain types from a clinical outbreak, as determined by MLST, whereas they bound to only 28% of the 35 less common strain types (6). The significance of this observation is unclear, but further research into the clinical importance of Bap in A. baumannii is clearly warranted.…”

“…medical devices and environmental surfaces (3)(4)(5)(6); (ii) its ability to adhere to, colonize and invade human epithelial cells (7,8); (iii) its repertoire of antibiotic resistance mechanisms that are able to be promptly up-regulated as required; and (iv) its ability to acquire foreign genetic material through lateral gene transfer to promote its own survival under antibiotic and host selection pressures (9,10). Specific virulence mechanisms identified to date are summarized in Table 1.…”

Acinetobacter baumannii: biology and drug resistance — role of carbapenemases

“…The expression level of csu operon is regulated by the two-component system -BfmS/BfmR, comprising a sensor kinase and a response regulator [39]. Biofilm-associated protein (Bap), expressed on the surface of the bacterial cells, is implicated in cell-to-cell adhesion providing biofilm development and maturation on different substrata [40]. Furthermore, one of the most important components of exopolysaccharides constituting biofilm matrix was polysaccharide polymer poly--beta-1,6-N-acetylglucosamine (PNAG) that is crucial for maintaining the integrity of A. baumannii biofilm under nutrient limitation and other environmental stresses [41].…”

Cell Membrane Damage of Prokaryotes and Analysis of the Cell Death Type in Eukaryotes by Fluorescence Microscopy

“…This kit provides a two dye fluorescence assay based on membrane integrity with extensive use in various microorganisms in the literature as Acinetobacter baumanni (Loehfelm et al, 2008), Mycoplasma putrefaciens (McAuliffe et al, 2006), Escherichia coli, and Micrococcus luteus (Ali et al, 2018). In Figure 3, it is possible to observe the visualization of the microorganisms with the kit which allows to distinguish between live and dead (Loehfelm et al, 2008;McAuliffe et al, 2006;Ali et al, 2018).…”