The csuC and csuE genes encode respectively a chaperone involved in pili assembly and the pilus major subunit. Expression of csu Serine/threonin kinase inhibitor genes was hardly detectable in all growth conditions (data not shown). Consistent with this result, we could not detect any production of csu pili in A.
baumannii SMAL by electron microscopy, regardless of growth conditions (Figure 3 and data not shown). This result would suggest that production of csu pili, and thus their contribution to selleck compound surface adhesion, might be limited in this strain. In addition to csu pili, A. baumannii 19606 biofilm is characterized by efficient binding to Calcofluor [17], a fluorescent dye which binds specifically to cellulose and chitin; this observation suggests that cellulose, which is produced as an extracellular polysaccharide (EPS) in many bacteria [29–32], might be a biofilm determinant in A. baumannii. To detect possible production of cellulose, we grew A. baumannii
SMAL on different solid media supplemented with Calcofluor. Interestingly, Calcofluor binding was detected on M9Glu/sup solid medium, but not on M9Suc/sup or in either CB-839 molecular weight peptone-based media (LB or LB1/4), suggesting that growth on glucose induces production of Calcofluor-binding EPS in A. baumannii SMAL (Figure 2B). In order to test the possible role of this EPS as an adhesion factor, we tested surface adhesion to polystyrene in different growth media in the presence of the cellulose-degrading enzyme cellulase (Figure 2C). Surface adhesion was efficiently
inhibited Tolmetin by low amounts of cellulase when A. baumannii SMAL was grown in M9Glu/sup (50% inhibition at 0.15 Units cellulase, Figure 2C), thus suggesting that surface adhesion is mediated by cellulose production. In contrast, cellulase was only able to impair surface adhesion at much higher concentrations when A. baumannii SMAL was grown either in M9Suc/sup or in LB1/4 media (50% inhibition at ca. 12 and 19 Units cellulase, respectively, Figure 2C). At these amounts of cellulase, inhibitory effects are likely due to non-specific effects such as changes in surface tension or other physico-chemical properties of the medium. Cellulase effects in LB medium were not tested due to the very inefficient biofilm formation in this medium (Figure 2A). To further verify the possible role of cellulose-related EPS as an adhesion factor, A. baumannii SMAL biofilm formed on microtiter plates by cells growing in M9Glu/sup medium was resuspended in 50 mM phosphate buffer pH 6.0 by vigorous pipetting and incubated 30 minutes either in the presence or in the absence of 1 U cellulase prior to fixation with gluteraldehyde and visualization by transmission electron microscopy. Figure 3 shows that A. baumannii SMAL cells recovered from the biofilm appear embedded in bundle-like filaments (Panel 3A), which disappear upon cellulase treatment (Panel 3B), further confirming direct involvement of cellulose in cell-cell aggregation. Figure 3 Transmission electron microscopy images of A.