Global assessment of small RNAs reveals a non-coding transcript involved in biofilm formation and attachment in Acinetobacter baumannii ATCC 17978

Abstract

Many strains of Acinetobacter baumannii have been described as being able to form biofilm. Small non-coding RNAs (sRNAs) control gene expression in many regulatory circuits in bacteria. The aim of the present work was to provide a global description of the sRNAs produced both by planktonic and biofilm-associated (sessile) cells of A. baumannii ATCC 17978, and to compare the corresponding gene expression profiles to identify sRNAs molecules associated to biofilm formation and virulence. sRNA was extracted from both planktonic and sessile cells and reverse transcribed. cDNA was subjected to 454-pyrosequencing using the GS-FLX Titanium chemistry. The global analysis of the small RNA transcriptome revealed different sRNA expression patterns in planktonic and biofilm associated cells, with some of the transcripts only expressed or repressed in sessile bacteria. A total of 255 sRNAs were detected, with 185 of them differentially expressed in the different types of cells. A total of 9 sRNAs were expressed only in biofilm cells, while the expression of other 21 coding regions were repressed only in biofilm cells. Strikingly, the expression level of the sRNA 13573 was 120 times higher in biofilms than in planktonic cells, an observation that prompted us to further investigate the biological role of this non-coding transcript. Analyses of an isogenic mutant and over-expressing strains revealed that the sRNA 13573 gene is involved in biofilm formation and attachment to A549 human alveolar epithelial cells. The present work serves as a basis for future studies examining the complex regulatory network that regulate biofilm biogenesis and attachment to eukaryotic cells in A. baumannii ATCC 17978.

Fig 1. Representation of the 255 putative sRNA expression regions found by deep sequencing in the genome of the A. baumannii strain ATCC 17978.

Bars indicate their location in the genome. no DE black bars indicate no expression differences of the sRNAs analyzed in the three growing conditions. Blue bars indicate sRNAs differentially expressed in stationary phase conditions related to exponential phase. Yellow bars indicate down regulated sRNAs in biofilm related to both types of planktonic cells. Light green bars indicate up regulated sRNAs in biofilm related to both types of planktonic cells. Red bars indicate sRNAs only expressed in planktonic cells and repressed in biofilm. Dark green bars indicate 9 sRNAs only expressed in biofilm and repressed in planktonic cells.

Fig 2. Biofilm formation assays.

A) Quantification of biofilm formation by A. baumannii ATCC 17978 (17978), A. baumannii ATCC 17978 Δ13573 (Δ13573), A. baumannii ATCC 17978 harbouring pETRA (17978 with empty pETRA), A. baumannii ATCC 17978 harbouring pETRA with sRNA 13575 (13573), and A. baumannii ATCC Δ13573 harbouring pETRA with sRNA 13575 (Δ13573 complemented). B) SEM analysis of A. baumannii ATCC 17978 (a), A. baumannii sRNA 13573 over-producing strain (b) and A. baumannii ATCC 17978 Δ13573 (c). Micrographs were taken at 5,000x and bars indicate the scale marks (2 μm).

Fig 3. Adhesion assays.

A) Attachment to A549 epithelial cells of A. baumannii wild type (17978), mutant derivative (Δ13573), mutant derivative over-expressing sRNA 13575 (Δ13573 complemented), harbouring empty pETRA vector (17978 with empty pETRA) and over-expressing sRNA 13575 (13573). The strain 17978 represents 100% of attached bacteria. B) SEM analysis of cells attached to A549 human alveolar cells of A. baumannii ATCC 17978 (a and d), A. baumannii ATCC 17978 over-expressing sRNA 13575 (b and e) and A. baumannii Δ13573 (c and f). Micrographs were taken at 5,000x (a, b and c) and 10,000x magnification (d, e and f). Bars indicate the scale marks (2 and 1 μm).