Expression patterns of BlsA, a photoreceptor involved in modulation of motility, biofilm formation and virulence against C. albicans in A. baumannii.

MATÍAS CABRUJA; LUIS ACTIS; MARÍA ALEJANDRA MUSSI

Colonia

Congreso; 9th International Symposium on the Biology of Acinetobacter.; 2013

Objectives: We have previously shown that A. baumannii ATCC 17978 is able to sense and respond to light, modulating motility, biofilm formation and virulence against C. albicans at environmental temperatures through the photoreceptor BlsA. Despite many aspects of this process have been characterized; no direct information about the presence or variation in the levels of BlsA within the cells under different conditions is available, which would be important to determine the conditions in which light regulation is important for these bacteria. Methods: We produced antibodies against A. baumannii ATCC 17978 BlsA in rabbit and performed western blot analysis under different conditions. Proteins were extracted from ATCC 17978 cultures grown in LB in agitation under blue light or in the dark, at 24 or 37°C. However, traditional western blotting with alkaline phosphatase using even 40 µg of total protein was not sensitive enough for detection of BlsA. After exhaustive conditions adjustment, we were able to obtain signal corresponding to BlsA when we loaded 400 µg of total protein and used a highly sensitive method able to detect as slight as fempto amounts of proteins (Thermo). Results: Our results show that BlsA is effectively expressed in the wild type, even though to very low levels. As expected, it is absent in the blsA mutant and over expressed (more than ten folds) in the mutant expressing blsA from the pWH1266 plasmid. Moreover, our results show that the levels of BlsA in the wild type strain under dark conditions are more than ten folds those observed for these cells under blue light at 24 °C. Conversely, the content of BlsA protein in the cells at 37 °C is negligible or null, at either temperature. Furthermore, we show fluctuations in its levels along the cell cycle that also depend on light and temperature conditions. Conclusion: The levels of BlsA levels are very low in the cells, compatible with its functioning as a regulator. The levels of this photoreceptor vary under light and temperature conditions along the cell cycle, showing that it may function as an activator or anti-repressor at environmental temperatures.