SERRATIA MARCESCENS PRTA: A KEY FACTOR TO STRUCTURE A MATURE BIOFILM

MOLINO, MV; BRUNA, R.; GARCÍA VÉSCOVI, E.

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress; 2019

SAIB-PABMB

Serratia marcescens (Sma) is an environmentally ubiquitous bacterium also acting as an opportunistic pathogen. Moreover, Sma displays biofilm formation capacity that has been shown to be related to its ability to colonize, persist, and proliferate on either biological or inert surfaces. This capacity to adapt and survive in either hostile or changing environments can be related to the expression of a myriad of secreted hydrolytic enzymes, including proteases. Genomic analysis of Sma clinical strain RM66262 identified four zinc-metalloprotease-encoding genes. Amongst them, we previously showed that PrtA is prominently secreted and its expression depends on the bacterial growth temperature, being transcriptionally upregulated at 30°C in comparison with 37°C. We also found that, in Sma, the CpxAR signal transduction system, which responds to envelope stress and bacterial surface adhesion, is activated at 37°C and able to down-regulate PrtA expression by CpxR at the transcriptional level. Taking into account that, in other enterobacteria, the CpxAR pathway is involved in modulating the ability of biofilm generation, we examined whether PrtA could influence Sma biofilm formation capacity. To that aim, we performed in vitro biofilm assays in polystyrene microwell plates, followed by biofilm quantitation using crystal violet staining. When the strains were grown at 30°C or 37°C in SLB medium the lack of PrtA expression in the prtA strain reduced the capacity of the bacteria to form biofilm compared with that of the wildtype strain, being more attenuated at 37°C. Results of confocal microscopy also showed this deficiency in biofilm formation in the prtA strain. The defect in biofilm formation of the prtA strain could be complemented to wild-type levels by adding purified, catalytically active, PrtA. To further understand PrtA influence on biofilm formation, we built a single-aminoacid-mutant protein that annuls the protease hydrolytic capacity and performed biofilm assays. Our results show a defective biofilm phenotype, equivalent to a prtA strain. The addition of purified catalytically active PrtA to this mutant restored biofilm formation to wild-type levels, demonstrating that PrtA expression and activity contribute to the ability of Sma to structure a biofilm community.