PrtA metalloprotease expression is regulated by CpxR and contributes to Serratia marcescens biofilm formation

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

Ciudad Autónoma de Buenos Aires

Congreso; LIII Reunión de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2017

SAIB

Serratia marcescens (Sma) is an environmentally ubiquitous bacterium also acting as an opportunistic pathogen. Its ability 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. In this study, we found that CpxRA -a canonical TCS typically involved in counteracting envelope stress- represses PrtA expression in a temperature dependent manner. Secreted proteolytic activity by azocaseinase assay was 50%-increased in a cpxR background at 37°C of growth temperature, but no significant difference was observed at 30ºC. In trans-expression of CpxR restored the activity of the mutant strain to wild type levels. This modulation at 37°C is attained at the transcriptional level, as shown by comparative fluorescence measurements from a PprtA-gfp reporter plasmid between wild type and cpxR mutant. Over-expression of outer membrane protein NlpE, a CpxRA-activating condition, diminished proteolytic activity at both growth temperatures (-64% at 30°C; -90% at 37°C) when compared with the control strain. Furthermore, we performed EMSA and DNAse I protection assays with radiolabeled DNA fragments and purified CpxR, and found that the regulator specifically binds to a previously in silico-predicted CpxR-binding site within prtA promoter region. Lastly, we examined if PrtA expression could influence biofilm control in Sma. Cristal violet assays indicated that prtA mutant is defective in biofilm production (-57% at 30°C; -30% at 37°C). In correlation with CpxR inhibitory action at 37 °C, cpxR inactivation enhanced 40% biofilm formation at 37 ºC, but no difference was observed at 30°C.