Supplementary Materials Supplemental material supp_198_1_66__index. biofilm maturation, as a substrate of
Supplementary Materials Supplemental material supp_198_1_66__index. biofilm maturation, as a substrate of LapG. We also demonstrated LapDG to be a minimal system sufficient to control CdrA localization in response to changes in the intracellular concentration of c-di-GMP. Our work establishes this biofilm signaling node as a regulator of a type Vb secretion system substrate in a clinically important pathogen. IMPORTANCE Here, the biological relevance of a conserved yet orphan signaling system in the opportunistic pathogen is revealed. In particular, we identified the adhesin CdrA, the cargo of a two-partner secretion system, as a substrate of a periplasmic protease whose activity is controlled by intracellular c-di-GMP levels and a corresponding transmembrane receptor via an inside-out signaling mechanism. The data indicate a posttranslational control mechanism of CdrA via c-di-GMP, in addition GSK1120212 manufacturer to its established transcriptional regulation via the same second messenger. INTRODUCTION Bacteria in nature exist as free-swimming motile organisms or as sessile communities adhered to solid surfaces that are enveloped in a self-produced matrix of adhesive proteins, polysaccharides, and nucleic acids (1). These biofilms protect bacterial communities from their surrounding environment, and as a result, infections caused by biofilm-forming pathogens are often tolerant to traditional antibiotic therapies and the immune system. Understanding the molecular mechanisms governing the regulation of bacterial biofilm formation is thus paramount to finding new avenues for treating such chronic infections. Biofilm formation is regulated via a bacterial second messenger, cyclic di-GMP (c-di-GMP), enzymes for its biosynthesis and degradation, and binding proteins that monitor levels of this cyclic dinucleotide. High levels GSK1120212 manufacturer of c-di-GMP are often associated with a switch to a sessile lifestyle through the binding of the second messenger to receptor proteins (2). Our previous studies identified a central c-di-GMP-specific receptor, LapD, and its associated signaling system in the environmental bacterium and model system for biofilm formation (3,C6) (Fig. 1A). In particular, we found that the inner membrane-localized LapD receptor is autoinhibited at low cellular c-di-GMP levels but switches into a signaling-active state when c-di-GMP levels rise and the dinucleotide binds to the intracellular module of LapD. The associated conformational change extends to LapD’s periplasmic output domain, which as a result recruits the periplasmic protease LapG. LapG’s substrate in is the large, cell surface-associated adhesin protein LapA. LapG cleaves the N-terminal domain of LapA, releasing this adhesin from the cell surface, which results in biofilm dispersal when c-di-GMP levels are low and LapG is not associated with LapD. When recruited to LapD under conditions of high c-di-GMP, LapG cannot process LapA, and Rabbit Polyclonal to TAS2R1 LapA is in turn retained at the cell surface, stably anchoring cells to substrate during the early stages of biofilm formation. Open in a separate window FIG 1 Mechanism of biofilm formation in accounts for 10% of hospital infections, notably in burn victims and surgical patients as well as those individuals with cystic fibrosis (8). These biofilm-based infections are notoriously difficult to treat, highlighting the importance of understanding the complex mechanisms controlling biofilm formation. Here, we follow on previous work by revealing the target of the LapD/LapG system in biofilm regulation. We show that LapG targets the outer GSK1120212 manufacturer membrane-anchored adhesin protein CdrA for posttranslational processing and in doing so alters cell-cell interactions and biofilm formation. CdrA is the passenger protein of a two-partner secretion system (also known as type Vb secretion system), and its expression is regulated by c-di-GMP (9,C11). Hence, our data reveal a second, c-di-GMP-regulated mechanism, in this case for the release of the adhesin from the cell surface, presumably when cellular second-messenger levels drop. This work validates the predicted conservation of the LapD/LapG receptor system but also uncovers a new class of adhesin proteins as targets for the LapG protease. MATERIALS AND METHODS Plasmid construction, protein expression, and protein purification. (i).