Modulation of the Epithelial Sodium Channel (ENaC) by Bacterial Metalloproteases and Protease Inhibitors

Abstract

The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP) from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC), leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens

“…AprI was recently shown to be able to inhibit purified PrtS protease activity in vitro (23). This was confirmed in our model system, where induced PrtS was inhibited by AprI in a dose-dependent manner (see Fig.…”

“…Molecular biology. The N-terminal polyhistidine-tagged slpB, slpC, and slpD genes under the control of the P BAD promoter on p15a-based plasmid pMQ125 (39) were generated by replacing prtS on pMQ356 (23) but maintaining the N-terminal His 7 tag. This was done by digesting pMQ356 with SalI and SmaI that cut in the prtS gene and then replacing the entire prtS gene using primers that amplify the slp genes and have regions of homology to the His 7 tag and vector backbone.…”

“…The cultures were grown in LB medium supplemented with gentamicin, and 0.2% (wt/vol) arabinose was added to the culture when it achieved an OD 600 of 0.8. Protein was expressed at 37°C for 4 to 6 h. The SlpB protein was purified from the insoluble material under denaturing conditions by following protocols previously described for PrtS and AprA (23). In vitro protein refolding and protease activity were assessed using a fluorescent peptide and casein substrates, as previously described (42).…”

“…In vitro protein refolding and protease activity were assessed using a fluorescent peptide and casein substrates, as previously described (42). The AprI protease inhibitor was expressed and purified as previously described (23).…”

“…Serralysin, and serralysin family metalloproteases, such as alkaline protease from Pseudomonas aeruginosa, can proteolyze mammalian cell surface proteins, thereby modulating cell physiology. A recent example is the protease-mediated activation of the epithelial sodium channel, leading to a cell surface that is more amenable to bacterial colonization (22,23). Several bacteria, including Serratia species, invade eukaryotic airway cells in a protease-dependent manner (24)(25)(26)(27).…”