Cleavage of the outer membrane protein OmpX by protealysin regulates Serratia proteamaculans invasion

Abstract

Protealysin is a thermolysin-like protease of Serratia proteamaculans capable of specifically cleaving actin, which correlates with the invasive activity of these bacteria. Here, we show that inactivation of the protealysin gene does not inhibit invasion but, in contrast, leads to a twofold increase in the S. proteamaculans invasive activity. By mass spectrometry, we identified the outer membrane protein OmpX as a substrate of protealysin. Recombinant E. coli carrying the OmpX gene truncated by 40 N-terminal residues or both the OmpX and protealysin genes, in contrast to the full-length OmpX, do not increase adhesion of these bacteria, indicating that the 40 N-terminal residues of OmpX are indispensable for S. proteamaculans invasion. Our results show that both protealysin and its substrates can stimulate Serratia invasion.

The Role of SprIR Quorum Sensing System in the Regulation of Serratia proteamaculans 94 Invasion

“…Previously, we have shown that inactivation of the AHL synthase sprI gene resulted in the increase in invasive activity of S. proteamaculans preceded by the increased bacterial adhesion to the cell surface [6]. By our data, the increased adhesion of S. proteamaculans SprI(-) could be caused by both an increase in the expression of the surface protein OmpX responsible for adhesion [6][7][8] and a decrease in the activity of protease protealysin, whose substrate is OmpX [7,8]. On the other hand, the AHL signaling molecules can directly affect host cells and their multiple signaling pathways by triggering calcium mobilization, activation of Rho GTPases, MAPK, and NFκB that control diverse host cells functions and behaviors, including cytoskeleton remodeling [9].…”

“…We have shown that inactivation of the AHL synthase sprI gene led to a more than fourfold increase in the invasive activity of S. proteamaculans as a result of increased adhesion [6]. This increase in the adhesion of S. proteamaculans with inactivated AHL synthase gene can be caused by an increase in the amount of the surface protein OmpX and a decrease in the activity of the protease protealysin, the substrate of which is OmpX [6][7][8]. These results allowed us to suggest that synthesis of AHLs controls the invasion of S. proteamaculans by accumulation on the bacterial surface of full-length OmpX not cleaved by protealysin [6].…”

“…Efficiency of invasion and adhesion was evaluated by the quantitative invasion assay [8,18]. Bacteria (1 mL) were pelleted at 9600× g for 10 min; the pellets were then resuspended in 100 µL DMEM and added to the host cells in a 1 mL fresh portion of DMEM.…”

“…To quantify the effectiveness of adhesion, suspension of the infected cells was quickly diluted and plated out on LB agar to determine the number of colony forming units (CFU) of adherent bacteria. To quantify the effectiveness of invasion, suspension of the infected cells was incubated in DMEM containing kanamycin to kill extracellular bacteria and then the cells were lysed with 1.5% sodium deoxycholate, quickly diluted with cold LB medium, and the aliquots of the resulting suspension were plated on LB agar to determine the number of colony forming units (CFU) of intracellular bacteria [8]. The results for each experiment are the average of an assay performed in triplicate and independently repeated three times.…”

“…The increase in adhesion of S. proteamaculans was previously shown to be associated with an increase in the expression of the bacterial surface protein OmpX [6][7][8] and a decrease in the activity of the bacterial protease protealysin, the substrate of which is OmpX [6,8]. Therefore, we evaluated the effects of the inactivation of the AHL receptor sprR gene on the level of the OmpX and protealysin expression.…”