Type I Signal Peptidase and Protein Secretion inStaphylococcus epidermidis

Abstract

Bacterial protein secretion is a highly orchestrated process that is essential for infection and virulence. Despite extensive efforts to predict or experimentally detect proteins that are secreted, the characterization of the bacterial secretome has remained challenging. A central event in protein secretion is the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein for secretion via the general secretory pathway, and the arylomycins are a class of natural products that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. Here, using an arylomycin derivative, along with two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identify 11 proteins whose secretion from stationary-phase Staphylococcus epidermidis is dependent on SPase activity, 9 of which are predicted to be translated with canonical N-terminal signal peptides. In addition, we find that the presence of extracellular domains of lipoteichoic acid synthase (LtaS) and the ␤-lactam response sensor BlaR1 in the medium is dependent on SPase activity, suggesting that they are cleaved at noncanonical sites within the protein. In all, the data define the proteins whose stationaryphase secretion depends on SPase and also suggest that the arylomycins should be valuable chemical biology tools for the study of protein secretion in a wide variety of different bacteria.

Activation of BlaR1 Protein of Methicillin-resistant Staphylococcus aureus, Its Proteolytic Processing, and Recovery from Induction of Resistance

“…This result indicated that the cleavage that releases the sensor domain occurs between amino acids Gly-331 and Glu-332. This type of processing was recently shown to be due to the action of type I signal peptidase in Staphylococcus epidermidis (27). BlaR1 from S. epidermidis has a high probability type I signal peptidase recognition sequence just to the C-terminal side of the last predicted transmembrane helix, and cleavage after Gly-331 was predicted for BlaR1 from S. epidermidis.…”

Proteolytic Cleavage Inactivates the Staphylococcus aureus Lipoteichoic Acid Synthase

“…Similarly, processed forms of LtaS-type enzymes were found in the supernatants of L. monocytogenes, Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Staphylococcus epidermidis cultures (2,18,37,44). In all cases where the cleavage site of LtaS-type enzymes has been mapped, it occurs after an Ala-X-Ala motif, and the location of the motif within the protein is conserved in LtaStype enzymes.…”

“…A proteomic study of B. subtilis showed that processing of the LtaS-type enzyme YfnI was diminished in the combined absence of two signal peptidases, SipT and SipV (1). More recently, it was reported that addition of the signal peptidasespecific inhibitor arylomycin to S. epidermidis cultures led to a reduction in the amount of a processed LtaS fragment in the culture supernatant (37). However, in the same study, an alternative LtaS processing site, located after residues 171 AlaPhe-Ala 173 and closer to the 5TM domain, was proposed based on the results of a computer prediction program (Fig.…”

“…However, in the same study, an alternative LtaS processing site, located after residues 171 AlaPhe-Ala 173 and closer to the 5TM domain, was proposed based on the results of a computer prediction program (Fig. 1A) (37).…”

Membrane Proteases in the Bacterial Protein Secretion and Quality Control Pathway

“…ternal signal peptides in a few polytopic membrane proteins (8,12,70,118,147). E. coli SPI is the best-studied signal peptidase in this family.…”