A SecA-associated protease modulates the extent of surface display of staphylococcal protein A

TL;DR

A bacterial protease called PepV interacts with a protein precursor, influencing its surface display and stability in Staphylococcus aureus.

Contribution

PepV is identified as a SecA-associated protease that modulates the surface display of proteins with YSIRK/GXXS signal peptides.

Findings

SpA deposition into cross-walls increases in bacteria lacking PepV.

PepV interacts with SecA and undergoes self-cleavage when incubated with SpA precursors.

SpA precursors destabilize PepV, possibly to counteract its inhibitory effect.

Abstract

In bacteria, signal peptides direct pre-proteins to the SecYEG secretion channel and are typically cleaved by signal peptidases during translocation across the membrane. In gram-positive bacteria, such as Staphylococcus aureus, some signal peptides have a pre-translocation function. Staphylococcal protein A (SpA) carries such an atypical signal sequence, with a YSIRK/GXXS motif that directs its precursor into the cross-wall of dividing cells for subsequent anchoring by sortase A. Here, we report that PepV—a member of the M20 peptidase family which has been described as a manganese-dependent dipeptidase in vitro—may influence the surface display of precursors with a YSIRK/GXXS motif. SpA deposition into cross-walls was increased in ΔpepV bacteria. Yet, in the absence of pepV, neither the kinetics of signal sequence processing nor the final product of the sorting reaction was altered. In pull-down experiments, PepV was identified as a ligand of SecA. When purified PepV was incubated with SpA precursors, this interaction triggered self-cleavage of the enzyme, an unexpected activity exacerbated by the presence of a chelating agent. In agreement with this finding, a pulse-chase experiment revealed that the half-life of PepV is extended in bacteria lacking spa. Collectively, these data reveal a mutually inhibitory relationship between SpA precursors and PepV, the net result suggesting that while PepV may reduce the surface display of SpA, SpA precursors destabilize PepV possibly to overcome such inhibition.

The “signal hypothesis” proposed that N-terminal sequences of secretory proteins contain targeting cues directing nascent polypeptides to the endoplasmic reticulum. This concept was later confirmed as broadly applicable, even to prokaryotes with a single membrane. In gram-positive bacteria, signal sequences bearing the YSIRK/GXXS motif are necessary and sufficient to direct precursors to septal membranes. However, trans-acting factors involved in this spatially restricted targeting remain largely unknown. Here, we identify a member of the M20 metalloprotease family as a potential contributor to the septal surface display of proteins containing YSIRK/GXXS signal peptides.

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