A conserved signal-peptidase antagonist modulates membrane homeostasis of actinobacterial sortase critical for surface morphogenesis.

Abstract

Most Actinobacteria encode a small transmembrane protein, whose gene lies immediately downstream of the housekeeping sortase coding for a transpeptidase that anchors many extracellular proteins to the Gram-positive bacterial cell wall. Here, we uncover the hitherto unknown function of this class of conserved proteins, which we name SafA, as a topological modulator of sortase in the oral Actinobacterium <i>Actinomyces oris</i>. Genetic deletion of <i>safA</i> induces cleavage and excretion of the otherwise predominantly membrane-bound SrtA in wild-type cells. Strikingly, the <i>safA</i> mutant, although viable, exhibits severe abnormalities in cell morphology, pilus assembly, surface protein localization, and polymicrobial interactions-the phenotypes that are mirrored by <i>srtA</i> depletion. The pleiotropic defect of the <i>safA</i> mutant is rescued by ectopic expression of <i>safA</i> from not only <i>A. oris</i>, but also <i>Corynebacterium diphtheriae</i> or <i>Corynebacterium matruchotii</i>. Importantly, the SrtA N terminus harbors a tripartite-domain feature typical of a bacterial signal peptide, including a cleavage motif AXA, mutations in which prevent SrtA cleavage mediated by the signal peptidase LepB2. Bacterial two-hybrid analysis demonstrates that SafA and SrtA directly interact. This interaction involves a conserved motif FPW within the exoplasmic face of SafA, since mutations of this motif abrogate SafA-SrtA interaction and induce SrtA cleavage and excretion as observed in the <i>safA</i> mutant. Evidently, SafA is a membrane-imbedded antagonist of signal peptidase that safeguards and maintains membrane homeostasis of the housekeeping sortase SrtA, a central player of cell surface assembly.