期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：28
DOI：10.1073/pnas.2203114119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Cell wall anchoring of surface proteins in Gram-positive bacteria requires a sortase enzyme. Here, we unveiled the hitherto unknown function of an evolutionarily conserved small transmembrane protein, named SafA, genetically linked to the housekeeping sortase in Actinobacteria. We show that
Actinomyces oris SafA interacts with the housekeeping sortase SrtA via the conserved FPW motif and prevents SrtA cleavage by the signal peptidase LepB2, hence maintaining membrane homeostasis of SrtA. This function is conserved as ectopic expression of SafA from
Corynebacterium diphtheriae and
Corynebacterium matruchotii in the
A. oris safA mutant rescues its defects in cell morphology, pilus assembly, surface protein localization, and polymicrobial interactions. Thus, SafA represents an archetypal antagonist of signal peptidase that modulates surface assembly in Actinobacteria.
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
Actinomyces oris. Genetic deletion of
safA induces cleavage and excretion of the otherwise predominantly membrane-bound SrtA in wild-type cells. Strikingly, the
safA mutant, although viable, exhibits severe abnormalities in cell morphology, pilus assembly, surface protein localization, and polymicrobial interactions—the phenotypes that are mirrored by
srtA depletion. The pleiotropic defect of the
safA mutant is rescued by ectopic expression of
safA from not only
A. oris, but also
Corynebacterium diphtheriae or
Corynebacterium matruchotii. 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
safA 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.
