Lethality of sortase depletion in A ctinomyces oris caused by excessive membrane accumulation of a surface glycoprotein

Summary Sortase, a cysteine‐transpeptidase conserved in G ram‐positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of the housekeeping sortase in several G ram‐positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for A ctinomyces oris ; yet, all of its predicted cell wall‐anchored protein substrates ( AcaA ‐ N ) were individually dispensable for cell viability. Using Tn 5 ‐transposon mutagenesis to identify factors that upend lethality of srtA deletion, we uncovered a set of genetic suppressors harbouring transposon insertions within genes of a locus encoding AcaC and a LytR ‐ CpsA ‐ Psr ( LCP )‐like protein. AcaC was shown to be highly glycosylated and dependent on LCP for its glycosylation. Upon SrtA depletion, the glycosylated form of AcaC , hereby renamed GspA , was accumulated in the membrane. Overexpression of GspA in a mutant lacking gspA and srtA was lethal; conversely, cells overexpressing a GspA mutant missing a membrane‐localization domain were viable. The results reveal a unique glycosylation pathway in A . oris that is coupled to cell wall anchoring catalysed by sortase SrtA . Significantly, this novel phenomenon of glyco‐stress provides convenient cell‐based assays for developing a new class of inhibitors against G ram‐positive pathogens.