title: Lethality of sortase depletion in Actinomyces oris caused by excessive membrane accumulation of a surface glycoprotein.
abstract: Sortase, a cysteine-transpeptidase conserved in Gram-positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of the housekeeping sortase in several Gram-positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for Actinomyces oris; yet, all of its predicted cell wall-anchored protein substrates (AcaA-N) were individually dispensable for cell viability. Using Tn5-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 Gram-positive pathogens.
authors.affiliation: Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, Houston, TX, USA.
authors.forename: Chenggang
authors.initials: C
authors.lastname: Wu
authors.forename: I-Hsiu
authors.initials: IH
authors.lastname: Huang
authors.forename: Chungyu
authors.initials: C
authors.lastname: Chang
authors.forename: Melissa Elizabeth
authors.initials: ME
authors.lastname: Reardon-Robinson
authors.forename: Asis
authors.initials: A
authors.lastname: Das
authors.forename: Hung
authors.initials: H
authors.lastname: Ton-That
mesh_terms: D000190:Actinomyces / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D019881:Aminoacyltransferases / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D020043:Genes, Essential; D005804:Genes, Lethal; D006031:Glycosylation; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; C090745:global stress protein A, bacteria; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases