Many bacterial species interact via direct cell-to-cell contact using CDI systems, which provide a mechanism to inject toxins that inhibit bacterial growth into one another. Here, we find that two CDI toxins, one that depolarizes membranes and another that degrades RNA, exploit the universally conserved SecY translocon machinery used to export proteins for target cell entry. ABSTRACT The C-terminal (CT) toxin domains of contact-dependent growth inhibition (CDI) CdiA proteins target Gram-negative bacteria and must breach both the outer and inner membranes of target cells to exert growth inhibitory activity. Here, we examine two CdiA-CT toxins that exploit the bacterial general protein secretion machinery after delivery into the periplasm. A Ser281Phe amino acid substitution in transmembrane segment 7 of SecY, the universally conserved channel-forming subunit of the Sec translocon, decreases the cytotoxicity of the membrane depolarizing orphan10 toxin from enterohemorrhagic Escherichia coli EC869. Target cells expressing secY ~(S281F) and lacking either PpiD or YfgM, two SecY auxiliary factors, are fully protected from CDI-mediated inhibition either by CdiA-CT _(o10) ~(EC869) or by CdiA-CT ~(GN05224), the latter being an EndoU RNase CdiA toxin from Klebsiella aerogenes GN05224 that has a related cytoplasm entry domain. RNase activity of CdiA-CT ~(GN05224) was reduced in secY ~(S281F) target cells and absent in secY ~(S281F) Δ ppiD or secY ~(S281F) Δ yfgM target cells during competition co-cultures. Importantly, an allele-specific mutation in secY ( secY ~(G313W) ) renders Δ ppiD or Δ yfgM target cells specifically resistant to CdiA-CT ~(GN05224) but not to CdiA-CT _(o10) ~(EC869), further suggesting a direct interaction between SecY and the CDI toxins. Our results provide genetic evidence of a unique confluence between the primary cellular export route for unfolded polypeptides and the import pathways of two CDI toxins.
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