Activity of acetyltransferase toxins involved in Salmonella persister formation during macrophage infection

Salmonella

Typhoidal

Non-Typhoidal

  • Only infect Human to Human.
  • Causing typhoidal fever and paratyphoid fever.
  • Infect animal to human, human to animal.
  • Causing gastroenteritis and food poisoning.
  • Primarily in sub-Saharan Africa and kills 3-4 folds more than typhoid.
  • Universally resistant to first, second line antibiotics.
  • 78% of reinfection may due to growth resuscitation by persister formation.

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Persister formation

  • In TA system, activation of Toxin could interfere major cellular function, such as RNA translation, DNA replication, causing growth inhibition and become dormant state.
  • In case of TacT toxin, through Acetylation of aminoacyl tRNA cause persister formation.

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Results

Non-typhoidal Salmonella form persisters in human macrophages.

Acetyltransferases TacT2SEn and TacT3 alter the translation.

Tac toxins block translation through aminoacyl-tRNA acetylation.

TacT2SEn acetylates aminoacyl-tRNAs more than TacT2STm

The Tac toxins target partially distinct subsets of aa-tRNAs.

  • Formation of antibiotic-tolerant persisters in response to internalization by host cell.
  • TacT, TacT2, TacT3 are conserved across serotype.
  • TacT2STm, TacT2SEn display a one amino acid change, lack of RelBE1, parDE.

  • TacT toxin induce persister state.
  • TacT2SEn, TacT3 counteracted by cognate antitoxin.
  • TacT2SEn shows greater extent of persister formation than TacT2STm.

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  • TacT2SEn and TacT3, but not TacT2STm toxins, prolong the bacterial non-growing state.
  • Growth inhibition counteracted by cognate antititoxin.
  • Abolished toxicity by substitution of catalytic site amino acid, responsible for transfer acetyl moiety.
  • Substitution of one amino acid, which show positively charged groove,** abolished toxicity.**
  • Measuring translation rates by pulse chase of radio-labelled Methionine, shows all active TacT toxins alter translation.

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  • DHFR production inhibited by Toxins.
  • Extent of acetylation mirrored extent of inhibited DHFR.
  • TacT3 shows different orientation of Acetyl-CoA compared to TacT.
  • Pth, detoxify the acetylated charged tRNAs.
  • Radioactive acetylation signal decreased by incubate Pth.
  • Also Pth counteracted effect of growth inhibitory and persister induction.

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  • TacT2STm, TacT2SEn show reversed charge amino acid E29K.
  • TacT2STm only acetylated the aminoacyl-tRNAs efficiently at a pH of 7.5
    - TacT2STm is more susceptible to pH changes than TacT2SEn.

  • TacT2STm is less stable protein than TacT2SEn.
    - TacT2SEn forms hydrogen bonds between side chain of K29 and Phe17, Tyr15.
  • TacT2STm cannot form hydrogen bonds.
  • TacT similarly stabilize by hydrogen bonds.
  • TacT3 form hydrogen bonds with Gln32, Arg17
  • Without hydrogen bonds, aa-tRNA is less accessible to toxin active site.

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  • Through pth, hydrolyze acetylated aa-tRNA and analyzed by LC-MS.
  • Even though each toxins show overlapped amino acids, but differed in their substrate specificity.