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.
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.
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.
- 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.
- 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.
- 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.
- 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.