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Deletion of both methionine sulfoxide reductase A and methionine sulfoxide reductase C genes renders Salmonella Typhimurium highly susceptible to hypochlorite stress and poultry macrophages

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Abstract

Salmonella Typhimurium survives and replicates inside the oxidative environment of phagocytic cells. Proteins, because of their composition and location, are the foremost targets of host inflammatory response. Among others, Met-residues are highly prone to oxidation. Methionine sulfoxide reductase (Msr), with the help of thioredoxin-thioredoxin reductase, can repair oxidized methionine (Met-SO) residues to Met. There are four methionine sulfoxide reductases localized in the cytosol of S. Typhimurium, MsrA, MsrB, MsrC and BisC. MsrA repairs both protein-bound and free ‘S’ Met-SO, MsrB repairs protein-bound ‘R’ Met-SO, MsrC repairs free ‘R’ Met-SO and BisC repairs free ‘S’ Met-SO. To assess the role(s) of various Msrs in Salmonella, few studies have been conducted by utilizing ΔmsrA, ΔmsrB, ΔmsrC, ΔmsrAΔmsrB, ΔmsrBΔmsrC and ΔbisC mutant strains of S. Typhimurium. Out of the above-mentioned mutants, ΔmsrA and ΔmsrC were found to play important role in the stress survival of this bacterium; however, the combined roles of these two genes have not been determined. In the current study, we have generated msrAmsrC double gene deletion strain (ΔmsrAΔmsrC) of S. Typhimurium and evaluated the effect of gene deletions on the survival of Salmonella against hypochlorite stress and intramacrophage replication. In in vitro growth curve analysis, ΔmsrAΔmsrC mutant strain showed a longer lag phase during the initial stages of the growth; however, it attained similar growth as the wild type strain of S. Typhimurium after 5 h. The ΔmsrAΔmsrC mutant strain has been highly (~ 3000 folds more) sensitive (p < 0.001) to hypochlorite stress. Further, ΔmsrA and ΔmsrAΔmsrC mutant strains showed more than 8 and 26 folds more susceptibility to poultry macrophages, respectively. Our data suggest that the deletion of both msrA and msrC genes severely affect the oxidative stress survival and intramacrophage proliferation of S. Typhimurium.

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Acknowledgements

The current study was funded by the Department of Biotechnology, India (Grant No.: BT/PR13689/BRB/10/1399/2015) and NASF, ICAR, India (Grant No.: NFBSFARA/BS-3012/2012-13). We thank our Director, ICAR-Indian Veterinary Research Institute (IVRI) for providing the necessary facilities.

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  1. Sonu S. Nair and Tapan Kumar Singh Chauhan contributed equally to this work

Authors and Affiliations

  1. Division of Bacteriology & Mycology, ICAR-Indian Veterinary Research Institute, Izatnagar, 243 122, India

    Sonu S. Nair, V. Athira &  Abhishek

  2. Division of Biochemistry, ICAR-Indian Veterinary Research Institute, Izatnagar, 243 122, India

    Tapan Kumar Singh Chauhan, Manoj Kumawat, Ratanti Sarkhel, Shekhar Apoorva, Arijit Shome & Manish Mahawar

  3. Division of Biological Products, ICAR-Indian Veterinary Research Institute, Izatnagar, 243 122, India

    Bablu Kumar

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Contributions

SSN and AS conducted the growth curve analysis experiments. SSN and SA conducted HOCl assays. TKSC, SSN and RS conducted macrophage assays. MK and MM created double gene deletion strain. SSN and MM wrote the manuscript and A, AV and BK edited the manuscript.

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Correspondence to Abhishek or Manish Mahawar.

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Ethical approval

The protocol for isolation of peripheral blood mononuclear cells from poultry blood was approved by the Institute Animal Ethics Committee, ICAR-IVRI, Izatnagar, India, vide letter number F.26-2/2019/JD(R) dated January 6th, 2020.

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Nair, S.S., Chauhan, T.K.S., Kumawat, M. et al. Deletion of both methionine sulfoxide reductase A and methionine sulfoxide reductase C genes renders Salmonella Typhimurium highly susceptible to hypochlorite stress and poultry macrophages. Mol Biol Rep 48, 3195–3203 (2021). https://doi.org/10.1007/s11033-021-06381-2

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