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The impact of inactivation of the purine biosynthesis genes, purN and purT, on growth and virulence in uropathogenic E. coli

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Abstract

De novo synthesis of purines has been suggested to be an important factor for the pathogenesis of uropathogenic E. coli (UPEC). We analyzed the role of the redundant purine biosynthesis genes purN and purT, responsible for the third step in the purine biosynthesis, during UPEC infection. Growth experiments in M9 (minimal media), MOPS (rich media), filtered urine, and human serum with E. coli UTI89 and ΔpurN, ΔpurT, and ΔpurN/T mutants revealed that UPEC relies on de novo purine synthesis for growth in minimal medium. Mutants in individual genes as well as the double mutant grew equally well as the wild type in urine, rich media, and serum. However, during competition for growth in urine, the wild type UTI89 strain significantly outcompeted the purine auxotrophic ΔpurN/T mutant from late exponential growth phase. Inactivation of purN and/or purT significantly affected UPEC invasion of human bladder cells, but not the intracellular survival. Cytotoxicity levels to bladder cells were also diminished when both purN and purT were deleted, while single gene mutants did not differ from the wild type. When infecting human macrophages, no differences were observed between UTI89 and mutants in uptake, survival or cytotoxicity. Finally, the lack of the pur-gene(s), whether analysed as single or double gene knock-out, did not affect recovery rates after in vivo infection in a mouse model of UTI. These findings suggest that de novo synthesis of purines might be required only when UPEC is fully deprived of nucleotides and when grown in competition with other microorganisms in urine.

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Acknowledgements

Authors would like to thank K. Wiberg Andersen, M. Hegstad Hansen and T. Poul Bønnelycke for their technical contribution/assistance. For the in-vivo studies conducted at Odense University Hospital, the authors also would like to thank Research Assistant C. Antoinette Asferg and PhD-fellow R. Grønnemose for good cooperation throughout the various procedures related to the animal experimentations.

Funding

This work has been funded by Danish Research Council for Independent research, Grant No. DFF – 4184-00050.

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Authors and Affiliations

  1. Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark

    Audrey Inge Schytz Andersen-Civil, Shahana Ahmed, Priscila Regina Guerra, Yaovi Mahuton Gildas Hounmanou, John Elmerdahl Olsen & Ana Herrero-Fresno

  2. Research Unit of Clinical Microbiology, Odense University Hospital, Odense, Denmark

    Thomas Emil Andersen

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  1. Audrey Inge Schytz Andersen-Civil
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  2. Shahana Ahmed
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Correspondence to Ana Herrero-Fresno.

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The authors declare that they have no conflict of interest.

Ethical approval

Experiments with mice were conducted according to the principles expressed in the Declaration of Helsinki. The animal trials were approved by the Danish Animal Experiments Inspectorate, license number 2015-15-0201-00480. Furthermore, we declare that the human urine was obtained from donors who have given their consent to participate in the study on the UTI89 purN and purT mutants.

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Andersen-Civil, A.I.S., Ahmed, S., Guerra, P.R. et al. The impact of inactivation of the purine biosynthesis genes, purN and purT, on growth and virulence in uropathogenic E. coli. Mol Biol Rep 45, 2707–2716 (2018). https://doi.org/10.1007/s11033-018-4441-z

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  • DOI: https://doi.org/10.1007/s11033-018-4441-z

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