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<p><i>Pantoea piersonii</i> IIIF1SW-P2<sup>T</sup> (basionym <i>Kalamiella piersonii</i> IIIF1SW-P2<sup>T</sup>), a bacterial species isolated from the International Space Station, was predicted to be non-pathogenic to humans, unlike its clinical counterpart <i>P. piersonii</i> YU22, which induces struvite crystallization and causes toxicity to Human Embryonic Kidney cell line (HEK 293&#xa0;T). Here, we identify L-asparaginase-mediated cytotoxicity of IIIF1SW-P2<sup>T</sup> on HEK 293&#xa0;T <i>in vitro</i> and demonstrate strain’s colonization ability in the reproductive tract <i>in vivo</i> using <i>Caenorhabditis elegans</i> as a model system. The recombinant L-asparaginase of IIIF1SW-P2<sup>T</sup> (Kp_AnsA, ~ 37&#xa0;kDa) generated significant amounts of NH<sub>4</sub><sup>+</sup> (4.1‒15.5&#xa0;μM, <i>P</i> &lt; 0.0001) and exerted cytotoxicity to HEK 293&#xa0;T (29.1‒36.0%, <i>P</i> &lt; 0.0001).&#xa0;NH<sub>4</sub><sup>+</sup>-driven cytotoxicity of HEK 293&#xa0;T was validated through the introduction of nearly equimolar amounts of standard NH<sub>4</sub><sup>+</sup> (12.7&#xa0;μM; 31% cytotoxicity, <i>P</i> &lt; 0.0001). Kp_AnsA found to be a halotolerant enzyme, highly active in alkaline pH (optimum pH 9), and exhibited <i>K</i><sub><i>m</i></sub>, <i>V</i><sub><i>max</i></sub> and <i>K</i><sub><i>cat</i></sub> values of 5.4&#xa0;mM, 8.4 U/mg and 135.6 μmoles s<sup>-1</sup>, respectively, in the presence of L-asparagine. Kp_AnsA displayed absolute amino acid sequence identity with that of YU22 and formed a tight phyletic association with that of marine bacteria. Analysis <i>in vivo</i> revealed the rapid and sustained colonization of green fluorescent protein (<i>gfp</i>)-tagged IIIF1SW-P2<sup>T</sup> in the reproductive tract with 5% mortality of <i>C. elegans</i>. <i>Gfp</i>-tagged IIIF1SW-P2<sup>T</sup> were localized at the vulvar and luminal regions, embryos and interembryonic space of <i>C. elegans</i>. The role of L-asparaginase in the colonization of IIIF1SW-P2<sup>T</sup> at the reproductive tract of <i>C. elegans</i> merits further investigation. Nonetheless, this study demonstrated cytotoxicity of bacterial L-asparaginase on HEK 293&#xa0;T and discovered the potential ability of IIIF1SW-P2<sup>T</sup> to colonize the reproductive tract of a eukaryote.</p>

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Pantoea piersonii IIIF1SW-P2T triggers cytotoxicity through L-asparaginase-driven ammonium secretion and colonizes reproductive tract of Caenorhabditis elegans

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  • 发表日期：

    2025年10月9日

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  • Asif Hameed,
  • Chiu-Chung Young,
  • Kokkarambath Vannadil Suchithra,
  • Ashwini Prabhu,
  • Honagodu Ravichandra Dhanyashree,
  • Rajesh Padumane Shastry

摘要

Pantoea piersonii IIIF1SW-P2T (basionym Kalamiella piersonii IIIF1SW-P2T), a bacterial species isolated from the International Space Station, was predicted to be non-pathogenic to humans, unlike its clinical counterpart P. piersonii YU22, which induces struvite crystallization and causes toxicity to Human Embryonic Kidney cell line (HEK 293 T). Here, we identify L-asparaginase-mediated cytotoxicity of IIIF1SW-P2T on HEK 293 T in vitro and demonstrate strain’s colonization ability in the reproductive tract in vivo using Caenorhabditis elegans as a model system. The recombinant L-asparaginase of IIIF1SW-P2T (Kp_AnsA, ~ 37 kDa) generated significant amounts of NH4+ (4.1‒15.5 μM, P < 0.0001) and exerted cytotoxicity to HEK 293 T (29.1‒36.0%, P < 0.0001). NH4+-driven cytotoxicity of HEK 293 T was validated through the introduction of nearly equimolar amounts of standard NH4+ (12.7 μM; 31% cytotoxicity, P < 0.0001). Kp_AnsA found to be a halotolerant enzyme, highly active in alkaline pH (optimum pH 9), and exhibited Km, Vmax and Kcat values of 5.4 mM, 8.4 U/mg and 135.6 μmoles s-1, respectively, in the presence of L-asparagine. Kp_AnsA displayed absolute amino acid sequence identity with that of YU22 and formed a tight phyletic association with that of marine bacteria. Analysis in vivo revealed the rapid and sustained colonization of green fluorescent protein (gfp)-tagged IIIF1SW-P2T in the reproductive tract with 5% mortality of C. elegans. Gfp-tagged IIIF1SW-P2T were localized at the vulvar and luminal regions, embryos and interembryonic space of C. elegans. The role of L-asparaginase in the colonization of IIIF1SW-P2T at the reproductive tract of C. elegans merits further investigation. Nonetheless, this study demonstrated cytotoxicity of bacterial L-asparaginase on HEK 293 T and discovered the potential ability of IIIF1SW-P2T to colonize the reproductive tract of a eukaryote.