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

Pantoea piersonii IIIF1SW-P2^T (basionym Kalamiella piersonii IIIF1SW-P2^T), 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-P2^T 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-P2^T (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 K_m, V_max and K_cat 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-P2^T in the reproductive tract with 5% mortality of C. elegans. Gfp-tagged IIIF1SW-P2^T 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-P2^T 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-P2^T to colonize the reproductive tract of a eukaryote.