Infection of Pseudomonas donghuensis HYS strain and its fur deletion mutant in Caenorhabditis elegans

Infection of Pseudomonas donghuensis HYS strain and its fur deletion mutant in Caenorhabditis elegans was conducted to assess changes in the expression profile of Caenorhabditis elegans, and potential core virulence factors were identified by measuring the gene expression levels of the HYS colonizing the nematode's intestine. Preliminary studies indicate that P. donghuensis HYS exhibits significant toxicity towards Caenorhabditis elegans, yet the underlying mechanisms of this pronounced toxicity remain unclear. Previous work identified several virulence factors contributing to the toxicity of HYS through detection and functional validation; however, the molecular mechanisms responsible for its strong toxicity have not been elucidated. Therefore, we aim to analyze the mechanisms underlying HYS's pronounced toxicity by examining the responses of infected Caenorhabditis elegans. The Ferric uptake regulator (Fur) is responsible for maintaining iron homeostasis in Gram-negative bacteria, and given that HYS possesses a greater iron uptake capacity than other common species in the same genus, such as Pseudomonas aeruginosa, we hypothesize that Fur may play a critical role in the strong toxicity exhibited by HYS. Consequently, we infected Caenorhabditis elegans with both HYS and its fur deletion mutant and analyzed the changes in the expression profile of Caenorhabditis elegans. We observed a significant reduction in toxicity following the deletion of fur, indicating that Fur regulates core virulence factors. To identify these core virulence factors, we conducted transcriptomic sequencing of the pathogenic bacteria under various conditions and performed a screening for virulence factors. Preliminary research in the laboratory revealed that the HYS strain produces substantial colonization within 24 hours of infecting the host, and unlike other pathogenic bacteria that infect C. elegans, HYS infection results in significant mortality of the nematodes within this timeframe. Therefore, to ensure the quantity of Caenorhabditis elegans and the quality of RNA extraction, we selected the 24-hour time point for nematode collection, which were rapidly frozen in liquid nitrogen for total RNA extraction. We utilized Escherichia coli OP50, which is minimally toxic to the nematodes, as a negative control, while the infected nematodes with HYS and fur mutant served as the experimental groups, with three biological replicates for each condition, comprising 1000 nematodes per sample. Following RNA extraction, high-throughput sequencing was conducted at Majorbio. For the pathogenic bacterial samples, we established the HYS strain grown on NGM plates as the control, while the HYS strain colonizing the nematodes constituted the experimental group, which, along with the HYS-infected nematodes, was processed as the same sample for dual-RNA seq. Due to the inability of the fur deletion mutant to form colonies, we included the fur mutant strain grown on NGM plates as another experimental group. The colonized HYS samples were obtained by homogenizing 1000 nematodes fed with the HYS strain for RNA extraction, while a total of 10^10 bacteria from those grown on NGM plates were required for total RNA extraction. Subsequently, all samples were sent to Majorbio for high-throughput sequencing. After sequencing, the results of the interactions between the nematodes and bacteria were identified using the Caenorhabditis elegans and HYS genomes, while other sequences were identified using a separate genome.