The construction of a genome‐wide transposon library of Glaesserella parasuis and its use to identify genes essential for in vitro and in vivo survival

Abstract Glaesserella parasuis is an important porcine pathogen that commonly colonizes the upper respiratory tract of pigs and is the etiological agent of Glässer's disease, causing substantial economic losses in the global swine industry. This study aimed to identify genes essential for both in vitro growth and in vivo pathogenicity of G. parasuis . We generated the most comprehensive transposon mutant library in G. parasuis to date, containing ~180,000 mutants in the serovar 4 strain H16SD2BY4, with a high TA‐site saturation of approximately 85%. This represents a successful application of transposon insertion sequencing (Tn‐seq) to define essential genes in G. parasuis and to screen for genes potentially associated with virulence using a mouse infection model. In vitro passaging assays confirmed the exceptional stability of the transposon mutant library and identified 435 genes essential for in vitro growth. A genome‐wide screen identified 74 genes specifically required for in vivo growth. Among these genes, 14 successfully constructed mutants all showed varying degrees of virulence attenuation in the mouse infection model, confirming the reliability of the transposon library. Additionally, four mutant strains (∆ serA , ∆ guaB , ∆ aroE , and ∆ dgkA ) showed significantly attenuated virulence, highlighting their important roles in G. parasuis pathogenesis. The transposon mutagenesis approach used in this work addresses a key gap in analyzing gene essentiality for G. parasuis and provides valuable new insights into its pathogenesis.