Extraintestinal pathogenic Escherichia coli (ExPEC) is an important zoonotic pathogen. Recently, ExPEC has been reported to be an emerging problem in pig farming. However, the mechanism of pathogenicity of porcine ExPEC remains to be revealed. In this study, we constructed a transposon (Tn) mutagenesis library covering Tn insertion in over 72% of the chromosome-encoded genes of a virulent and multi-drug resistant porcine ExPEC strain PCN033. By using a mouse infection model, a transposon-directed insertion site sequencing (TraDIS) assay was performed to identify in vivo fitness factors. By comparing the Tn insertion frequencies between the input Tn library and the recovered library from different organs, 64 genes were identified to be involved in fitness during systemic infection. 15 genes were selected and individual gene deletion mutants were constructed. The in vivo fitness was evaluated by using a competitive infection assay. Among them, Δ fimG was significantly outcompeted by the WT strain in vivo and showed defective adhesion to host cells. rfa which was involved in lipopolysaccharide biosynthesis was shown to be critical for in vivo fitness which may have resulted from its role in the resistance to serum killing. In addition, several metabolic genes including fepB , sdhC , fepG , gltS , dcuA , ccmH , ddpD , narU , glpD , malM , and yabL and two regulatory genes metJ and baeS were shown as important determinants of in vivo fitness of porcine ExPEC. Collectively, this study performed a genome-wide screening for in vivo fitness factors which will be important for understanding the pathogenicity of porcine ExPEC.

The Tat system is an important protein export system critical for bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility.

Live bacterial vector vaccines are one of the most promising vaccine types and have the advantages of low cost, flexibility, and good safety. Meanwhile, protein secretion systems have been reported as useful tools to facilitate the release of heterologous antigen proteins from bacterial vectors. The twin-arginine translocation (Tat) system is an important protein export system that transports fully folded proteins in a signal peptide-dependent manner. In this study, we constructed a live vector vaccine using an engineered commensal Escherichia coli strain in which amiA and amiC genes were deleted, resulting in a leaky outer membrane that allows the release of periplasmic proteins to the extracellular environment. The protective antigen proteins SLY, enolase, and Sbp against Streptococcus suis were targeted to the Tat pathway by fusing a Tat signal peptide. Our results showed that by exploiting the Tat pathway and the outer membrane-defective E. coli strain, the antigen proteins were successfully secreted. The strains secreting the antigen proteins were used to vaccinate mice. After S. suis challenge, the vaccinated group showed significantly higher survival and milder clinical symptoms compared with the vector group. Further analysis showed that the mice in the vaccinated group had lower burdens of bacteria load and slighter pathological changes. Our study reports a novel live bacterial vector vaccine that uses the Tat system and provides a new alternative for developing S. suis vaccine.