Comparative analysis of shared and unique mechanisms important for diverse strains of Pasteurella multocida to cause systemic infection in mice

Pasteurella multocida is a pathogen that causes a range of distinct diseases in livestock animals. Different P. multocida strains produce different capsule and lipopolysaccharide (LPS) structures. Different P. multocida diseases are associated with different capsule and LPS types, and little is known about what underpins this disease specificity. In this study, we utilised transposon-directed insertion site sequencing (TraDIS, also called Tn-Seq) to identify genes required for growth in rich media, and genes required for survival during systemic infections in BALB/c mice, for two diverse P. multocida strains, strain VP161 (capsule type A and LPS type L1) and strain M1404 (capsule type B and LPS type L2). Rich media analysis showed that both VP161 and M1404 shared 461 genes essential for growth in rich media, with comparison to the entire species showing that 95% of these rich media essential genes present in all publicly available closed P. multocida genomes. In vivo fitness analysis identified 63 and 94 genes important for VP161 and M1404 survival in BALB/c mice, respectively. Only 35 homologs were identified in both strains as important for survival, showing that conserved biological systems can be differentially important for different P. multocida strains. Investigation of proteins involved in the catabolite response showed that an active cyclic-adenosine monophosphate (cAMP) receptor protein (CRP) was required for maximal fitness in M1404. Furthermore, disrupting CRP or cAMP production also reduced capsule production in M1404, but increased capsule production in VP161, showing different strains of P. multocida have different regulatory systems for crucial virulence factors. Overall design: This study used transposon-directed insertion site sequencing (TraDIS, also called Tn-Seq) to investigate genes required for growth in rich media, and genes important for in vivo fitness, of two Pasteurella multocida strains, strain VP161 and strain M1404. A large VP161 Himar1 mutant library was grown in heart infusion broth, used to inject six-to-eight-week-old BALB/c mice via intraperitoneal injection with ~1 x 107 colony forming units. At the humane end point, surviving mutants were recovered from the bloodstream, liver and spleen, then plated on heart infusion agar and incubated overnight at 37ºC. The input library was also plated onto heart infusion agar and incubated overnight. The M1404 Himar1 mutant library was also used in the same way. Cells were recovered from the overnight growths, genomic DNA extracted and used to produce TraDIS libraries. TraDIS libraries were generated by shearing DNA to ~300 bp using Covaris sonication, using NEBNext kits to repair the ends of the DNA, A-tail, and then ligate adapters on the end. Specific amplification of transposon-chromosome junctions was performed using a Himar1-specific primer, and an adapter-specific primer. The adapter-specific primer contained index sequences specific for each dataset, and the primers contained Illumina P5 and P7 sequences to allow for Illumina sequencing. The TraDIS libraries were sequenced using an Illumina MiSeq, and the data analysed using the Bio-TraDIS toolkit. The data uploaded are the reads generated from the Illumina MiSeq, and the excel table that contains the analysed data shows the number of reads per gene, the number of unique Himar1 insertion sites per gene, and whether they were called as essential for growth in rich media or important for in vivo fitness in different tissues recovered from BALB/c mice.