Genomic and Transcriptomic Analysis of Bovine Pasteurella multocida Serogroup A Strain Reveals Insights Into Virulence Attenuation

Abstract

Pasteurella multocida is one of the primary pathogens of bovine respiratory disease (BRD), and causes huge losses in the cattle industry. The Pm3 strain was a natural isolate, which is a strong form of pathogen and is sensitive to fluoroquinolones antibiotics. A high fluoroquinolone resistant strain, Pm64 (MIC = 64 μg/mL), was formed after continuous induction with subinhibitory concentration (1/2 MIC) of enrofloxacin, with the enhanced growth characteristics and large attenuation of pathogenicity in mice. This study reports the whole genome sequence and the transcription profile by RNA-Seq of strain Pm3/Pm64. The results showed an ineffective difference between the two strains at the genome level. However, 32 genes could be recognized in the gene islands (GIs) of Pm64, in which 24 genes were added and 8 genes were lost. Those genes are involved in DNA binding, trehalose metabolism, material transportation, capsule synthesis, prophage, amino acid metabolism, and other functions. In Pm3 strain, 558 up-regulated and 568 down-regulated genes were found compared to Pm64 strain, from which 20 virulence factor-related differentially expressed genes (DEGs) were screened. Mainly differentially transcribed genes were associated with capsular polysaccharide (CPS), lipopolysaccharide (LPS), lipooligosaccharide (LOS). Iron utilization, and biofilm composition. We speculated that the main mechanism of virulence attenuation after the formation of resistance of Pm64 comes from the change of the expression profile of these genes. This report elucidated the toxicity targets of P. multocida serogroup A which provide fundamental information toward the understanding of the pathogenic mechanism and to decreasing antimicrobial drugs resistance.

Keywords: P. multocida serogroup A; complete genome sequencing; gene islands; transcriptomic sequencing; virulence gene.

Figure 1

Biological characteristics of Pm3 and Pm64. (A) Induction process of Pm64 from Pm3 strain under continuous increasing subinhibitory concentration (1/2MIC) enrofloxacin culture condition. (B) LD₅₀ of Pm3 and Pm64 to mice. (C) Growth curve of Pm3 and Pm64 (^***p < 0.001).

Figure 2

Statistical representation of gene distribution in GIs of Pm3 and Pm64. (A) Predicted GIs structure in Pm3 chromosome. (B) Predicted GIs structure in Pm64 chromosome. The horizontal coordinates are length scales and the lengh of gene islands shown in the figure are <15 kb.

Figure 3

Venn diagram of core and specific gene cluster of Pm3 and Pm64 gene islands. Each ellipse represents a sample, the data on each region represents the groups number occur in and only in the samples. A group represents a set of genes with more than 50% similarity and sequence length difference < 0.7.

Figure 4

Quinolone resistance determining region (QRDR) comparison of Pm3 and Pm64. Only part of the base mutations sequence (labeled in green) was shown.

Figure 5

RNA-Seq analysis of Pm3 and Pm64. (A) Correlation map of gene expression. (B) Differential analysis volcano map. (C) The clustering heat map of DEGs.

Figure 6

Top 10 up and down regulated DEGs screened from Pm3/Pm64.

Figure 7

Scatter plot of GO and KEGG enrichmen. (A) Top 30 enriched GO function and DEGs numbers enriched in each function of Pm3/Pm64. (B) Top 20 enriched KEGG pathways and the number of DEGs in of Pm3/Pm64. The abscissa represents the ratio of DEGs annotated to the total differential genes; the ordinate represents the GO Term and KEGG pathway, respectively; the size of the point represents the number of annotated genes; the color from red to purple represents the decreasing significance of enrichment.

Figure 8

Expression level validation of part of virulence factor-related DEGs (n = 3). (A) The mRNA expression (RNA-Seq) of virulence factor-related DEGs in Pm3/Pm64. (B) The mRNA expression (RNA-Seq) of virulence factor-related DEGs in Pm3/Pm64. Results were representative of two independent experiments, with three replicates per group, analyzed by multiple comparative analysis and expressed as means ± SD (^*p < 0.05, ^**p < 0.01).