Specific Integration of Temperate Phage Decreases the Pathogenicity of Host Bacteria

Abstract

Temperate phages are considered as natural vectors for gene transmission among bacteria due to the ability to integrate their genomes into a host chromosome, therefore, affect the fitness and phenotype of host bacteria. Many virulence genes of pathogenic bacteria were identified in temperate phage genomes, supporting the concept that temperate phages play important roles in increasing the bacterial pathogenicity through delivery of the virulence genes. However, little is known about the roles of temperate phages in attenuation of bacterial virulence. Here, we report a novel Bordetella bronchiseptica temperate phage, vB_BbrS_PHB09 (PHB09), which has a 42,129-bp dsDNA genome with a G+C content of 62.8%. Phylogenetic analysis based on large terminase subunit indicated that phage PHB09 represented a new member of the family Siphoviridae. The genome of PHB09 contains genes encoding lysogen-associated proteins, including integrase and cI protein. The integration site of PHB09 is specifically located within a pilin gene of B. bronchiseptica. Importantly, we found that the integration of phage PHB09 significantly decreased the virulence of parental strain B. bronchiseptica Bb01 in mice, most likely through disruption the expression of pilin gene. Moreover, a single shot of the prophage bearing B. bronchiseptica strain completely protected mice against lethal challenge with wild-type virulent B. bronchiseptica, indicating the vaccine potential of lysogenized strain. Our findings not only indicate the complicated roles of temperate phages in bacterial virulence other than simple delivery of virulent genes but also provide a potential strategy for developing bacterial vaccines.

Keywords: Bordetella bronchiseptica; attenuation; bacterial virulence; integration site; temperate phage.

Figure 1

Phylogenetic analysis of phage PHB09. The phylogenetic tree was generated with MEGA 6.0 using sequences of large terminase subunit. The numbers next to the branches are bootstrap values.

Figure 2

Genome organization and integration site of phage PHB09. (A) Genome organization of phage PHB09. ORFs are numbered consecutively from left to right. Colors denote different functional modules of genes. (B) The integration site of phage PHB09 in B. bronchiseptica Bb01 genome. The 14-bp integration site, highlighted in red, was located within pilin gene (yellow). (C) Analysis of the integration site of phage PHB09 in B. bronchiseptica Bb01+ genome. The phage PHB09 with 14-bp terminal repeat sequences was indicated. Four primers used for integration analysis and the size of corresponding PCR products were also indicated. (D) PCR analysis of 13 B. bronchiseptica strains containing prophage PHB09 with primers, 4F/4R (up panel) and 5F/5R (low panel). The B. bronchiseptica strain that doesn't have prophage PHB09 was used as a negative control.

Figure 3

The virulence of B. bronchiseptica strains Bb01 and Bb01+ in vitro. (A) Growth curve of B. bronchiseptica strains Bb01 and Bb01+ at 37°C in TSB medium. (B,C) The sensitivity of strains Bb01 and Bb01+ to mouse serum. The titer of strains after 1 h (B) or 2 h (C) incubation with mouse serum at 37°C. Inactive mouse serum and PBS were used as controls. (D,E) Adhesion and invasion of B. bronchiseptica strains Bb01 and Bb01+. The same amount of Bb01 and Bb01+ cells were incubated with HEp-2 cells at 37°C for 2 h (D) or 4 h (E) respectively. After washing out the unbound bacteria, the HEp-2 cells were lysed and the bacterial counts in the lysates were determined. (F) Anti-phagocytic ability of the strains Bb01 and Bb01+. The titer was determined after 2 h incubation with RAW264.7 cells at 37°C. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4

The virulence of B. bronchiseptica strains Bb01 and Bb01+ in vivo. (A) Mouse infection assay. Three groups of BALB/c mice were intranasally inoculated with Bb01, Bb01+, and PBS respectively and monitored daily for mortality. On day 3, three mice of each group were randomly selected and sacrificed for histopathologic analysis of tracheae (up panel) and lungs (low panel) of mice infected with Bb01 (B), Bb01+ (C), and PBS (D). See results for the details. Survival was analyzed by Kaplan-Meier analysis with a log-rank test.

Figure 5

Phage PHB09 lysogenized B. bronchiseptica strain protects mice against lethal challenges with B. bronchiseptica Bb01. (A) Immunization scheme. Two groups of BALB/c mice were intranasally immunized with Bb01+ and PBS, respectively, and challenged with B. bronchiseptica strain Bb01 on day 14. (B) The survival curve of mice.