Abstract
The Gram-negative bacterium Riemerella anatipestifer is an important waterfowl pathogen, causing major economic losses to the duck-producing industry. However, little is known of the virulence factors that mediate pathogenesis during R. anatipestifer infection. In this study, RAYM_RS09735 and RAYM_RS09740 were predicted to form a two-component signaling system (TCS) through bioinformatics analysis. This TCS was highly conserved across the Flavobacteriaceae. A mutant YMΔRS09735/RS09740 strain was constructed to investigate the role of the RAYM_RS09735/RAYM_RS09740 TCS in R. anatipestifer virulence and gene regulation. The median lethal dose (LD50) of YMΔRS09735/RS09740 was found to be >1011 CFU, equivalent to that of avirulent bacterial strains. The bacterial abundances of the YMΔRS09735/RS09740 strain in the heart, brain, liver, blood, and spleen were significantly lower than that of the wild-type R. anatipestifer YM strain. Pathological analysis using hematoxylin and eosin staining showed that, compared to the wild-type, the mutant YMΔRS09735/RS09740 strain caused significantly less virulence in infected ducklings. RNAseq and real-time PCR analysis indicated that the RAYM_RS09735/RAYM_RS09740 TCS is a PhoP/PhoR system. This is a novel type of TCS for Gram-negative bacteria. The TCS was also found to be a global regulator of expression in R. anatipestifer, with 112 genes up-regulated and 693 genes down-regulated in the YMΔRS09735/RS09740 strain (~33% genes demonstrated differential expression). In summary, we have reported the first PhoP/PhoR TCS identified in a Gram-negative bacterium and demonstrated that it is involved in virulence and gene regulation in R. anatipestifer.
Highlights
The disease riemerella anatipestifersis, caused by the Gram-negative bacterium Riemerella anatipestifer, occurs primarily in 1–8-week-old ducks but is most common in more susceptible 2– 3-week-old ducklings
Several virulence factors have been identified that associate with disease severity, including VapD (Chang et al, 1998), CAMP cohemolysin (Crasta et al, 2002), outer membrane protein A (OmpA; Hu et al, 2011), Nicotinamidase PncA (Wang et al, 2016), and putative genes associated with lipopolysaccharide (LPS) synthesis (Yu et al, 2016)
two-component signaling system (TCS) are typically composed of a sensor with histidine kinase activity and a cytoplasmic transcriptional regulator
Summary
The disease riemerella anatipestifersis, caused by the Gram-negative bacterium Riemerella anatipestifer, occurs primarily in 1–8-week-old ducks but is most common in more susceptible 2– 3-week-old ducklings. It is currently the most economically damaging bacterial infection affecting the global duck industry. R. anatipestifer serotypes 1, 2, and 10 are responsible for most major outbreaks in China. Many R. anatipestifer strains with differing virulence have been isolated from duck farms in China (Yuan et al, 2011; Wang et al, 2015; Zhang et al, 2015; Song et al, 2016). Several virulence factors have been identified that associate with disease severity, including VapD (Chang et al, 1998), CAMP cohemolysin (Crasta et al, 2002), outer membrane protein A (OmpA; Hu et al, 2011), Nicotinamidase PncA (Wang et al, 2016), and putative genes associated with lipopolysaccharide (LPS) synthesis (Yu et al, 2016)
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