Abstract
Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Genetic analyses suggest that this pathogen has a novel protein secretion system, known as the “type IX secretion system” (T9SS). We previously reported that deletion of the AS87_RS08465 gene significantly reduced the bacterial virulence of the R. anatipestifer strain Yb2, but the mechanism remained unclear. The AS87_RS08465 gene is predicted to encode the gliding motility protein GldM (GldM) protein, a key component of the T9SS complex. In this study, Western blotting analysis demonstrated that R. anatipestifer GldM was localized to the cytomembrane. Further study revealed that the adhesion and invasion capacities of the mutant strain RA2281 (designated Yb2ΔgldM) in Vero cells and the bacterial loads in the blood of infected ducks were significantly reduced. RNA-Seq and PCR analyses showed that six genes were upregulated and five genes were downregulated in the mutant strain Yb2ΔgldM and that these genes were mainly involved in the secretion of proteins. Yb2ΔgldM was also found to be defective in gliding motility and protein secretion. Liquid chromatography–tandem mass spectrometry analysis revealed that nine of the proteins had a conserved T9SS C-terminal domain and were differentially secreted by Yb2ΔgldM compared to Yb2. The complementation strain cYb2ΔgldM recovered the adhesion and invasion capacities in Vero cells and the bacterial loads in the blood of infected ducks as well as the bacterial gliding motility and most protein secretion in the mutant strain Yb2ΔgldM to the levels of the wild-type strain Yb2. Taken together, these results indicate that R. anatipestifer GldM is associated with T9SS and is important in bacterial virulence.
Highlights
Riemerella anatipestifer is a causative pathogen of diseases in ducks, geese, turkeys, and various other domestic and wild birds [1]
Polymerase chain reaction (PCR) amplification using the primers 16S rRNA-F/16S rRNA-R and RA_08465-F/RA_08465-R produced a 792-bp fragment of 16S rRNA and a 1560bp fragment of gldM, respectively, in the wild-type strain Yb2 and complementation strain cYb2ΔgldM, whereas no 1560-bp fragment was amplified from the mutant strain Yb2ΔgldM because of the insertion (Figure 1A)
No polar effect of the gldM mutation was found by quantitative PCR (qPCR) analyses on the adjacent genes (Figure 1B)
Summary
Riemerella anatipestifer is a causative pathogen of diseases in ducks, geese, turkeys, and various other domestic and wild birds [1]. The Riemerella anatipestifer is a member of the phylum Bacteroidetes. A novel protein secretion system, known as the “type IX secretion systems” (T9SSs) or “Por secretion system”, has recently been frequently found in members of the phylum Bacteroidetes [11, 12]. Many virulence factors of pathogenic bacteria are either secreted proteins or the secretion systems themselves [13]. T9SS is associated with bacterial gliding motility and protein secretion and is considered to be a virulence factor in many pathogens [14, 15].
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