Additional file of Response of swine spleen to Streptococcus suis infection revealed by transcription analysis

Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and human erysipeloid. Bacterial surface proteins are promising vaccine candidates. We recently identified 3 E. rhusiopathiae surface proteins (GAPDH, HP0728, and HP1472) and characterized their roles as virulence factors. However, their efficacy as protective antigens is still unknown. The N-terminal region of a previously identified surface protein, CbpB (CbpB-N), is speculated to be a protective antigen, but this needs to be verified. The aim of this study was to evaluate the protective efficacy of GAPDH, HP0728, HP1472, and CbpB-N. Immunization with recombinant GAPDH provided complete protection in a mouse model, recombinant CbpB-N provided partial protection, while recombinant HP0728 and HP1472 provided no protection. Recombinant GAPDH also provided good protection in a pig model. GAPDH antiserum exhibited significant blood bactericidal activity against E. rhusiopathiae. In conclusion, GAPDH and CbpB-N were found to be protective antigens of E. rhusiopathiae, and GAPDH is a promising vaccine candidate.

Erysipelothrix rhusiopathiae is a ubiquitous pathogen that has caused considerable economic losses to pig farmers. However, the mechanisms of E. rhusiopathiae pathogenesis remain unclear. To identify new virulence-associated factors, the differentially abundant cell wall-associated proteins (CWPs) between high- and low-virulence strains were investigated through isobaric Tags for Relative and Absolute Quantitation (iTRAQ) combined with liquid chromatography-quadrupole mass spectrometry (LC-MS/MS). In total, 100 CWPs showed significant differences in abundance. Selected differences were verified by western blotting to support the iTRAQ data. Among the differential proteins, the proteins with higher abundance in the high-virulence strain were mostly ABC transporter proteins and adhesion proteins, and the proteins with lower abundance in the high-virulence strain were mainly stress-response proteins. The more abundant proteins in the high-virulence strain may be related to bacterial virulence. The iTRAQ results showed that the abundance of the sugar ABC transporter substrate-binding protein Sbp (No. 5) was higher by 1.73-fold. We further constructed an sbp-deletion mutant. Experiments in animal models showed that the sbp-deletion mutant caused decreased mortality. Together, our data indicated that transporter proteins and adhesion proteins may play important roles in E. rhusiopathiae virulence and confirmed that sbp contributed to the virulence of E. rhusiopathiae. BIOLOGICAL SIGNIFICANCE:To our knowledge, this is the first proteomic analysis comparing differentially abundant CWPs between high- and low-virulence E. rhusiopathiae strains by iTRAQ. We generated comprehensive and accurate lists of E. rhusiopathiae CWPs proteomes and identified many differences at the protein level. Among the differential proteins with higher abundance in the high-virulence strain, sbp was verified to contribute to the virulence of E. rhusiopathiae through the construction of an sbp-deletion mutant. The differential proteins with higher abundance in the high-virulence strain identified in the present study should provide a foundation for future evaluation of virulence factors.

Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and human erysipeloid. The major protective antigen SpaA was suggested to play important roles in E. rhusiopathiae adhesion to host cells, but there is no specific study on SpaA pathogenic roles in adhesion. In this study we characterized direct and indirect roles of SpaA in E. rhusiopathiae adhesion to porcine endothelial cells. Recombinant E. rhusiopathiae SpaA (rSpaA) successfully binded to porcine iliac arterial endothelial cells. rSpaA protein pre-incubating endothelial cells or rSpaA antiserum pre-incubating E. rhusiopathiae significantly decreased E. rhusiopathiae adhesion to endothelial cells. rSpaA successfully binded host plasminogen and fibronectin, and rSpaA antiserum significantly decreased plasminogen-recruitment activity but not fibronectin-recruitment activity of E. rhusiopathiae. In conclusion, SpaA acts as adhesin in E. rhusiopathiae adhesion to host cells, and SpaA binding activity to host plasminogen highly likely play roles in this adhesion.

Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and human erysipeloid. Some pathogenic bacteria are able to recruit host plasminogen and then use the plasminogen system for migration across tissue barriers or for nutritional demands during infection. However, there is no study on E. rhusiopathiae recruitment of plasminogen. SpaA has long been known to be a major protective antigen of E. rhusiopathiae, but its roles in virulence have not yet been well clarified. The aim of this study was to detect the activity of E. rhusiopathiae to recruit host plasminogen and evaluate the ability of SpaA to act as a receptor in the recruitment process. It was found that E. rhusiopathiae could recruit host plasminogen. SpaA could specifically bind host plasminogen. Anti-SpaA serum could significantly decrease the activity of E. rhusiopathiae to recruit plasminogen. In addition, this binding activity was lysine dependent. In conclusion, E. rhusiopathiae was able to recruit host plasminogen via SpaA. To our knowledge, this is the first report on E. rhusiopathiae recruitment of host plasminogen and the receptor in the process.

A single nucleotide polymorphism–based PCR assay has been developed to differentiate the attenuated vaccine strain used in Japan from field isolates of Erysipelothrix rhusiopathiae found in pigs. However, this assay has been evaluated with only Japanese strains and isolates; therefore, it is unknown whether it could be used in other countries with E. rhusiopathiae strains and isolates of different genetic backgrounds. In our study, the PCR assay was evaluated using Chinese E. rhusiopathiae vaccine strains and field isolates. The PCR assay was able to differentiate the attenuated vaccine strains from the field isolates of E. rhusiopathiae in China but with a pattern different from that observed in Japan (only a single nucleotide polymorphism was detected in the Chinese vaccine strains compared with 5 in the Japanese vaccine strains). Importantly, either a DNA polymerase without 3′ to 5′ exonuclease activity or an exo+ polymerase with an antibody inhibiting the proofreading activity was required. In conclusion, after evaluation and improvement, this fast differentiation assay can be extended from Japan to China.

Strain F-1 was identified by morphological and cultural traits, physio-biochemical characteristics and 16 S rDNA sequence analysis. Antagonistic interaction between strain F-1 and Rhizoctonia solani, was tested using the dual cultural method. Cultural filtrate of strain F-1 was determined for control of R. solani on in vitro rice leaves and on rice plants. The results showed that strain F-1 was most closely related to Streptomyces platensis(100% si milarity) by the 16 S rDNA sequence analysis, the morphological and cultural traits, and physio-biochemical characte ristics of strain F-1 conformed with S. Platensis, so strain F-1 was affiliated to Streptomyces platensis F-1. It inhib ited growth of R. solani, resulting in hyphal malformation. Application of the culture filtrate of strain F-1 to rice d etached rice leaves and to potted rice plants reduced disease incidence caused by R. solani effectively. Strain F-1w as preliminarily identified as S. platensis F-1, and it is an effective agent against R. solani.