A novel approach for an immunogen against Corynebacterium pseudotuberculosis infection: An Escherichia coli bacterin expressing phospholipase D.

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CLA) in small ruminants. There is still needed an immunoprophylaxis model, which induces a protective and sustained immune response against the bacteria. In this study, we evaluated a recombinant Escherichia coli bacterin expressing the recombinant phospholipase D (rPLD) protein, the most relevant virulence factor of C. pseudotuberculosis, as a potential vaccine formulation. E. coli BL21 (DE3) Star strain was used for rPLD protein expression and was then inactivated by formaldehyde. Four groups with 10 Balb/c mice each were immunized twice within a 21 days interval: G1-control - 0.9% saline solution; G2- E. coli bacterin/pAE (naked plasmid); G3- E. coli bacterin/pAE/pld; G4-purified recombinant rPLD. Subsequently, the animals were challenged with a C. pseudotuberculosis virulent strain and evaluated for 40 days. The highest survival rate was observed for G3 with 40% protection, followed by 30% in the purified rPLD group (G4). These two groups also showed considerable IgG production when compared with the control group (G1). Also, a higher significant expression of interferon-γ was observed for the experimental groups G2, G3, and G4 when compared with a control group (G1) (p < 0.05). These results represent that a recombinant bacterin can be seen as a promising approach for vaccinal antigens against CLA, being possible to be used in association of different vaccine strategies.

Signal Amplification for Cell-Free Biosensors, an Analog-to-Digital Converter.

Toehold switches are biosensors useful for the detection of endogenous and environmental RNAs. They have been successfully engineered to detect virus RNAs in cell-free gene expression reactions. Their inherent sequence programmability makes engineering a fast and predictable process. Despite improvements in the design, toehold switches suffer from leaky translation in the OFF state, which compromises the fold change and sensitivity of the biosensor. To address this, we constructed and tested signal amplification circuits for three toehold switches triggered by Dengue and SARS-CoV-2 RNAs and an artificial RNA. The serine integrase circuit efficiently contained leakage, boosted the expression fold change from OFF to ON, and decreased the detection limit of the switches by 3-4 orders of magnitude. Ultimately, the integrase circuit converted the analog switches' signals into digital-like output. The circuit is broadly useful for biosensors and eliminates the hard work of designing and testing multiple switches to find the best possible performer.