Team:TMMU China/Project Results/Project4

Capsule production platform

Background

Capsular or extracellular polysaccharides produced by L. lactis

A wide range of lactic acid bacteria (LAB) is able to produce capsular or extracellular polysaccharides (CPS or EPS) with various chemical compositions and properties. Polysaccharides produced by LAB alter the rheological properties of the matrix in which they are dispersed, leading to typically viscous and Bropy products. Polysaccharides are involved in several mechanisms such as prebiosis and probiosis, tolerance to stress associated to food process, and technological properties of food. Many metabolic engineering approaches have been used to improve productivity and structure of CPS or EPS, though limited success were met. Nevertheless, the biosynthetic routes of EPS, CPS and other cell wall components can be exploited for the heterologous expression of other polysaccharides, including pathogenic bacteria capsules.

Pathogenic bacteria capsules and the Vi antigen of Salmonella enterica subspecies I serotype Typhi (S. Typhi)

Capsules are produced by diverse bacteria pathogens, including E.coli, Neisseria meningitidis, S. Typhi, Staphylococcus aureus and Streptococcus pneumoniae. Due to their surface association, capsules are frequently the first bacterial structure encountered by the immune system upon infection. Therefore, in many bacteria, the capsule is required for evasion of the host immune system and isolates without capsule are non-pathogenic. As a result, capsules have been exploited to develop vaccines against pathogenic bacteria. Effective capsule vaccines against N.meningitidis and S.pneumoniae have contributed a lot to public health.

S. Typhi is the causative agent of the human systemic infection typhoid fever, which is a public threat especially in developing countries. S. Typhi expresses a capsular polysaccharide known as Vi antigen (Vi). Vi is a linear, acidic homopolymer of a-1,4-linked N-acetylgalactosaminuronate (D-GalNAcA), variably O-acetylated at C-3. The viaB operon is responsible for the regulation and biosynthesis of Vi polysaccharide, but he enzyme responsible for O-acetylation of the Vi polysaccharide has not yet been identified. The viaB operon contains 10 genes involved in regulation of expression (tviA), biosynthesis (tviB totviE), and cell surface localization of the Vi polysaccharide (vexA to vexE).

The Vi capsule provides S. Typhi with mechanisms to avoid host defenses and enhancing infectivity and virulence. Besides being an important virulence factor, the Vi is also a protective antigen and a vaccine based on purified Vi polysaccharide has been developed and licensed for use as a parenteral vaccine against typhoid fever. However, large scale culturing S.Typhi and the following purification procedures of Vi poses a highly biosafety risk issue for workers. The potential of engineering L. lactis for Vi capsule production and delivery has never been tested.

Figure4.1:Overview of the viaB operon and Vi capsular polysaccharide biosynthesis pathway. A) Schematic diagram of the S. Typhi viaB operon. B) The Vi antigen is a linear polymer of a-1,4-linked N-acetylgalatosaminuronate, nonstoichiometrically esterified with acetyl groups at the C-3. A TviB-catalyzed oxidation of UDP-N-acetylglucosamine (UDP-GlcNAc) followed by the TviC-catalyzed epimerization at the C-4 of UDP-N-acetylglucosaminuronate (UDP-GlcNAcA) results in the formation of UDP-N-acetylgalactosaminuronate (UDP-GalNAcA), the building block for Vi polymer formation. The Vi polymer is synthesized in the cytoplasm and assembly is dependent on TviD and TviE. The enzyme catalyzing the O-acetylation of the capsular polysaccharide has not yet been identified. Subsequent translocation of the polysaccharide to the cell surface follows an ATP-binding cassette (ABC) transporter-dependent process. The transporter consists of VexA, VexB, VexC and VexD. The precise function of VexE is equivocal although it might be involved in anchoring the Vi to the cell surface.

Design

To test the potential of L. lactis to be a pathogenic bactieria capsules production platform, we took the Vi antigen as a proof of concept. The viaB operon is placed under the pNisZ promoter and followed by the nsr gene.

Figure4.2 Schematic representation of the NZ-Vi design

Result

We placed the viaB operon under the PnisZ promoter. After that, we introduced the pVi to the NZ-Blue strain, and white colonies were selected and verified by PCR. We successfully obtained the NZ-Vi strain. After induction by the nisin, we found that compared to NZ-Blue, the NZ-Vi strain exhibited as clumps, and the bacteria can quickly gathered together to form clumps after vigorous shaking. Since polysaccharides can spontaneously interact, we suspect that the Vi capsule is expressed and covered the cell wall surface of L. lactis. After staining and under microscopic, a clear transparent zone around the stained bacteria can be easily observed, which indicated that there are capsules around the bacteria, for capsule is refractory to be stained by dye. To demonstrate that Vi is indeed expressed, we used dot blot to confirm this. Using a polycolonal antibody against the Vi antigen, we found that the Vi-antigen is indeed expressed in NZ-Vi, but not NZ-Blue. In conclusion, we demonstrated that L. lactis can be used to express the Vi antigen of S. Typhi, indicating that L. lactis can be applied to other pathogenic bacteria capsule engineering for vaccine development.

Figure4.3 Expression of Vi antigen using L. lactis as a platform. A) View of the NZ-Blue and NZ-Vi culture. B) Microscopic view of NZ-Blue and NZ-Vi after staining. C) Dot blot of NZ-Blue and NZ-Vi culture using a polyclonal antibody against the Vi antigen.