Engineering for an HPV 9-valent vaccine candidate using genomic constitutive over-expression and low lipopolysaccharide levels in Escherichia coli cells.

BACKGROUND: The various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli. RESULTS: Of 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci-lpxM, lpxP, lpxL, eptA, gutQ and kdsD-were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity. CONCLUSIONS: Reengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.

Protection of rainbow trout against yersiniosis by lpxD mutant Yersinia ruckeri.

Yersinia ruckeri is a Gram negative bacteria causing yersiniosis in freshwater and marine fish. Lipid A, important for pathogenesis of Gram negative bacteria, biosynthesis pathway requires nine enzyme catalyzed steps. Although there are nine genes encoding lipid A biosynthesis in bacteria, biosynthesis of lipopolysaccharides relies on lpxD gene that encodes the third pathway enzyme. The roles of LpxD in Y. ruckeri virulence have not been studied. In the present study, in-frameshift deletion of lpxD gene and their role in Y. ruckeri virulence in rainbow trout were determined. For this purpose, 92% of the Y. ruckeri lpxD genes were deleted by homologous recombination. After running in SDS-PAGE and staining with silver stain, no LPS was detectable in the Y. ruckeri ΔlpxD mutant. Virulence and immunogenicity of the Y. ruckeri ΔlpxD mutant (YrΔlpxD) were determined in rainbow trout. Rainbow trout immunized with YrΔlpxD with immersion, or intraperitoneal injection method displayed superior protection (relative percentage survival ≥ 84%) after exposure to wild type Y. ruckeri. In conclusion, our results indicated that deletion of the lpxD gene causes significant attenuation of Y. ruckeri in rainbow trout, and LPS deficient YrΔlpxD could be used as a live attenuated vaccine against Y. ruckeri in rainbow trout. This vaccine can protect fish and it can be applied to fish with different methods such as immersion or injection.

Molecular mechanism of lipopolysaccharide (LPS) in promoting biomineralization on bacterial surface.

Biomineralization on bacterial surface is affected by biomolecules of bacterial cell surface. Lipopolysaccharide (LPS) is the main and outermost component on the extracellular membrane of Gram-negative bacteria. In the present study, the molecular mechanism of LPS in affecting biomineralization of Ag+/Cl- colloids was investigated by taking advantages of two LPS structural deficient mutants of Escherichia coli. The two mutants were generated by impairing the expression of waaP or wbbH genes with CRISPR/Cas9 technology and it induced deficient polysaccharide chain of O-antigen (ΔwbbH) or phosphate groups of core oligosaccharide (ΔwaaP) in LPS structures. There were significant changes of the cell morphology and surface charge of the two mutants in comparing with that of wild type cells. LPS from ΔwaaP mutant showed increased ΔHITC upon interacting with free Ag+ ions than LPS from wild type cells or ΔwbbH mutant, implying the binding affinity of LPS to Ag+ ions is affected by the phosphate groups in core oligosaccharide. LPS from ΔwbbH mutant showed decreased endotherm (ΔQ) upon interacting with Ag+/Cl- colloids than LPS from wild type or ΔwaaP mutant cells, implying LPS polysaccharide chain structure is critical for stabilizing Ag+/Cl- colloids. Biomineralization of Ag+/Cl- colloids on ΔwbbH mutant cell surface showed distinctive morphology in comparison with that of wild type or ΔwaaP mutant cells, which confirmed the critical role of O-antigen of LPS in biomineralization. The present work provided molecular evidence of the relationship between LPS structure, ions, and ionic colloids in biomineralization on bacterial cell surface.