持続可能な社会を支える環境バイオ技術創製に向けた異種微生物間相互作用の効果検証

九州工業大学博士学位論文（要旨）学位記番号：生工博甲第452号 学位授与年月日：令和4年12月27

Recovery of recombinant Mycobacterium tuberculosis antigens fused with cell wall anchoring motif (LysM) from inclusion bodies using non-denaturing reagent (N-lauryl sarcosine)

Background The current limitations of conventional BCG vaccines highlights the importance in developing novel and effective vaccines against tuberculosis (TB). The utilization of probiotics such as Lactobacillus plantarum for the delivery of TB antigens through in-trans surface display provides an effective and safe vaccine approach against TB. Such non-recombinant probiotic surface display strategy involves the fusion of candidate proteins with cell wall binding domain such as LysM, which enables the fusion protein to anchor the L. plantarum cell wall externally, without the need for vector genetic modification. This approach requires sufficient production of these recombinant fusion proteins in cell factory such as Escherichia coli which has been shown to be effective in heterologous protein production for decades. However, over expression in E. coli expression system resulted in limited amount of soluble heterologous TB-LysM fusion protein, since most of it are accumulated as insoluble aggregates in inclusion bodies (IBs). Conventional methods of denaturation and renaturation for solubilizing IBs are costly, time-consuming and tedious. Thus, in this study, an alternative method for TB antigen-LysM protein solubilization from IBs based on the use of non-denaturating reagent N-lauroylsarcosine (NLS) was investigated. Results Expression of TB antigen-LysM fusion genes was conducted in Escherichia coli, but this resulted in IBs deposition in contrast to the expression of TB antigens only. This suggested that LysM fusion significantly altered solubility of the TB antigens produced in E. coli. The non-denaturing NLS technique was used and optimized to successfully solubilize and purify ~ 55% of the recombinant cell wall-anchoring TB antigen from the IBs. Functionality of the recovered protein was analyzed via immunofluorescence microscopy and whole cell ELISA which showed successful and stable cell wall binding to L. plantarum (up to 5 days). Conclusion The presented NLS purification strategy enables an efficient and rapid method for obtaining higher yields of soluble cell wall-anchoring Mycobacterium tuberculosis antigens-LysM fusion proteins from IBs in E. coli

Analysis of Lactobacillus plantarum Pa21 with surface anchored heterologous protein microencapsulated with polysaccharide

Microencapsulation technology is retention of cells or active compounds within the microencapsulation matrix as a protection from harsh environments. The oral administration of microencapsulated probiotics carrying heterologous proteins through the Gastro intestinal tract (GIT) can be a novel approach for vaccine delivery. The peptidoglycan cell walls of Gram positive bacteria functions to transport and assemble of protein that capable to interact with a surrounding. These properties will enable nongenetically modified organism of Lactobacillus plantarum Pa21 to be manipulated as a carrier by anchoring externally added heterologous protein such as the AR antigens of Mycobacterium tuberculosis. To date, the protective effect of microencapsulation of Lb. plantarum Pa21 surface anchored with heterologous proteins is still unknown. The microencapsulation is believed to enhance the viability of cells in the intestine, this mechanism would also be capable to up-regulate the protection toward AR antigens anchored on Lb. plantarum Pa21 surface area. The purpose of this study is to investigate the protective effect of microencapsulation of Lb. plantarum Pa21 surface anchored with AR antigen againts simulated gastrointestinal fluid. Firstly, Lb. plantarum Pa21 without cell wall-anchored heterologous proteins were microencapsulated with different concentration of alginate and chitosan. The effect of 2% (w/v), 3% (w/v)and 4% (w/v) sodium alginate concentration coated with 0.2% w/v and 0.4% w/v chitosan concentration on the cell survival, hardness and cells released was studied. The beads produced were tested under stress conditions designed by incubation in Simulated Gastric Juice (SGF) solution at pH 1.8 followed by incubation in Simulated Intestinal Juice (SIF) solution at pH 7.45. Three percent alginate coated with 0.4 % (w/v) chitosan beads provided the best protection for Lb. plantarum in all treatments. Unencapsulated Lb. plantarum Pa21 survived the first 60 minute of the SGF solution treatment with 2.99x102 CFU/ml viable cells compared to 3% (w/v) alginate coated with0.4 % (w/v) chitosan microencapsulated cells with 6.25x108 CFU/ml viable cells after 120 minutes incubation. Secondly, a total amount of 50 μg/ml of AR protein was purified and attached to 2.19 x109 CFU/ml of Lb. plantarum Pa21. The confirmation of protein surface display was detected by SDS-PAGE and western blot before subsequently microencapsulated in 3% (w/v) alginate coated with 0.4 % (w/v) chitosan matrix. After SGF solution test, whole cell ELISA detection of microencapulated AR protein anchored to Lb. plantarum Pa21 in alginate coated chitosan has the higher value excitation of horseradish peroxide conjugate on secondary antibody compared to unencapsulated AR protein anchored to Lb plantarum Pa21. Subsequent microencapsulated AR protein anchoring to the cell wall of Lb. plantarum Pa21 also showed a microscopic excitation of FITC conjugate on secondary antibody which signified protection of microencapsulated AR protein binding on the surface of Lb. plantarum Pa21 along the GIT passage

Proof of concept in utilizing in-trans surface display system of Lactobacillus plantarum as mucosal tuberculosis vaccine via oral administration in mice

Abstract Background Tuberculosis is one of the most common and deadliest infectious diseases worldwide affecting almost a third of the world’s population. Although this disease is being prevented and controlled by the Bacille Calmette Guérin (BCG) vaccine, the protective efficacy is highly variable and substandard (0–80%) in adults. Therefore, novel and effective tuberculosis vaccine that can overcome the limitations from BCG vaccine need to be developed. Results A novel approach of utilizing an in-trans protein surface display system of Lactobacillus plantarum carrying and displaying combination of Mycobacterium tuberculosis subunit epitope antigens (Ag85B, CFP-10, ESAT-6, Rv0475 and Rv2031c) fused with LysM anchor motif designated as ACERL was constructed, cloned and expressed in Esherichia coli Rossetta expression host. Subsequently the binding capability of ACERL to the cell wall of L. plantarum was examined via the immunofluorescence microscopy and whole cell ELISA where successful attachment and consistent stability of cell wall binding up to 4 days was determined. The immunization of the developed vaccine of L. plantarum surface displaying ACERL (Lp ACERL) via the oral route was studied in mice for its immunogenicity effects. Lp ACERL immunization was able to invoke significant immune responses that favor the Th1 type cytokine response of IFN-γ, IL-12 and IL-2 as indicated by the outcome from the cytokine profiling of spleen, lung, gastrointestinal tract (GIT), and the re-stimulation of the splenocytes from the immunized mice. Co-administration of an adjuvant consisting of Lactococcus lactis secreting mouse IL-12 (LcIL-12) with Lp ACERL was also investigated. It was shown that the addition of LcIL-12 was able to further generate significant Th1 type cytokines immune responses, similar or better than that of Lp ACERL alone which can be observed from the cytokine profiling of the immunized mice’s spleen, lung and GIT. Conclusions This study represents a proof of concept in the development of L. plantarum as a carrier for a non-genetically modified organism (GMO) tuberculosis vaccine, which may be the strategy in the future for tuberculosis vaccine development