Toxoplasma gondii holds significant therapeutic potential; however, its nonspecific invasiveness results in off-target effects. The purpose of this study is to evaluate whether T. gondii specificity can be improved by surface display of scFv directed against dendritic cells\' endocytic receptor, DEC205, and immune checkpoint PD-L1. Anti-DEC205 scFv was anchored to the T. gondii surface either directly via glycosylphosphatidylinositol (GPI) or by fusion with the SAG1 protein. Both constructs were successfully expressed, but the binding results suggested that the anti-DEC-SAG1 scFv had more reliable functionality towards recombinant DEC protein and DEC205-expressing MutuDC cells. Two anti-PD-L1 scFv constructs were developed that differed in the localization of the HA tag. Both constructs were adequately expressed, but the localization of the HA tag determined the functionality by binding to PD-L1 protein. Co-incubation of T. gondii displaying anti-PD-L1 scFv with tumor cells expressing/displaying different levels of PD-L1 showed strong binding depending on the level of available biomarker. Neutralization assays confirmed that binding was due to the specific interaction between anti-PD-L1 scFv and its ligand. A mixed-cell assay showed that T. gondii expressing anti-PD-L1 scFv predominately targets the PD-L1-positive cells, with negligible off-target binding. The recombinant RH-PD-L1-C strain showed increased killing ability on PD-L1+ tumor cell lines compared to the parental strain. Moreover, a co-culture assay of target tumor cells and effector CD8+ T cells showed that our model could inhibit PD1/PD-L1 interaction and potentiate T-cell immune response. These findings highlight surface display of antibody fragments as a promising strategy of targeting replicative T. gondii strains while minimizing nonspecific binding.

Heavy metal resistance mechanisms and heavy metal response genes are crucial for microbial utilization in heavy metal remediation. Here, Corynebacterium crenatum was proven to possess good tolerance in resistance to copper. Then, the transcriptomic responses to copper stress were investigated, and the vital pathways and genes involved in copper resistance of C. crenatum were determined. Based on transcriptome analysis results, a total of nine significantly upregulated DEGs related to metal ion transport were selected for further study. Among them, GY20_RS0100790 and GY20_RS0110535 belong to transcription factors, and GY20_RS0110270, GY20_RS0100790, and GY20_RS0110545 belong to copper-binding peptides. The two transcription factors were studied for the function of regulatory gene expression. The three copper-binding peptides were displayed on the C. crenatum surface for a copper adsorption test. Furthermore, the nine related metal ion transport genes were deleted to investigate the effect on growth in copper stress. This investigation provided the basis for utilizing C. crenatum in copper bioremediation.

BACKGROUND: The accumulation of fast-growing polyethylene terephthalate (PET) wastes has posed numerous threats to the environments and human health. Enzymatic degradation of PET is a promising approach for PET waste treatment. Currently, the efficiency of various PET biodegradation systems requires further improvements.
RESULTS: In this work, we engineered whole cell systems with co-display of strong adhesive proteins and the most active PETase for PET biodegradation in E. coli cells. Adhesive proteins of cp52k and mfp-3 and Fast-PETase were simultaneously displayed on the surfaces of E. coli cells, and the resulting cells displaying mfp-3 showed 50% increase of adhesion ability compared to those without adhesive proteins. Consequently, the degradation rate of E. coli cells co-displaying mfp-3 and Fast-PETase for amorphous PET exceeded 15% within 24 h, exhibiting fast and thorough PET degradation.
CONCLUSIONS: Through the engineering of co-display systems in E. coli cells, PET degradation efficiency was significantly increased compared to E. coli cells with sole display of Fast-PETase and free enzyme. This feasible E. coli co-display system could be served as a convenient tool for extending the treatment options for PET biodegradation.

Bacterial cis-epoxysuccinic acid hydrolases (CESHs) are intracellular enzymes used in the industrial production of enantiomeric tartaric acids. The enzymes are mainly used as whole-cell catalysts because of the low stability of purified CESHs. However, the low cell permeability is the major drawback of the whole-cell catalyst. To overcome this problem, we developed whole-cell catalysts using various surface display systems for CESH[L] which produces L(+)-tartaric acid. Considering that the display efficiency depends on both the carrier and the passenger, we screened five different anchoring motifs in Escherichia coli. Display efficiencies are significantly different among these five systems and the InaPbN-CESH[L] system has the highest whole-cell enzymatic activity. Conditions for InaPbN-CESH[L] production were optimized and a maturation step was discovered which can increase the whole-cell activity several times. After optimization, the total activity of the InaPbN-CESH[L] surface display system is higher than the total lysate activity of an intracellular CESH[L] overexpression system, indicating a very high CESH[L] display level. Furthermore, the whole-cell InaPbN-CESH[L] biocatalyst exhibited good storage stability at 4 °C and considerable reusability. Thereby, an efficient whole-cell CESH[L] biocatalyst was developed in this study, which solves the cell permeability problem and provides a valuable system for industrial L(+)-tartaric acid production.

Recent studies have reported on the importance of RBCs in fish responses to viral infections and DNA vaccines. Surface-displaying recombinant bacterins (spinycterins) are a safe and adaptable prototype for viral vaccination of fish and represent an alternative method of aquaculture prophylaxis, since have been reported to enhance fish immune response. We evaluated the innate immune response of rainbow trout (Oncorhynchus mykiss) red blood cells (RBCs), head kidney, and spleen to spinycterins expressing a fragment of the glycoprotein G of viral haemorrhagic septicemia virus (VHSV), one of the most devastating world-wide diseases in farmed salmonids. We first selected an immunorelevant downsized viral fragment of VHSV glycoprotein G (frg16252-450). Then, spinycterins expressing frg16252-450 fused to Nmistic anchor-motif (Nmistic+frg16252-450) were compared to spinycterins expressing frg16252-450 internally without the anchor motif. Nmistic+frg16252-450 spinycterins showed increased attachment to RBCs in vitro and modulated the expression of interferon- and antigen presentation-related genes in RBCs in vitro and in vivo, after intravenous injection. In contrast, the head kidney and spleen of fish injected with frg16252-450, but not Nmistic+frg16252-450, spinycterins demonstrated upregulation of interferon and antigen-presenting genes. Intravenous injection of Nmistic+frg16252-450 spinycterins resulted in a higher innate immune response in RBCs while frg16252-450 spinycterins increased the immune response in head kidney and spleen. Although more studies are required to evaluate the practicality of using spinycterins as fish viral vaccines, these results highlight the important contribution of RBCs to the fish innate immune response to antiviral prophylactics.

Scytovirin (SVN) is a lectin from cyanobacteria which has a strong inhibitory activity against Ebola virus infection. We engineered scytovirin as the inhibitor for surface display of lactic acid bacteria to block Ebola virus infection. Two different bacterial strains (Lactobacillus casei and Lactococcus lactis) were successfully engineered for scytovirin expression on the bacterial surface. These bacteria were found to be effective at neutralizing pseudotyped Ebolavirus in a cell-based assay. This approach can be utilized for prophylactic prevention, as well as for treatment. Since lactic acid bacteria can colonize the human body, a long-term efficacy could be achieved. Furthermore, this approach is also simple and cost-effective and can be easily applied in the regions of Ebola outbreaks in the developing countries.

Avian influenza is a serious threat to both public health and the poultry industry worldwide. This respiratory virus can be combated by eliciting robust immune responses at the site of infection through mucosal immunization. Recombinant probiotics, specifically lactic acid bacteria, are safe and effective carriers for mucosal vaccines. In this study, we engineered recombinant fusion protein by fusing the hemagglutinin 1 (HA1) subunit of the A/Aquatic bird/Korea/W81/2005 (H5N2) with the Bacillus subtilis poly γ-glutamic acid synthetase A (pgsA) at the surface of Lactobacillus casei (pgsA-HA1/L. casei). Using subcellular fractionation and flow cytometry we confirmed the surface localization of this fusion protein. Mucosal administration of pgsA-HA1/L. casei in mice resulted in significant levels of HA1-specific serum IgG, mucosal IgA and neutralizing antibodies against the H5N2 virus. Additionally, pgsA-HA1/L. casei-induced systemic and local cell-mediated immune responses specific to HA1, as evidenced by an increased number of IFN-γ and IL-4 secreting cells in the spleens and higher levels of IL-4 in the local lymphocyte supernatants. Finally, mice inoculated with pgsA-HA1/L. casei were protected against a 10LD50 dose of the homologous mouse-adapted H5N2 virus. These results suggest that mucosal immunization with L. casei displaying HA1 on its surface could be a potential strategy for developing a mucosal vaccine against other H5 subtype viruses.

Surface display co-opts yeast\'s innate ability to embellish its cell wall with mannoproteins, thus converting the yeast\'s outer surface into a growing and self-sustaining catalyst. However, the efficient toolbox for converting the enzyme of interest into its surface-displayed isoform is currently lacking, especially if the isoform needs to be anchored to the cell wall near the isoform\'s N-terminus, e.g., through a short GPI-independent protein anchor. Aiming to advance such N-terminally anchored surface display, we employed in silico and machine-learning strategies to study the 3D structure, function, genomic organisation, and evolution of the Pir protein family, whose members evolved to covalently attach themselves near their N-terminus to the β-1,3-glucan of the cell wall. Through the newly-gained insights, we rationally engineered 14 S. cerevisiae Hsp150 (Pir2)-based fusion proteins. We quantified their performance, uncovering guidelines for efficient yeast surface display while developing a construct that promoted a 2.5-fold more efficient display of a reporter protein than the full-length Hsp150. Moreover, we developed a Pir-tag, i.e., a peptide spanning only 4.5 kDa but promoting as efficient surface display of a reporter protein as the full-length Hsp150. These constructs fortify the existing surface display toolbox, allowing for a prompt and routine refitting of intracellular proteins into their N-terminally anchored isoforms.

Coccidiosis is a parasitic disease in the intestine caused by the genus Eimeria that poses a substantial economic threat to the broiler breeding industry. The misuse of chemoprophylaxis and live oocyst vaccines has a negative impact on chicken reproductivity. Therefore, there is a pressing need to develop safe, convenient, and effective vaccines. Lactic acid bacteria can be used as a means to deliver mucosal vaccines against intestinal pathogens, which is a promising strategy. In this study, a recombinant Lactobacillus plantarum (L. plantarum) with surface-expressed antigens constructed from the fusion of Eimeria tenella (E. tenella) antigen profilin and the Salmonella enterica serovar Typhimurium flagellin protein FliC was created. After oral immunization with the recombinant L. plantarum, T-cell differentiation was analyzed by flow cytometry, and specific antibody levels were determined via indirect ELISA. Oocyst shedding, body weight, and cecum lesions were assessed as measures of protective immunity after challenge with E. tenella. The results of this study demonstrate the effectiveness of recombinant L. plantarum as an immunization agent for chickens. Specific IgA titers in the intestine and specific IgG antibody titers in the serum were significantly higher in chickens immunized with recombinant L. plantarum (P < 0.001). Additionally, the levels of IL-2 (P < 0.05) and IFN-γ (P < 0.01) in the serum were markedly increased. Recombinant L. plantarum induced T-cell differentiation, resulting in a higher proportion of CD4+ and CD8+ T cells in splenocytes (P < 0.001). Fecal oocyst shedding in the immunized group was significantly reduced (P < 0.001). Additionally, recombinant L. plantarum significantly relieved pathological damage in the cecum, as evidenced by lesion scores (P < 0.01) and histopathological cecum sections. In conclusion, the present study provides evidence to support the possibility of using L. plantarum as a promising carrier for the delivery of protective antigens to effectively protect chickens against coccidiosis.

Crimean-Congo hemorrhagic fever (CCHF), which has a fatality rate of 20-30%, is widely prevalent in several regions in Asia, Europe, and Africa and has spread to a wider range of areas in recent years. At present, there is a lack of safe and effective vaccines for the prevention of CCHF. In this study, we prepared three vaccine candidates, rvAc-Gn, rvAc-Np, and rvAc-Gn-Np, that encoded the CCHF virus (CCHFV) glycoprotein Gn and the nucleocapsid protein (Np) on the surface of baculovirus using an insect baculovirus vector expression system (BVES) and evaluated their immunogenicity in BALB/c mice. The experimental results showed that both CCHFV Gn and Np were expressed by the respective recombinant baculoviruses and anchored to the viral envelope. BALB/c mice were immunized, and all three recombinant baculoviruses showed significant humoral immunity. At the cellular level, the level of immunity in the rvAc-Gn group was significantly higher than that in the rvAc-Np and rvAc-Gn-Np groups, and the rvAc-Gn-Np coexpression group exhibited the lowest level of cellular immunity. In conclusion, the strategy of coexpressing Gn and Np in the baculovirus surface display system did not result in improvements in immunogenicity, whereas the recombinant baculovirus displaying Gn alone could induce significant humoral and cellular immunity in mice, indicating that rvAc-Gn has potential as a CCHF vaccine candidate. This study thus provides new ideas for the development of a CCHF baculovirus vaccine.