Co-expression of endoglucanase and cellobiohydrolase from yak rumen in lactic acid bacteria and its preliminary application in whole-plant corn silage fermentation.

Introduction: Endoglucanase (EG) and cellobiohydrolase (CBH) which produced by microorganisms, have been widely used in industrial applications. Methods: In order to construct recombinant bacteria that produce high activity EG and CBH, in this study, eg (endoglucanase) and cbh (cellobiohydrolase) were cloned from the rumen microbial genome of yak and subsequently expressed independently and co-expressed within Lactococcus lactis NZ9000 (L. lactis NZ9000). Results: The recombinant strains L. lactis NZ9000/pMG36e-usp45-cbh (L. lactis-cbh), L. lactis NZ9000/pMG36e-usp45-eg (L. lactis-eg), and L. lactis NZ9000/pMG36e-usp45-eg-usp45-cbh (L. lactis-eg-cbh) were successfully constructed and demonstrated the ability to secrete EG, CBH, and EG-CBH. The sodium carboxymethyl cellulose activity of the recombinant enzyme EG was the highest, and the regenerated amorphous cellulose (RAC) was the specific substrate of the recombinant enzyme CBH, and EG-CBH. The optimum reaction temperature of the recombinant enzyme CBH was 60°C, while the recombinant enzymes EG and EG-CBH were tolerant to higher temperatures (80°C). The optimum reaction pH of EG, CBH, and EG-CBH was 6.0. Mn2+, Fe2+, Cu2+, and Co2+ could promote the activity of CBH. Similarly, Fe2+, Ba2+, and higher concentrations of Ca2+, Cu2+, and Co2+ could promote the activity of EG-CBH. The addition of engineered strains to whole-plant corn silage improved the nutritional quality of the feed, with the lowest pH, acid detergent fiber (ADF), and neutral detergent fiber (NDF) contents observed in silage from the L. lactis-eg group (p < 0.05), and the lowest ammonia nitrogen (NH3-N), and highest lactic acid (LA) and crude protein (CP) contents in silage from the L. lactis-eg + L. lactis-cbh group (p < 0.05), while the silage quality in the L. lactis-cbh group was not satisfactory. Discussion: Consequently, the recombinant strains L. lactis-cbh, L. lactis-eg, and L. lactis-eg-cbh were successfully constructed, which could successfully expressed EG, CBH, and EG-CBH. L. lactis-eg promoted silage fermentation by degrading cellulose to produce sugar, enabling the secretory expression of EG, CBH, and EG-CBH for potential industrial applications in cellulose degradation.

Bis-benzylisoquinoline alkaloids inhibit African swine fever virus internalization and replication by impairing late endosomal/lysosomal function.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly infectious disease afflicting domestic pigs and wild boars. It exhibits an alarming acute infection fatality rate of up to 100%. Regrettably, no commercial vaccines or specific drugs for combating this disease are currently available. This study evaluated the anti-ASFV activities in porcine alveolar macrophages, 3D4/21 cells, and PK-15 cells of four bis-benzylisoquinoline alkaloids (BBAs): cepharanthine (CEP), tetrandrine, fangchinoline, and iso-tetrandrine. Furthermore, we demonstrated that CEP, which exhibited the highest selectivity index (SI = 81.31), alkalized late endosomes/lysosomes, hindered ASFV endosomal transport, disrupted virus uncoating signals, and thereby inhibited ASFV internalization. Additionally, CEP disrupted ASFV DNA synthesis, leading to the inhibition of viral replication. Moreover, berbamine was labeled with NBD to synthesize a fluorescent probe to study the cellular location of these BBAs. By co-staining with Lyso-Tracker and lysosome-associated membrane protein 1, we demonstrated that BBAs target the endolysosomal compartments for the first time. Our data together indicated that BBAs are a class of natural products with significant inhibitory effects against ASFV infection. These findings suggest their potential efficacy as agents for the prevention and control of ASF, offering valuable references for the identification of potential drug targets.IMPORTANCEThe urgency and severity of African swine fever (ASF) underscore the critical need for effective interventions against this highly infectious disease, which poses a grave threat to domestic pigs and wild boars. Our study reveals the potent anti-African swine fever virus (ASFV) efficacy of bis-benzylisoquinoline alkaloids (BBAs), particularly evident in the absence of progeny virus production under a 5 µM concentration treatment. The structural similarity among cepharanthine, tetrandrine, fangchinoline, and iso-tetrandrine, coupled with their analogous inhibitory stages and comparable selectivity indexes, strongly suggests a shared antiviral mechanism within this drug category. Further investigation revealed that BBAs localize to lysosomes and inhibit the internalization and replication of ASFV by disrupting the endosomal/lysosomal function. These collective results have profound implications for ASF prevention and control, suggesting the potential of the investigated agents as prophylactic and therapeutic measures. Furthermore, our study offers crucial insights into identifying drug targets and laying the groundwork for innovative interventions.

Development of a P30 protein-based indirect ELISA for detecting African swine fever antibodies utilizing the HEK293F expression system.

African swine fever (ASF) is an acute, febrile, and highly lethal infectious disease in pigs caused by the African swine fever virus (ASFV). Effective detection methods and strict biosecurity measures are crucial for preventing and controlling ASF, especially since there are currently no commercially available vaccines or antiviral drugs to combat ASFV infection effectively. However, the emergence of low-virulence strains of ASFV in recent years has led to false-positive results, highlighting the importance of early-produced antibody detection methods. Therefore, detecting antibodies against ASFV produced early in the infection can facilitate the prompt identification of infected pigs. This study focused on the p30 protein, an early expressed protein during ASFV infection, to develop an indirect ELISA. This method was established using the HEK293F suspension cell expression system, which has the ability to produce large quantities of correctly folded proteins with normal functionality. In this study, we developed an indirect ELISA test utilizing the p30 recombinant protein produced by the HEK293F suspension cell expression system as the antigen coating. The concentration of the p30 protein obtained from the HEK293F suspension cell expression system was measured at 4.668 mg/mL, serving as the foundation for establishing the indirect ELISA. Our findings indicate that the indirect ELISA method exhibits a sensitivity of 1:12800. Furthermore, it demonstrates high specificity and excellent reproducibility. Comparing our results to those obtained from the commercial kit, we found a coincidence rate of 98.148 % for the indirect ELISA. In summary, we have developed a sensitive method for detecting ASFV, providing a valuable tool for monitoring ASFV infection in pig herds.

Comparison of Structural Features of CRISPR-Cas Systems in Thermophilic Bacteria.

The clustered regularly interspaced short palindromic repeat (CRISPR) is an adaptive immune system that defends most archaea and many bacteria from foreign DNA, such as phages, viruses, and plasmids. The link between the CRISPR-Cas system and the optimum growth temperature of thermophilic bacteria remains unclear. To investigate the relationship between the structural characteristics, diversity, and distribution properties of the CRISPR-Cas system and the optimum growth temperature in thermophilic bacteria, genomes of 61 species of thermophilic bacteria with complete genome sequences were downloaded from GenBank in this study. We used CRISPRFinder to extensively study CRISPR structures and CRISPR-associated genes (cas) from thermophilic bacteria. We statistically analyzed the association between the CRISPR-Cas system and the optimum growth temperature of thermophilic bacteria. The results revealed that 59 strains of 61 thermophilic bacteria had at least one CRISPR locus, accounting for 96.72% of the total. Additionally, a total of 362 CRISPR loci, 209 entirely distinct repetitive sequences, 131 cas genes, and 7744 spacer sequences were discovered. The average number of CRISPR loci and the average minimum free energy (MFE) of the RNA secondary structure of repeat sequences were positively correlated with temperature whereas the average length of CRISPR loci and the average number of spacers were negatively correlated. The temperature did not affect the average number of CRISPR loci, the average length of repeats, or the guanine-cytosine (GC) content of repeats. The average number of CRISPR loci, the average length of the repeats, and the GC content of the repeats did not reflect temperature dependence. This study may provide a new basis for the study of the thermophilic bacterial adaptation mechanisms of thermophilic bacteria.

Expression of ß-Glucosidases from the Yak Rumen in Lactic Acid Bacteria: A Genetic Engineering Approach.

ß-glucosidase derived from microorganisms has wide industrial applications. In order to generate genetically engineered bacteria with high-efficiency ß-glucosidase, in this study two subunits (bglA and bglB) of ß-glucosidase obtained from the yak rumen were expressed as independent proteins and fused proteins in lactic acid bacteria (Lactobacillus lactis NZ9000). The engineered strains L. lactis NZ9000/pMG36e-usp45-bglA, L. lactis NZ9000/pMG36e-usp45-bglB, and L. lactis NZ9000/pMG36e-usp45-bglA-usp45-bglB were successfully constructed. These bacteria showed the secretory expression of BglA, BglB, and Bgl, respectively. The molecular weights of BglA, BglB, and Bgl were about 55 kDa, 55 kDa, and 75 kDa, respectively. The enzyme activity of Bgl was significantly higher (p < 0.05) than that of BglA and BglB for substrates such as regenerated amorphous cellulose (RAC), sodium carboxymethyl cellulose (CMC-Na), desiccated cotton, microcrystalline cellulose, filter paper, and 1% salicin. Moreover, 1% salicin appeared to be the most suitable substrate for these three recombinant proteins. The optimum reaction temperatures and pH values for these three recombinant enzymes were 50 °C and 7.0, respectively. In subsequent studies using 1% salicin as the substrate, the enzymatic activities of BglA, BglB, and Bgl were found to be 2.09 U/mL, 2.36 U/mL, and 9.4 U/mL, respectively. The enzyme kinetic parameters (Vmax, Km, Kcat, and Kcat/Km) of the three recombinant strains were analyzed using 1% salicin as the substrate at 50 °C and pH 7.0, respectively. Under conditions of increased K+ and Fe2+ concentrations, the Bgl enzyme activity was significantly higher (p < 0.05) than the BglA and BglB enzyme activity. However, under conditions of increased Zn2+, Hg2+, and Tween20 concentrations, the Bgl enzyme activity was significantly lower (p < 0.05) than the BglA and BglB enzyme activity. Overall, the engineered lactic acid bacteria strains generated in this study could efficiently hydrolyze cellulose, laying the foundation for the industrial application of ß-glucosidase.

Sequential Deletions of Interferon Inhibitors MGF110-9L and MGF505-7R Result in Sterile Immunity against the Eurasia Strain of Africa Swine Fever.

African swine fever virus (ASFV) causes significant morbidity and mortality in pigs worldwide. The lack of vaccines or therapeutic options warrants urgent further investigation. To this aim, we developed a rationally designed live attenuated ASFV-Δ110-9L/505-7R mutant based on the highly pathogenic Genotype II ASFV CN/GS/2018 backbone by deleting 2 well-characterized interferon inhibitors MGF110-9L and MGF505-7R. The mutant was slightly attenuated in vitro compared to parental ASFV but highly tolerant to genetic modifications even after 30 successive passages in vitro. Groups of 5 pigs were intramuscularly inoculated with increasing doses of the mutant, ranging from 103 to 106 hemadsorption units (HAD50). Thirty-five days later, all groups were challenged with 102 HAD50 of virulent parental ASFV. All the animals were clinically normal and devoid of clinical signs consistent with ASFV at the period of inoculation. In the virulent challenge, 2 animals from 103 HAD50-inoculated group and 1 animal from 104 HAD50-inoculated group were unprotected with severe postmortem and histological lesions. The rest of animals survived and manifested with relatively normal clinical appearance accompanied by tangible histological improvements in the extent of tissue damage. Meanwhile, antibody response, as represented by p30-specific antibody titers was positively correlated to protective efficacy, potentializing its usage as an indicator of protection. Moreover, compared to 1 dose, 2 doses provided additional protection, proving that 2 doses were better than 1 dose. The sufficiency in effectiveness supports the claim that our attenuated mutant may be a viable vaccine option with which to fight ASF. IMPORTANCE African swine fever virus (ASFV) is a causative agent of acute viral hemorrhagic disease of domestic swine which is associated with significant economic losses in the pig industry. The lack of vaccines or treatment options requires urgent further investigation. ASFV MGF110-9L and MGF505-7R, 2 well-characterized interferon inhibitors, were associated with viral virulence, host range, and immune modulation. In this study, a recombinant two-gene deletion ASFV mutant with deletion of MGF110-9L and MGF505-7R was constructed. The result showed that the mutant was safe, and also highly resistant to genetic modification even after 30 successive passages. High doses of our mutant (105 and 106 HAD50) provided sterile immunity and complete protection in a virulent challenge. Two doses were superior to 1 dose and provided additional protection. This study develops a new ASFV-specific live attenuated vaccine and may be a viable vaccine option against ASF.

Combinational Deletions of MGF360-9L and MGF505-7R Attenuated Highly Virulent African Swine Fever Virus and Conferred Protection against Homologous Challenge.

Multigene family (MGF) gene products are increasingly reported to be implicated in African swine fever virus (ASFV) virulence and attenuation of host defenses, among which the MGF360-9L and MGF505-7R gene products are characterized by convergent but distinct mechanisms of immune evasion. Herein, a recombinant ASFV mutant, ASFV-Δ9L/Δ7R, bearing combinational deletions of MGF360-9L and MGF505-7R, was constructed from the highly virulent ASFV strain CN/GS/2018 of genotype II that is currently circulating in China. Pigs inoculated intramuscularly with 104 50% hemadsorption doses (HAD50) of the mutant remained clinically healthy without any serious side effects. Importantly, in a virulence challenge, all four within-pen contact pigs demonstrated clinical signs and pathological findings consistent with ASF. In contrast, vaccinated pigs (5/6) were protected and clinical indicators tended to be normal, accompanied by extensive tissue repairs. Similar to most viral infections, innate immunity and both humoral and cellular immune responses appeared to be vital for protection. Notably, transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR) analysis revealed a regulatory function of the mutant in dramatic and sustained expression of type I/III interferons and inflammatory and innate immune genes in vitro. Furthermore, infection with the mutant elicited an early and robust p30-specific IgG response, which coincided and was strongly correlated with the protective efficacy. Analysis of the cellular response revealed a strong ASFV-specific interferon gamma (IFN-γ) response and immunostaining of CD4+ T cells coupled with a high level of CD163+ macrophage infiltration in spleens of vaccinated pigs. Our study identifies a new mechanism of immunological regulation by ASFV MGFs that rationalizes the design of live attenuated vaccine for implementation of improved control strategies to eradicate ASFV. IMPORTANCE Currently, the deficiency in commercially available vaccines or therapeutic options against African swine fever constitutes a matter of major concern in the swine industry globally. Here, we report the design and construction of a recombinant ASFV mutant harboring combinational deletions of interferon inhibitors MGF360-9L and MGF505-7R based on a genotype II ASFV CN/GS/2018 strain currently circulating in China. The mutant was completely attenuated when inoculated at a high dose of 104 HAD50. In the virulence challenge with homologous virus, sterile immunity was achieved, demonstrating the mutant's potential as a promising vaccine candidate. This sufficiency of effectiveness supports the claim that this live attenuated virus may be a viable vaccine option with which to fight ASF.