Capping Protein Research Articles

Prevalence, genotypes, and infection risk factors of psittacine beak and feather disease virus and budgerigar fledgling disease virus in captive birds in Hong Kong

Psittacine beak and feather disease virus (PBFDV) and budgerigar fledgling disease virus (BFDV) are significant avian pathogens that threaten both captive and wild birds, particularly parrots, which are common hosts. This study involved sampling and testing of 516 captive birds from households, pet shops, and an animal clinic in Hong Kong for PBFDV and BFDV. The results showed that PBFDV and BFDV were present in 7.17% and 0.58% of the samples, respectively. These rates were lower than those reported in most parts of Asia. Notably, the infection rates of PBFDV in pet shops were significantly higher compared to other sources, while no BFDV-positive samples were found in pet shops. Most of the positive samples came from parrots, but PBFDV was also detected in two non-parrot species, including Swinhoe’s white-eyes (Zosterops simplex), which had not been reported previously. The ability of PBFDV to infect both psittacine and passerine birds is concerning, especially in densely populated urban areas such as Hong Kong, where captive flocks come into close contact with wildlife. Phylogenetic analysis of the Cap and Rep genes of PBFDV revealed that the strains found in Hong Kong were closely related to those in Europe and other parts of Asia, including mainland China, Thailand, Taiwan, and Saudi Arabia. These findings indicate the presence of both viruses among captive birds in Hong Kong. We recommend implementing regular surveillance for both viruses and adopting measures to prevent contact between captive and wild birds, thereby reducing the transmission of introduced diseases to native species.

Tumor vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA): Suppresses macrophage capping protein beyond STING activation

The vascular disrupting agent (VDA) 5,6-dimethylxanthenone-4-acetic acid (DMXAA) induces apoptosis in vascular endothelial cells and leads to tumor hemorrhagic necrosis. While DMXAA has been proven to be a potent agonist of murine stimulator of interferon genes (mSTING), it has little effect on human-STING (hSTING). This species selectivity of DMXAA may explain its effectiveness against solid tumors in mice and its failure in clinical trials. However, DMXAA did reduce tumor volume in some patients during clinical trials. These paradoxical results have prompted us to investigate the anti-tumor mechanism of DMXAA beyond STING in the destruction of tumor vasculature in humans. In this study, we demonstrated that DMXAA binds to both human and mouse macrophage capping protein (CapG), with a KD of 5.839 μM for hCapG and a KD of 2.867 μM for mCapG, as determined by surface plasmon resonance (SPR) analysis. Homology modeling and molecular docking analysis of hCapG indicated that the critical residues involved in the hydrogen bond interaction of DMXAA with hCapG were Arg153, Thr151, and GLN141, Asn234. In addition, electrostatic pi-cation interaction occurred between DMXAA and hCapG. Further functional studies revealed that CapG protein plays a crucial role in the effects of DMXAA on human umbilical endothelial vein cell (HUEVC) angiogenesis and migration, as well as the expression of cytoskeletal proteins actin and tubulin, and the invasion of A549 lung adenocarcinoma cells. Our study has originally uncovered a novel cross-species pathway underlying the antitumor vascular disruption of DMXAA extends beyond STING activation. This finding deepens our understanding of the multifaceted actions of flavonoid VDAs in animal models and in clinical settings, and may provide insights for the precise therapy of DMXAA based on the biomarker CapG protein.

Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, β-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

Comparison of immune effects of porcine circovirus type 2d (PCV2d) capsid protein expressed by Escherichia coli and baculovirus-insect cells

Porcine circovirus type 2 (PCV2) is an important pathogen harmful to global pig production, which causes immunosuppression and serious economic losses. PCV2 capsid (Cap) protein expressed by E. coli or baculovirus-insect cells are often used in preparation of PCV2 subunit vaccines, but the latter is expensive to produce. It is therefore crucial to comparison of the immune effects of Cap protein expressed by the above two expression systems for reducing the production cost and guaranteeing PCV2 vaccine quality. In this study, the PCV2d-Cap protein lacking nuclear localization signal (NLS), designated as E. coli-Cap and Bac-Cap, was expressed by E. coli and baculovirus-Spodoptera frugiperda Sf9 (Bac-Sf9) cells, respectively. The expressed Cap proteins could self-assemble into virus-like particles (VLPs), but the Bac-Cap-assembled VLPs were more regular. The two system-expressed Cap proteins induced similar specific IgG responses in mice, but the neutralizing antibody levels of Bac-Cap-immunized mice was higher than those of E. coli-Cap. After PCV2 challenge, IL-10 in Bac-Cap immunized mice decreased significantly than that in E. coli-Cap. The lesions and PCV2 antigen positive cells in tissues of mice immunized with E. coli-Cap and Bac-Cap were significantly reduced, and Bac-Cap appeared mild lesions and fewer PCV2 antigen-positive cells compared with E. coli-Cap immunized mice. The study indicated that Cap proteins expressed by E. coli and Bac-Sf9 cells could induce specific protective immunity, but the latter induced more effective immunity, which provides valuable information for the research and development of PCV2 vaccine.

Investigation of porcine circovirus type 2 and porcine circovirus type 3 infections based on dual TaqMan fluorescent quantitative PCR method and genetic evolutionary analysis of these two viruses.

Porcine circovirus type 2 (PCV2) is the pathogen of Porcine Circovirus Associated Diseases. Porcine circovirus type 3 (PCV3) is a novel porcine circovirus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2 is clearly pathogenic, while the pathogenicity of PCV3 remains controversial, so it is crucial to monitor the prevalence of PCV2 and PCV3 in healthy and diseased pigs to investigate the effects of PCV3 and PCV2 on the health status of pigs. Here, we developed a PCV2 and PCV3 dual TaqMan quantitative PCR (qPCR) method to test samples from healthy and diseased pigs, to clarify the differences in the positive rates and viral copy numbers of PCV2 and PCV3, and to analyze the genetic evolution and molecular characterization of the viral genomes obtained with sequence alignment and phylogenetic analysis, homology and structural analysis of Cap proteins, and selection pressure analysis. We successfully established a dual TaqMan qPCR method for PCV2 and PCV3 with good repeatability, specificity and sensitivity. In total, 1,385 samples from 15 Chinese provinces were tested with the established qPCR. The total positive rates were 37.47% for PCV3 and 57.95% for PCV2, and the coinfection rate for was 25.49%. The positive rates of PCV3 and PCV2 in 372 healthy pigs were 15.05 and 69.89%, respectively, and the coinfection rate was 12.90%. The positive rates of PCV3 and PCV2 in 246 diseased pigs were 55.69 and 83.33%, respectively, and the coinfection rate was 47.97%. Eighteen PCV3 genomes and 64 PCV2 genomes were identified, including nine each of the PCV3a-1 and PCV3b genotypes, eight of PCV2a, 16 of PCV2b, and 40 of PCV2d. The amino acid identity within the PCV3 Cap proteins was 94.00-100.0%, whereas the PCV2 Cap proteins showed an identity of 81.30-100.0%. PCV3 Cap was most variable at amino acid sites 24, 27, 77, 104 and 150, whereas PCV2 Cap had 10-13 unique sites of variation between genotypes. These results clarify the prevalence and variations of PCV2 and PCV3 in healthy and diseased pigs, which will provide a basis for the prevention and control of the two viral infections.

Structural basis for nuclear import of bat adeno-associated virus capsid protein.

Adeno-associated viruses (AAV) are one of the world's most promising gene therapy vectors and as a result, are one of the most intensively studied viral vectors. Despite a wealth of research into these vectors, the precise characterisation of AAVs to translocate into the host cell nucleus remains unclear. Recently we identified the nuclear localization signals of an AAV porcine strain and determined its mechanism of binding to host importin proteins. To expand our understanding of diverse AAV import mechanisms we sought to determine the mechanism in which the Cap protein from a bat-infecting AAV can interact with transport receptor importins for translocation into the nucleus. Using a high-resolution crystal structure and quantitative assays, we were able to not only determine the exact region and residues of the N-terminal domain of the Cap protein which constitute the functional NLS for binding with the importin alpha two protein, but also reveal the differences in binding affinity across the importin-alpha isoforms. Collectively our results allow for a detailed molecular view of the way AAV Cap proteins interact with host proteins for localization into the cell nucleus.

A robust and flexible baculovirus-insect cell system for AAV vector production with improved yield, capsid ratios and potency

A robust and flexible baculovirus-insect cell system for AAV vector production with improved yield, capsid ratios and potency

Tuning capsid formation dynamics in recombinant adeno-associated virus producing synthetic cell lines to enhance full particle productivity.

Recombinant adeno-associated virus (rAAV) is widely used as an in vivo delivery vector for gene therapy. It is used in a very large dose, and the large quantities required for broad applications present manufacturing challenges. We have developed a synthetic biology platform of constructing cell lines integrated with essential viral genes which can be induced to produce rAAV without plasmid transfection or virus transduction. Through iterative design-construct-characterization cycles, we have showcased the potential of this synthetic cell production system. Systems characterization of the dynamics of viral transcripts and proteins as well as virus assembly and packaging revealed that the expression level and balance of viral genome and capsid protein are keys to not only the productivity but also the full particle content, an important product quality attribute. Boosting cap gene expression by sequential transfection and integration of multiple copies of the cap gene elevated the rAAV titer to levels on a par with traditional plasmid transfection and virus infection. However, overexpression of the cap gene shifted the balance and kinetics of the genome and capsid. We independently tuned the dynamics of genome amplification and capsid protein synthesis by modulating the induction concentration as well as the time profile, and significantly enhanced full particle content while maintaining a high productivity. This strategy of constructing an inducible stable producer cell line is readily adaptable to rAAV vectors of different serotypes and payloads. It can greatly facilitate scalable production of gene therapy vectors.

A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency.

Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the cap gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.

Differential Protein Expression in Set5p-Mediated Acetic Acid Stress Response and Novel Targets for Engineering Yeast Stress Tolerance.

Acetic acid is a prevalent inhibitor in lignocellulosic hydrolysate, which represses microbial growth and bioproduction. Histone modification and chromatin remodeling have been revealed to be critical for regulating eukaryotic metabolism. However, related studies in chronic acetic acid stress responses remain unclear. Our previous studies revealed that overexpression of the histone H4 methyltransferase Set5p enhanced acetic acid stress tolerance of the budding yeast Saccharomyces cerevisiae. In this study, we examined the role of Set5p in acetic acid stress by analyzing global protein expression. Significant activation of intracellular protein expression under the stress was discovered, and the functions of the differential proteins were mainly involved in chromatin modification, signal transduction, and carbohydrate metabolism. Notably, a substantial increase of Set5p expression was observed in response to acetic acid stress. Functional studies demonstrated that the restriction of the telomere capping protein Rtc3p, as well as Ies3p and Taf14p, which are related to chromatin regulation, was critical for yeast stress response. This study enriches the understanding of the epigenetic regulatory mechanisms underlying yeast stress response mediated by histone-modifying enzymes. The results also benefit the development of robust yeast strains for lignocellulosic bioconversion.

The Nuclear Localization Signal of Porcine Circovirus Type 4 Affects the Subcellular Localization of the Virus Capsid and the Production of Virus-like Particles.

Porcine circovirus 4 (PCV4) is a newly identified virus belonging to PCV of the Circoviridae family, the Circovirus genus. We previously found that PCV4 is pathogenic in vitro, while the virus's replication in cells is still unknown. In this study, we evaluated the N-terminal of the PCV4 capsid (Cap) and identified an NLS at amino acid residues 4-37 of the N-terminus of the PCV4 Cap, 4RSRYSRRRRNRRNQRRRGLWPRASRRRYRWRRKN37. The NLS was further divided into two fragments (NLS-A and NLS-B) based on the predicted structure, including two α-helixes, which were located at 4RSRYSRRRRNRRNQRR19 and 24PRASRRRYRWRRK36, respectively. Further studies showed that the NLS, especially the first α-helixes formed by the NLS-A fragment, determined the nuclear localization of the Cap protein, and the amino acid 4RSRY7 in the NLS of the PCV4 Cap was the critical motif affecting the VLP packaging. These results will provide a theoretical basis for elucidating the infection mechanism of PCV4 and developing subunit vaccines based on VLPs.

The surface morphology of Atractylodes macrocephala polysaccharide and its inhibitory effect on PCV2 replication.

Porcine infection with Porcine circovirus type 2 (PCV2) causes immunosuppression, which is easy to cause concurrent or secondary infection, making the disease complicated and difficult to treat, and causing huge economic losses to the pig industry. Total polysaccharide from the rhizoma of Atractylodes macrocephala Koidz. (PAMK) is outstanding in enhancing non-specific immunity and cellular immunity, and effectively improving the body's disease resistance, indicating its potential role in antiviral immunotherapy. PAMK had the characteristics of compact, polyporous and agglomerated morphology, but does not have triple helix conformation. PCV2 infection led to the increase in LC3-II, degradation of p62 and the increase of viral Cap protein expression and viral copy number. PAMK treatment significantly alleviated PCV2-induced autophagy and inhibited PCV2 replication. Moreover, PAMK treatment significantly attenuated the increase of PINK1 protein expression and the decrease of TOMM20 protein expression caused by PCV2 infection, alleviated Parkin recruitment from cytoplasm to mitochondria and intracellular reactive oxygen species accumulation, restored mitochondrial membrane charge, alleviated viral Cap protein expression. PAMK alleviates PCV2-induced mitophagy to suppress PCV2 replication by inhibiting the Pink 1/Parkin pathway. These findings may provide new insights into the prevention and treatment of PCV2. © 2023 Society of Chemical Industry.

DEAD-box RNA helicase 21 interacts with porcine circovirus type 2 Cap protein and facilitates viral replication.

Porcine circovirus type 2 (PCV2) is the etiological agent of PCV2-associated diseases that pose a serious threat to the swine industry. PCV2 capsid (Cap) protein has been shown to interact with DEAD-box RNA helicase 21 (DDX21), an important protein that regulates RNA virus replication. However, whether the interaction between DDX21 and the PCV2 Cap regulates PCV2 replication remains unclear. Herein, by using western blotting, interaction assays, and knockdown analysis, we found that PCV2 infection induced the cytoplasmic relocation of DDX21 from the nucleolus in cultured PK-15 cells. Moreover, the nuclear localization signal (NLS) of PCV2 Cap interacted directly with DDX21. The NLS of PCV2 Cap and 763GSRSNRFQNK772 residues at the C-terminal domain (CTD) of DDX21 were essential for the dual interaction. Upon shRNA-mediated DDX21 depletion in PK-15 cells, we observed impaired PCV2 replication via a lentivirus-delivered system, as evidenced by decreased levels of viral protein expression and virus production. In contrast, the replication of PCV2 increased in transiently DDX21-overexpressing cells. Our results indicate that DDX21 interacts with PCV2 Cap and plays a crucial role in virus replication. These results provide a reference for developing novel potential targets for prevention and control of PCV2 infection.

Abstract B071: A genetic approach to selectively inhibit macropinocytosis while sparing autophagy

Abstract Macropinocytosis is a bulk endocytic process that can fuel tumor growth and drug resistance by providing access to extracellular protein and necrotic cell debris. This process is primarily studied by utilizing the chemical inhibitor, 5-(N-ethyl-N-isopropyl)-Amiloride (EIPA), but this inhibitor is nonselective can hinder proliferation even in non-macropinocytic breast cancer cells. Hence, a more selective, genetic approach is needed. Formation of macropinosomes is dependent on the actin cytoskeleton, which in turn is regulated by CARMIL1, a large multimeric protein that regulates the actin capping protein. CARMIL1 is essential for macropinocytosis, and mutation of two residues in the Capping-Protein Interaction (CPI) region of CARMIL1 to alanine (CARMIL1-AA) limits the CARMIL1-capping protein interaction and more importantly, inhibits macropinocytosis upon introduction of exogenous CARMIL1-AA in tumor cells. Therefore, CARMIL1-AA is a genetic tool to study the effects of macropinocytosis on other cellular processes. However, autophagy, a lysosome-dependent recycling system that enables cell survival, additionally utilizes actin to form autophagosomes. The overall goal of this project is to determine whether CARMIL1-AA specifically inhibits macropinocytosis while still supporting autophagy. CARMIL1-WT and -AA mutant cells were generated using the MMTV-PyMT tumor model and HeLa cell lines and validated for CARMIL1 expression by western blot and quantitative PCR and for macropinocytosis by dextran analysis. These cells were evaluated for autophagic flux by western blot for LC3 I/II and by utilizing a GFP-LC3-RFP-LC3ΔG reporter. Our observation that CARMIL1-AA does not impact autophagy enables further investigation of the importance of macropinocytosis in therapeutic resistance and in shaping the tumor microenvironment. Citation Format: Rebecca M Lim, Brandon Chu, Aimee Lara Edinger. A genetic approach to selectively inhibit macropinocytosis while sparing autophagy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B071.

Multi-omics kinetic analysis of recombinant adeno-associated virus production by plasmid transfection of HEK293 cells.

Recombinant adeno-associated virus (rAAV) is among the most commonly used vectors for gene therapy. It is commonly produced by transfection of HEK293 cells with three plasmids each containing the vector genome including gene of interest (GOI), helper functions, and rep and cap genes for genome replication and capsid formation. To meet the potential clinical needs, the productivity of the production system needs to be enhanced. A better process characterization of the production system will further advance our insights into ways to enhance productivity. Here, we employed transcriptomic analysis to quantify the dynamics of different isoforms of viral transcripts and to assess the shift of cellular physiology, and deployed targeted proteomic analysis for absolute quantification of viral proteins and tandem mass tags (TMTs) for assessing cellular responses at the protein level. Functional analysis at transcriptome and proteome levels identified defense and immune response, unfolded protein response, p53 signaling as enriched. The small molecule additive intervention study based on functional analysis showed the potential of such omics-guided productivity enhancement. Together, multi-omics analysis advanced understanding of rAAV production and provided insight into enhancing rAAV production by plasmid transfection.

The temporal association of CapZ with early endosomes regulates endosomal trafficking and viral entry into host cells

BackgroundMany viruses enter host cells by hijacking endosomal trafficking. CapZ, a canonical actin capping protein, participates in endosomal trafficking, yet its precise role in endocytosis and virus infection remains elusive.ResultsHere, we showed that CapZ was transiently associated with early endosomes (EEs) and was subsequently released from the matured EEs after the fusion of two EEs, which was facilitated by PI(3)P to PI(3,5)P2 conversion. Vacuolin-1 (a triazine compound) stabilized CapZ at EEs and thus blocked the transition of EEs to late endosomes (LEs). Likewise, artificially tethering CapZ to EEs via a rapamycin-induced protein–protein interaction system blocked the early-to-late endosome transition. Remarkably, CapZ knockout or artificially tethering CapZ to EEs via rapamycin significantly inhibited flaviviruses, e.g., Zika virus (ZIKV) and dengue virus (DENV), or beta-coronavirus, e.g., murine hepatitis virus (MHV), infection by preventing the escape of RNA genome from endocytic vesicles.ConclusionsThese results indicate that the temporal association of CapZ with EEs facilitates early-to-late endosome transition (physiologically) and the release of the viral genome from endocytic vesicles (pathologically).

Helicobacter pylori FlgN binds its substrate FlgK and the flagellum ATPase FliI in a similar manner observed for the FliT chaperone.

In bacterial flagellum biogenesis, secretion of the hook-filament junction proteins FlgK and FlgL and completion of the flagellum requires the FlgN chaperone. Similarly, the related FliT chaperone is necessary for the secretion of the filament cap protein FliD and binds the flagellar export gate protein FlhA and the flagellum ATPase FliI. FlgN and FliT require FliJ for effective substrate secretion. In Helicobacter pylori, neither FlgN, FliT, nor FliJ have been annotated. We demonstrate that the genome location of HP1120 is identical to that of flgN in other flagellated bacteria and that HP1120 is the homolog of Campylobacter jejuni FlgN. A modeled HP1120 structure contains three α-helices and resembles the FliT chaperone, sharing a similar substrate-binding pocket. Using pulldowns and thermophoresis, we show that both HP1120 and a HP1120Δ126-144 deletion mutant bind to FlgK with nanomolar affinity, but not to the filament cap protein FliD, confirming that HP1120 is FlgN. Based on size-exclusion chromatography and multi-angle light scattering, H. pylori FlgN binds to FlgK with 1:1 stoichiometry. Overall structural similarities between FlgN and FliT suggest that substrate recognition on FlgN primarily involves an antiparallel coiled-coil interface between the third helix of FlgN and the C-terminal helix of the substrate. A FlgNΔ126-144 N100A, Y103A, S111I triple mutant targeting this interface significantly impairs the binding of FlgK. Finally, we demonstrate that FlgNΔ126-144 , like FliT, binds with sub-micromolar affinity to the flagellum ATPase FliI or its N-terminal domain. Hence FlgN and FliT likely couple delivery of low-abundance export substrates to the flagellum ATPase FliI.

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. Currently, there is a growing need for rapid and accurate diagnostic methods in disease monitoring. In this study, four monoclonal antibodies (mAbs) against PCV3 capsid proteins were prepared (mAbs 2F6, 2G8, 6E2, and 7E3). MAb 7E3, which had the highest binding affinity for the Cap protein, was chosen for further investigation. A novel B cell epitope 110DLDGAW115 was identified using mAb 7E3. An epitope-blocking (EB) enzyme-linked immunosorbent assay (ELISA) was successfully developed using horseradish-peroxidase-labeled mAb 7E3 to detect PCV3 antibodies in porcine sera. Moreover, the EB-ELISA showed no specific reaction with other porcine disease sera, and the cut-off value was defined as 35%. Compared with the commercial ELISA, the percentage agreement was 95.59%. Overall, we have developed a novel EB-ELISA method that accurately and conveniently detects PCV3 in serum, making it a valuable tool for the clinical detection of PCV3 infection.

Expression regulation of type III secretion system 2 in Vibrio parahaemolyticus by catabolite activator protein.

Vibrio parahaemolyticus has two sets of type III secretion systems that are major pathogenic factors: T3SS1 (cytotoxicity) and T3SS2 (enterotoxicity). V. parahaemolyticus mainly colonizes the distal small intestine after oral infection and may be exposed to carbon-limiting stress due to the lack of readily available carbohydrates in this environment. Catabolite activator protein (CAP), a transcription factor involved in carbon-limiting metabolism in many Gram-negative bacteria, is well known to be involved in the regulation of the expression of many virulence factors. In this study, we determined the effects of CAP on the expression of T3SSs in this bacterium. Based on a lactate dehydrogenase-based cytotoxicity assay, CAP was found to have a greater contribution to the expression of T3SS2-dependent cytotoxicity than to that of T3SS1. Reverse transcription quantitative PCR revealed decreased expression of many T3SS2-related genes, including vpa1348, in the cap gene deletion mutant compared to the parent strain. CAP was demonstrated to bind near the T-rich elements within the vpa1348 promoter region in an electrophoretic mobility shift assay and DNase I footprinting. CAP also enhanced the expression of vpa1348 in a β-galactosidase reporter assay. Collectively, these results suggest that CAP is involved in T3SS2-mediated virulence by regulating the expression of vpa1348 in V. parahaemolyticus.

A DUF3494 ice-binding protein with a root cap domain in a streptophyte glacier ice alga.

Ice-binding proteins (IBPs) of the DUF3494 type have been found in many ice-associated unicellular photoautotrophs, including chlorophytes, haptophytes, diatoms and a cyanobacterium. Unrelated IBPs have been found in many land plants (streptophytes). Here we looked for IBPs in two streptophyte algae that grow only on glaciers, a group in which IBPs have not previously been examined. The two species, Ancylonema nordenskioeldii and Ancylonema. alaskanum, belong to the class Zygnematophyceae, whose members are the closest relatives to all land plants. We found that one of them, A. nordenskioeldii, expresses a DUF3494-type IBP that is similar to those of their chlorophyte ancestors and that has not previously been found in any streptophytes. The protein is unusual in having what appears to be a perfect array of TXT motifs that have been implicated in water or ice binding. The IBP strongly binds to ice and almost certainly has a role in mitigating the daily freeze-thaw cycles that the alga is exposed to during late summer. No IBP was found in the second species, A. alaskanum, which may rely more on glycerol production for its freeze-thaw tolerance. The IBP is also unusual in having a 280-residue domain with a β sandwich structure (which we designate as the DPH domain) that is characteristic of root cap proteins of land plants, and that may have a role in forming IBP oligomers. We also examined existing transcriptome data obtained from land plants to better understand the tissue and temperature dependence of expression of this domain.