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There is no currently approved human vaccine against leishmaniasis. Utilization of immunogenic antigens and their epitopes capable of enhancing immune responses against leishmaniasis is a crucial step for rational in silico vaccine design. The objective of this study was to generate and evaluate a potential vaccine candidate against leishmaniasis, designed by immunodominant proteins from gp46 and gp63 of Leishmania major, which can stimulate helper T-lymphocytes (HTL) and cytotoxic T-lymphocytes (CTL). For this aim, the IFN-γ-inducing MHC-I and MHC-II binders were predicted for each examined protein (gp46 and gp63) and connected with appropriate linkers, along with an adjuvant (Mycobacterium tuberculosis L7/L12) and a histidine tag. The vaccine’s stability, antigenicity, structure, and interaction with the TLR-4 receptor were evaluated in silico. The resulting chimeric vaccine was composed of 344 amino acids and had a molecular weight of 35.64 kDa. Physico-chemical properties indicated that it was thermotolerant, soluble, highly antigenic, and non-allergenic. Predictions of the secondary and tertiary structures were made, and further analyses confirmed that the vaccine construct could interact with the human TLR-4 receptor. Virtual immune simulation demonstrated strong stimulation of T-cell responses, particularly by an increase in IFN-γ, following vaccination. In summary, the in silico data indicated that the vaccine candidate showed high antigenicity in humans. It was also found to trigger significant levels of clearance mechanisms and other components of the cellular immune profile. Nevertheless, further wet experiments are required to properly assess the efficacy of this multi-epitope vaccine candidate against leishmaniasis.

The nucleoid-associated protein HU is a common bacterial transcription factor, whose role in pathogenesis and virulence has been described in many bacteria. Our recent studies showed that the HU protein is an indispensable virulence factor in the human pathogenic bacterium Francisella tularensis, a causative agent of tularemia disease, and that this protein can be a key target in tularemia treatment or vaccine development. Here, we show that Francisella HU protein is inhibited by Gp46, a protein of Bacillus subtilis bacteriophage SPO1. We predicted that Gp46 could occupy the F. tularensis HU protein DNA binding site, and subsequently confirmed the ability of Gp46 to abolish the DNA-binding capacity of HU protein. Next, we showed that the growth of Francisella wild-type strain expressing Gp46 in trans corresponded to that of a deletion mutant strain lacking the HU protein. Similarly, the efficiency of intracellular proliferation in mouse macrophages resembled that of the deletion mutant strain, but not that of the wild-type strain. These results, in combination with findings from a recent study on Gp46, enabled us to confirm that Gp46 could be a universal inhibitor of HU proteins among bacterial species.

Human T-cell leukemia virus type-1 (HTLV-1) establishes a long-term persistent infection in humans and causes malignant T-cell leukemia, adult T-cell leukemia (ATL). HTLV-1-specific cytotoxic T lymphocytes have been suggested to play a major role in the immunosurveillance of HTLV-1-infected T cells. However, it remains unclear whether HTLV-1-specific functional antibodies are also involved in the host defense. To explore the role of antibodies in the course of HTLV-1 infection, we quantitated HTLV-1-specific neutralizing and antibody-dependent cellular cytotoxicity (ADCC)-inducing antibody levels in plasma from asymptomatic carriers (ACs) and ATL patients. The levels of neutralizing antibodies, as determined by a syncytium inhibition assay, were significantly lower in acute and chronic ATL patients than in ACs. The levels of ADCC-inducing activity were tested using an autologous pair of HTLV-1-producing cells and cultured natural killer (NK) cells, which showed that the ADCC-inducing activity of IgG at a concentration of 100 µg/ml was comparable between ACs and acute ATL patients. The anti-gp46 antibody IgG levels, determined by ELISA, correlated with those of the neutralizing and ADCC-inducing antibodies. In contrast, the proviral loads did not correlate with any of these antibody levels. NK cells and a monoclonal anti-gp46 antibody reduced the number of HTLV-1 Tax-expressing cells in cultured peripheral blood mononuclear cells from patients with aggressive ATL. These results suggest a protective role for HTLV-1 neutralizing and ADCC-inducing antibodies during the course of HTLV-1 infection. [ABSTRACT FROM AUTHOR]

The architectural protein histone-like protein from Escherichia coli strain U93 (HU) is the most abundant bacterial DNA binding protein and highly conserved among bacteria and Apicomplexan parasites. It not only binds to double-stranded DNA (dsDNA) to maintain DNA stability but also, interacts with RNAs to regulate transcription and translation. Importantly, HU is essential to cell viability for many bacteria; hence, it is an important antibiotic target. Here, we report that Gp46 from bacteriophage SPO1 of Bacillus subtilis is an HU inhibitor whose expression prevents nucleoid segregation and causes filamentous morphology and growth defects in bacteria. We determined the solution structure of Gp46 and revealed a striking negatively charged surface. An NMR-derived structural model for the Gp46–HU complex shows that Gp46 occupies the DNA binding motif of the HU and therefore, occludes DNA binding, revealing a distinct strategy for HU inhibition. We identified the key residues responsible for the interaction that are conserved among HUs of bacteria and Apicomplexans, including clinically significant Mycobacterium tuberculosis, Acinetobacter baumannii, and Plasmodium falciparum, and confirm that Gp46 can also interact with these HUs. Our findings provide detailed insight into a mode of HU inhibition that provides a useful foundation for the development of antibacteria and antimalaria drugs. [ABSTRACT FROM AUTHOR]

There is no currently approved human vaccine against leishmaniasis. Utilization of immunogenic antigens and their epitopes capable of enhancing immune responses against leishmaniasis is a crucial step for rational in silico vaccine design. The objective of this study was to generate and evaluate a potential vaccine candidate against leishmaniasis, designed by immunodominant proteins from gp46 and gp63 of Leishmania major , which can stimulate helper T-lymphocytes (HTL) and cytotoxic T-lymphocytes (CTL). For this aim, the IFN-γ-inducing MHC-I and MHC-II binders were predicted for each examined protein (gp46 and gp63) and connected with appropriate linkers, along with an adjuvant (Mycobacterium tuberculosis L7/L12) and a histidine tag. The vaccine's stability, antigenicity, structure, and interaction with the TLR-4 receptor were evaluated in silico. The resulting chimeric vaccine was composed of 344 amino acids and had a molecular weight of 35.64 kDa. Physico-chemical properties indicated that it was thermotolerant, soluble, highly antigenic, and non-allergenic. Predictions of the secondary and tertiary structures were made, and further analyses confirmed that the vaccine construct could interact with the human TLR-4 receptor. Virtual immune simulation demonstrated strong stimulation of T-cell responses, particularly by an increase in IFN-γ, following vaccination. In summary, the in silico data indicated that the vaccine candidate showed high antigenicity in humans. It was also found to trigger significant levels of clearance mechanisms and other components of the cellular immune profile. Nevertheless, further wet experiments are required to properly assess the efficacy of this multi-epitope vaccine candidate against leishmaniasis. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a chronic disease that is characterized by the excessive deposition of scar tissue in the lungs. As currently available treatments are unable to restore lung function in patients, there is an urgent medical need for more effective drugs. Developing such drugs, however, is challenging because IPF has a complex pathogenesis. Emerging evidence indicates that heat shock protein 47 (HSP47), which is encoded by the gene Serpinh1, may be a suitable therapeutic target as it is required for collagen synthesis. Pharmacological inhibition or knockdown of HSP47 could therefore be a promising approach to treat fibrosis. The objective of this study was to assess the therapeutic potential of Serpinh1-targeting small interfering RNA (siRNA) in fibrogenic precision-cut lung slices prepared from murine tissue. To enhance fibrogenesis, slices were cultured for up to 144 h with transforming growth factor β1. Self-deliverable siRNA was used to knockdown mRNA and protein expression, without affecting the viability and morphology of slices. After silencing HSP47, only the secretion of fibronectin was reduced while other aspects of fibrogenesis remained unaffected (e.g., myofibroblast differentiation as well as collagen secretion and deposition). These observations are surprising as others have shown that Serpinh1-targeting siRNA suppressed collagen deposition in animals. Further studies are therefore warranted to elucidate downstream effects on fibrosis upon silencing HSP47.

Background: Cutaneous leishmaniasis (CL) is a parasitic disease with a significant burden in the Old World countries., Objective: In the current study, some of the primary biochemical properties and IFN-γ inducing epitopes with specific binding capacity to human and mouse MHC alleles were predicted for Leishmania major gp46 antigenic protein., Methods: Several online servers were used to predict physico-chemical traits, allergenicity, antigenicity, transmembrane domain and signal peptide, subcellular localization, post-translational modifications (PTMs), secondary and tertiary structures, tertiary model refining with validations. Also, IEDB web server was used to predict mouse/human cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes., Results: The 33.25 kDa protein was stable, hydrophilic, antigenic, while non-allergenic, with enhanced thermotolerance and 45 PTM sites. The secondary structure encompassed a random coil, followed by extended strands and helices. Ramachandran-based analysis of the refined model showed 73.1%, 21.6%, 3.4% and 1.9% of residues in the most favored, additional allowed, generously-allowed and disallowed regions, respectively. Epitope screening demonstrated 4 HTL epitopes against seemingly protective HLA alleles, 5 HTL epitopes against the HLA reference set, 3 human CTL epitopes and a number of mouse MHC-restricted epitopes., Conclusion: This paper provides insights into the bioinformatics characteristics of the L. major gp46 protein as a promising vaccine candidate., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The nucleoid-associated protein HU is a common bacterial transcription factor, whose role in pathogenesis and virulence has been described in many bacteria. Our recent studies showed that the HU protein is an indispensable virulence factor in the human pathogenic bacterium Francisella tularensis , a causative agent of tularemia disease, and that this protein can be a key target in tularemia treatment or vaccine development. Here, we show that Francisella HU protein is inhibited by Gp46, a protein of Bacillus subtilis bacteriophage SPO1. We predicted that Gp46 could occupy the F. tularensis HU protein DNA binding site, and subsequently confirmed the ability of Gp46 to abolish the DNA-binding capacity of HU protein. Next, we showed that the growth of Francisella wild-type strain expressing Gp46 in trans corresponded to that of a deletion mutant strain lacking the HU protein. Similarly, the efficiency of intracellular proliferation in mouse macrophages resembled that of the deletion mutant strain, but not that of the wild-type strain. These results, in combination with findings from a recent study on Gp46, enabled us to confirm that Gp46 could be a universal inhibitor of HU proteins among bacterial species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Spidlova, Sokolova and Pavlik.)

Bacterial antibiotic resistance is a serious threat to public health and bacteriophage therapy is an alternative for antibiotics in the era of multidrug resistance. While phage draws attention in fighting bacterial infection and is used in protein display to study macromolecular interactions, the molecular machinery of the host invasion mechanism remains largely unclear for many bacteriophages. Despite recent studies on T4 and T7 phages of Gram-negative model organism Escherichia coli revealing many interesting features of their invasive strategies, the studies on Gram-positive bacterial phages still lag far behind their counterparts. SPO1 is a lytic phage of model organism Bacillus subtilis and one of the best studied Gram-positive bacterial phages. SPO1 features a unique Host Takeover Module coding for 24 proteins which show little similarity to any previously known proteins. Gp46, located in this module, is an acidic protein that is produced by SPO1 presumably during the host takeover event. Here we describe the complete resonance assignment of Gp46 as the basis for the first structure determination of SPO1 phage protein and further mechanism study. [ABSTRACT FROM AUTHOR]

To prevent the spread of HTLV-I (Human T-lymphotropic virus type 1), a safe and effective vaccine is required. To increase immune responses against the peptide antigens can be potentiated with polymer-based nanoparticles, like chitosan (CHT) and trimethylchitosan (TMC), as delivery system/adjuvant. CHT and TMC nanoparticles loaded with recombinant proteins (env23 & env13) of gp46 were prepared by direct coating of antigens with positively charged polymers. The size of CHT and TMC nanoparticles (NPs) loaded with each antigen was about 400 nm. The physical stability of NPs was followed for 4 weeks. Both formulations showed to be stable for about 15 days. The immunogenicity of NPs loaded with antigens was studied after nasal and subcutaneous immunization in mice. Three immunizations (7.5 μg antigen) were performed with 2 weeks intervals. Two weeks after the last booster dose, sera IgG subtypes were measured. After subcutaneous administration, for both nanoparticulate antigens, serum IgG1 and IgGtotal levels were higher than antigen solution (P < 0.001). After nasal administration, for env23, IgG2a levels and IgG2a/IgG1 ratio was significantly higher than groups with subcutaneous administration (P < 0.001). Both nanoparticles showed good immunoadjuvant potential. Env23 antigen was a better candidate for vaccination against HTLV-I, as it induced higher cellular immune responses, compared with env13. [ABSTRACT FROM AUTHOR]

The architectural protein histone-like protein from Escherichia coli strain U93 (HU) is the most abundant bacterial DNA binding protein and highly conserved among bacteria and Apicomplexan parasites. It not only binds to double-stranded DNA (dsDNA) to maintain DNA stability but also, interacts with RNAs to regulate transcription and translation. Importantly, HU is essential to cell viability for many bacteria; hence, it is an important antibiotic target. Here, we report that Gp46 from bacteriophage SPO1 of Bacillus subtilis is an HU inhibitor whose expression prevents nucleoid segregation and causes filamentous morphology and growth defects in bacteria. We determined the solution structure of Gp46 and revealed a striking negatively charged surface. An NMR-derived structural model for the Gp46-HU complex shows that Gp46 occupies the DNA binding motif of the HU and therefore, occludes DNA binding, revealing a distinct strategy for HU inhibition. We identified the key residues responsible for the interaction that are conserved among HUs of bacteria and Apicomplexans, including clinically significant Mycobacterium tuberculosis , Acinetobacter baumannii , and Plasmodium falciparum , and confirm that Gp46 can also interact with these HUs. Our findings provide detailed insight into a mode of HU inhibition that provides a useful foundation for the development of antibacteria and antimalaria drugs., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Idiopathic pulmonary fibrosis (IPF) is a chronic disease that is characterized by the excessive deposition of scar tissue in the lungs. As currently available treatments are unable to restore lung function in patients, there is an urgent medical need for more effective drugs. Developing such drugs, however, is challenging because IPF has a complex pathogenesis. Emerging evidence indicates that heat shock protein 47 (HSP47), which is encoded by the gene Serpinh1 , may be a suitable therapeutic target as it is required for collagen synthesis. Pharmacological inhibition or knockdown of HSP47 could therefore be a promising approach to treat fibrosis. The objective of this study was to assess the therapeutic potential of Serpinh1 -targeting small interfering RNA (siRNA) in fibrogenic precision-cut lung slices prepared from murine tissue. To enhance fibrogenesis, slices were cultured for up to 144 h with transforming growth factor β1. Self-deliverable siRNA was used to knockdown mRNA and protein expression, without affecting the viability and morphology of slices. After silencing HSP47, only the secretion of fibronectin was reduced while other aspects of fibrogenesis remained unaffected (e.g., myofibroblast differentiation as well as collagen secretion and deposition). These observations are surprising as others have shown that Serpinh1 -targeting siRNA suppressed collagen deposition in animals. Further studies are therefore warranted to elucidate downstream effects on fibrosis upon silencing HSP47., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ruigrok, El Amasi, Leeming, Sand, Frijlink, Hinrichs and Olinga.)

Vocal fold (VF) mucosal fibrosis results in substantial voice impairment and is recalcitrant to current treatments. To reverse this chronic disorder, anti-fibrotic therapies should target the molecular pathology of aberrant collagen accumulation in the extracellular matrix. We investigated the therapeutic potential of siRNA against Serpinh1, a collagen-specific chaperone that enables cotranslational folding and assembly of procollagens in the endoplasmic reticulum. We implemented a previously validated siRNA construct, conducted transfection experiments using in vitro and in vivo rat models, and measured knockdown efficiency, dose responses, delivery strategies, and therapeutic outcomes. Liposome-mediated delivery of Serpinh1-siRNA downregulated collagen production in naive and scar VF fibroblasts as well as naive VF mucosa; moreover, sustained Serpinh1 knockdown in fibrotic VF mucosa reversed scar-associated collagen accumulation within 4 weeks. Analysis of therapeutic effects at the transcriptome level showed evidence of cell cycle upregulation, catabolism, matrix disassembly, and morphogenesis. These findings indicate that Serpinh1-siRNA holds potential as a molecular therapy for chronic VF mucosal fibrosis., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bacteriophage T4 encodes orthologs of the proteins Rad50 (gp46) and Mre11 (gp47), which form a heterotetrameric complex (MR) that is responsible for host genome degradation and the processing of DNA ends for recombination-dependent DNA repair. In this chapter, we describe the ensemble methods currently employed by our laboratory to characterize the exonuclease activity of the T4 MR complex. DNA exonucleases play a vital role in maintaining the integrity of DNA through their participation in DNA repair pathways and as proofreaders for DNA polymerases. Methods for quantifying the general features of the exonuclease, and for determining steady-state kinetic parameters (K m , k cat ), the polarity of exonuclease activity, and processivity are presented. These methods should be applicable to all DNA exonucleases, and to some extent endonucleases., (© 2018 Elsevier Inc. All rights reserved.)