Spore Coat Proteins Research Articles

Molecular architecture of the assembly of Bacillus spore coat protein GerQ revealed by cryo-EM

Protein filaments are ubiquitous in nature and have diverse biological functions. Cryo-electron microscopy (cryo-EM) enables the determination of atomic structures, even from native samples, and is capable of identifying previously unknown filament species through high-resolution cryo-EM maps. In this study, we determine the structure of an unreported filament species from a cryo-EM dataset collected from Bacillus amyloiquefaciens biofilms. These filaments are composed of GerQ, a spore coat protein known to be involved in Bacillus spore germination. GerQ assembles into a structurally stable architecture consisting of rings containing nine subunits, which stacks to form filaments. Molecular dockings and model predictions suggest that this nine-subunit structure is suitable for binding CwlJ, a protein recruited by GerQ and essential for Ca2+-DPA induced spore germination. While the assembly state of GerQ within the spores and the direct interaction between GerQ and CwlJ have yet to be validated through further experiments, our findings provide valuable insights into the self-assembly of GerQ and enhance our understanding of its role in spore germination.

Laccase surface-display for environmental tetracycline removal: From structure to function

Facing the increasingly prominent tetracycline pollution and the resulting environmental problems, how to find environmental and efficient treatment means is one of the current research hotspots. In this study, the laccase surface-display technology for tetracycline treatment was investigated. Via study, the type of anchoring protein had a minor influence on the laccase ability, while the type of laccase showed a major impact. Bacillus subtilis spore coat protein (CotA) exhibited higher laccase activity, stability, and efficiency in degrading tetracycline than Pleurotus ostreatus laccase 6 (Lacc6). The superiority of bacterial laccase over fungal laccase was elucidated from the perspective of crystal structure. Besides, a variety of technical means were used to verify the success of surface-display. pGSA-CotA surface-displayed bacteria exhibited good tolerance to high temperature, pH, and various heavy metals. Importantly, surface-displayed bacteria showed faster degradation efficiency and better treatment effects than the intracellular expression bacteria in tetracycline degradation. This implies that surface display technology has greater potential for laccase-mediated environmental remediation. Due to the adverse impacts of tetracycline on soil enzyme activity and microorganisms, our study found that pGSA-CotA surface-displayed bacteria can alleviate tetracycline stress in soil and partially activate the soil, thereby increasing soil enzyme activity and certain nitrogen cycling genes.

SpoIVA is an essential morphogenetic protein for the formation of heat- and lysozyme-resistant spores in Clostridium sporogenes NBRC 14293.

Clostridium sporogenes is an anaerobic spore-forming bacterium genetically related to Clostridium botulinum but lacks toxin genes. The sporulation mechanism and spore structures of anaerobic bacteria, including C. sporogenes, have not been comprehensively analyzed. Based on 16S rRNA gene analysis, it has been determined that C. sporogenes NBRC 14293 belongs to C. botulinum Group I. Moreover, SpoIVA is highly conserved in Bacillus and Clostridium species. Therefore, the aim of the present study is to investigate the mechanism of spore formation in C. sporogenes by performing a functional analysis of spoIVA encoding SpoIVA, a protein involved in the early development of the spore coat and cortex in Bacillus subtilis. Inactivation of spoIVA in C. sporogenes resulted in the loss of resistance of sporulating cells to lysozyme and heat treatments. Phase-contrast microscopy indicated that the inactivation of spoIVA caused the development of abnormal forespores and production of only a few immature spores. In the spoIVA mutant, abnormal swirl structures were detected in the mother cell using both phase-contrast and transmission electron microscopy. These swirls were stained with auramine O, pararosaniline hydrochloride, and 2-(4-aminophenyl)benzothiazole to examine the surface of mature spores of the wild-type strain. We found that the spore coat and exosporium proteins were misassembled and that they accumulated in the mother cells of the mutant. The results of this study indicate that SpoIVA is a spore morphogenetic protein, providing novel insights into spore morphogenesis in C. sporogenes.

Integrating In-silico and In-vitro approaches to identify plant-derived bioactive molecules against spore coat protein CotH3 and high affinity iron permease FTR1 of Rhizopus oryzae

Rhizopus oryzae is one of the major causative agents of mucormycosis. The disease has a poor prognosis with a high mortality rate, and resistance towards current antifungal drugs poses additional concern. The disease treatment is complicated with antifungals; therefore, surgical approach is preferred in many cases. A comprehensive understanding of the pathogenicity-associated virulence factors of R. oryzae is essential to develop new antifungals against this fungus. Virulence factors in R. oryzae include cell wall proteins, spore germination proteins and enzymes that evade host immunity. The spore coat protein (CotH3) and high-affinity iron permease (FTR1) have been identified as promising therapeutic targets in R. oryzae. In-silico screening is a preferred approach to identify hit molecules for further in-vitro studies. In the present study, twelve bioactive molecules were docked within the active site of CotH3 and FTR1. Further, molecular dynamics simulation analysis of best-docked protein-ligand structures revealed the dynamics information of their stability in the biological system. Eugenol and isoeugenol exhibited significant binding scores with both the protein targets of R. oryzae and followed the Lipinski rule of drug-likeness. To corroborate the in-silico results, in-vitro studies were conducted using bioactive compounds eugenol, isoeugenol, and myristicin against R. oryzae isolated from the soil sample. Eugenol, isoeugenol exhibited antifungal activity at 156 µg/mL whereas myristicin at 312 µg/mL. Hence, the study suggested that eugenol and isoeugenol could be explored further as potential antifungal molecules against R. oryzae.

Detoxification of aflatoxin B1 by a Bacillus subtilis spore coat protein through formation of the main metabolites AFQ1 and epi-AFQ1.

A variety of important agricultural crops host fungi from the Aspergillus genus can produce cancerogenic secondary metabolites such as aflatoxins. Consequently, novel strategies for detoxification and their removal from food and feed chains are required. Here, detoxification of Aflatoxin B1 (AFB1) by the Bacillus subtilis multi-copper oxidase CotA (BsCotA) was investigated. This laccase was recombinantly produced in E. coli while codon optimization led to duplication of the amount of active protein obtained. CuCl2 was added to the cultivation medium leading to a 25-fold increase of V max corresponding to improved incorporation of Cu2+ into the enzyme protein which is essential for the catalytic reaction. To avoid potential cytotoxicity of Cu2+, cultivation was performed at microaerobic conditions indeed leading to 100x more functional protein when compared to standard aerobic conditions. This was indicated by an increase of V max from 0.30 ± 0.02 to 33.56 ± 2.02 U/mg. Degradation kinetics of AFB1 using HPLC with fluorescence detection (HPLC-FLD) analysis indicated a theoretical substrate saturation above solubility in water. At a relatively high concentration of 500 μg/L, AFB1 was decomposed at 10.75 μg/Lh (0.17 nmol*min-1*mg-1) at a dosage of 0.2 μM BsCotA. AFQ1 and epi-AFQ1 were identified as the initial oxidation products according to mass spectrometry (i.e., HPLC-MS, HPLC-QTOF). None of these molecules were substrates for laccase but both decomposed in buffer. However, decomposition does not seem to be due to hydration of the vinyl ether in the terminal furan ring. Genotoxicity of the formed AFB1 was assessed in several dilutions based on the de-repression of the bacterial SOS response to DNA damage indicating about 80-times reduction in toxicity when compared to AFQ1. The results of this study indicate that BsCotA has high potential for the biological detoxification of aflatoxin B1.

An intravenous pancreatic cancer therapeutic: Characterization of CRISPR/Cas9n-modified Clostridium novyi-Non Toxic.

Clostridium novyi has demonstrated selective efficacy against solid tumors largely due to the microenvironment contained within dense tumor cores. The core of a solid tumor is typically hypoxic, acidic, and necrotic-impeding the penetration of current therapeutics. C. novyi is attracted to the tumor microenvironment and once there, can both lyse and proliferate while simultaneously re-activating the suppressed immune system. C. novyi systemic toxicity is easily mitigated by knocking out the phage DNA plasmid encoded alpha toxin resulting in C. novyi-NT; but, after intravenous injection spores are quickly cleared by phagocytosis before accomplishing significant tumor localization. C. novyi-NT could be designed to accomplish intravenous delivery with the potential to target all solid tumors and their metastases in a single dose. This study characterizes CRISPR/Cas9 modified C. novyi-NT to insert the gene for RGD, a tumor targeting peptide, expressed within the promoter region of a spore coat protein. Expression of the RGD peptide on the outer spore coat of C. novyi-NT indicates an increased capacity for tumor localization of C. novyi upon intravenous introduction based on the natural binding of RGD with the αvβ3 integrin commonly overexpressed on the epithelial tissue surrounding a tumor, and lead to immune stimulation.

Environmentally Responsible Bioengineering for Spore Surface Expression of Helicobacter pylori Antigen

The development of genetic technologies and bioengineering are creating an increasing number of genetically engineered microorganisms with new traits for diverse industrial applications such as vaccines, drugs and pollutant degraders. However, the destiny of genetically engineered bacterial spores released into the environment as long-life organisms has remained a big environmental challenge. In this study, an environmentally responsible and sustainable gene technology solution based on the concept of thymine starvation is successfully applied for cloning and expression of a Helicobacter pylori antigen on Bacillus subtilis spore surface. As an example, a recombinant Bacillus subtilis strain A1.13 has been created from a gene fusion of the corresponding N-terminal fragment of spore coat protein CotB in B. subtilis and the entire urease subunit A (UreA) in H. pylori and the fusion showed a high stability of spore surface expression. The outcomes can open the door for developing highly safe spore vectored vaccines against this kind of pathogen and contributing to reduced potential risks of genetically engineered microorganisms released in the environment.

Creating a Vaccine-like Supplement against Respiratory Infection Using Recombinant Bacillus subtilis Spores Expressing SARS-CoV-2 Spike Protein with Natural Products.

Vaccination is the most effective method of combating COVID-19 infection, but people with a psychological fear of needles and side effects are hesitant to receive the current vaccination, and alternative delivery methods may help. Bacillus subtilis, a harmless intestinal commensal, has recently earned a strong reputation as a vaccine production host and delivery vector, with advantages such as low cost, safety for human consumption, and straightforward oral administration. In this study, we have succeeded generating "S spores" by engineering B. subtilis with spore coat proteins resembling the spike (S) protein of the ancestral SARS-CoV-2 coronavirus. With the addition of two immunostimulating natural products as adjuvants, namely Astragalus membranaceus (Fisch.) Bge (AM) and Coriolus versicolor (CV), oral administration of S spores could elicit mild immune responses against COVID-19 infection without toxicity. Mucosal IgA against the S protein was enhanced by co-feeding with AM and CV in an S spores-inoculated mouse model. Faster and stronger IgG responses against the S protein were observed when the mice were fed with S spores prior to vaccination with the commercial COVID-19 vaccine CoronaVac. In vitro studies demonstrated that AM, CV, and B. subtilis spores could dose-dependently activate both macrophages and dendritic cells by secreting innate immunity-related IL-1β, IL-6, and TNF-α, and some other proinflammatory chemokines and cytokines. In conclusion, the combination of S spores with AM and CV may be helpful in developing a vaccine-like supplement against respiratory infection.

Isolation, Genomic, and Proteomic Characterization of a Novel Neotropical Strain of Bacillus thuringiensis with Mosquitocidal Activities

The combination of genomic and proteomic analyses is a useful tool for the study of novel Bacillus thuringiensis (Bt) strains, as these approaches allow the accurate identification of pesticidal proteins and virulence factors produced. Here, we isolated and evaluated the potential of a novel Neotropical Bt strain (TOD651) for controlling larvae of Aedes aegypti and Culex quinquefasciatus mosquitoes. Aiming for the full comprehension of the TOD651 larvicidal potential, we further evaluated the whole TOD651 genome and conducted the proteomic analysis of the TOD651 spore–crystal mixtures. Our results showed that Bt TOD651 similarly killed both A. aegypti (0.011 µg/mL) and C. quinquefasciatus (0.023 µg/mL) larvae, exhibiting similar potency to the commercial Bt strain. The genome sequence revealed that Bt TOD651 harbors cry11Aa3, cry10Aa4, cry4Aa4, cry4Ba5, cyt1Aa5, cyt1Ca1, cyt2Ba13, mpp60Aa3, and mpp60Ba3. The proteomic analysis revealed no expression of Mpp60Aa3, while all the other pesticidal proteins were expressed (Cry4Ba5 was more abundant than Cyt1Aa5). The expression of the Mppe showed the major proportions between proteases. The virulent factor neutral protease B and spore coat proteins were also expressed. The expression of relevant pesticidal proteins (e.g., Cry, Cyt, Mpp, and other pathogenic factors), whose actions can occur in a synergic relation, indicates that the biocontrol using Bt TOD651 may contribute to delaying the selection of resistant individuals.

Protein S-palmitoylation regulates different stages of meiosis in Schizosaccharomyces pombe.

Posttranslational protein S-palmitoylation regulates the localization and function of its target proteins involved in diverse cellular processes including meiosis. In this study, we demonstrate that S-palmitoylation mediated by Erf2-Erf4 and Akr1 palmitoylacyltransferases is required at multiple meiotic stages in the fission yeast Schizosaccharomyces pombe We find that S-palmitoylation by Erf2-Erf4 is required for Ras1 localization at the cell periphery to enrich at the cell conjugation site for mating pheromone response. In the absence of Erf2 or Erf4, mutant cells are sterile. A role of Akr1 S-palmitoylating the nuclear fusion protein Tht1 to function in karyogamy is identified. We demonstrate that S-palmitoylation stabilizes and localizes Tht1 to ER, interacting with Sey1 ER fusion GTPase for proper meiotic nuclear fusion. In akr1, tht1, or sey1 mutant, meiotic cells, haploid nuclei are unfused with subsequent chromosome segregation defects. Erf2-Erf4 has an additional substrate of the spore coat protein Isp3. In the absence of Erf2, Isp3 is mislocalized from the spore coat. Together, these results highlight the versatility of the cellular processes in which protein S-palmitoylation participates.

Bacillus subtilis spores displaying RBD domain of SARS-CoV-2 spike protein

Bacillus subtilis spores are considered to be efficient and useful vehicles for the surface display and delivery of heterologous proteins. In this study, we prepared recombinant spores with the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein displayed on their surface in fusion with the CotZ or CotY spore coat proteins as a possible tool for the development of an oral vaccine against the SARS-CoV-2 virus. The RBD was attached to the N-terminus or C-terminus of the coat proteins. We also directly adsorbed non-recombinantly produced RBD to the spore surface. SDS-PAGE, western blot and fluorescence microscopy were used to analyze RBD surface expression on purified spores. Results obtained from both display systems, recombinant and non-recombinant, demonstrated that RBD was present on the spore surfaces.

Transcriptional responses of human intestinal epithelial HT-29 cells to spore-displayed p40 derived from Lacticaseibacillus rhamnosus GG

BackgroundsThe aims of this study were to construct spore-displayed p40, a Lacticaseibacillus rhamnosus GG-derived soluble protein, using spore surface display technology and to evaluate transcriptional responses in human intestinal epithelial cells.Resultsp40 was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchor protein. Effects of spore-displayed p40 (CotG-p40) on gene expression of intestinal epithelial cell line HT-29 were evaluated by transcriptome analysis using RNA-sequencing. As a result of differentially expressed gene (DEG) analysis, 81 genes were up-regulated and 82 genes were down-regulated in CotG-p40 stimulated cells than in unstimulated cells. Gene ontology enrichment analysis showed that CotG-p40 affected biological processes such as developmental process, metabolic process, cell surface receptor linked signaling pathway, and retinoic acid metabolic process. Gene-gene network analysis suggested that 10 DEGs (EREG, FOXF1, GLI2, PTGS2, SPP1, MMP19, TNFRSF1B, PTGER4, CLDN18, and ALDH1A3) activated by CotG-p40 were associated with probiotic action.ConclusionsThis study demonstrates the regulatory effects of CotG-p40 on proliferation and homeostasis of HT-29 cells. This study provided comprehensive insights into the transcriptional response of human intestinal epithelial cells stimulated by CotG-p40.

Pathogenesis and Pathology of COVID-Associated Mucormycosis: What Is New and Why.

Purpose of ReviewThere is global increase in the incidence of mucormycosis. However, a sudden increase in the COVID-associated mucormycosis (CAM) was noted, particularly in India, during the second wave of the COVID-19 pandemic. The interplay of factors involved in the pathogenesis is complex. In this review, the influence of pre-existing disease, exaggerated risk factors, altered milieu due to COVID-19 itself and the consequences of its treatment on the host pathogen interactions leading to the disease and morphology of the fungus will be highlighted.Recent FindingsHyperglycemia, acidosis, available free iron, lowered host defenses, and the fungal virulence factors promote the growth of Mucorales. There is a high background prevalence of diabetes mellitus (DM) in India. Uncontrolled or undiagnosed DM, COVID-19 itself, and inappropriate administration of corticosteroids in high doses and for prolonged periods result in hyperglycemia. Diabetic ketoacidosis (DKA) and metabolic acidosis due to hypoxia or renal failure contribute to acidic pH and dissociate bound iron from serum proteins. The host defenses are lowered due to COVID-19-induced immune dysregulation, hyperglycemia itself, and administration of corticosteroids and immune suppressants for the treatment of COVID-19. The altered metabolic milieu in the local microenvironment of nose and paranasal sinuses (PNS) promotes specific interaction of glucose-regulated protein-78 (GRP-78) on host cells with spore coat protein homologue (CotH 3) on Mucorales resulting in rhino-orbito-cerebral mucormycosis (ROCM) as the predominant clinical form in CAM. The pathology is extensive soft tissue involvement with angioinvasion and perineural invasion. Melanized hyphae and sporangia were seen on histopathology, which is unique to CAM. While many factors favor the growth of Mucorales in CAM, hyperglycemia, hyperferritinemia, and administration of hyperbaric oxygen result in reactive oxygen species (ROS) and inadequate humidification results in dehydration. Melanization is possibly the adaptive and protective mechanism of Mucorales to escape the unfavorable conditions due to the treatment of COVID-19.SummaryHigh background prevalence of DM, inappropriate administration of corticosteroids and immune dysregulation due to COVID-19 favor the growth of Mucorales in CAM. Melanization of Mucorales hyphae and sporangia on histopathology probably represent adaptive and protective mechanism due to the treatment with hyperbaric oxygen with inadequate humidification as well as the metabolic alterations.

Role of the Spore Coat Proteins CotA and CotB, and the Spore Surface Protein CDIF630_02480, on the Surface Distribution of Exosporium Proteins in Clostridioides difficile 630 Spores.

Clostridioides difficile is Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea. During disease, C. difficile forms metabolically dormant spores that persist in the host and contribute to recurrence of the disease. The outermost surface of C. difficile spores, termed the exosporium, plays an essential role in interactions with host surfaces and the immune system. The main exosporium proteins identified to date include three orthologues of the BclA family of collagen-like proteins, and three cysteine-rich proteins. However, how the underlying spore coat influences exosporium assembly remains unclear. In this work, we explore the contribution of spore coat proteins cotA and cotB, and the spore surface protein, CDIF630_02480, to the exosporium ultrastructure, formation of the polar appendage and the surface accessibility of exosporium proteins. Transmission electron micrographs of spores of insertional inactivation mutants demonstrate that while cotB contributes to the formation of thick-exosporium spores, cotA and CDIF630_02480 contribute to maintain proper thickness of the spore coat and exosporium layers, respectively. The effect of the absence of cotA, cotB and CDIF630_02480 on the surface accessibility of the exosporium proteins CdeA, CdeC, CdeM, BclA2 and BclA3 to antibodies was affected by the presence of the spore appendage, suggesting that different mechanisms of assembly of the exosporium layer might be implicated in each spore phenotype. Collectively, this work contributes to our understanding of the associations between spore coat and exosporium proteins, and how these associations affect the assembly of the spore outer layers. These results have implications for the development of anti-infecting agents targeting C. difficile spores.

P343 Novel hydrophobic binding surface proteins are instrumental for phagocytosis of Lichtheimia corymbifera by macrophages

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesPerform proteomic analysis of the spore surface alongside secretome analysis of two strains of Lichtheimia corymbifera.Evaluate interaction, recognition, and phagocytosis of Lichtheimia spores by murine alveolar macrophages.MethodsTwo strains of L. corymbifera (JMRC: FSU: 09682 and JMRC: FSU: 10164) were used in this study. For phagocytosis assay, the spores were labeled with 0.1 mg/ml Fluorescein isothiocyanate (FITC) (Sigma Aldrich Chemie) in 0.1 M Na2CO3 for 30 min 146 at 30°C. Murine alveolar macrophages MH-S (ATCC:CRL-2019) were cultivated in RPMI-1640 (Sigma, 30-2001) supplemented.Identification of the surface proteins of L. corymbifera was carried out as described before with minor modification.1 The supernatants were stored at −80°C for liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Secreted proteins of L. corymbifera were determined as described in a previous study with minor modification2.The raw files generated by the LC-MS/MS were further processed by the software Proteome Discoverer v1.4.0.288 (Thermo). Tandem mass spectra were searched against the NCBI L. corymbifera protein database.3 Approximately 10 000 MH-S cells were cultivated onto 96-well microplates (NUNC 163320) in 100 μL RPMI-1640 medium (Sigma, 30-2001). Images were acquired by the Zeiss Axio Observer 7 Spinning Disk Confocal Microscope (ZEISS, Jena, Germany) and processed with ZEN 2.1 Software (ZEISS) by 63x objective lens.ResultsAbundant surface proteins were found which serve as Candidates for secretome analysis. A total of 113 proteins were identified. Thirty proteins were confirmed to be on the spore surface based on the presence of signal peptides3. The following proteins were predominantly identified: Spore coat protein (CotH), hydrophobic surface binding protein A (HsbA), aconitase, ricin-like lectin, two transition elongation factors, multi-copper oxidase, heat shock protein 70 family (Hsp70), malate synthase, putative allergen Candidates, etc.These proteins were heterologsly overexpressed in yeast. Successful overexpression was confirmed by LC-MS/MS. The yeast mutants were subjected to phagocytosis assays (Fig. 1). The role of surface proteins in macrophages is discussed.ConclusionThe surface proteins are instrumental for recognition of L. corymbifera by macrophages and for intracellular survival in macrophages. The role of surface proteins is discussed in the light of evolution from environmental to human pathogenic fungus.Sources:Voltersen V, Blango MG, Herrmann S, et al. Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic Fungus Aspergillus fumigatus. mBio. 2018;9(5). doi:10.1128/MBIO.01557-18Thürich J, Meichsner D, Furch ACU, et al. Arabidopsis thaliana responds to colonisation of Piriformospora indica by secretion of symbiosis-specific proteins. PLOS ONE. 2018;13(12):e0209658. doi:10.1371/JOURNAL. PONE.0209658.Schwartze VU, Winter S, Shelest E, et al. Gene Expansion Shapes Genome Architecture in the Human Pathogen Lichtheimia corymbifera: An Evolutionary Genomics Analysis in the Ancient Terrestrial Mucorales (Mucoromycotina). PLOS Genetics. 2014;10(8):e1004496. doi:10.1371/JOURNAL.PGEN.1004496.

Key molecules of Mucorales for COVID-19-associated mucormycosis: a narrative review

Mucormycosis is a lethal human disease caused by fungi of the order Mucorales. Mucormycosis is caused by fungi mainly belonging to the genera Mucor, Rhizopus, and Lichtheimia, all of which belong to the order Mucorales. The number of individuals with mucormycosis-causing disorders has increased in recent years, hence, leading to the spread of mucormycosis. Throughout the coronavirus disease 2019 (COVID-19) pandemic, numerous cases of mucormycosis in COVID-19-infected patients have been reported worldwide, and the illness is now recognized as COVID-19-associated mucormycosis, with most of the cases being reported from India. Immunocompromised patients such as those with bone marrow sickness and uncontrolled diabetes are at a greater risk of developing mucormycosis. Genes, pathways, and other mechanisms have been studied in Mucorales, demonstrating a direct link between virulence and prospective therapeutic and diagnostic targets. This review discusses several proteins such as high-affinity iron permease (FTR1), calcineurin, spore coat protein (CotH), and ADP-ribosylation factors involved in the pathogenesis of mucormycosis that might prove to be viable target(s) for the development of novel diagnostic and therapeutic methods.

Surface Display of Peptides Corresponding to the Heptad Repeat 2 Domain of the Feline Enteric Coronavirus Spike Protein on Bacillus subtilis Spores Elicits Protective Immune Responses Against Homologous Infection in a Feline Aminopeptidase-N-Transduced Mouse Model.

Although feline coronavirus (FCoV) infection is extremely common in cats, there are currently few effective treatments. A peptide derived from the heptad repeat 2 (HR2) domain of the coronavirus (CoV) spike protein has shown effective for inhibition of various human and animal CoVs in vitro, but further use of FCoV-HR2 in vivo has been limited by lack of practical delivery vectors and small animal infection model. To overcome these technical challenges, we first constructed a recombinant Bacillus subtilis (rBSCotB-HR2P) expressing spore coat protein B (CotB) fused to an HR2-derived peptide (HR2P) from a serotype II feline enteric CoV (FECV). Immunogenic capacity was evaluated in mice after intragastric or intranasal administration, showing that recombinant spores could trigger strong specific cellular and humoral immune responses. Furthermore, we developed a novel mouse model for FECV infection by transduction with its primary receptor (feline aminopeptidase N) using an E1/E3-deleted adenovirus type 5 vector. This model can be used to study the antiviral immune response and evaluate vaccines or drugs, and is an applicable choice to replace cats for the study of FECV. Oral administration of rBSCotB-HR2P in this mouse model effectively protected against FECV challenge and significantly reduced pathology in the digestive tract. Owing to its safety, low cost, and probiotic features, rBSCotB-HR2P is a promising oral vaccine candidate for use against FECV/FCoV infection in cats.

Pathogenesis of Mucormycosis: The Spores on a Sail

Fungi of the order Mucorales are capable to infect human beings and animals. Mucormycosis is a well-known, life-threatening infection, primarily occurring in immune-compromised conditions like diabetic ketoacidosis, organ transplantation, increased serum iron and neutropenia. The mortality and morbidity rate due to Mucormycosis is rapidly rising despite of surgical debridement and antifungal aids. In this article, we will review (i) the existing knowledge about fungi (ii) interaction of Mucorales with host immune system (iii) assessment of virulence factors of Mucorales which play a key role in infection like high-affinity iron permease FTR1, spore coat protein CotH, reductase permease system. Since Mucormycosis infection is angioinvasive in nature, emphasis is placed on the ways by which organisms acquire iron from the host and its interaction with blood vessels. The present mini-review attempts to spread awareness regarding difficult to treat infection of mucormycosis and to provide possible targets for future research for therapeutic measures.

Insights into the Structure and Protein Composition of Moorella thermoacetica Spores Formed at Different Temperatures.

The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a lower temperature than the optimal temperature. Here, we used electron microcopy to characterize the ultrastructure of the coat of the spores formed at different sporulation temperatures; we found that spores produced at 55 °C mainly exhibited a lamellar inner coat tightly associated with a diffuse outer coat, while spores produced at 45 °C showed an inner and an outer coat separated by a less electron-dense zone. Moreover, misarranged coat structures were more frequently observed when spores were produced at the lower temperature. We then analyzed the proteome of the spores obtained at either 45 °C or 55 °C with respect to proteins putatively involved in the spore coat, exosporium, or in spore resistance. Some putative spore coat proteins, such as CotSA, were only identified in spores produced at 55 °C; other putative exosporium and coat proteins were significantly less abundant in spores produced at 45 °C. Altogether, our results suggest that sporulation temperature affects the structure and protein composition of M. thermoacetica spores.

Looking into mucormycosis coinfections in COVID-19 patients using computational analysis

<abstract> <p>Mucormycosis infection may develop after using steroids treatment to improve the severely of the symptoms in coronavirus patients. The rising in the infection rate of mucormycosis has been noticed in patients after COVID-19 infection. To understand the high morbidity mucormycosis coinfection, the cell surface Glucose Regulated Protein 78 (CS-GRP78) was docked to the virus ACE2-SARS-CoV-2 RBD to create the ACE2-SARS-CoV-2 RBD-GRP78 complex which facilitates the virus entrance into the cell. The spore coat protein homolog 3 (CotH3) of mucormycosis was modeled and docked to the ACE2-SARS-CoV-2 RBD-GRP78 complex. The binding energies of CotH3 with RBD, ACE2, and GRP78 were calculated. The binding results show that GRP78 substrate-binding domain β weakly binds to the spike RBD combined with ACE2 of the spike RBD-ACE2 complex. Its main function is to stabilize the binding between RBD and ACE2, while CotH3 has a strong affinity for the SARS-CoV-2 RBD, but not for ACE2 or GRP78. The CotH3 appeared to have the same affinity to RBD in the SARS-CoV-2 lineages with some preference to the lineage B.1.617.2 (Delta variant). The complex design illustrates that the coat protein of the fungi is more likely linked to the spike protein of the SARS-CoV-2 virus, which would explain the increased mortality mucormycosis coinfections in COVID-19 patients.</p> </abstract>