Just Seeing Is Not Enough for Believing: Immunolabelling as Indisputable Proof of SARS-CoV-2 Virions in Infected Tissue

Background: There is increasing evidence that identification of SARS-CoV-2 virions by transmission electron microscopy could be misleading due to the similar morphology of virions and ubiquitous cell structures. This study thus aimed to establish methods for indisputable proof of the presence of SARS-CoV-2 virions in the observed tissue. Methods: We developed a variant of the correlative microscopy approach for SARS-CoV-2 protein identification using immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels. We also performed immunogold labelling of SARS-CoV-2 virions. Results: Immunohistochemistry (IHC) of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of SARS-CoV-2 virions in the analysed human nasopharyngeal tissue. The presence of SARS-CoV-2 virions was also confirmed by immunogold labelling for the first time. Conclusions: Immunoelectron microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections. Both methodological approaches indisputably proved the presence of SARS-CoV-2 virions in cells.

E-cadherin redistribution mediated by Clostridioides difficile toxins increases spore-association to adherens junctions

Nearly ~20% of patients with C. difficile infection (CDI) manifest recurrence of CDI (R-CDI). During CDI, C. difficile forms spores essential for R-CDI. Interactions of C. difficile spores with intestinal epithelial cells (IECs) contribute to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence that C. difficile spores interact with E-cadherin, contributing to spore-adherence and internalization into IECs. C. difficile toxins TcdA/TcdB lead to adherens junctions opening and increase spore-adherence to IECs. Confocal micrographs demonstrate that C. difficile spores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA/TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By ELISA, immunofluorescence, and immunogold labelling, we observed that E-cadherin binds to C. difficile spores, specifically to the hair-like projections of the spore. Overall, these results expand our knowledge of how C. difficile spores bind to IECs and how toxin-mediated damage affects spore interactions with IECs.

Identification of the receptor-binding protein of Clostridium difficile phage CDHS-1 reveals a new class of receptor-binding domains.

Phage-bacterial recognition is species-specific, determined by interactions between phage receptor-binding proteins (RBPs) and corresponding bacterial receptors. RBPs are diverse, and we present data demonstrating the identification and characterisation of a novel C. difficile phage RBP. Putative RBP was identified for CDHS-1, and overexpressed, purified, and polyclonal antibodies were raised and used in phage neutralization assays. Anti-gp22 neutralised CDHS-1, indicating it is the RBP. Immunogold-labelling and transmission electron microscopy confirmed this, enabling visualization of the protein locations. A detailed structural understanding was obtained from determining the three-dimensional structure of gp22 by X-ray crystallography. gp22 is a new RBP class consisting of an N-terminal L-shaped α-helical superhelix domain and a C-terminal Mg2+-binding domain. The protein is a stable homodimer in solution mediated via reciprocal contacts between an α-helical hairpin located within the superhelix domain and additional asymmetrical contacts between the ends of the short arm of each L-shaped protomer. The dimer resembles U-shape with a crossbar formed from the hairpin of each partner. C. difficile binding is Mg2+-dependent. CDHS-1 could not infect a C. difficile S-layer mutant suggesting the bacterial receptors are within the S-layer. These findings provide novel insights into phage biology and extend our knowledge of RBPs.

Role of collagen-like protein BclA3 in the assembly of the exosporium layer of Clostridioides difficile spores.

Newly formed spores are essential for persistence of C. difficile in the host, transmission to a new susceptible host (Deakin et al., 2012b) and recurrence of CDI. BclA3 and BclA2 Spore surface proteins are expressed during sporulation under the control of mother-cell specific sigma factors of the RNA polymerase, SigE and SigK. Deletion of bclA3 leads to spores with an electron-dense exosporium layer that lacks bump-like structures in the electron-dense layer and hair-like projections, both structures typically found in the wild type spore. Therefore, in this work, we have addressed the role of the exosporium collagen-like BclA3 glycoprotein in the assembly of the exosporium layer. Immunogold labelling of BclA2CTD and BclA3CTD indicates that both proteins are located in the hairs, with BclA2 located outermost of BclA3. Absence of BclA3 leads to spores with no hair-like projections, and absence of bumps in thick exosporium spores, a phenotype also expressed in by the deletion of the collagen-like region of BclA3. Overall, these results provide insights into the role of BclA3 in the assembly of the exosporium layer of C. difficile spores.

Immunogold-labelling localization of chlorophyllase at different developmental stages of Pachira macrocarpa leaves

Background: Chlorophyllases (Chlases) are housekeeping proteins in plant cells. The dephytylating enzymes can catalyze chlorophyll (Chl) to form chlorophyllide, but the distribution of Chlases in plant cells is still an interesting debate. In this study, antibody of PmCLH2 was made and used by immunogold-labelling technique to detect the location of Chlase of Pachira macrocarpa (Pm) leaves at four developmental stages, including young, mature, yellowing, and senesced stages. Results: The transmission electron microscopy results show that Chlases were comprehensively found in portions of chloroplast, such as the inner membrane of the envelope, grana, and the thylakoid membrane of the chloroplast, cytosol, and vacuoles at young, mature, and yellowing stages of Pm leaves, but not in the cell wall, plasma membrane, mitochondria, and nucleus. Conclusions: PmChlases were mainly detected in vacuoles at the senescent stage, but a few were found in the chloroplasts. A pathway is proposed to explain the birth and death of Chl, Chlase, and chloroplasts in higher plants.

Regulation of photosynthetic electron flow on dark to light transition by ferredoxin:NADP(H) oxidoreductase interactions

During photosynthesis, electron transport is necessary for carbon assimilation and must be regulated to minimize free radical damage. There is a longstanding controversy over the role of a critical enzyme in this process (ferredoxin:NADP(H) oxidoreductase, or FNR), and in particular its location within chloroplasts. Here we use immunogold labelling to prove that FNR previously assigned as soluble is in fact membrane associated. We combined this technique with a genetic approach in the model plant Arabidopsis to show that the distribution of this enzyme between different membrane regions depends on its interaction with specific tether proteins. We further demonstrate a correlation between the interaction of FNR with different proteins and the activity of alternative photosynthetic electron transport pathways. This supports a role for FNR location in regulating photosynthetic electron flow during the transition from dark to light.

An activity-based labelling method for the detection of ammonia and methane-oxidizing bacteria

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia- and methane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. However, the in situ detection and phylogenetic identification of novel ammonia- and methane-oxidizing bacteria remains a challenge. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and methane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labelling of cells harboring active ammonia and methane monooxygenases. The biotinylation of these enzymes in combination with immunogold labelling reveals the subcellular localization of the tagged proteins, while the fluorescent labelling of cells harboring active ammonia or methane monooxygenases provides a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labelling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and methanotrophs from complex environmental samples, facilitating the retrieval of their high quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and methane-oxidizing bacteria present in complex microbial communities.

Streptozotocin induces alpha-2u globulin nephropathy in male rats during diabetic kidney disease

Background: Alpha-2u globulin nephropathy mainly shows toxicological pathology only in male rats induced by certain chemicals and drugs, such as levamisole (antiparasitic and anticancer drugs). Streptozotocin (STZ) is also an anticancer-antibiotic agent that has been used for decades to induce a diabetic kidney disease model in rodents. The purpose of this study is to determine if STZ causes alpha-2u globulin nephropathy in male rats during an advanced stage of diabetic kidney disease.Methods: To test this hypothesis, the present study used a male diabetic Wistar rat model with 45 mg/kg of STZ injected intraperitoneally. Hyperglycaemic rats were divided into 2 groups: with and without alpha-2u globulin deposition in proximal tubule. Alpha-2u globulin nephropathy was examined by histopathological and electron microscope studies. Water absorption and filtration capacities (via aquaporin [AQP]-1, -2, -4 and -5) and mitochondrial function (through haloacid dehalogenase-like hydrolase domain-containing protein [HDHD]-3 and NADH-ubiquinone oxidoreductase 75 kDa subunit [NDUFS]-1 proteins) were determined using immunohistochemistry, immunofluorescence and immunogold labelling techniques.Results: More than 80% of severe clinical illness rats induced by STZ injection simultaneously exhibited alpha-2u globulin nephropathy with mitochondrial degeneration and filtration apparatus especially pedicels impairment. They also showed significantly upregulated AQP-1, -2, -4 and -5, HDHD-3 and NDUFS-1 compared with those of the rats without alpha-2u globulin nephropathy.Conclusions: STZ-induced alpha-2u globulin nephropathy during diabetic kidney disease in association with deterioration of pedicels, renal tubular damage with adaptation and mitochondrial driven apoptosis.

Identification of putative viroplasms within banana cells infected by banana streak MY virus

The badnavirus replication cycle is poorly understood and most knowledge is based on extrapolations from model viruses such as Cauliflower mosaic virus (CaMV). However, in contrast to CaMV, badnaviruses are thought not to produce viroplasms and therefore it has been a mystery as to where virion assembly occurs. In this study, ultrathin sections of a banana leaf infected with a badnavirus, banana streak MY virus (BSMYV), were examined by transmission electron microscopy. Electron-dense inclusion bodies (EDIBs) were sporadically distributed in parenchymatous tissues of the leaf, most commonly in the palisade and spongy mesophyll cells. These EDIBs had a characteristic structure, comprising an electron-dense core, a single, encircling lacuna and an outer ring of electron-dense material. However, much less frequently, EDIBs with two or three lacunae were observed. In the outer ring, densely packed virions were visible with a shape and size consistent with that expected for badnaviruses. Immunogold labelling was done with primary antibodies that detected the N-terminus of the capsid protein and strong labelling of the outer ring but not the central core or lacuna was observed. It is concluded that the EDIBs that were observed are equivalent in function to the viroplasms of CaMV, although obviously different in composition as there is not a paralogue of the transactivation/viroplasm protein in the badnavirus genome. It is postulated that production of a viroplasm could be a conserved characteristic of all members of the Caulimoviridae.