Rapid oxidative release of fungal mannan for detection by immunoassay.

Detection of fungal cells in infected tissue by procedures such as potassium hydroxide (KOH) microscopy and histopathology are well-established methods in medical mycology. However, microscopy requires skilled personnel, specialized equipment, and may take considerable time to a result. An alternative approach is immunoassay for detection of fungal mannans in tissue as a biomarker for the presence of fungal cells. However, mannan is a component of the fungal cell wall, and detection of mannan would require a facile means for mannan extraction prior to detection by immunoassay. In this study, we evaluated a broad spectrum of extraction reagents using Trichophyton rubrum mycelia and Saccharomyces cerevisiae Mnn2 blastoconidia as model fungi. Oxidative release by treatment with dilute bleach proved to be a novel and highly effective procedure. Complete extraction occurred in as little as 2-4 min. Detergents, chaotropes, and acid were ineffective. Strong base released mannan but was less efficient than oxidative release and required the use of highly corrosive reagents. Oxidative release of cell wall mannans from fungal mycelia and blastoconidia may be an effective first step in immunodetection of fungi in tissues from infected humans, animals, or plants that could be done at or near the diagnostic point of need.

Mannans are components of the fungal cell wall that play a role in disease production and are potential biomarkers for the diagnosis of infection. Oxidative release of mannans from intact cell walls is a novel method for mannan extraction that is rapid, uses relatively mild reagents, and yields soluble mannans that are readily detected by immunoassay.

Mitochondrial O-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells.

O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics.

Point-of-Care Testing for Infectious Diseases: Past, Present, and Future.

Point-of-care (POC) diagnostics provide rapid actionable information for patient care at the time and site of an encounter with the health care system. The usual platform has been the lateral flow immunoassay. Recently, emerging molecular diagnostics have met requirements for speed, low cost, and ease of use for POC applications. A major driver for POC development is the ability to diagnose infectious diseases at sites with a limited infrastructure. The potential use in both wealthy and resource-limited settings has fueled an intense effort to build on existing technologies and to generate new technologies for the diagnosis of a broad spectrum of infectious diseases.

Conserved ion and amino acid transporters identified as phosphorylcholine-modified N-glycoproteins by metabolic labeling with propargylcholine in Caenorhabditis elegans cells.

Phosphorylcholine (PC) modification of proteins by pathogens has been implicated in mediating host-pathogen interactions. Parasitic nematodes synthesize PC-modified biomolecules that can modulate the host's antibody and cytokine production to favor nematode survival, contributing to long-term infections. Only two nematode PC-modified proteins (PC-proteins) have been unequivocally identified, yet discovering the protein targets of PC modification will be paramount to understanding the role(s) that this epitope plays in nematode biology. A major hurdle in the field has been the lack of techniques for selective purification of PC-proteins. The nonparasitic nematode Caenorhabditis elegans expresses PC-modified N-linked glycans, offering an attractive model to study the biology of PC-modification. We developed a robust method to identify PC-proteins by metabolic labeling of primary embryonic C. elegans cells with propargylcholine, an alkyne-modified choline analog. Cu(I)-catalyzed cycloaddition with biotin-azide enables streptavidin purification and subsequent high-throughput LC-MS identification of propargyl-labeled proteins. All proteins identified using stringent criteria are known or predicted to be membrane or secreted proteins, consistent with the model of a Golgi-resident, putative PC-transferase. Of the 55 PC-N-glycosylation sites reported, 33 have been previously observed as N-glycosylation sites in high-throughput screens of C. elegans. Several identified PC-proteins are nematode-specific proteins, but 10 of the PC-proteins are widely conserved ion transporters and amino acid transporters, while eight are conserved proteins involved in synaptic function. This finding suggests a functional role for PC-modification beyond immunomodulation. The approach presented in this study provides a method to identify PC-proteins in C. elegans and related nematodes.

Size-matched alkyne-conjugated cyanine fluorophores to identify differences in protein glycosylation.

Currently, there are few methods to detect differences in posttranslational modifications (PTMs) in a specific manner from complex mixtures. Thus, we developed an approach that combines the sensitivity and specificity of click chemistry with the resolution capabilities of 2D-DIGE. In "Click-DIGE", posttranslationally modified proteins are metabolically labeled with azido-substrate analogs, then size- and charge-matched alkyne-Cy3 or alkyne-Cy5 dyes are covalently attached to the azide of the PTM by click chemistry. The fluorescently-tagged protein samples are then multiplexed for 2DE analysis. Whereas standard DIGE labels all proteins, Click-DIGE focuses the analysis of protein differences to a targeted subset of posttranslationally modified proteins within a complex sample (i.e. specific labeling and analysis of azido glycoproteins within a cell lysate). Our data indicate that (i) Click-DIGE specifically labels azido proteins, (ii) the resulting Cy-protein conjugates are spectrally distinct, and (iii) the conjugates are size- and charge-matched at the level of 2DE. We demonstrate the utility of this approach by detecting multiple differentially expressed glycoproteins between a mutant cell line defective in UDP-galactose transport and the parental cell line. We anticipate that the diversity of azido substrates already available will enable Click-DIGE to be compatible with analysis of a wide range of PTMs.

Antigenic Relatedness between Mannans from Coccidioides immitis and Coccidioides posadasii Spherules and Mycelia.

Immunoassays for cell wall mannans that are excreted into serum and urine have been used as an aid in the diagnosis of many disseminated fungal infections, including coccidioidomycosis. Antigen-detection immunoassays are critically dependent on the detection of an analyte, such as mannan, by antibodies that are specific to the analyte. The goal of this study was to evaluate the extent of cross-reactivity of polyclonal antibodies raised against Coccidioides spp. Analysis of antigenic relatedness between mannans from C. posadasii and C. immitis spherules and mycelia showed complete relatedness when evaluated by the method of Archetti and Horsfall, which was originally used to study the antigenic relationships between Influenzae virus isolates. In a further effort to validate the suitability of the antigenic relatedness calculation methodology for polysaccharide antigens, we also applied the method of Archetti and Horsfall to published results that had previously identified the major capsular serotypes of Cryptococcus species. The results of this analysis showed that Archetti and Horsfall's antigenic relatedness calculation correctly identified the major cryptococcal serotypes. Together, these results suggest that the method is applicable to polysaccharide antigens, and that immunoassays that detect Coccidioides mannans are likely to have good reactivity across Coccidioides species (inclusivity) due to the species' high level of antigenic relatedness.

Comparison of a Prototype SARS-CoV-2 Lateral Flow IMMUNOASSAY with the BinaxNOWTM COVID-19 Antigen CARD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the COVID-19 pandemic. From the onset of the pandemic, rapid antigen tests have quickly proved themselves to be an accurate and accessible diagnostic platform. The initial (and still most commonly used antigen tests) for COVID-19 diagnosis were constructed using monoclonal antibodies (mAbs) specific to severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP). These mAbs are able to bind SARS-CoV-2 NP due to high homology between the two viruses. However, since first being identified in 2019, SARS-CoV-2 has continuously mutated, and a multitude of variants have appeared. These mutations have an elevated risk of leading to possible diagnostic escape when using tests produced with SARS-CoV-derived mAbs. Here, we established a library of 18 mAbs specific to SARS-CoV-2 NP and used two of these mAbs (1CV7 and 1CV14) to generate a prototype antigen-detection lateral flow immunoassay (LFI). A side-by-side analysis of the 1CV7/1CV14 LFI and the commercially available BinaxNOWTM COVID-19 Antigen CARD was performed. Results indicated the 1CV7/1CV14 LFI outperformed the BinaxNOWTM test in the detection of BA.2, BA.2.12.1, and BA.5 Omicron sub-variants when testing remnant RT-PCR positive patient nasopharyngeal swabs diluted in viral transport media.

Tracheal colonization factor A (TcfA) is a biomarker for rapid and specific detection of Bordetella pertussis.

Pertussis is a highly contagious disease for which prompt, point-of-care diagnosis remains an unmet clinical need. Results from conventional test modalities (nucleic acid detection, serology, and culture) take hours to days. To overcome this challenge, we identified a new biomarker (tracheal colonization factor A, TcfA) for detection of Bordetella pertussis infection by lateral flow immunoassay (LFIA). We developed a library of 28 epitope-mapped monoclonal antibodies against TcfA and incorporated three antibodies into a LFIA. The LFIA did not cross-react with common bacterial or fungal organisms, but did react with nine distinct B. pertussis strains. The minimal linear epitope sequences targeted by the LFIA were conserved in 98% of 954 B. pertussis isolates collected across 12 countries from 1949-2017. The LFIA's limit of detection was 3.0 × 105 CFU/mL with B. pertussis cells in buffer, 6.2 × 105 CFU/mL with nasopharyngeal washes from a non-human primate model, and 2.3 ng/mL with recombinant TcfA. The LFIA reacted with patient nasopharyngeal swab specimens containing as few as 1.8 × 106 B. pertussis genomes/mL and showed no false-positives. Rapid (< 20 min) LFIA detection of TcfA as a biomarker for B. pertussis infection is feasible and may facilitate early detection of pertussis.

Conservation of Mannan Synthesis in Fungi of the Zygomycota and Ascomycota Reveals a Broad Diagnostic Target.

Ascomycetes and zygomycetes account for the majority of (i) fungi responsible for cutaneous, subcutaneous, and invasive human fungal infections, (ii) plant fungal pathogens, (iii) fungi that threaten global biodiversity, (iv) fungal agents of agricultural spoilage, and (v) fungi in water-damaged buildings. Rapid recognition of fungal infection (or contamination) enables early treatment (or remediation). A bioinformatics search found homologues of Saccharomyces cerevisiae Mnn9p present in members of the Zygomycota and Ascomycota phyla and absent in members of the Chytridiomycota and Basidiomycota. Mnn9p is a component of the yeast mannan polymerization complex and is necessary for α-1,6 mannan production. A monoclonal antibody (2DA6) was produced that was reactive with purified mannans of Mucor, Rhizopus, Aspergillus, Fusarium, and Candida species. Experimentation using a 2DA6 antigen capture enzyme-linked immunosorbent assay (ELISA) and extracts of fungi from the four phyla found agreement between the presence or absence of Mnn9p homologues and production or lack of production of mannan reactive with 2DA6. Studies of cell extracts from yeast mannan mutants identified α-1,6 mannan as the epitope recognized by 2DA6. To translate this finding into a point-of-use diagnostic, a 2DA6 lateral flow immunoassay was constructed that detected mannan in (i) extracts of dermatophytes and fungi that produce trauma-related infection and (ii) tissue from plants infected with Grosmannia clavigera or Sclerotium cepivorum These studies (i) revealed that the conservation of α-1,6-linked mannan in fungi of the Zygomycota and Ascomycota can be exploited as a broad diagnostic target and (ii) have provided a means to detect that target in an immunoassay platform that is well suited for clinic or field use.IMPORTANCE A key question asked when faced with an infection, an infestation, or environmental damage is whether it is a fungus. Identification of fungi as the cause of the problem can lead to remediation or treatment. Zygomycetes and ascomycetes account for the vast majority of fungal causes of human, animal, and plant disease, large-scale biodiversity loss, agricultural spoilage, and contamination of water-damaged buildings. These studies revealed the conservation of a common cell wall structural component of zygomycetes and ascomycetes to be a diagnostic target applicable to multiple pathogenic fungi and have leveraged that insight for practical use. Monoclonal antibodies reactive with this pan-fungal structure were produced and used to construct immunoassays (including ELISA and lateral flow assay) for detection of a broad range of pathogenic fungi.

Prevalence of Sickle Cell Trait and Reliability of Self-Reported Status among Expectant Parents in Nigeria: Implications for Targeted Newborn Screening.

BACKGROUND/AIMS: Sickle cell disease (SCD) is a life-threatening, autosomal recessive blood disorder prevalent in sub-Saharan Africa. We identified the prevalence of sickle cell trait (SCT) among pregnant women and their male partners in Enugu State, Nigeria, and determined the accuracy of self-reported sickle cell status and its reliability for identifying high-risk newborns for targeted screening. METHODS: We conducted a nested cohort study of expectant parents enrolled in the Healthy Beginning Initiative (HBI). The HBI is a cluster-randomized trial of a congregation-based approach designed to increase HIV testing. Participants completed a survey regarding self-awareness of their sickle cell genotype and consented to genotype screening by cellulose acetate electrophoresis. RESULTS: SCT prevalence (HbAS) was 22% (746/3,371). Only 50% of participants provided an accurate self-report. Self-report accuracy was significantly different (p < 0.0001) between individuals who reported having SCT or SCD (61% accuracy) versus those who reported not having SCT or SCD (86% accuracy). Demographic variables including gender, age, household size, employment, education, and home location were significantly associated with providing an accurate self-report. CONCLUSIONS: Low numbers of accurate parental self-reports, coupled with a high SCT prevalence in Nigeria, could limit the efficacy of targeted newborn screening. However, our data indicate that it is feasible to integrate sickle cell screening for pregnant women with existing, community-based health care programs developed by the President's Emergency Plan for AIDS Relief (PEPFAR), such as the HBI. Expanding screening programs could enable the development of targeted newborn screening based on maternal genotype that could identify all newborns with SCD in resource-limited settings.

Multiple proteins with essential mitochondrial functions have glycosylated isoforms.

Nucleocytosolic and secreted proteins are commonly glycosylated. However, reports of glycosylated mitochondrial proteins are rare. Using lectin chromatography on bovine heart, we detected low-abundance glycoforms of nuclear-encoded proteins with well-established mitochondrial function: pyruvate dehydrogenase E1α, NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, ADP/ATP translocase, ATP synthase d and oligomycin sensitivity-conferring protein. Notably, the latter two have been previously detected at the plasma membrane. Our findings indicate that glycosylation of classic mitochondrial proteins may be more common than previously appreciated. We discuss the implication that glycosylation could represent an unexplored mechanism for regulating these proteins' functions within mitochondria or at extra-mitochondrial locations.

Metabolic labeling of Caenorhabditis elegans primary embryonic cells with azido-sugars as a tool for glycoprotein discovery.

Glycobiology research with Caenorhabditis elegans (C. elegans) has benefitted from the numerous genetic and cell biology tools available in this system. However, the lack of a cell line and the relative inaccessibility of C. elegans somatic cells in vivo have limited the biochemical approaches available in this model. Here we report that C. elegans primary embryonic cells in culture incorporate azido-sugar analogs of N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc), and that the labeled glycoproteins can be analyzed by mass spectrometry. By using this metabolic labeling approach, we have identified a set of novel C. elegans glycoprotein candidates, which include several mitochondrially-annotated proteins. This observation was unexpected given that mitochondrial glycoproteins have only rarely been reported, and it suggests that glycosylation of mitochondrially-annotated proteins might occur more frequently than previously thought. Using independent experimental strategies, we validated a subset of our glycoprotein candidates. These include a mitochondrial, atypical glycoprotein (ATP synthase α-subunit), a predicted glycoprotein (aspartyl protease, ASP-4), and a protein family with established glycosylation in other species (actin). Additionally, we observed a glycosylated isoform of ATP synthase α-subunit in bovine heart tissue and a primate cell line (COS-7). Overall, our finding that C. elegans primary embryonic cells are amenable to metabolic labeling demonstrates that biochemical studies in C. elegans are feasible, which opens the door to labeling C. elegans cells with other radioactive or azido-substrates and should enable the identification of additional post-translationally modified targets and analysis of the genes required for their modification using C. elegans mutant libraries.