Correlation of COVID-19 Severity and Immunoglobulin Presence Against Spike and Nucleocapsid Proteins in SARS-CoV-2.

Data on the human immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins have been applied to vaccine development and diagnosing coronavirus disease 2019 (COVID-19), but little research has been done on the relationship between the human immune response and COVID-19 severity. We herein sought to determine whether there is a correlation between the immunoglobulin level and COVID-19 severity. Clinical samples were collected from 102 patients with COVID-19. Of these, 65 and 37 patients had mild and severe symptoms, respectively. An enzyme-linked immunosorbent assay using the recombinant SARS-CoV-2 nucleocapsid (N) protein, spike (S) protein, and synthetic peptides covering N and S as antigens was performed to measure the IgM and IgG levels. The correlation between the immunoglobulin level and COVID-19 severity was then analyzed. A significant difference in the level of IgG antibodies against N and of IgM antibodies against the receptor binding domain of the S protein was observed between patients with nonsevere and severe COVID-19 symptoms, and the level of IgG antibodies against N was found to be higher in patients with severe symptoms whereas the level of IgM antibodies against the S peptides was higher in patients with nonsevere symptoms. The level of specific antibodies against SARS-CoV-2 structural proteins might correlate with COVID-19 severity. If so, this fact may be useful for predicting the prognosis of the disease and in determining the appropriate treatment with greater precision.

Emergence and Spread of Carbapenem-Resistant and Aminoglycoside-Panresistant Enterobacter cloacae Complex Isolates Coproducing NDM-Type Metallo-ß-Lactamase and 16S rRNA Methylase in Myanmar.

Surveillance of 10 hospitals and a regional public health laboratory in Myanmar identified 31 isolates of carbapenem-resistant Enterobacter cloacae complex harboring blaNDM-type Of these isolates, 19 were highly resistant to aminoglycosides and harbored one or more genes encoding 16S rRNA methylases, including armA, rmtB, rmtC, and/or rmtE Of the 19 isolates, 16 were Enterobacter xiangfangensis ST200, with armA on the chromosome and a plasmid harboring blaNDM-1 and rmtC, indicating that these isolates were clonally disseminated nationwide in Myanmar.IMPORTANCE The emergence of multidrug-resistant E. cloacae complex has become a public health threat worldwide. E. xiangfangensis is a recently classified species belonging to E. cloacae complex. Here, we report a clonal dissemination of multidrug-resistant E. xiangfangensis ST200 producing two types of New Delhi metallo-ß-lactamase (NDM-type MBL), NDM-1 and -4, and three types of 16S rRNA methylases, ArmA, RmtC, and RmtE, in hospitals in Myanmar. The observation of these multidrug-resistant E. xiangfangensis ST200 isolates stresses the urgency to continue molecular epidemiological surveillance of these pathogens in Myanmar and in South Asian countries.

Characterization of sulfur-compound metabolism underlying wax-ester fermentation in Euglena gracilis.

Euglena gracilis is a microalga, which has been used as a model organism for decades. Recent technological advances have enabled mass cultivation of this species for industrial applications such as feedstock in nutritional foods and cosmetics. E. gracilis degrades its storage polysaccharide (paramylon) under hypoxic conditions for energy acquisition by an oxygen-independent process and accumulates high amount of wax-ester as a by-product. Using this sequence of reactions referred to as wax-ester fermentation, E. gracilis is studied for its application in biofuel production. Although the wax-ester production pathway is well characterized, little is known regarding the biochemical reactions underlying the main metabolic route, especially, the existence of an unknown sulfur-compound metabolism implied by the nasty odor generation accompanying the wax-ester fermentation. In this study, we show sulfur-metabolomics of E. gracilis in aerobic and hypoxic conditions, to reveal the biochemical reactions that occur during wax-ester synthesis. Our results helped us in identifying hydrogen sulfide (H2S) as the nasty odor-producing component in wax-ester fermentation. In addition, the results indicate that glutathione and protein degrades during hypoxia, whereas cysteine, methionine, and their metabolites increase in the cells. This indicates that this shift of abundance in sulfur compounds is the cause of H2S synthesis.

Improved fermentative L-cysteine overproduction by enhancing a newly identified thiosulfate assimilation pathway in Escherichia coli.

Sulfate (SO42-) is an often-utilized and well-understood inorganic sulfur source in microorganism culture. Recently, another inorganic sulfur source, thiosulfate (S2O32-), was proposed to be more advantageous in microbial growth and biotechnological applications. Although its assimilation pathway is known to depend on O-acetyl-L-serine sulfhydrylase B (CysM in Escherichia coli), its metabolism has not been extensively investigated. Therefore, we aimed to explore another yet-unidentified CysM-independent thiosulfate assimilation pathway in E. coli. ΔcysM cells could accumulate essential L-cysteine from thiosulfate as the sole sulfur source and could grow, albeit slowly, demonstrating that a CysM-independent thiosulfate assimilation pathway is present in E. coli. This pathway is expected to consist of the initial part of the thiosulfate to sulfite (SO32-) conversion, and the latter part might be shared with the final part of the known sulfate assimilation pathway [sulfite → sulfide (S2-) â†’ L-cysteine]. This is because thiosulfate-grown ΔcysM cells could accumulate a level of sulfite and sulfide equivalent to that of wild-type cells. The catalysis of thiosulfate to sulfite is at least partly mediated by thiosulfate sulfurtransferase (GlpE), because its overexpression could enhance cellular thiosulfate sulfurtransferase activity in vitro and complement the slow-growth phenotype of thiosulfate-grown ΔcysM cells in vivo. GlpE is therefore concluded to function in the novel CysM-independent thiosulfate assimilation pathway by catalyzing thiosulfate to sulfite. We applied this insight to L-cysteine overproduction in E. coli and succeeded in enhancing it by GlpE overexpression in media containing glucose or glycerol as the main carbon source, by up to ~1.7-fold (1207 mg/l) or ~1.5-fold (1529 mg/l), respectively.

Development and evaluation of immunochromatography to detect Gram-negative bacteria producing ArmA 16S rRNA methylase responsible for aminoglycoside resistance.

Rapid and reliable detection of aminoglycoside-resistant bacteria is an important infection-control measure and a crucial aspect of antimicrobial chemotherapy. The enzyme 16S rRNA methylase has been shown to mediate aminoglycoside resistance in bacteria. This study describes a newly developed immunochromatographic assay using novel monoclonal antibodies (mAbs) that recognize ArmA 16S rRNA methylase. Epitope mapping showed that these mAbs recognized amino acids 1-93 of ArmA, which consists of 257 amino acids. Evaluation of the assay using ArmA producing and non-producing bacterial species, as well as bacteria producing other types of 16S rRNA methylases, indicated that immunochromatographic detection of the ArmA-type 16S rRNA methylase was fully consistent with PCR analysis for armA genes, with all immunochromatographically positive strains being resistant to aminoglycosides (MIC≥128µg/mL). The detection limit of the assay was 12ng ArmA. These findings indicate that this assay can be used for the rapid and reliable detection of the production of ArmA 16S rRNA methylase by Gram-negative bacteria, including Acinetobacter baumannii and Escherichia coli.

Physicochemically stable cholera toxin B subunit pentamer created by peripheral molecular constraints imposed by de novo-introduced intersubunit disulfide crosslinks.

We attempted to generate a physicochemically stable cholera toxin B subunit (CTB) by de novo-introduction of intersubunit disulfide bonds between adjacent subunits. Genes encoding double mutant CTB (dmCTB) encompassing a pair of amino acids to be replaced with cysteine residues either at the N-terminal (T1C/T92C, Q3C/T47C), C-terminal (F25C/N103C, Y76C/N103C), or at the internal α-helix region (L77C/T78C), were engineered. One mutant with the N-terminal constraint [dmCTB(T1C/T92C)], expressed as pentamer retained monosialoganglioside G(M1) (GM1) binding affinity, and exhibited robust thermostability. However, when the mutant CTB was heat-treated in the presence of a reducing agent, the thermostable phenotype was abolished, indicating the observed phenotype is due to the introduction of intersubunit disulfide bonds. The mutant CTB also exhibited a strong acid stability at a pH as low as 1.2, as well as stability against incubation with sodium dodecyl sulfate at concentrations as high as 10%. Furthermore, intranasal administration of the mutant CTB to mice induced CTB-specific serum IgG even after heat treatment, while the wildtype CTB failed to show such heat-resistant mucosal immunogenicity. This study demonstrated that an enterotoxin B subunit could be transformed into a physicochemically stable pentamer by the de novo-introduction of peripherally arranged intersubunit disulfide crosslinks, which may prove to be a useful strategy for the development of molecularly stable enterotoxin B subunit-based vaccines and delivery molecules.