Expanding polysaccharide–protein coupling of glycoconjugate vaccines

For the preparation of glycoconjugate vaccines, polysaccharide antigens can usually be chemically modified to generate reactive functional groups (e.g., the formation of aldehyde groups by periodate oxidation of adjacent diols) for covalent coupling with proteins. In a recent issue of JBC, Duke et al. showed that an alternative agent, galactose oxidase (GOase) isolated from the fungus Fusarium sp. can generate aldehyde groups in a unique chemoenzymatic approach to prepare a conjugate vaccine against Streptococcus pneumoniae. These findings introduce a new strategy for the design and development of glycoconjugate vaccines.     

Engineering a genome‐reduced bacterium to eliminate Staphylococcus aureus biofilms in vivo

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome‐reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm‐associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome‐reduced bacterium that can fight against clinically relevant biofilm‐associated bacterial infections.     

Analysis of diverse β-lactamases presenting high-level resistance in association with OmpK35 and OmpK36 porins in ESBL-producing Klebsiella pneumoniae

Emerging extensively drug-resistant (XDR) Klebsiella pneumoniae due to the production of β-lactamases and porin loss is a substantial worldwide concern. This study aimed to elucidate the role of outer membrane porin (OMP) loss, AmpC, and carbapenemases among extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae strains with XDR phenotype. This study analyzed 79 K. pneumoniae from several clinical sources and detected ESBLs in 29 strains co-harbored with other β-lactamases using standard microbiological practices and phenotypic procedures. Minimum inhibitory concentrations (MICs) were determined against several antibiotics using Microscan WalkAway plus. OMP analysis was carried out using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ESBL, AmpC, and carbapenemase genes were detected using molecular methods. The microbiological analysis discovered 29 (36.7%) ESBL strains of K. pneumoniae, which showed the co-existence of 7 (24.1%) AmpC β-lactamases and 22 (75.9%) carbapenemases. Porin loss of OmpK35 was observed in 13 (44.8%) and OmpK36 in 8 (27.5%) K. pneumoniae strains. The strains were significantly associated with the intensive care unit (ICU) (p = 0.006) and urinary sources (p = 0.004). The most commonly detected gene variants in each β-lactamase class included 16 (55.2%) bla_CTX-M−1, 7 (100%) bla_CYM-2, 11 (50%) bla_NDM-1, and integron-1 was detected in 21/29 (72.4%) strains. MICs of cephalosporin, fluoroquinolone, carbapenem, aminoglycoside, and β-lactam combinations demonstrated a high number of XDR strains. Tigecycline (2 µg/mL MIC₅₀ and >32 µg/mL MIC₉₀) and colistin (1 µg/mL MIC₅₀ and 8 µg/mL MIC₉₀) presented lower resistance. ESBL K. pneumoniae strains with OmpK35 and OmpK36 porin loss demonstrate conglomerate resistance mechanisms with AmpC and carbapenemases, leading to emerging XDR and pan drug resistance.     

PKGIα is activated by metal-dependent oxidation in vitro but not in intact cells

Type I cGMP-dependent protein kinases (PKGIs) are important components of various signaling pathways and are canonically activated by nitric oxide– and natriuretic peptide–induced cGMP generation. However, some reports have shown that PKGIα can also be activated in vitro by oxidizing agents. Using in vitro kinase assays, here, we found that purified PKGIα stored in PBS with Flag peptide became oxidized and activated even in the absence of oxidizing agent; furthermore, once established, this activation could not be reversed by reduction with DTT. We demonstrate that activation was enhanced by addition of Cu²⁺ before storage, indicating it was driven by oxidation and mediated by trace metals present during storage. Previous reports suggested that PKGIα Cys⁴³, Cys¹¹⁸, and Cys¹⁹⁶ play key roles in oxidation-induced kinase activation; we show that activation was reduced by C118A or C196V mutations, although C43S PKGIα activation was not reduced. In contrast, under the same conditions, purified PKGIβ activity only slightly increased with storage. Using PKGIα/PKGIβ chimeras, we found that residues throughout the PKGIα-specific autoinhibitory loop were responsible for this activation. To explore whether oxidants activate PKGIα in H9c2 and C2C12 cells, we monitored vasodilator-stimulated phosphoprotein phosphorylation downstream of PKGIα. While we observed PKGIα Cys⁴³ crosslinking in response to H₂O₂ (indicating an oxidizing environment in the cells), we were unable to detect increased vasodilator-stimulated phosphoprotein phosphorylation under these conditions. Taken together, we conclude that while PKGIα can be readily activated by oxidation in vitro, there is currently no direct evidence of oxidation-induced PKGIα activation in vivo.     

A new variant of the capsule 3 cluster occurs in Streptococcus pneumoniae from deceased wild chimpanzees

The presence of new Streptococcus pneumoniae clones in dead wild chimpanzees from the Taï National Park, Côte d''Ivoire, with previous respiratory problems has been demonstrated recently by DNA sequence analysis from samples obtained from the deceased apes. In order to broadenour understanding on the relatedness of these pneumococcal clones to those from humans, the gene locus responsible for biosynthesis of the capsule polysaccharide (CPS) has now been characterized. DNA sequence analysis of PCR fragments identified a cluster named cps3_Taï containing the four genes typical for serotype 3 CPS, but lacking a 5′-region of ≥2 kb which is degenerated in other cps3 loci and not required for type 3 biosynthesis. CPS3 is composed of a simple disaccharide repeat unit comprising glucose and glucuronic acid (GlcUA). The two genes ugd responsible for GlcUA synthesis and wchE encoding the type 3 synthase are essential for CPS3 biosynthesis, whereas both, galU and the 3′-truncated gene pgm are not required due to the presence of homologues elsewhere in the genome. The DNA sequence of cps3_Taï diverged considerably from those of other cps3 loci. Also, the gene pgm _Taï represents a full length version with a nonsense mutation at codon 179. The two genes ugd _Taï and wchE _Taï including the promoter region were transformed into a nonencapsulated laboratory strain S. pneumoniae R6. Transformants which expressed type 3 capsule polysaccharide were readily obtained, documenting that the gene products are functional. In summary, the data indicate that cps3_Taï evolved independent from other cps3 loci, suggesting the presence of specialized serotype 3 S. pneumoniae clones endemic to the Taï National Park area.     

Periodate-induced lymphocyte transformation. 3. Effects of temperature and pH

The optimum conditions for sodium periodate induced lymphocyte transformation occur at low concentrations of NaIO₄, low temperature, pH between 6 and 7.5, and with short periods of exposure to the NaIO₄. Exposures for prolonged times at elevated temperatures or to NaIO₄ at pH above 7.5 result in diminished responses. The finding of maximum lymphocyte response under these conditions is compatible with the proposal that the triggering event in NaIO₄ transformation of lymphocytes is the oxidation and cleavage of the two terminal carbon atoms of sialic acid. Exposure of lymphocytes to NaIO₄ under conditions of higher temperature, higher pH or longer exposure times does not significantly reduce their ability to respond to phytohemagglutinin (PHA) or pokeweed mitogen (PWM). This finding suggests that these mitogens have different target sites from that of NaIO₄ or that they are acting on different populations of lymphocytes.     

Improvement in in?vitro fertilization outcome following in?vivo synchronization of oocyte maturation in mice

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.     

SitA contributes to the virulence of Klebsiella pneumoniae in a mouse infection model

Klebsiella pneumoniae is an opportunistic pathogen, which causes a wide range of nosocomial infections. Recently, antibiotic resistance makes K. pneumoniae infection difficult to deal with. Investigation on virulence determinants of K. pneumoniae can provide more information about pathogenesis and unveil new targets for treatment or vaccine development. In this study, SitA, a Fur-regulated divalent cation transporter, was found significantly increased when K. pneumoniae was cultured in a nutrient-limited condition. A sitA-deletion strain (ΔsitA) was created to characterize the importance of SitA in virulence. ΔsitA showed higher sensitivity toward hydroperoxide than its parental strain. In a mouse intraperitoneal infection model, the survival rate of mice infected with ΔsitA strain increased greatly when compared with that of mice infected with the parental strain, suggesting that sitA deletion attenuates the bacterial virulence in vivo. To test whether ΔsitA strain is a potential vaccine candidate, mice were immunized with inactivated bacteria and then challenged with the wild-type strain. The results showed that using ΔsitA mutant protected mice better than using the wild-type strain or the capsule-negative congenic bacteria. In summary, SitA was found being important for the growth of K. pneumoniae in vivo and deleting sitA might be a potential approach to generate vaccines against K. pneumoniae.     

Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication

Zinc pyrithione (1a), together with its analogues 1b–h and ruthenium pyrithione complex 2a, were synthesised and evaluated for the stability in biologically relevant media and anti-SARS-CoV-2 activity. Zinc pyrithione revealed potent in vitro inhibition of cathepsin L (IC₅₀=1.88 ± 0.49 µM) and PL^Pro (IC₅₀=0.50 ± 0.07 µM), enzymes involved in SARS-CoV-2 entry and replication, respectively, as well as antiviral entry and replication properties in an ex vivo system derived from primary human lung tissue. Zinc complexes 1b–h expressed comparable in vitro inhibition. On the contrary, ruthenium complex 2a and the ligand pyrithione a itself expressed poor inhibition in mentioned assays, indicating the importance of the selection of metal core and structure of metal complex for antiviral activity. Safe, effective, and preferably oral at-home therapeutics for COVID-19 are needed and as such zinc pyrithione, which is also commercially available, could be considered as a potential therapeutic agent against SARS-CoV-2.     

Wheat germ in?vitro translation to produce one of the most toxic sodium channel specific toxins

Envenoming following scorpion sting is a common emergency in many parts of the world. During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim causing serious medical problems. The exploration of toxin structure-function relationship would benefit from the generation of soluble recombinant scorpion toxins in Escherichia coli. We developed an in vitro wheat germ translation system for the expression of the highly toxic Aah (Androctonus australis hector)II protein that requires the proper formation of four disulphide bonds. Soluble, recombinant N-terminal GST (glutathione S-transferase)-tagged AahII toxin is obtained in this in vitro translation system. After proteolytic removal of the GST-tag, purified rAahII (recombinant AahII) toxin, which contains two extra amino acids at its N terminal relative to the native AahII, is highly toxic after i.c.v. (intracerebroventricular) injection in Swiss mice. An LD₅₀ (median lethal dose)-value of 10 ng (or 1.33 pmol), close to that of the native toxin (LD₅₀ of 3 ng) indicates that the wheat germ in vitro translation system produces properly folded and biological active rAahII. In addition, NbAahII10 (Androctonus australis hector nanobody 10), a camel single domain antibody fragment, raised against the native AahII toxin, recognizes its cognate conformational epitope on the recombinant toxin and neutralizes the toxicity of purified rAahII upon injection in mice.     

Structure and Molecular Characterization of Streptococcus pneumoniae Capsular Polysaccharide 10F by Carbohydrate Engineering in Streptococcus oralis

Although closely related at the molecular level, the capsular polysaccharide (CPS) of serotype 10F Streptococcus pneumoniae and coaggregation receptor polysaccharide (RPS) of Streptococcus oralis C104 have distinct ecological roles. CPS prevents phagocytosis of pathogenic S. pneumoniae, whereas RPS of commensal S. oralis functions as a receptor for lectin-like adhesins on other members of the dental plaque biofilm community. Results from high resolution NMR identified the recognition region of S. oralis RPS (i.e. Galfβ1–6GalNAcβ1–3Galα) in the hexasaccharide repeat of S. pneumoniae CPS10F. The failure of this polysaccharide to support fimbriae-mediated adhesion of Actinomyces naeslundii was explained by the position of Galf, which occurred as a branch in CPS10F rather than within the linear polysaccharide chain, as in RPS. Carbohydrate engineering of S. oralis RPS with wzy from S. pneumoniae attributed formation of the Galf branch in CPS10F to the linkage of adjacent repeating units through sub terminal GalNAc in Galfβ1–6GalNAcβ1–3Galα rather than through terminal Galf, as in RPS. A gene (wcrD) from serotype 10A S. pneumoniae was then used to engineer a linear surface polysaccharide in S. oralis that was identical to RPS except for the presence of a β1–3 linkage between Galf and GalNAcβ1–3Galα. This polysaccharide also failed to support adhesion of A. naeslundii, thereby establishing the essential role of β1–6-linked Galf in recognition of adjacent GalNAcβ1–3Galα in wild-type RPS. These findings, which illustrate a molecular approach for relating bacterial polysaccharide structure to function, provide insight into the possible evolution of S. oralis RPS from S. pneumoniae CPS.     

Evaluation of synthetic schemes to prepare immunogenic conjugates of Vibrio cholerae O139 capsular polysaccharide with chicken serum albumin

Vibrio cholerae serotype O139 is a new etiologic agent of epidemic cholera. There is no vaccine available against cholera caused by this serotype. V. cholerae O139 is an encapsulated bacterium, and its polysaccharide capsule is an essential virulent factor and likely protective antigen.This study evaluated several synthetic schemes for preparation of conjugates of V. cholerae O139 capsular polysaccharide (CPS) with chicken serum albumin as the carrier protein (CSA) using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents. Four conjugates described here as representative of many experiments were synthesized in 2 steps: 1) preparation of adipic acid hydrazide derivative of CPS (CPS_AH) or of CSA (CSA_AH), and 2) binding of CPS_AH to CSA or of CPS to CSA_AH. Although all conjugates induced CPS antibodies, the conjugate prepared by EDC-mediated binding of CPS and CSA_AH (EDC:CPS-CSA_AH) was statistically significantly less immunogenic than the other three conjugates. Representative sera from mice injected with these three conjugates contained antibodies that mediated the lysis of V. cholerae O139 inoculum.Evaluation of the different synthetic schemes and reaction conditions in relation to the immunogenicity of the resultant conjugates provided the basis for the preparation of a V. cholerae O139 conjugate vaccine with a medically useful carrier protein such as diphtheria toxin mutant.     

Coagulansin-A has beneficial effects on the development of bovine embryos in?vitro via HSP70 induction

Coagulansin-A (withanolide) is the steroidal lactone obtained from Withania coagulans which belong to Solanaceae family. The present study investigated the effects of coagulansin-A on bovine oocyte maturation and embryo development in vitro. All these oocytes were aspirated from the ovaries obtained from Korean Hanwoo cows at a local abattoir. To determine whether coagulansin-A has beneficial effects on bovine oocyte maturation in vitro, 355 oocytes per group (control and treated) in seven replicates were subjected with different concentrations (1, 2.5, 5, 7.5 and 10 μM) of coagulansin-A. The coagulansin-A was added in the in vitro maturation (IVM) media followed by in vitro fertilization (IVF) and then in vitro culture (IVC). Only treatment with 5 μM coagulansin-A remarkably (P<0.05) improved embryos development (Day 8 blastocyst) having 27.30 and 40.01% for control and coagulansin-A treated groups respectively. Treatment with 5 μM coagulansin-A significantly induced activation of heat shock protein 70 (HSP70) (P<0.05). Immunofluorescence analysis revealed that 5 μM coagulansin-A treatment also significantly inhibited oxidative stress and inflammation during bovine embryo development in vitro by decreasing 8-oxoguanosine (8-OxoG) (P<0.05) and nuclear factor-κB (NF-κB) (P<0.05). The expressions of HSP70 and NF-κB were also conformed through real-time PCR (RT-PCR). Additionally, the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay confirmed that coagulansin-A treatment significantly improved the embryo quality and reduced bovine embryo DNA damage (P<0.05). The present study provides new information regarding the mechanisms by which coagulansin-A promotes bovine embryo development in vitro.     

Slow Axonemal Dynein e Facilitates the Motility of Faster Dynein c

We highly purified the Chlamydomonas inner-arm dyneins e and c, considered to be single-headed subspecies. These two dyneins reside side-by-side along the peripheral doublet microtubules of the flagellum. Electron microscopic observations and single particle analysis showed that the head domains of these two dyneins were similar, whereas the tail domain of dynein e was short and bent in contrast to the straight tail of dynein c. The ATPase activities, both basal and microtubule-stimulated, of dynein e (k_cat = 0.27 s^–1 and k_cat,MT = 1.09 s^–1, respectively) were lower than those of dynein c (k_cat = 1.75 s^–1 and k_cat,MT = 2.03 s^–1, respectively). From in vitro motility assays, the apparent velocity of microtubule translocation by dynein e was found to be slow (V_ap = 1.2 ± 0.1 μm/s) and appeared independent of the surface density of the motors, whereas dynein c was very fast (V_max = 15.8 ± 1.5 μm/s) and highly sensitive to decreases in the surface density (V_min = 2.2 ± 0.7 μm/s). Dynein e was expected to be a processive motor, since the relationship between the microtubule landing rate and the surface density of dynein e fitted well with first-power dependence. To obtain insight into the in vivo roles of dynein e, we measured the sliding velocity of microtubules driven by a mixture of dynein e and c at various ratios. The microtubule translocation by the fast dynein c became even faster in the presence of the slow dynein e, which could be explained by assuming that dynein e does not retard motility of faster dyneins. In flagella, dynein e likely acts as a facilitator by holding adjacent microtubules to aid dynein c’s power stroke.     

Irreversible inhibition of the labellar sugar receptor of the blowfly, Phormia regina, by periodate-activated starch

Activated starch bearing aldehyde groups, which is prepared by oxidation of soluble starch with sodium periodate (NaIO₄) and binds amino groups strongly, irreversibly inhibited the responses of taste receptors in the blowfly, Phormia regina. After highly activated starch (oxidized with 0.2 M NaIO₄) was applied through the tip opening of the chemosensillum, the responses of the sugar, salt and water receptors were nonspecifically depressed. The chemoreceptor membranes were nonspecifically protected by all the sugars tested against the treatment with highly activated starch, but in the case of the response to fructose, the membrane was specifically protected by fructose. Mildly activated starch (oxidized with 0.04 M NaIO₄), however, selectively depressed the sugar response. The depression depended on the pH of the solution containing the mildly activated starch. After treatment at pH 8.6, the response to sucrose, maltose or glucose was not depressed at all, but that to fructose or galactose was depressed. After treatment at pH 9.0, however, the responses to all sugars were depressed. Such depression of the sugar response lasted for 20 h or more after treatment.     

Metformin inhibits MAPK signaling and rescues pancreatic aquaporin 7 expression to induce insulin secretion in type 2 diabetes mellitus

Metformin is the first-line antidiabetic agent for type 2 diabetes mellitus (T2DM) treatment. Although accumulated evidence has shed light on the consequences of metformin action, the precise mechanisms of its action, especially in the pancreas, are not fully understood. Aquaporin 7 (AQP7) acts as a critical regulator of intraislet glycerol content, which is necessary for insulin production and secretion. The aim of this study was to investigate the effects of different doses of metformin on AQP7 expression and explore the possible mechanism of its protective effects in the pancreatic islets. We used an in vivo model of high-fat diet in streptozocin-induced diabetic rats and an in vitro model of rat pancreatic β-cells (INS-1 cells) damaged by hyperglycemia and hyperlipidemia. Our data showed that AQP7 expression levels were decreased, whereas p38 and JNK mitogen-activated protein kinases (MAPKs) were activated in vivo and in vitro in response to hyperglycemia and hyperlipidemia. T2DM rats treated with metformin demonstrated a reduction in blood glucose levels and increased regeneration of pancreatic β-cells. In addition, metformin upregulated AQP7 expression as well as inhibited activation of p38 and JNK MAPKs both in vivo and in vitro. Overexpression of AQP7 increased glycerol influx into INS-1 cells, whereas inhibition of AQP7 reduced glycerol influx, thereby decreasing subsequent insulin secretion. Our findings demonstrate a new mechanism by which metformin suppresses the p38 and JNK pathways, thereby upregulating pancreatic AQP7 expression and promoting glycerol influx into pancreatic β-cells and subsequent insulin secretion in T2DM.