Anti-prion protein (PrP) monoclonal antibody 132, which recognizes mouse PrP amino acids 119-127, enables us to reliably detect abnormal isoform prion protein (PrPSc) in cells or frozen tissue ...sections by immunofluorescence assay, although treatment with guanidinium salts is a prerequisite. Despite the benefit of this mAb, the mechanism of PrPSc-specific detection remains unclear. Therefore, to address this mechanism, we analyzed the reactivities of mono- and bivalent mAb 132 to recombinant mouse PrP (rMoPrP) by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). In ELISA, binding of the monovalent form was significantly weaker than that of the bivalent form, indicating that bivalent binding confers a higher binding stability to mAb 132. Compared with other anti-PrP mAbs tested, the reactivity of bivalent mAb 132 was easily affected by a decrease in antigen concentration. The binding kinetics of mAb 132 assessed by SPR were consistent with the results of ELISA. The dissociation constant of the monovalent form was approximately 260 times higher than that of the bivalent form, suggesting that monovalent binding is less stable than bivalent binding. Furthermore, the amount of mAb 132 that bound to rMoPrP decreased if the antigen density was too low to allow bivalent binding. If two cellular PrP (PrPC) are close enough to allow bivalent binding, mAb 132 binds to PrPC. These results indicate that weak monovalent binding to monomeric PrPC diminishes PrPC signals to background level, whereas after exposure of the epitope, mAb 132 binds stably to oligomeric PrPSc in a bivalent manner.
Molecules that inhibit the formation of an abnormal isoform of prion protein (PrP(Sc)) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules ...inhibit PrP(Sc) formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrP(C)) and PrP(Sc) in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrP(Sc) levels without altering intracellular distribution of PrP(Sc). PPS did not change the distribution and levels of PrP(C), whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrP(C) to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrP(Sc) formation. In contrast, CPZ and U18666A initiated the redistribution of PrP(Sc) from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrP(C). The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrP(Sc) redistribution by CPZ or U18666A partly antagonized PrP(Sc) degradation, suggesting that the transfer of PrP(Sc) to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrP(Sc) degradation. This study revealed that precise analysis of the intracellular dynamics of PrP(C) and PrP(Sc) provides important information for understanding the mechanism of anti-prion agents.
Conversion of cellular prion protein (PrP.sup.C) into the pathogenic isoform of prion protein (PrP.sup.Sc) in neurons is one of the key pathophysiological events in prion diseases. However, the ...molecular mechanism of neurodegeneration in prion diseases has yet to be fully elucidated because of a lack of suitable experimental models for analyzing neuron-autonomous responses to prion infection. In the present study, we used neuron-enriched primary cultures of cortical and thalamic mouse neurons to analyze autonomous neuronal responses to prion infection. PrP.sup.Sc levels in neurons increased over the time after prion infection; however, no obvious neuronal losses or neurite alterations were observed. Interestingly, a finer analysis of individual neurons co-stained with PrP.sup.Sc and phosphorylated protein kinase RNA-activated-like endoplasmic reticulum (ER) kinase (p-PERK), the early cellular response of the PERK-eukaryotic initiation factor 2 (eIF2alpha) pathway, demonstrated a positive correlation between the number of PrP.sup.Sc granular stains and p-PERK granular stains, in cortical neurons at 21 dpi. Although the phosphorylation of PERK was enhanced in prion-infected cortical neurons, there was no sign of subsequent translational repression of synaptic protein synthesis or activations of downstream unfolded protein response (UPR) in the PERK-eIF2alpha pathway. These results suggest that PrP.sup.Sc production in neurons induces ER stress in a neuron-autonomous manner; however, it does not fully activate UPR in prion-infected neurons. Our findings provide insights into the autonomous neuronal responses to prion propagation and the involvement of neuron-non-autonomous factor(s) in the mechanisms of neurodegeneration in prion diseases.
Abstract MraY (phospho- N -acetylmuramoyl-pentapeptide-transferase) inhibitory natural products are attractive molecules as candidates for a new class of antibacterial agents to combat ...antimicrobial-resistant bacteria. Structural optimization of these natural products is required to improve their drug-like properties for therapeutic use. However, chemical modifications of these natural products are painstaking tasks due to complex synthetic processes, which is a bottleneck in advancing natural products to the clinic. Here, we develop a strategy for a comprehensive in situ evaluation of the build-up library, which enables us to streamline the preparation of the analogue library and directly assess its biological activities. We apply this approach to a series of MraY inhibitory natural products. Through construction and evaluation of the 686-compound library, we identify promising analogues that exhibit potent and broad-spectrum antibacterial activity against highly drug-resistant strains in vitro as well as in vivo in an acute thigh infection model. Structures of the MraY-analogue complexes reveal distinct interaction patterns, suggesting that these analogues represent MraY inhibitors with unique binding modes. We further demonstrate the generality of our strategy by applying it to tubulin-binding natural products to modulate their tubulin polymerization activities.
The total synthesis of the depsipeptide natural product plusbacin A3 (1) utilizing solid-phase peptide synthesis (SPPS) was disclosed. A 3-hydroxy-proline derivative compatible with Fmoc SPPS was ...prepared by a diastereoselective Joullié–Ugi three-component reaction (JU-3CR)/hydrolysis sequence. After peptide elongation on the solid support, cleavage of the peptide from the resin, followed by macrolactamization and global deprotection, gave plusbacin A3 (1).
Chronic wasting disease (CWD) is a prion disease affecting cervid species primarily in the United States of America and Canada; however, it is now emerging in Scandinavian countries. Although CWD ...cases have not been reported in Japan, in case of a CWD outbreak occuring, it is critical to prepare for testing a large number of specimens. The present study showed that a rapid post‐mortem test kit, which is used for bovine spongiform encephalopathy surveillance in Japan, is valid for the detection of CWD prion.
The development of new antibacterial drugs with different mechanisms of action is urgently needed to address antimicrobial resistance. MraY is an essential membrane enzyme required for bacterial cell ...wall synthesis. Sphaerimicins are naturally occurring macrocyclic nucleoside inhibitors of MraY and are considered a promising target in antibacterial discovery. However, developing sphaerimicins as antibacterials has been challenging due to their complex macrocyclic structures. In this study, we construct their characteristic macrocyclic skeleton via two key reactions. Having then determined the structure of a sphaerimicin analogue bound to MraY, we use a structure-guided approach to design simplified sphaerimicin analogues. These analogues retain potency against MraY and exhibit potent antibacterial activity against Gram-positive bacteria, including clinically isolated drug resistant strains of S. aureus and E. faecium. Our study combines synthetic chemistry, structural biology, and microbiology to provide a platform for the development of MraY inhibitors as antibacterials against drug-resistant bacteria.
•The first report on the characterization of STEC isolated from cattle in Mongolia.•A variety of STEC with various O-genotypes and H-genotypes were isolated.•STEC with a higher risk of causing severe ...human disease exists in Mongolian cattle.
Shiga toxin-producing Escherichia coli (STEC) are associated with severe infections including hemorrhagic colitis and hemolytic uremic syndrome in humans. Ruminants are known as reservoirs of STEC; however, no data are available on STEC in ruminants in Mongolia, where more than 5 million cattle and 25 million sheep are raised. To disclose the existence and characteristics of STEC in Mongolia, in this study, we isolated and characterized STEC from cattle in Mongolia. We collected 350 rectal swabs of cattle from 30 farms near Ulaanbaatar city and isolated 45 STEC from 21 farms. Rectal swabs were precultured with modified Escherichia coli broth and then inoculated to Cefixime-Tellurite Sorbitol MacConkey agar plate and/or CHROMagar STEC agar plate for the isolation of STEC. The isolation ratios in each farm were from 0% to 40%. Multiplex PCR for the estimation of O- and H-serotypes identified 12 O-genotypes (Og-types) and 11 H-genotypes (Hg-types) from 45 isolates; however, Og-types of 19 isolates could not be determined. Stx gene subtyping by PCR identified 2 stx1 subtypes (1a and 1c) and 4 stx2 subtypes (2a, 2c, 2d, and 2g). Forty-five isolates were divided into 21 different groups based on the Og- and Hg-types, stx gene subtypes and the existence of virulence factors, ehxA, eae, and saa, which includes several major serotypes associated with human illness such as O26:H11 and O157:H7. The most dominant isolate, OgUT:H19 stx1a (+), stx2a (+), ehxA (+) and saa (+), was isolated from eight farms. This is the first report on the characterization of STEC in cattle in Mongolia, and the results suggest the importance of further monitoring of STEC contamination in the food chains as well as STEC infection in humans.
Transmission of colistin-resistant
Enterobacterales
from companion animals to humans poses a clinical risk as colistin is a last-line antimicrobial agent for treatment of multidrug-resistant ...Gram-negative bacteria including
Enterobacterales
. In this study, we investigated the colistin susceptibility of 285
Enterobacterales
(including 140
Escherichia coli
, 86
Klebsiella
spp., and 59
Enterobacter
spp.) isolated from companion animals in Japan. We further characterized colistin-resistant isolates by multilocus sequence typing (MLST), phylogenetic analysis of
hsp60
sequences, and population analysis profiling, to evaluate the potential clinical risk of companion animal-derived colistin-resistant
Enterobacterales
to humans in line with the One Health approach. All
E. coli
isolates were susceptible to colistin, and only one
Klebsiella
spp. isolate (1.2%, 1/86 isolates) was colistin resistant.
Enterobacter
spp. isolates were frequently colistin resistant (20.3%, 12/59 isolates). In colistin-resistant
Enterobacter
spp., all except one isolate exhibited colistin heteroresistance by population analysis profiling. These colistin-heteroresistant isolates belonged to clusters I, II, IV, VIII, and XII based on
hsp60
phylogeny. MLST analysis revealed that 12 colistin-resistant
Enterobacter
spp. belonged to the
Enterobacter cloacae
complex; five
Enterobacter kobei
(four ST591 and one ST1577), three
Enterobacter asburiae
(one ST562 and two ST1578), two
Enterobacter roggenkampii
(ST606 and ST1576), and
Enterobacter hormaechei
(ST1579) and
E. cloacae
(ST765) (each one strain). Forty-two percent of the colistin-resistant
E. cloacae
complex isolates (predominantly ST562 and ST591) belonged to lineages with human clinical isolates. Four
E. kobei
ST591 isolates were resistant to third-generation cephalosporines, aminoglycosides, and fluroquinolones but remained susceptible to carbapenems. In conclusion, our study is the first to our knowledge to report the frequent isolation of the colistin-resistant
E. cloacae
complex from companion animals. Furthermore, a subset of isolates belonged to human-associated lineages with resistance to multiple classes of antibiotics. These data warrant monitoring carriage of the colistin-resistant
E. cloacae
complex in companion animals as part of a domestic infection control procedure in line with the One Health approach.
There has been no report on Chronic wasting disease (CWD) cases in Japan to date; however, there is concern about the geographic spread of CWD. To clarify the CWD status in Japan, we conducted CWD ...monitoring using real-time quaking-induced conversion (RT-QuIC) assay which can detect the low level of CWD prions. A total of 690 obex samples collected from sika deer and Reeves’s muntjac in Hokkaido and Honshu was tested for CWD prions. No CWD-positive cases were found, suggesting that CWD is nonexistent in Japan. Our results also indicate that RT-QuIC assay is useful for continuous monitoring of CWD. Furthermore, nucleotide sequence analysis of the PrP gene revealed sika deer in Japan harbor CWD susceptible allele.
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