ZN148 Is a Modular Synthetic Metallo-ß-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo.

Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (ß-lactamases able to inactivate carbapenems) have been identified in both serine ß-lactamase (SBL) and metallo-ß-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 µM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

Effects of dietary n-3 fatty acids on Toll-like receptor activation in primary leucocytes from Atlantic salmon (Salmo salar).

The shortage of the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the international markets has led to increasing substitution of fish oil by plant oils in Atlantic salmon (Salmo salar) feed and thereby reducing the EPA and DHA content in salmon. However, the minimum required levels of these fatty acids in fish diets for securing fish health are unknown. Fish were fed with 0, 1 or 2% EPA or DHA alone or in combination of both over a period, growing from 50 to 400 g. Primary head kidney leucocytes were isolated and stimulated with Toll-like receptor (TLR) ligands to determine if EPA and DHA deficiency can affect expression of important immune genes and eicosanoid production. Several genes related to viral immune response did not vary between groups. However, there was a tendency that the high-level EPA and DHA groups expressed lower levels of IL-1ß in non-stimulated leucocytes. These leucocytes were also more responsive to the TLR ligands, inducing higher expression levels of IL-1ß and Mx1 after stimulation. The levels of prostaglandin E2 and leukotriene B4 in serum and media from stimulated leucocytes were lower in both low and high EPA and DHA groups. In conclusion, leucocytes from low EPA and DHA groups seemed to be less responsive towards immunostimulants, like TLR ligands, indicating that low levels or absence of dietary EPA and DHA may have immunosuppressive effects.

Short-term effect of bisphenol-a on oxidative stress responses in Atlantic salmon kidney cell line: a transcriptional study.

Bisphenol A (BPA) is regularly detected in aquatic ecosystems due to increased use of products based on polycarbonate plastics and epoxy resins. It migrates from these products directly into rivers and marine waters or indirectly through effluents from wastewater treatment plants and landfilled sites. BPA can affect aquatic organisms both chronically and acutely at sensitive live stages. Despite reports indicating harmful effects of BPA, little is known about its role in oxidative stress responses in fish. In this study, we investigated the transcriptional effect of BPA (0, 1, 10, 100 µM) on an Atlantic salmon kidney (ASK) cell line for 6 h and 24 h by monitoring expression of 11 genes: elongation factor 1-alpha (ef1a), 18S ribosomal RNA (18s), gluthation (gsh), superoxide dismutase (sod), thioredoxin (txd), Salmo salar oxidative stress-responsive serine-rich 1 (oxr), glucose-regulated protein 78 (grp78), heat shock protein 70 (hsp70), sequestosome1 (p62), interleukin-1 beta (il-1beta) and toll-like receptor 8 (tlr8). In general, only the 100 µM concentration treatment altered the mRNA expression. BPA down-regulated the expression of gsh and sod genes for both exposure-times while txd gene was the only down-regulated after 6-h exposure. The up-regulation of genes in the ASK cell line exposed for 6 h was only observed in il-1beta, while the 24-h exposure resulted in the up-regulation of oxr, tlr8, hsp70, p62 and il-1beta genes. The last three genes increased several fold compared to the others. The results showed that BPA exposure at 100 µM imposed oxidative stress on the ASK cell line and longer exposure time involved transcriptional responses of immune-related genes. This may indicate the possible role of BPA-associated oxidative stress in induction of inflammatory response in this macrophage-like cell type.

The infectious salmon anemia virus esterase prunes erythrocyte surfaces in infected Atlantic salmon and exposes terminal sialic acids to lectin recognition.

Many sialic acid-binding viruses express a receptor-destroying enzyme (RDE) that removes the virus-targeted receptor and limits viral interactions with the host cell surface. Despite a growing appreciation of how the viral RDE promotes viral fitness, little is known about its direct effects on the host. Infectious salmon anemia virus (ISAV) attaches to 4-O-acetylated sialic acids on Atlantic salmon epithelial, endothelial, and red blood cell surfaces. ISAV receptor binding and destruction are effectuated by the same molecule, the haemagglutinin esterase (HE). We recently discovered a global loss of vascular 4-O-acetylated sialic acids in ISAV-infected fish. The loss correlated with the expression of viral proteins, giving rise to the hypothesis that it was mediated by the HE. Here, we report that the ISAV receptor is also progressively lost from circulating erythrocytes in infected fish. Furthermore, salmon erythrocytes exposed to ISAV ex vivo lost their capacity to bind new ISAV particles. The loss of ISAV binding was not associated with receptor saturation. Moreover, upon loss of the ISAV receptor, erythrocyte surfaces became more available to the lectin wheat germ agglutinin, suggesting a potential to alter interactions with endogenous lectins of similar specificity. The pruning of erythrocyte surfaces was inhibited by an antibody that prevented ISAV attachment. Furthermore, recombinant HE, but not an esterase-silenced mutant, was sufficient to induce the observed surface modulation. This links the ISAV-induced erythrocyte modulation to the hydrolytic activity of the HE and shows that the observed effects are not mediated by endogenous esterases. Our findings are the first to directly link a viral RDE to extensive cell surface modulation in infected individuals. This raises the questions of whether other sialic acid-binding viruses that express RDEs affect host cells to a similar extent, and if such RDE-mediated cell surface modulation influences host biological functions with relevance to viral disease.

Chitosan nanoparticle formulation attenuates poly (I:C) induced innate immune responses against inactivated virus vaccine in Atlantic salmon (Salmo salar).

Many vaccine formulations, in particular vaccines based on inactivated virus, needs adjuvants to boost immunogenicity. In aquaculture, mineral and plant oil are used as adjuvant in commercial vaccines, and the advent of oil-adjuvanted vaccines was crucial to aquaculture development. Nevertheless, some of these approved vaccines display suboptimal performance in the field compared to experimental conditions. Therefore, there is a need to improve adjuvants and delivery methods for fish vaccines against viruses. We used RNA sequencing of Atlantic salmon head kidney to analyse the difference in gene expression 24 h after injection of different experimental vaccine formulations. We compared five different formulations in addition to a PBS control: inactivated virus alone (group V), soluble poly (I:C) (group P), nanoparticles containing poly (I:C) (group N), soluble poly (I:C) + inactivated virus (group PV) and finally nanoparticles containing poly (I:C) + inactivated virus (group NV). Our results showed poly (I:C)'s ability as adjuvant and its capacity influence innate immune genes expression in Atlantic salmon. Soluble poly (I:C) upregulated multiple immune related genes and was more effective compared to poly (I:C) formulated into chitosan nanoparticles (more than 10 fold increase in differentially expressed genes, DEGs). However, inclusion of inactivated ISA virus in the nanoparticle vaccine, increased the number of DEGs fivefold suggesting a synergistic effect of adjuvant and antigen. Our results indicate that the way poly (I:C) is formulated and the presence of antigen is important for the magnitude of the innate immune response in Atlantic salmon.

Kinetics of transcriptional response against poly (I:C) and infectious salmon anemia virus (ISAV) in Atlantic salmon kidney (ASK) cell line.

Vaccine adjuvants induce host innate immune responses improving long-lasting adaptive immunity against vaccine antigens. In vitro models can be used to compare these responses between adjuvants and the infection targeted by the vaccine. We utilized transcriptomic profiling of an Atlantic salmon cell line to compare innate immune responses against ISAV and an experimental viral vaccine adjuvant: poly (I:C). Induction of interferon and interferon induced genes were observed after both treatments, but often with different amplitude and kinetics. Using KEGG ortholog database and available software from Bioconductor we could specify a complete bioinformatic pipeline for analysis of transcriptomic data from Atlantic salmon, a feature not previously available. We have identified important differences in the transcriptional profile of Atlantic salmon cells exposed to viral infection and a viral vaccine adjuvant candidate, poly (I:C). This report increases our knowledge of viral host-pathogen interaction in salmon and to which extent these can be mimicked by adjuvant compounds.

Interaction between dietary fatty acids and genotype on immune response in Atlantic salmon (Salmo salar) after vaccination: A transcriptome study.

A pivotal matter to aquaculture is the sourcing of sustainable resources as ingredients to aquafeeds. Levels of plant delivered oils as source of fatty acids (FA) in aquafeeds have reached around 70% resulting in reduced levels of long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in salmon fillet composition. EPA and DHA can modulate inflammation and immune response, so it is crucial to understand how fish immune response is affected by low LC n-3 PUFA diet and if this diet can have a detrimental effect on vaccine response. Atlantic salmon (Salmo salar) can produce EPA/DHA from α-linolenic acid (ALA) and this endogenous capacity can be explored to develop families with higher tolerance to low LC n-3 PUFA diets. Here we analyze innate and adaptive immune response in Atlantic salmon to a commercial vaccine after being fed low levels of EPA and DHA, and we also compare three strains of salmon selected by their endogenous capacity of synthesizing LC- n-3 PUFA. A total of 2,890 differentially expressed genes (DEGs) were identified (p-value adjusted < 0.1) when comparing vaccinated fish against control non-vaccinated. Gene ontology (GO) and KEGG analysis with 442 up/downregulated genes revealed that most DEGs were both related to immune response as well as part of important immune related pathways, as "Toll-like receptor" and "Cytokine-Cytokine interaction". Adaptive response was also addressed by measuring antigen specific IgM, and titers were significantly higher than in the pre-immune fish at 62 days post-immunization. However, diet and strain had no/little effect on vaccine-specific IgM or innate immune responses. Atlantic salmon therefore display robustness in its response to vaccination even when feed low levels of LC n-3 PUFA.

Synthesis and biological evaluation of zinc chelating compounds as metallo-ß-lactamase inhibitors.

The syntheses of metallo-ß-lactamase inhibitors comprising chelating moieties, with varying zinc affinities, and peptides partly inspired from bacterial peptide sequences, have been undertaken. The zinc chelator strength was varied using the following chelators, arranged in order of ascending binding affinity: dipicolylamine (DPA, tridentate), dipicolyl-1,2,3-triazolylmethylamine (DPTA, tetradentate) dipicolyl ethylenediamine (DPED, tetradentate) and trispicolyl ethylenediamine (TPED, pentadentate). The chosen peptides were mainly based on the known sequence of the C-terminus of the bacterial peptidoglycan precursors. Biological evaluation on clinical bacterial isolates, harbouring either the NDM-1 or VIM-2 metallo-ß-lactamase, showed a clear relationship between the zinc chelator strength and restoration of meropenem activity. However, evaluation of toxicity on different cancer cell lines demonstrated a similar trend, and thus inclusion of the bacterial peptides did possess rather high toxicity towards eukaryotic cells.

Synthesis and Preclinical Evaluation of TPA-Based Zinc Chelators as Metallo-ß-lactamase Inhibitors.

The rise of antimicrobial resistance (AMR) worldwide and the increasing spread of multi-drug-resistant organisms expressing metallo-ß-lactamases (MBL) require the development of efficient and clinically available MBL inhibitors. At present, no such inhibitor is available, and research is urgently needed to advance this field. We report herein the development, synthesis, and biological evaluation of chemical compounds based on the selective zinc chelator tris-picolylamine (TPA) that can restore the bactericidal activity of Meropenem (MEM) against Pseudomonas aeruginosa and Klebsiella pneumoniae expressing carbapenemases Verona integron-encoded metallo-ß-lactamase (VIM-2) and New Delhi metallo-ß-lactamase 1 (NDM-1), respectively. These adjuvants were prepared via standard chemical methods and evaluated in biological assays for potentiation of MEM against bacteria and toxicity (IC50) against HepG2 human liver carcinoma cells. One of the best compounds, 15, lowered the minimum inhibitory concentration (MIC) of MEM by a factor of 32-256 at 50 µM within all tested MBL-expressing clinical isolates and showed no activity toward serine carbapenemase expressing isolates. Biochemical assays with purified VIM-2 and NDM-1 and 15 resulted in inhibition kinetics with kinact/ KI of 12.5 min-1 mM-1 and 0.500 min-1 mM-1, respectively. The resistance frequency of 15 at 50 µM was in the range of 10-7 to 10-9. 15 showed good tolerance in HepG2 cells with an IC50 well above 100 µM, and an in vivo study in mice showed no acute toxic effects even at a dose of 128 mg/kg.