Emergence of highly pathogenic avian influenza viruses H5N1 and H5N5 in white-tailed eagles, 2021-2023.

Highly pathogenic avian influenza (HPAI) poses a substantial threat to several raptors. Between 2021 and 2023, HPAI viruses (HPAIVs) of the Goose/Guangdong lineage H5 clade 2.3.4.4b became widespread in wild birds in Norway, and H5N1 and H5N5 viruses were detected in 31 white-tailed eagles (Haliaeetus albicilla, WTEs). Post-mortem examinations of four WTEs revealed no macroscopic pathological findings. Microscopic examinations showed the presence of myocardial and splenic necroses and a few lesions in the brain. In situ hybridization revealed the presence of the virus in several organs, suggesting a multisystemic infection. The detection of HPAIV H5N5 in a WTE in February 2022 marked the first recorded occurrence of this subtype in Norway. Since then, the virus has persisted, sporadically being detected in WTEs and other wild bird species. Phylogenetic analyses reveal that at least two distinct incursions of HPAIV H5N1 Eurasian (EA) genotype C affected WTEs, likely introduced by migratory birds from Eurasia and seabirds entering from Western and Central Europe. Some WTE isolates from 2021 to 2022 clustered with those from Canada and Ireland, aligning with the transatlantic spread of H5N1. Others were related to the 2021 mass mortality of great skuas in the UK or outbreaks in seabird populations, including gannets, gulls and terns, during 2022 in the North Sea region. This suggests that the WTEs were likely preying on the affected birds. Our study highlights that WTEs can act as sentinels for some HPAIV strains, but the absence of several known circulating genotypes in WTEs suggests varying pathogenic effects on this species.

A novel proteinaceous molecule produced by Lysinibacillus sp. OF-1 depends on the Ami oligopeptide transporter to kill Streptococcus pneumoniae.

Infections caused by antibiotic-resistant Streptococcus pneumoniae are of growing concern for healthcare systems, which need new treatment options. Screening microorganisms in terrestrial environments has proved successful for discovering antibiotics, while production of antimicrobials by marine microorganisms remains underexplored. Here we have screened microorganisms sampled from the Oslo Fjord in Norway for production of molecules that prevent the human pathogen S. pneumoniae from growing. A bacterium belonging to the genus Lysinibacillus was identified. We show that this bacterium produces a molecule that kills a wide range of streptococcal species. Genome mining in BAGEL4 and AntiSmash suggested that it was a new antimicrobial compound, and we therefore named it lysinicin OF. The compound was resistant to heat (100 °C) and polymyxin acylase but susceptible to proteinase K, showing that it is of proteinaceous nature, but most probably not a lipopeptide. S. pneumoniae became resistant to lysinicin OF by obtaining suppressor mutations in the ami locus, which encodes the AmiACDEF oligo peptide transporter. We created ΔamiC and ΔamiEF mutants to show that pneumococci expressing a compromised Ami system were resistant to lysinicin OF. Furthermore, by creating mutants expressing an intact but inactive Ami system (AmiED184A and AmiFD175A) we could conclude that the lysinicin OF activity depended on the active form (ATP-hydrolysing) of the Ami system. Microscopic imaging and fluorescent labelling of DNA showed that S. pneumoniae treated with lysinicin OF had an average reduced cell size with condensed DNA nucleoid, while the integrity of the cell membrane remained intact. The characteristics and possible mode of action of lysinicin OF are discussed.

Stress-induced inhibition of translation independently of eIF2α phosphorylation.

Exposure of fission yeast cells to ultraviolet (UV) light leads to inhibition of translation and phosphorylation of the eukaryotic initiation factor-2α (eIF2α). This phosphorylation is a common response to stress in all eukaryotes. It leads to inhibition of translation at the initiation stage and is thought to be the main reason why stressed cells dramatically reduce protein synthesis. Phosphorylation of eIF2α has been taken as a readout for downregulation of translation, but the role of eIF2α phosphorylation in the downregulation of general translation has not been much investigated. We show here that UV-induced global inhibition of translation in fission yeast cells is independent of eIF2α phosphorylation and the eIF2α kinase general control nonderepressible-2 protein (Gcn2). Also, in budding yeast and mammalian cells, the UV-induced translational depression is largely independent of GCN2 and eIF2α phosphorylation. Furthermore, exposure of fission yeast cells to oxidative stress generated by hydrogen peroxide induced an inhibition of translation that is also independent of Gcn2 and of eIF2α phosphorylation. Our findings show that stress-induced translational inhibition occurs through an unknown mechanism that is likely to be conserved through evolution.

Decreased susceptibility to viscosin in Streptococcus pneumoniae.

Growing numbers of infections caused by antibiotic-resistant Streptococcus pneumoniae strains are a major concern for healthcare systems that will require new antibiotics for treatment as well as preventative measures that reduce the number of infections. Lipopeptides are antimicrobial molecules, of which some are used as antibiotics, including the last resort antibiotics daptomycin and polymyxins. Here we have studied the antimicrobial effect of the cyclic lipopeptide viscosin on S. pneumoniae growth and morphology. Most lipopeptides function as surfactants that create pores in membrane layers, which is regarded as their main antimicrobial activity. We show that viscosin can inhibit growth of S. pneumoniae without disintegration of the cytoplasmic membrane. Instead, the cells developed abnormal shapes and misplaced new division sites. The cell wall of these bacteria appeared less dense in electron microscopy images, suggesting that viscosin interfered with normal cell wall synthesis. Corroborating this observation, a luciferase reporter assay was used to show that the two-component systems LiaFSR and CiaRH, which are known to be activated upon cell wall stress, were strongly induced by viscosin. Furthermore, a mutant displaying 1.8-fold decreased susceptibility to viscosin was generated by sequential exposure to increasing concentrations of the lipopeptide. The mutant suffered from significant fitness loss and had mutations in genes involved in fatty acid synthesis, teichoic acid synthesis, and cell wall synthesis as well as transcription and translation. How these mutations might be linked to decreased viscosin susceptibility is discussed.IMPORTANCEStreptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis, and meningitis in children, and the incidence of infections caused by antibiotic-resistant strains is increasing. Development of new antibiotics is therefore necessary to treat these types of infections in the future. Here, we have studied the activity of the antimicrobial lipopeptide viscosin on S. pneumoniae and show that in addition to having the typical membrane destabilizing activity of lipopeptides, viscosin inhibits pneumococcal growth by obstructing normal cell wall synthesis. This suggests a more specific mode of action than just the surfactant activity. Furthermore, we show that S. pneumoniae does not easily acquire resistance to viscosin, which makes it a promising molecule to explore further, for example, by synthesizing less toxic derivates that can be tested for therapeutic potential.

Multiple transatlantic incursions of highly pathogenic avian influenza clade 2.3.4.4b A(H5N5) virus into North America and spillover to mammals.

Highly pathogenic avian influenza (HPAI) viruses have spread at an unprecedented scale, leading to mass mortalities in birds and mammals. In 2023, a transatlantic incursion of HPAI A(H5N5) viruses into North America was detected, followed shortly thereafter by a mammalian detection. As these A(H5N5) viruses were similar to contemporary viruses described in Eurasia, the transatlantic spread of A(H5N5) viruses was most likely facilitated by pelagic seabirds. Some of the Canadian A(H5N5) viruses from birds and mammals possessed the PB2-E627K substitution known to facilitate adaptation to mammals. Ferrets inoculated with A(H5N5) viruses showed rapid, severe disease onset, with some evidence of direct contact transmission. However, these viruses have maintained receptor binding traits of avian influenza viruses and were susceptible to oseltamivir and zanamivir. Understanding the factors influencing the virulence and transmission of A(H5N5) in migratory birds and mammals is critical to minimize impacts on wildlife and public health.

Tetraploid Ancestry Provided Atlantic Salmon With Two Paralogue Functional T Cell Receptor Beta Regions Whereof One Is Completely Novel.

Protective cellular immune responses have been difficult to study in fish, due to lack of basic understanding of their T cell populations, and tools to study them. Cellular immunity is thus mostly ignored in vaccination and infection studies compared to humoral responses. High throughput sequencing, as well as access to well assembled genomes, now advances studies of cellular responses. Here we have used such resources to describe organization of T cell receptor beta genes in Atlantic salmon. Salmonids experienced a unique whole genome duplication approximately 94 million years ago, which provided these species with many functional duplicate genes, where some duplicates have evolved new functions or sub-functions of the original gene copy. This is also the case for T cell receptor beta, where Atlantic salmon has retained two paralogue T cell receptor beta regions on chromosomes 01 and 09. Compared to catfish and zebrafish, the genomic organization in both regions is unique, each chromosomal region organized with dual variable- diversity- joining- constant genes in a head to head orientation. Sequence identity of the chromosomal constant sequences between TRB01 and TRB09 is suggestive of rapid diversification, with only 67 percent as opposed to the average 82-90 percent for other duplicated genes. Using virus challenged samples we find both regions expressing bona fide functional T cell receptor beta molecules. Adding the 292 variable T cell receptor alpha genes to the 100 variable TRB genes from 14 subgroups, Atlantic salmon has one of the most diverse T cell receptor alpha beta repertoire of any vertebrate studied so far. Perhaps salmonid cellular immunity is more advanced than we have imagined.

Engineering Lactococcus lactis for Increased Vitamin K2 Production.

Cheese produced with Lactococcus lactis is the main source of vitamin K2 in the Western diet. Subclinical vitamin K2 deficiency is common, calling for foods with enhanced vitamin K2 content. In this study we describe analyses of vitamin K2 (menaquinone) production in the lactic acid bacterium L. lactis ssp. cremoris strain MG1363. By cloning and expression from strong promoters we have identified genes and bottlenecks in the biosynthetic pathways leading to the long-chained menaquinones, MK-8 and MK-9. Key genes of the biosynthetic menaquinone pathway were overexpressed, singly or combined, to examine how vitamin K2 production can be enhanced. We observed that the production of the long menaquinone polyprenyl side chain, rather than production of the napthoate ring (1,4-dihydroxy-2-naphtoic acid), limits total menaquinone synthesis. Overexpression of genes causing increased ring formation (menF and menA) led to overproduction of short chained MK-3, while overexpression of other key genes (mvk and llmg_0196) resulted in enhanced full-length MK-9 production. Of two putatively annotated prenyl diphosphate synthases we pinpoint llmg_0196 (preA) to be important for menaquinone production in L. lactis. The genes mvk, preA, menF, and menA were found to be important contributors to menaquinone levels as single overexpression of these genes double and more than triple the total menaquinone content in culture. Combined overexpression of mvk, preA, and menA increased menaquinone levels to a higher level than obtained individually. When the overproducing strains were applied for milk fermentations vitamin K2 content was effectively increased 3-fold compared to the wild type. The results provide a foundation for development of strains to ferment foods with increased functional value i.e., higher vitamin K2 content.