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In contrast to most fishes, salmonids exhibit the unique ability to hold their eggs for several days after ovulation without significant loss of viability. During this period, eggs are held in the body cavity in a biological fluid, the coelomic fluid (CF) that is responsible for preserving egg viability. To identify CF proteins responsible for preserving egg viability, a proteomic comparison was performed using 3 salmonid species and 3 non-salmonid species to identify salmonid-specific highly abundant proteins. In parallel, rainbow trout CF fractions were purified and used in a biological test to estimate their egg viability preservation potential. The most biologically active CF fractions were then subjected to mass spectrometry analysis. We identified 50 proteins overabundant in salmonids and present in analytical fractions with high egg viability preservation potential. The identity of these proteins illuminates the biological processes participating in egg viability preservation. Among identified proteins of interest, the ovarian-specific expression and abundance in CF at ovulation of N-acetylneuraminic acid synthase a (Nansa) suggest a previously unsuspected role. We show that salmonid CF is a complex biological fluid containing a diversity of proteins related to immunity, calcium binding, lipid metabolism, proteolysis, extracellular matrix and sialic acid metabolic pathway that are collectively responsible for preserving egg viability., (© 2024. The Author(s).)

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens that cause human diseases ranging from diarrhea to life-threatening complications including hemolytic-uremic syndrome. Virulence of STEC strains and their ability to cause severe diseases are associated with the activity of prophage-encoded Shiga toxins (Stxs). The first objective of this work was to isolate and characterize the Stx2d phage from STEC O80:H2 and to study the transfer of this phage in non-STEC strains. The second objective was to assess the survival of Galleria mellonella larvae inoculated with these transduced strains. Firstly, one bacteriophage isolated from a STEC O80:H2 strain was used to infect six non-STEC strains, resulting in the conversion of three strains. Then, stability assays were performed, showing that this phage was stable in the new STEC strains after three successive subculturing steps, as confirmed by a combination of short and long read genome sequencing approaches. This phage, vB_EcoS_ULI-O80_Stx2d, is resistant to moderate temperature and pH. It belongs to a currently unclassified genus and family within the Caudoviricetes class, shares 98% identity with Stx2_112808 phage and encodes several proteins involved in the lysogenic cycle. The yecE gene was identified at the insertion site. Finally, G. mellonella experiments showed that the transduced strains caused significantly higher mortality rates than the corresponding non-STEC strains. In conclusion, this study showed that stx2d gene from O80:H2 E. coli can be transferred to non-STEC strains and contributes to their virulence., (© 2022. The Author(s).)

Understanding the composition of the oviduct fluid (OF) is crucial to better comprehend the microenvironment in which sperm capacitation, fertilization and early embryo development take place. Therefore, our aim was to determine the spatiotemporal changes in the OF proteome according to the anatomical region of the oviduct (ampulla vs. isthmus), the proximity of the ovulating ovary (ipsilateral vs. contralateral side) and the peri-ovulatory stage (pre-ovulatory or Pre-ov vs. post-ovulatory or Post-ov). Oviducts from adult cyclic cows were collected at a local slaughterhouse and pools of OF were analyzed by nanoLC-MS/MS and label-free protein quantification (n = 32 OF pools for all region × stage × side conditions). A total of 3760 proteins were identified in the OF, of which 65% were predicted to be potentially secreted. The oviduct region was the major source of variation in protein abundance, followed by the proximity of the ovulating ovary and finally the peri-ovulatory stage. Differentially abundant proteins between regions, stages and sides were involved in a broad variety of biological functions, including protein binding, response to stress, cell-to-cell adhesion, calcium homeostasis and the immune system. This work highlights the dynamic regulation of oviduct secretions and provides new protein candidates for interactions between the maternal environment, the gametes and the early embryo., (© 2022. The Author(s).)

Aminobacter sp. MSH1 (CIP 110285) can use the pesticide dichlobenil and its recalcitrant transformation product, 2,6-dichlorobenzamide (BAM), as sole source of carbon, nitrogen, and energy. The concentration of BAM in groundwater often exceeds the threshold limit for drinking water, requiring additional treatment in drinking water treatment plants or closure of the affected abstraction wells. Biological treatment with MSH1 is considered a potential sustainable alternative to remediate BAM-contamination in drinking water production. We present the complete genome of MSH1, which was determined independently in two institutes at Aarhus University and KU Leuven. Divergences were observed between the two genomes, i.e. one of them lacked four plasmids compared to the other. Besides the circular chromosome and the two previously described plasmids involved in BAM catabolism, pBAM1 and pBAM2, the genome of MSH1 contained two megaplasmids and three smaller plasmids. The MSH1 substrain from KU Leuven showed a reduced genome lacking a megaplasmid and three smaller plasmids and was designated substrain MK1, whereas the Aarhus variant with all plasmids was designated substrain DK1. A plasmid stability experiment indicate that substrain DK1 may have a polyploid chromosome when growing in R2B medium with more chromosomes than plasmids per cell. Finally, strain MSH1 is reassigned as Aminobacter niigataensis MSH1., (© 2021. The Author(s).)

The Paenibacillus larvae infecting phage API480 (vB_PlaP_API480) is the first reported podovirus for this bacterial species, with an 58 nm icosahedral capsid and a 12 × 8 nm short, non-contractile tail. API480 encodes 77 coding sequences (CDSs) on its 45,026 bp dsDNA genome, of which 47 were confirmed using mass spectrometry. This phage has got very limited genomic and proteomic similarity to any other known ones registered in public databases, including P. larvae phages. Comparative genomics indicates API480 is a new species as it's a singleton with 28 unique proteins. Interestingly, the lysis module is highly conserved among P. larvae phages, containing a predicted endolysin and two putative holins. The well kept overall genomic organisation (from the structural and morphogenetic modules to the host lysis, DNA replication and metabolism related proteins) confirms a common evolutionary ancestor among P. larvae infecting phages. API480 is able to infect 69% of the 61 field strains with an ERIC I genotype, as well as ERIC II strains. Furthermore, this phage is very stable when exposed to high glucose concentrations and to larval gastrointestinal conditions. This highly-specific phage, with its broad lytic activity and stability in hive conditions, might potentially be used in the biocontrol of American Foulbrood (AFB).

Pseudomonas phage LKA1 of the subfamily Autographivirinae encodes a tailspike protein (LKA1gp49) which binds and cleaves B-band LPS (O-specific antigen, OSA) of Pseudomonas aeruginosa PAO1. The crystal structure of LKA1gp49 catalytic domain consists of a beta-helix, an insertion domain and a C-terminal discoidin-like domain. The putative substrate binding and processing site is located on the face of the beta-helix whereas the C-terminal domain is likely involved in carbohydrates binding. NMR spectroscopy and mass spectrometry analyses of degraded LPS (OSA) fragments show an O5 serotype-specific polysaccharide lyase specificity. LKA1gp49 reduces virulence in an in vivo Galleria mellonella infection model and sensitizes P. aeruginosa to serum complement activity. This enzyme causes biofilm degradation and does not affect the activity of ciprofloxacin and gentamicin. This is the first comprehensive report on LPS-degrading lyase derived from a Pseudomonas phage. Biological properties reveal a potential towards its applications in antimicrobial design and as a microbiological or biotechnological tool.

Mantle cell lymphoma (MCL) is a hematologic neoplasm characterised by the t(11;14)(q13;q32) translocation leading to aberrant cyclin D1 expression. The cell functions of cyclin D1 depend on its partners and/or subcellular distribution, resulting in different oncogenic properties. We observed the accumulation of cyclin D1 in the cytoplasm of a subset of MCL cell lines and primary cells. In primary cells, this cytoplasmic distribution was correlated with a more frequent blastoid phenotype. We performed immunoprecipitation assays and mass spectrometry on enriched cytosolic fractions from two cell lines. The cyclin D1 interactome was found to include several factors involved in adhesion, migration and invasion. We found that the accumulation of cyclin D1 in the cytoplasm was associated with higher levels of migration and invasiveness. We also showed that MCL cells with high cytoplasmic levels of cyclin D1 engrafted more rapidly into the bone marrow, spleen, and brain in immunodeficient mice. Both migration and invasion processes, both in vivo and in vitro, were counteracted by the exportin 1 inhibitor KPT-330, which retains cyclin D1 in the nucleus. Our data reveal a role of cytoplasmic cyclin D1 in the control of MCL cell migration and invasion, and as a true operator of MCL pathogenesis.

The ability of bacteriophages to kill bacteria is well known, as is their potential use as alternatives to antibiotics. As such, bacteriophages reach high doses locally through infection of their bacterial host in the human body. In this study we assessed the gene expression profile of peripheral blood monocytes from six donors for twelve immunity-related genes (i.e. CD14, CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, IL10, LYZ, SOCS3, TGFBI and TNFA) induced by Staphylococcus aureus phage ISP and four Pseudomonas aeruginosa phages (i.e. PNM, LUZ19, 14-1 and GE-vB_Pae-Kakheti25). The phages were able to induce clear and reproducible immune responses. Moreover, the overall immune response was very comparable for all five phages: down-regulation of LYZ and TGFBI, and up-regulation of CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, SOCS3 and TNFA. The observed immune response was shown to be endotoxin-independent and predominantly anti-inflammatory. Addition of endotoxins to the highly purified phages did not cause an immune response comparable to the one induced by the (endotoxin containing) phage lysate. In addition, the use of an intermediate level of endotoxins tipped the immune response to a more anti-inflammatory response, i.e. up-regulation of IL1RN and a strongly reduced expression of CXCL1 and CXCL5.

Morganella morganii is a common but frequent neglected environmental opportunistic pathogen which can cause deadly nosocomial infections. The increased number of multidrug-resistant M. morganii isolates motivates the search for alternative and effective antibacterials. We have isolated two novel obligatorily lytic M. morganii bacteriophages (vB&#95;MmoM&#95;MP1, vB&#95;MmoP&#95;MP2) and characterized them with respect to specificity, morphology, genome organization and phylogenetic relationships. MP1's dsDNA genome consists of 163,095 bp and encodes 271 proteins, exhibiting low DNA (<40%) and protein (<70%) homology to other members of the Tevenvirinae. Its unique property is a >10 kb chromosomal inversion that encompass the baseplate assembly and head outer capsid synthesis genes when compared to other T-even bacteriophages. MP2 has a dsDNA molecule with 39,394 bp and encodes 55 proteins, presenting significant genomic (70%) and proteomic identity (86%) but only to Morganella bacteriophage MmP1. MP1 and MP2 are then novel members of Tevenvirinae and Autographivirinae, respectively, but differ significantly from other tailed bacteriophages of these subfamilies to warrant proposing new genera. Both bacteriophages together could propagate in 23 of 27 M. morganii clinical isolates of different origin and antibiotic resistance profiles, making them suitable for further studies on a development of bacteriophage cocktail for potential therapeutic applications.

Endolysins constitute a promising class of antibacterials against Gram-positive bacteria. Recently, endolysins have been engineered with selected peptides to obtain a new generation of lytic proteins, Artilysins, with specific activity against Gram-negative bacteria. Here, we demonstrate that artilysation can also be used to enhance the antibacterial activity of endolysins against Gram-positive bacteria and to reduce the dependence on external conditions. Art-240, a chimeric protein of the anti-streptococcal endolysin λSa2lys and the polycationic peptide PCNP, shows a similar species specificity as the parental endolysin, but the bactericidal activity against streptococci increases and is less affected by elevated NaCl concentrations and pH variations. Time-kill experiments and time-lapse microscopy demonstrate that the killing rate of Art-240 is approximately two-fold higher compared to wildtype endolysin λSa2lys, with a reduction in viable bacteria of 3 log units after 10 min. In addition, lower doses of Art-240 are required to achieve the same bactericidal effect., Competing Interests: R.L. and Y.B. act as scientific advisors of Lisando GmbH. W.-L.C., C.H. and M.B. are employees of Lisando GmbH. Artilysin is a registered trademark in the European Union, United States, and other countries.

Bacteriophage therapy is currently resurging as a potential complement/alternative to antibiotic treatment. However, preclinical evaluation lacks streamlined approaches. We here focus on preclinical approaches which have been implemented to assess bacteriophage efficacy against Pseudomonas biofilms and infections. Laser interferometry and profilometry were applied to measure biofilm matrix permeability and surface geometry changes, respectively. These biophysical approaches were combined with an advanced Airway Surface Liquid infection model, which mimics in vitro the normal and CF lung environments, and an in vivo Galleria larvae model. These assays have been implemented to analyze KTN4 (279,593 bp dsDNA genome), a type-IV pili dependent, giant phage resembling phiKZ. Upon contact, KTN4 immediately disrupts the P. aeruginosa PAO1 biofilm and reduces pyocyanin and siderophore production. The gentamicin exclusion assay on NuLi-1 and CuFi-1 cell lines revealed the decrease of extracellular bacterial load between 4 and 7 logs and successfully prevents wild-type Pseudomonas internalization into CF epithelial cells. These properties and the significant rescue of Galleria larvae indicate that giant KTN4 phage is a suitable candidate for in vivo phage therapy evaluation for lung infection applications.

A major limitation for better understanding the role of the human gut virome in health and disease is the lack of validated methods that allow high throughput virome analysis. To overcome this, we evaluated the quantitative effect of homogenisation, centrifugation, filtration, chloroform treatment and random amplification on a mock-virome (containing nine highly diverse viruses) and a bacterial mock-community (containing four faecal bacterial species) using quantitative PCR and next-generation sequencing. This resulted in an optimised protocol that was able to recover all viruses present in the mock-virome and strongly alters the ratio of viral versus bacterial and 16S rRNA genetic material in favour of viruses (from 43.2% to 96.7% viral reads and from 47.6% to 0.19% bacterial reads). Furthermore, our study indicated that most of the currently used virome protocols, using small filter pores and/or stringent centrifugation conditions may have largely overlooked large viruses present in viromes. We propose NetoVIR (Novel enrichment technique of VIRomes), which allows for a fast, reproducible and high throughput sample preparation for viral metagenomics studies, introducing minimal bias. This procedure is optimised mainly for faecal samples, but with appropriate concentration steps can also be used for other sample types with lower initial viral loads.