Vom Huhn abgeleitete Antikörper für Diagnostik und Immuntherapie

Due to the large evolutionary distance between birds (Aves) und humans, immunization of chickens with human proteins results in a strong response of the bird’s adaptive immune system to proteins of mammalian origin. Additionally, chicken-derived antibodies display less undesired cross-reactivity in analytical setups than conventional rodent-derived antibodies. Due to these features as well as the facile amplification of antibody-coding genes, chicken-derived antibodies emerged as promising molecules for the immunotherapy and various biotechnological applications.

Glycoproteins of Predicted Amphibian and Reptile Lyssaviruses can Mediate Infection of Mammalian and Reptile Cells

Lyssaviruses are neurotropic rhabdoviruses thought to be restricted to mammalian hosts, and to originate from bats. The identification of lyssavirus sequences from amphibians and reptiles by metatranscriptomics thus comes as a surprise and challenges the mammalian origin of lyssaviruses. The novel sequences of the proposed American tree frog lyssavirus (ATFLV) and anole lizard lyssavirus (ALLV) reveal substantial phylogenetic distances from each other and from bat lyssaviruses, with ATFLV being the most distant. As virus isolation has not been successful yet, we have here studied the functionality of the authentic ATFLV- and ALLV-encoded glycoproteins in the context of rabies virus pseudotype particles. Cryogenic electron microscopy uncovered the incorporation of the plasmid-encoded G proteins in viral envelopes. Infection experiments revealed the infectivity of ATFLV and ALLV G-coated RABV pp for a broad spectrum of cell lines from humans, bats, and reptiles, demonstrating membrane fusion activities. As presumed, ATFLV and ALLV G RABV pp escaped neutralization by human rabies immune sera. The present findings support the existence of contagious lyssaviruses in poikilothermic animals, and reveal a broad cell tropism in vitro, similar to that of the rabies virus.

Structural and biophysical aspects of L-asparaginases: a growing family with amazing diversity

L-Asparaginases have remained an intriguing research topic since their discovery ∼120 years ago, especially after their introduction in the 1960s as very efficient antileukemic drugs. In addition to bacterial asparaginases, which are still used to treat childhood leukemia, enzymes of plant and mammalian origin are now also known. They have all been structurally characterized by crystallography, in some cases at outstanding resolution. The structural data have also shed light on the mechanistic details of these deceptively simple enzymes. Yet, despite all this progress, no better therapeutic agents have been found to beat bacterial asparaginases. However, a new option might arise with the discovery of yet another type of asparaginase, those from symbiotic nitrogen-fixing Rhizobia, and with progress in the protein engineering of enzymes with desired properties. This review surveys the field of structural biology of L-asparaginases, focusing on the mechanistic aspects of the well established types and speculating about the potential of the new members of this amazingly diversified family.

Screening of Mosquitoes for West Nile Virus and Usutu Virus in Croatia, 2015–2020

In the period from 2015 to 2020, an entomological survey for the presence of West Nile virus (WNV) and Usutu virus (USUV) in mosquitoes was performed in northwestern Croatia. A total of 20,363 mosquitoes were sampled in the City of Zagreb and Međimurje county, grouped in 899 pools and tested by real-time RT-PCR for WNV and USUV RNA. All pools were negative for WNV while one pool each from 2016 (Aedes albopictus), 2017 (Culex pipiens complex), 2018 (Cx. pipiens complex), and 2019 (Cx. pipiens complex), respectively, was positive for USUV. The 2018 and 2019 positive pools shared 99.31% nucleotide homology within the USUV NS5 gene and both clustered within USUV Europe 2 lineage. The next-generation sequencing of one mosquito pool (Cx. pipiens complex) collected in 2018 in Zagreb confirmed the presence of USUV and revealed several dsDNA and ssRNA viruses of insect, bacterial and mammalian origin.

Biological Functions of Prokaryotic Amyloids in Interspecies Interactions: Facts and Assumptions

Amyloids are fibrillar protein aggregates with an ordered spatial structure called “cross-β”. While some amyloids are associated with development of approximately 50 incurable diseases of humans and animals, the others perform various crucial physiological functions. The greatest diversity of amyloids functions is identified within prokaryotic species where they, being the components of the biofilm matrix, function as adhesins, regulate the activity of toxins and virulence factors, and compose extracellular protein layers. Amyloid state is widely used by different pathogenic bacterial species in their interactions with eukaryotic organisms. These amyloids, being functional for bacteria that produce them, are associated with various bacterial infections in humans and animals. Thus, the repertoire of the disease-associated amyloids includes not only dozens of pathological amyloids of mammalian origin but also numerous microbial amyloids. Although the ability of symbiotic microorganisms to produce amyloids has recently been demonstrated, functional roles of prokaryotic amyloids in host–symbiont interactions as well as in the interspecies interactions within the prokaryotic communities remain poorly studied. Here, we summarize the current findings in the field of prokaryotic amyloids, classify different interspecies interactions where these amyloids are involved, and hypothesize about their real occurrence in nature as well as their roles in pathogenesis and symbiosis.

Unexpected Prevalence of eae-Positive Escherichia coli in the Animas River, Durango, Colorado

Since 2014, biology students at Fort Lewis College have studied the water quality of the Animas River in Durango, Colorado. Environmental microbiology and molecular biology techniques have been employed to study Escherichia coli isolates from the river and to define characteristics of the bacteria related to public health. E. coli was found in the river, as well as in culverts and tributary creeks that drain into the river within the Durango city limits. Concentrations of E. coli in the river occasionally exceeded the US EPA guideline of 126 CFU per 100 mL for recreational water use. Many of the E. coli isolates were able to be grown at 45 °C, an indication of mammalian origin. Unexpectedly, 8% of the isolates contained the intimin (eae) gene, a virulence gene characteristic of two pathotypes of E. coli, the enterohemorrhagic and enteropathogenic E. coli. Several isolates tested were resistant to multiple antibiotics commonly used in animal and human medicine. Further study is warranted to determine the source of these bacteria entering the Animas River, and to further characterize the possible disease potential of multi-antibiotic resistant and virulence gene-containing isolates found in a semi-rural/urban setting.

Cytoplasmic glycoengineering enables biosynthesis of nanoscale glycoprotein assemblies

Glycosylation of proteins profoundly impacts their physical and biological properties. Yet our ability to engineer novel glycoprotein structures remains limited. Established bacterial glycoengineering platforms require secretion of the acceptor protein to the periplasmic space and preassembly of the oligosaccharide substrate as a lipid-linked precursor, limiting access to protein and glycan substrates respectively. Here, we circumvent these bottlenecks by developing a facile glycoengineering platform that operates in the bacterial cytoplasm. The Glycoli platform leverages a recently discovered site-specific polypeptide glycosyltransferase together with variable glycosyltransferase modules to synthesize defined glycans, of bacterial or mammalian origin, directly onto recombinant proteins in the E. coli cytoplasm. We exploit the cytoplasmic localization of this glycoengineering platform to generate a variety of multivalent glycostructures, including self-assembling nanomaterials bearing hundreds of copies of the glycan epitope. This work establishes cytoplasmic glycoengineering as a powerful platform for producing glycoprotein structures with diverse future biomedical applications.

Lipase-Catalyzed Chemoselective Ester Hydrolysis of Biomimetically Coupled Aryls for the Synthesis of Unsymmetric Biphenyl Esters

Lipases are among the most frequently used biocatalysts in organic synthesis, allowing numerous environmentally friendly and inexpensive chemical transformations. Here, we present a biomimetic strategy based on iron(III)-catalyzed oxidative coupling and selective ester monohydrolysis using lipases for the synthesis of unsymmetric biphenyl-based esters under mild conditions. The diverse class of biphenyl esters is of pharmaceutical and technical relevance. We explored the potency of a series of nine different lipases of bacterial, fungal, and mammalian origin on their catalytic activities to cleave biphenyl esters, and optimized the reaction conditions, in terms of reaction time, temperature, pH, organic solvent, and water–organic solvent ratios, to improve the chemoselectivity, and hence control the ratio of unsymmetric versus symmetric products. Elevated temperature and increased DMSO content led to an almost exclusive monohydrolysis by the four lipases Candida rugosa lipase (CRL), Mucor miehei lipase (MML), Rhizopus niveus lipase (RNL), and Pseudomonas fluorescens lipase (PFL). The study was complemented by in silico binding predictions to rationalize the observed differences in efficacies of the lipases to convert biphenyl esters. The optimized reaction conditions were transferred to the preparative scale with high yields, underlining the potential of the presented biomimetic approach as an alternative strategy to the commonly used transition metal-based strategies for the synthesis of diverse biphenyl esters.

Biofilm production of coagulase-negative staphylococci isolated from rescued wild animals in the Republic of Korea

Biofilm production is a well-known causative factor of catheter- and medical device-related sepsis. Its high prevalence in coagulase-negative staphylococci (CoNS) has recently been reported. Information on biofilm production in CoNS isolated from wild animals is lacking. Herein, we studied the biofilm formation capabilities of CoNS isolated from rescued wild animals in the Republic of Korea. Swab samples were collected from the conjunctiva, nasal cavity, perianal area, and rectum for mammals while the sampling was done from the conjunctiva, oral mucosa, pericloacal area, and cloaca for birds. Isolation of CoNS was based on morphological and biochemical analyses along with molecular typing. Biofilm production was analyzed using 96-well plate based quantitative adherence assays. The studies demonstrated that CoNS of mammalian origin have higher biofilm-producing ability (70.4%) than the isolates from birds (62.5%). In particular, all methicillin-resistant (MR) CoNS isolated from mammals were capable of biofilm formation while only 63.3% of MR CoNS isolated from birds could produce biofilms. The MR CoNS isolated from mammals also had a significantly higher ability to form biofilms (100%) than methicillin susceptible CoNS (60.0%) than those isolates from birds. The findings show that wild animals may act as reservoirs as well as possible transmitters of biofilm-mediated antibiotic resistant genes.