Generation and characterization of hagfish variable lymphocyte receptor B against glycoprotein of viral hemorrhagic septicemia virus (VHSV).

Variable lymphocyte receptors B (VLRBs) are non-immunoglobulin components of the humoral immune system in jawless vertebrates including hagfish (Eptatretus burgeri) and lamprey (Petromyzon marinus). Hagfish VLRBs consist of leucine rich repeat (LRR) modules with a superhydrophobic C-terminal tail, the latter of which leads to extremely low expression levels in recombinant protein technology. Here, we present an artificially oligomerized VLRB (arVLRB) that conjugates via the C4bp oligomerization domain derived from human C4b-binding protein (hC4bp) rather than the superhydrophobic tail. The resulting arVLRB had a tightly multimerized form with seven monomeric VLRB arms and showed high expression and secretion levels in a mammalian expression system. To isolate antigen-specific arVLRB, we constructed large VLRB libraries from hagfish immunized with the fish pathogen, viral hemorrhagic septicemia virus (VHSV). The selected arVLRBs were found to recognize various types of antigens, including the recombinant target protein, purified viruses, and progeny viruses, with high antigen binding abilities and specificities. We also performed in vitro affinity maturation of the arVLRBs through LRRCT mutagenesis, and found that this enhanced their antigen-binding properties by at least 125-fold. Our epitope mapping analysis revealed that 37DWDTPL42, which is located in a region conserved among the glycoproteins of all VHSV isolates, is the recognition epitope of the arVLRBs. Thus, our newly developed arVLRB could prove useful in the development of universal diagnostic tools and/or therapeutic agents for the virus. Together, our novel findings provide valuable insights into hagfish VLRB and its potential use as a novel alternative to conventional antibodies for biotechnological applications.

Outer membrane vesicles from ß-lactam-resistant Escherichia coli enable the survival of ß-lactam-susceptible E. coli in the presence of ß-lactam antibiotics.

Outer membrane vesicles (OMVs) containing various bacterial compounds are released from mainly gram-negative bacteria. Secreted OMVs play important roles in the ability of a bacterium to defend itself, and thus contribute to the survival of bacteria in a community. In this study, we collected OMVs from ß-lactam antibiotic-resistant Escherichia coli established by conjugation assay and the parental ß-lactam antibiotic-susceptible strain, and performed comparative proteomic analysis to examine whether these OMVs carried ß-lactam-resistant compounds. We also investigated whether both types of OMVs could protect susceptible cells from ß-lactam-induced death and/or directly degrade ß-lactam antibiotics. Several proteins that can be involved in degrading ß-lactam antibiotics were more abundant in OMVs from ß-lactam-resistant E. coli, and thus OMVs from ß-lactam resistant E. coli could directly and dose-dependently degrade ß-lactam antibiotics and fully rescue ß-lactam-susceptible E. coli and other bacterial species from ß-lactam antibiotic-induced growth inhibition. Taken together, present study demonstrate that OMVs from ß-lactam-resistant E. coli play important roles in survival of antibiotic susceptible bacteria against ß-lactam antibiotics. This finding may pave the way for new efforts to combat the current global spread of antibiotic resistances, which is considered to be a significant public health threat.