Toxin:antitoxin ratio sensing autoregulation of the Vibrio cholerae parDE2 module.

The parDE family of toxin-antitoxin (TA) operons is ubiquitous in bacterial genomes and, in Vibrio cholerae, is an essential component to maintain the presence of chromosome II. Here, we show that transcription of the V. cholerae parDE2 (VcparDE) operon is regulated in a toxin:antitoxin ratio-dependent manner using a molecular mechanism distinct from other type II TA systems. The repressor of the operon is identified as an assembly with a 6:2 stoichiometry with three interacting ParD2 dimers bridged by two ParE2 monomers. This assembly docks to a three-site operator containing 5'- GGTA-3' motifs. Saturation of this TA complex with ParE2 toxin results in disruption of the interface between ParD2 dimers and the formation of a TA complex of 2:2 stoichiometry. The latter is operator binding-incompetent as it is incompatible with the required spacing of the ParD2 dimers on the operator.

Entropic pressure controls the oligomerization of the Vibrio cholerae ParD2 antitoxin.

ParD2 is the antitoxin component of the parDE2 toxin-antitoxin module from Vibrio cholerae and consists of an ordered DNA-binding domain followed by an intrinsically disordered ParE-neutralizing domain. In the absence of the C-terminal intrinsically disordered protein (IDP) domain, V. cholerae ParD2 (VcParD2) crystallizes as a doughnut-shaped hexadecamer formed by the association of eight dimers. This assembly is stabilized via hydrogen bonds and salt bridges rather than by hydrophobic contacts. In solution, oligomerization of the full-length protein is restricted to a stable, open decamer or dodecamer, which is likely to be a consequence of entropic pressure from the IDP tails. The relative positioning of successive VcParD2 dimers mimics the arrangement of Streptococcus agalactiae CopG dimers on their operator and allows an extended operator to wrap around the VcParD2 oligomer.

Alternative dimerization is required for activity and inhibition of the HEPN ribonuclease RnlA.

The rnlAB toxin-antitoxin operon from Escherichia coli functions as an anti-phage defense system. RnlA was identified as a member of the HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) superfamily of ribonucleases. The activity of the toxin RnlA requires tight regulation by the antitoxin RnlB, the mechanism of which remains unknown. Here we show that RnlA exists in an equilibrium between two different homodimer states: an inactive resting state and an active canonical HEPN dimer. Mutants interfering with the transition between states show that canonical HEPN dimerization via the highly conserved RX4-6H motif is required for activity. The antitoxin RnlB binds the canonical HEPN dimer conformation, inhibiting RnlA by blocking access to its active site. Single-alanine substitutions mutants of the highly conserved R255, E258, R318 and H323 show that these residues are involved in catalysis and substrate binding and locate the catalytic site near the dimer interface of the canonical HEPN dimer rather than in a groove located between the HEPN domain and the preceding TBP-like domain. Overall, these findings elucidate the structural basis of the activity and inhibition of RnlA and highlight the crucial role of conformational heterogeneity in protein function.

The Escherichia coli RnlA-RnlB toxin-antitoxin complex: production, characterization and crystallization.

The Escherichia coli rnlAB operon encodes a toxin-antitoxin module that is involved in protection against infection by bacteriophage T4. The full-length RnlA-RnlB toxin-antitoxin complex as well as the toxin RnlA were purified to homogeneity and crystallized. When the affinity tag is placed on RnlA, RnlB is largely lost during purification and the resulting crystals exclusively comprise RnlA. A homogeneous preparation of RnlA-RnlB containing stoichiometric amounts of both proteins could only be obtained using a His tag placed C-terminal to RnlB. Native mass spectrometry and SAXS indicate a 1:1 stoichiometry for this RnlA-RnlB complex. Crystals of the RnlA-RnlB complex belonged to space group C2, with unit-cell parameters a = 243.32, b = 133.58, c = 55.64 Å, ß = 95.11°, and diffracted to 2.6 Šresolution. The presence of both proteins in the crystals was confirmed and the asymmetric unit is likely to contain a heterotetramer with RnlA2:RnlB2 stoichiometry.

In planta expression of nanobody-based designer chicken antibodies targeting Campylobacter.

Campylobacteriosis is a widespread infectious disease, leading to a major health and economic burden. Chickens are considered as the most common infection source for humans. Campylobacter mainly multiplies in the mucus layer of their caeca. No effective control measures are currently available, but passive immunisation of chickens with pathogen-specific maternal IgY antibodies, present in egg yolk of immunised chickens, reduces Campylobacter colonisation. To explore this strategy further, anti-Campylobacter nanobodies, directed against the flagella and major outer membrane proteins, were fused to the constant domains of chicken IgA and IgY, combining the benefits of nanobodies and the effector functions of the Fc-domains. The designer chimeric antibodies were effectively produced in leaves of Nicotiana benthamiana and seeds of Arabidopsis thaliana. Stable expression of the chimeric antibodies in seeds resulted in production levels between 1% and 8% of the total soluble protein. These in planta produced antibodies do not only bind to their purified antigens but also to Campylobacter bacterial cells. In addition, the anti-flagellin chimeric antibodies are reducing the motility of Campylobacter bacteria. These antibody-containing Arabidopsis seeds can be tested for oral passive immunisation of chickens and, if effective, the chimeric antibodies can be produced in crop seeds.