Functional and structural characterization of PaeM, a colicin M-like bacteriocin produced by Pseudomonas aeruginosa.

Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg(2+) ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.

Repetitive mRNA vaccination is required to improve the quality of broad-spectrum anti-SARS-CoV-2 antibodies in the absence of CXCL13.

Since the initial spread of severe acute respiratory syndrome coronavirus 2 infection, several viral variants have emerged and represent a major challenge for immune control, particularly in the context of vaccination. We evaluated the quantity, quality, and persistence of immunoglobulin G (IgG) and IgA in individuals who received two or three doses of messenger RNA (mRNA) vaccines, compared with previously infected vaccinated individuals. We show that three doses of mRNA vaccine were required to match the humoral responses of preinfected vaccinees. Given the importance of antibody-dependent cell-mediated immunity against viral infections, we also measured the capacity of IgG to recognize spike variants expressed on the cell surface and found that cross-reactivity was also strongly improved by repeated vaccination. Last, we report low levels of CXCL13, a surrogate marker of germinal center activation and formation, in vaccinees both after two and three doses compared with preinfected individuals, providing a potential explanation for the short duration and low quality of Ig induced.

Structural and functional analysis of the fibronectin-binding protein FNE from Streptococcus equi spp. equi.

Streptococcus equi is a horse pathogen belonging to Lancefield group C. Infection by S. equi ssp. equi causes strangles, a serious and highly contagious disease of the upper respiratory tract. S. equi ssp. equi secretes a fibronectin (Fn)-binding protein, FNE, that does not contain cell wall-anchoring motifs. FNE binds to the gelatin-binding domain (GBD) of Fn, composed of the motifs (6) FI (12) FII (789) FI . FNE lacks the canonical Fn-binding peptide repeats observed in many microbial surface components recognizing adhesive matrix molecules. We found that the interaction between FNE and the human GBD is mediated by the binding of the disordered C-terminal region (residues 208-262) of FNE to the (789) FI GBD subfragment. The crystal structure of FNE showed that it is similar to the minor pilus protein Spy0125 of Streptococcus pyogenes, found at the end of pilus polymers and responsible for adhesion. FNE and Spy0125 both have a superimposable internal thioester bond between highly conserved Cys and Gln residues. Small-angle X-ray scattering of the FNE-(789) FI complex provided a model that aligns the C-terminal peptide of FNE with the E-strands of the FI domains, adopting the ß-zipper extension model observed in previous structures of microbial surface components recognizing adhesive matrix molecule adhesion peptides bound to FI domains.