Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30 to 76% of the cases of neonatal meningitis. In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low-, and non-biofilm-forming strains, and to facilitate interpretation of data. This protocol was used to screen the biofilm-forming abilities of 366 GBS clinical isolates from pregnant women and from neonatal infections of different serotypes in relation to medium composition and pH. The results identified a subset of isolates of serotypes III and V that formed strong biofilms under acidic conditions. Importantly, the best biofilm formers belonged to serotype III hypervirulent clone ST-17. Moreover, the abilities of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm initiation and contribute to biofilm structural stability. Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a leading cause of invasive neonatal infections worldwide. It is a common colonizer of the gastrointestinal and urogenital tracts of up to 40% of healthy individuals (1). However, under certain circumstances, GBS can become a life-threatening pathogen causing invasive infections in human neonates (2, 3). Earlyonset group B streptococcal disease occurs in infants less than 7 days old, and late-onset disease (LOD) occurs in infants between 7 and 89 days old. GBS is usually transmitted from mothers to newborns during childbirth (4), but it can also penetrate the human placenta (5), and in the case of LOD, it can be nosocomially acquired.Historically, GBS isolates have been classified into 10 different serotypes according to their capsular polysaccharide composition (6, 7). Multiple surveillance studies have indicated that all serotypes are able to colonize the vagina and perianal region of pregnant women, but five serotypes (Ia, Ib, II, III, and V) are predominant and are also the most frequent in human infections (8-12). In particular, serotype III accounts for 30 to 76% of neonatal disease cases (13,14). The use of multilocus sequence typing (MLST) allowed the classification of GBS isolates independently from their capsular serotypes and the identification of the bacterial genogroups more often associated with invasive infections in newborns (15). Serotype III isolates of a particular genotype cluster, sequence type 17 (ST-17), disproportionately cause late-onset GBS disease (15-19) and more frequently cause meningitis than other STs do (20). The precise mechanism by which ST-17 causes LOD more frequently than other STs do is not well unde...

Clostridium difficile toxin A (TcdA) is a member of the large clostridial toxin family, and is responsible, together with C. difficile toxin B (TcdB), for many clinical symptoms during human infections. Like other large clostridial toxins, TcdA catalyzes the glucosylation of GTPases, and is able to inactivate small GTPases within the host cell. Here, we report the crystal structures of the TcdA glucosyltransferase domain (TcdA‐GT) in the apo form and in the presence of Mn2+ and hydrolyzed UDP‐glucose. These structures, together with the recently reported crystal structure of TcdA‐GT bound to UDP‐glucose, provide a detailed understanding of the conformational changes of TcdA that occur during the catalytic cycle. Indeed, we present a new intermediate conformation of a so‐called ‘lid’ loop (residues 510–522 in TcdA), concomitant with the absence of glucose in the catalytic domain. The recombinant TcdA was expressed in Brevibacillus in the inactive apo form. High thermal stability of wild‐type TcdA was observed only after the addition of both Mn2+ and UDP‐glucose. The glucosylhydrolase activity, which is readily restored after reconstitution with both these cofactors, was similar to that reported for TcdB. Interestingly, we found that ammonium, like K+, is able to activate the UDP‐glucose hydrolase activities of TcdA. Consequently, the presence of ammonium in the crystallization buffer enabled us to obtain the first crystal structure of TcdA‐GT bound to the hydrolysis product UDP. Database • Coordinates of apo‐TcdA‐GT and Mn2+–UDP–TcdA‐GT are available in the Protein Data Bank under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4DMV and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4DMW, respectively

bClostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.