Attaching and effacing Escherichia coli cause diarrhea and typically produce lymphostatin (LifA), an inhibitor of mitogen-activated proliferation of lymphocytes and pro-inflammatory cytokine synthesis. A near-identical factor (Efa1) has been reported to mediate adherence of E. coli to epithelial cells. An amino-terminal region of LifA shares homology with the catalytic domain of the large clostridial toxins, which are retaining glycosyltransferases with a DXD motif involved in binding of a metal ion. Understanding the mode(s) of action of lymphostatin has been constrained by difficulties obtaining a stably transformed plasmid expression clone. We constructed a tightly inducible clone of enteropathogenic E. coli O127:H6 lifA for affinity purification of lymphostatin. The purified protein inhibited mitogen-activated proliferation of bovine T lymphocytes in the femtomolar range. It is a monomer in solution and the molecular envelope was determined using both transmission electron microscopy and small-angle x-ray scattering. Domain architecture was further studied by limited proteolysis. The largest proteolytic fragment containing the putative glycosyltransferase domain was tested in isolation for activity against T cells, and was not sufficient for activity. Tryptophan fluorescence studies indicated thatlymphostatin binds uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) but not UDP-glucose (UDP-Glc). Substitution of the predicted DXD glycosyltransferase motif with alanine residues abolished UDP-GlcNAc binding and lymphostatin activity, although other biophysical properties were unchanged. The data indicate that lymphostatin has UDP-sugar binding potential that is critical for activity, and is a major leap toward identifying the nature and consequences of modifications of host cell factors.

Cellulose, chitin and peptidoglycan are major long‐chain carbohydrates in living organisms, and constitute a substantial fraction of the biomass. Characterization of the biochemical basis of dynamic changes and degradation of these β,1–4‐linked carbohydrates is therefore important for both functional studies of biological polymers and biotechnology. Here, we investigated the functional role of multiplicity of the carbohydrate‐binding lysin motif (LysM) domain that is found in proteins involved in bacterial peptidoglycan synthesis and remodelling. The Bacillus subtilis peptidoglycan‐hydrolysing NlpC/P60 d,l‐endopeptidase, cell wall‐lytic enzyme associated with cell separation, possesses four LysM domains. The contribution of each LysM domain was determined by direct carbohydrate‐binding studies in aqueous solution with microscale thermophoresis. We found that bacterial LysM domains have affinity for N‐acetylglucosamine (GlcNac) polymers in the lower‐micromolar range. Moreover, we demonstrated that a single LysM domain is able to bind carbohydrate ligands, and that LysM domains act additively to increase the binding affinity. Our study reveals that affinity for GlcNAc polymers correlates with the chain length of the carbohydrate, and suggests that binding of long carbohydrates is mediated by LysM domain cooperativity. We also show that bacterial LysM domains, in contrast to plant LysM domains, do not discriminate between GlcNAc polymers, and recognize both peptidoglycan fragments and chitin polymers with similar affinity. Finally, an Ala replacement study suggested that the carbohydrate‐binding site in LysM‐containing proteins is conserved across phyla.

The oxidation resistance proteins (OXR) help to protect eukaryotes from reactive oxygen species. The sole C-terminal domain of the OXR, named TLDc is sufficient to perform this function. However, the mechanism by which oxidation resistance occurs is poorly understood. We present here the crystal structure of the TLDc domain of the oxidation resistance protein 2 from zebrafish. The structure was determined by X-ray crystallography to atomic resolution (0.97Å) and adopts an overall globular shape. Two antiparallel β-sheets form a central β-sandwich, surrounded by two helices and two one-turn helices. The fold shares low structural similarity to known structures.

Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile. The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory ␣-helix. A conserved glutamate residue in the inhibitory helix motif was previously shown in other Fic domain proteins to prevent proper binding of the ATP ␥-phosphate. However, here we demonstrate that both ATP binding and autoadenylylation activity of the C. difficile Fic domain protein are independent of the inhibitory motif. In support of this, the crystal structure of a mutant of this Fic protein in complex with ATP reveals that the ␥-phosphate adopts a conformation unique among Fic domains that seems to override the effect of the inhibitory helix. These results provide important structural insight into the adenylylation reaction mechanism catalyzed by Fic domains. Our findings reveal the presence of a class II Fic domain protein in the human pathogen C. difficile that is not regulated by autoinhibition and challenge the current dogma that all class I-III Fic domain proteins are inhibited by the inhibitory ␣-helix.Clostridium difficile is a Gram-positive, anaerobic, and spore-forming pathogen recognized as the leading cause of healthcare-associated diarrhea (1-3). The incidence and severity of these infections have been increasing in the past decade, and treatment is difficult and expensive. The virulence is primarily attributed to increased expression of two large toxins, TcdA and TcdB, which have both been shown to inactivate Rho GTPases in host cells by glycosylation of a threonine residue in the switch I region (4, 5). However, there are contrasting reports on the pathological importance of these toxins. In one hamster study, TcdB was found to be essential for C. difficile virulence (6). Another group found that both the A ϩ B Ϫ and A Ϫ B ϩ strains are able to cause disease in hamsters (7), whereas a third study concluded that TcdA was the major determinant for virulence (8). Adding to the confusion, a highly virulent strain was recently identified where the pathogenic phenotype could not be attributed to increased toxin production. Instead, this strain was shown to hold additional laterally acquired DNA containing genes of hypothetical function (9). Therefore, increased virulence is also likely associated with accessory virulence factors, which have not yet been described.Recently, two bacterial virulence factors, IbpA from Histophilus somni and VopS from Vibrio parahaemolyticus, were also discovered to inactivate host cell Rho GTPases by generating a phosphodiester bond with the AMP moiety on either a conserved ty...

Mycobacterium abscessus is an emerging human pathogen that is notorious for being one of the most drug‐resistant species of Mycobacterium. It has developed numerous strategies to overcome the antibiotic stress response, limiting treatment options and leading to frequent therapeutic failure. The panel of aminoglycosides (AG) usually used in the treatment of M. abscessus pulmonary infections is restricted by chemical modification of the drugs by the N‐acetyltransferase Eis2 protein (Mabs_Eis2). This enzyme acetylates the primary amine of AGs, preventing these antibiotics from binding ribosomal RNA and thereby impairing their activity. In this study, the high‐resolution crystal structures of Mabs_Eis2 in its apo‐ and cofactor‐bound forms were solved. The structural analysis of Mabs_Eis2, supported by the kinetic characterization of the enzyme, highlights the large substrate specificity of the enzyme. Furthermore, in silico docking and biochemical approaches attest that Mabs_Eis2 modifies clinically relevant drugs such as kanamycin and amikacin, with a better efficacy for the latter. In line with previous biochemical and in vivo studies, our work suggests that Mabs_Eis2 represents an attractive pharmacological target to be further explored. The high‐resolution crystal structures presented here may pave the way to the design of Eis2‐specific inhibitors with the potential to counteract the intrinsic resistance levels of M. abscessus to an important class of clinically important antibiotics. Database Structural data are available in the PDB database under the accession numbers: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6RFY, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6RFX and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6RFT.