Legionella pneumophila is the agent of Legionnaires' disease. It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells. The molecular mechanisms of the Legionella interaction with target cells are not fully defined. In an attempt to discover novel virulence factors of L. pneumophila, we searched for bacterial enzymes with transferase activity. Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells. An approximately 60-kDa UDP-glucosyltransferase was purified from L. pneumophila and subjected to microsequencing. An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished L. pneumophila genome database. The intact gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase. We designated this gene ugt (UDP-glucosyltransferase). The Legionella enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function. Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in L. pneumophila strains but was absent from the other Legionella species. This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote L. pneumophila proliferation.Legionella pneumophila is the principal agent of Legionnaires' disease, a pneumonic illness of humans. This bacterium is a facultative intracellular parasite that invades and proliferates in both professional and nonprofessional phagocytes (54). The host cells for L. pneumophila include freshwater protozoans and human alveolar macrophages and epithelial cells. Bacterial factors that have been identified as crucial for intracellular infection include, among others, the major outer membrane protein (25), Mip (13), Hsp60 (17, 18), PilD and type II protein secretion (4, 43), type IV pili (29, 51), flagella (41), the Dot/Icm type IV protein secretion system, and the products of the enh, lvh, mil, and pmi loci (2,11,14,16,35,49). In addition to these virulence factors, a variety of enzymes have been identified in Legionella cultures, including a lowmolecular-mass cytotoxin (30), a metalloprotease cytolysin (8, 9), phospholipases (3, 4, 6), lipases (3), phosphatases (5, 44) and phosphokinases (45). For many of these activities, the role in virulence is unclear and/or the eukaryotic substrate(s) is unknown. Thus, further studies aimed at identifying and characterizing novel L. pneumophila enzymes should be important for understanding both Legionnaires' disease and bacterial interactions with eukary...

Clostridium difficile and Clostridium perfringens type A are infectious agents of enteric diseases. The main virulence factors of these microorganisms include toxins A and B of C. difficile (ToxA and ToxB) and enterotoxin of C. perfringens (Cpe). In this study genetic constructions have been created for the expression of ToxA, ToxB and Cpe fragments either as individual components or as a hybrid multidomain (ToxA-ToxB-Cpe) protein. Rabbit monospecific sera raised against individual peptides reacted with the chimeric product indicating that the corresponding antigenic determinants were correctly expressed on the hybrid molecule. Furthermore, mice immunized with the fusion protein produced antibodies specific to each of the three separate components. These data suggest that the constructed three-domain molecule could be used in future studies for development of a vaccine against enteric clostridial diseases.

Uptake of microorganisms by eukaryotic cells depends on proper functioning of the actin machinery. It creates a driving force for the cell membrane deformations necessary for ingestion and killing of microbes by phagocytes. Therefore, specific alterations in the activity of the actin apparatus could be favorable for pathogenic bacteria, representing an efficient mechanism in their virulence. Such alterations are supposed to be achieved in two principle ways. One is accomplished via binding of bacterial ligands to certain surface receptors, which initiate subsequent actin cytoskeleton rearrangements. Another is to introduce cytoskeleton-targeted products directly into eukaryotic cells and in this way modulate the activity of the actin apparatus. Indeed, Legionella and some other intracellular parasites possess ligands able to stimulate certain receptors on the surface of phagocytes and possess devices suitable for translocation of effector molecules into eukaryotic cytoplasm. The results of such events could be increased uptake of these microbes and their subsequent transportation to permit multiplication in their intracellular niche. On the contrary, representatives of Clostridium and a number of other extracellular pathogens create products which penetrate eukaryotic cells and disorganize the actin cytoskeleton network, thus making uptake of these pathogens by phagocytes impossible.