The crystal structure of dihydrodipicolinate reductase from the human-pathogenic bacterium Bartonella henselae strain Houston-1 at 2.3†Šresolution.

In bacteria, the second committed step in the diaminopimelate/lysine anabolic pathways is catalyzed by the enzyme dihydrodipicolinate reductase (DapB). DapB catalyzes the reduction of dihydrodipicolinate to yield tetrahydrodipicolinate. Here, the cloning, expression, purification, crystallization and X-ray diffraction analysis of DapB from the human-pathogenic bacterium Bartonella henselae, the causative bacterium of cat-scratch disease, are reported. Protein crystals were grown in conditions consisting of 5%(w/v) PEG 4000, 200 mM sodium acetate, 100 mM sodium citrate tribasic pH 5.5 and were shown to diffract to ∼2.3 Šresolution. They belonged to space group P4322, with unit-cell parameters a = 109.38, b = 109.38, c = 176.95 Å. Rr.i.m. was 0.11, Rwork was 0.177 and Rfree was 0.208. The three-dimensional structural features of the enzymes show that DapB from B. henselae is a tetramer consisting of four identical polypeptides. In addition, the substrate NADP+ was found to be bound to one monomer, which resulted in a closed conformational change in the N-terminal domain.

Comparative analysis of glutaredoxin domains from bacterial opportunistic pathogens.

Glutaredoxin proteins (GLXRs) are essential components of the glutathione system that reductively detoxify substances such as arsenic and peroxides and are important in the synthesis of DNA via ribonucleotide reductases. NMR solution structures of glutaredoxin domains from two Gram-negative opportunistic pathogens, Brucella melitensis and Bartonella henselae, are presented. These domains lack the N-terminal helix that is frequently present in eukaryotic GLXRs. The conserved active-site cysteines adopt canonical proline/tyrosine-stabilized geometries. A difference in the angle of α-helix 2 relative to the ß-sheet surface and the presence of an extended loop in the human sequence suggests potential regulatory regions and/or protein-protein interaction motifs. This observation is consistent with mutations in this region that suppress defects in GLXR-ribonucleotide reductase interactions. These differences between the human and bacterial forms are adjacent to the dithiol active site and may permit species-selective drug design.

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA.

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10-302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α-[(32)P]-ATP. Its crystal structure, determined to 2.9-Å resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional ß-rich domain at the C-terminus. On crystal soaking with ATP/Mg(2+), additional electron density indicated the presence of a PP(i) /Mg(2+) moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg(2+) and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the α-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence-independent mechanism of target positioning through antiparallel ß-strand interactions between enzyme and target is suggested.

Structure of the head of the Bartonella adhesin BadA.

Trimeric autotransporter adhesins (TAAs) are a major class of proteins by which pathogenic proteobacteria adhere to their hosts. Prominent examples include Yersinia YadA, Haemophilus Hia and Hsf, Moraxella UspA1 and A2, and Neisseria NadA. TAAs also occur in symbiotic and environmental species and presumably represent a general solution to the problem of adhesion in proteobacteria. The general structure of TAAs follows a head-stalk-anchor architecture, where the heads are the primary mediators of attachment and autoagglutination. In the major adhesin of Bartonella henselae, BadA, the head consists of three domains, the N-terminal of which shows strong sequence similarity to the head of Yersinia YadA. The two other domains were not recognizably similar to any protein of known structure. We therefore determined their crystal structure to a resolution of 1.1 A. Both domains are beta-prisms, the N-terminal one formed by interleaved, five-stranded beta-meanders parallel to the trimer axis and the C-terminal one by five-stranded beta-meanders orthogonal to the axis. Despite the absence of statistically significant sequence similarity, the two domains are structurally similar to domains from Haemophilus Hia, albeit in permuted order. Thus, the BadA head appears to be a chimera of domains seen in two other TAAs, YadA and Hia, highlighting the combinatorial evolutionary strategy taken by pathogens.

Type IV secretion systems and their effectors in bacterial pathogenesis.

Type IV secretion systems (T4SSs) are membrane-associated transporter complexes used by various bacteria to deliver substrate molecules to a wide range of target cells. T4SSs are involved in horizontal DNA transfer to other bacteria and eukaryotic cells, in DNA uptake from or release into the extracellular milieu, in toxin secretion and in the injection of virulence factors into eukaryotic host target cells by several mammalian pathogens. Rapid progress has been made towards defining the structures and functions of T4SSs, identifying the translocated effector molecules and elucidating the mechanisms by which the effectors subvert eukaryotic cellular processes during infection. These findings have had an important impact on our understanding of how these pathogens manipulate host cell functions to trigger bacterial uptake, facilitate intracellular growth and suppress defence mechanisms, thus facilitating bacterial colonization and disease development.

Structure and biological activity of the short-chain lipopolysaccharide from Bartonella henselae ATCC 49882T.

The facultative intracellular pathogen Bartonella henselae is responsible for a broad range of clinical manifestations, including the formation of vascular tumors as a result of increased proliferation and survival of colonized endothelial cells. This remarkable interaction with endotoxin-sensitive endothelial cells and the apparent lack of septic shock are considered to be due to a reduced endotoxic activity of the B. henselae lipopolysaccharide. Here, we show that B. henselae ATCC 49882(T) produces a deep-rough-type lipopolysaccharide devoid of O-chain and report on its complete structure and Toll-like receptor-dependent biological activity. The major short-chain lipopolysaccharide was studied by chemical analyses, electrospray ionization, and matrix-assisted laser desorption/ionization mass spectrometry, as well as by NMR spectroscopy after alkaline deacylation. The carbohydrate portion of the lipopolysaccharide consists of a branched trisaccharide containing a glucose residue attached to position 5 of an alpha-(2-->4)-linked 3-deoxy-d-manno-oct-2-ulosonic acid disaccharide. Lipid A is a pentaacylated beta-(1'-->6)-linked 2,3-diamino-2,3-dideoxy-glucose disaccharide 1,4'-bisphosphate with two amide-linked residues each of 3-hydroxydodecanoic and 3-hydroxyhexadecanoic acids and one residue of either 25-hydroxyhexacosanoic or 27-hydroxyoctacosanoic acid that is O-linked to the acyl group at position 2'. The lipopolysaccharide studied activated Toll-like receptor 4 signaling only to a low extent (1,000-10,000-fold lower compared with that of Salmonella enterica sv. Friedenau) and did not activate Toll-like receptor 2. Some unusual structural features of the B. henselae lipopolysaccharide, including the presence of a long-chain fatty acid, which are shared by the lipopolysaccharides of other bacteria causing chronic intracellular infections (e.g. Legionella and Chlamydia), may provide the molecular basis for low endotoxic potency.

Outer membrane proteins of Bartonella henselae and their interaction with human endothelial cells.

Members of the genus Bartonella are unique in that they are bacteria which cause proliferation of microvascular endothelial cells and neovascularization (angiogenesis). The mechanisms by which Bartonella henselae causes these processes are unknown. Given the importance of surface-exposed determinants in the pathogenesis of many organisms, outer membrane proteins (OMPs) of B. henselae were identified. Enrichment of the outer membrane fraction of B. henselae by sarkosyl treatment of total membranes, together with radioiodination and biotinylation of intact organisms, suggest that at least nine proteins, with molecular weights of 28, 30, 35, 43, 58, 61, 79, 92 and 171 kDa, are located in the outer membrane. Triton X-100-extracted biotinylated human umbilical vein endothelial cell (HUVEC) surface proteins bound to the 43 kDa B. henselae OMP after B. henselae whole-cell lysates and sarkosyl-fractionated OMPs were separated by SDS-PAGE and transferred onto nylon. Biotinylated B. henselae surface proteins of 28, 32, 43, 52 and 58 kDa were shown to bind intact HUVEC, with the 43 kDa protein being the major adhesin. Preincubation of HUVEC with an increasing concentration (20 microg/ml to 4 mg/ml) of sarkosyl-fractionated unlabelled B. henselae outer membrane proteins inhibited the attachment of all identified HUVEC binding proteins. The identification of B. henselae OMPs, as well as adhesins, should provide a basis for further investigation of the role of adherence in the pathogenesis of B. henselae.

Comparison of the abilities of proteins from Bartonella bacilliformis and Bartonella henselae to deform red cell membranes and to bind to red cell ghost proteins.

Infections in humans by Bartonella bacilliformis, but not Bartonella henselae, are characterized by invasion of red cells. Supernatants of culture medium from B. bacilliformis and B. henselae each contain a protein which causes invagination of membranes of human red cells and formation of intracellular vacuoles. These two proteins are very similar in molecular mass, heat stability and mechanism of action. B. henselae does not bind to human red cells, but human red cell ghost membrane proteins were recognized by both bacteria, five by B. bacilliformis and the same five, and one additional protein by B. henselae. Two of these proteins had molecular masses consistent with actin and spectrin. Actin binds to five electroblotted outer membrane proteins from B. henselae and four of these proteins are retained on an actin-Sepharose column.