The BID Domain of Type IV Secretion Substrates Forms a Conserved Four-Helix Bundle Topped with a Hook.

The BID (Bep intracellular delivery) domain functions as secretion signal in a subfamily of protein substrates of bacterial type IV secretion (T4S) systems. It mediates transfer of (1) relaxases and the attached DNA during bacterial conjugation, and (2) numerous Bartonella effector proteins (Beps) during protein transfer into host cells infected by pathogenic Bartonella species. Furthermore, BID domains of Beps have often evolved secondary effector functions within host cells. Here, we provide crystal structures for three representative BID domains and describe a novel conserved fold characterized by a compact, antiparallel four-helix bundle topped with a hook. The conserved hydrophobic core provides a rigid scaffold to a surface that, despite a few conserved exposed residues and similarities in charge distribution, displays significant variability. We propose that the genuine function of BID domains as T4S signal may primarily depend on their rigid structure, while the plasticity of their surface may facilitate adaptation to secondary effector functions.

Structural Insight into How Bacteria Prevent Interference between Multiple Divergent Type IV Secretion Systems.

UNLABELLED: Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species (vir, vbh, and trw). In a unique case, most rickettsial species encode a T4SS (rvh) enriched with gene duplication. Within single genomes, the evolutionary and functional implications of cross-system interchangeability of analogous T4SS protein components remains poorly understood. To lend insight into cross-system interchangeability, we analyzed the VirB8 family of T4SS channel proteins. Crystal structures of three VirB8 and two TrwG Bartonella proteins revealed highly conserved C-terminal periplasmic domain folds and dimerization interfaces, despite tremendous sequence divergence. This implies remarkable structural constraints for VirB8 components in the assembly of a functional T4SS. VirB8/TrwG heterodimers, determined via bacterial two-hybrid assays and molecular modeling, indicate that differential expression of trw and vir systems is the likely barrier to VirB8-TrwG interchangeability. We also determined the crystal structure of Rickettsia typhi RvhB8-II and modeled its coexpressed divergent paralog RvhB8-I. Remarkably, while RvhB8-I dimerizes and is structurally similar to other VirB8 proteins, the RvhB8-II dimer interface deviates substantially from other VirB8 structures, potentially preventing RvhB8-I/RvhB8-II heterodimerization. For the rvh T4SS, the evolution of divergent VirB8 paralogs implies a functional diversification that is unknown in other T4SSs. Collectively, our data identify two different constraints (spatiotemporal for Bartonella trw and vir T4SSs and structural for rvh T4SSs) that mediate the functionality of multiple divergent T4SSs within a single bacterium. IMPORTANCE: Assembly of multiprotein complexes at the right time and at the right cellular location is a fundamentally important task for any organism. In this respect, bacteria that express multiple analogous type IV secretion systems (T4SSs), each composed of around 12 different components, face an overwhelming complexity. Our work here presents the first structural investigation on factors regulating the maintenance of multiple T4SSs within a single bacterium. The structural data imply that the T4SS-expressing bacteria rely on two strategies to prevent cross-system interchangeability: (i) tight temporal regulation of expression or (ii) rapid diversification of the T4SS components. T4SSs are ideal drug targets provided that no analogous counterparts are known from eukaryotes. Drugs targeting the barriers to cross-system interchangeability (i.e., regulators) could dysregulate the structural and functional independence of discrete systems, potentially creating interference that prevents their efficient coordination throughout bacterial infection.

Clinical, histologic, microbiologic, and biochemical characterization of the causative agent of bacillary (epithelioid) angiomatosis: a rickettsial illness with features of bartonellosis.

It has been suggested that bacillary (epithelioid) angiomatosis (BEA) is a manifestation of cat scratch disease (CSD). Because of clinical similarity between this condition and the verruga peruana phase of bartonellosis, we sought to further characterize this disease as well as its causative agent and to compare it to bartonellosis. We isolated a small flagellated pleomorphic bacillus from skin lesions of two patients with BEA. Organisms were stained successfully with Warthin-Starry silver stains, but immunohistochemistry failed to demonstrate binding with a polyclonal antibody directed against the cat scratch bacillus. Whole cell fatty-acid gas chromatography performed on both BEA organisms and Bartonella bacilliformis demonstrated marked similarity between the two. Electron microscopy of BEA organisms in tissue and in suspension revealed features characteristic of a gram negative bacillus. Based on these findings, we propose that this unusual rickettsial infectious disease with vascular proliferation may represent an unusual variant of infection with a bartonella-like organism rather than a manifestation of cat scratch disease.

Bartonella quintana lipopolysaccharide effects on leukocytes, CXC chemokines and apoptosis: a study on the human whole blood and a rat model.

Bartonella quintana, an emerging gram-negative pathogen, may cause trench fever, endocarditis, cerebral abscess and bacillary angiomatosis usually with the absence of septic shock in humans. B. quintana lipopolysaccharide (LPS), a deep rough endotoxin with strong reactivity in the limulus amebocyte lysate (LAL)-assay, was studied in human whole blood and in a rat model. A significant (P<0.05) increase of interleukin-8 (IL-8) concentration, comparable to the level induced by enterobacterial LPS, was stimulated in the human whole blood by B. quintana LPS. Isolated human neutrophils delayed their apoptotic behavior in the presence of B. quintana LPS. In the rat, B. quintana LPS induced a significant (P<0.001) increase in white blood cell count, both 30 and 60 min after intravenous injection. Such leukocytosis was inhibited by pretreatment with prazosin, an alpha-adrenergic antagonist. B. quintana LPS did not significantly change heart rate (HR), hematocrit (HCT) and platelet count in the above reported in vivo model, and regarding mean blood pressure (MAP) only a very early (5 min after LPS) and mild (yet significant) hypotension was observed. In contrast, a long-lasting decrease of MAP was found in Salmonella minnesota R595 LPS-treated animals. Blood TNFalpha levels did not change significantly from the baseline in rats injected with either saline or with B. quintana LPS, on the contrary S. minnesota R595 LPS-injected animals showed substantial increase of TNFalpha levels up to 2924 pg/ml at 60 min after LPS injection. B. quintana LPS as well as Salmonella LPS-injected rats exhibited an increase of the blood levels of GRO/CINC-1, particularly at 240 min after LPS administration. Apical part of rat gut villi showed several TUNEL-positive cells in tissue sections from B. quintana LPS-treated animals. Taken together, our data demonstrates that B. quintana LPS is able to selectively stimulate some inflammatory mediators. B. quintana LPS-induced leukocytosis appears mediated by an alpha-adrenergic receptor. The delayed apoptotic process of leukocytes and the chemokine increase may explain the apoptotic cells found in the rat gut and the inflammatory reactions in some human Bartonella diseases. This peculiar inflammatory pattern induced by B. quintana LPS, may partially account for the lack of severe septic shock, observed in human B. quintana infections.

Horizontal transfers and gene losses in the phospholipid pathway of bartonella reveal clues about early ecological niches.

Bartonellae are mammalian pathogens vectored by blood-feeding arthropods. Although of increasing medical importance, little is known about their ecological past, and host associations are underexplored. Previous studies suggest an influence of horizontal gene transfers in ecological niche colonization by acquisition of host pathogenicity genes. We here expand these analyses to metabolic pathways of 28 Bartonella genomes, and experimentally explore the distribution of bartonellae in 21 species of blood-feeding arthropods. Across genomes, repeated gene losses and horizontal gains in the phospholipid pathway were found. The evolutionary timing of these patterns suggests functional consequences likely leading to an early intracellular lifestyle for stem bartonellae. Comparative phylogenomic analyses discover three independent lineage-specific reacquisitions of a core metabolic gene-NAD(P)H-dependent glycerol-3-phosphate dehydrogenase (gpsA)-from Gammaproteobacteria and Epsilonproteobacteria. Transferred genes are significantly closely related to invertebrate Arsenophonus-, and Serratia-like endosymbionts, and mammalian Helicobacter-like pathogens, supporting a cellular association with arthropods and mammals at the base of extant Bartonella spp. Our studies suggest that the horizontal reacquisitions had a key impact on bartonellae lineage specific ecological and functional evolution.

The Bartonella vinsonii subsp. arupensis immunodominant surface antigen BrpA gene, encoding a 382-kilodalton protein composed of repetitive sequences, is a member of a multigene family conserved among bartonella species.

Bartonella proteins that elicit an antibody response during an infection are poorly defined; therefore, to characterize antigens recognized by the host, a Bartonella genomic expression library was screened with serum from an infected mouse. This process led to the discovery of a Bartonella vinsonii subsp. arupensis gene encoding a 382-kDa protein, part of a gene family encoding large proteins, each containing multiple regions of repetitive segments. The genes were termed brpA to -C (bartonella repeat protein) and bore significant similarity to genes encoding the BadA adhesin protein and members of the variably expressed outer membrane protein family of proteins from Bartonella henselae and Bartonella quintana, respectively.

Identification of outer membrane proteins of Bartonella bacilliformis.

Purification of the outer membrane of Bartonella bacilliformis by sucrose step gradient centrifugation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) suggest that 14 proteins, ranging from 11.2 to 75.3 kDa, are located in the outer membrane of the pathogen. On the basis of M(r)s, eleven of these proteins have counterparts which are labeled by extrinsic radioiodination of intact bartonellae, and two of the proteins are visibly sensitive to extrinsic proteinase K digestion in analysis by SDS-PAGE. While nearly all the extrinsically radioiodinated proteins could be immunoprecipitated with rabbit antibartonella hyperimmune serum, proteins of 31.5, 42, and 45 kDa were prominent immunoprecipitants. Purified lipopolysaccharide from the outer membrane of B. bacilliformis produced a diffuse band of approximately 5 kDa on SDS-PAGE and was not detectable on immunoblots developed with rabbit antibartonella hyperimmune antiserum.

Bacterial persistence within erythrocytes: a unique pathogenic strategy of Bartonella spp.

The genus Bartonella comprises human-specific and zoonotic pathogens responsible for a wide range of clinical manifestations, including Carrion's disease, trench fever, cat scratch disease, bacillary angiomatosis and peliosis, endocarditis and bacteremia. These arthropod-borne pathogens typically parasitise erythrocytes in their mammalian reservoir host(s), resulting in a long-lasting haemotropic infection. We have studied the process of Bartonella erythrocyte parasitism by tracking green fluorescent protein-expressing bacteria in the blood of experimentally infected animals. Following intravenous infection, bacteria colonise a yet enigmatic primary niche, from where they are seeded into the blood stream in regular intervals of approximately five days. Bacteria invade mature erythrocytes, replicate temporarily and persist in this unique intracellular niche for the remaining life span of the infected erythrocytes. A triggered antibody response typically results in an abrogation of bacteremia within 3 months of infection, likely by blocking new waves of bacterial invasion into erythrocytes. The recent establishment of genetic tools for Bartonella spp. permitted us to identify several putative pathogenicity determinants. Application of differential fluorescence induction technology resulted in the isolation of bacterial genes differentially expressed during infection in vitro and in vivo, including an unknown family of autotransporter proteins as well as a novel type IV secretion system homologous to the conjugation system of E. coli plasmid R388. Mutational analysis of a previously described type IV secretion system displaying homology to the virB locus of Agrobacterium tumefaciens provided the first example of an essential pathogenicity locus in Bartonella. Though required for establishing haemotropic infection, it remains to be demonstrated if this type IV secretion system is necessary for colonisation of the primary niche or for the subsequent colonisation of erythrocytes.

Deformin, a substance found in Bartonella bacilliformis culture supernatants, is a small, hydrophobic molecule with an affinity for albumin.

Culture supernatants of Bartonella bacilliformis were previously shown to contain a factor, called deforming factor or deformin, which causes deformation and invagination of red cell membranes and formation of intracellular vacuoles. This factor is here shown to be a small water-soluble molecule, approximately 1400 Da as estimated by gel-filtration chromatography. Deforming factor binds tightly to albumin, especially albumin dimers and multimers, present in the growth medium. It can be released from albumin with 50% ethanol and has been partially purified by filtration and HPLC.

Deformation factor: an extracellular protein synthesized by Bartonella bacilliformis that deforms erythrocyte membranes.

Bartonella bacilliformis, a hemotropic bacterium and the causative agent of the human disease bartonellosis, when incubated in a tryptone-based medium produces an extracellular factor, termed deformation factor (DF), which induces extensive indentations and trenches in trypsinized erythrocyte membranes. The factor is stable during storage at 4 degrees C. It can be inactivated by proteases or brief heating to 70 to 80 degrees C, can be precipitated by ammonium sulfate, is nondialyzable, and is retained by membranes with a 30,000-molecular-weight cutoff. These properties suggest that DF is probably a protein. Incubation of erythrocytes with phospholipase D renders them resistant to deformation by DF.

Mitogenic effect of Bartonella bacilliformis on human vascular endothelial cells and involvement of GroEL.

Bartonellae are bacterial pathogens for a wide variety of mammals. In humans, bartonellosis can result in angioproliferative lesions that are potentially life threatening to the patient, including bacillary angiomatosis, bacillary peliosis, and verruga peruana. The results of this study show that Bartonella bacilliformis, the agent of Oroya fever and verruga peruana, produces a proteinaceous mitogen for human vascular endothelial cells (HUVECs) that acts in a dose-dependent fashion in vitro with maximal activity at >or=72 h of exposure and results in a 6- to 20-fold increase in cell numbers relative to controls. The mitogen increases bromodeoxyuridine (BrdU) incorporation into HUVECs by almost twofold relative to controls. The mitogen is sensitive to heat and trypsin but is not affected by the lipopolysaccharide inhibitor polymyxin B. The mitogen does not affect caspase 3 activity in HUVECs undergoing serum starvation-induced apoptosis. The Bartonella mitogen was found in bacterial culture supernatants, the soluble cell lysate fraction, and, to a lesser degree, in insoluble cell fractions of the bacterium. In contrast, soluble cell lysate fractions from closely related B. henselae, although possessing significant mitogenicity for HUVECs, resulted in only about a twofold increase in cell numbers. Biochemical and immunological analyses identified GroEL as a participant in the observed HUVEC mitogenicity. A B. bacilliformis strain containing the intact groES-groEL operon on a multicopy plasmid was generated and used to demonstrate a correlation between HUVEC mitogenicity and GroEL levels in the lysate (r(2) = 0.85). Antiserum to GroEL significantly inhibited mitogenicity of the lysate. Data also show that GroEL is located in the soluble and insoluble fractions (including inner and outer membranes) of the cell and is actively secreted by B. bacilliformis.