The Passenger Domain of Bartonella bacilliformis BafA Promotes Endothelial Cell Angiogenesis via the VEGF Receptor Signaling Pathway.

Bartonella bacilliformis is a Gram-negative bacterial pathogen that provokes pathological angiogenesis and causes Carrion's disease, a neglected tropical disease restricted to South America. Little is known about how B. bacilliformis facilitates vasoproliferation resulting in hemangioma in the skin in verruga peruana, the chronic phase of Carrion's disease. Here, we demonstrate that B. bacilliformis extracellularly secrets a passenger domain of the autotransporter BafA exhibiting proangiogenic activity. The B. bacilliformis-derived BafA passenger domain (BafABba) increased the number of human umbilical endothelial cells (HUVECs) and promoted tube-like morphogenesis. Neutralizing antibody against BafABba detected the BafA derivatives from the culture supernatant of B. bacilliformis and inhibited the infection-mediated hyperproliferation of HUVECs. Moreover, stimulation with BafABba promoted phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and extracellular-signal-regulated kinase 1/2 in HUVECs. Suppression of VEGFR2 by anti-VEGFR2 antibody or RNA interference reduced the sensitivity of cells to BafABba. In addition, surface plasmon resonance analysis confirmed that BafABba directly interacts with VEGFR2 with lower affinity than VEGF or Bartonella henselae-derived BafA. These findings indicate that BafABba acts as a VEGFR2 agonist analogous to the previously identified B. henselae- and Bartonella quintana-derived BafA proteins despite the low sequence similarity. The identification of a proangiogenic factor produced by B. bacilliformis that directly stimulates endothelial cells provides an important insight into the pathophysiology of verruga peruana. IMPORTANCE Bartonella bacilliformis causes life-threatening bacteremia or dermal eruption known as Carrion's disease in South America. During infection, B. bacilliformis promotes endothelial cell proliferation and the angiogenic process, but the underlying molecular mechanism has not been well understood. We show that B. bacilliformis induces vasoproliferation and angiogenesis by producing the proangiogenic autotransporter BafA. As the cellular/molecular basis for angiogenesis, BafA stimulates the signaling pathway of vascular endothelial growth factor receptor 2 (VEGFR2). Identification of functional BafA protein from B. bacilliformis in addition to B. henselae and B. quintana, the causes of cat scratch disease and trench fever, raises the possibility that BafA is a common virulence factor for human-pathogenic Bartonella.

Subtractive proteomics and immunoinformatics approaches to explore Bartonella bacilliformis proteome (virulence factors) to design B and T cell multi-epitope subunit vaccine.

Bartonella bacilliformis a gram-negative facultative aerobe responsible for the Carrion's disease widely distributed in Ecuador, Peru, and Colombia with a high mortality rate when no specific treatment is received. B bacilliformis is transmitted by Sand fly (Lutzomyia verrucarum) to healthy individuals. Immunoinformatic and subtractive proteomics approaches were employed in this study to prioritize the best candidates for vaccine designing. These approaches resulted in five vaccine candidates, flagellar biosynthetic protein (Uniprot ID: A1UTU1), heme exporter protein C (UniProt ID: A1UU82), Cytochrome c-type biogenesis protein (Uniprot ID: A1URZ7), Hemin ABC transporter (Uniprot ID: A1US20) and Phosphatidate cytidylyltransferase (Uniprot ID: A1USE3). The mentioned proteins are antigenic and essential for pathogen survival. A range of immune-informatics tools was applied for the prediction of B and T cell epitopes for the vaccine candidate proteins. In-silico vaccine was constructed using carefully evaluated epitopes and consequently modeled for docking with human Toll-like receptor 4. TLR-4 agonist 50S ribosomal protein L7/L12 (UniproKB ID; P9WHE3) was linked to the vaccine as an adjuvant to boost immune response towards the vaccine. For stability evaluation of the vaccine-TLR-4 docked complex, MD simulations were performed. The final vaccine was back-translated and cloned in Eschericia coli to attain the maximal expression of the vaccine protein. The maximal expression was ensured, and the CAI score of 0.96 was reported. The current vaccine requires future experimental validation to confirm its effectiveness. The vaccine developed will be helpful to protect against B bacilliformis associated infections.

In silico analysis of Pap31 from Bartonella bacilliformis and other Bartonella spp.

Pap31 is an outer membrane protein of Bartonella bacilliformis which is considered to be a potential antigenic candidate for the development of diagnostic tools. The present study aimed to compare Pap31 from B. bacilliformis with that of other Bartonella spp. The results showed the presence of at least 5 different B. bacilliformis Pap31 alleles, with the strain Ver097 being the most divergent (89.7% of identity with the reference strain KC583). The most significant finding was the presence of a variable number (1 to 3) of 6 amino acid tandem repeats (GTEGGG) in the different B. bacilliformis Pap31 alleles, with no similar structure in other established Bartonella spp., except for Bartonella ancashensis, another Bartonella spp. isolated from chronic cases of Carrion's disease. In both B. bacilliformis and B. ancashensis this repetitive region was coincident with the most predicted immunogenic region of the protein. In other microorganisms, the presence of amino acid tandem repeats has been related to the development of poorly functional antibodies. The findings of this study also suggest a utility of Pap31 amino acid tandem repeats as potential contributors to the immune evasion of Carrion's disease-related Bartonella spp. and the establishment of asymptomatic B. bacilliformis / B. ancashensis infections.

The protein-protein interactions required for assembly of the Tn3 resolution synapse.

The site-specific recombinase Tn3 resolvase initiates DNA strand exchange when two res recombination sites and six resolvase dimers interact to form a synapse. The detailed architecture of this intricate recombination machine remains unclear. We have clarified which of the potential dimer-dimer interactions are required for synapsis and recombination, using a novel complementation strategy that exploits a previously uncharacterized resolvase from Bartonella bacilliformis ("Bart"). Tn3 and Bart resolvases recognize different DNA motifs, via diverged C-terminal domains (CTDs). They also differ substantially at N-terminal domain (NTD) surfaces involved in dimerization and synapse assembly. We designed NTD-CTD hybrid proteins, and hybrid res sites containing both Tn3 and Bart dimer binding sites. Using these components in in vivo assays, we demonstrate that productive synapsis requires a specific "R" interface involving resolvase NTDs at all three dimer-binding sites in res. Synapses containing mixtures of wild-type Tn3 and Bart resolvase NTD dimers are recombination-defective, but activity can be restored by replacing patches of Tn3 resolvase R interface residues with Bart residues, or vice versa. We conclude that the Tn3/Bart family synapse is assembled exclusively by R interactions between resolvase dimers, except for the one special dimer-dimer interaction required for catalysis.

Characterization and expression analysis of the groESL operon of Bartonella bacilliformis.

The groESL operon of Bartonella bacilliformis, a facultative intracellular, Gram-negative bacterium and etiologic agent of Oroya Fever, was characterized. Sequence analysis revealed an operon containing two genes of 294 (groES) and 1632 nucleotides (groEL) separated by a 55-nt intergenic spacer. The operon is preceded by a 72-nt ORF (ORF1) that encodes a hypothetical protein with homology to a portion of the HrcA repressor for groESL. A divergent fumarate hydratase C (fumC) gene lies further upstream. Deduced amino acid sequences for B. bacilliformis GroEL and GroES revealed a high degree of identity with homologues from other Bartonella and alpha-Protebacteria. A single transcriptional start site (TSS) was mapped 79 nucleotides upstream of the groES start codon, regardless of incubation temperature. The TSS was located immediately 5' to a potential controlling inverted repeat of chaperonin expression (CIRCE) element and is preceded by a sigma70-like promoter. The operon is followed by a predicted rho-independent transcriptional terminator. Northern blot analysis indicated that groES and groEL are co-transcribed as a single mRNA of approximately 2.4 kb. A 6-h time course analysis by qRT-PCR showed that groEL expression increases 1.3-fold within 30 min of a temperature upshift from 30 to 37 degrees C, with maximum transcription reached after 60 min (approximately 4.3-fold), followed by a steady decrease to background (30 degrees C) transcription levels by 6 h. Western blot analysis revealed a 1.4- and 1.5-fold increase in GroEL synthesis following a temperature upshift or by inhibiting DNA supercoiling with coumermycin A1, respectively. Functional expression and complementation of temperature-sensitive Escherichia coli groES or groEL mutants with the cloned operon allowed them to grow at otherwise restrictive temperatures.