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1.
mSphere ; 7(2): e0008122, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35379004

RESUMO

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.


Assuntos
Infecções por Bartonella , Bartonella bacilliformis , Infecções por Bartonella/microbiologia , Infecções por Bartonella/patologia , Bartonella bacilliformis/genética , Bartonella bacilliformis/metabolismo , Células Endoteliais/patologia , Humanos , Morfogênese , Neovascularização Patológica/metabolismo , Neovascularização Patológica/microbiologia , Transdução de Sinais , Sistemas de Secreção Tipo V , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
2.
Infect Genet Evol ; 85: 104551, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32931955

RESUMO

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.


Assuntos
Vacinas Bacterianas/imunologia , Infecções por Bartonella/microbiologia , Bartonella bacilliformis/metabolismo , Biologia Computacional , Epitopos , Proteoma , Proteômica , Vacinas Bacterianas/genética , Infecções por Bartonella/imunologia , Infecções por Bartonella/prevenção & controle , Bartonella bacilliformis/imunologia , Bartonella bacilliformis/patogenicidade , Biologia Computacional/métodos , Epitopos/química , Epitopos/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Modelos Moleculares , Relação Estrutura-Atividade , Vacinologia , Fatores de Virulência/imunologia
3.
Infect Genet Evol ; 84: 104482, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32738366

RESUMO

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.


Assuntos
Antígenos de Bactérias/metabolismo , Bartonella bacilliformis/metabolismo , Simulação por Computador , Regulação Bacteriana da Expressão Gênica/fisiologia , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Bartonella bacilliformis/genética , Sequência de Bases , DNA Bacteriano/genética , Humanos , Especificidade da Espécie
4.
Mol Microbiol ; 114(6): 952-965, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33405333

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Bartonella bacilliformis/metabolismo , Transposon Resolvases/metabolismo , Proteínas de Bactérias/genética , Bartonella bacilliformis/genética , Sítios de Ligação , DNA Nucleotidiltransferases/metabolismo , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/metabolismo , Dimerização , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transposon Resolvases/genética
5.
Gene ; 359: 53-62, 2005 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-16126349

RESUMO

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.


Assuntos
Proteínas de Bactérias/genética , Bartonella bacilliformis/genética , Chaperoninas/genética , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Bartonella bacilliformis/metabolismo , Sequência de Bases , Northern Blotting , Western Blotting , Chaperoninas/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Teste de Complementação Genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Sequências Reguladoras de Ácido Nucleico/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Temperatura , Transcrição Gênica/genética
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