Baculovirus-Assisted Production of Bartonella bacilliformis Proteins: A Potential Strategy for Improving Serological Diagnosis of Carrion's Disease.

Carrion's disease, caused by Bartonella bacilliformis, is a neglected tropical disease prevalent in the Andean region of South America. Without antimicrobial treatment, this disease has a mortality rate of up to 88% in infected patients. The most common method for diagnosing B. bacilliformis infection is serological testing. However, the current serological assays are limited in sensitivity and specificity, underscoring the need for the development of novel and more accurate diagnostic tools. Recombinant proteins have emerged as promising candidates to improve the serological diagnosis of Carrion's disease. So, we focused on evaluating the conditions for producing two previously predicted proteins of B. bacilliformis using the baculovirus-insect cell expression system, mainly the flashBAC ULTRA technology. We assessed various parameters to identify the conditions that yield the highest protein production, including cell lines, temperature, and hours post-infection (hpi). The results showed that the expression conditions for achieving the highest yields of the Prot_689 and Prot_504 proteins were obtained using High Five™ cells at 21 °C and harvesting at 120 hpi. Subsequently, the seroreactivity of recombinant proteins was evaluated using positive sera from patients diagnosed with Carrion's disease. These findings offer valuable insights into the production conditions of B. bacilliformis recombinant proteins using the baculovirus system, which could significantly contribute to developing more precise diagnostic tools for Carrion's disease. Therefore, this research provides implications for improving diagnostics and potentially developing therapeutic strategies.

Single-nucleotide polymorphisms in ialB, gltA and rpoB genes of Bartonella bacilliformis isolated from patients in endemic Peruvian regions.

Bartonella bacilliformis is a Gram-negative, aerobic bacterium and the known causal agent of Carrion's disease, still considered a neglected disease. There is limited information about the nucleotide sequences of this bacterium in international databases, and few studies have addressed the genetic diversity of B. bacilliformis. We analyzed a total of 20 isolates of B. bacilliformis from the Peruvian regions of Ancash and Cajamarca. Three genes (ialB, gltA, and rpoB) were sequenced in each isolate and nucleotide sequences retrieved from GenBank (16 B. bacilliformis genomes) were also included in the study. All this information was merged in order to obtain clearer evidence of the phylogenetic relationships of B. bacilliformis. In the phylogenetic analysis conducted with the concatenated markers, four isolates (B.b-1, B. b-3, B. b- 7, B.b-8) from the Ancash region were observed to form a subgroup different from B. bacilliformis type strain KC583, showing dissimilarity levels of 5.96% (ialB), 3.69% (gltA) and 3.04% (rpoB). Our results suggest that B. bacilliformis consists of two different subgroups. Future investigations are needed to establish the taxonomic status of these subgroups.

In-silico identification of linear B-cell epitopes in specific proteins of Bartonella bacilliformis for the serological diagnosis of Carrion's disease.

Carrion´s disease is caused by Bartonella bacilliformis, it is a Gram-negative pleomorphic bacterium. B. bacilliformis is transmitted by Lutzomyia verrucarum in endemic areas of the Peruvian Inter-Andean valleys. Additionally, the pathogenicity of B. bacilliformis involves an initial infection of erythrocytes and the further infection of endothelial cells, which mainly affects children and expectant women from extreme poverty rural areas. Therefore, the implementation of serological diagnostic methods and the development of candidate vaccines for the control of CD could be facilitated by the prediction of linear b-cell epitopes in specific proteins of B. bacilliformis by bioinformatics analysis. In this study, We used an in-silico analysis employing six web servers for the identification of epitopes in proteins of B. bacilliformis. The selection of B. bacilliformis-specific proteins and their analysis to identify epitopes allowed the selection of seven protein candidates that are expected to have high antigenic activity.