Whole-Genome Sequencing of Two Bartonella bacilliformis Strains

Bartonella bacilliformis is the causative agent of Carrion's disease, a highly endemic human bartonellosis in Peru. We performed a whole-genome assembly of two B. bacilliformis strains isolated from the blood of infected patients in the acute phase of Carrion's disease from the Cusco and Piura regions in Peru

Prioritisation of potential drug targets against bartonella bacilliformis by an integrative in-silico approach

BACKGROUND Carrion’s disease (CD) is a neglected biphasic illness caused by Bartonella bacilliformis, a Gram-negative bacteria found in the Andean valleys. The spread of resistant strains underlines the need for novel antimicrobials against B. bacilliformis and related bacterial pathogens. OBJECTIVE The main aim of this study was to integrate genomic-scale data to shortlist a set of proteins that could serve as attractive targets for new antimicrobial discovery to combat B. bacilliformis. METHODS We performed a multidimensional genomic scale analysis of potential and relevant targets which includes structural druggability, metabolic analysis and essentiality criteria to select proteins with attractive features for drug discovery. FINDINGS We shortlisted seventeen relevant proteins to develop new drugs against the causative agent of Carrion’s disease. Particularly, the protein products of fabI, folA, aroA, trmFO, uppP and murE genes, meet an important number of desirable features that make them attractive targets for new drug development. This data compendium is freely available as a web server (http://target.sbg.qb.fcen.uba.ar/). MAIN CONCLUSION This work represents an effort to reduce the costs in the first phases of B. bacilliformis drug discovery.Fil: Farfán López, Mariella. Universidad Nacional Mayor de San Marcos; PerúFil: Espinoza Culupú, Abraham. Universidad Nacional Mayor de San Marcos; PerúFil: García De la guarda, Ruth. Universidad Nacional Mayor de San Marcos; PerúFil: Serral, Federico. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; ArgentinaFil: Sosa, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Palomino, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Fernández Do Porto, Darío Augusto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; Argentin

Caracterización molecular de la nucleoproteína del virus de la rabia en canes procedentes de Arequipa, Perú

Objetivos. Caracterizar la nucleoproteína (N) y establecer el origen del virus de la rabia en canes procedentes de Arequipa. Materiales y métodos. Se analizaron 30 muestras de tejido nervioso procedentes de los departamentos de Arequipa y Puno. Se extrajo el ARN total de las muestras y se sintetizó ADNc para amplificar el gen de la nucleoproteína, secuenciarlo y realizar el análisis bioinformático. Resultados. Se obtuvo la formación de un grupo definido con respecto al grupo externo (European bat lyssavirus). Este grupo fue clasificado en dos subgrupos, uno constituido por muestras procedentes de Puno y Arequipa (subgrupo A), y otro por muestras de Puno (subgrupo B), observándose una identidad nucleotídica de 99,9% en el subgrupo A. Conclusiones. Los agrupamientos de las secuencias virales muestran que los casos de rabia canina notificados en Arequipa son el resultado de la expansión de rabia canina procedente de la región endémica de Puno

Caracterización molecular de la región determinante de resistencia a quinolonas (QRDR) de la topoisomerasa IV de Bartonella bacilliformis en aislados clínicos

Bartonella bacilliformis is the etiologic agent of Carrion's disease, which if endemic to Peru. Studies on antimicrobial resistance genes from clinical isolates of this pathogen are scarce, and the molecular characteristics of these genes and their region resistance-associated are currently unknown. In this work we made the molecular characterization of the quinolone-resistance, and establish the region (QRDR) for the topoisomerase IV, which is encoded by the parC and parE genes, as well as develop an antimicrobial susceptibility test for B. bacilliformis. 65 Blood samples from La Libertad, Cusco, Ancash and Piura were processed on Blood Agar plates and incubated at 30 °C, 5% CO2. The antimicrobial susceptibility was determined, then the genomic DNA extracted, aforementioned genes amplified, their sequence determined and it analyzed using bioinformatics tools. Six positive cultures were obtained. The isolates were susceptible to Ciprofloxacin (except one strain from Quillabamba – Cusco, which showed decreased susceptibility) and were resistant to Nalidixic Acid. From the sequence analysis of B. bacilliformis ParC and ParE there have been shown amino acid differences compared to the respective protein sequences from E. coli K12 MG1655, which is likely to confer resistance to Nalidixic Acid but not to Ciprofloxacin. It was determined that B. bacilliformis ParC and ParE proteins QRDRs are comprised between amino acids 67 to 118 and 473 to 530, respectively. The antibiogram and the minimal inhibitory concentration are best assessed using the #1 McFarland standards after a 6-day incubation period.Bartonella bacilliformis es el agente etiológico de la Enfermedad de Carrión, endémica del Perú. Pocas investigaciones han sido realizadas acerca de los genes asociados a la resistencia antimicrobiana en aislados clínicos de este patógeno. Estos genes no están caracterizados molecularmente, ni se conoce la región asociada a dicha resistencia. Por ello, el objetivo del este trabajo fue caracterizar molecularmente la región determinante de la resistencia a las quinolonas (QRDR) en la topoisomerasa IV, que está codificada por los genes parC y parE, así como también desarrollar una prueba de susceptibilidad antimicrobiana para B. bacilliformis. Las muestras sanguíneas de 65 pacientes procedentes de La Libertad, Cusco, Ancash y Piura, se sembraron en placas de agar sangre e incubaron a 30 °C con 5% CO2. Luego se procedió a: (1) determinar la susceptibilidad antimicrobiana y (2) extraer el DNA genómico, amplificar los genes mencionados, secuenciarlos y analizarlos mediante herramientas bioinformáticas. Se obtuvieron 6 cultivos positivos. Los aislados fueron sensibles a la ciprofloxacina (excepto uno procedente de Quillabamba-Cusco, que presentó susceptibilidad disminuida) y resistentes al ácido nalidíxico. Del análisis de las secuencias aminoacídicas de ParC y ParE de B. bacilliformis se concluye que presentan diferencias aminoacídicas en comparación con las secuencias de las proteínas respectivas de E. coli K12 MG1655, que probablemente confieran resistencia al ácido nalidíxico pero no a la ciprofloxacina. Se determinó que las QRDR de las proteínas ParC y ParE de B. bacilliformis están comprendidas entre los aminoácidos 67 al 118 y 473 al 530, respectivamente. El antibiograma y la concentración mínima inhibitoria se evalúan mejor usando inóculos a escala 1 de McFarland y a los 6 días de incubación

Acylpolyamine Mygalin as a TLR4 Antagonist Based on Molecular Docking and In Vitro Analyses

Toll-like receptors (TLRs) are transmembrane proteins that are key regulators of innate and adaptive immune responses, particularly TLR4, and they have been identified as potential drug targets for the treatment of disease. Several low-molecular-weight compounds are being considered as new drug targets for various applications, including as immune modulators. Mygalin, a 417 Da synthetic bis-acylpolyamine, is an analog of spermidine that has microbicidal activity. In this study, we investigated the effect of mygalin on the innate immune response based on a virtual screening (VS) and molecular docking analysis. Bone marrow-derived macrophages and the cell lines J774A.1 and RAW 264.7 stimulated with lipopolysaccharide (LPS) were used to confirm the data obtained in silico. Virtual screening and molecular docking suggested that mygalin binds to TLR4 via the protein myeloid differentiation factor 2 (MD-2) and LPS. Macrophages stimulated by mygalin plus LPS showed suppressed gene expression of tumor necrosis factor (TNF-α), interleukine 6 (IL-6), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as inhibition of signaling protein p65 of the nuclear factor κB (NF-κB), resulting in decreased production of nitric oxide (NO) and TNF-α. These results indicate that mygalin has anti-inflammatory potential, being an attractive option to be explored. In addition, we reinforce the importance of virtual screening analysis to assist in the discovery of new drugs

Molecular mechanisms mediated by cellular activation induced by Mygalin, effects on TLR receptors and their contribution in the control of bacterial infections

O aumento de bactérias resistentes aos antibióticos é um grave problema de saúde pública que requer a identificação de moléculas que atuem em alvos moleculares importantes e específicos dos patógenos favorecendo seu controle. Vários análogos de poliaminas foram construídos e usados no controle de microrganismos e células tumorais. Migalina é uma acilpoliamina, derivada da hemolinfa da aranha Acanthoscurria gomesiana, sendo um análogo da espermidina e a presença de grupos acil na sua estrutura pode favorecer sua função efetora, cujos mecanismos são desconhecidos. Neste trabalho foi avaliado o mecanismo molecular da interação da migalina sintética com E. coli DH5α e células fagocíticas, da linhagem celular J774A.1, RAW 264.7 e macrófagos primários derivados de medula óssea murina. Paralelamente foi realizada uma análise in silico usando acoplamento molecular e similaridade com outras moléculas. Nossos dados mostram que a migalina tem efeito bactericida dose e tempo dependentes em E. coli. Houve redução da viabilidade celular após 4 e 6 horas de tratamento com 1 mM deste composto. Além disso, o tratamento de E. coli com migalina (250 e 500 μM) induziu a produção de altos níveis de produtos derivados do oxigênio (ROS), fragmentação e dano oxidativo no DNA. Em outro análise foi comprovado que houve desestruturação da membrana bacteriana, de modo similar ao ocorrido com alguns antibióticos, havendo o mesmo nível de incorporação do iodeto de propídio. Estes dados foram reforçados pelo encontro de níveis elevados de fluorescência no ensaio de atividade de esterase, indicando alteração na membrana celular de E. coli após tratamento com a migalina. Ao avaliar o nível de glutationa redutase nas bactérias tratadas, houve redução de 20% em relação aos grupos não tratados. Estes resultados comprovam que o mecanismo de ação da migalina sobre E. coli envolve a geração de ROS, quebra do DNA e desestruturação da membrana bacteriana. A migalina não apresentou citotoxicidade, nem induziu a expressão de mediadores inflamatórios em estudos in vitro usando células fagocíticas. Em modelo usando células fagocíticas ativadas com LPS, foi definido que os alvos dessa molécula foi o receptores Toll-like 4, uma vez que a adição de migalina às células tratadas com LPS reduziu o nível de NO e TNF-α e a expressão dos genes para iNOS, TNF-α, IL-6 e COX-2, assim como a expressão da proteína p65 do fator NF-kB em doses superiores a 150 μM. Foi também comprovado que a migalina se liga ao LPS, sendo que a pré-incubação dessas moléculas antes da ativação dos macrófagos neutralizou a atividade do LPS, reduzindo a produção de NO de modo dose dependente. A triagem virtual e a modelagem molecular confirmaram que um dos mecanismos de ação da migalina ocorre via TLR4, através da interação com a molécula adaptadora MD2. Os dados mostraram que a migalina pode neutralizar a ação do LPS bloqueando a interação LPS/TLR4-MD2. Os resultados indicam que a migalina tem potencial antibacteriano, além de efeito supressor da resposta inflamatória induzida por LPS, sendo uma nova molécula atrativa a ser estudada para o controle de infecções.The increase in antibiotic-resistant bacteria is a serious public health problem, requiring the identification of molecules that act on important and specific molecular targets of pathogens that control bacterial survival and help fight infections. Several polyamine analogues have been constructed and used to control microorganisms and tumour cells. Mygalin is a synthetic acylpolyamine, analogous to spermidine, derived from the hemolymph of the spider Acanthoscurria gomesiana and the presence of acyl groups in its structure can differentiate its effector function. However, the mechanisms involved are unknown. In this work, the molecular mechanism of the interaction of mygalin with E. coli and phagocytic cells, J774A.1 cell line, RAW and primary macrophages derived from murine bone marrow was evaluated. In parallel, an in silico analysis was performed using molecular docking and similarity with other molecules. Our data show that mygalin has a bactericidal effect that is dose and time-dependent on E. coli. There was a reduction in cell viability after 4 and 6 hours of treatment with 1 mM of the compound. In addition, the treatment of E. coli with mygalin (250 and 500 μM) induced the production of high levels of reactive oxygen species (ROS), fragmentation and oxidative damage to DNA. Another analysis showed that the compound disrupted the bacterial membrane, similar to the action mechanism of some antibiotics, with the same level of incorporation of propidium iodide. The high fluorescence levels observed in the esterase activity assay reinforce these data, indicating a change in the E. coli cell membrane after treatment with mygalin. Additionally, the glutathione reductase levels were 20% decreased as compared to the untreated groups. These results prove that the mechanism of action of mygalin on E. coli involves the generation of ROS, DNA breakdown and damage to the bacterial membrane. In vitro studies using phagocytic cells have shown that mygalin does not present cytotoxicity, nor does it induce the expression of inflammatory mediators. In a model using LPS-activated phagocytic cells, the addition of mygalin in doses greater than 150 μM to LPS-treated cells reduced the levels of NO and TNF-α, the expression of genes for iNOS, TNF-α, IL-6, and COX2, as well as the expression of the NF-kB factor p65 protein. These results suggest that Toll-like 4 receptors are the target of mygalin. Our data also indicate that mygalin binds to LPS and that its pre-incubation with LPS before the activation of macrophages neutralized LPS activity, reducing NO production in a dose-dependent manner. The virtual screening and molecular docking confirmed that one of the mechanisms of action of Mygalin occurs via TLR4, through the interaction with the adapter molecule MD2. The data show that mygalin can act via the neutralization of LPS and blocking the LPS/TLR4-MD2 interaction. The results indicate that mygalin has antibacterial potential, in addition to suppressing the inflammatory response induced by LPS, being a new attractive molecule to be studied for infection control

Caracterización de las regiones determinantes de resistencia a antimicrobianos de cepas de Bartonella bacilliformis aisladas de zonas endémicas del Perú

TesisBartonella bacilliformis, agente causal de la Enfermedad de Carrión transmitida por insectos flebotominos del género Lutzomyia y otros, es una endemia ancestral que afecta especialmente a la población más pobre de nuestros valles interandinos. En el Perú está presente en 12 de 25 departamentos. Es endémica en Ancash, Cajamarca, Amazonas, Cusco, La Libertad y Piura. Pocas son las investigaciones acerca de la susceptibilidad in vitro a antimicrobianos de Bartonella bacilliformis, no se cuenta con una prueba estandarizada de antibiograma para esta bacteria, como tampoco se conocen los mecanismos de resistencia ni las secuencias de los genes asociados a dicha resistencia. Por consiguiente, se ha planteado evaluar la resistencia antimicrobiana in vitro de cepas de Bartonella bacilliformis aisladas de zonas endémicas, mediante métodos convencionales y métodos moleculares, con la finalidad de conocer la resistencia a drogas de las cepas circulantes en las zonas endémicas. En el presente trabajo se obtuvieron muestras sanguíneas de 47 pacientes procedentes de Piura, Cusco y Ancash las cuales se sembraron en placas de agar sangre e incubaron a 30°C con 5% CO2. Se ha estandarizado un método de difusión por disco (para el antibiograma) y la prueba de Épsilon (para determinar la concentración mínima inhibitoria) para el estudio in vitro de la susceptibilidad antimicrobiana de B. bacilliformis, empleando los aislados clínicos y una cepa del Instituto Pasteur de Francia. Luego se procedió a extraer el DNA genómico, amplificar los genes mencionados, secuenciarlos y analizarlos mediante herramientas bioinformáticas. Se obtuvieron 3 cultivos positivos. Las cepas fueron sensibles a la ciprofloxacina, gentamicina, rifampicina, eritromicina, cloranfenicol, ceftriaxona y amoxicilina y resistentes al ácido nalidíxico. Del análisis de las secuencias aminoacídicas de las proteínas de GyrA, GyrB, ParC y ParE de B. bacilliformis, asociados a la resistencia de las quinolonas (ciprofloxacina, ácido nalidíxico) se concluye que presentan diferencias aminoacídicas de Ser por Ala, en comparación con las secuencias de las proteínas respectivas de E. coli K12 MG1655. Esta diferencia probablemente confiera resistencia al ácido nalidíxico pero no a la ciprofloxacina en B. bacilliformis. Se determinó que las QRDR de las proteínas GyrA, GyrB, ParC y ParE de B. bacilliformis están comprendidas entre los aminoácidos 74 a 124, 428 a 426, 67 a 118, y 473 a 530, respectivamente. Para el antibiótico rifampicina se determinó la RRDR en el gen rpoB comprendida entre los aminoácidos 521 a 547, y para el gen rplD de la proteína L4 relacionado con resistencia a eritromicina reportamos la ERDR nunca antes mencionado y comprende los aminoácidos 60 a 79, siendo el primer reporte para este gen en Bartonella bacilliformis. Finalmente en el modelamiento de las estructuras terciarias de las regiones determinantes de resistencia se apreciaron cambios en las posiciones de Ser por Ala, y estos cambios permiten una débil interacción con la droga. Al evaluar los perfiles de hidrofobicidad de los aminoácidos pertenecientes a la región determinante de resistencia, nos permitió inferir que los cambios de aminoácidos con propiedades diferentes como Ser por Ala contribuyen a la resistencia

Antimicrobial Activity of <i>Apis mellifera</i> Bee Venom Collected in Northern Peru

Due to the emergence of microorganisms resistant to antibiotics and the failure of antibiotic therapies, there is an urgent need to search for new therapeutic options, as well as new molecules with antimicrobial potential. The objective of the present study was to evaluate the in vitro antibacterial activity of Apis mellifera venom collected in the beekeeping areas of the city of Lambayeque in northern Peru against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Bee venom extraction was performed by electrical impulses and separated using the Amicon ultra centrifugal filter. Subsequently, the fractions were quantified by spectrometric 280 nm and evaluated under denaturant conditions in SDS-PAGE. The fractions were pitted against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853. A purified fraction (PF) of the venom of A. mellifera and three low molecular weight bands of 7 KDa, 6 KDa, and 5 KDa were identified that showed activity against E. coli with a MIC of 6.88 µg/mL, while for P. aeruginosa and S. aureus, it did not present a MIC. No hemolytic activity at a concentration lower than 15.6 µg/mL and no antioxidant activity. The venom of A. mellifera contains a potential presence of peptides and a predilection of antibacterial activity against E. coli.</i

Whole-Genome Sequencing of Two Bartonella bacilliformis Strains

Bartonella bacilliformis is the causative agent of Carrion's disease, a highly endemic human bartonellosis in Peru. We performed a whole-genome assembly of two B. bacilliformis strains isolated from the blood of infected patients in the acute phase of Carrion's disease from the Cusco and Piura regions in Peru