RESUMEN
Fluoroquinolone antibiotics have been a mainstay in the treatment of bacterial diseases. The most notable representative, ciprofloxacin, possesses potent antimicrobial activity; however, a rise in resistance to this agent necessitates development of novel derivatives to prolong the clinical lifespan of these antibiotics. Herein we have synthesized and analyzed the antimicrobial properties of a library of N-acylated ciprofloxacin analogues. We find that these compounds are broadly effective against Gram-positive and Gram-negative bacteria, with many proving more effective than the parental drug, and several possessing MICs ≤1.0 µg/ml against methicillin-resistant Staphylococcus aureus and Bartonella species. An analysis of spontaneous mutation frequencies reveals very low potential for resistance in MRSA compared to existing fluoroquinolones. Mode of action profiling reveals that modification of the piperazinyl nitrogen by acylation does not alter the effect of these molecules towards their bacterial target. We also present evidence that these N-acylated compounds are highly effective at killing intracellular bacteria, suggesting the suitability of these antibiotics for therapeutic treatment.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Ciprofloxacina/análogos & derivados , Ciprofloxacina/farmacología , Acilación , Infecciones Bacterianas/tratamiento farmacológico , Bartonella/efectos de los fármacos , Infecciones por Bartonella/tratamiento farmacológico , Farmacorresistencia Bacteriana , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/tratamiento farmacológicoRESUMEN
Six broad-host-range plasmid vectors were developed to study gene expression in Bartonella henselae. The vectors were used to express a beta-galactosidase reporter gene in B. henselae and to generate antisense RNA for gene knockdown. When applied to ompR, a putative transcription response regulator of B. henselae, this antisense RNA gene knockdown strategy reduced bacterial invasion of human endothelial cells by over 60%.
Asunto(s)
Bartonella henselae/patogenicidad , Células Endoteliales/microbiología , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Plásmidos/genética , beta-Galactosidasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bartonella henselae/genética , Bartonella henselae/metabolismo , Técnicas de Silenciamiento del Gen , Vectores Genéticos , Humanos , ARN sin Sentido , beta-Galactosidasa/genéticaRESUMEN
The facultative intracellular bacterium Bartonella henselae induces unique angiogenic lesions in immunocompromised hosts. To determine the role of intracellular calcium pools in B. henselae-induced endothelial cell proliferation, we generated B. henselae-conditioned medium (BCM) and tested the ability of these cell-free proteins to induce human umbilical vein endothelial cell (HUVEC) proliferation, CXCL8 production, and intracellular Ca2+ signals. HUVECs incubated with BCM for 3 days had higher cell numbers than controls. In addition, HUVECs produced increased amounts of CXCL8 in response to BCM when compared to medium controls. When BCM was added to HUVECs and the intracellular Ca2+ response measured with the calcium-sensitive dye fura-2/AM, a Ca2+ rise was demonstrated. It was determined that this Ca2+ rise originated from intracellular Ca2+ stores through the use of the Ca2+ ATPase inhibitor thapsigargin. Further, it was demonstrated that BCM enhanced CXCL8 production and HUVEC proliferation in a Ca2+-dependent manner. Conditioned medium from B. henselae causes an intracellular Ca2+ rise in HUVECs, which is involved in B. henselae-induced HUVEC proliferation and CXCL8 production. These results implicate intracellular Ca2+ pools in B. henselae-induced angiogenesis and may lead to increased understanding of the mechanisms of pathogen-induced angiogenesis.
Asunto(s)
Bartonella henselae/fisiología , Calcio/metabolismo , Proliferación Celular , Endotelio Vascular/citología , Western Blotting , Células Cultivadas , Chaperonina 60/metabolismo , Medios de Cultivo Condicionados , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Interleucina-8/metabolismo , Tapsigargina/farmacología , Venas Umbilicales/citologíaRESUMEN
Bartonella henselae can infect humans resulting in a wide range of disease syndromes including cat-scratch disease, fever with bacteremia, endocarditis, bacillary angiomatosis, and bacillary peliosis hepatis, among others. The nature and severity of the clinical presentation correlates well with the status of the hosts' immune system. Individuals with impaired immune function, including HIV infection, progress to systemic infections more often. Patients with intact immune function who become infected with B. henselae usually get cat-scratch disease, a disease that usually involves lymphadenopathy resulting from a strong cellular immune response to the bacterium. However, immunocompromised patients often progress to bacillary angiomatosis or bacillary peliosis hepatis. The reduced ability of the hosts immune response to control bacterial infection apparently results in a bacteremia of longer duration, and in some patients the presence of angiogenic lesions that are unique among bacterial infections to Bartonella. Recently, the role of immune effector cells that produce angiogenic cytokines upon stimulation with B. henselae has been proposed. Here, the current status of the role of the immune response in both controlling infection and in B. henselae-triggered immunopathogenesis is presented.
Asunto(s)
Infecciones por Bartonella/inmunología , Bartonella henselae/patogenicidad , Animales , Infecciones por Bartonella/patología , Enfermedad por Rasguño de Gato/inmunología , Modelos Animales de Enfermedad , Interacciones Huésped-Parásitos/inmunología , Humanos , Huésped Inmunocomprometido , Ratones , Neovascularización Patológica/microbiologíaRESUMEN
Bartonella bacilliformis is the bacterial agent of Carrión's disease and is presumed to be transmitted between humans by phlebotomine sand flies. Carrión's disease is endemic to high-altitude valleys of the South American Andes, and the first reported outbreak (1871) resulted in over 4,000 casualties. Since then, numerous outbreaks have been documented in endemic regions, and over the last two decades, outbreaks have occurred at atypical elevations, strongly suggesting that the area of endemicity is expanding. Approximately 1.7 million South Americans are estimated to be at risk in an area covering roughly 145,000 km2 of Ecuador, Colombia, and Peru. Although disease manifestations vary, two disparate syndromes can occur independently or sequentially. The first, Oroya fever, occurs approximately 60 days following the bite of an infected sand fly, in which infection of nearly all erythrocytes results in an acute hemolytic anemia with attendant symptoms of fever, jaundice, and myalgia. This phase of Carrión's disease often includes secondary infections and is fatal in up to 88% of patients without antimicrobial intervention. The second syndrome, referred to as verruga peruana, describes the endothelial cell-derived, blood-filled tumors that develop on the surface of the skin. Verrugae are rarely fatal, but can bleed and scar the patient. Moreover, these persistently infected humans provide a reservoir for infecting sand flies and thus maintaining B. bacilliformis in nature. Here, we discuss the current state of knowledge regarding this life-threatening, neglected bacterial pathogen and review its host-cell parasitism, molecular pathogenesis, phylogeny, sand fly vectors, diagnostics, and prospects for control.
Asunto(s)
Infecciones por Bartonella , Bartonella bacilliformis , Enfermedades Desatendidas , Animales , Interacciones Huésped-Patógeno , Humanos , Insectos Vectores , Psychodidae , América del SurRESUMEN
Francisella tularensis is a zoonotic bacterium that must exist in diverse environments ranging from arthropod vectors to mammalian hosts. To better understand how virulence genes are regulated in these different environments, a transcriptional response regulator gene (genome locus FTL0552) was deleted in F. tularensis live vaccine strain (LVS). The FTL0552 deletion mutant exhibited slightly reduced rates of extracellular growth but was unable to replicate or survive in mouse macrophages and was avirulent in the mouse model using either BALB/c or C57BL/6 mice. Mice infected with the FTL0552 mutant produced reduced levels of inflammatory cytokines, exhibited reduced histopathology, and cleared the bacteria quicker than mice infected with LVS. Mice that survived infection with the FTL0552 mutant were afforded partial protection when challenged with a lethal dose of the virulent SchuS4 strain (4 of 10 survivors, day 21 postinfection) when compared to naive mice (0 of 10 survivors by day 7 postinfection). Microarray experiments indicate that 148 genes are regulated by FTL0552. Most of the genes are downregulated, indicating that FTL0552 controls transcription of genes in a positive manner. Genes regulated by FTL0552 include genes located within the Francisella pathogenicity island that are essential for intracellular survival and virulence of F. tularensis. Further, a mutant in FTL0552 or the comparable locus in SchuS4 (FTT1557c) may be an alternative candidate vaccine for tularemia.
Asunto(s)
Vacunas Bacterianas , Francisella tularensis/genética , Francisella tularensis/inmunología , Genes Bacterianos , Proteínas Mutantes/inmunología , Tularemia/terapia , Vacunas Atenuadas , Animales , Vacunas Bacterianas/inmunología , Células Cultivadas , Citocinas/metabolismo , Femenino , Francisella tularensis/patogenicidad , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Mutantes/genética , Mutación , Análisis de Supervivencia , Tularemia/inmunología , Tularemia/metabolismo , Tularemia/mortalidad , Replicación Viral/genéticaRESUMEN
The gram-negative bacterium Bartonella henselae is capable of causing angiogenic lesions as a result of infection. Previously, it has been shown that B. henselae infection can result in production of the chemokine interleukin-8 (IL-8). In this study, we demonstrated that monocytes, endothelial cells, and hepatocytes produce IL-8 in response to B. henselae infection. We also investigated the role of IL-8 in B. henselae-induced endothelial cell proliferation and capillary tube formation. Both in vitro angiogenesis assays were IL-8 dependent. B. henselae-mediated inhibition of apoptosis, as indicated by gene expression of Bax and Bcl-2, was also shown to be IL-8 dependent in endothelial cells. Furthermore, infection of endothelial cells with B. henselae stimulated upregulation of the IL-8 chemokine receptor CXCR2. Infection of human endothelial cells by B. henselae resulting in IL-8 production likely plays a central role in the ability of this organism to cause angiogenesis during infection.
Asunto(s)
Angiomatosis Bacilar/inmunología , Bartonella henselae , Interleucina-8/fisiología , Neovascularización Patológica/inmunología , Receptores de Interleucina-8B/metabolismo , Angiomatosis Bacilar/genética , Angiomatosis Bacilar/patología , Apoptosis/genética , Comunicación Autocrina , Capilares/crecimiento & desarrollo , Proliferación Celular , Células Cultivadas , Endotelio Vascular/inmunología , Endotelio Vascular/microbiología , Endotelio Vascular/patología , Expresión Génica , Hepatocitos/inmunología , Humanos , Inmunoglobulina G/farmacología , Interleucina-8/antagonistas & inhibidores , Interleucina-8/genética , Monocitos/inmunología , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores de Interleucina-8B/genética , Regulación hacia Arriba , Proteína X Asociada a bcl-2/genéticaRESUMEN
Bacillary angiomatosis (BA), one of the many clinical manifestations resulting from infection with the facultative intracellular bacterium Bartonella henselae, is characterized by angiogenic lesions. Macrophages have been identified as important effector cells contributing to the angiogenic process during B. henselae infection by infiltrating BA lesions and secreting vascular endothelial growth factor. Monocyte-macrophage chemoattractant protein 1 (MCP-1) recruits macrophages to sites of inflammation. In this study, we investigated the ability of B. henselae to upregulate MCP-1 gene expression and protein production in the human microvascular endothelial cell line HMEC-1. MCP-1 mRNA was induced at 6 and 24 h after treatment with bacteria, whereas protein production was elevated at 6, 24, and 48 h. This induction was not dependent on the presence of bacterial lipopolysaccharide or endothelial cell toll-like receptor 4. However, MCP-1 production was dependent on NF-kappaB activity. Outer membrane proteins of low molecular weight were able to upregulate MCP-1 production. Furthermore, supernatants from B. henselae-infected HMEC-1 were able to induce chemotaxis of THP-1 monocytes. These data suggest a mechanism by which the macrophage effector cell is recruited to the endothelium during B. henselae infection and then contributes to bacterial-induced angiogenesis.
Asunto(s)
Bartonella henselae/fisiología , Comunicación Celular/inmunología , Movimiento Celular/fisiología , Quimiocina CCL2/genética , Endotelio Vascular/microbiología , Monocitos/fisiología , Proteínas de la Membrana Bacteriana Externa/fisiología , Línea Celular Transformada , Línea Celular Tumoral , Quimiocina CCL2/biosíntesis , Quimiotaxis/fisiología , Humanos , Glicoproteínas de Membrana/fisiología , Monocitos/citología , Monocitos/metabolismo , FN-kappa B/fisiología , Receptores de Superficie Celular/fisiología , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
Bartonella henselae is responsible for various disease syndromes that loosely correlate with the immune status of the host. In the immunocompromised individual, B. henselae-induced angiogenesis, or bacillary angiomatosis, is characterized by vascular proliferative lesions similar to those in Kaposi's sarcoma. We hypothesize that B. henselae-mediated interaction with immune cells, namely, macrophages, induces potential angiogenic growth factors and cytokines which contribute in a paracrine manner to the proliferation of endothelial cells. Vascular endothelial growth factor (VEGF), a direct inducer of angiogenesis, and interleukin-1beta (IL-1beta), a potentiator of VEGF, were detected within 12 and 6 h, respectively, in supernatants from phorbol 12-myristate 13-acetate-differentiated human THP-1 macrophages exposed to live B. henselae. Pretreatment of macrophages with cytochalasin D, a phagocytosis inhibitor, yielded comparable results, suggesting that bacterium-cell attachment is sufficient for VEGF and IL-1beta induction. IL-8, an angiogenic cytokine with chemotactic properties, was induced in human microvascular endothelial cells (HMEC-1) within 6 h of infection, whereas no IL-8 induction was observed in infected THP-1 cells. In addition, conditioned medium from infected macrophages induced the proliferation of HMEC-1, thus demonstrating angiogenic potential. These data suggest that Bartonella modulation of host or target cell cytokines and growth factors, rather than a direct role of the bacterium as an endothelial cell mitogen, is the predominant mechanism responsible for angiogenesis. B. henselae induction of VEGF, IL-1beta, and IL-8 outlines a broader potential paracrine angiogenic loop whereby macrophages play the predominant role as the effector cell and endothelial cells are the final target cell, resulting in their proliferation.