What Is in a Cat Scratch? Growth of Bartonella henselae in a Biofilm.

Bartonella henselae (B. henselae) is a gram-negative bacterium that causes cat scratch disease, bacteremia, and endocarditis, as well as other clinical presentations. B. henselae has been shown to form a biofilm in vitro that likely plays a role in the establishment and persistence of the bacterium in the host. Biofilms are also known to form in the cat flea vector; hence, the ability of this bacterium to form a biofilm has broad biological significance. The release of B. henselae from a biofilm niche appears to be important in disease persistence and relapse in the vertebrate host but also in transmission by the cat flea vector. It has been shown that the BadA adhesin of B. henselae is critical for adherence and biofilm formation. Thus, the upregulation of badA is important in initiating biofilm formation, and down-regulation is important in the release of the bacterium from the biofilm. We summarize the current knowledge of biofilm formation in Bartonella species and the role of BadA in biofilm formation. We discuss the evidence that defines possible mechanisms for the regulation of the genes required for biofilm formation. We further describe the regulation of those genes in the conditions that mimic both the arthropod vector and the mammalian host for B. henselae. The treatment for persistent B. henselae infection remains a challenge; hence, a better understanding of the mechanisms by which this bacterium persists in its host is critical to inform future efforts to develop drugs to treat such infections.

A non-coding RNA controls transcription of a gene encoding a DNA binding protein that modulates biofilm development in Bartonella henselae.

Bartonella henselae (Bh) is a Gram-negative zoonotic bacterium that can grow as large aggregates and form biofilms in vitro dependent upon the adhesin BadA. Previously, we reported that the Houston-1 strain of Bh has a family of nine small, highly-expressed intergenic transcripts called Bartonellaregulatory transcripts, Brt1-9. Each of the Brts bears a stem and loop structure on the 3' end followed by a gene encoding a DNA binding protein called the Transcriptional regulatory proteins, Trp1-9. RNA-seq analysis of laboratory-grown bacteria revealed the trps were poorly transcribed suggesting that the 3' stem and loop on the Brts results in transcript termination upstream of the trp genes under these conditions. Here we demonstrate that transcription of brt1 continues into trp1 when Bh is grown in a biofilm. Deletion of brt1, or just the 3' terminus of brt1 (containing the stem and loop structure), resulted in increased transcription of both trp1 and badA and increased biofilm formation. Trp1 was shown to directly bind the putative badA promoter region as demonstrated by an electrophoretic mobility shift assay (EMSA). Our data suggest that the 3' end of brt1 responds to a stimulus generated by growth of Bh in an in vitro biofilm to allow increased trp1 transcription. We further show that transcription of trp1 increases under conditions consistent with the mammalian host but is not highly expressed in the cat flea vector until the bacterium is excreted into the flea feces. Based on these data, we hypothesize that the 3' end of Brt1 functions to control trp1 transcription and Trp1 in turn results in increased badA expression and enhanced biofilm formation.

The trimeric autotransporter adhesin BadA is required for in vitro biofilm formation by Bartonella henselae.

Bartonella henselae (Bh) is a Gram-negative rod transmitted to humans by a scratch from the common house cat. Infection of humans with Bh can result in a range of clinical diseases including lymphadenopathy observed in cat-scratch disease and more serious disease from persistent bacteremia. It is a common cause of blood-culture negative endocarditis as the bacterium is capable of growing as aggregates, and forming biofilms on infected native and prosthetic heart valves. The aggregative growth requires a trimeric autotransporter adhesin (TAA) called Bartonella adhesin A (BadA). TAAs are found in all Bartonella species and many other Gram-negative bacteria. Using Bh Houston-1, Bh Houston-1 ∆badA and Bh Houston-1 ∆badA/pNS2PTrc badA (a partial complement of badA coding for a truncated protein of 741 amino acid residues), we analyze the role of BadA in adhesion and biofilm formation. We also investigate the role of environmental factors such as temperature on badA expression and biofilm formation. Real-time cell adhesion monitoring and electron microscopy show that Bh Houston-1 adheres and forms biofilm more efficiently than the Bh Houston-1 ∆badA. Deletion of the badA gene significantly decreases adhesion, the first step in biofilm formation in vitro, which is partially restored in Bh Houston-1 ∆badA/pNS2PTrc badA. The biofilm formed by Bh Houston-1 includes polysaccharides, proteins, and DNA components and is susceptible to enzymatic degradation of these components. Furthermore, both pH and temperature influence both badA expression and biofilm formation. We conclude that BadA is required for optimal adhesion, agglutination and biofilm formation.

Bartonella Species, an Emerging Cause of Blood-Culture-Negative Endocarditis.

Since the reclassification of the genus Bartonella in 1993, the number of species has grown from 1 to 45 currently designated members. Likewise, the association of different Bartonella species with human disease continues to grow, as does the range of clinical presentations associated with these bacteria. Among these, blood-culture-negative endocarditis stands out as a common, often undiagnosed, clinical presentation of infection with several different Bartonella species. The limitations of laboratory tests resulting in this underdiagnosis of Bartonella endocarditis are discussed. The varied clinical picture of Bartonella infection and a review of clinical aspects of endocarditis caused by Bartonella are presented. We also summarize the current knowledge of the molecular basis of Bartonella pathogenesis, focusing on surface adhesins in the two Bartonella species that most commonly cause endocarditis, B. henselae and B. quintana. We discuss evidence that surface adhesins are important factors for autoaggregation and biofilm formation by Bartonella species. Finally, we propose that biofilm formation is a critical step in the formation of vegetative masses during Bartonella-mediated endocarditis and represents a potential reservoir for persistence by these bacteria.

A family of genus-specific RNAs in tandem with DNA-binding proteins control expression of the badA major virulence factor gene in Bartonella henselae.

Bartonella henselae is a gram-negative zoonotic bacterium that causes infections in humans including endocarditis and bacillary angiomatosis. B. henselae has been shown to grow as large aggregates and form biofilms in vitro. The aggregative growth and the angiogenic host response requires the trimeric autotransporter adhesin BadA. We examined the transcriptome of the Houston-1 strain of B. henselae using RNA-seq revealing nine novel, highly-expressed intergenic transcripts (Bartonella regulatory transcript, Brt1-9). The Brt family of RNAs is unique to the genus Bartonella and ranges from 194 to 203 nucleotides with high homology and stable predicted secondary structures. Immediately downstream of each of the nine RNA genes is a helix-turn-helix DNA-binding protein (transcriptional regulatory protein, Trp1-9) that is poorly transcribed under the growth conditions used for RNA-seq. Using knockdown or overexpressing strains, we show a role of both the Brt1 and Trp1 in the regulation of badA and also in biofilm formation. Based on these data, we hypothesize that Brt1 is a trans-acting sRNA that also serves as a cis-acting riboswitch to control the expression of badA. This family of RNAs together with the downstream Trp DNA-binding proteins represents a novel coordinated regulatory circuit controlling expression of virulence-associated genes in the bartonellae.

Characterization of the general stress response in Bartonella henselae.

Bacteria utilize a general stress response system to combat stresses from their surrounding environments. In alpha-proteobacteria, the general stress response uses an alternate sigma factor as the main regulator and incorporates it with a two-component system into a unique regulatory circuit. This system has been described in several alpha-proteobacterial species, including the pathogens Bartonella quintana and Brucella abortus. Most of the studies have focused on characterizing the PhyR anti-anti-sigma factor, the NepR anti-sigma factor, and the alternate sigma factor. However, not enough attention is directed toward studying the role of histidine kinases in the general stress response. Our study identifies the general stress response system in Bartonella henselae, where the gene synteny is conserved and both the PhyR and alternate sigma factor have similar sequence and domain structures with other alpha-proteobacteria. Our data showed that the general stress response genes are up-regulated under conditions that mimic the cat flea vector. Furthermore, we showed that both RpoE and PhyR positively regulate this system and that RpoE also affects transcription of genes encoding heme-binding proteins and the gene encoding the BadA adhesin. Finally, we identified a histidine kinase, annotated as BH13820 that can potentially phosphorylate PhyR.

Oroya fever and verruga peruana: bartonelloses unique to South America.

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.

Zebrafish embryo model of Bartonella henselae infection.

Bartonella henselae (Bh) is an emerging zoonotic pathogen that has been associated with a variety of human diseases, including bacillary angiomatosis that is characterized by vasoproliferative tumor-like lesions on the skin of some immunosuppressed individuals. The study of Bh pathogenesis has been limited to in vitro cell culture systems due to the lack of an animal model. Therefore, we wanted to investigate whether the zebrafish embryo could be used to model human infection with Bh. Our data showed that Tg(fli1:egfp)(y1) zebrafish embryos supported a sustained Bh infection for 7 days with >10-fold bacterial replication when inoculated in the yolk sac. We showed that Bh recruited phagocytes to the site of infection in the Tg(mpx:GFP)uwm1 embryos. Infected embryos showed evidence of a Bh-induced angiogenic phenotype and an increase in the expression of genes encoding pro-inflammatory factors and pro-angiogenic markers. However, infection of zebrafish embryos with a deletion mutant in the major adhesin (BadA) resulted in little or no bacterial replication and a diminished host response, providing the first evidence that BadA is critical for in vivo infection. Thus, the zebrafish embryo provides the first practical model of Bh infection that will facilitate efforts to identify virulence factors and define molecular mechanisms of Bh pathogenesis.

Studies on the antimicrobial properties of N-acylated ciprofloxacins.

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.

Synthesis and antimicrobial activities of structurally novel S,S'-bis(heterosubstituted) disulfides.

The central focus of this study is on the antibacterial and antifungal properties of synthetically produced S,S'-bis(heterosubstituted) disulfides as a means to control the growth of various infection-causing pathogens. Staphylococcus aureus, Francisella tularensis and Candida albicans were each found to be highly susceptible to several of these compounds by agar or broth dilution and Kirby-Bauer diffusion assays. These structurally simple, low molecular weight disulfides have shown promising bioactivities and may serve as leads to the development of effective new antibacterials for pathogenic bacteria such as methicillin-resistant S. aureus and F. tularensis.

Plasmid-based system for high-level gene expression and antisense gene knockdown in Bartonella henselae.

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%.

Generation and characterization of an attenuated mutant in a response regulator gene of Francisella tularensis live vaccine strain (LVS).

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.

Bartonella-induced endothelial cell proliferation is mediated by release of calcium from intracellular stores.

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.

Autocrine role for interleukin-8 in Bartonella henselae-induced angiogenesis.

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.

Interaction of Bartonella henselae with endothelial cells promotes monocyte/macrophage chemoattractant protein 1 gene expression and protein production and triggers monocyte migration.

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.

Rapid and efficient transposon mutagenesis of Bartonella henselae by transposome technology.

Molecular genetics are difficult to perform in Bartonella henselae, the causative agent of cat scratch disease and the vasculoproliferative disorders bacillary angiomatosis and bacillary peliosis. To elucidate the underlying bacterial pathogenic mechanisms, genetic manipulation of B. henselae is the method of choice. We describe how to perform transposon mutagenesis in B. henselae using transposome technology. B. henselae mutants revealed by this technique showed random transpositional insertion into the chromosome. In contrast to transposon mutagenesis by conjugational transfer, transposome technology allows transposon mutagenesis of early passaged Bartonella spp. with approximately 100-fold higher efficiency. The results show that transposome technique is a rapid, efficient and simple method to generate transposon mutants of B. henselae.

The role of the host immune response in pathogenesis of Bartonella henselae.

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.

Induction of a potential paracrine angiogenic loop between human THP-1 macrophages and human microvascular endothelial cells during Bartonella henselae infection.

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.

Role of Bartonella henselae in the etiology of Henoch-Schönlein purpura.

BACKGROUND: The etiology of Henoch-Schönlein purpura (HSP) has been ascribed to a variety of infectious and noninfectious agents. Because we encountered a patient with HSP who had evidence of Bartonella henselae infection and a prior report of a patient with systemic cat-scratch disease presenting as leukoclastic vasculitis, we investigated the association of B. henselae infection with HSP. METHODS: We determined the antibody titers to B. henselae on the sera of 18 patients with HSP and on 57 controls. All patients presented with the characteristic leukoclastic rash of HSP. About one-half of the patients had joint or abdominal symptoms, and four had hematuria at presentation. An indirect immunofluorescent assay was used to determine serum antibody titers to B. henselae. Sera that were reactive at a dilution of 1/64 were considered positive. RESULTS: Eight of the 57 (14%) control sera and 12 of the 18 (67%) patient sera were positive for B. henselae antibody (P < 0.0001). CONCLUSION: The results of this study indicate a significant association of antecedent B. henselae infection with HSP. The frequency of this association (67%) exceeds that of previously ascribed etiologic agents for this disease, such as the group A Streptococcus.

Ehrlichiosis humana: reporte del primer caso en Venezuela / Human ehrlichiosis: report of the first case in Venezuela

La ehrlichiosis humana es una enfermedad zoonótica, causada por una rickettsia leucocitaria descrita por primera vez en los Estados Unidos de Norteamérica en 1986. Más de 300 casos han sido reportados en ese país, así como un caso en Portugal, dos en Francia y uno en un turista procedente de Malí (Africa). En Venezuela, un país tropical, se reporta el primer caso en una niña de 17 meses de edad, quien presentó inicialmente sintomatología compatible con una virosis, progresando con exantema, complicaciones pulmonares, hepáticas, esplénicas, renales y hematológicas incluyendo pancitopenia y coagulación intravascular diseminada (CID). Diferentes diagnósticos fueron planteados antes de concluir que se trataba de una ehrliochiosis. Ante tal evidencia se indicó tratamiento con tretraciclina y se obtuvo rápida recuperación. El diagnóstico inicial fue realizado usando frotis de capa blanca teñido con Diff Quick Stain. La presencia de anticuerpos específicos contra Ehrlichia chaffeensis, fue demostrada utilizando la técnica Inmunofluorescencia indirecta (IFI). Estos hallazgos en humanos no se habían descritos con anterioridad. No se pudo demostrar que la enfermedad fue transmitida por pidaduras de garrapatas