Scalp eschar and neck lymphadenopathy after tick bite (SENLAT) caused by Bartonella henselae in Korea: a case report.

BACKGROUND: Tick-borne lymphadenopathy (TIBOLA) is an infectious disease, mainly caused by species from the spotted fever group rickettsiae and is characterized by enlarged lymph nodes following a tick bite. Among cases of TIBOLA, a case of scalp eschar and neck lymphadenopathy after tick bite (SENLAT) is diagnosed when an eschar is present on the scalp, accompanied by peripheral lymphadenopathy (LAP). Only a few cases of SENLAT caused by Bartonella henselae have been reported. CASE PRESENTATION: A 58-year-old male sought medical advice while suffering from high fever and diarrhea. Three weeks before the visit, he had been hunting a water deer, and upon bringing the deer home discovered a tick on his scalp area. Symptoms occurred one week after hunting, and a lump was palpated on the right neck area 6 days after the onset of symptoms. Physical examination upon presentation confirmed an eschar-like lesion on the right scalp area, and cervical palpation revealed that the lymph nodes on the right side were non-painful and enlarged at 2.5 × 1.5 cm. Fine needle aspiration of the enlarged lymph nodes was performed, and results of nested PCR for the Bartonella internal transcribed spacer (ITS) confirmed B. henselae as the causative agent. CONCLUSION: With an isolated case of SENLAT and a confirmation of B. henselae in Korea, it is pertinent to raise awareness to physicians in other Asian countries that B. henselae could be a causative agent for SENLAT.

Ahnak-knockout mice show susceptibility to Bartonella henselae infection because of CD4+ T cell inactivation and decreased cytokine secretion.

The present study evaluated the role of AHNAK in Bartonella henselae infection. Mice were intraperitoneally inoculated with 2 × 108 colony-forming units of B. henselae Houston-1 on day 0 and subsequently on day 10. Blood and tissue samples of the mice were collected 8 days after the final B. henselae injection. B. henselae infection in the liver of Ahnak-knockout and wild-type mice was confirmed by performing polymerase chain reaction, with Bartonella adhesion A as a marker. The proportion of B. henselaeinfected cells increased in the liver of the Ahnak-knockout mice. Granulomatous lesions, inflammatory cytokine levels, and liver enzyme levels were also higher in the liver of the Ahnak-knockout mice than in the liver of the wild-type mice, indicating that Ahnak deletion accelerated B. henselae infection. The proportion of CD4＋interferon-γ (IFN-γ)＋ and CD4＋interleukin (IL)-4＋ cells was significantly lower in the B. henselae-infected Ahnak-knockout mice than in the B. henselae-infected wild-type mice. In vitro stimulation with B. henselae significantly increased IFN-γ and IL-4 secretion in the splenocytes obtained from the B. henselae-infected wild-type mice, but did not increase IFN-γ and IL-4 secretion in the splenocytes obtained from the B. henselae-infected Ahnak-KO mice. In contrast, IL-1α, IL-1ß, IL-6, IL-10, RANTES, and tumor necrosis factor-α secretion was significantly elevated in the splenocytes obtained from both B. henselae-infected wild-type and Ahnak-knockout mice. These results indicate that Ahnak deletion promotes B. henselae infection. Impaired IFN-γ and IL-4 secretion in the Ahnak-knockout mice suggests the impairment of Th1 and Th2 immunity in these mice. [BMB Reports 2019; 52(4): 289-294].

Bartonella henselae infection in diverse clinical conditions in a tertiary care hospital in north India.

BACKGROUND & OBJECTIVES: : Bartonella henselae causes infections which closely resemble febrile illness and chronic diseases such as tuberculosis and haematological malignancies. There are not many studies on Bartonella infections from India. The present study was undertaken to diagnose B. henselae infection in diverse clinical conditions in a tertiary care hospital in north India. METHODS: A total of 145 patients including those with fever and lymphadenopathy, infective endocarditis and neuroretinitis were enrolled in the study. Whole blood, serum and lymph node aspirate and valvular vegetations if available, were obtained. Samples were plated on chocolate agar and brain-heart infusion agar containing five per cent fresh rabbit blood and were incubated at 35°C for at least four weeks in five per cent CO2with high humidity. Immunofluorescent antibody assay (IFA) was done for the detection of IgM antibodies in the serum using a commercial kit. Whole blood was used to perform polymerase chain reaction (PCR) for the citrate synthase gene (gltA). RESULTS: IFA was positive in 11 of 140 (7.85%) patients and PCR was positive in 3 of 140 (2.14%) patients. Culture was negative in all the cases. A higher incidence of Bartonella infection was seen in patients with fever and lymphadenopathy (n=30), seven of whom were children. In ophthalmological conditions, four cases were IFA positive. INTERPRETATION & CONCLUSIONS: The present study shows that the threat of Bartonella infection is a reality in India. It is also an important treatable cause of fever and lymphadenopathy in children. Serology and PCR are useful tests for its diagnosis. Clinicians should consider. BARTONELLA: infection in the differential diagnosis of febrile illnesses and chronic diseases.

Multiorgan Involvement Confounding the Diagnosis of Bartonella henselae Infective Endocarditis in Children With Congenital Heart Disease.

Two children with congenital heart disease status post surgical correction presented with prolonged constitutional symptoms, hepatosplenomegaly and pancytopenia. Concern for malignancy prompted bone marrow biopsies that were without evidence thereof. In case 1, echocardiography identified a multilobulated vegetation on the conduit valve. In case 2, transthoracic, transesophageal and intracardiac echocardiography were performed and were without evidence of cardiac vegetations; however, pulmonic emboli raised concern for infective endocarditis. Both patients underwent surgical resection of the infected material and had histopathologic evidence of infective endocarditis. Further diagnostics identified elevated cytoplasmic antineutrophil cytoplasmic antibodies and antiproteinase 3 antibodies in addition to acute kidney injury with crescentic glomerulonephritis on renal biopsy. Serologic evidence of infection with Bartonella henselae was observed in both patients. These 2 cases highlight the potential multiorgan involvement that may confound the diagnosis of culture-negative infective endocarditis caused by B. henselae.

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.

Bartonella henselae transmission by blood transfusion in mice.

BACKGROUND: Bartonella spp. are neglected fastidious Gram-negative bacilli. We isolated Bartonella henselae from 1.2% of 500 studied blood donors and demonstrated that the bacteria remain viable in red blood cell units after 35 days of experimental infection. Now, we aim to evaluate the possibility of B. henselae transmission by blood transfusion in a mouse model. STUDY DESIGN AND METHODS: Eight BALB/c mice were intraperitoneal inoculated with a 30 µL of suspension with 10(4) CFU/mL of B. henselae and a second group of eight mice were inoculated with saline solution and used as control. After 96 hours of inoculation, the animals were euthanized. We collected blood and tissue samples from skin, liver, and spleen. Thirty microliters of blood from four Bartonella-inoculated animals were transfused into a new group (n = 4). Another group received blood from the control animals. B. henselae infection was investigated by conventional and nested polymerase chain reaction (PCR). RESULTS: Blood samples from all 24 mice were negative by molecular tests though half of the tissue samples were positive by nested PCR in the intraperitoneal Bartonella-investigated animals. Tissues from two of the four mice that received blood transfusions from Bartonella-inoculated animals were also nested PCR positives. CONCLUSIONS: Transmission of B. henselae by transfusion is possible in mice even when donor animals have undetectable bloodstream infection. The impact of human Bartonella sp. transmission through blood transfusion recipients must be evaluated.

Prolonged course of hepatic granulomatous disease due to Bartonella henselae infection.

Cat-scratch disease (CSD) is an emerging zoonosis caused by Bartonella henselae. The disease is usually self-limiting and typically presents in about 90% of all cases as a subacute regional lymphadenopathy. We present a case report of an unusual CSD presentation, persistent hepatic granulomatous disease due to Bartonella henselae infection despite combination therapy with doxycycline and rifampicin. Furthermore, a review of literature was conducted. (Acta gastroenterol. belg., 2016, 79, 497-499).

Infección por Bartonella henselae como presentación de fiebre de origen desconocida en niños peruanos / Infection with bartonella henselae in patients with fever of unknown origin

Objetivo. Describir los casos por infección por Bartonella henselae como presentación de fiebre de origen desconocida (FOD) en el Instituto Nacional de Salud del Niño (INSN) de Perú. Material y métodos. Estudio de serie de casos de niños atendidos en el INSN en el año 2012. Se definió infección por B. henselae si el caso presentaba serología positiva. Resultados. En el año 2012 se diagnosticaron 26 casos de FOD, de los cuales 12 fueron por infección por B. henselae. De estos 12, 6 fueron masculinos, con una mediana para la edad de 5 años (RIQ 3,5-4,0); 11 tenían una historia de exposición a gatos; 6 tuvieron lesión lineal ocasionada por el gato. Dentro de los exámenes de laboratorio, 4 de 12 tuvieron leucocitosis; 5 presentaron una PCR mayor de 10. El examen ecográfico reveló que 8 de 12 presentaron lesiones hipoecoicas en bazo; existió coinfección con otras infecciones; 2 casos tuvieron manifestación articular y 1, convulsión. Conclusión. Se concluye que los casos de FOD por infección por B. henselae en niños, están relacionados con exposición a gatos y lesiones hipoecoicas en bazo e hígado, con una evolución de la enfermedad con pronóstico bueno.

Objective. To describe the cases of infection with Bartonella henselae as presentation of fever of unknown origin (FUO) at the National Institute of Child Health (NICH) of Peru. Material and Methods. Study of a series of cases of children cared for in the NICH in 2012, infection was defined by B. henselae if presented positive serology. Results. In 2012, 26 cases were diagnosed of FUO of which 12 were due to infection by B. henselae, 06 were male, with a median age of 5 years (IQR 3,5-4,0), 11 of 12 had a history of exposure to cats, 6 of 12 had linear lesion caused by the cat. Within the lab tests 4 out of 12 had leukocytosis, 5 of 12 presented a CRP of greater than 10. The ultrasound examination revealed that 8 out of 12 lesions were hypoechoics in the spleen; there was co-infection with other infections; 02 cases had demonstration articulate and one with seizure. We conclude that the cases of FUO by infection by B. henselae in children are related to school-age, with the exposure of cats and injury hypoechoic in the spleen. Conclusions. We conclude that the cases of FUO by infection by B. henselae in children are related to the exposure of cats and injury hypoechoic in the spleen and liver and good prognosis.

Strategy for identification & characterization of Bartonella henselae with conventional & molecular methods.

BACKGROUND & OBJECTIVES: Bartonella henselae is a fastidious gram-negative bacterium usually causing self limiting infections in immunocompetent individuals but often causes potentially life threatening infection, such as bacillary angiomatosis in immunocompromised patients. Both diagnosis of infections and research into molecular mechanisms of pathogenesis have been hindered by lack of appropriate and reliable diagnostic techniques. We undertook this study to standardize methods to characterize B. henselae in clinical samples to diagnose Bartonella infection correctly. METHODS: B. henselae ATCC 49882 strain was procured from American type culture collection, USA. This strain was revived and maintained in the laboratory, and identification and characterization of this strain was done by conventional and molecular techniques, which included culture on various media, staining by different methods including electron microscopy, biochemical analysis by conventional methods and API, polymerase chain reaction (PCR) for amplification of citrate synthase gene followed by restriction fragment length polymorphism (RFLP). RESULTS: This organism was biochemically inert due to slow growth and generated unique identification code with API. The amplification of the citrate-synthase gene with primers yielded a 381 bp product followed by specific RFLP profile for B. henselae. INTERPRETATION & CONCLUSIONS: Bartonella is fastidious and fragile organism and should be handled carefully. Extra effort and careful observation are required to isolate and characterize this organism.

Depolymerization of cytokeratin intermediate filaments facilitates intracellular infection of HeLa cells by Bartonella henselae.

Bartonella henselae is capable of invading epithelial and endothelial cells by modulating the function of actin-dependent cytoskeleton proteins. Although understanding of the pathogenesis has been increased by the development of an in vitro infection model involving endothelial cells, little is known about the mechanism of interaction between B. henselae and epithelial cells. This study aims to identify the binding candidates of B. henselae in epithelial cells and explores their effect on B. henselae infection. Pull-down assays and mass spectrometry analysis confirmed that some of the binding proteins (keratin 14, keratin 6, and F-actin) are cytoskeleton associated. B. henselae infection significantly induces the expression of the cytokeratin genes. Chemical disruption of the keratin network by using ethylene glycol tetraacetic acid promotes the intracellular persistence of B. henselae in HeLa cells. However, cytochalasin B and phalloidin treatment inhibits B. henselae invasion. Immunofluorescent staining demonstrates that B. henselae infection induces an F-actin-dependent rearrangement of the cytoskeleton. However, we demonstrated via immunofluorescent staining and whole-mount cell electron microscopy that keratin intermediate filaments are depolymerized by B. henselae. The results indicate that B. henselae achieves an intracellular persistence in epithelial cells through the depolymerization of cytokeratin intermediate filaments that are protective against B. henselae invasion.

Bartonella henselae trimeric autotransporter adhesin BadA expression interferes with effector translocation by the VirB/D4 type IV secretion system.

The Gram-negative, zoonotic pathogen Bartonella henselae is the aetiological agent of cat scratch disease, bacillary angiomatosis and peliosis hepatis in humans. Two pathogenicity factors of B. henselae - each displaying multiple functions in host cell interaction - have been characterized in greater detail: the trimeric autotransporter Bartonella adhesin A (BadA) and the type IV secretion system VirB/D4 (VirB/D4 T4SS). BadA mediates, e.g. binding to fibronectin (Fn), adherence to endothelial cells (ECs) and secretion of vascular endothelial growth factor (VEGF). VirB/D4 translocates several Bartonella effector proteins (Beps) into the cytoplasm of infected ECs, resulting, e.g. in uptake of bacterial aggregates via the invasome structure, inhibition of apoptosis and activation of a proangiogenic phenotype. Despite this knowledge of the individual activities of BadA or VirB/D4 it is unknown whether these major virulence factors affect each other in their specific activities. In this study, expression and function of BadA and VirB/D4 were analysed in a variety of clinical B. henselae isolates. Data revealed that most isolates have lost expression of either BadA or VirB/D4 during in vitro passages. However, the phenotypic effects of coexpression of both virulence factors was studied in one clinical isolate that was found to stably coexpress BadA and VirB/D4, as well as by ectopic expression of BadA in a strain expressing VirB/D4 but not BadA. BadA, which forms a dense layer on the bacterial surface, negatively affected VirB/D4-dependent Bep translocation and invasome formation by likely preventing close contact between the bacterial cell envelope and the host cell membrane. In contrast, BadA-dependent Fn binding, adhesion to ECs and VEGF secretion were not affected by a functional VirB/D4 T4SS. The obtained data imply that the essential virulence factors BadA and VirB/D4 are likely differentially expressed during different stages of the infection cycle of Bartonella.

Persistence of Bartonella spp. stealth pathogens: from subclinical infections to vasoproliferative tumor formation.

Bartonella spp. are facultative intracellular bacteria that typically cause a long-lasting intraerythrocytic bacteremia in their mammalian reservoir hosts, thereby favoring transmission by blood-sucking arthropods. In most cases, natural reservoir host infections are subclinical and the relapsing intraerythrocytic bacteremia may last weeks, months, or even years. In this review, we will follow the infection cycle of Bartonella spp. in a reservoir host, which typically starts with an intradermal inoculation of bacteria that are superficially scratched into the skin from arthropod feces and terminates with the pathogen exit by the blood-sucking arthropod. The current knowledge of bacterial countermeasures against mammalian immune response will be presented for each critical step of the pathogenesis. The prevailing models of the still-enigmatic primary niche and the anatomical location where bacteria reside, persist, and are periodically seeded into the bloodstream to cause the typical relapsing Bartonella spp. bacteremia will also be critically discussed. The review will end up with a discussion of the ability of Bartonella spp., namely Bartonella henselae, Bartonella quintana, and Bartonella bacilliformis, to induce tumor-like vascular deformations in humans having compromised immune response such as in patients with AIDS.

Bartonella henselae engages inside-out and outside-in signaling by integrin ß1 and talin1 during invasome-mediated bacterial uptake.

The VirB/D4 type IV secretion system (T4SS) of the bacterial pathogen Bartonella henselae (Bhe) translocates seven effector proteins (BepA-BepG) into human cells that subvert host cellular functions. Two redundant pathways dependent on BepG or the combination of BepC and BepF trigger the formation of a bacterial uptake structure termed the invasome. Invasome formation is a multi-step process consisting of bacterial adherence, effector translocation, aggregation of bacteria on the cell surface and engulfment, and eventually, complete internalization of the bacterial aggregate occurs in an F-actin-dependent manner. In the present study, we show that Bhe-triggered invasome formation depends on integrin-ß1-mediated signaling cascades that enable assembly of the F-actin invasome structure. We demonstrate that Bhe interacts with integrin ß1 in a fibronectin- and VirB/D4 T4SS-independent manner and that activated integrin ß1 is essential for both effector translocation and the actin rearrangements leading to invasome formation. Furthermore, we show that talin1, but not talin2, is required for inside-out activation of integrin ß1 during invasome formation. Finally, integrin-ß1-mediated outside-in signaling by FAK, Src, paxillin and vinculin is necessary for invasome formation. This is the first example of a bacterial entry process that fully exploits the bi-directional signaling capacity of integrin receptors in a talin1-specific manner.

BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures.

The gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.

Transfer of R388 derivatives by a pathogenesis-associated type IV secretion system into both bacteria and human cells.

Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies. B. henselae codes for two T4SSs: VirB/VirD4 and Trw. A ΔvirB mutant strain was transfer deficient, while a ΔtrwE mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels. B. henselae bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient Escherichia coli under certain conditions, underscoring the versatility of T4SSs.

Adhesion and host cell modulation: critical pathogenicity determinants of Bartonella henselae.

Bartonella henselae, the agent of cat scratch disease and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis, contains to date two groups of described pathogenicity factors: adhesins and type IV secretion systems. Bartonella adhesin A (BadA), the Trw system and possibly filamentous hemagglutinin act as promiscous or specific adhesins, whereas the virulence locus (Vir)B/VirD4 type IV secretion system modulates a variety of host cell functions. BadA mediates bacterial adherence to endothelial cells and extracellular matrix proteins and triggers the induction of angiogenic gene programming. The VirB/VirD4 type IV secretion system is responsible for, e.g., inhibition of host cell apoptosis, bacterial persistence in erythrocytes, and endothelial sprouting. The Trw-conjugation system of Bartonella spp. mediates host-specific adherence to erythrocytes. Filamentous hemagglutinins represent additional potential pathogenicity factors which are not yet characterized. The exact molecular functions of these pathogenicity factors and their contribution to an orchestral interplay need to be analyzed to understand B. henselae pathogenicity in detail.

Doença da arranhadura do gato: relato de caso / Cat scratch disease: case report

O diagnóstico diferencial das linfadenomegalias supuradas deve se basear na historia clínica e em exames complementares, principalmente na análise histopatólogica. A Bartonella henselae, bastonete gram-negativo fastidioso que frequentemente causa bacteriemia em gatos, constitui agente que sempre deve ser associado a linfadenomegalia. Os gatos são reservatórios importantes desse microrganismo. A doença da arranhadura do gato normalmente cursa com poliadenopatias relacionadas ao local de inoculação (por arranhadura ou lambedura) e sintomatologia geral leve, sendo normalmente autolimitada. Raramente a doença complica com acometimento visceral, ocular e neurológico. Em alguns casos, há poliadenopatia persistente e supurada. Este relato apresenta as alterações descritas por mulher de 79 anos, com poliadenopatia cervical ulcerada, surgida após contato próximo com felino. As alterações clínicas e histopatológicas foram sugestivas de doença da arranhadura do gato, de evolução prolongada. Houve boa resposta a antimicrobianos. Este trabalho ilustra o amplo diagnóstico diferencial de linfadenomegalias persistentes, que deve incluir sempre a infecção pela Bartonella henselae entre as prováveis etiologias.

Differential diagnosis of suppurative lymphadenomegalies must be based on clinical history and laboratory tests, particularly in histhopatological analysis. The Bartonella henselae is a fastidious gram-negative rod-shaped bacterium that commonly causes bacteremia in cats, frequently associated with lymphadenopathy. Cats are important reservoirs of this organism. Cat scratch disease usually happens with polyadenopathies related to the inoculation local (by scratching or licking) and general light symptoms, besides being selflimiting. This disease is rarely complicated with visceral, ocular and neurological injuries. Some cases show persistent and festering polyadenopathy. This report shows changes described by a 79 old-year-woman, who presents ulcerated cervical polyadenopathy, acquired after close contact with cats. Clinical and histopathological changes were suggestive of cat scratch disease, of long time evolution. There was good response to antibiotics. This paper illustrates the wide differential diagnosis of persistent lymphadenomegalies, which should always include infection with Bartonella henselae among several etiologies.

Combined action of the type IV secretion effector proteins BepC and BepF promotes invasome formation of Bartonella henselae on endothelial and epithelial cells.

Bartonella henselae (Bhe) can invade human endothelial cells (ECs) by two distinguishable entry routes: either individually by endocytosis or as large bacterial aggregates by invasome-mediated internalization. Only the latter process is dependent on a functional VirB/VirD4 type IV secretion system (T4SS) and the thereby translocated Bep effector proteins. Here, we introduce HeLa cells as a new cell system suitable to study invasome formation. We describe a novel route to trigger invasome formation by the combined action of the effectors BepC and BepF. Co-infections of either HUVEC or HeLa cells with the Bep-deficient ΔbepA-G mutant expressing either BepC or BepF restores invasome formation. Likewise, ectopic expression of a combination of BepC and BepF in HeLa cells enables invasome-mediated uptake of the Bhe ΔbepA-G mutant strain. Further, eGFP-BepC and eGFP-BepF fusion proteins localize to the cell membrane and, upon invasome formation, to the invasome. Furthermore, the combined action of BepC and BepF inhibits endocytic uptake of inert microspheres. Finally, we show that BepC and BepF-triggered invasome formation differs from BepG-triggered invasome formation in its requirement for cofilin1, while the Rac1/Scar1/WAVE/Arp2/3 and Cdc42/WASP/Arp2/3 signalling pathways are required in both cases.

The Bartonella henselae VirB/Bep system interferes with vascular endothelial growth factor (VEGF) signalling in human vascular endothelial cells.

The vasculotropic pathogen Bartonella henselae (Bh) intimately interacts with human endothelial cells (ECs) and subverts multiple cellular functions. Here we report that Bh specifically interferes with vascular endothelial growth factor (VEGF) signalling in ECs. Bh infection abrogated VEGF-induced proliferation and wound closure of EC monolayers as well as the capillary-like sprouting of EC spheroids. On the molecular level, Bh infection did not alter VEGF receptor 2 (VEGFR2) expression or cell surface localization, but impeded VEGF-stimulated phosphorylation of VEGFR2 at tyrosine(1175) . Consistently, we observed that Bh infection diminished downstream events of the tyrosine(1175) -dependent VEGFR2-signalling pathway leading to EC proliferation, i.e. phospholipase-Cγ activation, cytosolic calcium fluxes and mitogen-activated protein kinase ERK1/2 phosphorylation. Pervanadate treatment neutralized the inhibitory activity of Bh on VEGF signalling, suggesting that Bh infection may activate a phosphatase that alleviates VEGFR2 phosphorylation. Inhibition of VEGFR2 signalling by Bh infection was strictly dependent on a functional VirB type IV secretion system and thereby translocated Bep effector proteins. The data presented in this study underscore the role of the VirB/Bep system as important factor controlling EC proliferation in response to Bh infection; not only as previously reported by counter-acting an intrinsic bacterial mitogenic stimulus, but also by restricting the exogenous angiogenic stimulation by Bh-induced VEGF.