Volkhard A.J. Kempf

Bartonella Adhesin A Mediates a Proangiogenic Host Cell Response

Bartonella henselae causes vasculoproliferative disorders in humans. We identified a nonfimbrial adhesin of B. henselae designated as Bartonella adhesin A (BadA). BadA is a 340-kD outer membrane protein encoded by the 9.3-kb badA gene. It has a modular structure and contains domains homologous to the Yersinia enterocolitica nonfimbrial adhesin (Yersinia adhesin A). Expression of BadA was restored in a BadA-deficient transposon mutant by complementation in trans. BadA mediates the binding of B. henselae to extracellular matrix proteins and to endothelial cells, possibly via β1 integrins, but prevents phagocytosis. Expression of BadA is crucial for activation of hypoxia-inducible factor 1 in host cells by B. henselae and secretion of proangiogenic cytokines (e.g., vascular endothelial growth factor). BadA is immunodominant in B. henselae–infected patients and rodents, indicating that it is expressed during Bartonella infections. Our results suggest that BadA, the largest characterized bacterial protein thus far, is a major pathogenicity factor of B. henselae with a potential role in the induction of vasculoproliferative disorders.

Hypoxia-Independent Activation of HIF-1 by Enterobacteriaceae and Their Siderophores

BACKGROUND & AIMSnHypoxia inducible factor-1 (HIF-1) is the key transcriptional regulator during adaptation to hypoxia. Recent studies provide evidence for HIF-1 activation during bacterial infections. However, molecular details of how bacteria activate HIF-1 remain unclear. Here, we pursued the role of bacterial siderophores in HIF-1 activation during infection with Enterobacteriaceae.nnnMETHODSnIn vivo, HIF-1 activation and HIF-1-dependent gene induction in Peyers patches were analyzed after orogastric infection with Yersinia enterocolitica. The course of an orogastric Y enterocolitica infection was determined using mice with a deletion of HIF-1alpha in the intestine. In vitro, the mechanism of HIF-1 activation was analyzed in infections with Y enterocolitica, Salmonella enterica subsp enterica, and Enterobacter aerogenes.nnnRESULTSnInfection of mice with Y enterocolitica led to functional activation of HIF-1 in Peyers patches. Because mice with deletion of HIF-1alpha in the intestinal epithelium showed a significantly higher susceptibility to orogastric Y enterocolitica infections, bacterial HIF-1 activation appears to represent a host defense mechanism. Additional studies with Y enterocolitica, S enterica subsp enterica, or E aerogenes, and, moreover, application of their siderophores (yersiniabactin, salmochelin, aerobactin) caused a robust, dose-dependent HIF-1 response in human epithelia and endothelia, independent of cellular hypoxia. HIF-1 activation occurs most likely because of inhibition of prolylhydroxylase activity and is abolished upon infection with siderophore uptake deficient bacteria.nnnCONCLUSIONSnTaken together, this study reveals what we believe to be a previously unrecognized role of bacterial siderophores for hypoxia-independent activation of HIF-1 during infection with human pathogenic bacteria.

Oxygen-Independent Stabilization of Hypoxia Inducible Factor (HIF)-1 during RSV Infection

Background Hypoxia-inducible factor 1 (HIF)-1α is a transcription factor that functions as master regulator of mammalian oxygen homeostasis. In addition, recent studies identified a role for HIF-1α as transcriptional regulator during inflammation or infection. Based on studies showing that respiratory syncytial virus (RSV) is among the most potent biological stimuli to induce an inflammatory milieu, we hypothesized a role of HIF-1α as transcriptional regulator during infections with RSV. Methodology, Principal Findings We gained first insight from immunohistocemical studies of RSV-infected human pulmonary epithelia that were stained for HIF-1α. These studies revealed that RSV-positive cells also stained for HIF-1α, suggesting concomitant HIF-activation during RSV infection. Similarly, Western blot analysis confirmed an approximately 8-fold increase in HIF-1α protein 24 h after RSV infection. In contrast, HIF-1α activation was abolished utilizing UV-treated RSV. Moreover, HIF-α-regulated genes (VEGF, CD73, FN-1, COX-2) were induced with RSV infection of wild-type cells. In contrast, HIF-1α dependent gene induction was abolished in pulmonary epithelia following siRNA mediated repression of HIF-1α. Measurements of the partial pressure of oxygen in the supernatants of RSV infected epithelia or controls revealed no differences in oxygen content, suggesting that HIF-1α activation is not caused by RSV associated hypoxia. Finally, studies of RSV pneumonitis in mice confirmed HIF-α-activation in a murine in vivo model. Conclusions/Significance Taking together, these studies suggest hypoxia-independent activation of HIF-1α during infection with RSV in vitro and in vivo.

Structure of the head of the Bartonella adhesin BadA.

Trimeric autotransporter adhesins (TAAs) are a major class of proteins by which pathogenic proteobacteria adhere to their hosts. Prominent examples include Yersinia YadA, Haemophilus Hia and Hsf, Moraxella UspA1 and A2, and Neisseria NadA. TAAs also occur in symbiotic and environmental species and presumably represent a general solution to the problem of adhesion in proteobacteria. The general structure of TAAs follows a head-stalk-anchor architecture, where the heads are the primary mediators of attachment and autoagglutination. In the major adhesin of Bartonella henselae, BadA, the head consists of three domains, the N-terminal of which shows strong sequence similarity to the head of Yersinia YadA. The two other domains were not recognizably similar to any protein of known structure. We therefore determined their crystal structure to a resolution of 1.1 Å. Both domains are β-prisms, the N-terminal one formed by interleaved, five-stranded β-meanders parallel to the trimer axis and the C-terminal one by five-stranded β-meanders orthogonal to the axis. Despite the absence of statistically significant sequence similarity, the two domains are structurally similar to domains from Haemophilus Hia, albeit in permuted order. Thus, the BadA head appears to be a chimera of domains seen in two other TAAs, YadA and Hia, highlighting the combinatorial evolutionary strategy taken by pathogens.

Analysis of Bartonella Adhesin A Expression Reveals Differences between Various B. henselae Strains

ABSTRACT Bartonella henselae causes cat scratch disease and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis in humans. One of the best known pathogenicity factors of B. henselae is Bartonella adhesin A (BadA), which is modularly constructed, consisting of head, neck/stalk, and membrane anchor domains. BadA is important for the adhesion of B. henselae to extracellular-matrix proteins and endothelial cells (ECs). In this study, we analyzed different B. henselae strains for BadA expression, autoagglutination, fibronectin (Fn) binding, and adhesion to ECs. We found that the B. henselae strains Marseille, ATCC 49882, Freiburg 96BK3 (FR96BK3), FR96BK38, and G-5436 express BadA. Remarkably, BadA expression was lacking in a B. henselae ATCC 49882 variant, in strains ATCC 49793 and Berlin-1, and in the majority of bacteria of strain Berlin-2. Adherence of B. henselae to ECs and Fn reliably correlated with BadA expression. badA was present in all tested strains, although the length of the gene varied significantly due to length variations of the stalk region. Sequencing of the promoter, head, and membrane anchor regions revealed only minor differences that did not correlate with BadA expression, apart from strain Berlin-1, in which a 1-bp deletion led to a frameshift in the head region of BadA. Our data suggest that, apart from the identified genetic modifications (frameshift deletion and recombination), other so-far-unknown regulatory mechanisms influence BadA expression. Because of variations between and within different B. henselae isolates, BadA expression should be analyzed before performing infection experiments with B. henselae.

Do plant and human pathogens have a common pathogenicity strategy

Recently, a novel two-step model of pathogenicity has been described that suggests host-cell-derived vasculoproliferative factors play a crucial role in the pathogenesis of bacillary angiomatosis, a disease caused by the human pathogenic bacterium Bartonella henselae. The resulting proliferation of endothelial cells could be interpreted as bacterial pathogens triggering the promotion of their own habitat: the host cell. Similar disease mechanisms are well known in the plant pathogen Agrobacterium tumefaciens, which causes crown gall disease. There are notable similarities between the pathogenicity of A. tumefaciens leading to tumourous disease in plants and to the B. henselae-triggered proliferation of endothelial cells in humans. Here, we hypothesize that this pathogenicity strategy might be common to several bacterial species in different hosts owing to shared pathogenicity factors.

Influence of Nebivolol and Metoprolol on Inflammatory Mediators in Human Coronary Endothelial or Smooth Muscle Cells. Effects on Neointima Formation After Balloon Denudation in Carotid Arteries of Rats Treated with Nebivolol

Objective and Background: Inflammation plays a critical role in all stages of atherogenesis. Proliferating vascular smooth muscle cells (SMC) and endothelial cells (EC) enhancing the inflammatory response, both contribute to the progression of atherosclerosis. Anti-proliferative, anti-inflammatory and anti-oxidative therapy seems to be a promising therapeutic strategy. The aim of this study was to assess the anti-proliferative and anti-inflammatory effect of the β-blocker nebivolol in comparison to metoprolol in vitro and to find out whether nebivolol inhibits neointima formation in vivo. Methods and Results: Real-time-RT-PCR revealed a decrease in VCAM-1, ICAM-1, PDGF-B, E-selectin and P-selectin mRNA expression in human coronary artery EC and SMC incubated with nebivolol for 72 hours while metoprolol did not have this effect. Nebivolol reduced MCP-1 and PDGF-BB protein in the culture supernatant of SMC and EC, respectively. Sprague-Dawley rats were treated with nebivolol for 0 or 35 days before and 28 days after carotid balloon injury. Immunohistological analyses showed that pre-treatment with nebivolol was associated with a decreased number of SMC layers and macrophages and an increased lumen area at the site of the arterial injury. The intima area was reduced by 43% after pre-treatment. Conclusion: We found that nebivolol reduced the expression of proinflammatory genes in endothelial cells and vascular smooth muscle cells in vitro whereas metoprolol did not. In vivo, nebivolol inhibited neointima formation by reducing SMC proliferation and macrophage accumulation.

Unusual trafficking pattern of Bartonella henselae ‐containing vacuoles in macrophages and endothelial cells

Bartonella henselae, the agent of cat‐scratch disease and vasculoproliferative disorders in humans, is a fastidious facultative intracellular pathogen, whose interaction with macrophages and endothelial cells (ECs) is crucial in the pathogenesis of these diseases. However, little is known about the subcellular compartment in which B. henselae resides. Two hours after infection of murine macrophages and human ECs, the majority of B. henselae‐containing vacuoles (BCVs) lack typical endocytic marker proteins, fail to acidify, and do not fuse with lysosomes, suggesting that B. henselae resides in a non‐endocytic compartment. In contrast to human umbilical vein endothelial cells, bacterial death and lysosomal fusion with BCVs is apparent in J774A.1 macrophages at 24u2003h. This phenomenon of delayed lysosomal fusion requires bacterial viability, and is confined to the BCV itself. Using magnetic selection, we enriched for transposon‐mutagenized B. henselae trapped in lysosomes of macrophages 2u2003h after infection. Genes affected appear to be relevant to the intracellular lifestyle in macrophages and ECs and include some previously implicated in Bartonella pathogenicity. We conclude that B. henselae has a specific capacity to actively avoid the host endocytic pathway after entry of macrophages and ECs, from within a specialized non‐endocytic membrane‐bound vacuole.

Presumed Tuberculosis-induced Retinal Vasculitis, Diagnosed with Positron Emission Tomography (18F-FDG-PET/CT), Aspiration Biopsy, and Culture

Purpose: The diagnosis of tuberculosis as an etiological factor in patients with uveitis is difficult because of lack of specific diagnostic tests. The authors report 2 cases of occlusive retinal vasculitis, in which 18F-FDG-PET/CT was helpful for the diagnosis of tuberculosis as a presumptive cause of intraocular inflammation. Methods: In 2 patients with severe occlusive retinal vasculitis and positive QuantiFERON TB-Gold test, 18F-FDG-PET/CT, transbronchial needle-aspiration biopsy, and microbiological investigation were performed. Results: 18F-FDG-PET/CT showed increased fluorodeoxyglucose uptake in some mediastinal and hilar lymph nodes. After needle-aspiration biopsy of PET-positive lymph nodes, M. tuberculosis was recovered in culture in both cases. Remission of uveitis was achieved only after a combination therapy with 3 anti-tubercular agents and systemic steroids. Conclusion: The authors favor the use of 18F-FDG-PET/CT in patients with sight-threatening intraocular inflammation and positive interferon-gamma release assay. Anti-tubercular therapy, together with anti-inflammatory treatment, may lead to a remission in such patients.

Secondary syphilis after liver transplantation: case report and review of the literature

Abstract:u2002 Syphilitic disease is uncommon, but its incidence has increased worldwide in the last few years. An unusual manifestation of secondary syphilis after orthotopic liver transplantation is described which confirms that lues should be considered in patients with immune deficiency and abnormal liver function tests.