Search PubMed⌕ Search

Biomedical subjects

Volkhard A J Kempf

Publications and source records attributed to Volkhard A J Kempf.

At least 19 recordsLinked to original sources
PubMed2006

Analysis of Bartonella adhesin A expression reveals differences between various B. henselae strains.

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.

Adhesins, Bacterial↗
PubMed2006

Trimeric autotransporter adhesins: variable structure, common function.

Trimeric autotransporter adhesins (TAAs) are important virulence factors in gram-negative pathogens. Despite the variety of hosts ranging from plants to mammals and the specialized regulation of TAAs, their molecular organization follows surprisingly simple rules: they form trimeric surface structures with a head-stalk-anchor architecture. The head and stalk are composed of a small set of domains, building blocks that are frequently arranged repetitively. We propose that this repetitive arrangement facilitates recombination of domains to modulate the specificity of the common function: adhesion to the host.

Adhesins, Bacterial↗
PubMed2006

Orthopoxvirus infection transmitted by a domestic cat.

The variola virus was declared eradicated by the World Health Organization in 1980 but human infections by cowpox virus, another member of the genus Orthopoxvirus, are still observed, mainly in European countries. We report a woman who presented with two umbilicated vesicles surrounded by an indurated erythematous edema within cat scratch injuries on her thigh. The diagnosis of an Orthopoxvirus infection was based on the visualization of characteristic virus particles by electron microscopy and the detection of the A27L gene (14-kd fusion protein gene) of the genus Orthopoxvirus by polymerase chain reaction from a lesional skin biopsy specimen. Differential diagnoses of cat scratch disease, pustula maligna, and bullous impetigo were excluded by microbiologic investigation of the biopsy specimen. Both lesions scarred after 6 weeks of a continuous local antiseptic treatment.

Adult↗
PubMed2006

Bartonella quintana variably expressed outer membrane proteins mediate vascular endothelial growth factor secretion but not host cell adherence.

Bartonella quintana causes trench fever, endocarditis, and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis in humans. Little is known about the interaction of this pathogen with host cells. We attempted to elucidate the interaction of B. quintana with human macrophages (THP-1) and epithelial cells (HeLa 229). Remarkably, only B. quintana strain JK-31 induced secretion of vascular endothelial growth factor (VEGF) from THP-1 and HeLa 229 cells upon infection similar to the secretion induced by B. henselae Marseille, whereas other strains (B. quintana 2-D70, B. quintana Toulouse, and B. quintana Munich) did not induce such secretion. Immunofluorescence testing and electron microscopy revealed that the B. quintana strains unable to induce VEGF secretion did not express the variable outer membrane proteins (Vomps) on their surfaces. Surprisingly, the increase in VEGF secretion mediated by B. quintana JK-31 was not paralleled by elevated host cell adherence rates compared with the rates for Vomp-negative B. quintana strains. Our results suggest that the Vomps play a leading role in the angiogenic reprogramming of host cells by B. quintana but not in the adherence to host cells.

Amino Acid Sequence↗
PubMed2005

Infection of human CD34+ progenitor cells with Bartonella henselae results in intraerythrocytic presence of B. henselae.

Although there is evidence that endothelial cells are important targets for human pathogenic Bartonella species, the primary niche of infection is unknown. Here we elucidated whether human CD34+ hematopoietic progenitor cells (HPCs) internalize B. henselae and may serve as a potential niche of the pathogen. We showed that B. henselae does not adhere to or invade human erythrocytes. In contrast, B. henselae invades and persists in HPCs as shown by gentamicin protection assays, confocal laser scanning microscopy (CLSM), and electron microscopy (EM). Fluorescence-activated cell sorting (FACS) analysis of glycophorin A expression revealed that erythroid differentiation of HPCs was unaffected following infection with B. henselae. The number of intracellular B. henselae continuously increased over a 13-day period. When HPCs were infected with B. henselae immediately after isolation, intracellular bacteria were subsequently detectable in differentiated erythroid cells on day 9 and day 13 after infection, as shown by CLSM, EM, and FACS analysis. Our data provide, for the first time, evidence that a bacterial pathogen is able to infect and persist in differentiating HPCs, and suggest that HPCs might serve as a potential primary niche in Bartonella infections.

Angiomatosis, Bacillary↗
PubMed2005

Activation of hypoxia-inducible factor-1 in bacillary angiomatosis: evidence for a role of hypoxia-inducible factor-1 in bacterial infections.

BACKGROUND: Bartonella species are the only known bacterial pathogens causing vasculoproliferative disorders in humans (bacillary angiomatosis [BA]). Cellular and bacterial pathogenetic mechanisms underlying the induction of BA are largely unknown. METHODS AND RESULTS: Activation of hypoxia-inducible factor-1 (HIF-1), the key transcription factor involved in angiogenesis, was detected in Bartonella henselae-infected host cells in vitro by immunofluorescence, Western blotting, electrophoretic mobility shift, and reporter gene assays and by immunohistochemistry in BA tissue lesions in vivo. Gene microarray analysis revealed that a B henselae infection resulted in the activation of genes typical for the cellular response to hypoxia. HIF-1 was essential for B henselae-induced expression of vascular endothelial growth factor as shown by inhibition with the use of HIF-1-specific short-interfering RNA. Moreover, infection with B henselae resulted in increased oxygen consumption, cellular hypoxia, and decreased ATP levels in host cells. Infection with a pilus-negative variant of B henselae did not lead to cellular hypoxia or activation of HIF-1 or vascular endothelial growth factor secretion, suggesting a crucial role of this bacterial surface protein in the angiogenic reprogramming of the host cells. CONCLUSIONS: B henselae induces a proangiogenic host cell response via HIF-1. Our data provide for the first time evidence that HIF-1 may play a role in bacterial infections.

Adenosine Triphosphate↗
PubMed2005

Rapid detection and identification of pathogens in blood cultures by fluorescence in situ hybridization and flow cytometry.

Septicemia is one of the leading causes of death in hospitalized patients. The timely detection and identification of microorganisms from the patient's blood has great diagnostic, prognostic and economic significance. Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has been proven to be a fast method for the identification of human pathogenic bacteria and yeasts. Data presented herein reveal that the combination of FISH and flow cytometry (FC-FISH) is a rapid and reliable technique for identification of pathogens (Gram-negative rods, Candida spp.) directly from blood cultures without further cultivation and biotyping. Moreover, detection of growing pathogens (e.g., Stenotrophomonas maltophilia) in blood cultures is achieved more rapidly by FC-FISH compared to standard detection methods. Therefore, FC-FISH allows rapid detection and identification of pathogens in blood cultures.

Bacteria↗
PubMed2005

Bartonella henselae inhibits apoptosis in Mono Mac 6 cells.

Bartonella henselae causes the vasculoproliferative disorders bacillary angiomatosis and peliosis probably resulting from the release of vasculoendothelial growth factor (VEGF) from infected epithelial or monocytic host cells. Here we demonstrate that B. henselae in addition to VEGF induction was also capable of inhibiting the endogenous sucide programme of monocytic host cells. Our results show that B. henselae inhibits pyrrolidine dithiocarbamate (PDTC)-induced apoptosis in Mono Mac 6 cells. B. henselae was observed to be present in a vacuolic compartment of Mono Mac 6 cells. Direct contact of B. henselae with Mono Mac 6 cells was crucial for inhibition of apoptosis as shown by the use of a two-chamber model. Inhibition of apoptosis was paralleled by diminished caspase-3 activity which was significantly reduced in PDTC-stimulated and B. henselae-infected cells. The anti-apoptotic effect of B. henselae was accompanied by (i) the activation of the transcription factor NF-kappaB and (ii) the induction of cellular inhibitor of apoptosis proteins-1 and -2 (cIAP-1, -2). Our results suggest a new synergistic mechanism in B. henselae pathogenicity by (i) inhibition of host cell apoptosis via activation of NF-kappaB and (ii) induction of host cell VEGF secretion.

Antioxidants↗
PubMed2005

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 24 h. 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 2 h 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.

Animals↗
PubMed2005

DltABCD- and MprF-mediated cell envelope modifications of Staphylococcus aureus confer resistance to platelet microbicidal proteins and contribute to virulence in a rabbit endocarditis model.

The DltABCD and MprF proteins contribute a net positive charge to the Staphylococcus aureus surface envelope by alanylating and lysinylating teichoic acids and membrane phosphatidylglycerol, respectively. These surface charge modifications are associated with increased in vitro resistance profiles of S. aureus to a number of endogenous cationic antimicrobial peptides (CAPs), such as alpha-defensins. The current study investigated the effects of dltA and mprF mutations on the following host factors relevant to endovascular infections: (i) in vitro susceptibility to the CAP thrombin-induced platelet microbicidal protein 1 (tPMP-1), (ii) in vitro adherence to endothelial cells (EC) and matrix proteins, and (iii) in vivo virulence in an endovascular infection model (rabbit endocarditis) in which tPMP-1 is felt to play a role in limiting S. aureus pathogenesis. Both mutations resulted in substantial increases in the in vitro susceptibility to tPMP-1 compared to that of the parental strain. The dltA (but not the mprF) mutation resulted in a significantly reduced capacity to bind to EC in vitro, while neither mutation adversely impacted in vitro binding to fibronectin, fibrinogen, or platelets. In vivo, both mutations significantly attenuated virulence in terms of early colonization of sterile vegetations and subsequent proliferation at this site (versus the parental strain). However, only the dltA mutation significantly reduced metastatic infections in kidneys and spleens compared to those in animals infected with the parental strain. These data underscore the importance of resistance to distinct CAPs and of teichoic acid-dependent EC interactions in the context of endovascular infection pathogenesis.

Aminoacyltransferases↗
PubMed2005

Rapid identification of Staphylococcus aureus in blood cultures by a combination of fluorescence in situ hybridization using peptide nucleic acid probes and flow cytometry.

Fluorescence in situ hybridization (FISH) using peptide nucleic acid probes (PNAs) allows the identification of Staphylococcus aureus from human blood culture samples. We present data revealing that the combination of PNA FISH and flow cytometry is a possible approach for the noncultural identification of staphylococci in blood cultures.

Bacteremia↗
PubMed2004

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 beta1 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.

Adhesins, Bacterial↗
PubMed2004

Host cell modulation by human, animal and plant pathogens.

Members of the alpha-proteobacteria display a broad range of interactions with higher eukaryotes. Some are pathogens of humans, such as Rickettsia and Bartonella that are associated with diseases like epidemic typhus, trench fever, cat scratch disease and bacillary angiomatosis. Others like the Brucella cause abortions in pregnant animals. Yet other species have evolved elaborate interactions with plants; in this group we find both plant symbionts and parasites. Despite radically different host preferences, extreme genome size variations and the absence of toxin genes, similarities in survival strategies and host cell interactions can be recognized among members of the alpha-proteobacteria. Here, we review some of these similarities, with a focus on strategies for modulation of the host target cell.

Animals↗
PubMed2003

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.

Bartonella henselae↗
PubMed2003

Hemin binding, functional expression, and complementation analysis of Pap 31 from Bartonella henselae.

Growth of Bartonella henselae is strongly heme dependent, and B. henselae is unable to synthesize heme itself. At least five outer membrane-associated proteins from B. henselae bind hemin, including the 31-kDa protein designated Pap31. The gene of this protein was heterologously expressed in Escherichia coli M15(pREP4) and detected with monoclonal antibodies in the outer membrane fraction. Complementation of the hemA-deficient mutant E. coli K-12 EB53 (aroB tsx malT hemA) with pap31 demonstrated that this protein is involved in heme acquisition and may be an important virulence factor in the pathogenesis of B. henselae.

Antibodies, Monoclonal↗
PubMed2003

Rapid screening for Streptococcus agalactiae in vaginal specimens of pregnant women by fluorescent in situ hybridization.

Group B streptococci (GBS) are the most frequent pathogens in neonates with sepsis. A rapid screening method is required to identify carriage of GBS in pregnant women at the time of delivery. In order to detect GBS in vaginal specimens, the efficiency of the standard culture versus fluorescent in situ hybridization (FISH) was investigated. In 258 examined vaginal specimens, FISH identified 58 of the 59 GBS-positive samples (98.3%), whereas by means of standard culture only 38 specimens were positive (64.4%). We recommend FISH as a rapid, specific, highly sensitive screening technique for the detection of GBS in pregnant women at delivery.

Base Sequence↗