Kinesin-like protein CENP-E is upregulated in rheumatoid synovial fibroblasts

INTRODUCTION: Articular destruction by invading synovial fibroblasts is a typical feature in rheumatoid arthritis (RA). Recent data support the hypothesis that key players in this scenario are transformed-appearing synovial fibroblasts at the site of invasion into articular cartilage and bone. They maintain their aggressive phenotype toward cartilage, even when first cultured and thereafter coimplanted together with normal human cartilage into severe combined immunodeficient mice for an extended period of time. However, little is known about the upregulation of genes that leads to this aggressive fibroblast phenotype. To inhibit this progressive growth without interfering with pathways of physiological matrix remodelling, identification of pathways that operate specifically in RA synovial fibroblasts is required. In order to achieve this goal, identification of genes showing upregulation restricted to RA synovial fibroblasts is essential. AIMS: To identify specifically expressed genes using RNA arbitrarily primed (RAP)-polymerase chain reaction (PCR) for differential display in patients with RA. METHODS: RNA was extracted from cultured synovial fibroblasts from 10 patients with RA, four patients with osteoarthritis (OA), and one patient with psoriatic arthritis. RAP-PCR was performed using different arbitrary primers for first-strand and second-strand synthesis. First-strand and second-strand synthesis were performed using arbitrary primers: US6 (5' -GTGGTGACAG-3') for first strand, and Nuclear 1+ (5' -ACGAAGAAGAG-3'), OPN28 (5' -GCACCAGGGG-3'), Kinase A2+ (5' -GGTGCCTTTGG-3')and OPN24 (5' -AGGGGCACCA-3') for second-strand synthesis. PCR reactions were loaded onto 8 mol/l urea/6% polyacrylamide-sequencing gels and electrophoresed.Gel slices carrying the target fragment were then excised with a razor blade, eluated and reamplified. After verifying their correct size and purity on 4% agarose gels, the reamplified products derived from the single-strand confirmation polymorphism gel were cloned, and five clones per transcript were sequenced. Thereafter, a GenBank(®) analysis was performed. Quantitative reverse transcription PCR of the segments was performed using the PCR MIMIC(®) technique.In-situ expression of centromere kinesin-like protein-E (CENP-E) messenger (m)RNA in RA synovium was assessed using digoxigenin-labelled riboprobes, and CENP-E protein expression in fibroblasts and synovium was performed by immunogold-silver immunohistochemistry and cytochemistry. Functional analysis of CENP-E was done using different approaches (eg glucocorticoid stimulation, serum starvation and growth rate analysis of synovial fibroblasts that expressed CENP-E). RESULTS: In RA, amplification of a distinct PCR product suitable for sequencing could be observed. The indicated complementary DNA fragment of 434 base pairs from RA mRNA corresponded to nucleotides 6615-7048 in the human centromere kinesin-like protein CENP-E mRNA (GenBank(®) accession No. emb/Z15005).The isolated sequence shared greater than 99% nucleic acid (P = 2.9e(-169)) identity with the human centromere kinesin-like protein CENP-E. Two base changes at positions 6624 (A to C) and 6739 (A to G) did not result in alteration in the amino acid sequence, and therefore 100% amino acid identity could be confirmed. The amplification of 10 clones of the cloned RAP product revealed the presence of CENP-E mRNA in every fibroblast culture examined, showing from 50% (271.000 ± 54.000 phosphor imager arbitrary units) up to fivefold (961.000 ± 145.000 phosphor imager arbitrary units) upregulation when compared with OA fibroblasts. Neither therapy with disease-modifying antirheumatic drugs such as methotrexate, gold, resochine or cyclosporine A, nor therapy with oral steroids influenced CENP-E expression in the RA fibroblasts. Of the eight RA fibroblast populations from RA patients who were receiving disease-modifying antirheumatic drugs, five showed CENP-E upregulation; and of the eight fibroblast populations from RA patients receiving steroids, four showed CENP-E upregulation. Numerous synovial cells of the patients with RA showed a positive in situ signal for the isolated CENP-E gene segment, confirming CENP-E mRNA production in rheumatoid synovium, whereas in OA synovial tissue CENP-E mRNA could not be detected. In addition, CENP-E expression was independent from medication. This was further confirmed by analysis of the effect of prednisolone on CENP-E expression, which revealed no alteration in CENP-E mRNA after exposure to different (physiological) concentrations of prednisolone. Serum starvation also could not suppress CENP-E mRNA completely. DISCUSSION: Since its introduction in 1992, numerous variants of the differential display method and continuous improvements including RAP-PCR have proved to have both efficiency and reliability in examination of differentially regulated genes. The results of the present study reveal that RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts. The mRNA, which has been found to be upregulated in rheumatoid synovial fibroblasts, codes for a kinesin-like motor protein named CENP-E, which was first characterized in 1991. It is a member of a family of centromere-associated proteins, of which six (CENP-A to CENP-F) are currently known. CENP-E itself is a kinetochore motor, which accumulates transiently at kinetochores in the G(2) phase of the cell cycle before mitosis takes place, appears to modulate chromosome movement and spindle elongation,and is degraded at the end of mitosis. The presence or upregulation of CENP-E has never been associated with RA. The three-dimensional structure of CENP-E includes a coiled-coil domain. This has important functions and shows links to known pathways in RA pathophysiology. Coiled-coil domains can also be found in jun and fos oncogene products, which are frequently upregulated in RA synovial fibroblasts. They are also involved in DNA binding and transactivation processes resembling the situation in AP-1 (Jun/Fos)-dependent DNA-binding in rheumatoid synovium. Most interestingly, these coiled-coil motifs are crucial for the assembly of viral proteins, and the upregulation of CENP-E might reflect the influence of infectious agents in RA synovium. We also performed experiments showing that serum starvation decreased, but did not completely inhibit CENP-E mRNA expression. This shows that CENP-E is related to, but does not completely depend on proliferation of these cells. In addition, we determined the growth rate of CENP-E high and low expressors, showing that it was independent from the amount of CENP-E expression. supporting the statement that upregulation of CENP-E reflects an activated RA fibroblast phenotype. In summary, the results of the present study support the hypothesis that CENP-E, presumably independently from medication, may not only be upregulated, but may also be involved in RA pathophysiology

Molecular detection of Bartonella henselae in 11 Ixodes ricinus ticks extracted from a single cat

Background: Bartonella henselae is a highly prevalent, vector-borne pathogen. Transmission to humans and animals by ticks is discussed controversially. Here, we present a case report, where eleven Ixodes ricinus ticks all harbouring B. henselae DNA were removed from one single cat. Results: The first feeding tick was tested positive for B. henselae DNA. The cat was also found to be seropositive for anti-B. henselae IgG antibodies (titer 1:640). Bartonella henselae was not cultivatable from cat blood. Ten more feeding ticks removed 7 months later contained also B. henselae DNA. Sequence analysis of the 16SrDNA and the 16S-23S internal transcribed spacer (ITS) region revealed 100% sequence homology between all ticks. Bartonella adhesin A (badA) and VirB/VirD4 type IV secretion system (virB) DNA were also detected in all ticks. Conclusions: Our results indicate that cats may serve as a reservoir for adult ticks to acquire B. henselae. Whether this observation implies an increased threat for human and animal health needs to be resolved

Adhesion of Bartonella henselae to fibronectin is mediated via repetitive motifs present in the stalk of Bartonella adhesin A

Adhesion to host cells is the first and most crucial step in infections with pathogenic Gram-negative bacteria and is often mediated by trimeric autotransporter adhesins (TAAs). Bartonella henselae targets the extracellular matrix glycoprotein fibronectin (Fn) via the Bartonella adhesin A (BadA) attaching the bacteria to the host cell. The TAA BadA is characterized by a highly repetitive passenger domain consisting of 30 neck/stalk domains with various degrees of similarity. To elucidate the motif sequences mediating Fn binding, we generated 10 modified BadA constructs and verified their expression via Western blotting, confocal laser scanning, and electron microscopy. We analyzed their ability to bind human plasma Fn using quantitative whole-cell enzyme-linked immunosorbent assays (ELISAs) and fluorescence microscopy. Polyclonal antibodies targeting a 15-mer amino acid motif sequence proved to reduce Fn binding. We suggest that BadA adheres to Fn in a cumulative effort with quick saturation primarily via unpaired β-strands appearing in motifs repeatedly present throughout the neck/stalk region. In addition, we demonstrated that the length of truncated BadA constructs correlates with the immunoreactivity of human patient sera. The identification of BadA-Fn binding regions will support the development of new “antiadhesive” compounds inhibiting the initial adherence of B. henselae and other TAA-expressing pathogens to host cells

Long-read sequencing reveals genetic adaptation of Bartonella adhesin a among different Bartonella henselae isolates

Bartonella henselae is the causative agent of cat scratch disease and other clinical entities such as endocarditis and bacillary angiomatosis. The life cycle of this pathogen, with alternating host conditions, drives evolutionary and host-specific adaptations. Human, feline, and laboratory adapted B. henselae isolates often display genomic and phenotypic differences that are related to the expression of outer membrane proteins, for example the Bartonella adhesin A (BadA). This modularly-structured trimeric autotransporter adhesin is a major virulence factor of B. henselae and is crucial for the initial binding to the host via the extracellular matrix proteins fibronectin and collagen. By using next-generation long-read sequencing we demonstrate a conserved genome among eight B. henselae isolates and identify a variable genomic badA island with a diversified and highly repetitive badA gene flanked by badA pseudogenes. Two of the eight tested B. henselae strains lack BadA expression because of frameshift mutations. We suggest that active recombination mechanisms, possibly via phase variation (i.e., slipped-strand mispairing and site-specific recombination) within the repetitive badA island facilitate reshuffling of homologous domain arrays. The resulting variations among the different BadA proteins might contribute to host immune evasion and enhance long-term and efficient colonisation in the differing host environments. Considering the role of BadA as a key virulence factor, it remains important to check consistently and regularly for BadA surface expression during experimental infection procedures

Adhesion of human pathogenic bacteria to endothelial cells is facilitated by fibronectin interaction

Human pathogenic bacteria circulating in the bloodstream need to find a way to interact with endothelial cells (ECs) lining the blood vessels to infect and colonise the host. The extracellular matrix (ECM) of ECs might represent an attractive initial target for bacterial interaction, as many bacterial adhesins have reported affinities to ECM proteins, in particular to fibronectin (Fn). Here, we analysed the general role of EC-expressed Fn for bacterial adhesion. For this, we evaluated the expression levels of ECM coding genes in different ECs, revealing that Fn is the highest expressed gene and thereby, it is highly abundant in the ECM environment of ECs. The role of Fn as a mediator in bacterial cell-host adhesion was evaluated in adhesion assays of Acinetobacter baumannii, Bartonella henselae, Borrelia burgdorferi, and Staphylococcus aureus to ECs. The assays demonstrated that bacteria colocalised with Fn fibres, as observed by confocal laser scanning microscopy. Fn removal from the ECM environment (FN1 knockout ECs) diminished bacterial adherence to ECs in both static and dynamic adhesion assays to varying extents, as evaluated via absolute quantification using qPCR. Interactions between adhesins and Fn might represent the crucial step for the adhesion of human-pathogenic Gram-negative and Gram-positive bacteria targeting the ECs as a niche of infection

Interaction of Bartonella henselae with Fibronectin Represents the Molecular Basis for Adhesion to Host Cells

Bacterial adhesion to the host is the most decisive step in infections. Trimeric autotransporter adhesins (TAA) are important pathogenicity factors of Gram-negative bacteria. The prototypic TAA Bartonella adhesin A (BadA) from human-pathogenic Bartonella henselae mediates bacterial adherence to endothelial cells (ECs) and extracellular matrix proteins. Here, we determined the interaction between BadA and fibronectin (Fn) to be essential for bacterial host cell adhesion. BadA interactions occur within the heparin-binding domains of Fn. The exact binding sites were revealed by mass spectrometry analysis of chemically cross-linked whole-cell bacteria and Fn. Specific BadA interactions with defined Fn regions represent the molecular basis for bacterial adhesion to ECs and these data were confirmed by BadA-deficient bacteria and CRISPR-Cas knockout Fn host cells. Interactions between TAAs and the extracellular matrix might represent the key step for adherence of human-pathogenic Gram-negative bacteria to the host. IMPORTANCE Deciphering the mechanisms of bacterial host cell adhesion is a clue for preventing infections. We describe the underestimated role that the extracellular matrix protein fibronectin plays in the adhesion of human-pathogenic Bartonella henselae to host cells. Fibronectin-binding is mediated by a trimeric autotransporter adhesin (TAA) also present in many other human-pathogenic Gram-negative bacteria. We demonstrate that both TAA and host-fibronectin contribute significantly to bacterial adhesion, and we present the exact sequence of interacting amino acids from both proteins. Our work shows the domain-specific pattern of interaction between the TAA and fibronectin to adhere to host cells and opens the perspective to fight bacterial infections by inhibiting bacterial adhesion which represents generally the first step in infections