Borrelia burgdorferi Outer Membrane Vesicles Contain Antigenic Proteins, but Do Not Induce Cell Death in Human Cells.

Like many bacterial species, Borrelia burgdorferi, the pleomorphic bacterium that causes Lyme borreliosis, produces outer membrane vesicles (OMVs). Borrelial OMVs (BbOMVs) have been identified as containing virulence factors, such as outer surface proteins (Osps) A, B, and C, as well as DNA. However, the pathogenicity of BbOMVs in disease development is still unclear. In this study, we characterized purified BbOMVs by analyzing their size and immunolabeling for known antigenic markers: OspA, OspC, p39, and peptidoglycan. In addition, BbOMVs were cocultured with human non-immune cells for cytotoxicity analysis. The results demonstrated that, on average, the vesicles were small, ranging between 11 and 108 nm in diameter. In addition, both OspA and OspC, as well as Lyme arthritis markers p39 and peptidoglycan, were detected from BbOMVs. Furthermore, BbOMVs were cocultured with non-immune cells, which did not result in cell death. Combined, these results suggested that BbOMVs could participate in the induction of infection by functioning as a decoy for the host immune system. Furthermore, BbOMVs might serve as a means for persistent antigens to remain in the host for prolonged periods of time.

Distinctive Evasion Mechanisms to Allow Persistence of Borrelia burgdorferi in Different Human Cell Lines.

Lyme borreliosis is a multisystemic disease caused by the pleomorphic bacteria of the Borrelia burgdorferi sensu lato complex. The exact mechanisms for the infection to progress into a prolonged sequelae of the disease are currently unknown, although immune evasion and persistence of the bacteria in the host are thought to be major contributors. The current study investigated B. burgdorferi infection processes in two human cell lines, both non-immune and non-phagocytic, to further understand the mechanisms of infection of this bacterium. By utilizing light, confocal, helium ion, and transmission electron microscopy, borrelial infection of chondrosarcoma (SW1353) and dermal fibroblast (BJ) cells were examined from an early 30-min time point to a late 9-days post-infection. Host cell invasion, viability of both the host and B. burgdorferi, as well as, co-localization with lysosomes and the presence of different borrelial pleomorphic forms were analyzed. The results demonstrated differences of infection between the cell lines starting from early entry as B. burgdorferi invaded BJ cells in coiled forms with less pronounced host cell extensions, whereas in SW1353 cells, micropodial interactions with spirochetes were always seen. Moreover, infection of BJ cells increased in a dose dependent manner throughout the examined 9 days, while the percentage of infection, although dose dependent, decreased in SW1353 cells after reaching a peak at 48 h. Furthermore, blebs, round body and damaged B. burgdorferi forms, were mostly observed from the infected SW1353 cells, while spirochetes dominated in BJ cells. Both infected host cell lines grew and remained viable after 9 day post-infection. Although damaged forms were noticed in both cell lines, co-localization with lysosomes was low in both cell lines, especially in BJ cells. The invasion of non-phagocytic cells and the lack of cytopathic effects onto the host cells by B. burgdorferi indicated one mechanism of immune evasion for the bacteria. The differences in attachment, pleomorphic form expressions, and the lack of lysosomal involvement between the infected host cells likely explain the ability of a bacterium to adapt to different environments, as well as, a strategy for persistence inside a host.

Parvovirus B19V Nonstructural Protein NS1 Induces Double-Stranded Deoxyribonucleic Acid Autoantibodies and End-Organ Damage in Nonautoimmune Mice.

BACKGROUND: Viral infection is implicated in development of autoimmunity. Parvovirus B19 (B19V) nonstructural protein, NS1, a helicase, covalently modifies self double-stranded deoxyribonucleic acid (dsDNA) and induces apoptosis. This study tested whether resulting apoptotic bodies (ApoBods) containing virally modified dsDNA could induce autoimmunity in an animal model. METHODS: BALB/c mice were inoculated with (1) pristane-induced, (2) B19V NS1-induced, or (3) staurosporine-induced ApoBods. Serum was tested for dsDNA autoantibodies by Crithidia luciliae staining and enzyme-linked immunosorbent assay. Brain, heart, liver, and kidney pathology was examined. Deposition of self-antigens in glomeruli was examined by staining with antibodies to dsDNA, histones H1 and H4, and TATA-binding protein. RESULTS: The B19V NS1-induced ApoBod inoculation induced dsDNA autoantibodies in a dose-dependent fashion. Histopathological features of immune-mediated organ damage were evident in pristane-induced and NS1-induced ApoBod groups; severity scores were higher in these groups than in staurosporine-treated groups. Tissue damage was dependent on NS1-induced ApoBod dose. Nucleosomal antigens were deposited in target tissue from pristane-induced and NS1-induced ApoBod inoculated groups, but not in the staurosporine-induced ApoBod inoculated group. CONCLUSIONS: This study demonstrated proof of principle in an animal model that virally modified dsDNA in apoptotic bodies could break tolerance to self dsDNA and induce dsDNA autoantibodies and end-organ damage.

Role of mitochondria in parvovirus pathology.

Proper functioning of the mitochondria is crucial for the survival of the cell. Viruses are able to interfere with mitochondrial functions as they infect the host cell. Parvoviruses are known to induce apoptosis in infected cells, but the role of the mitochondria in parvovirus induced cytopathy is only partially known. Here we demonstrate with confocal and electron microscopy that canine parvovirus (CPV) associated with the mitochondrial outer membrane from the onset of infection. During viral entry a transient depolarization of the mitochondrial transmembrane potential and increase in ROS level was detected. Subsequently, mitochondrial homeostasis was normalized shortly, as detected by repolarization of the mitochondrial membrane and decrease of ROS. Indeed, activation of cell survival signalling through ERK1/2 cascade was observed early in CPV infected cells. At 12 hours post infection, concurrent with the expression of viral non-structural protein 1, damage to the mitochondrial structure and depolarization of its membrane were apparent. Results of this study provide additional insight of parvovirus pathology and also more general information of virus-mitochondria association.

Mechanisms of cell death in canine parvovirus-infected cells provide intuitive insights to developing nanotools for medicine.

Viruses have great potential as nanotools in medicine for gene transfer, targeted gene delivery, and oncolytic cancer virotherapy. Here we have studied cell death mechanisms of canine parvovirus (CPV) to increase the knowledge on the CPV life cycle in order to facilitate the development of better parvovirus vectors. Morphological studies of CPV-infected Norden laboratory feline kidney (NLFK) cells and canine fibroma cells (A72) displayed characteristic apoptotic events. Apoptosis was further confirmed by activation of caspases and cellular DNA damage. However, results from annexin V-propidium iodide (PI) labeling and membrane polarization assays indicated disruption of the plasma membrane uncommon to apoptosis. These results provide evidence that secondary necrosis followed apoptosis. In addition, two human cancer cell lines were found to be infected by CPV. This necrotic event over apoptotic cell death and infection in human cells provide insightful information when developing CPV as a nanotool for cancer treatments.

Late steps of parvoviral infection induce changes in cell morphology.

Previously, virus-induced non-filopodial extensions have not been encountered in connection with viral infections. Here, we report emergence of long extensions protruding from Norden laboratory feline kidney (NLFK) and A72 (canine fibroma) cells infected with canine parvovirus for 72 h. These extensions significantly differ in length and number from those appearing in control cells. The most striking feature in the extensions is the length, reaching up to 130 microm, almost twice the average length of a healthy NLFK cell. In A72 cells, the extensions were even longer, up to 200 microm. The results presented here also suggest that the events leading to the growth of these extensions start earlier in infection and abnormal extension growth is detectable already at 24-h post-infection (p.i.). These extensions may have a vital role in the cell-to-cell transmission of the virus.

Cholesterol supports the retinoic acid-induced synaptic vesicle formation in differentiating human SH-SY5Y neuroblastoma cells.

Synaptic vesicle formation, vesicle activation and exo/endocytosis in the pre-synaptic area are central steps in neuronal communication. The formation and localization of synaptic vesicles in human SH-SY5Y neuroblastoma cells, differentiated with 12-o-tetradecanoyl-phorbol-13-acetate, dibutyryl cyclic AMP, all-trans-retinoic acid (RA) and cholesterol, was studied by fluorescence microscopy and immunocytochemical methods. RA alone or together with cholesterol, produced significant neurite extension and formation of cell-to-cell contacts. Synaptic vesicle formation was followed by anti-synaptophysin (SypI) and AM1-43 staining. SypI was only weakly detected, mainly in cell somata, before 7 days in vitro, after which it was found in neurites. Depolarization of the differentiated cells with high potassium solution increased the number of fluorescent puncta, as well as SypI and AM1-43 co-localization. In addition to increase in the number of synaptic vesicles, RA and cholesterol also increased the number and distribution of lysosome-associated membrane protein 2 labeled lysosomes. RA-induced Golgi apparatus fragmentation was partly avoided by co-treatment with cholesterol. The SH-SY5Y neuroblastoma cell line, differentiated by RA and cholesterol and with good viability in culture, is a valuable tool for basic studies of neuronal metabolism, specifically as a model for dopaminergic neurons.