The ISBsu2 Mobile Element Is Present in a Plasmid of a Soil Strain and in the Chromosomes of Several Other Strains of Bacillus subtilis

Chromosomes of several Bacillus subtilis strains were shown to contain homologs of the ISBsu2 mobile genetic element, which was earlier revealed in a cryptic plasmid of a soil strain of B. subtilis.     

Plasmid transfer in bacilli by a self-transmissible plasmid p19 from a Bacillus subtilis soil strain

The cryptic 95-kb plasmid p19 of the Bacillus subtilis 19 soil strain promotes the transfer of a small kanamycin resistance plasmid pUB110. To facilitate direct selection for p19 transfer, a plasmid derivative carrying the chloramphenicol resistance gene was constructed. The frequency of transfer of the large plasmid between cells of B. subtilis 19 approached 100% but was more than two orders of magnitude lower when the strain B. subtilis 168 was a recipient. However, when the restriction-deficient strain B. subtilis 168 was a recipient, the transfer efficiency was almost completely recovered. The effectiveness of pUB110 mobilization was virtually not altered in all these cases. pC194 was not mobilized by p19. The kinetics of p19 conjugative transfer is also presented.     

Molecular analysis of some genes from plasmid p19 of the soil strain Bacillus subtilis 19 involved in conjugation

Two fragments of conjugative plasmid p19 (95 kb) from the soil strain Bacillus subtilis 19 were cloned and sequenced; these fragments carry genes, products of which are indispensable for the conjugative transfer. One of the fragments 4518 bp in size carries five open reading frames and their fragments (ORF1-ORF5). The protein corresponding to ORF4 is homologous to proteins from the family VirD4. Inactivation of ORF4 and ORF1 by insertional mutagenesis caused a three-to-fivefold decrease in the frequency of plasmid p19 conjugative transfer. Another 2932-bp fragment of p19 was shown to possess a rep region homologous to the rep region of plasmid pBS72 from the soil strain B. subtilis 72 and a novel ORF (ORF6); the protein corresponding to this ORF contains the HTH motif typical for DNA-binding proteins.     

Generation of cytotoxic T cells against virus-infected human brain macrophages in a murine model of HIV-1 encephalitis

HIV-1 encephalitis (HIVE) and its associated dementia can occur in up to 20% of infected individuals, usually when productive viral replication in brain mononuclear phagocytes (macrophages and microglia) and depletion of CD4(+) T lymphocytes are most significant. T cells control viral replication through much of HIV-1 disease, but how this occurs remains incompletely understood. With this in mind, we studied HIV-1-specific CTL responses in a nonobese diabetic (NOD)-SCID mouse model of HIVE. HIV-1-infected monocyte-derived macrophages (MDM) were injected into the basal ganglia after syngeneic immune reconstitution by HLA-A*0201-positive human PBL to generate a human PBL-NOD-SCID HIVE mouse. Engrafted T lymphocytes produced HIV-1gag- and HIV-1pol-specific CTL against virus-infected brain MDM within 7 days. This was demonstrated by tetramer staining of human PBL in mouse spleens and by IFN-gamma ELISPOT. CD8, granzyme B, HLA-DR, and CD45R0 Ag-reactive T cells and CD79alpha-positive B cells migrated to and were in contact with human MDM in brain areas where infected macrophages were abundant. The numbers of productively infected MDM were markedly reduced (>85%) during 2 wk of observation. The human PBL-NOD-SCID HIVE mouse provides a new tool for studies of cellular immune responses against HIV-1-infected brain mononuclear phagocytes during natural disease and after vaccination.     

Copolymer-1 induces adaptive immune anti-inflammatory glial and neuroprotective responses in a murine model of HIV-1 encephalitis

Copolymer-1 (COP-1) elicits neuroprotective activities in a wide range of neurodegenerative disorders. This occurs, in part, by adaptive immune-mediated suppression of microglial inflammatory responses. Because HIV infection and immune activation of perivascular macrophages and microglia drive a metabolic encephalopathy, we reasoned that COP-1 could be developed as an adjunctive therapy for disease. To test this, we developed a novel animal model system that reflects HIV-1 encephalitis in rodents with both innate and adaptive arms of the immune system. Bone marrow-derived macrophages were infected with HIV-1/vesicular stomatitis-pseudotyped virus and stereotactically injected into the basal ganglia of syngeneic mice. HIV-1 pseudotyped with vesicular stomatitis virus envelope-infected bone marrow-derived macrophages induced significant neuroinflammation, including astrogliosis and microglial activation with subsequent neuronal damage. Importantly, COP-1 immunization reduced astro- and microgliosis while diminishing neurodegeneration. Hippocampal neurogenesis was, in part, restored. This paralleled reductions in proinflammatory cytokines, including TNF-alpha and IL-1beta, and inducible NO synthase, and increases in brain-derived neurotrophic factor. Ingress of Foxp3- and IL-4-expressing lymphocytes into brains of COP-1-immunized animals was observed. We conclude that COP-1 may warrant therapeutic consideration for HIV-1-associated cognitive impairments.     

Human immunodeficiency virus type 1 pathobiology studied in humanized BALB/c-Rag2-/-gammac-/- mice

The specificity of human immunodeficiency virus type 1 (HIV-1) for human cells precludes virus infection in most mammalian species and limits the utility of small animal models for studies of disease pathogenesis, therapy, and vaccine development. One way to overcome this limitation is by human cell xenotransplantation in immune-deficient mice. However, this has proved inadequate, as engraftment of human immune cells is limited (both functionally and quantitatively) following transplantation of mature human lymphocytes or fetal thymus/liver. To this end, a human immune system was generated from umbilical cord blood-derived CD34(+) hematopoietic stem cells in BALB/c-Rag2(-/-)gamma(c)(-/-) mice. Intrapartum busulfan administration followed by irradiation of newborn pups resulted in uniform engraftment characterized by human T-cell development in thymus, B-cell maturation in bone marrow, lymph node development, immunoglobulin M (IgM)/IgG production, and humoral immune responses following ActHIB vaccination. Infection of reconstituted mice by CCR5-coreceptor utilizing HIV-1(ADA) and subtype C 1157 viral strains elicited productive viral replication and lymphadenopathy in a dose-dependent fashion. We conclude that humanized BALB/c-Rag2(-/-)gamma(c)(-/-) mice represent a unique and valuable resource for HIV-1 pathobiology studies.     

Characterization of the transfer-related <Emphasis Type="Italic">tra</Emphasis> region of the conjugative plasmid p19 from a <Emphasis Type="Italic">Bacillus subtilis</Emphasis> soil strain

The nucleotide sequences of three DNA fragments (total size 30574 bp) of the plasmid p19 from the Bacillus subtilis 19 soil strain have been determined. Thirty open reading frames (ORFs) have been identified in these fragments. oriT of the plasmid has also been identified. As shown by the search for homologs of hypothetical protein products of these ORFs in databases, such homology exists for 18 ORFs. The protein products of nine ORFs can be assumed to have specific functions. Several ORFs were inactivated via insertional mutagenesis, and the conjugation capacity of the mutant plasmids was estimated. According to the data on homology of protein products and the results of ORF inactivation, regions of a total size of about 20 kb from the DNA fragments sequenced by us were inferred to belong to the tra region of p19. As follows from the analysis of the identified ORFs of the p19 tra region, it differs from the earlier described tra regions of other plasmids, irrespective of a certain similarity with the corresponding regions of plasmids of gram-positive bacteria from the genera Bacillus, Clostridium, and Listeria.     

Soil Strain of Bacillus subtilis Harboring a Large Plasmid That Mediates High-Frequency Conjugal Mobilization

The ability of a soil strain of Bacillus subtilis harboring a large plasmid, p19, to mobilize a small staphylococcal plasmid, pUB110, was studied. The latter plasmid was transferred to the recipient cells of Bacillus subtilis168 at a high frequency (about 10^–2 per recipient cell) both on the filter surface and in liquid medium. Mobilization was initiated 40 min after the beginning of the contact between donor and recipient cells.     

High Frequency Conjugative Mobilization Accomplished by a Natural Bacillus subtilisStrain Carrying a Large Plasmid

Conjugative properties of the strain Bacillus subtiliscarrying a large plasmid approximately 95 kb in size and isolated in Belarus from forest soil were described. The staphylococcal plasmid pUB110 that had previously been introduced into this strain was transferred to recipient cells of the Bacillus subtilis168 strain with a frequency of approximately 10^–2. The transfer occurred with approximately the same frequency both upon donor and recipient cell contact on the surface of membranes and in a liquid medium. The latter fact makes this system suitable as a model for studying conjugative mobilization in bacilli. A large plasmid cannot be transferred to recipients. An optimal temperature for conjugation of donor and recipient cells was 37°C, but conjugation also proceeded at lower temperatures, up to 21°C.