Specific control of endogenous cCF10 pheromone by a conserved domain of the pCF10-encoded regulatory protein PrgY in Enterococcus faecalis

Conjugative transfer of Enterococcus faecalis plasmid pCF10 is induced by the heptapeptide pheromone cCF10. cCF10 produced by plasmid-free recipient cells is detected by pCF10-containing donor cells, which respond by induction of plasmid-encoded transfer functions. The pCF10-encoded membrane protein PrgY is essential to prevent donor cells from responding to endogenously produced pheromone while maintaining the ability to respond to pheromone from an exogenous source; this function has not been identified in any nonenterococcal prokaryotic signaling system. PrgY specifically inhibited endogenous cCF10 and cPD1 (a pheromone that induces transfer of closely related plasmid pPD1) but not cAD1 (which is specific for less-related plasmid pAD1). Ectopic expression of PrgY in plasmid-free recipient cells reduced pheromone activity in culture supernatants and reduced the ability of these cells to acquire pCF10 by conjugation but did not have any effect on the interaction of these cells with exogenously supplied cCF10. The cloned prgY gene could complement a pCF10 prgY null mutation, and complementation was used to identify point mutations impairing PrgY function. Such mutations also abolished the inhibitory effect of PrgY expression in recipients on pheromone production and on acquisition of pCF10. Most randomly generated point mutations identified in the genetic screen mapped to a predicted extracellular domain in the N terminus of PrgY that is conserved in a newly identified family of related proteins from disparate species including Borrelia burgdorferi, Archaeoglobus fulgidus, Arabidopsis thaliana, and Homo sapiens. The combined genetic and physiological data suggest that PrgY may sequester or inactivate cCF10 as it is released from the membrane.     

Acquired cellular immunity: extracellular killing of Listeria monocytogenes by a product of immunologically activated macrophages

A listericidal factor was released by monolayers of peritoneal cells from BCG-immune guinea pigs following incubation with PPD in cell culture. Significantly less listericidal factor was released by monolayers of BCG-immune cells incubated without PPD, or by monolayers of nonimmune cells incubated in the presence or absence of antigen. Culture supernatants containing listericidal factor were active at a dilution of 1:10, but supernatants diluted 1:100 were not listericidal. Dialysis of supernatants against fresh tissue culture medium did not affect their ability to kill Listeria, although heating at 60 °C for 30 min destroyed all activity. Supernatants from cultures of guinea pig fibroblasts or ascites-form hepatoma were not listericidal.     

Antigen-initiated B lymphocyte differentiation. XIV. Nonspecific effects of antigen stimulation cause proliferation in the "pre-progenitor" subset of primary B cells.

The effect of specific and nonspecific stimuli on the cycle status of subsets of primary B lymphocytes was assessed by preinjecting donor CBA mice 1 to 2 days previously with various substances, and then incubating the isolated spleen cells with high specific activity 3H-TdR before assay. AFC-progenitor activity was assessed as a response to NIP-POL antigen, either by adoptive transfer to irradiated recipients or by cell culture. Previous studies showed these assays reflected the activity of different subsets of B cells, termed "pre-progenitors" (adoptive assay) and "direct progenitors" (culture assay). Most functional primary B cells, whether assayed in culture or by adoptive transfer, were not initially in rapid cell cycle in normal adult mice. However, nonspecific stimulation for 1 day caused NIP-specific adoptive transfer IgM AFC-progenitors to enter rapid cell cycle. This effect was independent of T cells and not related to the antigenicity of the stimulus: particulate peritoneal irritants were the most effective stimulants. In contrast to adoptive transfer results. AFC-progenitors assayed in cell culture were unaffected by nonspecific stimuli, but were activated into cell cycle by specific antigen.     

Inhibition of the lymphocyte blastogenic response to antigen by serum-free culture supernatants of leukemic B cells

Suppressive factors were detected in culture supernatants of the guinea pig B-cell L₂C leukemia. Dialyzed culture supernatants (DCS) inhibited the blastogenic response of sensitized lymph node cells (LNC) to a wide dose range of the sensitizing antigen (ovalbumin or PPD) but failed to inhibit the proliferative response to PHA or Con A. In addition, DCS inhibited the response of blast cells to preformed T-cell growth factor (TCGF). The inhibitor(s) in DCS was noncytotoxic, heat stable (30 min at 80 °C), resistant to treatment with trypsin, and exerted its effect subsequent to activation of sensitized LNC by antigen. Washing of DCS-treated cells restored normal reactivity to a subsequent antigen challenge. The target cell for the inhibitor may be cells responding to amplification signals produced by activated T cells. KCl (3 M) extracts of L₂C cells behaved like DCS in inhibiting only antigen responses. Both undialyzed culture supernatants (UCS) and leukemic sera inhibited mitogen, allogeneic, and antigen-stimulated proliferative responses by greater than 80%. These soluble factors may participate in the depression of cell-mediated immunity associated with lymphoid malignancies.     

Functional differentiation of B lymphocytes in congenital agammaglobulinemia. II. Immunochemical analysis of the in vitro primary immune response.

Cultures of peripheral blood lymphocytes (PBL) in which specific hemolytic plaque-forming cells (HcPFC) had been induced were labeled with 14C-amino acids. Antigen-specific products in the culture supernatants were characterized by using indirect immune precipitation in conjunction with specific immunoabsorbents and/or gel filtration followed by SDS-polyacrylamide gel electrophoresis. After 5 days of culture with antigen (sheep red blood cells or ovalbumin) newly synthesized IgM and specific IgM antibody were demonstrated in culture supernatants from normal donors and from four out of five patients with congenital agammaglobulinemia (cAgamma). Secreted products bound specifically to antigen and pretreatment of labeled supernatants with anti-mu and anti-L chain antisera, but not with anti-gamma antiserum, prevented binding. Typical mu- and L chains constituted only a proportion of the anigen-binding peptides recognized by the anti-mu reagents. Induction of IgM antibody synthesis was dependent on the presence of antigen and was correlated with the generation of HcPFC. No major differences between the antigen-induced products of cAgamma and normal PBL were observed. These findings suggest that in the absence of terminal B cell differentiation in vivo, certain patients with cAgamma possess precursor cells that can respond to antigen in vitro with the synthesis of specific humoral products, including IgM antibody.     

Antigen-specific augmentation of murine immediate hypersensitivity-like footpad reaction by a T-cell factor in the culture supernatant of immune spleen cells

An antigen-specific factor capable of augmenting delayed-type hypersensitivity (DH) in the culture supernatants from immune spleen cells and erythrocyte antigen has been found. These culture supernatants also augmented an immediate hypersensitivity-like reaction which appeared in advance of the classical DH reaction. In this paper, the basic characteristics and cells producing the augmentation factor (IAF) involved in immediate hypersensitivity-like reaction were investigated. Maximum activity of IAF was detected in a supernatant from 24-hr culture of immune spleen cells and antigen. In vitro antigen stimulation was essentially required for the production or release of IAF. IAF showed antigen-specificity. IAF was produced or released by T cells. In addition to these facts, the DH-augmentation factor proved to be a T-cell product.     

Leishmania donovani: generation of monospecific antibody reagents to soluble acid phosphatase

Four monoclonal antibodies (McAbs) were generated against the soluble extracellular acid phosphatase (EC 3.1.3.2) (S-AcP) of Leishmania donovani. These were detected in the primary screen using an ELISA with promastigote culture supernatants as antigen. Three of the McAbs demonstrated bound S-AcP from such culture supernatants in an enzyme activity binding assay. All immunoprecipitated metabolically labeled S-AcP but none showed any binding to the promastigote surface by indirect immunofluorescence. Moreover, none reacted with Triton X-100 solubilized plasma membranes by immunoprecipitation or Western blotting. These results demonstrated that the McAbs did not recognize the surface membrane bound acid phosphatase, but were specific for the extracellular soluble enzyme. Further, none of the antibodies immunoprecipitated any of the five human acid phosphatase isozymes or reacted with them in Western blots or the enzyme activity binding assay. Therefore, they are specific for the parasite-derived enzyme. One of these was used to affinity purify sufficient L. donovani S-AcP to immunize a rabbit and generate a specific, polyvalent antiserum. This polyvalent antibody immunoprecipitated S-AcP activity but did not cross-react with the surface membrane acid phosphatase, indicating that these two parasite enzymes are separate gene products.     

Interaction between cytokines and 8-mercaptoguanosine in humoral immunity: synergy with interferon

In previous studies it has been demonstrated that a T cell-like differentiation signal is transmitted by C8-substituted guanine ribonucleosides such as 8-mercaptoguanosine (8MGuo) to antigen-stimulated B cells. A large subset of potentially reactive B cells remains unresponsive to antigen even in the presence of signals provided by these nucleosides except when this signal is preceded by a soluble activity present in mixed lymphocyte culture supernatants. Studies with purified preparations of interleukin (IL)-1, IL-2, IL-3, granulocyte-macrophage colony stimulating factor, B cell stimulatory factor 1 (IL-4), and B cell growth factor II (IL-5) indicated that none of these activities is capable of synergizing with 8MGuo to augment B cell responsiveness to antigen. Therefore, supernatants from a number of cloned cell lines were examined for activity that could synergize with 8MGuo, in order to determine the cellular source of this activity. Soluble products secreted by cloned 24/G1 T cells act synergistically with 8MGuo to evoke enhanced antibody responses to specific antigen in populations of purified B cells. Because concanavalin (Con) A-activated 24/G1 cells produce large quantities of interferon-gamma (IFN-gamma), the possibility that interferons might mediate synergy with 8MGuo was investigated. Purified murine IFN-gamma is unable to interact synergistically with 8MGuo; moreover, treatment of active 24/G1 supernatants with monoclonal anti-IFN-gamma antibodies or at pH 2 fails to abrogate their ability to synergize. In contrast to IFN-gamma, when B cells were supplemented with either IFN-alpha or IFN-beta, antigen-dependent synergy with 8MGuo was observed. However, abrogation of IFN-alpha and IFN-beta activity with specific antibodies fails to interfere with synergy between 8MGuo and mixed lymphocyte culture or Con A supernatants. Therefore, it appears that although IFN-alpha and IFN-beta are not responsible for the synergizing activity present in activated T cell supernatants, they nonetheless represent a previously unrecognized source of synergizing activity.     

A labile antigen complex at the lymphocyte surface: the effect of the substratum on its expression

Human T lymphocytes carry a surface antigen, detectable by a monoclonal antifibronectin antibody, which appears to consist of 150 and 55 kd components as revealed by SDS-PAGE. After in vitro culture of the lymphocytes on a plastic substratum for 48 hr comparatively few cells (40 +/- 18% in separate individuals) express the antigen. In contrast, the vast majority of lymphocytes cultured on a collagen matrix for the same time period maintains surface expression of the antigen (76 +/- 14% in separate individuals). Conditioned media from lymphocytes on plastic contain substantial amounts of antigenicity detectable by the same antibody, whereas conditioned media from lymphocytes on collagen are devoid of such antigenicity. The expression at the cell surface of other T lymphocyte antigens (Leu-4, Leu-3, and OKT8) is identical during culture on plastic and collagen for 48 hr. Collagen does not activate the cells to DNA synthesis or expression of IL 2 receptors, and consequently the potentiation of antigen expression by this substratum cannot be attributed to a mitogenic effect. The composition of subsets of T lymphocytes and the viability of the cells are the same on plastic and collagen, which excludes that the substratum-dependent variations in antigenicity reflect selection or loss of antigen-bearing cells. Thus, substratum-dependent regulation of the expression at the cell surface appears to be a unique property of the 150/55 kd T cell surface antigen. Culture on collagen substrata augments the number of lymphocytes showing motile behavior two to four times compared with culture on plastic.     

Inhibitory influence of IL-4 on human B cell responsiveness

The role of IL-4 in human B cell activation, proliferation, and differentiation was examined. rIL-2, but not rIL-4, was able to promote maximum proliferation and generation of Ig-secreting cells in cultures of highly purified B cells stimulated with Cowan I Staphylococcus aureus (SA). Addition of rIL-4 to rIL-2-supported cultures of SA-stimulated peripheral blood, spleen, or lymph node B cells dramatically suppressed both proliferation and differentiation. Results from experiments in which rIL-4 was added to culture at progressively later times indicated a requirement for rIL-4 to be present during the first 2 days of a 5-day incubation to cause inhibition of responsiveness. When a two-stage culture system was utilized, rIL-4 was found to support proliferation or differentiation of B cells initially activated with SA for 2 days only minimally. However, rIL-4 did not inhibit responses of SA preactivated B cells supported by IL-2. The presence of rIL-4 during the initial 48-h activation of B cells with SA and rIL-2 resulted in a profound inhibition of the ability of the activated B cells to respond subsequently to rIL-2 or lymphokine-rich T cell supernatants. A similar 48-h incubation with rIL-4 alone without SA had no effect on subsequent B cell responsiveness. The presence of rIFN-gamma during B cell activation decreased the inhibitory effect of IL-4. Other cytokines including IFN-alpha, IL-1, and commercially available low m.w. B cell growth factor also diminished the inhibitory effect of IL-4. These results indicate that IL-4 inhibits the capacity of human B cells to be activated maximally by SA and rIL-2 and therefore suggest a new immunomodulatory role for this cytokine.     

Release of E-rosette augmenting factor (E-RAF) after stimulation of human leukocytes with mitogens or antigens.

Stimulation of human peripheral blood leukocytes (HPBL) with mitogens such as phytohemagglutinin or concanavalin A increases the total number of lymphocytes that form rosettes with sheep erythrocytes. A similar effect is seen when HPBL from skin test-positive, but not skin test-negative, donors are stimulated by a specific antigen. It was also found that the culture fluids from mitogen-stimulated lymphocytes contained a substance that significantly increased the percentage of E-rosette forming cells (85 to 95%) over that of control culture fluids (40%). A similar phenomenon was also observed with supernatant fluids derived from antigenic stimulation of cells from skin test-positive donors, but not from skin test-negative subjects. The factor that produces this effect has been designated E-rosette augmenting factor (E-RAF). It is nondialyzable; it appears in the supernatants of antigen or mitogen-stimulated cells within 12 hr; and its production is not blocked by mitomycin C. It is produced by cells that do not adhere to glass wool columns and by mitogen-stimulated thymocytes. Sephadex G-100 chromatography showed that mitogen-induced E-RAF eluted from the column in advance of antigen-induced E-RAF. E-RAF has many properties that are characteristic of lymphokines.     

Genetic and physiological analysis of conjugation in Streptococcus faecalis.

In an effort to elucidate the mechanisms of conjugal plasmid transfer in Streptococcus faecalis, a genetic analysis of the sex pheromone-dependent tetracycline resistance plasmid pCF-10 was initiated. Rare transconjugants obtained from short matings with wild-type donors not exposed to sex pheromones were screened for increased donor potential in a subsequent mating. From this screening, a mutant plasmid, designated pCF-11, whose transfer functions are expressed in the absence of pheromone induction was isolated. Cells carrying pCF-11 spontaneously clump when grown in broth culture but do not excrete sex pheromones active against wild-type donors. In the course of initial experiments, it was observed that physiological conditions could affect plasmid transfer frequency. Therefore, a set of standardized optimal mating conditions was defined. The experiments carried out to determine these conditions revealed that a transient increase in transfer frequency of about 2 order of magnitude occurred in early-exponential-phase donor cells. This peak of activity is independent of sex pheromone response, since it was observed with induced or uninduced donor cells carrying either pCF-11 or pCF-10.     

Pertussigen enhances antigen-driven interferon-gamma production by sensitized lymphoid cells

We have studied the effects on interferon-gamma (IFN-gamma) production of pertussigen, a protein toxin from Bordetella pertussis that augments and prolongs delayed-type hypersensitivity (DTH) reactions. Lymphoid cell suspensions from immunized mice were incubated with antigen or mitogen, and the culture supernatants were assayed for IFN-gamma. The production of IFN-gamma on exposure to specific antigen or concanavalin A was greatly enhanced if mice were given pertussigen at the time of immunization. There was no detectable IFN-gamma production when cells were exposed to saline or to an irrelevant antigen. The effect of pertussigen on antigen-driven IFN-gamma production correlated with its effect on the capacity of the same cell populations to transfer DTH. The enhanced IFN-gamma production by cells from mice given pertussigen could not be attributed to an increased antigen-presenting capacity of this cell population. Production of IFN-gamma was abolished if the cells were pretreated with emetine, but not with mitomycin C, and the release of IFN-gamma was not detected in the first 8 hr of culture. After immunization with pertussigen, IFN-gamma was produced by lymph node and spleen cells from 7 days onward and both cell types produced IFN-gamma until at least 30 days after immunization. It is suggested that the augmentation of antigen-specific IFN-gamma production may contribute to the prolonged DTH reactions induced by pertussigen in vivo.     

Murine cutaneous leishmaniasis: resistance correlates with the capacity to generate interferon-gamma in response to Leishmania antigens in vitro

The kinetics of cell-mediated immunity developed during the course of Leishmania major infection in resistant (C57BL/6) and susceptible (BALB/c) mice were determined by using in vitro bioassays. Cells isolated from the lymph nodes draining the infected footpads were assayed for their proliferative responses to leishmania antigens (promastigote and amastigote) or to concanavalin A (Con A). Although lymph node cells (LNC) from both mouse strains proliferated to mitogen and antigen early after infection, both C57BL/6 and BALB/c mice developed diminished in vitro proliferative reactivity within 3 to 5 wk after infection. LNC from both mouse strains recovered lymphoproliferative reactivity to Con A (week 6), but only C57BL/6 mice regained reactivity to leishmania antigens. BALB/c cells remained unresponsive to leishmania antigens throughout the subsequent course of the infection. Supernatants derived from cultures of LNC that had been stimulated with Con A or leishmania antigens were assayed for interferon-gamma (IFN-gamma) by analyzing three distinct activities associated with IFN-gamma. Culture supernatants derived from leishmania antigen stimulation of LNC from infected C57BL/6 mice, but not BALB/c mice, were able to induce surface Ia on murine P388D1 cells. Ia-inducing activity was detectable in supernatants from C57BL/6 cells as early as 3 wk, and peaked by 5 wk after infection. Although cells from infected BALB/c mice never produced detectable IFN-gamma in response to leishmania antigens, LNC from both mouse strains produced readily detectable IFN-gamma in response to Con A throughout the course of infection. Culture supernatants that induced Ia on P388D1 cells were also capable of activating resident peritoneal macrophages to display leishmanicidal activity and of inhibiting encephalomyocarditis virus replication in murine fibroblasts. Each of these activities could be removed by prior incubation of the supernatants with rabbit heterologous anti-murine IFN-gamma sera or monoclonal rat-anti-murine IFN-gamma. The correlation of healer status with the capacity to generate IFN-gamma in vitro in response to leishmania antigens was examined in BALB/c mice that had been exposed to sublethal irradiation (550 rad) before infection. These animals have been previously shown to effectively resist L. major infection. Consistent with observations in the genetically resistant C57BL/6 mice, LNC from these animals demonstrated the capacity to respond to in vitro leishmania antigen stimulation with lymphoproliferation, and more importantly, by producing IFN-gamma.(ABSTRACT TRUNCATED AT 400 WORDS)     

A phorbol ester (TPA) can replace macrophages in human lymphocyte cultures stimulated with a mitogen but not with an antigen

A culture system was developed in which human peripheral blood mononuclear cells (PBM) depleted of macrophages did not proliferate in response to the lectin mitogen PHA or to the soluble antigen of tetanus toxoid. These cells were able to respond to both mitogen and antigen if purified autologous macrophages were added back to the culture. The response to PHA was partially restored by supplementing the cultures with supernatants from LPS-stimulated macrophages or with partially purified human interleukin 1 (IL 1). The response to tetanus was not restored by reconstitution with these materials. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), has been shown to have IL 1-like effects in other species and is a polyclonal activator of human T and B lymphocytes. In this study, we tested the ability of TPA to replace macrophages in human lymphocyte cultures stimulated with mitogen or with antigen. Small doses of TPA (50 ng/ml) completely replaced macrophages in the PHA-stimulated cultures; however, in doses of up to 400 ng/ml, TPA was not able to replace macrophages in cultures stimulated with tetanus. Thus, TPA appears to mimic the macrophage-replacing ability of soluble factors (IL 1, macrophage supernatants) in the triggering of human lymphocytes.     

Plasmodium falciparum: characterization of a 33-kDa soluble antigen

A 33-kDa soluble antigen identified in the culture supernatant by patient serum and monoclonal antibodies was present in rings, trophozoites, schizonts, and merozoites of Plasmodium falciparum. The antigen which is released into the culture supernatant by growing parasites was also observed in the host cells of trophozoites and schizonts and could be localized on the host cell surface. Its specificity for the surface of trophozoites and schizonts was observed to decrease with increased duration without subculture. The antigen could then be detected on the surface of noninfected erythrocytes. The antigenicity of the 33-kDa antigen was destroyed by heating at 65 degrees C. Monoclonal and polyclonal specific antibodies weakly inhibited parasite growth in vitro. The antigen was present in both knob positive and knob negative parasites in all the P. falciparum isolates tested.     

In vitro infection of natural killer cells with different human immunodeficiency virus type 1 isolates.

Natural killer (NK) cells are a discrete subset of leukocytes, distinct from T and B lymphocytes. NK cells mediate spontaneous non-MHC-restricted killing of a wide variety of target cells without prior sensitization and appear to be involved in initial protection against certain viral infections. Depressed NK cell-mediated cytotoxicity, one of the many immunological defects observed in AIDS patients, may contribute to secondary virus infections. Here we report that clonal and purified polyclonal populations of NK cells, which expressed neither surface CD4 nor CD4 mRNA, were susceptible to infection with various isolates of human immunodeficiency virus type 1 (HIV-1). Viral replication was demonstrated by detection of p24 antigen intracellularly and in culture supernatants, by the presence of HIV DNA within infected cells, and by the ability of supernatants derived from HIV-infected NK cells to infect peripheral blood mononuclear cells or CD4+ cell lines. Infection of NK cells was not blocked by anti-CD4 or anti-Fc gamma RIII monoclonal antibodies. NK cells from HIV-infected and uninfected cultures were similar in their ability to lyse three different target cells. Considerable numbers of cells died in HIV-infected NK cell cultures. These results suggest that loss of NK cells in AIDS patients is a direct effect of HIV infection but that reduced NK cell function involves another mechanism. The possibility that NK cells serve as a potential reservoir for HIV-1 must be considered.     

Extracellular identification of a processed type II ComR/ComS pheromone of Streptococcus mutans

The competence-stimulating peptide (CSP) and the sigX-inducing peptide (XIP) are known to induce Streptococcus mutans competence for genetic transformation. For both pheromones, direct identification of the native peptides has not been accomplished. The fact that extracellular XIP activity was recently observed in a chemically defined medium devoid of peptides, as mentioned in an accompanying paper (K. Desai, L. Mashburn-Warren, M. J. Federle, and D. A. Morrison, J. Bacteriol. 194:3774-3780, 2012), provided ideal conditions for native XIP identification. To search for the XIP identity, culture supernatants were filtered to select for peptides of less than 3 kDa, followed by C(18) extraction. One peptide, not detected in the supernatant of a comS deletion mutant, was identified by tandem mass spectrometry (MS/MS) fragmentation as identical to the ComS C-terminal sequence GLDWWSL. ComS processing did not require Eep, a peptidase involved in processing or import of bacterial small hydrophobic peptides, since eep deletion had no inhibitory effect on XIP production or on synthetic XIP response. We investigated whether extracellular CSP was also produced. A reporter assay for CSP activity detection, as well as MS analysis of supernatants, revealed that CSP was not present at detectable levels. In addition, a mutant with deletion of the CSP-encoding gene comC produced endogenous XIP levels similar to those of a nondeletion mutant. The results indicate that XIP pheromone production is a natural phenomenon that may occur in the absence of natural CSP pheromone activity and that the heptapeptide GLDWWSL is an extracellular processed form of ComS, possibly the active XIP pheromone. This is the first report of direct identification of a ComR/ComS pheromone.