Functional Analysis of the Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase (CobT) Enzyme,Involved in the Late Steps of Coenzyme B12 Biosynthesis in Salmonella enterica

In Salmonella enterica, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M cobT mutations were deleterious, we determined the nature of three class M cobT alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobT^WT) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobT^WT. The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M cobT alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.Cobalamin (Cbl, also known as B₁₂) is a structurally complex cyclic tetrapyrrole with a cobalt ion coordinated by equatorial bonds with pyrrolic nitrogen atoms and is unique among cyclic tetrapyrroles (e.g., heme, chlorophylls, coenzyme F₄₃₀) in that it has an upper and a lower axial ligand (Fig. ​(Fig.1).1). The coenzymic form of Cbl is known as adenosylcobalamin (AdoCbl) or coenzyme B₁₂.Open in a separate windowFIG. 1.Role of CobT in the late steps of AdoCbl biosynthesis. This branch of the AdoCbl biosynthetic pathway is known as the NLA pathway. The black box in the inner membrane represents the corrinoid-specific ABC transporter BtuCD. The inset shows the chemical structure of AdoCbl; the coring ring in the scheme is represented by the rhomboid cartoon with the Co ion in the middle.Some bacteria and archaea synthesize AdoCbl de novo or from preformed precursors such as cobyric acid (Cby) or cobinamide (Cbi) (Fig. ​(Fig.1)1) (11, 32). The enterobacterium Salmonella enterica serovar Typhimurium LT2 (hereafter referred to as S. Typhimurium) synthesizes the corrin ring of AdoCbl de novo only under anoxic conditions (15). Although oxygen blocks de novo corrin ring biosynthesis in this bacterium, it does not block the assembly of adenosylcobalamin (AdoCbl, coenzyme B₁₂) if cells are provided with preformed, incomplete corrinoids such as Cbi or Cby (11, 13-15).The late steps in AdoCbl biosynthesis can be divided into two different branches that comprise the nucleotide loop assembly (NLA) pathway (19). One of the branches of the NLA pathway activates the lower ligand base, while the other one activates adenosylcobinamide (AdoCbi) to AdoCbi-GDP (Fig. ​(Fig.11).In this paper, we focus on the activation of 5,6-dimethylbenzimidazole (DMB), the lower ligand base of AdoCbl. There are two ways in which DMB can be activated. In both cases, the CobT enzyme (EC 2.4.2.21) catalyzes the reaction, but the product of the reaction varies, depending on whether the cosubstrate of CobT is NAD⁺ or its precursor nicotinate mononucleotide (NaMN). If NaMN is the substrate, CobT synthesizes α-ribazole-phosphate (α-RP) (reaction: DMB + NaMN → α-RP + Na) (30). If NAD⁺ substitutes for NaMN, CobT synthesizes α-DMB adenine dinucleotide (α-DAD) (reaction: DMB + NAD⁺ → α-DAD + Nm) (18). α-RP is a good substrate for the AdoCbl-5′-phosphate (AdoCbl-P) synthase (CobS; EC 2.7.8.26) enzyme (19, 34), suggesting that an unidentified enzyme cleaves α-DAD into α-RP and AMP (reaction: α-DAD → α-RP + AMP). Although it is possible that CobS may use α-DAD as a substrate, to date, data are not available to support this idea. The AdoCbl-P phosphatase (CobC; EC 3.1.3.73) enzyme catalyzes the last step of the NLA pathway to yield AdoCbl (Fig. ​(Fig.1)1) (34).Early genetic studies identified four classes of cobT alleles, namely, classes J, K, L, and M (12); class M was an intriguing class of mutations because they did not display intragenic complementation (12). Here we identify the nature of class M cobT mutations, report the initial in vitro and in vivo characterization of class M CobT variant proteins, and propose structural explanations for the observed deficiencies in CobT activity caused by class M mutations.     

Effective Downregulation of HLA-A*2 and HLA-B*57 by Primary Human Immunodeficiency Virus Type 1 Isolates Cultured from Elite Suppressors

Elite controllers or suppressors (ES) are human immunodeficiency virus type 1 (HIV-1)-infected patients who control viral replication to <50 copies/ml without antiretroviral therapy. Downregulation of HLA class I molecules is an important mechanism used by HIV-1 to evade the immune system. In this study, we showed that primary isolates from ES are as effective as isolates obtained from patients with progressive HIV-1 disease at downregulating HLA-A*2 and HLA-B*57 molecules on primary CD4⁺ T cells. Thus, a diminished ability of viral isolates from ES to evade HIV-specific immune responses probably does not contribute to the control of viral replication in these patients.Long-term nonprogressors (LTNP) are human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain normal CD4⁺ T-cell counts and remain asymptomatic for longer than 10 years without therapy (7). Although many LTNP have detectable levels of HIV-1 RNA in their plasma, patients known as elite suppressors (ES) have viral loads of <50 RNA copies/ml. Understanding the factors involved in the maintenance of LTNP and ES statuses may be critical for the development of effective vaccines and immunotherapeutic treatments. One such factor under investigation is the role of cytotoxic-T-lymphocyte (CTL) responses. Several studies have shown that the HLA-B*27 and -B*57 alleles are overrepresented in cohorts of ES (13, 16, 19, 28, 29, 34). These findings suggest important roles for major histocompatibility complex class I (MHC-I) restriction and CD8⁺ T-cell responses in the control of viremia. Indeed, multiple studies have documented qualitatively superior CD8⁺ T cell function in ES compared to that in chronic HIV progressors (CP) (2, 5, 12, 27, 28, 37, 47).Other studies suggest that some ES and LTNP are infected with attenuated viruses. One illustrative example comes from studies done on the Sydney Blood Bank Cohort, in which an LTNP donor transmitted an HIV-1 isolate with a large deletion in nef and the U3 region of the long terminal repeat to multiple recipients, all of whom became LTNP (11, 21). As in the Sydney Blood Bank Cohort studies, several other investigators have detected viruses with defective nef genes in LTNP and ES (1, 8, 18, 23, 25, 35, 36, 38, 43). In contrast, other studies showed that CD4⁺ T cells from ES could produce Gag when they were stimulated in vitro (20, 26), and full-length sequence analyses of plasma and proviral genomes revealed no evidence of significant deletions (30). Recent studies have suggested that plasma isolates (31) and replication-competent viruses (32) from HLA-B*57/B58*01 ES and LTNP, respectively, are less fit than isolates from B*57/B*5801 CP, but the difference in fitness observed is unlikely to fully explain the control of viral replication in these patients. Furthermore, we recently performed detailed genotypic and phenotypic analyses of replication-competent viruses isolated from ES and showed that these viruses were fully replication competent (6) Although nef is not required for viral replication in vitro, it has been strongly associated with pathogenesis in vivo (reviewed in reference 14). It is thus possible that some ES isolates are replication competent but have mutations in nef that result in diminished pathogenesis.nef has been shown to be involved in the downregulation of both CD4 (15) and MHC-I (41). Several studies have shown that nef-induced MHC-I downregulation has a major impact on CTL function. In a seminal study, a dramatic reduction in HLA-A*2 expression by CD4⁺ T cells infected with wild-type virus but not by those infected with a virus carrying a defective nef gene was demonstrated. This downregulation resulted in diminished killing of HIV-1-infected cells by CTL clones specific for an HLA-A*2-restricted HIV-1 Gag epitope (10). Similarly, nef-mediated MHC-I downregulation was shown to impair the ability of HIV-1-specific CTL clones to suppress viral replication (42, 44). While these findings strongly suggest that HIV-1 partially evades the immune response by inducing MHC-I downregulation, other studies have demonstrated that primary CD8⁺ T cells from some ES and CP could effectively respond to autologous viral replication in autologous CD4⁺ T cells (26).We tested the hypothesis that ES are infected with HIV-1 isolates that are less capable of downregulating MHC-I molecules. This could potentially cause the isolates to be more susceptible to CD8⁺ T-cell suppression of replication and may explain the superior CD8⁺ T-cell responses reported in prior ES studies (2, 5, 12, 27, 28, 37, 47). To date, fully characterized replication-competent isolates have been reported from just six ES subjects (1, 3, 6). We compared the MHC-I downregulation capacity of isolates from five of these ES to that of isolates obtained from resting CD4⁺ T cells of eight patients with progressive disease (viral load, >10,000 copies/ml). In order to develop a physiological model for HIV-1-induced MHC-I downregulation, we enriched primary CD4⁺ T cells from peripheral blood mononuclear cells (PBMC) from donors who were HLA-A*2 and/or HLA-B*57 positive by CD8⁺ T cell depletion with magnetic beads (Dynal), followed by activation in vitro with phytohemagglutinin for 3 days. For evaluation of HLA-A*2 downregulation, CD4⁺ T cells were obtained from HIV-seronegative donors. CD4⁺ T cells from ES were used for the evaluation of HLA-B*57 downregulation. This allele was as effectively downregulated in these ES as it was in multiple HLA-B*57 CP (data not shown). Following activation, the cells were infected with primary HIV-1 isolates from ES or CP by spinoculation (33). The primary isolates were obtained as previously described from latently infected CD4⁺ T cells (9). The median peak viral load and CD4⁺ T-cell nadir of the CP from whom viral isolates were obtained was 81,000 copies/ml and 279 cells/μl, respectively, and thus these isolates should be effective at HLA downregulation (22).At different time points, the cells were harvested and stained with either fluorescein isothiocyanate (FITC)-conjugated anti-HLA-A*2 (Becton Dickinson) and tricolor-conjugated anti-CD4 antibodies (Caltag) or biotinylated anti-HLA-B*57 antibody (One Lambda) followed by FITC-conjugated streptavidin, peridinin chlorophyll protein-Cy5.5-conjugated anti-CD4 antibody (Becton Dickenson), and allophycocyanin-conjugated anti-CD3 antibody. The cells were fixed and permeabilized with Cytofix/Cytoperm solution (Becton Dickenson). Intracellular staining was then performed with the phycoerythrin-conjugated Gag-specific monoclonal antibody Kc57 or an immunoglobulin G1 mouse isotype control (Beckman Coulter). A total of 100,000 to 500,000 events were analyzed for each sample. HLA typing of ES was performed as previously described (4). The HLA-specific antibodies were tested on cells from a panel of ES with known HLA types to confirm specificity.MHC-I downregulation was measured by comparing the mean fluorescence intensities (MFI) of HLA-A*2 and HLA-B*57 on HIV-1-infected versus noninfected CD4⁺ T cells. Infected cells were defined as cells that stained positive for intracellular Gag and had downregulated CD4 (Fig. ​(Fig.1).1). Uninfected CD4⁺ T cells were defined as cells that expressed high levels of CD4 and were negative for intracellular Gag protein. In order to standardize values, we determined relative MFI by dividing the MFI of the infected population by that of the CD4-positive, uninfected population. The Wilcoxon Mann-Whitney test was used to analyze the data.Open in a separate windowFIG. 1.Analysis of HLA-B*57 downregulation on HIV-1-infected cells. (A) CD8⁺ T-cell-depleted PBMC were stained with anti-HLA-B*57 and anti-CD4 monoclonal antibodies 3 days after infection with primary isolates from an ES (ES8) or a CP (CP2). Cells in quadrant 1 are uninfected CD4⁺ T cells, and cells in quadrant 4 (Gag-positive, low levels of CD4) are infected cells that have downregulated CD4. (PE, phycoerythrin; IgG, immunoglobulin G.) (B) The MFI of HLA-B*57 were compared for uninfected (quadrant 1) and infected (quadrant 4) cells from each sample.To determine if there was a difference in the ability of HIV-1 isolates cultured from ES versus CP to downregulate HLA-A*2, we measured the MFI of this molecule on infected CD4⁺ T cells that had downregulated CD4. On average, primary CD4⁺ T cells infected by ES viruses had levels of MHC-I downregulation of about two- to threefold, with relative MFI of 0.51, 0.37, and 0.30 on days 2, 3, and 4, respectively. Similarly, cells infected by isolates cultured from CP had relative MFI of 0.46, 0.36, and 0.33 on days 2, 3, and 4, respectively (Fig. ​(Fig.2B).2B). These differences were not significantly different at any time point.Open in a separate windowFIG. 2.(A) Relative MFIs of HLA-A*02 on cells infected with isolates from five ES (triangles) and eight CP (squares) on days 2 to 4 postinfection. The relative MFI is defined as the MFI of the infected cells divided by the MFI of the uninfected CD4⁺ T cells in each sample. The horizontal bars represent the median for each group. (B) Average relative MFI of HLA-A*02 for cells infected with isolates from ES and CP on each day. (C) Average relative MFI of HLA-A*02 for cells infected with the wild-type NL4-3-green fluorescent protein virus (diamonds) or the Nef⁻ Vpr⁻ mutant virus (circles).In order to rule out nonspecific downregulation of MHC-I on infected cells, we determined the MFI of HLA-DR and CD45 RO on cells infected with isolates from two subjects. The average relative MFI of the two proteins were 1.28 and 1.48, respectively, indicating that the MHC-I was in fact specifically downregulated. Since mutations in Nef have been shown to abrograte HLA downregulation, we also compared HLA-A2 downregulation by the HIV-1-based reporter construct NL4-3-green fluorescent protein and a Nef⁻ Vpr⁻ mutant vector (45, 46). As shown in Fig. ​Fig.2C,2C, no downregulation of HLA-A2 was seen at any point after infection with the Nef⁻ Vpr⁻ mutant virus, whereas infection with wild-type virus caused a degree of downregulation that was similar to that seen with primary isolates from ES and CP. Finally, we also looked at CD3 downregulation, as this molecule has been shown to be downregulated by Nef from HIV-2 and many simian immunodeficiency virus (SIV) isolates but not from HIV-1 (39). Furthermore, since SIVsmm nef isolated from sooty mangabeys with preserved CD4⁺ T-cell counts causes significantly more downregulation than SIVsmm nef from sooty mangabeys with CD4⁺ T-cell depletion (40), we determined whether isolates from ES also selectively downregulated this molecule. As shown in Fig. ​Fig.3A,3A, there was no significant downmodulation of CD3 after infection of cells with isolates from ES or CP.Open in a separate windowFIG. 3.(A) Relative MFI of CD3 on cells infected with isolates from five ES (triangles) and five CP (squares) on day 3 postinfection. The horizontal bars represent the median for each group. (B) Relative MFI of HLA-B*57 on cells infected with isolates from ES and CP.Epidemiologic studies have suggested that HLA-B alleles play a larger role than HLA-A alleles in determining the outcome of infection (17). Furthermore, while HLA-B*57 is the most overrepresented allele seen in ES, there have not been any studies looking at downregulation of this MHC-I protein. Activated CD4⁺ T cells from an HLA-B*5703-positive ES were infected with isolates from five ES and five CP, and the degree of HLA-B*57 downregulation was measured on day 3. As shown in Fig. ​Fig.3B,3B, the average relative MFI of cells infected with isolates from five ES was 0.53, which was not significantly different from the average relative MFI of 0.64 that was seen in cells infected with isolates from five progressors.While it appeared that there was generally more downregulation of HLA-A*2 than HLA-B*57, the studies were performed in cells from different donors, and this precluded a direct comparison of the MFI of the two MHC-I alleles. Two ES in our cohort were positive for both HLA alleles, and the degrees of downregulation of these proteins could thus be compared. CD4⁺ T cells from ES8 were infected with autologous virus (6), and cells from ES9 were infected with a primary isolate from the CP who transmitted virus to her (3). For patient ES8, HLA-A2 showed a greater degree of downregulation than HLA-B57 at day 3 (a relative MFI of 0.36 versus 0.62) (Fig. ​(Fig.4).4). In contrast, in ES9 the degrees of downregulation of the two proteins were nearly identical (a relative MFI of 0.35 for HLA-A2 versus 0.31 for HLA-B*57).Open in a separate windowFIG. 4.Comparison of the relative MFI of HLA-A*02 and HLA-B*57 on CD8⁺ T-cell-depleted PBMC from ES8 and ES9 that were infected with autologous virus (ES8) or with the primary isolate from the CP who transmitted the virus to ES9. The MFI of HLA-A*2 or HLA-B*57 on uninfected CD4⁺ T cells (top panels) and infected cells that had downregulated CD4 (bottom panels) are shown.This is the first study to look at downregulation of MHC-I proteins on CD4⁺ T cells infected with HIV-1 isolates cultured from ES CD4⁺ T cells. We used a physiological model where primary CD4⁺ T cells were infected with primary HIV-1 isolates. One advantage of this approach is that it accounts for HLA downregulation mediated by viral proteins such as Tat (24), as well as Nef. Similar amounts of MHC-I downregulation were seen for cells infected with replication-competent isolates cultured from ES and progressors. These results demonstrate that most ES are not infected by HIV-1 virions that are deficient in downregulating MHC-I compared to those of CP. Thus, it is likely that other factors enable ES to control viremia. The identification of these factors will have implications for the design of HIV-1 vaccines.     

Simian Immunodeficiency Virus Infection Induces Expansion of α4β7+ and Cytotoxic CD56+ NK Cells

Herein we demonstrate that chronic simian immunodeficiency virus (SIV) infection induces significant upregulation of the gut-homing marker α4β7 on macaque NK cells, coupled with downregulation of the lymph node-trafficking marker, CCR7. Interestingly, in naïve animals, α4β7 expression was associated with increased NK cell activation and, on CD16⁺ NK cells, delineated a unique dual-function cytotoxic-CD107a⁺/gamma interferon (IFN-γ)-secreting population. However, while SIV infection increased CD107a expression on stimulated CD56⁺ NK cells, α4β7⁺ and α4β7⁻ NK cells were affected similarly. These findings suggest that SIV infection redirects NK cells away from the lymph nodes to the gut mucosae but alters NK cell function independent of trafficking repertoires.Human peripheral blood contains two primary subsets of natural killer (NK) cells—a major CD16⁺ CD56^dim subset and a minor CD16⁻ CD56^bright subset. We have defined subsets of rhesus macaque (Macaca mulatta) NK cells and found that, similarly, macaque peripheral blood is dominated by a CD16⁺ CD56⁻ subset but contains two minor CD16⁻ CD56⁺ and CD16⁻ CD56⁻ subpopulations (34). As in humans, macaque CD16⁻ CD56⁺ NK cells are the primary producers of gamma interferon (IFN-γ) and express cell surface markers such as CCR7 and CD62L that mediate homing to lymph nodes, whereas CD16⁺ CD56⁻ NK cells do not express CCR7 or CD62L and primarily mediate cytolytic functions (7, 12, 30, 34). In both humans and macaques, the distribution of NK subsets in peripheral blood is distinct from that observed in lymph nodes and mucosal tissues, where NK cells are primarily CD56⁺ (9, 12, 30, 35).NK cells are important for the control of multiple viral infections, and increasing evidence suggests that NK cells play a significant role in controlling human immunodeficiency virus (HIV) infection (3, 5, 13, 14, 19, 21, 22, 24, 33), as well as simian immunodeficiency virus (SIV) infection of rhesus macaques and sooty mangabeys (6, 16, 26). HIV and SIV primarily replicate in the gut mucosa (18), and although we and others have demonstrated the presence of NK cells in the gastrointestinal tracts of both humans and rhesus macaques (8, 9, 25, 30), the nature of these NK cells is still poorly understood. Interestingly, the integrin α4β7, which directs lymphocyte trafficking to the gut (4), has been shown to be expressed on peripheral blood NK cells in humans and rhesus macaques (11, 27). This finding suggests that the gut mucosa is a site of active NK cell trafficking.Despite the importance of gut-associated lymphoid tissue in HIV/SIV pathogenesis, little is known about the effects of infection on NK cell homing to these tissues. In order to address this deficit, a total of 47 Indian rhesus macaques were studied, 27 of which were SIV naïve and 20 infected with either SIVmac239 (5) or SIVmac251 (15) for more than 150 days (mean duration of infection, 293 days). All animals were housed at the New England Primate Research Center and maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (23a).PBMC isolation and polychromatic flow cytometry staining were carried out using protocols described previously for our laboratory (29, 31); the antibodies used are listed in Table ​Table1.1. NK cells were defined as CD3⁻ CD8α⁺ NKG2A⁺ (30, 34), and CD16 and CD56 expression were used to delineate three primary subsets: CD56⁻ CD16⁺ (CD16⁺), the dominant subset; CD56⁺ CD16⁻ (CD56⁺); and CD56⁻ CD16⁻ (double negative [DN]) (Fig. ​(Fig.11 A). The results of polychromatic flow cytometry analyses demonstrated that α4β7 was expressed at the highest levels on CD16⁺ NK cells and that, while expression on this subset was not altered during SIV infection, α4β7 was significantly upregulated on both CD56⁺ and DN NK cells in SIV-infected animals (Fig. 1B and C). Interestingly, CCR7, which is expressed only on the CD56⁺ and DN NK cell subsets in macaques (30, 34), was concomitantly downregulated on these subsets of NK cells during chronic SIV infection (Fig. ​(Fig.1B).1B). The relationship between the two markers delineated a dichotomous expression pattern between naïve and SIV-infected macaques (Fig. ​(Fig.1D).1D). This dramatic shift in CD56⁺ and DN NK cell trafficking repertoires is likely indicative of increased homing of these NK subsets to the gut coupled to decreased homing to lymph nodes. Also, as shown in Fig. ​Fig.1E,1E, the absolute numbers of both CD16⁺ and DN NK cells increased during chronic SIV infection, resulting in increased absolute numbers of gut-homing α4β7⁺ cells in both subsets. Interestingly, while the absolute numbers of all CD56⁺ NK cells tended to decrease during chronic SIV infection, the absolute numbers of the α4β7⁺ CD56⁺ NK cell subset increased slightly (Fig. ​(Fig.1E,1E, middle panel), further suggesting that multiple subsets of α4β7⁺ NK cells increase during chronic SIV infection.Open in a separate windowFIG. 1.Comparison of α4β7 expression on NK cell subsets in naïve and SIV-infected macaques. (A) Macaque NK cell subsets were defined as CD3⁻ CD8α⁺ NKG2A⁺ (30, 34) and then further delineated into CD56⁺, CD16⁺, and DN subsets. (B) Representative flow cytometry plots of α4β7 and CCR7 expression on NK cell subsets in naïve and SIV-infected macaques. (C) Percentages of α4β7⁺ cells above the background level were compared between naïve and SIV-infected macaques for CD56⁺, CD16⁺, and DN NK subsets. (D) Relationships between α4β7 and CCR7 expression on CD56⁺ and DN NK cells in naïve and SIV-infected macaques. (E) Absolute numbers of total circulating NK cells were determined by using a bead-based flow cytometric assay as described previously (29, 30), and α4β7⁺ NK cell subset counts were extrapolated using these data combined with NK cell frequency data determined by polychromatic flow cytometry (panel A). Horizontal bars indicate median values for 20 to 27 animals. Student''s t tests were used to compare naive and SIV-infected animal groups; P values of >0.05 are considered statistically significant.

TABLE 1.

Antibodies used in polychromatic flow cytometry analyses

Mononucleosis and Antigen-Driven T Cell Responses Have Different Requirements for Interleukin-2 Signaling in Murine Gammaherpesvirus Infection

Interleukin-2 (IL-2) has been implicated as being necessary for the optimal formation of primary CD8⁺ T cell responses against various pathogens. Here we have examined the role that IL-2 signaling plays in several aspects of a CD8⁺ T cell response against murine gammaherpesvirus 68 (MHV-68). Exposure to MHV-68 causes a persistent infection, along with infectious mononucleosis, providing a model for studying these processes in mice. Our study indicates that CD25 is necessary for optimal expansion of the antigen-specific CD8⁺ T cell response but not for the long-term memory response. Contrastingly, IL-2 signaling through CD25 is absolutely required for CD8⁺ T cell mononucleosis.Members of the gammaherpesvirus family are associated with significant diseases, such as nasopharyngeal carcinoma, lymphoid malignancies, and infectious mononucleosis (16). Murine gammaherpesvirus 68 (MHV-68) is a γ2-herpesvirus related to the human pathogens Epstein-Barr virus (EBV) and Kaposi''s sarcoma virus (19, 21). Intranasal (i.n.) infection of mice with MHV-68 results in acute infection of the lung epithelium, which is eventually controlled; however, the virus also establishes a latent infection in B cells, dendritic cells, and macrophages that is maintained throughout the life of the host (8, 9). Infection with MHV-68 generates a broad array of antigen-specific CD8⁺ T cells that can control the virus without eliminating persistent infection (5, 12, 13). Additionally, CD4⁺ T cells and neutralizing antibodies are thought to be critical for the prevention of virus reactivation (3, 6).A major complication of EBV infection is infectious mononucleosis (16), which occurs when infection is delayed until puberty. Signs of disease include dramatic lymph node enlargement and the presence of large numbers of activated CD8⁺ T cells in the peripheral blood. Similarly to EBV infection, MHV-68 induces a polyclonal activation of B cells upon establishment of latency. Concurrently, a CD8⁺ T cell-dominated lymphocytosis of the peripheral blood occurs, as seen with EBV. However, there are distinct differences between the two types of infectious mononucleosis. CD8⁺ T cell lymphocytosis seen with EBV consists of a broad array of T cell receptor specificities, a large proportion of which are specific for EBV epitopes. In contrast, MHV-68-induced mononucleosis is dominated by oligoclonal Vβ4⁺ CD8⁺ T cells that are not reactive to MHV-68 epitopes. With MHV-68, the expansion of this population is dramatic, with levels reaching upwards of 60% of the peripheral blood CD8⁺ T cell population (20). This occurs in different mouse strains, across at least five different major histocompatibility complex (MHC) class I haplotypes. However, it is important to note that infection of wood mice (Apodemus sylvaticus) does not induce splenomegaly, as seen with laboratory strains of mice, indicating a potential lack of Vβ4 expansion that may be species related (14). Interestingly, evidence suggests that Vβ4⁺ CD8⁺ T cell expansion does not require classical MHC class Ia antigen presentation (4). Recent studies instead implicate a secreted viral protein, M1, capable of stimulating the Vβ4+ T cell population in a novel manner, and the authors propose a role for Vβ4+ T cells in control of MHV-68 infection (7).We and others have recently shown that IL-2 signaling during the early stages of a response to acute viral and bacterial pathogens is required for optimal expansion and differentiation of CD8⁺ T cells (15, 17, 18). However, reports with other viruses have shown IL-2-independent primary CD8⁺ T cell responses (1, 22). Therefore, we wished to determine whether IL-2 signals are necessary for the expansion, maintenance, and/or recall of CD8⁺ T cell responses during murine gammaherpesvirus infection.We generated chimeric mice through lethal irradiation of C57BL/6 mice followed by adoptive transfer of mixed bone marrow from C57BL/6 wild-type (WT) and CD25^−/− donors, as previously described (17). Following previous described protocols, mice were given bone marrow in a 2:1 ratio of CD25^−/−/WT to generate equally proportioned congenic populations in recipient mice (see Fig. S1 in the supplemental material) (1, 17). The resultant mice contained CD8⁺ T cells of both WT and CD25^−/− origin, which could be distinguished by congenic markers. Chimeric mice were infected intranasally with 400 PFU of MHV-68, and the kinetics of the CD8⁺ T cell response were followed by antibody and tetramer staining of peripheral blood for CD8⁺ T cells specific for the epitopes ORF6₄₈₇ (p56) and ORF61₅₂₄ (p79), as previously described (13). While antigen-specific CD25^−/− CD8⁺ T cells were initially able to proliferate in response to infection, the peak response was significantly lower than that of the wild-type cells (Fig. ​(Fig.11 A and B). This indicates that while CD25 is dispensable for early activation of CD8⁺ T cells, IL-2 signaling is required for full expansion of the antigen-specific response to MHV-68. Despite this deficit in the acute antiviral response, the resultant memory populations were not statistically different between the groups (Fig. 1A and B). In our previous report, CD25^−/− CD8⁺ T cells were unable to fully differentiate into short-lived effector cells (SLECs), defined as KLRG1^high CD127^low (17). To determine if MHV-68-specific responses were also unable to fully differentiate, we infected chimeric mice and stained p79⁺ CD8⁺ T cells for the cell surface markers KLRG1 and CD127. At the peak of the response (14 days postinfection [p.i.]), p79⁺ WT cells had differentiated into SLEC (KLRG1^high CD127^low), memory precursor (MPEC) (KLRG1^low CD127^high), and doubly positive populations. However, the p79⁺ CD25^−/− cells failed to form the SLEC population and instead had a corresponding increase in the MPEC population, indicating that CD25 is necessary for full effector differentiation of gammaherpesvirus-specific CD8⁺ T cell responses (Fig. 1C and D).Open in a separate windowFIG. 1.IL-2 signals are necessary for the optimal expansion of MHV-68-specific CD8⁺ T cells. WT/CD25^−/− chimeric mice were infected with MHV-68 intranasally and bled at set time points. The antigen-specific responses against two dominant epitopes, p79 (A) and p56 (B), were determined via tetramer staining of peripheral blood. p79-specific CD8⁺ T cells from the WT and CD25^−/− populations were stained at the peak of the response (day 14 p.i.) for KLRG1 and CD127 to determine their ability to differentiate into short-lived and memory precursor effector cells (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent standard deviations from the means. Four mice were used per group, and data are representative of at least two experiments.To determine whether antigen-specific CD25^−/− CD8⁺ T cells were capable of optimally responding to a secondary challenge, we infected chimeric mice with MHV-68 and waited 60 days before challenging with recombinant vaccinia virus (rVV) expressing the ORF61₅₂₄ epitope (2 × 10⁶ PFU, intraperitoneal). It is necessary to use a heterologous virus to induce a recall CD8⁺ T cell response since MHV-68 generates a robust neutralizing antibody response, preventing secondary infection. Previous studies with rVV indicate that the recall response of MHV-68-specific CD8⁺ T cells is antigen dependent, since administration of rVV expressing an irrelevant epitope had no effect upon the MHV-68-specific populations (2). WT and CD25^−/− cells were able to respond to the secondary challenge with similar kinetics (Fig. ​(Fig.22 A and B), indicating that MHV-68 memory CD8⁺ T cells are capable of a generating a recall response in the absence of IL-2 signaling. These data, together with our previous report (17), show that the dependence on CD25 for formation of the SLEC population is conserved between both persistent and acute virus infections.Open in a separate windowFIG. 2.CD25^−/− CD8⁺ T cells can respond to secondary challenge. WT/CD25^−/− chimeric mice were infected with MHV-68 i.n. After 60 days, the percentage of peripheral blood CD8⁺ T cells specific for p79 was determined. Mice were then challenged with rVV p79, and the p79⁺ CD8⁺ population was determined 5 days postchallenge (A). The numbers in the box represent the averages ± standard deviations. The average fold increase was calculated to determine the ability of WT and CD25^−/− CD8⁺ T cells to respond to a secondary challenge (B). Error bars represent standard deviations from the means. Four mice were used per group, and data are representative of at least two experiments.WT CD8⁺ T cells underwent a dramatic expansion between days 15 and 21 p.i. (Fig. ​(Fig.3A),3A), consistent with infectious mononucleosis (10). Interestingly, we did not observe a similar expansion of CD25^−/− CD8⁺ T cells, indicating a role for IL-2 signaling in the expansion of CD8⁺ T cells during mononucleosis (Fig. ​(Fig.3A).3A). Since mononucleosis is dominated by Vβ4+ CD8⁺ T cells, we analyzed these T cells from both naive and infected mice (17 days p.i.) for expression of CD25 by flow cytometry. While Vβ4⁺ CD8⁺ T cells from the spleen and peripheral blood of naive mice did not express detectable levels of CD25, mice infected with MHV-68 expressed intermediate levels of CD25 during the time period when dramatic expansion of Vβ4⁺ T cells occurs (Fig. 3B and C). Consistent with a role for IL-2 signaling in Vβ4 expansion, we observed a severe deficit in expansion in the CD25^−/− population of chimeric mice, since the percentage of WT Vβ4⁺ cells increased dramatically between days 14 and 36 p.i., accompanied by only a small expansion of the CD25^−/− Vβ4⁺ population over the same period (Fig. ​(Fig.44 A and B).Open in a separate windowFIG. 3.Vβ4⁺ CD8⁺ T cells express CD25 upon infection with MHV-68. WT/CD25^−/− chimeric mice were infected with MHV-68 i.n., and the percentage of peripheral blood cells that were CD8⁺ was determined over time for each congenic population (A). Vβ4⁺ CD8⁺ T cells from naive and MHV-68-infected mice (day 17 p.i.) were analyzed for expression of CD25 (B and C). Isotype control, filled histogram; naive mice, dashed line; infected mice, solid line (**, P < 0.01). Error bars represent standard deviations from the means. Four mice were used per group, and data are representative of at least two experiments.Open in a separate windowFIG. 4.CD8⁺ T cell-based infectious mononucleosis does not occur in the absence of IL-2 signaling in MHV-68-infected mice. WT/CD25^−/− chimeric mice were infected with MHV-68 i.n., and the percentage of Vβ4⁺ CD8⁺ T cells was determined over time for each congenic population. Representative plots from day 36 p.i. (A) or the averages over time (B) are shown. WT and CD25^−/− CD8⁺ T cells from chimeric mice were analyzed for expression of CD62L over time. Representative plots from day 24 p.i. (C) or the averages over time (D) are shown. *, P < 0.05; **, P < 0.01). Error bars represent standard deviations from the means. Four mice were used per group, and data are representative of at least two experiments.During infectious mononucleosis, CD8⁺ T cells are in a highly activated state and thus express low levels of CD62L (20). Therefore, we analyzed CD8⁺ T cells from chimeric mice for expression of CD62L. After MHV-68 infection, the majority of WT CD8⁺ T cells in the peripheral blood were CD62L^low, as previously reported (Fig. 4C and D) (20). Interestingly, CD25^−/− CD8⁺ T cells failed to develop this dominant CD62L^low population, indicating that CD25 is necessary for the activation of the CD8⁺ T cell compartment in addition to cell expansion during mononucleosis (Fig. 4C and D). When we analyzed the Vβ4⁺ CD8⁺ T cell compartment, we observed that WT cells downregulated expression of CD62L. While Vβ4⁺ cells from the CD25^−/− compartment also decreased expression of CD62L, they did so to a lesser extent both as a percentage and on a per-cell basis (see Fig. S2 in the supplemental material).In these studies, we have shown that signaling through CD25 is necessary for the generation of an optimal primary CD8⁺ T cell response against a gammaherpesvirus, since virus-specific CD8⁺ T cells were unable to expand as robustly as WT cells and did not fully differentiate into short-lived effector cells. These observations are consistent with previous results from our lab and findings of others using a variety of acute infection models (17, 18). However, not all persistent infections appear to require CD25, since the m45-specific response to murine cytomegalovirus (MCMV) infection occurs normally in the absence of IL-2 signals (1). What allows for some responses to be independent of IL-2 remains unknown. Potential explanations could involve differences in tropism, the route of infection, or the amount of proinflammatory cytokines induced by each infection. Despite the dependence on CD25 for the short-term effector response, the memory CD8⁺ T cell response remained intact in the absence of IL-2 signaling. In contrast, Vβ4 expansion and mononucleosis never attained normal levels. Unlike the antigen-specific response, which relies upon peptide/MHC interactions for induction, mononucleosis does not rely upon conventional antigen presentation (4). Instead, the M1 protein of MHV-68, expressed during the establishment and expansion of latency in the spleen, appears to drive Vβ4 expansion (7). Interestingly, our evidence shows that both antigen-dependent and -independent CD8⁺ T cell expansion require CD25. Antigen-specific T cells also undergo an apoptotic contraction phase, followed by a lower frequency of cells surviving as relatively quiescent memory cells. In contrast, during mononucleosis caused by MHV-68, CD8⁺ T cells remain in an activated state and do not undergo a marked contraction, providing a potential explanation as to why the WT and CD25^−/− Vβ4 populations continue to differ in both number and phenotype later in the response.Earlier studies have also identified CD4⁺ T cells as being critical for the development of MHV-68-induced infectious mononucleosis (11, 20). We have previously shown that CD4⁺ T cell help was critical for robust expression of CD25 on activated antigen-specific CD8⁺ T cells. Interestingly, when we measured CD25 expression on Vβ4⁺ cells from mice lacking CD4⁺ T cells, we saw a moderate decrease in the level of CD25 expressed (data not shown), indicating one potential reason why CD4-deficient mice do not experience infectious mononucleosis. However, it is likely that other factors involving CD4⁺ T cells and activation of B cells are also involved (10).In conclusion, the significance of these studies is twofold. First, they shed light on the requirements for MHV-68-induced mononucleosis. Second, our data illustrate that CD25 is required for both antigen-specific and non-antigen-specific activation of CD8⁺ T cell responses, while being dispensable for memory cell formation. This knowledge may be useful in developing new T cell-based immune therapies to enhance control of persistent gammaherpesvirus infections.      

Gag p27-Specific B- and T-Cell Responses in Simian Immunodeficiency Virus SIVagm-Infected African Green Monkeys

Nonpathogenic simian immunodeficiency virus SIVagm infection of African green monkeys (AGMs) is characterized by the absence of a robust antibody response against Gag p27. To determine if this is accompanied by a selective loss of T-cell responses to Gag p27, we studied CD4⁺ and CD8⁺ T-cell responses against Gag p27 and other SIVagm antigens in the peripheral blood and lymph nodes of acutely and chronically infected AGMs. Our data show that AGMs can mount a T-cell response against Gag p27, indicating that the absence of anti-p27 antibodies is not due to the absence of Gag p27-specific T cells.Simian immunodeficiency virus (SIV) infection in African green monkeys (AGM) is nonpathogenic, even though it is characterized by plasma viral load (PVL) levels similar to those found during acute and chronic pathogenic infection of humans with human immunodeficiency virus type 1 and macaques with SIVmac (14). This feature is shared with other African nonhuman primates, such as sooty mangabeys (SM) and mandrills (19, 20). SIV-infected AGMs also display high viral loads in the gastrointestinal mucosa (11), a transient decline of circulating CD4⁺ T cells during acute infection (13), and longer-lasting CD4⁺ T-cell depletion in the intestinal lamina propia (10). Concomitant with the peak viral load during acute infection, SIVagm-infected AGMs display transient increases of CD4⁺ and CD8⁺ T cells expressing activation, and proliferation markers, such as MHC-II DR and Ki-67 (4, 13), and anti-SIVagm antibodies (Ab) are induced with kinetics similar to those found in SIVmac infection (5). Interestingly, however, the Ab response against Gag p27 is weak, if present at all (1, 2, 12, 15, 17, 18). This observation is surprising since, in the context of human immunodeficiency virus type 1 and SIVmac infections, Ab responses to Gag p27 are usually quite strong. Weak or low reactivity to Gag p27 has also been observed in some other natural SIV infections (7, 8, 20) but not in all of them (21). We wondered whether such a selective lack of Ab reactivity in the SIV-infected AGM might be related to a lack of Gag p27-specific T cells. With this hypothesis in mind, we first confirmed and extended the studies of humoral responses against Gag p27 by characterizing the antigen-specific immunoglobulin G (IgG) responses and mid-point titers against total SIVagm antigens (SIVagm virions) and recombinant Gag p27 (rP27; SIVagm) in naturally and experimentally SIVagm-infected AGMs. Second, we searched for the presence of Gag p27-specific T-cell responses in SIVagm infection by analyzing the CD4⁺ and CD8⁺ T-cell responses specific for Gag p27 and other SIVagm proteins in blood and lymph nodes (LNs) of acutely and chronically infected animals.Humoral responses against SIV were analyzed in 50 wild-born AGMs (Chlorocebus sabaeus) and 17 rhesus macaques (RMs). The animals were housed at the Institut Pasteur in Dakar, Senegal, and the California National Primate Research Center, Davis, CA, respectively, according to institutional and national guidelines. RMs were either noninfected (n = 5) or intravenously infected with SIVmac251 (n = 12). AGMs were noninfected (n = 23), naturally infected (n = 17), or intravenously infected with wild-type SIVagm.sab92018 (n = 10) (5, 9). IgG titers against SIVagm.sab92018 virions or rP27 were determined by an enzyme-linked immunosorbent assay (ELISA) using monkey anti-IgG as secondary Ab (Fig. 1A and B). The virions had been purified by ultracentrifugation on an iodixanol cushion from cell-free supernatants of SIVagm.sab92018-infected SupT1 cells. The His-tagged rP27 was constructed using DNA from gut cells of an SIVagm.sab92018-infected AGM 96011 (11). A Gag p27 PCR product was subcloned into pET-14b, and the recombinant protein was produced in Escherichia coli BL21(DE3)(pLysS) and purified on nitrilotriacetic acid columns. SIV-infected macaques showed high IgG titers cross-reacting with both SIVagm virions (Fig. 1A and B, left panels) and rP27 (Fig. 1A and B, right panels). In contrast, only 2 out of 27 SIV-infected AGMs showed detectable IgG responses against rP27 (Fig. 1A and B, right panels), while 21 out of 27 displayed significant responses against SIVagm virions (Fig. 1A and B, left panels). Two AGMs out of 23 from the negative control group showed weak responses at the limit of detection against SIVagm and two against rP27, suggesting a natural response against SIVagm proteins, cross-reactivity with unknown pathogens, maternal Ab, or recent SIV infection. Of note, the titers against whole SIV in the infected monkeys were higher in macaques than in AGMs, which may be due to a lack of anti-p27 Ab in most AGMs.Open in a separate windowFIG. 1.Cross-sectional analysis of IgG Ab responses against SIVagm or Gag p27 in SIV-infected AGMs and RMs. (A and B) Cross-sectional analysis by ELISA. IgG Ab against SIVagm.sab₉₂₀₁₈ virions or recombinant p27-Gag antigens were determined in SIV-negative (Rh SIV−) and chronically SIVmac₂₅₁-infected (Rh SIV+) RMs and in SIV-negative and chronically SIVagm-infected AGMs that were either naturally (AGM Nat SIV+) or experimentally (AGM Exp SIV+) infected with SIVagm.sab₉₂₀₁₈. Ab titers were calculated for each animal by limited dilution of plasma on coated ELISA plates with 5 μg/ml of (p27 equivalent) virions (left) or 1 μg/ml of the monomeric recombinant protein (rP27) (right). IgG detection by ELISA displayed a high background for rP27, especially at the highest plasma concentration (e.g., 1/100 and 1/400 plasma dilution) in SIV-negative RMs and AGMs. To discriminate between positive responses and background, calculated dose-response curves were compared to theoretical sigmoid-dose response curves corresponding to the 95% confidence interval of SIV-negative animals. By convention, responses were considered background when sigmoid dose-response curves were graphically within the 95% confidence interval of SIV-negative animals and when the calculated negative log 50% effective concentration (EC₅₀) was lower than the top theoretical sigmoid dose-response curve from SIV-negative animals (corresponding to a threshold of negative log EC₅₀ of 2.8). (A) Results (optical density at 450 nm [OD₄₅₀]) are represented for both virions (left) and rP27 (right) over plasma dilution (log₁₀) on a per animal basis (data points) and for each group (lines). Lines represent the sigmoid dose-response curves for each group (Prism 4; Graphpad). (B) Mid-point IgG titers were determined for each animal from individual sigmoid dose-response curves, and presented as the log₁₀ value from the reciprocal of the effective concentration that corresponds to 50% response between minimum and maximum OD₄₅₀ (negative log EC₅₀). Horizontal bars represent the median mid-point titer per each group. Mann-Whitney nonparametric tests were applied for statistical analysis (n.s., nonsignificant, with P values of >0.1) (C) Cross-sectional analysis of Ab against SIVagm proteins by Western blot analysis using denatured SIVagm.sab₉₂₀₁₈. For the positive controls on the left, we used sera from an SIVmac₂₅₁-infected macaque and a SIVagm.sab₉₂₀₁₈-infected AGM. Development times and reagents were identical for all Western blots. Mo, months of infection; y, years of infection; C−, negative control; C+, positive control.The study of IgGs by Western blot analysis using denatured SIVagm.sab92018 virions showed no or weak anti-Gag responses in SIV-infected AGMs, yet the anti-Env responses were often strong (Fig. ​(Fig.1C).1C). In contrast, SIV-infected macaques showed a dominant IgG cross-reactive response against the SIVagm Gag p27 protein. Even if responses in AGMs were detected more frequently with the Western blot analyses than with the ELISAs, these responses were different in magnitude and considerably weaker than those in macaques.To compare B- and T-cell responses over time, five simian T-cell leukemia virus-seronegative AGMs were infected with SIVagm.sab92018, and the animals were followed longitudinally during the acute and postacute phases of infection until day 90 postinfection (p.i.). Sequential blood samples were collected and biopsies of auxiliary and inguinal LNs were performed on day −5 and at three times p.i. (days 14, 43, and 62). PVL was measured by real-time PCR (5). Since we searched for Gag p27-specific responses, we also quantified Gag p27 antigen in the plasma (SIV p27 antigen assay; Coulter, Miami, FL). Viral RNA and p27 antigenemia peaks were observed between days 7 and 14 p.i. (Fig. 2A and B, respectively). The Gag p27 levels were variable among the animals but in a range similar to those reported previously in AGMs and macaques (3, 5). As has also been observed in SIVmac infection (except for rapid progressors), plasma Gag p27 levels fell below the detection level in the postacute phase (i.e., after day 28 p.i.) (Fig. ​(Fig.2B2B and data not shown). There were significant increases in circulating CD8⁺ DR⁺ T cells at days 7 and 14 p.i. and in CD8⁺ Ki-67⁺ T cells at days 14 and 28 p.i. (Fig. 2C and D, left panels). After day 28 p.i., the percentages were no longer statistically different from baseline levels. In LN cells (LNCs), the percentage of CD8⁺ Ki-67⁺ T cells rose from 3.1% ± 1.1% before infection to 6.1% ± 0.3% at day 62 p.i., but the difference was not statistically significant (Fig. ​(Fig.2D,2D, right panel). The levels of blood CD4⁺ DR⁺ Ki-67⁺, CD8⁺ DR⁺ Ki-67⁺, CD8⁺ Ki-67⁺ T cells, and LNC CD8⁺ Ki-67⁺ T cells were positively correlated with viremia (P values of 0.002 for DR⁺ cells and P values of <0.02 for Ki-67⁺ cells). Altogether, these results confirm previous data showing early, transient T-cell activation in the peripheral blood of SIVagm-infected AGMs (13).Open in a separate windowFIG. 2.Plasma viremia and T-cell activation in blood and LNs of five longitudinally followed SIVagm.sab₉₂₀₁₈-infected African green monkeys. (A) SIVagm.sab RNA copy numbers in plasma. (B) Plasma Gag p27 concentrations. (C) Percentages of MHC-II DR-positive CD4⁺ (•) and CD8⁺ (○) T cells within, respectively, total CD4⁺ and CD8⁺ T cells from PBMCs and LNCs. (D) Percentages of Ki-67⁺ CD4⁺ (•) and CD8⁺ (○) T cells within, respectively, total CD4⁺ and CD8⁺ T cells from PBMCs and LNCs. Results are shown as the mean ± the standard error of the mean. Asterisks indicate statistically significant differences compared to levels before infection (P < 0.05).We next looked for the presence of Ab responses against rP27 in these animals. No Ab were detected before infection. After infection, all five AGMs developed anti-SIVagm IgGs within 4 to 9 weeks p.i., with AGM 02001 showing the fastest response (Fig. ​(Fig.3A).3A). While the humoral responses against whole virions were significant (Fig. ​(Fig.3B),3B), the anti-rP27 responses were below the threshold for positivity (Fig. ​(Fig.3B),3B), with the exception of one animal (AGM 02001). The anti-rP27 response in this animal was only transient since it was no longer detectable at week 75 p.i., in contrast to the anti-SIV Ab that were sustained (Fig. ​(Fig.3B3B and data not shown).Open in a separate windowFIG. 3.Longitudinal analysis of IgG titers and T-cell proliferative responses against SIVagm and Gag p27 in five AGMs experimentally infected with SIVagm.sab₉₂₀₁₈. (A and B) Ab responses were analyzed by ELISA. (A) IgG dose-response curves against SIVagm (top) and rP27 (bottom) are shown over time (week −1 to week 24 p.i.). O.D.450, optical density at 450 nm. (B) Mid-point titers were calculated as described in the legend to Fig. ​Fig.1A.1A. Continuous lines correspond to median titers from all five animals. Red, anti-SIVagm IgGs; green, anti-p27 IgGs. (C) Proliferative responses of CD4⁺ and CD8⁺ T cells were assessed by flow cytometry using carboxy fluorescein succinimidyl ester staining (CFSE). CD4⁺ and CD8⁺ T-cell responses in PBMCs (left) and LNCs (right) after stimulation with peptide pools (Gag without P27, P27, and Tat) and Gag rP27 are shown for each animal. All data are reported after background subtraction. Results are presented in columns as the mean ± the standard error of the mean. Asterisks indicate statistically significant differences compared to individual values before infection (P < 0.05).We next searched for T-cell responses against Gag p27 compared to other SIVagm antigens in these animals. Gag p27 epitopes were presented in the following two ways: in the context of rP27 and as synthetic peptides. The peptide pools (comprised of overlapping 15-mers) spanned the following SIVagm proteins: Gag p27, Gag without p27, Env, and Tat. The amino acid sequences of the Gag and Env peptides corresponded to the autologous wild-type SIVagm.sab92018 sequence, and those of the Tat peptides corresponded to an SIVagm.sab consensus sequence. The latter was determined using Tat sequences of other SIVagm viruses from Senegal that are available in the databases (SIVagm.sab1c, SIVagm.sabD42, and SIVagm.sabD30). We measured T-cell responses by investigating the antigen-induced proliferation. T cells from blood (peripheral blood mononuclear cells [PBMCs]) and LNs were analyzed. All assays were performed with fresh cells that were stimulated with 10 μg/ml of Gag rP27 and 5 μg/ml of peptides over a period of 4 days. Dead cells were gated out using 7-amino-actinomycin D, and dividing (CFSE^low) cells were analyzed after stimulation with medium alone, SIV antigens, or concanavalin A as a positive control. We detected significant Gag p27-specific proliferative responses for CD8⁺ T cells in PBMCs and for CD4⁺ and CD8⁺ T cells in LNCs (Fig. ​(Fig.3C).3C). The animal with the detectable anti-p27 Ab (AGM 02001) did not show stronger p27-specific T-cell responses than the other animals. Thus, all SIV-infected AGMs were able to mount a proliferative T-cell response against p27, while anti-p27 IgGs were lacking in four of the animals. However, the SIVagm-specific T-cell responses were detected at only a few time points p.i.We then analyzed the T-cell responses in the chronic phase of AGMs naturally and experimentally infected with SIVagm.sab92018. PVL, peripheral blood cell counts (CD4⁺ and CD8⁺ T cells; CD20⁺ B cells), and immune activation (Ki-67⁺ CD4⁺ and CD8⁺ T cells) were similar in naturally infected and in experimentally infected AGMs (Fig. ​(Fig.4A).4A). As expected, cell counts and immune activation levels were also not different from SIV-negative AGMs (Fig. ​(Fig.4A).4A). Again, we measured SIV-specific responses first by a proliferation assay (Fig. ​(Fig.4B).4B). One out of five animals tested had a proliferative SIV-specific CD4⁺ T-cell response (against Gag without p27, P27, rP27, Env GP120, and Tat), and two animals had a CD8⁺ T-cell response (against P27 in both animals and against Env GP120 and Tat in one). Two animals (one naturally infected and one experimentally infected with SIVagm.sab92018) did not show any detectable antigen-specific proliferative CD4⁺ or CD8⁺ T-cell response.Open in a separate windowFIG. 4.Immune parameters and SIVagm-specific proliferative and cytokine T-cell responses in chronically infected AGMs. (A) Cell counts (CD4⁺ and CD8⁺ T cells; B cells) and immune activation levels (percent of Ki-67⁺ in CD4⁺ and CD8⁺ T cells) in AGMs (n = 4) naturally infected with SIVagm (Nat SIV⁺) and AGMs (n = 6) experimentally infected with SIVagm.sab₉₂₀₁₈ (Exp SIV⁺) compared to uninfected AGMs (n = 10) (SIV⁻). PVL, if known, is indicated. Green, blue, and orange symbols correspond, respectively, to noninfected, naturally infected, and experimentally infected AGMs. (B) Proliferative response to SIVagm antigens in chronically infected AGMs (n = 5) compared to those in uninfected AGMs (n = 3). PBMCs were stimulated with the same antigens as those described in the legend to Fig. ​Fig.3.3. (C) Analysis of cytokine responses (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]) by SIVagm-specific T cells. ConA was used as a positive control. Representative results from a single animal are shown here. (D) Cumulative values of SIVagm-specific TNF-α and IFN-γ responses in chronically infected animals. The responses to SIVagm antigens were analyzed in peripheral blood specimens of 4 naturally and 5 experimentally infected AGMs as well as 10 uninfected AGMs. The data are reported after background subtraction corresponding to the subtraction of the frequency of positive events from the unstimulated samples to the frequency of positive events from the antigen-specific stimulation. Proliferative T-cell responses and cytokine T-cell responses in SIV-infected AGMs were defined as positive when higher than 3 standard deviations above the mean responses for uninfected animals. Freq, frequency; w/o, without.These results were extended to an analysis of SIV-specific T-cell cytokine responses, e.g., the production of IFN-γ and TNF-α in nine chronically infected compared to 10 noninfected AGMs (Fig. 4C and D). Fresh cells were stimulated for 8 h with the antigens described above. SIV-specific cytokine responses were detected in CD8⁺ but not in CD4⁺ T cells. Seven animals out of nine showed a response against at least one antigen. The two animals showing no response were among the four naturally infected animals tested. We therefore cannot exclude that the absence of response in these two animals is due to the presence of highly divergent viruses. However, a precise epitope mapping in SIVagm sequences would be necessary to confirm this. In those animals showing a SIVagm-specific cytokine T-cell response, the responses were directed against Gag p27 (four out of nine animals), other Gag proteins than p27 (two out of nine animals), and Env GP120 (four out of nine animals). In the experimentally infected animals, we might have underestimated the responses against Tat compared to Gag and Env antigens, since the Tat peptides corresponded to an SIVagm.sab consensus sequence and not to the autologous virus (SIVagm.sab92018). There was no correlation between the magnitude or breadth of SIV-specific T-cell responses and immune activation or PVL.Altogether, our study demonstrates that AGMs can mount T-cell proliferative and cytokine responses against Gag p27. The T-cell response was variable among the animals. In general, it appeared moderate, comparable to chronically SIV-infected RMs (9). Of note, T-cell responses were not consistently detected at all time points and not in all animals. We cannot exclude the possibility that we underestimated the magnitude of the cytokine responses. For instance, we did not costimulate the cells during the assays. However, cytokine responses were also variable in vervet AGMs, with a trend for reduced levels compared to those for RMs, even when more-sensitive assays were used (23). In SM, the responses were also reported to be not stronger than in RMs. This is in line with the lack of efficient control of viral replication in natural hosts (6, 22).In our study, we show that IgG responses against Gag p27 are either lacking, weak, or transient, while Ab against other SIVagm proteins are present. The mechanisms underlying this selective lack of Gag p27 Ab responses are unclear. It could be related to moderate and/or dysfunctional CD4⁺ T-cell responses and/or due to an unknown suppressive regulatory mechanism. SIV-specific T-cell cytokine responses were indeed principally found at the CD8⁺ T-cell level. This was also reported in SIVsm-infected SM (6, 22). Here, we also searched for SIVagm Gag p27-specific proliferative responses. Interestingly, they were detected for CD4⁺ T cells, indicating the presence of p27-specific CD4⁺ memory cells in AGMs. Moreover, AGMs can potentially mount a strong and sustained anti-Gag p27 humoral response, when appropriately immunized (D. Favre et al., unpublished data). This suggests that there is neither a central B-cell tolerance against p27 Gag protein in AGMs nor an inherent inability for CD4⁺ T cells to provide helper B-cell functions. The transient nature of anti-p27 Ab in one animal would be in favor of regulatory mechanisms, but that needs to be confirmed. Another explanation could be that AGMs are able to mount Ab responses against some p27 epitopes but not to those exposed by the native protein, which would explain why we and others detect more frequently humoral responses in Western blot analysis than in ELISAs (16).In conclusion, we characterized the IgG responses against SIVagm and confirmed a lower humoral response against p27 than in RMs. Moreover, our study reveals that cytokine and proliferative T-cell responses against SIVagm Gag p27 are detectable in AGMs. Thus, the reduced ability of the AGM to produce Ab against Gag p27 p.i. is not related to a lack of Gag p27-specific T cells.     

Metabolic Flux Control at the Pyruvate Node in an Anaerobic Escherichia coli Strain with an Active Pyruvate Dehydrogenase

During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. A pfl mutant with LDH and PDH* activities, as well as a pfl ldh double mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (Y_ATP) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lower Y_ATP suggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering of E. coli and related bacteria for the production of various biobased products at high rates and yields.In Escherichia coli as well as in other aerobic organisms, sugars such as glucose are metabolized in two separate steps: glycolysis, which converts glucose to pyruvate, and tricarboxylic acid (TCA) cycle enzymes, which oxidize acetyl coenzyme A (acetyl-CoA) to CO₂ (5, 9). The pyruvate dehydrogenase complex (PDH) connects the glycolytic reactions to TCA cycle enzymes by catalyzing the production of acetyl-CoA from pyruvate. Because of its unique central role in metabolism, PDH is regulated at both the genetic and the biochemical level (7, 12, 27, 33, 34). The NADH generated during the complete oxidation of sugar is reoxidized to NAD⁺ by O₂ through the respiratory electron transport pathway with accompanying energy production (11). Optimum coupling of these enzyme reactions helps to maintain the internal ratios of [NADH] to [NAD⁺] (redox balance) and of [ATP] to [ADP] plus [AMP] in order to support growth at the highest rate.The absence of O₂ or another external electron acceptor during the growth of E. coli (anaerobic conditions) forces the bacterium to minimize the contribution of the TCA cycle enzymes to biosynthesis from catabolism (4, 14). Under these conditions, pyruvate or acetyl-CoA derived from pyruvate serves as the electron acceptor (reduced to lactate or ethanol, respectively) to maintain the redox balance. The enzymes responsible for redox balance in anaerobic E. coli are pyruvate formate-lyase (PFL), lactate dehydrogenase (LDH), and alcohol/aldehyde dehydrogenase (adhE; ADH-E). The main products of the fermentation of E. coli are a mixture of organic acids, such as acetate, lactate, and formate, in addition to ethanol (2, 4). Succinate, derived from phosphoenolpyruvate (PEP), is a minor product of fermentation and normally accounts for less than 5% of the total products produced from glucose by the culture.Anaerobic growth of E. coli, compared to aerobic growth, is also limited by energy, leading to an increase in glycolytic flux (19). The conversion of pyruvate to acetate and ethanol yields an additional ATP per glucose, suggesting that this would be the preferred route for pyruvate oxidation during anaerobic growth. This is accomplished by the PFL-dependent production of acetyl-CoA and further conversion to acetate (Fig. ​(Fig.1).1). This preference for PFL has been demonstrated `with several bacteria under carbon limitation conditions imposed either in a chemostat or in the presence of a poor carbon source (10, 20, 23). This additional ATP also elevates the ATP yield per glucose to 3, with an increase in the growth rate, and has been shown to be essential for the anaerobic growth of E. coli in xylose-mineral salts medium (13). The absence of this third ATP in a pfl mutant has been reported to increase glycolytic flux to lactate to compensate for this decrease in ATP yield per glucose (39). However, the flow of pyruvate carbon to acetate is tempered by the need to maintain redox balance, and this is achieved by the conversion of a second acetyl-CoA to ethanol by ADH-E. Under conditions of energy excess due to a declining growth rate, lactate production is expected to support redox balance maintenance without the additional ATP from the PFL-ADH-E pathway (Fig. ​(Fig.1).1). The production of this mixture of products in an appropriate ratio helps to maintain the redox balance under anaerobic conditions while also maximizing the ATP yield per glucose to support high growth rates and cell yields.Open in a separate windowFIG. 1.Anaerobic metabolic pathways of E. coli carrying the lpd101 mutation (PDH*).No PDH-based fermentation reaction to ethanol that can also help maintain cellular redox balance in an anaerobic cell has evolved in E. coli or other closely related bacteria. PDH activity is inhibited by NADH, normally found at higher levels in anaerobically growing cultures than in aerobic cultures (12, 18, 34, 35). Based on genome sequences available in GenBank, the genes encoding the components of PDH are not found in strictly anaerobic bacteria.We have recently described a mutation (lpd101) in the dihydrolipoamide dehydrogenase (LPD) of the PDH that allowed the enzyme to function in anaerobic cells (designated PDH* here) (17, 18). With this altered PDH*, an anaerobic cell can have three different pathways for pyruvate metabolism (Fig. ​(Fig.1).1). The three main enzymes that utilize pyruvate as a substrate, PDH*, PFL, and LDH, have different apparent K_m values for pyruvate (0.4, 2.0, and 7.2 mM, respectively) (1, 18, 37, 41). PDH requires NAD⁺ for activity (apparent K_m, 0.07 mM), while LDH is dependent on NADH (apparent K_m, 0.2 mM) as the second substrate (18, 37).The PDH* serves as the first enzyme in a pathway that oxidatively decarboxylates pyruvate to acetyl-CoA and NADH, followed by reduction of the acetyl-CoA by alcohol dehydrogenase to ethanol in a two-step process using 2 NADHs (Fig. ​(Fig.1).1). The NADH produced during the conversion of glucose to acetyl-CoA dictates that the acetyl-CoA generated by PDH be used for redox balance (ethanol) and not for ATP generation (acetate), unless some of the NADH is used for biosynthesis by the growing cell (17). PDH* and LDH serve essentially the same physiological role in the cell, oxidizing NADH to support continued operation of glycolysis, although it is not readily apparent with PDH*. Although PDH* contributes to an increase in NADH pool, the redox balance is still maintained by coupling PDH* to NADH-dependent reduction of acetyl-CoA to ethanol by ADH-E (Fig. ​(Fig.1).1). This potential competition between LDH and PDH has been eliminated in the wild type through inhibition of the activity of PDH by NADH (12, 18, 32). However, the in vivo role of PDH* in a mutant that has all three pathways has not been investigated, since the flow of pyruvate through any of the three reactions during growth and postgrowth fermentation of sugars to products is expected to be dependent on the redox state, the ATP requirement, and other physiological conditions of the anaerobic cell. Using a combination of metabolic flux analysis and mutations in one or more of the genes encoding these enzymes, we have evaluated the flow of pyruvate carbon among the three potential pathways. The results are presented in this communication.     

Identification of the Biosynthetic Gene Cluster for the Pseudomonas aeruginosa Antimetabolite l-2-Amino-4-Methoxy-trans-3-Butenoic Acid

l-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of human infections and is considered the main pathogen responsible for chronic pneumonia in cystic fibrosis patients (7, 23). P. aeruginosa also infects other organisms, such as insects (4), nematodes (6), plants (18), and amoebae (20). Its ability to thrive as a pathogen and to compete in aquatic and soil environments can be partly attributed to the production and interplay of secreted virulence factors and secondary metabolites. While the importance of many of these exoproducts has been studied, the antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid (AMB; methoxyvinylglycine) (Fig. ​(Fig.1)1) has received only limited attention. Identified during a search for new antibiotics, AMB was found to reversibly inhibit the growth of Bacillus spp. (26) and Escherichia coli (25) and was later shown to inhibit the growth and metabolism of cultured Walker carcinosarcoma cells (28). AMB is a γ-substituted vinylglycine, a naturally occurring amino acid with a β,γ-C=C double bond. Other members of this family are aminoethoxyvinylglycine from Streptomyces spp. (19) and rhizobitoxine, made by Bradyrhizobium japonicum (16) and Pseudomonas andropogonis (15) (Fig. ​(Fig.1).1). As inhibitors of pyridoxal phosphate-dependent enzymes (13, 17, 21, 22), γ-substituted vinylglycines have multiple targets in bacteria, animals, and plants (3, 5, 10, 21, 22, 29). However, the importance of AMB as a toxin in biological interactions with P. aeruginosa has not been addressed, as AMB biosynthesis and the genes involved have not been elucidated.Open in a separate windowFIG. 1.Chemical structures of the γ-substituted vinylglycines AMB, aminoethoxyvinylglycine, and rhizobitoxine.