Induction of HIV-1 long terminal repeat-mediated transcription by Neisseria gonorrhoeae

Gonorrhoea enhances the transmission of HIV through increased viral shedding and the increased probability of seroconversion among previously HIV-negative individuals. However, the mechanism(s) underlying these influences remain poorly understood. We demonstrated that exposure toNeisseria gonorrhoeaeinduces the nuclear factor kappa B-dependent transcription from the HIV-1 long terminal repeat in derivatives of the Jurkat CD4 T cell line. These data suggest that gonococcal infection directly impacts HIV-1 transmission through the localized stimulation of viral expression. Although ulcerative sexually transmitted diseases probably influence the transmission of HIV by increasing exposure to infected bodily secretions, gonorrhoea enhances HIV-1 transmission in the absence of genital lesions. The reason for this effect remains poorly understood, but Neisseria gonorrhoeae infection appears to increase both viral shedding by HIV-1-positive individuals and a recipient's susceptibility to HIV-1 after exposure [1]. As any influence on HIV-1 expression could, presumably, influence both viral shedding and the chance of the virus establishing a productive infection, we have tested whether gonococcal infection can directly impact HIV-1 long terminal repeat (HIV-LTR)-dependent transcription in CD4 T lymphocytes. 1G5 cells are a derivative of the Jurkat CD4 T cell line containing a stably integrated reporter consisting of a luciferase gene driven by the HIV-LTR (Fig. 1a) [2]. Infection of these lymphocytes with N. gonorrhoeae encoding the heparan sulfate proteoglycan-specific Opa50 protein caused a clear, dose-dependent, increase in luciferase activity (Fig. 1b), with peak expression occurring at a multiplicity of infection (MOI) of 100. The decline in luciferase activity at MOI greater than 100 may be attributable to non-specific infection-induced toxicity; however, no significant increase in lymphocyte apoptosis or necrosis was apparent in response to MOI of 200 or less gonococci/cell, as determined by staining with propidium iodide and fluorescein-labelled annexin (data not shown).Fig. 1.: Neisseria gonorrhoeae induction of transcription from HIV-1 long terminal repeat. (a) Schematic diagram illustrating the luciferase reporter construct that is stably integrated in the chromosome of 1G5 cells. Transcription factor binding sites are indicated. 1G5 cells used herein were generously supplied by Michel Tremblay (Laval University, Quebec, Canada). (b) 1G5 cells respond to Neisseria gonorrhoeae in a dose-dependent manner. 1G5 cells were left untreated, treated with 100 nM phytohaemagglutinin (PHA) and 0.5 μg/ml PHA (positive control), or infected with N. gonorrhoeae strain N303 (P−Opa50 +) at increasing multiplicities of infection (MOI). After cellular lysis, luciferase activity was measured using a microplate scintillation counter. Results are the mean plus standard deviation (SD) for triplicate samples. Asterisks indicate values significantly different from the uninfected control (P < 0.01). (c) Influence of gonococcal adhesins on HIV-long terminal repeat (LTR)-mediated transcription. Infections were carried out essentially as described above except that recombinant N. gonorrhoeae and Escherichia coli strains expressing either pilus (P+Opa−), the heparan sulphate proteoglycan receptor-specific Opa50, CEACAM-specific Opa57, or no adhesin (P−Opa−) were used (MOI 100). Recombinant bacterial strains employed in this study were generously supplied by Professor T.F. Meyer (Max-Planck-Institut fur Infektionsbiologie, Berlin, Germany). Results are the mean plus SD for five replicate samples. Asterisks indicate paired values (as indicated by arrows) that are significantly different (P < 0.01). (d) Gonococcal culture filtrates induce HIV-LTR-dependent expression. 1G5 cells were resuspended in 0.22 μm filtrates of gonococcal culture supernatant that were either left untreated or pre-treated with either 30 U/ml benzonase, 200 μg/ml proteinase K, or both. As controls, cells were also treated with PMA/PHA or infected with a non-piliated, Opa+ N. gonorrhoeae strain (Ngo; MOI 100). The results for each experimental condition are the mean ± SD of five replicate samples. Asterisks denote values from enzymatically modified filtrate-treated samples that are significantly different (P < 0.01) from the untreated Ngo filtrate control. (e) Gonococcal infection induces nuclear factor kappa B (NF-κB)-dependent transcription in Jurkat T cells. Jurkat cells were transiently transfected with plasmid encoding tandem NF-κB binding sites upstream of the gene encoding chloramphenicol acetyltransferase (CAT), as illustrated schematically in the top panel. Plasmid encoding the NF-κB-CAT reporter construct was generously provided by Dr K. Copeland (Ottawa Hospital Research Institute, Ottawa, Ontario, Canada). Twenty-four hours post-transfection, cells were either left untreated, treated with 100 nM PMA and 0.5 μg/ml PHA, or infected with a non-piliated Opa+ N. gonorrhoeae strain (MOI 100). Cells were harvested, and lysed 48 h post-transfection, and supernatants assayed for CAT activity by determining acetylation after 1 h. The results are the mean plus SD of triplicate samples. Asterisks indicate values that are significantly different (P < 0.01) from the uninfected (untreated) control. (f) N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) inhibits luciferase expression by 1G5 T cells. 1G5 cells were treated with 40 μM ALLN for 2 h at 37°C before stimulation. Cells were then either left untreated, treated with PMA/PHA, or infected with N. gonorrhoeae strain N303 (P− Opa50 +) for 24 h, after which cells were harvested for luciferase assay as described above. The results are the mean ± SD of triplicate samples. Asterisks indicate samples that are significantly different (P < 0.01) from the parallel untreated samples. Data shown are representative of at least three independent experiments.Several different adhesins can mediate gonococcal binding to host cells [3]. To assess the influence of these adhesins on HIV-LTR-directed transcription, we infected 1G5 cells with recombinant N. gonorrhoeae and Escherichia coli expressing defined gonococcal adhesins (Fig. 1c). Although bacterial expression of either pilus or Opa proteins marginally augment HIV-LTR-dependent transcription compared with bacteria expressing no adhesin, gonococci lacking adhesins still stimulated a marked expression of the luciferase reporter. Exposure to E. coli also increased luciferase activity (Fig. 1c), indicating that transcription was at least partly mediated by a general response to bacteria rather than requiring a Neisseria-specific factor. The induction of HIV-LTR was not dependent on bacterial viability, as gentamicin-killed gonococci also had a stimulatory effect (data not shown). This effect could presumably occur via any of the receptors of the innate immune system, which allow host recognition of bacterial products based upon their conserved molecular patterns [4,5]. Consequently, we tested the influence of various well-described inducers of innate immunity on HIV-LTR activity. The treatment of 1G5 cells with either bacterial lipopolysaccharide, formylated peptide, or bacterial (CpG-containing) DNA had no impact on luciferase expression (data not shown), indicating that these cells are unresponsive to each of these factors. This prompted us to ascertain whether any soluble components found in gonococcal culture supernatants influence HIV-LTR-mediated transcription, or if contact with intact bacteria was instead required for this effect. Compared with untreated controls, a clear increase in luciferase expression was observed when 1G5 cells were resuspended in filtrates of gonococcal culture supernatants (Fig. 1d). Treatment of these filtrates with benzonase endonuclease did not alter the magnitude of this effect, consistent with the lack of response to purified chromosomal DNA (data not shown). In contrast, treatment with proteinase K, either alone or with benzonase, reduced expression of the luciferase reporter by approximately 50%, indicating that at least part of this activity is caused by bacterial protein(s) present in the extracellular milieu. Although gonococci express several proteins known to influence the cellular response to infection, including a secreted endopeptidase that induces expression of pro-inflammatory cytokines [6] and the PorB porin [7], such Neisseria-specific factors could not explain the effect of E. coli DH5α on 1G5 cells (Fig. 1c). The recent demonstration of Toll-like receptor 2 expression in murine T lymphocytes [8] suggests that bacterial lipoproteins may activate a Toll-like receptor 2-dependent signalling cascade that elevates HIV-LTR activity. However, as gonococci are also prone to lysis because of the expression of a growth phase-dependent autolysin [9], the possibility that the primary inducer of HIV-LTR-mediated expression may instead be protein(s) released upon bacterial autolysis cannot be ignored. Nuclear factor kappa B (NF-κB) is an immediate early transcription factor involved in the regulation of immunomodulatory genes in response to infection or other stresses [10]. The binding of NF-κB to the HIV-1 enhancer region (nucleotides −109 to −79; Fig. 1e) increases the expression of HIV-1 in T cells [11] and macrophage [12]. Although N. gonorrhoeae has been observed to increase NF-κB activity in epithelial and endothelial cells [13], whether it also influences NF-κB activity in immune cells remains unknown. Jurkat T cells expressing a reporter construct that contains tandem NF-κB binding sites upstream of the gene encoding chloramphenicol acetyltransferase were infected with Opa-expressing variants of N. gonorrhoeae. The induction of chloramphenicol acetyltransferase activity evident upon infection (Fig. 1e) indicates that gonococci can induce NF-κB-mediated transcription in T lymphocytes. To assess whether NF-κB is required for the gonococcal induction of HIV-LTR-driven expression, we tested the effect of N-acetyl-leucinyl-leucinyl-norleucinal, a proteasome inhibitor that prevents NF-κB activation [14–16]. Treatment with N-acetyl-leucinyl-leucinyl-norleucinal abrogated the response of 1G5 cells to both N. gonorrhoeae and the phorbol myristate acetate/phytohaemagglutinin control (Fig. 1f), consistent with the involvement of NF-κB in bacterial-induced luciferase expression. To our knowledge, this study represents the first clear association between gonococcal infection and HIV-LTR-dependent transcription. Such activity would probably impact viral transmission by increasing shedding from the mucosa of infected individuals. As higher HIV-1-RNA loads early in infection are predictive of faster progression to AIDS [17], bacterial co-infection may also influence the progression of disease immediately after exposure to virus. Such effects should interact with other manifestations of gonococcal infection to elevate HIV production in vivo. For example, CD4 T lymphocyte counts in the endocervix are elevated during gonococcal infection [18], potentially allowing increased viral replication at the primary site of transmission. Furthermore, the transient decline in the number of CD4 T cells [19] and HIV-1-specific CD8 T cells [20] coincident with gonococcal infection of HIV-positive patients, perhaps attributable to gonococcal Opa protein-mediated ligation of the CEACAM1 co-inhibitory receptor [21], suggests that HIV-specific immunity may be depressed. Ongoing studies aimed at dissecting the relative contribution of these and other aspects will further our understanding of the synergy that exists between these two important sexually transmitted pathogens.

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.