Effect of Deletion or Overexpression of the 19-Kilodalton Lipoprotein Rv3763 on the Innate Response to Mycobacterium tuberculosis
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感染与免疫杂志 2005年第10期
Center for Molecular Microbiology and Infection and Wellcome Trust Center for Research in Clinical Tropical Medicine, Imperial College London, London, United Kingdom
ABSTRACT
The 19-kDa lipoprotein of Mycobacterium tuberculosis is an important target of the innate immune response. To investigate the immune biology of this antigen in the context of the whole bacillus, we derived a recombinant M. tuberculosis H37Rv that lacked the 19-kDa-lipoprotein gene (19) and complemented this strain by reintroduction of the 19-kDa-lipoprotein gene on a multicopy vector to produce 19::pSMT181. The 19 strain multiplied less well than 19::pSMT181 in human monocyte-derived macrophages (MDM) (P = 0.039). Surface expression of major histocompatibility complex class II molecules was reduced in phagocytes infected with M. tuberculosis; this effect was not seen in cells infected with 19. 19 induced lower interleukin 1 (IL-1) secretion from monocytes and MDM. Overexpression of the 19-kDa protein increased IL-1, IL-12p40, and tumor necrosis factor alpha secretion irrespective of phagocyte maturity. These data support reports that the 19-kDa lipoprotein has pleiotropic effects on the interaction of M. tuberculosis with phagocytes. However, this analysis indicates that in the context of the whole bacillus, the 19-kDa lipoprotein is only one of a number of molecules that mediate the innate response to M. tuberculosis.
INTRODUCTION
The host response to infection is initiated by recognition of microbial components by signaling receptors on the surfaces of host cells. This results in generation of an innate immune response that includes direct antimicrobial function together with expression of molecules that shape the subsequent adaptive response (12). Although the recognition repertoire of innate immune receptors is limited in comparison to that of the adaptive immune system, the ability to combine multiple signals allows a degree of flexibility that is a crucial determinant both of the outcome of infection and of the effect of vaccination. Differences in the virulence of Mycobacterium tuberculosis isolates in a mouse model, for example, can be ascribed to differences in the very early response, even though their effect becomes physiologically apparent only at later stages of disease (21).
Mycobacteria induce a strong proinflammatory response, and considerable effort has been invested in fractionating bacteria to identify the individual components that trigger innate immune signaling. Potent stimuli include the nonprotein components of the mycobacterial cell wall, such as phosphatidylinositol mannosides, phenolic glycolipids, phthiocerol dimycoserate, lipoarabinomannan, sulfatides, and trehalose dimycolate (2, 4, 7, 8, 17, 18, 21, 24). These molecules contribute to host-pathogen interactions and exhibit immunomodulatory activities.
In addition to glycolipids, mycobacterial lipoproteins have also been implicated in the innate response. Attention has focused particularly on the 19-kDa lipoprotein of M. tuberculosis, which induces Toll-like receptor 2 (TLR2)-dependent production of interleukin-12 p40 (IL-12p40) when added to a macrophage cell line (3). Downstream signaling is dependent on myeloid differentiation factor 88 (MyD88) (9, 14, 16). Purified 19-kDa protein added to cultures of human monocytes or alveolar macrophages reduced the intracellular growth of M. tuberculosis during the first 48 h of infection by a mechanism that was dependent on TLR2 but independent of nitric oxide (25). Subsequent studies have suggested that such 19-kDa-lipoprotein-induced suppression of intracellular growth may involve apoptosis of infected cells (5, 14). By contrast, other reports suggest that the 19-kDa protein can contribute to pathogenesis. Disruption of the 19-kDa-lipoprotein gene was associated with moderately reduced bacterial survival in mice in a high-throughput mutagenesis screen (22); overexpression of the 19-kDa-lipoprotein gene in a recombinant strain of Mycobacterium vaccae resulted in suppression of IL-12p40 secretion by human macrophages (20); and the purified 19-kDa protein was found to downregulate the expression of major histocompatibility complex (MHC) molecules and to inhibit gamma interferon (IFN-) activation of phagocytes (9, 10, 16, 19, 26). As had been found with the protective effects associated with the 19-kDa protein, the latter effects were also dependent on TLR2/MyD88 signaling.
The studies described above suggest that innate immune signaling in response to mycobacterial infection involves recognition of multiple components and that at least some of these have pleiotropic effects. To explore how these multiple signals combine to determine the outcome of infection, it is important to complement experiments using the isolated molecules with experiments in which individual components are manipulated within the context of the whole bacterium. The effect of addition of purified 19-kDa lipoprotein to cells may differ from its effect during infection, for example, as a consequence of differences in local concentration, orientation, and exposure of different parts of the molecule or of copresentation with competing TLR2 agonists and other signaling ligands. We have addressed this issue in the present study, first by deleting the gene encoding the 19-kDa lipoprotein from M. tuberculosis and then by reintroducing multiple copies of the gene. We have analyzed the effects of 19-kDa-lipoprotein expression on growth of the bacteria in vitro and in macrophages and on the cytokine response of innate immune cells from human peripheral blood.
MATERIALS AND METHODS
Generation of mutant and complemented mutant strains of M. tuberculosis lacking the 19-kDa-lipoprotein gene (Rv3763). The 19-kDa-lipoprotein gene was deleted from M. tuberculosis H37Rv as described previously for its deletion from Mycobacterium bovis BCG (31). Briefly, a counterselectable suicide vector (p19KO) was constructed to deliver a hygromycin resistance gene (hyg) to replace the 19-kDa-lipoprotein gene. p19KO contained a pUC18 backbone along with the sacB gene, for counterselection against single-crossover integration, and the hyg gene flanked by two 2-kb regions of mycobacterial DNA from upstream and downstream of the 19-kDa-lipoprotein gene. Approximately 2 μg of plasmid was UV irradiated and electroporated into M. tuberculosis. Gene replacement transformants (19) were selected on 50 μg/ml hygromycin and 2% sucrose and were confirmed by Southern blotting. The 19-kDa-lipoprotein gene was reintroduced into 19 on a multicopy mycobacterial/Escherichia coli shuttle vector, pSMT181. This plasmid carries a pUC19 backbone with a pAL5000-based mycobacterial origin of replication, the kanamycin resistance gene from Tn903, and the 19-kDa-lipoprotein open reading frame preceded by 200 bp of upstream untranslated sequence.
Isolation and culture of monocytes, MDM, and DC. Buffy coats from healthy donors were obtained from the National Blood Transfusion Service (Colindale, London, United Kingdom). Following dilution in RPMI medium (1/3, vol/vol), peripheral blood mononuclear cells were separated by centrifugation over Ficoll-Paque Plus (Pharmacia, Uppsala, Sweden). Cells were washed in RPMI medium, pooled, and counted. Cells were suspended at 1.2 x 107/ml in RPMI medium-10% fetal calf serum (R10 medium), and aliquots of 25 ml were added to 150-cm2 tissue culture flasks. Flasks were placed flat in a 5% CO2 incubator, and monocytes were allowed to adhere for 2 h at 37°C. Nonadherent cells were removed by three washes with 10 ml of prewarmed RPMI medium. Finally, 10 ml of ice-cold phosphate-buffered saline was added, and the flasks were incubated at 4°C for 20 min. By using a scraper, monocytes were gently dislodged from the bottoms of the flasks and pooled in a 50-ml Falcon tube for counting. For experiments requiring monocytes, cells were plated in RPMI medium containing 10% serum at 106/well in a 24-well tissue culture plate and were cultured overnight before infection. Monocyte-derived macrophages (MDM) were derived from monocytes by culture for 7 days in RPMI medium-10% serum in the absence of antigen. Dendritic cells (DC) were derived from monocytes by culture in X-VIVO 15 (Biowhittaker, Walkersville, MD) for 6 days in the presence of 50 ng/ml granulocyte-macrophage colony-stimulating factor (PeproTech EC Ltd., London, United Kingdom) and 1,000 U/ml IL-4 (PeproTech). Fresh cytokines were added to the cultures on day 3. In some experiments, MDM were activated for 16 h prior to, and during, infection with 1 ng/ml recombinant IFN- (Imukin; Boehringer-Ingelheim). The following antibodies (and appropriate isotype controls) were used to characterize the various preparations of mononuclear cells using a BD FACSCalibur flow cytometer and CellQuest software for analysis. phycoerythrin (PE)-conjugated anti- HLA-DR, fluorescein isothiocyanate (FITC)-conjugated CD11b, FITC-conjugated CD86, and a PE-conjugated mouse immunoglobulin G1 (IgG1) isotype control (Serotec Ltd., Kidlington, United Kingdom); FITC-conjugated TLR-2 (clone TL2.1) and mouse IgG2a (clone MOPC-173) from eBioscience (San Diego, CA); PE-conjugated CD80, FITC-conjugated CD83, PE-conjugated CD14, and an FITC-conjugated mouse IgG1 isotype control from BD Biosciences (San Jose, CA). Maturation of monocytes to MDM was associated with a fall in the percentage of cells staining positive for CD11b (from 76% ± 3.7% to 57% ± 7%) and TLR2 expression (from 39% ± 8.2% to 8% ± 2%). Maturation to DC markedly decreased the percentage of cells staining positive for CD14 (from 87% ± 7% to 7% ± 0.9%) and increased the numbers of CD80-positive cells from 4% ± 1.4% to 57% ± 6.3%.
Infection of cells and CFU analysis. Bacilli used to infect cells were grown in Middlebrook 7H9 broth supplemented with albumin-dextrose-catalase to mid-log phase (optical density [OD], 0.4 to 0.8) and then frozen in aliquots in 15% glycerol. The CFU content of aliquots was determined by serial dilution and plating on Middlebrook 7H11 agar supplemented with oleic acid-albumin-dextrose-catalase. To analyze the growth of strains within cells, duplicate wells of a 24-well plate containing 106 phagocytes/well in R10 medium were infected at a multiplicity of infection of 1. After 2 h, nonphagocytosed bacilli were removed by three washes in prewarmed RPMI medium. The medium was then replaced, and cells were cultured for 0, 72, or 144 h. The antibiotics for plasmid selection (hygromycin and kanamycin) were not used in cell culture. To determine CFU, the supernatant was harvested and cells were lysed for 15 min in sterile H2O with additional disruption by pipetting. Lysates were serially diluted in H2O, and three consecutive dilutions were plated on 7H11 medium. The plates were then incubated for 17 to 21 days at 37°C. At the end of this culture period, CFU was enumerated for at least two consecutive dilutions and averaged. We have previously determined that extracellular growth of mycobacteria, as assessed by culture of the supernatants, is consistently >1 log unit lower than intracellular growth (29). To measure cytokine secretion, cells were cocultured with bacilli at a 1:1 ratio in RPMI-10% serum. The duration of culture was 72 h, at which time supernatants were aspirated, filtered (pore size, 0.22 μm), and stored at –80°C pending analysis by enzyme-linked immunosorbent assay (ELISA). To obtain an in vitro growth curve, aliquots of bacilli were defrosted and diluted in 7H9 medium to an OD at 600 nm (OD600) of 0.05. The ODs of cultures were monitored periodically thereafter.
RNA extraction and quantitative RT-PCR for M. tuberculosis genes. M. tuberculosis RNA was extracted from infected cells exactly as described previously (28). We used quantitative real-time reverse transcription-PCR (RT-PCR) with internal fluorescent hybridization probes in an ABI (Foster City, CA) Prism 7000 sequence detection system. Specific RT and PCR primers and a probe for the 19-kDa-lipoprotein gene were designed using Primer Express software (ABI). These sequences were as follows: RT primer (–477RT), 5'-GGAACAGGTCACCT-3'; forward primer (299F), 5'-TTGGGCTCGGTAACGTCAAC-3'; reverse primer (409R), 5'-TAGCGGTCCCAGTGATCTTGTAG-3'; probe (344T), 5'-FAM-CACCGGACAGGGTAACGCCTCG-3'-TAMRA. Sequences used to detect M. tuberculosis 16S RNA were as previously published (6). Probes were dually labeled with 5-carboxyfluorescein (FAM) at the 5' end and N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA) at the 3' end. Multiplex reverse transcription was performed for 1 h at 37°C using 1 μM of each specific RT primer and 0.25 U/μl of avian myeloblastosis virus reverse transcriptase (ABgene, Epsom, Surrey, United Kingdom) in the supplier's reaction buffer in the presence of 500 μM of each deoxynucleoside triphosphate. For PCR, 5 μl of each cDNA was assayed in a total reaction volume of 25 μl containing TaqMan Universal PCR master mix (Applied Biosystems). For 16S RNA, the probe was used at 100 nM and the primers at 250 nM; for the 19-kDa-lipoprotein gene, the probe was used at 100 nM, the forward primer at 300 nM, and the reverse primer at 50 nM. Reaction conditions consisted of 1 cycle of 50°C for 2 min and 1 cycle of 95°C for 10 min, then 40 cycles of 95°C for 15 s followed by annealing and elongation at 65°C (16S RNA) or 60°C (19-kDa-lipoprotein gene) for 1 min. The cycle threshold (CT) for each sample was compared with CT values of known amounts of a standard DNA from M. tuberculosis. These standards also allowed us to estimate PCR efficiency over a wide range of template concentrations (1 to 106 copies/μl). The efficiency of each reaction was similar in all assays. To ensure lack of DNA contamination of the RNA samples, a duplicate tube of sample with no reverse transcriptase enzyme was included as a control. Results are expressed as values normalized to the 16S rRNA content.
Cytokine ELISA. Cytokine ELISA was performed using the commercially available DuoSet ELISA Development Systems (R&D Systems, Minneapolis, MN) according to the manufacturer's recommendations. The sensitivity of the assays was 15 pg/ml for IL-12p40, 10 pg/ml for IL-1, and 50 pg/ml for tumor necrosis factor alpha (TNF-).
Statistical methods. Paired and unpaired parametric variables were compared by Student's t test. Paired and unpaired nonparametric variables were compared by the Wilcoxon signed-rank test or the Mann-Whitney U test, respectively. Significance was inferred for P values of 0.05.
RESULTS
Characterization of M. tuberculosis 19 and 19::pSMT181. PCR analysis confirmed the presence of the Rv3763 gene in M. tuberculosis and 19::pSMT181 and its absence from 19 (Fig. 1A). Western blotting of protein from cell pellets showed that production of the 19-kDa protein was moderately higher in 19::pSMT181 than in wild-type M. tuberculosis (Fig. 1B). Similar results were also obtained from culture supernatants, and densitometric analysis of evenly loaded Western blots from broth cultures indicated that the protein level was at least 1.4 times higher in the complemented strain in repeated experiments.
Effect of 19-kDa-lipoprotein expression on mycobacterial growth. To screen for a possible role for the 19-kDa lipoprotein in mycobacterial physiology, we compared the growth rate of wild-type M. tuberculosis to that of the 19-kDa knockout and complemented strains in 7H9 Middlebrook medium. We were unable to detect any difference either in doubling time or in the level of the stationary-phase plateau (Fig. 2A). The lack of an essential growth function in laboratory media is consistent with saturation transposon mutagenesis studies (22) and with the fact that the 19-kDa-lipoprotein gene has been spontaneously deleted from some clinical isolates of M. tuberculosis (13, 27). We next compared the growth of the different strains in IFN--activated MDM from four healthy donors infected at a 1:1 ratio. The growth of the 19 strain was reduced in comparison to the growth of wild-type M. tuberculosis and the complemented 19::pSMT181 strain; the difference between knockout and complemented strains attained significance (P = 0.039) after 7 days of infection (Fig. 2B). By contrast, there was no difference in growth or survival of any of the strains over a 1-week incubation in monocyte cultures (Fig. 2C). A twofold difference was observed in the uptake of the complemented strain by monocytes, but this was not statistically significant.
Expression of the 19-kDa-lipoprotein gene within cells. Inside murine macrophages, expression of the 19-kDa-lipoprotein gene decreases to approximately one-fifth of its extracellular level (23). To test for a similar effect in human cells, peripheral blood monocytes or monocyte-derived macrophages were infected with M. tuberculosis at a 1:1 ratio, and 19-kDa-lipoprotein RNA was measured relative to 16S rRNA over 4 days. By comparison with bacilli grown extracellularly, there was a very slight increase (barely twofold; not significant) in 19-kDa-lipoprotein RNA expression 1 h after infection of monocytes. By 10 h, 19-kDa-lipoprotein RNA expression had dropped back to the starting level and remained constant up to 96 h. In bacteria incubated in RPMI medium in the absence of cells, 19-kDa-lipoprotein gene expression increased slightly, but not significantly, over the course of the experiment (Fig. 3A). Thus, the 19-kDa-lipoprotein gene is appreciably expressed within cells but, by comparison with bacilli grown in RPMI medium without cells, not specifically upregulated during the first 96 h of infection. Using cells from a single donor, we also tested the intracellular expression of the 19-kDa-lipoprotein gene by the recombinant strains at 6 h. No expression of the 19-kDa-lipoprotein gene was found in cells infected with the 19 strain (Fig. 3B), consistent with our PCR and Western blot analysis (Fig. 1). Approximately five times as much 19-kDa-lipoprotein RNA was detected in cells infected by the complemented 19::pSMT181 strain as in cells infected by wild-type H37Rv (Fig. 3B), again consistent with the increase in the protein level that we had observed (Fig. 1).
Effect of 19-kDa-lipoprotein expression on cytokine induction. The 19-kDa lipoprotein was initially identified as a mediator of innate immune signaling on the basis of its ability to induce the secretion of IL-12p40 by a human monocytic cell line (3). To test whether the protein had an analogous effect when presented as part of the whole mycobacterium, we infected mononuclear phagocytes at a ratio of 1 bacterium per cell and determined the secretion of cytokines. Preliminary experiments indicated that the effect of deletion of the 19-kDa-lipoprotein gene on cytokine secretion differed within the same donor depending on the maturity of the mononuclear phagocytes. We undertook a systematic analysis of cytokine secretion for 10 donors by using innate immune cells that had been allowed to mature under different in vitro culture conditions: there were not always sufficient cells to investigate all cell phenotypes for all donors. In monocytes, there was no difference in IL-12p40 secretion between cells infected with the wild-type and those infected with the 19 strain, but a significant elevation in cytokine secretion was observed with the complemented strain (Fig. 4A). M. tuberculosis infection induced different levels of IL-12p40 in the different cell types, with DC producing the largest amounts (Fig. 4C), but the pattern of response to different strains was similar in each case. There was a tendency toward a decrease in IL-12p40 production in response to the 19 strain (this reached statistical significance in the case of the MDM cultures) and a significant increase in IL-12p40 induction by the strain overexpressing the 19-kDa-lipoprotein in all cell types (Fig. 4A to C).
All of the strains elicited similar secretion of TNF- from infected monocytes, although, again, there was a significant increase in induction by the 19::pSMT181 strain in MDM and DC (Fig. 4D to F). Production of IL-1 in response to M. tuberculosis was highest for cells with a high level of TLR2 expression (monocytes and MDM) and undetectable for DC (Fig. 4G and H). IL-1 levels were significantly reduced in monocyte and MDM cultures infected with 19 (P = 0.01 and 0.015, respectively). The strain overexpressing the 19-kDa lipoprotein elicited significantly larger amounts of IL-1 than wild-type M. tuberculosis in MDM but not in monocytes.
Effect of the 19-kDa protein on surface expression of HLA-DR. Purified 19-kDa protein has been reported to decrease MHC class II expression and thereby antigen presentation to T cells, an effect that would favor bacillary survival (10, 16, 19). We determined the effect of deleting the 19-kDa-lipoprotein gene on MHC class II expression in eight donors. Monocytes from six donors were isolated by adherence, and the presence and intensity of HLA-DR staining were determined after 48 h of coculture at 1 bacillus per cell. Infection of monocytes with M. tuberculosis led to a modest but significant decrease in the numbers of HLA-DR-positive cells (P = 0.016) (Fig. 5). The mean fluorescence intensity (MFI) of staining on HLA-DR-positive cells showed a similar decrement (from 554 ± 76 to 454 ± 46). These effects were not observed with the 19 strain, which, if anything, tended to increase HLA-DR expression (MFI, 652 ± 33; percent positive cells, 88% ± 1.5%; both effects were significant by comparison with wild-type M. tuberculosis [P = 0.008 and 0.034, respectively]). The complemented 19::pSMT181 strain displayed an intermediate phenotype and did not differ significantly from either the wild type or 19. For two further donors, HLA-DR expression in IFN--activated MDM was similarly investigated. Infection with wild-type M. tuberculosis led to a similar decrease in the percentage of positive cells, from 98 to 84.3%; the values for 19- and 19::pSMT181-infected cells were 90.8 and 88.3%, respectively.
DISCUSSION
The 19-kDa lipoprotein of M. tuberculosis has been extensively studied in terms of its ability to trigger responses in innate immune cells and to act as an antigen for cellular and humoral arms of the adaptive response (1, 15, 30). Its physiological function in M. tuberculosis itself is unknown. Although conservation of related genes throughout the mycobacterial genus suggests some essential function outside of any specialized role in infection, the absence of any growth defect in Middlebrook media suggests that this function is dispensable for growth in the laboratory. The reduced growth of the deletion strain in IFN--activated MDM but not monocyte cultures may, however, reflect a physiological requirement under the growth conditions encountered within an intact animal. Consistent with these in vitro results, disruption of the 19-kDa-lipoprotein gene has been shown to moderately reduce the growth of M. tuberculosis in a murine model (22).
A robust increase in IL-12p40 secretion was seen in all cell types following infection with a strain engineered to express the protein at a level higher than that of the wild type. While these results are consistent with the initial description of the 19-kDa lipoprotein as an important early trigger for IL-12p40 production (3), it is clearly not the sole, nor perhaps the dominant, component of M. tuberculosis responsible for this activity. Even when consideration is restricted to the 118 putative or confirmed lipoproteins of M. tuberculosis, there are others, such as PstS1 and LprG, with known effects on the innate response (3, 11). Our data suggest that the strongest response occurs when the concentration of the 19-kDa ligand rises above that of the physiological level encountered during wild-type infection. It has been reported previously that overexpression of the 19-kDa lipoprotein in rapidly growing mycobacteria caused downregulation of IL-12p40 secretion (20); it is possible that the effect of 19-kDa-lipoprotein signaling is dependent on the pattern of parallel signaling interactions involving other ligands that differ between pathogenic and nonpathogenic species.
The effect of 19-kDa-lipoprotein deletion on cytokine secretion was most marked for IL-1. As with IL-12p40, there was evidence of increased IL-1 secretion from MDM infected with the complemented strain compared to the wild type. Ciaramella et al. also found that the dominant effect of the 19-kDa lipoprotein expressed by intact mycobacteria was evident in the IL-1 response (5). These results suggest a differential role for particular mycobacterial ligands in induction of particular cytokines. The 19-kDa lipoprotein seems to have a dominant role in the induction of IL-1, particularly in the context of high levels of TLR2 expression, and a less pronounced role in IL-12p40 production. We observed no significant effect of 19-kDa-lipoprotein gene deletion on TNF-, although there was evidence of increased TNF- secretion by MDM and DC infected with the overexpressing 19::pSMT181 strain.
Monocytes infected with the 19 strain were characterized by a higher level of surface expression of MHC class II protein than those infected with wild-type M. tuberculosis. This result is consistent with previous reports that the purified 19-kDa protein is able to suppress MHC class II expression (9, 16). The decrease that we observed in the number of monocytes staining positive for MHC class II in cultures infected with M. tuberculosis (13% over 48 h) was relatively modest. This may be due to the use of a low multiplicity of infection (to avoid excessive cytopathic effects), which resulted in only around 10% of the cells being infected. The suppressive effect of M. tuberculosis was completely reversed following deletion of the 19-kDa-lipoprotein gene and was partially restored by reintroduction of that gene in 19::pSMT181. We suggest that the intermediate phenotype of the complemented strain with respect to MHC class II expression arose as a consequence of the increased proinflammatory response, which would tend to increase MHC class II expression, countered by the tendency of the 19-kDa lipoprotein to decrease it. Our data will also tend to reflect the accumulation of protein in the culture supernatant, which may not necessarily reflect the true state of the cells at later time points.
Our results from deletion and addition of the 19-kDa protein in the context of infection with whole M. tuberculosis demonstrate a range of effects that can be viewed as beneficial either to the host or to the pathogen. On the one hand, induction of IL-1 (resulting in recruitment of antimicrobial effector cells) and induction of IL-12p40 (priming a macrophage-activating Th1 response) are both associated with a protective immune response. Conversely, increased bacterial growth in MDM and suppression of antigen-presenting function would seem to favor progression of infection. At a teleological level, it can be argued that, since the vast majority of clinical isolates have maintained the 19-kDa-lipoprotein gene, the balance of these effects falls in favor of the pathogen. This is indeed the case in the mouse model of tuberculosis, in which disruption or deletion of the 19-kDa-lipoprotein gene reduces the level of infection (22; G. R. Stewart and A. S. Apt, unpublished data). As we progress in our understanding of the complex signaling events associated with the innate immune response to M. tuberculosis infection, the ability to dissect positive and negative effects, via the analysis of immune responses to bacilli with defined mutations, could open important new avenues for vaccine development.
ACKNOWLEDGMENTS
This work was supported by the Wellcome Trust (grants 064261, 060079, and 038997).
G.R.S. and K.A.W. contributed equally to this work.
Present address: School of Biological and Medical Sciences, University of Surrey, Guildford, United Kingdom.
Present address: Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, South Africa.
Present address: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109.
|| Present address: Unite de Genetique Mycobacterienne, Institut Pasteur, Paris, France.
# Present address: University of Warwick Medical School, Coventry, United Kingdom.
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ABSTRACT
The 19-kDa lipoprotein of Mycobacterium tuberculosis is an important target of the innate immune response. To investigate the immune biology of this antigen in the context of the whole bacillus, we derived a recombinant M. tuberculosis H37Rv that lacked the 19-kDa-lipoprotein gene (19) and complemented this strain by reintroduction of the 19-kDa-lipoprotein gene on a multicopy vector to produce 19::pSMT181. The 19 strain multiplied less well than 19::pSMT181 in human monocyte-derived macrophages (MDM) (P = 0.039). Surface expression of major histocompatibility complex class II molecules was reduced in phagocytes infected with M. tuberculosis; this effect was not seen in cells infected with 19. 19 induced lower interleukin 1 (IL-1) secretion from monocytes and MDM. Overexpression of the 19-kDa protein increased IL-1, IL-12p40, and tumor necrosis factor alpha secretion irrespective of phagocyte maturity. These data support reports that the 19-kDa lipoprotein has pleiotropic effects on the interaction of M. tuberculosis with phagocytes. However, this analysis indicates that in the context of the whole bacillus, the 19-kDa lipoprotein is only one of a number of molecules that mediate the innate response to M. tuberculosis.
INTRODUCTION
The host response to infection is initiated by recognition of microbial components by signaling receptors on the surfaces of host cells. This results in generation of an innate immune response that includes direct antimicrobial function together with expression of molecules that shape the subsequent adaptive response (12). Although the recognition repertoire of innate immune receptors is limited in comparison to that of the adaptive immune system, the ability to combine multiple signals allows a degree of flexibility that is a crucial determinant both of the outcome of infection and of the effect of vaccination. Differences in the virulence of Mycobacterium tuberculosis isolates in a mouse model, for example, can be ascribed to differences in the very early response, even though their effect becomes physiologically apparent only at later stages of disease (21).
Mycobacteria induce a strong proinflammatory response, and considerable effort has been invested in fractionating bacteria to identify the individual components that trigger innate immune signaling. Potent stimuli include the nonprotein components of the mycobacterial cell wall, such as phosphatidylinositol mannosides, phenolic glycolipids, phthiocerol dimycoserate, lipoarabinomannan, sulfatides, and trehalose dimycolate (2, 4, 7, 8, 17, 18, 21, 24). These molecules contribute to host-pathogen interactions and exhibit immunomodulatory activities.
In addition to glycolipids, mycobacterial lipoproteins have also been implicated in the innate response. Attention has focused particularly on the 19-kDa lipoprotein of M. tuberculosis, which induces Toll-like receptor 2 (TLR2)-dependent production of interleukin-12 p40 (IL-12p40) when added to a macrophage cell line (3). Downstream signaling is dependent on myeloid differentiation factor 88 (MyD88) (9, 14, 16). Purified 19-kDa protein added to cultures of human monocytes or alveolar macrophages reduced the intracellular growth of M. tuberculosis during the first 48 h of infection by a mechanism that was dependent on TLR2 but independent of nitric oxide (25). Subsequent studies have suggested that such 19-kDa-lipoprotein-induced suppression of intracellular growth may involve apoptosis of infected cells (5, 14). By contrast, other reports suggest that the 19-kDa protein can contribute to pathogenesis. Disruption of the 19-kDa-lipoprotein gene was associated with moderately reduced bacterial survival in mice in a high-throughput mutagenesis screen (22); overexpression of the 19-kDa-lipoprotein gene in a recombinant strain of Mycobacterium vaccae resulted in suppression of IL-12p40 secretion by human macrophages (20); and the purified 19-kDa protein was found to downregulate the expression of major histocompatibility complex (MHC) molecules and to inhibit gamma interferon (IFN-) activation of phagocytes (9, 10, 16, 19, 26). As had been found with the protective effects associated with the 19-kDa protein, the latter effects were also dependent on TLR2/MyD88 signaling.
The studies described above suggest that innate immune signaling in response to mycobacterial infection involves recognition of multiple components and that at least some of these have pleiotropic effects. To explore how these multiple signals combine to determine the outcome of infection, it is important to complement experiments using the isolated molecules with experiments in which individual components are manipulated within the context of the whole bacterium. The effect of addition of purified 19-kDa lipoprotein to cells may differ from its effect during infection, for example, as a consequence of differences in local concentration, orientation, and exposure of different parts of the molecule or of copresentation with competing TLR2 agonists and other signaling ligands. We have addressed this issue in the present study, first by deleting the gene encoding the 19-kDa lipoprotein from M. tuberculosis and then by reintroducing multiple copies of the gene. We have analyzed the effects of 19-kDa-lipoprotein expression on growth of the bacteria in vitro and in macrophages and on the cytokine response of innate immune cells from human peripheral blood.
MATERIALS AND METHODS
Generation of mutant and complemented mutant strains of M. tuberculosis lacking the 19-kDa-lipoprotein gene (Rv3763). The 19-kDa-lipoprotein gene was deleted from M. tuberculosis H37Rv as described previously for its deletion from Mycobacterium bovis BCG (31). Briefly, a counterselectable suicide vector (p19KO) was constructed to deliver a hygromycin resistance gene (hyg) to replace the 19-kDa-lipoprotein gene. p19KO contained a pUC18 backbone along with the sacB gene, for counterselection against single-crossover integration, and the hyg gene flanked by two 2-kb regions of mycobacterial DNA from upstream and downstream of the 19-kDa-lipoprotein gene. Approximately 2 μg of plasmid was UV irradiated and electroporated into M. tuberculosis. Gene replacement transformants (19) were selected on 50 μg/ml hygromycin and 2% sucrose and were confirmed by Southern blotting. The 19-kDa-lipoprotein gene was reintroduced into 19 on a multicopy mycobacterial/Escherichia coli shuttle vector, pSMT181. This plasmid carries a pUC19 backbone with a pAL5000-based mycobacterial origin of replication, the kanamycin resistance gene from Tn903, and the 19-kDa-lipoprotein open reading frame preceded by 200 bp of upstream untranslated sequence.
Isolation and culture of monocytes, MDM, and DC. Buffy coats from healthy donors were obtained from the National Blood Transfusion Service (Colindale, London, United Kingdom). Following dilution in RPMI medium (1/3, vol/vol), peripheral blood mononuclear cells were separated by centrifugation over Ficoll-Paque Plus (Pharmacia, Uppsala, Sweden). Cells were washed in RPMI medium, pooled, and counted. Cells were suspended at 1.2 x 107/ml in RPMI medium-10% fetal calf serum (R10 medium), and aliquots of 25 ml were added to 150-cm2 tissue culture flasks. Flasks were placed flat in a 5% CO2 incubator, and monocytes were allowed to adhere for 2 h at 37°C. Nonadherent cells were removed by three washes with 10 ml of prewarmed RPMI medium. Finally, 10 ml of ice-cold phosphate-buffered saline was added, and the flasks were incubated at 4°C for 20 min. By using a scraper, monocytes were gently dislodged from the bottoms of the flasks and pooled in a 50-ml Falcon tube for counting. For experiments requiring monocytes, cells were plated in RPMI medium containing 10% serum at 106/well in a 24-well tissue culture plate and were cultured overnight before infection. Monocyte-derived macrophages (MDM) were derived from monocytes by culture for 7 days in RPMI medium-10% serum in the absence of antigen. Dendritic cells (DC) were derived from monocytes by culture in X-VIVO 15 (Biowhittaker, Walkersville, MD) for 6 days in the presence of 50 ng/ml granulocyte-macrophage colony-stimulating factor (PeproTech EC Ltd., London, United Kingdom) and 1,000 U/ml IL-4 (PeproTech). Fresh cytokines were added to the cultures on day 3. In some experiments, MDM were activated for 16 h prior to, and during, infection with 1 ng/ml recombinant IFN- (Imukin; Boehringer-Ingelheim). The following antibodies (and appropriate isotype controls) were used to characterize the various preparations of mononuclear cells using a BD FACSCalibur flow cytometer and CellQuest software for analysis. phycoerythrin (PE)-conjugated anti- HLA-DR, fluorescein isothiocyanate (FITC)-conjugated CD11b, FITC-conjugated CD86, and a PE-conjugated mouse immunoglobulin G1 (IgG1) isotype control (Serotec Ltd., Kidlington, United Kingdom); FITC-conjugated TLR-2 (clone TL2.1) and mouse IgG2a (clone MOPC-173) from eBioscience (San Diego, CA); PE-conjugated CD80, FITC-conjugated CD83, PE-conjugated CD14, and an FITC-conjugated mouse IgG1 isotype control from BD Biosciences (San Jose, CA). Maturation of monocytes to MDM was associated with a fall in the percentage of cells staining positive for CD11b (from 76% ± 3.7% to 57% ± 7%) and TLR2 expression (from 39% ± 8.2% to 8% ± 2%). Maturation to DC markedly decreased the percentage of cells staining positive for CD14 (from 87% ± 7% to 7% ± 0.9%) and increased the numbers of CD80-positive cells from 4% ± 1.4% to 57% ± 6.3%.
Infection of cells and CFU analysis. Bacilli used to infect cells were grown in Middlebrook 7H9 broth supplemented with albumin-dextrose-catalase to mid-log phase (optical density [OD], 0.4 to 0.8) and then frozen in aliquots in 15% glycerol. The CFU content of aliquots was determined by serial dilution and plating on Middlebrook 7H11 agar supplemented with oleic acid-albumin-dextrose-catalase. To analyze the growth of strains within cells, duplicate wells of a 24-well plate containing 106 phagocytes/well in R10 medium were infected at a multiplicity of infection of 1. After 2 h, nonphagocytosed bacilli were removed by three washes in prewarmed RPMI medium. The medium was then replaced, and cells were cultured for 0, 72, or 144 h. The antibiotics for plasmid selection (hygromycin and kanamycin) were not used in cell culture. To determine CFU, the supernatant was harvested and cells were lysed for 15 min in sterile H2O with additional disruption by pipetting. Lysates were serially diluted in H2O, and three consecutive dilutions were plated on 7H11 medium. The plates were then incubated for 17 to 21 days at 37°C. At the end of this culture period, CFU was enumerated for at least two consecutive dilutions and averaged. We have previously determined that extracellular growth of mycobacteria, as assessed by culture of the supernatants, is consistently >1 log unit lower than intracellular growth (29). To measure cytokine secretion, cells were cocultured with bacilli at a 1:1 ratio in RPMI-10% serum. The duration of culture was 72 h, at which time supernatants were aspirated, filtered (pore size, 0.22 μm), and stored at –80°C pending analysis by enzyme-linked immunosorbent assay (ELISA). To obtain an in vitro growth curve, aliquots of bacilli were defrosted and diluted in 7H9 medium to an OD at 600 nm (OD600) of 0.05. The ODs of cultures were monitored periodically thereafter.
RNA extraction and quantitative RT-PCR for M. tuberculosis genes. M. tuberculosis RNA was extracted from infected cells exactly as described previously (28). We used quantitative real-time reverse transcription-PCR (RT-PCR) with internal fluorescent hybridization probes in an ABI (Foster City, CA) Prism 7000 sequence detection system. Specific RT and PCR primers and a probe for the 19-kDa-lipoprotein gene were designed using Primer Express software (ABI). These sequences were as follows: RT primer (–477RT), 5'-GGAACAGGTCACCT-3'; forward primer (299F), 5'-TTGGGCTCGGTAACGTCAAC-3'; reverse primer (409R), 5'-TAGCGGTCCCAGTGATCTTGTAG-3'; probe (344T), 5'-FAM-CACCGGACAGGGTAACGCCTCG-3'-TAMRA. Sequences used to detect M. tuberculosis 16S RNA were as previously published (6). Probes were dually labeled with 5-carboxyfluorescein (FAM) at the 5' end and N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA) at the 3' end. Multiplex reverse transcription was performed for 1 h at 37°C using 1 μM of each specific RT primer and 0.25 U/μl of avian myeloblastosis virus reverse transcriptase (ABgene, Epsom, Surrey, United Kingdom) in the supplier's reaction buffer in the presence of 500 μM of each deoxynucleoside triphosphate. For PCR, 5 μl of each cDNA was assayed in a total reaction volume of 25 μl containing TaqMan Universal PCR master mix (Applied Biosystems). For 16S RNA, the probe was used at 100 nM and the primers at 250 nM; for the 19-kDa-lipoprotein gene, the probe was used at 100 nM, the forward primer at 300 nM, and the reverse primer at 50 nM. Reaction conditions consisted of 1 cycle of 50°C for 2 min and 1 cycle of 95°C for 10 min, then 40 cycles of 95°C for 15 s followed by annealing and elongation at 65°C (16S RNA) or 60°C (19-kDa-lipoprotein gene) for 1 min. The cycle threshold (CT) for each sample was compared with CT values of known amounts of a standard DNA from M. tuberculosis. These standards also allowed us to estimate PCR efficiency over a wide range of template concentrations (1 to 106 copies/μl). The efficiency of each reaction was similar in all assays. To ensure lack of DNA contamination of the RNA samples, a duplicate tube of sample with no reverse transcriptase enzyme was included as a control. Results are expressed as values normalized to the 16S rRNA content.
Cytokine ELISA. Cytokine ELISA was performed using the commercially available DuoSet ELISA Development Systems (R&D Systems, Minneapolis, MN) according to the manufacturer's recommendations. The sensitivity of the assays was 15 pg/ml for IL-12p40, 10 pg/ml for IL-1, and 50 pg/ml for tumor necrosis factor alpha (TNF-).
Statistical methods. Paired and unpaired parametric variables were compared by Student's t test. Paired and unpaired nonparametric variables were compared by the Wilcoxon signed-rank test or the Mann-Whitney U test, respectively. Significance was inferred for P values of 0.05.
RESULTS
Characterization of M. tuberculosis 19 and 19::pSMT181. PCR analysis confirmed the presence of the Rv3763 gene in M. tuberculosis and 19::pSMT181 and its absence from 19 (Fig. 1A). Western blotting of protein from cell pellets showed that production of the 19-kDa protein was moderately higher in 19::pSMT181 than in wild-type M. tuberculosis (Fig. 1B). Similar results were also obtained from culture supernatants, and densitometric analysis of evenly loaded Western blots from broth cultures indicated that the protein level was at least 1.4 times higher in the complemented strain in repeated experiments.
Effect of 19-kDa-lipoprotein expression on mycobacterial growth. To screen for a possible role for the 19-kDa lipoprotein in mycobacterial physiology, we compared the growth rate of wild-type M. tuberculosis to that of the 19-kDa knockout and complemented strains in 7H9 Middlebrook medium. We were unable to detect any difference either in doubling time or in the level of the stationary-phase plateau (Fig. 2A). The lack of an essential growth function in laboratory media is consistent with saturation transposon mutagenesis studies (22) and with the fact that the 19-kDa-lipoprotein gene has been spontaneously deleted from some clinical isolates of M. tuberculosis (13, 27). We next compared the growth of the different strains in IFN--activated MDM from four healthy donors infected at a 1:1 ratio. The growth of the 19 strain was reduced in comparison to the growth of wild-type M. tuberculosis and the complemented 19::pSMT181 strain; the difference between knockout and complemented strains attained significance (P = 0.039) after 7 days of infection (Fig. 2B). By contrast, there was no difference in growth or survival of any of the strains over a 1-week incubation in monocyte cultures (Fig. 2C). A twofold difference was observed in the uptake of the complemented strain by monocytes, but this was not statistically significant.
Expression of the 19-kDa-lipoprotein gene within cells. Inside murine macrophages, expression of the 19-kDa-lipoprotein gene decreases to approximately one-fifth of its extracellular level (23). To test for a similar effect in human cells, peripheral blood monocytes or monocyte-derived macrophages were infected with M. tuberculosis at a 1:1 ratio, and 19-kDa-lipoprotein RNA was measured relative to 16S rRNA over 4 days. By comparison with bacilli grown extracellularly, there was a very slight increase (barely twofold; not significant) in 19-kDa-lipoprotein RNA expression 1 h after infection of monocytes. By 10 h, 19-kDa-lipoprotein RNA expression had dropped back to the starting level and remained constant up to 96 h. In bacteria incubated in RPMI medium in the absence of cells, 19-kDa-lipoprotein gene expression increased slightly, but not significantly, over the course of the experiment (Fig. 3A). Thus, the 19-kDa-lipoprotein gene is appreciably expressed within cells but, by comparison with bacilli grown in RPMI medium without cells, not specifically upregulated during the first 96 h of infection. Using cells from a single donor, we also tested the intracellular expression of the 19-kDa-lipoprotein gene by the recombinant strains at 6 h. No expression of the 19-kDa-lipoprotein gene was found in cells infected with the 19 strain (Fig. 3B), consistent with our PCR and Western blot analysis (Fig. 1). Approximately five times as much 19-kDa-lipoprotein RNA was detected in cells infected by the complemented 19::pSMT181 strain as in cells infected by wild-type H37Rv (Fig. 3B), again consistent with the increase in the protein level that we had observed (Fig. 1).
Effect of 19-kDa-lipoprotein expression on cytokine induction. The 19-kDa lipoprotein was initially identified as a mediator of innate immune signaling on the basis of its ability to induce the secretion of IL-12p40 by a human monocytic cell line (3). To test whether the protein had an analogous effect when presented as part of the whole mycobacterium, we infected mononuclear phagocytes at a ratio of 1 bacterium per cell and determined the secretion of cytokines. Preliminary experiments indicated that the effect of deletion of the 19-kDa-lipoprotein gene on cytokine secretion differed within the same donor depending on the maturity of the mononuclear phagocytes. We undertook a systematic analysis of cytokine secretion for 10 donors by using innate immune cells that had been allowed to mature under different in vitro culture conditions: there were not always sufficient cells to investigate all cell phenotypes for all donors. In monocytes, there was no difference in IL-12p40 secretion between cells infected with the wild-type and those infected with the 19 strain, but a significant elevation in cytokine secretion was observed with the complemented strain (Fig. 4A). M. tuberculosis infection induced different levels of IL-12p40 in the different cell types, with DC producing the largest amounts (Fig. 4C), but the pattern of response to different strains was similar in each case. There was a tendency toward a decrease in IL-12p40 production in response to the 19 strain (this reached statistical significance in the case of the MDM cultures) and a significant increase in IL-12p40 induction by the strain overexpressing the 19-kDa-lipoprotein in all cell types (Fig. 4A to C).
All of the strains elicited similar secretion of TNF- from infected monocytes, although, again, there was a significant increase in induction by the 19::pSMT181 strain in MDM and DC (Fig. 4D to F). Production of IL-1 in response to M. tuberculosis was highest for cells with a high level of TLR2 expression (monocytes and MDM) and undetectable for DC (Fig. 4G and H). IL-1 levels were significantly reduced in monocyte and MDM cultures infected with 19 (P = 0.01 and 0.015, respectively). The strain overexpressing the 19-kDa lipoprotein elicited significantly larger amounts of IL-1 than wild-type M. tuberculosis in MDM but not in monocytes.
Effect of the 19-kDa protein on surface expression of HLA-DR. Purified 19-kDa protein has been reported to decrease MHC class II expression and thereby antigen presentation to T cells, an effect that would favor bacillary survival (10, 16, 19). We determined the effect of deleting the 19-kDa-lipoprotein gene on MHC class II expression in eight donors. Monocytes from six donors were isolated by adherence, and the presence and intensity of HLA-DR staining were determined after 48 h of coculture at 1 bacillus per cell. Infection of monocytes with M. tuberculosis led to a modest but significant decrease in the numbers of HLA-DR-positive cells (P = 0.016) (Fig. 5). The mean fluorescence intensity (MFI) of staining on HLA-DR-positive cells showed a similar decrement (from 554 ± 76 to 454 ± 46). These effects were not observed with the 19 strain, which, if anything, tended to increase HLA-DR expression (MFI, 652 ± 33; percent positive cells, 88% ± 1.5%; both effects were significant by comparison with wild-type M. tuberculosis [P = 0.008 and 0.034, respectively]). The complemented 19::pSMT181 strain displayed an intermediate phenotype and did not differ significantly from either the wild type or 19. For two further donors, HLA-DR expression in IFN--activated MDM was similarly investigated. Infection with wild-type M. tuberculosis led to a similar decrease in the percentage of positive cells, from 98 to 84.3%; the values for 19- and 19::pSMT181-infected cells were 90.8 and 88.3%, respectively.
DISCUSSION
The 19-kDa lipoprotein of M. tuberculosis has been extensively studied in terms of its ability to trigger responses in innate immune cells and to act as an antigen for cellular and humoral arms of the adaptive response (1, 15, 30). Its physiological function in M. tuberculosis itself is unknown. Although conservation of related genes throughout the mycobacterial genus suggests some essential function outside of any specialized role in infection, the absence of any growth defect in Middlebrook media suggests that this function is dispensable for growth in the laboratory. The reduced growth of the deletion strain in IFN--activated MDM but not monocyte cultures may, however, reflect a physiological requirement under the growth conditions encountered within an intact animal. Consistent with these in vitro results, disruption of the 19-kDa-lipoprotein gene has been shown to moderately reduce the growth of M. tuberculosis in a murine model (22).
A robust increase in IL-12p40 secretion was seen in all cell types following infection with a strain engineered to express the protein at a level higher than that of the wild type. While these results are consistent with the initial description of the 19-kDa lipoprotein as an important early trigger for IL-12p40 production (3), it is clearly not the sole, nor perhaps the dominant, component of M. tuberculosis responsible for this activity. Even when consideration is restricted to the 118 putative or confirmed lipoproteins of M. tuberculosis, there are others, such as PstS1 and LprG, with known effects on the innate response (3, 11). Our data suggest that the strongest response occurs when the concentration of the 19-kDa ligand rises above that of the physiological level encountered during wild-type infection. It has been reported previously that overexpression of the 19-kDa lipoprotein in rapidly growing mycobacteria caused downregulation of IL-12p40 secretion (20); it is possible that the effect of 19-kDa-lipoprotein signaling is dependent on the pattern of parallel signaling interactions involving other ligands that differ between pathogenic and nonpathogenic species.
The effect of 19-kDa-lipoprotein deletion on cytokine secretion was most marked for IL-1. As with IL-12p40, there was evidence of increased IL-1 secretion from MDM infected with the complemented strain compared to the wild type. Ciaramella et al. also found that the dominant effect of the 19-kDa lipoprotein expressed by intact mycobacteria was evident in the IL-1 response (5). These results suggest a differential role for particular mycobacterial ligands in induction of particular cytokines. The 19-kDa lipoprotein seems to have a dominant role in the induction of IL-1, particularly in the context of high levels of TLR2 expression, and a less pronounced role in IL-12p40 production. We observed no significant effect of 19-kDa-lipoprotein gene deletion on TNF-, although there was evidence of increased TNF- secretion by MDM and DC infected with the overexpressing 19::pSMT181 strain.
Monocytes infected with the 19 strain were characterized by a higher level of surface expression of MHC class II protein than those infected with wild-type M. tuberculosis. This result is consistent with previous reports that the purified 19-kDa protein is able to suppress MHC class II expression (9, 16). The decrease that we observed in the number of monocytes staining positive for MHC class II in cultures infected with M. tuberculosis (13% over 48 h) was relatively modest. This may be due to the use of a low multiplicity of infection (to avoid excessive cytopathic effects), which resulted in only around 10% of the cells being infected. The suppressive effect of M. tuberculosis was completely reversed following deletion of the 19-kDa-lipoprotein gene and was partially restored by reintroduction of that gene in 19::pSMT181. We suggest that the intermediate phenotype of the complemented strain with respect to MHC class II expression arose as a consequence of the increased proinflammatory response, which would tend to increase MHC class II expression, countered by the tendency of the 19-kDa lipoprotein to decrease it. Our data will also tend to reflect the accumulation of protein in the culture supernatant, which may not necessarily reflect the true state of the cells at later time points.
Our results from deletion and addition of the 19-kDa protein in the context of infection with whole M. tuberculosis demonstrate a range of effects that can be viewed as beneficial either to the host or to the pathogen. On the one hand, induction of IL-1 (resulting in recruitment of antimicrobial effector cells) and induction of IL-12p40 (priming a macrophage-activating Th1 response) are both associated with a protective immune response. Conversely, increased bacterial growth in MDM and suppression of antigen-presenting function would seem to favor progression of infection. At a teleological level, it can be argued that, since the vast majority of clinical isolates have maintained the 19-kDa-lipoprotein gene, the balance of these effects falls in favor of the pathogen. This is indeed the case in the mouse model of tuberculosis, in which disruption or deletion of the 19-kDa-lipoprotein gene reduces the level of infection (22; G. R. Stewart and A. S. Apt, unpublished data). As we progress in our understanding of the complex signaling events associated with the innate immune response to M. tuberculosis infection, the ability to dissect positive and negative effects, via the analysis of immune responses to bacilli with defined mutations, could open important new avenues for vaccine development.
ACKNOWLEDGMENTS
This work was supported by the Wellcome Trust (grants 064261, 060079, and 038997).
G.R.S. and K.A.W. contributed equally to this work.
Present address: School of Biological and Medical Sciences, University of Surrey, Guildford, United Kingdom.
Present address: Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, South Africa.
Present address: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109.
|| Present address: Unite de Genetique Mycobacterienne, Institut Pasteur, Paris, France.
# Present address: University of Warwick Medical School, Coventry, United Kingdom.
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