Infection Biology of a Novel -Crystallin of Mycobacterium tuberculosis: Acr2
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免疫学杂志 2005年第7期
Abstract
Heat shock proteins assist the survival of Mycobacterium tuberculosis (MTB) but also provide a signal to the immune response. The gene most strongly induced by heat shock in MTB is Rv0251c, which encodes Acr2, a novel member of the -crystallin family of molecular chaperones. The expression of acr2 increased within 1 h after infection of monocytes or macrophages, reaching a peak of 18- to 55-fold by 24 h. Inhibition of superoxide action reduced the intracellular increase in acr2. Despite this contribution to the stress response of MTB, the gene for acr2 appears dispensable; a deletion mutant (acr2) was unimpaired in log phase growth and persisted in IFN--activated human macrophages. Acr2 protein was strongly recognized by cattle with early primary Mycobacterium bovis infection and by healthy MTB-sensitized people. Within the latter group, those with recent exposure to infectious tuberculosis had, on average, 2.6 times the frequency of Acr2-specific IFN--secreting T cells than those with more remote exposure (p = 0.009). These data show that, by its up-regulation early after entry to cells, Acr2 gives away the presence of MTB to the immune response. The demonstration that there is infection stage-specific immunity to tuberculosis has implications for vaccine design.
Introduction
Increased knowledge of the function and antigenic properties of Mycobacterium tuberculosis (MTB)4 proteins will assist the design of vaccines and immunodiagnostic reagents against tuberculosis, a disease that kills up to 2 million people per year (1). The heat shock proteins of MTB are of particular interest because their elevated expression is required for bacterial adaptation to adverse conditions encountered during infection, while at the same time providing a signal for host immune recognition (2, 3, 4). Previous work in our laboratory showed that constitutive overexpression of Hsp70 and related heat shock proteins as a result of deletion of the HspR transcriptional regulator induced an increased immune response that significantly impaired the ability of MTB to persist during the chronic phase of infection in mice (5). Whole-genome expression analysis of the HspR deletion strain, and heat-shocked wild-type MTB, identified Rv0251c as a novel major heat shock gene (6). This finding has been confirmed and extended by the comprehensive transcriptomic analysis of Schnappinger et al. (7). Annotated as hsp in the MTB H37Rv genome sequence (8), Rv0251c was found to be induced in naive and activated murine macrophages, and by heat shock, H2O2, SDS, high-dose NO, and palmitic acid in broth cultures (7). On this basis, they categorized Rv0251c as belonging to a group of seven MTB genes that are up-regulated in response to multiple stresses.
Rv0251c encodes a protein with 30% amino acid sequence identity to the well-characterized 16-kDa -crystallin homolog Acr (encoded by Rv2031c, and referred to as acr or hspX) (6, 8). The identity increases to 41% based on comparison of residues present in the -crystallin core. The acr gene is known to be required for log phase growth of MTB and during the transition to stationary phase, at which time it is suspected to contribute via chaperonin activity to the stabilization of intracellular structures (9, 10, 11). In addition to this important role in bacillary physiology, the protein is also immunodominant in humans (12, 13, 14). In contrast to acr2, the gene for acr is induced by oxygen deprivation or by exposure to low concentrations of NO in vitro, and its induction in murine macrophages is clearly NOS2 dependent (7, 15, 16). We have previously named Rv0251c acr2 because it is the second member of the -crystallin family to be identified in MTB. From this point in this report we therefore refer to Acr1 and Acr2 (6).
We investigated the biology of acr2 by measurement of its RNA accumulation in human mononuclear phagocytes, phenotypic analysis of a mutant MTB that lacks the acr2 gene (acr2), and analysis of immune recognition of the recombinant Acr2 protein. We found that the level of acr2 RNA increases shortly after phagocytosis of MTB in response to exposure to host reactive oxygen intermediates (ROI). However, the acr2 strain was only mildly compromised in its ability to resist various in vivo and in vitro stress stimuli. By virtue of its early expression, we found that Acr2 is a dominant immune target of the early bovine and human T cell responses to mycobacterial infection. The results suggest that, although acr2 plays a role in bacillary defense, its early expression evokes a vigorous early immune response. Thus, Acr2 may be a rare Achilles’ heel for MTB, giving away its presence early after infection.
Materials and Methods
Isolation, infection, and culture of monocytes and monocyte-derived macrophages (MDM)
Buffy coats from healthy donors were obtained from the National Blood Transfusion Service (Colindale, London, U.K.). Following dilution in RPMI 1640 (1/3 v/v) PBMC were separated by centrifugation over Ficoll-Paque Plus (Pharmacia). Cells were washed extensively in RPMI 1640, pooled, and counted. Cells were suspended at 1.2 x 107/ml in RPMI 1640/10% FCS medium (R10), and aliquots of 25 ml were added to 150-cm2 tissue culture flasks. Flasks were placed flat in the incubator, and monocytes were allowed to adhere for 2 h at 37°C. Nonadherent cells were removed by washing three times with 10 ml of prewarmed RPMI 1640. Finally, 10 ml of ice-cold PBS was added, and the flasks were incubated at 4°C for 20 min. Using a scraper, monocytes were gently dislodged from the bottom of the flasks and pooled in a 50-ml Falcon tube to count. FACS analysis showed the resultant cell population to be 82 ± 4% CD14 and 76 ± 7% CD11b positive. For experiments requiring monocytes, cells were plated in RPMI 1640 containing 10% non-heat-inactivated FCS at 106/well in a 24-well tissue culture plate, and cultured overnight before infection. For experiments requiring MDM, cells were suspended in X-VIVO 15 (BioWhittaker) at 106/well in a 24-well plate and cultured for 7 days before infection. This maturation procedure resulted in an increase in HLA-DR expression (95 ± 0.3%), retention of CD14 positivity (84 ± 3%), and a decrease in CD11b (15 ± 1.3%). For the analysis of intracellular growth of mutant strains, MDM were activated with IFN- at 1 ng/ml 24 h before infection (added on day 6).
Monocytes or MDM were infected in 24-well culture plates. Before infection, the medium was removed from the wells using a Pasteur pipette and replaced with fresh RPMI 1640 supplemented with L-glutamine plus 10% non-heat-inactivated FCS. Cells were infected at a multiplicity of infection of 1:1 (bacillus:cell) for 4 h, followed by washing three times using warm RPMI 1640 to remove nonphagocytosed bacteria. No antibiotics were used. Finally, RPMI 1640 containing L-glutamine and 10% heat-inactivated FCS was added to the infected cells. On average, this procedure led to infection of between 5 and 10% mononuclear phagocytes (data not shown). The washing procedure ensures that 90% of bacilli subsequently recovered from cultures are in the cell lysate rather than supernatant (17). For some experiments, monocytes were preincubated with 10 mM N-acetyl cysteine (NAC; Sigma-Aldrich) before infection.
Bacterial strains and growth curve
MTB H37Rv (supplied by Prof. S. Cole, Institut Pasteur, Paris, France) and mutant strains of MTB were grown with shaking to mid-log phase in Middlebrook 7H9 broth (Difco) containing 0.2% glycerol, 0.05% Tween 80, and 10% albumin-dextrose-catalase. Aliquots were prepared in 15% glycerol and stored at –80°C. The CFU content of aliquots was determined by serial dilution and plating on to Middlebrook 7H11 agar (Difco) containing 0.5% glycerol and 10% oleic acid-albumin-dextrose-catalase. The construction and characterization of a strain lacking Rv0251c/acr2 (acr2) and its complementation by a single copy of the acr2 gene under its own promoter (acr2::pSMT184) is described in detail elsewhere (18). To analyze the growth rate of strains, duplicate cultures were set up and sampled for CFU analysis at intervals. The mean of four to six estimations of CFU was recorded by serial dilution and plating on 7H11 agar.
RNA extraction and quantitative RT-PCR
The extraction of MTB RNA from infected cells was performed exactly as described previously (19). We used quantitative real-time RT-PCR in an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Specific reverse transcription (RT) and PCR primers and a probe for Acr2 were as follows: RT primer (–266RT), 5'-GCGTGTGCTCGTCG-3'; forward primer (160F), 5'-GCGGTGGTCCGTTTGGA-3'; reverse primer (227R), 5'-CCAGGGTCAAGCTCGACGTT-3'; probe (181T), 5'-FAM-CCCGGCATTGACGTCGACAAGG-3'-TAMRA. Sequences used to detect MTB acr1 and 16S RNA were as previously published (16, 20). All probes were dually labeled with FAM at the 5' end and TAMRA at the 3' end. Multiplex RT was performed for 1 h at 37°C using 1 μM each specific RT primer, 0.25 U/μl avian myeloblastosis virus-RT (ABgene) in the presence of 500 μM each dNTP. 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 primers at 250 nM; for Acr1, the probe at 200 nM, forward primer at 150 nM, and the reverse at 50 nM; for Acr2, the probe at 100 nM, forward primer at 50 nM, and the reverse at 300 nM. Reaction conditions consisted of 1 cycle of 50°C for 2 min and 1 cycle of 95°C for 10 min and then 40 cycles of 95°C for 15 s followed by annealing and elongation at 65°C (16S), 67°C (acr1), or 60°C (acr2) for 1 min. The cycle threshold (CT) for each sample was compared with CT values of known amounts of a standard DNA from MTB H37Rv. To assure lack of DNA contamination of the RNA samples, a duplicate tube of sample with no RT enzyme was included as control. Results are expressed as values normalized to the 16S rRNA content.
Experimental infection of cattle with Mycobacterium bovis and analysis of in vitro IFN- responses
Five-month-old calves (Holstein-Friesians) were obtained from herds free of bovine tuberculosis and kept in category 3 biosafety accommodation at Veterinary Laboratories Agency Weybridge. Five animals were infected with the sequenced M. bovis strain (AF 2122/97) by endobronchial instillation of 1000 CFU (21). Blood samples were collected postinfection, and IFN- assays were performed as described previously (22). Briefly, 0.25 ml of heparinized whole blood was aliquotted in 96-well plates and 25 μl of Ag solution was added at the following final concentrations: bovine PPD (PPD-B) (10 μg/ml), Acr1 and Acr2 (20 μg/ml). Supernatants were harvested after 48 h of culture and IFN- was determined using the BOVIGAM EIA kit (CSL). Data are expressed as OD450 units (OD450 values multiplied by 1000) with responses of >100 OD450 U classified as positive (22).
Human subjects
This study was conducted with ethical approval from the Harrow Local Research Ethics Committee (LREC 1646 and 2414). Newly diagnosed tuberculosis patients (TP) and healthy sensitized subjects (HS) were recruited from Northwick Park Hospital, Harrow. TP had untreated overt (i.e., culture- or biopsy-positive) tuberculosis (n = 29; 18 male, 11 female; average age, 33.9 years). The HS group comprised asymptomatic adults with normal chest radiographs who nevertheless exhibited positive purified protein derivative (PPD) skin test reactions (Heaf grade 2 and above; equivalent to a Mantoux of >10 mm) who were considered clinically to have latent tuberculosis infection (n = 69; 38 male, 31 female; average age, 36.6 years). All subjects were subsequently advised and, if indicated, treated according to British Thoracic Society guidelines (23, 24).
Recombinant Ags
Recombinant early secreted antigenic target-6 (rESAT-6) was prepared as previously described (25). Recombinant culture filtrate protein-10 (rCFP-10) and rAcr1 were obtained from Lionex. N-terminal sequencing confirmed the identity of the cloned Ags. Acr2 was cloned and expressed as an N-terminal His.Tag and purified as described by Stewart et al. (18). Endotoxin levels were as follows (endotoxin units (EU)/mg): ESAT-6, 18.65; CFP-10, 47.41; Acr2, 1.33; PPD, 18.65; Acr1, 11.01. Twenty-mer synthetic peptides covering the entire sequence of the Acr1 and Acr2 proteins and overlapping by 12 residues were prepared by multipin synthesis by Mimotopes (26). Peptides were used individually in ELISPOT analysis at a concentration of 20 μg/ml.
ELISPOT assay for single-cell IFN- release
Ninety-six-well polyvinylidene difluoride-backed plates (MAIPS45; Millipore), precoated with 15 μg/ml anti-IFN- mAb 1-D1K (Mabtech), were blocked with R10 for 2 h. A total of 3 x 105 PBMC was added in 100 μl of R10/well. Duplicate wells containing rAcr1, rAcr2, rESAT-6, or rCFP-10 at previously determined optimum concentrations (2.5–20 μg/ml) were set up. PPD (Evans Medical) at 100 U/ml, and PHA (ICN) at 5 μg/ml were added to positive control wells. No Ag was added to the negative control wells. After 16-h incubation at 37°C in 5% CO2, plates were washed with PBS containing 0.05% Tween 20. Fifty microliters of 1 μg/ml biotinylated anti-IFN- mAb, 7-B6-1-biotin (Mabtech), was added for 2 h. Plates were then washed and streptavidin-alkaline phosphatase toxoid was added at 1/1000 dilution. After 1 h and further washing, 50 μl of chromogenic alkaline phosphatase substrate (Bio-Rad), diluted 1/25 with deionized water, was added. Ten minutes later, plates were washed and allowed to dry, and spot-forming cells (SFC) were enumerated with a magnifying glass. A positive response was defined as 15 IFN- SFC/106 PBMC.
ELISA for Abs to Acr2
Polyvinyl microtiter plates (Nunc) were coated overnight at 4°C with 10 μg/ml rAcr2 in 0.05 M carbonate buffer (pH 9.6). The plates were blocked with 5% (w/v) fat-free skimmed milk powder in PBS for 30 min at 37°C. Serum was diluted 1/100 in PBS/milk containing 0.05% (v/v) Tween 20 and applied for 2 h at room temperature with agitation. The wells were washed three times in PBS/Tween, and then HRP-conjugated rabbit anti-human IgG (DakoCytomation) was applied at a dilution of 1/3000 in PBS/milk/Tween 20 for 2 h at room temperature. The wells were washed three times in PBS/Tween 20, and Ab binding was detected using o-phenylenediamine as a substrate, and the reaction product was measured at 492 nm.
Statistical analysis
Contingency analysis was performed by Fisher’s exact test of probability. Paired and unpaired parametric variables were compared by Student’s t test. Paired and unpaired nonparametric variables were compared by Wilcoxon signed rank or Mann-Whitney U test, respectively. Significance was inferred for values of p 0.05.
Results
Expression of Acr2 within human mononuclear phagocytes
The kinetics of acr2 RNA accumulation within monocytes and MDM from two donors was studied by quantitative RT-PCR. Duplicate wells of 5 x 106 cells were infected with MTB at 1:1 bacillus:cell. For the 1-h time point, nonphagocytosed bacilli were washed away immediately before lysis and RNA extraction; for all other time points, nonphagocytosed bacteria were removed after 4 h. Experiments were controlled by inoculating bacilli into parallel wells containing culture medium but not cells. The ratio of acr2 RNA to MTB 16S rRNA increased 5.18-fold within 1 h of infection of monocytes, reaching a peak of 16.27-fold after 24 h, after which there was a decline (Fig. 1A). In MDM, acr2 induction was slightly less rapid but reached a higher peak of 62.3-fold at 48 h. In contrast, the ratio of acr1 RNA to 16S rRNA progressively declined over the 96-h time course in the same cells (Fig. 1B). A marked induction of acr1 was observed in the control cultures that contained only RPMI 1640/10% FCS, possibly reflecting hypoxia in these unshaken cultures.
FIGURE 1. Expression of acr2 within human mononuclear phagocytes. Cells were infected with MTB H37Rv at 1:1. Nonphagocytosed bacilli were washed away before lysis, and in any case at 4 h, for RNA extraction and quantitative RT-PCR. acr2 and acr1 RNA levels were normalized to the 16S rRNA content of the same sample. A and B, RPMI 1640/10% FCS (?); monocytes (); MDM (). Intracellular acr2 RNA accumulated rapidly in both monocytes and MDM from the same donors. In contrast, the acr1 RNA level within cells gradually fell over 96 h, although in cell culture medium without cells, there was a marked increase between 12 and 96 h. Data show the mean ± SD of two donors. C, At 24 h, the mean intracellular induction of acr2 in monocytes (n = 5) was 17.8-fold, and 54.7-fold in MDM (n = 7) (p 0.0182). D, Overnight preincubation of monocytes with NAC in two donors completely abrogated the increase in acr2 RNA level.
The increase in acr2/16S ratio at 24 h was further investigated in monocytes and MDM from five and seven donors, respectively (Fig. 1C). This confirmed a 17.8-fold increase in monocytes and a 54.7-fold increase in MDM (p 0.0182 for both cell types). In two donors, the effect of overnight preincubation of monocytes with an inhibitor of superoxide action (NAC; 10 mM) was tested. Results were calculated as the mean of the ratio (acr2/16S in cells plus inhibitor:acr2/16S in medium plus inhibitor) at 24 h. This design allowed us to control the possible effects of inhibitor alone on bacillary gene expression outside cells. The presence of NAC almost completely abolished the intracellular increase in acr2 (Fig. 1D). Together, these data show that acr2 gene expression is up-regulated shortly after entry to cells in response to ROI.
Phenotypic characterization of a mutant MTB lacking the gene for Acr2 (acr2)
Further investigation of the role of acr2 was pursued by analysis of a mutant MTB strain that lacked the acr2 gene (acr2). We also examined a strain in which the mutation was complemented by reinsertion of a single copy of the acr2 gene at the attB phage integration site in the chromosome (acr2::pSMT184). Doubling times calculated over 144 h of log phase growth in Middlebrook 7H9 broth were similar (24.3, 24.7, and 32.3 h for wild type, acr2, and acr2::pSMT184, respectively). Monocytes or MDM from four donors, respectively, were infected at 1:1 (bacillus:cell), and nonphagocytosed bacilli were washed away at 4 h. The uptake into, and growth of strains over 6.5 days within monocytes did not differ. Even in IFN--activated MDM, growth was similar up to 4 days, after which the acr2 mutant showed no further increase, although the difference from H37Rv at 6.5 days was not statistically significant (Fig. 2A). In other experiments, we confirmed that, although the acr2 strain was moderately more susceptible to H2O2 in 7H9 broth, its growth in vivo over 60 days in the organs of mice was unimpaired (data not shown). Interestingly, in IFN--activated MDM at the 24-h time point, the expression of acr within cells infected with acr2 was slightly (2.08-fold) higher than in cells infected with wild type (Fig. 2B). This raises the possibility that the absence of acr2 could be partially compensated by increased expression of acr. The overall expression of acr within IFN--activated MDM greatly increased such that the acr2/acr1 ratio, which had been >100 in monocyte or MDM cultures, was reduced to near equality (2.5 ± 0.2) in IFN--activated MDM (Fig. 2C).
FIGURE 2. A, Intracellular growth of a strain of MTB lacking the gene for Acr2 (H37Rv (); acr2::pSMT184 (?); acr2 ()). IFN--activated MDM were infected at 1:1, with nonphagocytosed bacilli being washed off after 4 h. The uptake into, and growth within, cells was similar up to 4 days, after which the acr2 showed a slight decline (p = 0.068 by comparison with H37Rv at 6.5 days). Mean ± SD of four donors. B, Intracellular expression of Acr in IFN--activated MDM. IFN--activated MDM were infected at 1:1, with nonphagocytosed bacilli being washed off after 4 h. At the 24-h time point, the expression of Acr within cells infected with acr2 was slightly higher. Mean ± SD of two donors. C, Ratio of intracellular Acr2/Acr1 in various cell types. Monocytes (Monos), MDM, or IFN--activated MDM (-MDM) from two donors were infected at 1:1 with MTB H37Rv. After 4 h, nonphagocytosed bacilli were washed off, and at 24 h, the cultures were lysed for analysis of RNA content. The Acr2/Acr1 ratio was greatly reduced by induction of the Acr1 gene in activated cells.
The immune response to Acr2
Because we had demonstrated that the acr2 gene is up-regulated early after infection of mononuclear phagocytes, we hypothesized that it could be a target of early T cell responses directed toward MTB. We measured the primary immune response to rAcr2 in a cattle model of experimental M. bovis infection (27). M. bovis possesses an acr2 gene identical with that of MTB H37Rv (28). Because experimental infection allows control over the time and dose of infection, precise documentation of immune responses postinfection is possible. Five calves were infected with a low dose of M. bovis and in vitro IFN- responses were determined in blood samples taken at intervals up to 5 wk postinfection. All animals at this stage of experimental infection remained free of clinical signs of tuberculosis, and may therefore be akin to recently exposed, asymptomatic humans. Our results demonstrate that Acr2 was a T cell target in recently infected cattle, in contrast to Acr1, which was recognized only later in the infection (Fig. 3). Responses to Acr2 were detected as early as 2 wk postinfection in four of five calves, and by wk 3 postinfection, all five calves exhibited strong Acr2-specific responses. These responses remained high at 4 and 5 wk postinfection. In contrast, none of the calves responded to Acr1 2 wk after infection, and only one responded at wk 3 (p = 0.04 at both time points). By wk 4 and 5, three of five and five of five calves responded to Acr1. However, even at wk 5 postinfection, significantly lower amounts of IFN- were induced by Acr1 stimulation than by Acr2 (p = 0.0003 at wk 5).
FIGURE 3. IFN- responses to Acr1 and Acr2 in cattle experimentally infected with M. bovis. The IFN- responses after in vitro stimulation with bovine PPD, Acr1, and Acr2 proteins were determined postinfection. Horizontal bars show median values. The frequency of Acr2 responders at 2 and 3 wk postinfection was significantly higher than frequencies of Acr1 responders (p = 0.04 at both time points); Acr2 responses were quantitatively higher than Acr1 responses even at wk 5 postinfection (p = 0.0003).
An unusual opportunity to test the human recall response presented itself by the precisely timed inadvertent exposure of a bacillus Calmette-Guérin (BCG)-vaccinated person to pathogenic M. bovis. We compared the IFN- ELISPOT response to Acr2 with that made in response to Acr1, ESAT-6, CFP-10, and PPD. Because ESAT-6 and CFP-10 are encoded by the RD1 genomic segment of MTB and M. bovis, these genes are not present in any M. bovis-BCG strains and can thus specifically detect T cells sensitized by members of the pathogenic MTB complex (29, 30). The response to PPD reflects accumulated history of BCG vaccination and other mycobacterial exposure. In a blood sample taken 1 day after exposure, there were 100 PPD-specific IFN--producing T cells/106 PBMC detectable by ELISPOT analysis but no significant response to the other Ags (Fig. 4). After 1 wk, the frequency of Acr2-specific cells increased greatly to 173 per 106 PBMC, whereas the PPD-specific response modestly increased to 132 per 106. There was a subsequent fall in the Acr2-specific response to a plateau (42–55 per 106) accompanied by a gradual rise in specific responses to ESAT-6 and CFP-10. At no time point was there a significant response to Acr1. The subject remained asymptomatic throughout and was healthy 28 mo after exposure. Isoniazid preventive therapy was not used. Thus, a timed exposure to M. bovis resulted, in this person, in a rapid expansion of Acr2-reactive T cells accompanied by a steady rise in M. bovis-specific responses.
FIGURE 4. Longitudinal analysis of IFN--secreting T cells in a person accidentally exposed to M. bovis. The subject of this analysis was inadvertently exposed to pathogenic M. bovis. One day after this exposure, there were no Acr2- or CFP-10-specific, and 100 PPD-specific IFN--producing T cells/106 PBMC detectable. After 1 wk, the frequency of Acr2-specific cells increased greatly to 173 per 106, whereas the PPD-specific response modestly increased to 132 per 106. There was a subsequent fall in the Acr2-specific response to a plateau (42–55 per 106) accompanied by a gradual rise in the ESAT-6- and CFP-10-specific response. There was no response to Acr1 at any time point.
We extended ELISPOT analysis of the T cell response to Acr2 to 98 adults sensitized by exposure or by active tuberculosis. HS (n = 69) were persons who were PPD skin test positive but were free of symptoms of active tuberculosis and had normal chest radiographs. This group was stratified into 13 individuals with documented recent (<6 mo ago) contact with infectious tuberculosis and 56 individuals from whom no history of recent contact with infectious tuberculosis could be elicited. TP (n = 29) had untreated active tuberculosis at the time of sampling. The frequency of positive responses and also the numbers of IFN--secreting T cells responding to both -crystallins tended to be higher in HS compared with TP, significantly so for Acr2 (p = 0.044; Table I). In HS who responded, the median IFN- SFC was 43, whereas it was 33 among responding TP. The number of PPD-reactive IFN--secreting T cells was also significantly higher in HS. By contrast, the response to the RD1-encoded ESAT-6 and CFP-10 proteins tended to be higher in TP, although not significantly so. Stratification of HS by recent or remote exposure to tuberculosis revealed that 92% of those with recent exposure had a response to Acr2 in comparison with 63% with more remote contact (p = 0.029). In addition, the median frequency of IFN- SFC in recent contacts was 65 in comparison with 25 in the remote group (p = 0.009). The relationship between T cell reactivity and recency of contact was seen only for Acr2 response (Fig. 5A).
Table I. Frequency and magnitude of response to Ags of MTBa
FIGURE 5. The human immune response to Acr2 of MTB. A, The T cell response to Acr2 in 69 HS adults who were PPD skin test positive but free of symptoms of active tuberculosis. This group was stratified into 13 individuals with documented recent (<6 mo ago) contact with infectious tuberculosis (, , , ) and 56 individuals from whom no recent history of contact with infectious tuberculosis could be elicited (, , ?, ). The median frequency of IFN- SFC in recent contacts was 65 by comparison with 25 in the remote group (p = 0.009). This relationship between T cell reactivity and recency of infectious contact held only for Acr2. B, Comparison of the T cell and B cell response to Acr2. The Ab response (as determined by ELISA) was compared with the frequency of IFN--secreting cells in 41 subjects. HS had lower Ab levels with higher frequencies of IFN--secreting cells, such that 9 of 20 had Ab OD values <0.3 and IFN--secreting cells >40 per 106 PBMC in comparison with just 2 of 21 patients (p = 0.0148).
In a subset of 41 subjects (20 HS and 21 TP), the availability of a serum sample allowed us to compare T and B cell Ab responses to Acr2. HS had lower Ab levels with higher frequencies of IFN--secreting cells, such that 9 of 20 HS had a response characterized by Ab OD values <0.3 and IFN- secreting cells >40 per 106 PBMC; only 2 of 21 TP shared this profile (p = 0.0148; Fig. 5B). However, responses were heterogeneous, and there was no overall inverse correlation between the Ab and T cell response, nor was there any correlation between Acr1- and Acr2-specific responses (data not shown). Because Acr2 shares 30% amino acid sequence identity with Acr1, we were also interested to investigate whether T cell recognition of the -crystallins was specific or cross-reactive. Of all the subjects in the study, 34% responded to both molecules, 14% to Acr1 alone, 30% to Acr2 alone, and 22% to neither. Thus, T cell recognition of one molecule was more common than recognition of both, implying specificity. This impression was reinforced by epitope mapping of both molecules in a subset of 24 responding donors (Fig. 6). Dominant epitopes in Acr1 were found between residues 81–124, consistent with our previous reports of two permissively recognized epitopes in this region (13, 14, 31). In contrast, no single peptide within Acr2 stimulated the T cells of >50% of the subjects, implying that Acr2 contains multiple epitopes restricted by specific MHC class II molecules. Recognition of peptides that contained >40% identical residues in both Acr1 and Acr2 was exceptional, occurring only for sequence 97–116 (10 identical residues: 50%) at a frequency of 7 of 11 for Acr1 and just 1 of 13 for Acr2. These data strongly imply specific recognition even of this, the most homologous peptide.
FIGURE 6. Epitope mapping of -crystallins in 24 responding donors. The T cell response to overlapping 20-mer peptides was assayed by the overnight IFN- ELISPOT assay. Only donors who responded to either or both -crystallins were included in this analysis. Dominant epitopes in Acr1 are found between residues 81 and 124, whereas no single peptide within Acr2 stimulated the T cells of >50% subjects. Sequence numbers in bold type indicate >40% identity between the Acr1 and Acr2 sequence. *, 125–144 for Acr1 sequence.
Discussion
We have investigated the gene expression, effect of deletion from the genome, and the immune response to Acr2, a novel -crystallin of MTB. MTB has two members of the -crystallin family of molecular chaperones. Although neither is absolutely required for growth in vivo or in vitro (32), Acr1 has been extensively characterized in the context of its role in the bacterial response to hypoxia/NO and as an immunodominant Ag. Our study demonstrates that Acr2 also makes a contribution to pathogen biology but more so to the host immune response. We found that acr2 gene expression was up-regulated shortly after entry into cells in response to exposure to host reactive oxygen. As a consequence, Acr2 is a prominent immune target, especially of T cells in individuals who are asymptomatic but recently exposed to infectious tuberculosis.
Expression of the acr2 gene is controlled by the heat shock regulator, HspR (5), and by the alternative factor, E, which is itself regulated by heat shock and by oxidative stress (33). It has recently been shown that acr2 is strongly induced following uptake of MTB by quiescent mouse macrophages, in contrast to acr1 that is induced only in activated macrophages (7). Consistent with findings in the murine system, we have observed strong induction of acr2 expression following uptake of MTB by human monocytes or MDM (Fig. 1). There was no induction of acr1 under these conditions. Preincubation of monocytes with NAC showed that acr2 induction was dependent on the generation of ROI, consistent with in vitro studies of acr2 regulation (7).
Building on our characterization of the role of acr2 in the pathogen response to infection, we reasoned that the early induction of acr2 expression could result in its availability as an attractive target for an early response on the part of the host immune system. Three lines of evidence suggest that this is indeed the case. First, the Acr2 Ag was strongly recognized by cattle infected by M. bovis, which contains an identical Acr2 gene (Fig. 3). Second, we had the opportunity to monitor the kinetics of T cell responses to different Ags in a single individual following accidental exposure to virulent M. bovis (Fig. 4). A strong response to Acr2 was observed within 1 wk of exposure, significantly in advance of responses to two other well-characterized immunodominant Ags, ESAT-6 and CFP-10 (34). Although the interpretation of data from a single donor has to be cautious, we take the data to indicate that infection with M. bovis did occur and that this infection rapidly re-expanded a population of previously undetectable Acr2-specific IFN--producing T cells. The kinetics of immune response to acr2 in this person, together with the transient response to ESAT-6, may indicate that the infection was cleared very rapidly. The third less direct evidence is derived from analysis of responses in groups of TP and HS who had clinical and immunological evidence of recent or past exposure to MTB. The frequency and number of IFN--producing T cells specific for Acr2 were higher among HS than among TP, with the highest responses seen in individuals with the most recent exposure to infection. These correlations were not seen in the case of responses to Acr1, ESAT-6, or CFP-10 (Fig. 5A).
Although BCG vaccination is of insufficient efficacy to control tuberculosis in human populations, only a few experimental approaches have improved its efficacy in animal models (3). Thus, attention has been drawn to the possibility of augmenting BCG by overexpression of its existing Ags or by combining it with Ags delivered either as DNA, protein subunit, or other live vector in a prime-boost strategy (35, 36, 37, 38). It is recognized that an early immune evasion strategy adopted by MTB is retardation of phagosomal maturation, crucially delaying its degradation and thus immune recognition (39). Nevertheless, our study indicates the early expression of acr2 gives away the presence of MTB to the immune response. The demonstration that Acr2 is an early immune target is encouraging, but insufficient to qualify it as a vaccine candidate. Our ongoing work is therefore directed toward determining whether Acr2 can be used to augment the vaccine efficacy of BCG in experimental models.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
Dr. Mark Nicol of the University of Cape Town is thanked for his critical reading of the manuscript. We thank the medical, microbiological, and nursing staff of Northwick Park Hospital for assistance in recruiting and characterizing subjects for the study, particularly Drs. Robert Davidson, Robert Wall, and Stuart Dickson, and Prof. Geoffrey Pasvol.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Wellcome Trust of Great Britain (Refs. 064261, 060079, 038997, 059141). H.M.V. is supported by Department for Environment Food and Rural Affairs.
2 K.A.W. and G.R.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Robert J. Wilkinson, Wellcome Trust Center for Research in Clinical Tropical Medicine, Faculty of Medicine, Imperial College London, Wright Fleming Institute, Norfolk Place, London W2 1PG, U.K. E-mail address: r.j.wilkinson@imperial.ac.uk
4 Abbreviations used in this paper: MTB, Mycobacterium tuberculosis; ROI, reactive oxygen intermediate; MDM, monocyte-derived macrophage; NAC, N-acetyl cysteine; RT, reverse transcription; CT, cycle threshold; TP, tuberculosis patient; HS, healthy sensitized subject; PPD, purified protein derivative; SFC, spot-forming cell; BCG, bacillus Calmette-Guérin; ESAT-6, early secreted antigenic target-6; CFP-10, culture filtrate protein-10.
Received for publication May 26, 2004. Accepted for publication December 29, 2004.
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Heat shock proteins assist the survival of Mycobacterium tuberculosis (MTB) but also provide a signal to the immune response. The gene most strongly induced by heat shock in MTB is Rv0251c, which encodes Acr2, a novel member of the -crystallin family of molecular chaperones. The expression of acr2 increased within 1 h after infection of monocytes or macrophages, reaching a peak of 18- to 55-fold by 24 h. Inhibition of superoxide action reduced the intracellular increase in acr2. Despite this contribution to the stress response of MTB, the gene for acr2 appears dispensable; a deletion mutant (acr2) was unimpaired in log phase growth and persisted in IFN--activated human macrophages. Acr2 protein was strongly recognized by cattle with early primary Mycobacterium bovis infection and by healthy MTB-sensitized people. Within the latter group, those with recent exposure to infectious tuberculosis had, on average, 2.6 times the frequency of Acr2-specific IFN--secreting T cells than those with more remote exposure (p = 0.009). These data show that, by its up-regulation early after entry to cells, Acr2 gives away the presence of MTB to the immune response. The demonstration that there is infection stage-specific immunity to tuberculosis has implications for vaccine design.
Introduction
Increased knowledge of the function and antigenic properties of Mycobacterium tuberculosis (MTB)4 proteins will assist the design of vaccines and immunodiagnostic reagents against tuberculosis, a disease that kills up to 2 million people per year (1). The heat shock proteins of MTB are of particular interest because their elevated expression is required for bacterial adaptation to adverse conditions encountered during infection, while at the same time providing a signal for host immune recognition (2, 3, 4). Previous work in our laboratory showed that constitutive overexpression of Hsp70 and related heat shock proteins as a result of deletion of the HspR transcriptional regulator induced an increased immune response that significantly impaired the ability of MTB to persist during the chronic phase of infection in mice (5). Whole-genome expression analysis of the HspR deletion strain, and heat-shocked wild-type MTB, identified Rv0251c as a novel major heat shock gene (6). This finding has been confirmed and extended by the comprehensive transcriptomic analysis of Schnappinger et al. (7). Annotated as hsp in the MTB H37Rv genome sequence (8), Rv0251c was found to be induced in naive and activated murine macrophages, and by heat shock, H2O2, SDS, high-dose NO, and palmitic acid in broth cultures (7). On this basis, they categorized Rv0251c as belonging to a group of seven MTB genes that are up-regulated in response to multiple stresses.
Rv0251c encodes a protein with 30% amino acid sequence identity to the well-characterized 16-kDa -crystallin homolog Acr (encoded by Rv2031c, and referred to as acr or hspX) (6, 8). The identity increases to 41% based on comparison of residues present in the -crystallin core. The acr gene is known to be required for log phase growth of MTB and during the transition to stationary phase, at which time it is suspected to contribute via chaperonin activity to the stabilization of intracellular structures (9, 10, 11). In addition to this important role in bacillary physiology, the protein is also immunodominant in humans (12, 13, 14). In contrast to acr2, the gene for acr is induced by oxygen deprivation or by exposure to low concentrations of NO in vitro, and its induction in murine macrophages is clearly NOS2 dependent (7, 15, 16). We have previously named Rv0251c acr2 because it is the second member of the -crystallin family to be identified in MTB. From this point in this report we therefore refer to Acr1 and Acr2 (6).
We investigated the biology of acr2 by measurement of its RNA accumulation in human mononuclear phagocytes, phenotypic analysis of a mutant MTB that lacks the acr2 gene (acr2), and analysis of immune recognition of the recombinant Acr2 protein. We found that the level of acr2 RNA increases shortly after phagocytosis of MTB in response to exposure to host reactive oxygen intermediates (ROI). However, the acr2 strain was only mildly compromised in its ability to resist various in vivo and in vitro stress stimuli. By virtue of its early expression, we found that Acr2 is a dominant immune target of the early bovine and human T cell responses to mycobacterial infection. The results suggest that, although acr2 plays a role in bacillary defense, its early expression evokes a vigorous early immune response. Thus, Acr2 may be a rare Achilles’ heel for MTB, giving away its presence early after infection.
Materials and Methods
Isolation, infection, and culture of monocytes and monocyte-derived macrophages (MDM)
Buffy coats from healthy donors were obtained from the National Blood Transfusion Service (Colindale, London, U.K.). Following dilution in RPMI 1640 (1/3 v/v) PBMC were separated by centrifugation over Ficoll-Paque Plus (Pharmacia). Cells were washed extensively in RPMI 1640, pooled, and counted. Cells were suspended at 1.2 x 107/ml in RPMI 1640/10% FCS medium (R10), and aliquots of 25 ml were added to 150-cm2 tissue culture flasks. Flasks were placed flat in the incubator, and monocytes were allowed to adhere for 2 h at 37°C. Nonadherent cells were removed by washing three times with 10 ml of prewarmed RPMI 1640. Finally, 10 ml of ice-cold PBS was added, and the flasks were incubated at 4°C for 20 min. Using a scraper, monocytes were gently dislodged from the bottom of the flasks and pooled in a 50-ml Falcon tube to count. FACS analysis showed the resultant cell population to be 82 ± 4% CD14 and 76 ± 7% CD11b positive. For experiments requiring monocytes, cells were plated in RPMI 1640 containing 10% non-heat-inactivated FCS at 106/well in a 24-well tissue culture plate, and cultured overnight before infection. For experiments requiring MDM, cells were suspended in X-VIVO 15 (BioWhittaker) at 106/well in a 24-well plate and cultured for 7 days before infection. This maturation procedure resulted in an increase in HLA-DR expression (95 ± 0.3%), retention of CD14 positivity (84 ± 3%), and a decrease in CD11b (15 ± 1.3%). For the analysis of intracellular growth of mutant strains, MDM were activated with IFN- at 1 ng/ml 24 h before infection (added on day 6).
Monocytes or MDM were infected in 24-well culture plates. Before infection, the medium was removed from the wells using a Pasteur pipette and replaced with fresh RPMI 1640 supplemented with L-glutamine plus 10% non-heat-inactivated FCS. Cells were infected at a multiplicity of infection of 1:1 (bacillus:cell) for 4 h, followed by washing three times using warm RPMI 1640 to remove nonphagocytosed bacteria. No antibiotics were used. Finally, RPMI 1640 containing L-glutamine and 10% heat-inactivated FCS was added to the infected cells. On average, this procedure led to infection of between 5 and 10% mononuclear phagocytes (data not shown). The washing procedure ensures that 90% of bacilli subsequently recovered from cultures are in the cell lysate rather than supernatant (17). For some experiments, monocytes were preincubated with 10 mM N-acetyl cysteine (NAC; Sigma-Aldrich) before infection.
Bacterial strains and growth curve
MTB H37Rv (supplied by Prof. S. Cole, Institut Pasteur, Paris, France) and mutant strains of MTB were grown with shaking to mid-log phase in Middlebrook 7H9 broth (Difco) containing 0.2% glycerol, 0.05% Tween 80, and 10% albumin-dextrose-catalase. Aliquots were prepared in 15% glycerol and stored at –80°C. The CFU content of aliquots was determined by serial dilution and plating on to Middlebrook 7H11 agar (Difco) containing 0.5% glycerol and 10% oleic acid-albumin-dextrose-catalase. The construction and characterization of a strain lacking Rv0251c/acr2 (acr2) and its complementation by a single copy of the acr2 gene under its own promoter (acr2::pSMT184) is described in detail elsewhere (18). To analyze the growth rate of strains, duplicate cultures were set up and sampled for CFU analysis at intervals. The mean of four to six estimations of CFU was recorded by serial dilution and plating on 7H11 agar.
RNA extraction and quantitative RT-PCR
The extraction of MTB RNA from infected cells was performed exactly as described previously (19). We used quantitative real-time RT-PCR in an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Specific reverse transcription (RT) and PCR primers and a probe for Acr2 were as follows: RT primer (–266RT), 5'-GCGTGTGCTCGTCG-3'; forward primer (160F), 5'-GCGGTGGTCCGTTTGGA-3'; reverse primer (227R), 5'-CCAGGGTCAAGCTCGACGTT-3'; probe (181T), 5'-FAM-CCCGGCATTGACGTCGACAAGG-3'-TAMRA. Sequences used to detect MTB acr1 and 16S RNA were as previously published (16, 20). All probes were dually labeled with FAM at the 5' end and TAMRA at the 3' end. Multiplex RT was performed for 1 h at 37°C using 1 μM each specific RT primer, 0.25 U/μl avian myeloblastosis virus-RT (ABgene) in the presence of 500 μM each dNTP. 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 primers at 250 nM; for Acr1, the probe at 200 nM, forward primer at 150 nM, and the reverse at 50 nM; for Acr2, the probe at 100 nM, forward primer at 50 nM, and the reverse at 300 nM. Reaction conditions consisted of 1 cycle of 50°C for 2 min and 1 cycle of 95°C for 10 min and then 40 cycles of 95°C for 15 s followed by annealing and elongation at 65°C (16S), 67°C (acr1), or 60°C (acr2) for 1 min. The cycle threshold (CT) for each sample was compared with CT values of known amounts of a standard DNA from MTB H37Rv. To assure lack of DNA contamination of the RNA samples, a duplicate tube of sample with no RT enzyme was included as control. Results are expressed as values normalized to the 16S rRNA content.
Experimental infection of cattle with Mycobacterium bovis and analysis of in vitro IFN- responses
Five-month-old calves (Holstein-Friesians) were obtained from herds free of bovine tuberculosis and kept in category 3 biosafety accommodation at Veterinary Laboratories Agency Weybridge. Five animals were infected with the sequenced M. bovis strain (AF 2122/97) by endobronchial instillation of 1000 CFU (21). Blood samples were collected postinfection, and IFN- assays were performed as described previously (22). Briefly, 0.25 ml of heparinized whole blood was aliquotted in 96-well plates and 25 μl of Ag solution was added at the following final concentrations: bovine PPD (PPD-B) (10 μg/ml), Acr1 and Acr2 (20 μg/ml). Supernatants were harvested after 48 h of culture and IFN- was determined using the BOVIGAM EIA kit (CSL). Data are expressed as OD450 units (OD450 values multiplied by 1000) with responses of >100 OD450 U classified as positive (22).
Human subjects
This study was conducted with ethical approval from the Harrow Local Research Ethics Committee (LREC 1646 and 2414). Newly diagnosed tuberculosis patients (TP) and healthy sensitized subjects (HS) were recruited from Northwick Park Hospital, Harrow. TP had untreated overt (i.e., culture- or biopsy-positive) tuberculosis (n = 29; 18 male, 11 female; average age, 33.9 years). The HS group comprised asymptomatic adults with normal chest radiographs who nevertheless exhibited positive purified protein derivative (PPD) skin test reactions (Heaf grade 2 and above; equivalent to a Mantoux of >10 mm) who were considered clinically to have latent tuberculosis infection (n = 69; 38 male, 31 female; average age, 36.6 years). All subjects were subsequently advised and, if indicated, treated according to British Thoracic Society guidelines (23, 24).
Recombinant Ags
Recombinant early secreted antigenic target-6 (rESAT-6) was prepared as previously described (25). Recombinant culture filtrate protein-10 (rCFP-10) and rAcr1 were obtained from Lionex. N-terminal sequencing confirmed the identity of the cloned Ags. Acr2 was cloned and expressed as an N-terminal His.Tag and purified as described by Stewart et al. (18). Endotoxin levels were as follows (endotoxin units (EU)/mg): ESAT-6, 18.65; CFP-10, 47.41; Acr2, 1.33; PPD, 18.65; Acr1, 11.01. Twenty-mer synthetic peptides covering the entire sequence of the Acr1 and Acr2 proteins and overlapping by 12 residues were prepared by multipin synthesis by Mimotopes (26). Peptides were used individually in ELISPOT analysis at a concentration of 20 μg/ml.
ELISPOT assay for single-cell IFN- release
Ninety-six-well polyvinylidene difluoride-backed plates (MAIPS45; Millipore), precoated with 15 μg/ml anti-IFN- mAb 1-D1K (Mabtech), were blocked with R10 for 2 h. A total of 3 x 105 PBMC was added in 100 μl of R10/well. Duplicate wells containing rAcr1, rAcr2, rESAT-6, or rCFP-10 at previously determined optimum concentrations (2.5–20 μg/ml) were set up. PPD (Evans Medical) at 100 U/ml, and PHA (ICN) at 5 μg/ml were added to positive control wells. No Ag was added to the negative control wells. After 16-h incubation at 37°C in 5% CO2, plates were washed with PBS containing 0.05% Tween 20. Fifty microliters of 1 μg/ml biotinylated anti-IFN- mAb, 7-B6-1-biotin (Mabtech), was added for 2 h. Plates were then washed and streptavidin-alkaline phosphatase toxoid was added at 1/1000 dilution. After 1 h and further washing, 50 μl of chromogenic alkaline phosphatase substrate (Bio-Rad), diluted 1/25 with deionized water, was added. Ten minutes later, plates were washed and allowed to dry, and spot-forming cells (SFC) were enumerated with a magnifying glass. A positive response was defined as 15 IFN- SFC/106 PBMC.
ELISA for Abs to Acr2
Polyvinyl microtiter plates (Nunc) were coated overnight at 4°C with 10 μg/ml rAcr2 in 0.05 M carbonate buffer (pH 9.6). The plates were blocked with 5% (w/v) fat-free skimmed milk powder in PBS for 30 min at 37°C. Serum was diluted 1/100 in PBS/milk containing 0.05% (v/v) Tween 20 and applied for 2 h at room temperature with agitation. The wells were washed three times in PBS/Tween, and then HRP-conjugated rabbit anti-human IgG (DakoCytomation) was applied at a dilution of 1/3000 in PBS/milk/Tween 20 for 2 h at room temperature. The wells were washed three times in PBS/Tween 20, and Ab binding was detected using o-phenylenediamine as a substrate, and the reaction product was measured at 492 nm.
Statistical analysis
Contingency analysis was performed by Fisher’s exact test of probability. Paired and unpaired parametric variables were compared by Student’s t test. Paired and unpaired nonparametric variables were compared by Wilcoxon signed rank or Mann-Whitney U test, respectively. Significance was inferred for values of p 0.05.
Results
Expression of Acr2 within human mononuclear phagocytes
The kinetics of acr2 RNA accumulation within monocytes and MDM from two donors was studied by quantitative RT-PCR. Duplicate wells of 5 x 106 cells were infected with MTB at 1:1 bacillus:cell. For the 1-h time point, nonphagocytosed bacilli were washed away immediately before lysis and RNA extraction; for all other time points, nonphagocytosed bacteria were removed after 4 h. Experiments were controlled by inoculating bacilli into parallel wells containing culture medium but not cells. The ratio of acr2 RNA to MTB 16S rRNA increased 5.18-fold within 1 h of infection of monocytes, reaching a peak of 16.27-fold after 24 h, after which there was a decline (Fig. 1A). In MDM, acr2 induction was slightly less rapid but reached a higher peak of 62.3-fold at 48 h. In contrast, the ratio of acr1 RNA to 16S rRNA progressively declined over the 96-h time course in the same cells (Fig. 1B). A marked induction of acr1 was observed in the control cultures that contained only RPMI 1640/10% FCS, possibly reflecting hypoxia in these unshaken cultures.
FIGURE 1. Expression of acr2 within human mononuclear phagocytes. Cells were infected with MTB H37Rv at 1:1. Nonphagocytosed bacilli were washed away before lysis, and in any case at 4 h, for RNA extraction and quantitative RT-PCR. acr2 and acr1 RNA levels were normalized to the 16S rRNA content of the same sample. A and B, RPMI 1640/10% FCS (?); monocytes (); MDM (). Intracellular acr2 RNA accumulated rapidly in both monocytes and MDM from the same donors. In contrast, the acr1 RNA level within cells gradually fell over 96 h, although in cell culture medium without cells, there was a marked increase between 12 and 96 h. Data show the mean ± SD of two donors. C, At 24 h, the mean intracellular induction of acr2 in monocytes (n = 5) was 17.8-fold, and 54.7-fold in MDM (n = 7) (p 0.0182). D, Overnight preincubation of monocytes with NAC in two donors completely abrogated the increase in acr2 RNA level.
The increase in acr2/16S ratio at 24 h was further investigated in monocytes and MDM from five and seven donors, respectively (Fig. 1C). This confirmed a 17.8-fold increase in monocytes and a 54.7-fold increase in MDM (p 0.0182 for both cell types). In two donors, the effect of overnight preincubation of monocytes with an inhibitor of superoxide action (NAC; 10 mM) was tested. Results were calculated as the mean of the ratio (acr2/16S in cells plus inhibitor:acr2/16S in medium plus inhibitor) at 24 h. This design allowed us to control the possible effects of inhibitor alone on bacillary gene expression outside cells. The presence of NAC almost completely abolished the intracellular increase in acr2 (Fig. 1D). Together, these data show that acr2 gene expression is up-regulated shortly after entry to cells in response to ROI.
Phenotypic characterization of a mutant MTB lacking the gene for Acr2 (acr2)
Further investigation of the role of acr2 was pursued by analysis of a mutant MTB strain that lacked the acr2 gene (acr2). We also examined a strain in which the mutation was complemented by reinsertion of a single copy of the acr2 gene at the attB phage integration site in the chromosome (acr2::pSMT184). Doubling times calculated over 144 h of log phase growth in Middlebrook 7H9 broth were similar (24.3, 24.7, and 32.3 h for wild type, acr2, and acr2::pSMT184, respectively). Monocytes or MDM from four donors, respectively, were infected at 1:1 (bacillus:cell), and nonphagocytosed bacilli were washed away at 4 h. The uptake into, and growth of strains over 6.5 days within monocytes did not differ. Even in IFN--activated MDM, growth was similar up to 4 days, after which the acr2 mutant showed no further increase, although the difference from H37Rv at 6.5 days was not statistically significant (Fig. 2A). In other experiments, we confirmed that, although the acr2 strain was moderately more susceptible to H2O2 in 7H9 broth, its growth in vivo over 60 days in the organs of mice was unimpaired (data not shown). Interestingly, in IFN--activated MDM at the 24-h time point, the expression of acr within cells infected with acr2 was slightly (2.08-fold) higher than in cells infected with wild type (Fig. 2B). This raises the possibility that the absence of acr2 could be partially compensated by increased expression of acr. The overall expression of acr within IFN--activated MDM greatly increased such that the acr2/acr1 ratio, which had been >100 in monocyte or MDM cultures, was reduced to near equality (2.5 ± 0.2) in IFN--activated MDM (Fig. 2C).
FIGURE 2. A, Intracellular growth of a strain of MTB lacking the gene for Acr2 (H37Rv (); acr2::pSMT184 (?); acr2 ()). IFN--activated MDM were infected at 1:1, with nonphagocytosed bacilli being washed off after 4 h. The uptake into, and growth within, cells was similar up to 4 days, after which the acr2 showed a slight decline (p = 0.068 by comparison with H37Rv at 6.5 days). Mean ± SD of four donors. B, Intracellular expression of Acr in IFN--activated MDM. IFN--activated MDM were infected at 1:1, with nonphagocytosed bacilli being washed off after 4 h. At the 24-h time point, the expression of Acr within cells infected with acr2 was slightly higher. Mean ± SD of two donors. C, Ratio of intracellular Acr2/Acr1 in various cell types. Monocytes (Monos), MDM, or IFN--activated MDM (-MDM) from two donors were infected at 1:1 with MTB H37Rv. After 4 h, nonphagocytosed bacilli were washed off, and at 24 h, the cultures were lysed for analysis of RNA content. The Acr2/Acr1 ratio was greatly reduced by induction of the Acr1 gene in activated cells.
The immune response to Acr2
Because we had demonstrated that the acr2 gene is up-regulated early after infection of mononuclear phagocytes, we hypothesized that it could be a target of early T cell responses directed toward MTB. We measured the primary immune response to rAcr2 in a cattle model of experimental M. bovis infection (27). M. bovis possesses an acr2 gene identical with that of MTB H37Rv (28). Because experimental infection allows control over the time and dose of infection, precise documentation of immune responses postinfection is possible. Five calves were infected with a low dose of M. bovis and in vitro IFN- responses were determined in blood samples taken at intervals up to 5 wk postinfection. All animals at this stage of experimental infection remained free of clinical signs of tuberculosis, and may therefore be akin to recently exposed, asymptomatic humans. Our results demonstrate that Acr2 was a T cell target in recently infected cattle, in contrast to Acr1, which was recognized only later in the infection (Fig. 3). Responses to Acr2 were detected as early as 2 wk postinfection in four of five calves, and by wk 3 postinfection, all five calves exhibited strong Acr2-specific responses. These responses remained high at 4 and 5 wk postinfection. In contrast, none of the calves responded to Acr1 2 wk after infection, and only one responded at wk 3 (p = 0.04 at both time points). By wk 4 and 5, three of five and five of five calves responded to Acr1. However, even at wk 5 postinfection, significantly lower amounts of IFN- were induced by Acr1 stimulation than by Acr2 (p = 0.0003 at wk 5).
FIGURE 3. IFN- responses to Acr1 and Acr2 in cattle experimentally infected with M. bovis. The IFN- responses after in vitro stimulation with bovine PPD, Acr1, and Acr2 proteins were determined postinfection. Horizontal bars show median values. The frequency of Acr2 responders at 2 and 3 wk postinfection was significantly higher than frequencies of Acr1 responders (p = 0.04 at both time points); Acr2 responses were quantitatively higher than Acr1 responses even at wk 5 postinfection (p = 0.0003).
An unusual opportunity to test the human recall response presented itself by the precisely timed inadvertent exposure of a bacillus Calmette-Guérin (BCG)-vaccinated person to pathogenic M. bovis. We compared the IFN- ELISPOT response to Acr2 with that made in response to Acr1, ESAT-6, CFP-10, and PPD. Because ESAT-6 and CFP-10 are encoded by the RD1 genomic segment of MTB and M. bovis, these genes are not present in any M. bovis-BCG strains and can thus specifically detect T cells sensitized by members of the pathogenic MTB complex (29, 30). The response to PPD reflects accumulated history of BCG vaccination and other mycobacterial exposure. In a blood sample taken 1 day after exposure, there were 100 PPD-specific IFN--producing T cells/106 PBMC detectable by ELISPOT analysis but no significant response to the other Ags (Fig. 4). After 1 wk, the frequency of Acr2-specific cells increased greatly to 173 per 106 PBMC, whereas the PPD-specific response modestly increased to 132 per 106. There was a subsequent fall in the Acr2-specific response to a plateau (42–55 per 106) accompanied by a gradual rise in specific responses to ESAT-6 and CFP-10. At no time point was there a significant response to Acr1. The subject remained asymptomatic throughout and was healthy 28 mo after exposure. Isoniazid preventive therapy was not used. Thus, a timed exposure to M. bovis resulted, in this person, in a rapid expansion of Acr2-reactive T cells accompanied by a steady rise in M. bovis-specific responses.
FIGURE 4. Longitudinal analysis of IFN--secreting T cells in a person accidentally exposed to M. bovis. The subject of this analysis was inadvertently exposed to pathogenic M. bovis. One day after this exposure, there were no Acr2- or CFP-10-specific, and 100 PPD-specific IFN--producing T cells/106 PBMC detectable. After 1 wk, the frequency of Acr2-specific cells increased greatly to 173 per 106, whereas the PPD-specific response modestly increased to 132 per 106. There was a subsequent fall in the Acr2-specific response to a plateau (42–55 per 106) accompanied by a gradual rise in the ESAT-6- and CFP-10-specific response. There was no response to Acr1 at any time point.
We extended ELISPOT analysis of the T cell response to Acr2 to 98 adults sensitized by exposure or by active tuberculosis. HS (n = 69) were persons who were PPD skin test positive but were free of symptoms of active tuberculosis and had normal chest radiographs. This group was stratified into 13 individuals with documented recent (<6 mo ago) contact with infectious tuberculosis and 56 individuals from whom no history of recent contact with infectious tuberculosis could be elicited. TP (n = 29) had untreated active tuberculosis at the time of sampling. The frequency of positive responses and also the numbers of IFN--secreting T cells responding to both -crystallins tended to be higher in HS compared with TP, significantly so for Acr2 (p = 0.044; Table I). In HS who responded, the median IFN- SFC was 43, whereas it was 33 among responding TP. The number of PPD-reactive IFN--secreting T cells was also significantly higher in HS. By contrast, the response to the RD1-encoded ESAT-6 and CFP-10 proteins tended to be higher in TP, although not significantly so. Stratification of HS by recent or remote exposure to tuberculosis revealed that 92% of those with recent exposure had a response to Acr2 in comparison with 63% with more remote contact (p = 0.029). In addition, the median frequency of IFN- SFC in recent contacts was 65 in comparison with 25 in the remote group (p = 0.009). The relationship between T cell reactivity and recency of contact was seen only for Acr2 response (Fig. 5A).
Table I. Frequency and magnitude of response to Ags of MTBa
FIGURE 5. The human immune response to Acr2 of MTB. A, The T cell response to Acr2 in 69 HS adults who were PPD skin test positive but free of symptoms of active tuberculosis. This group was stratified into 13 individuals with documented recent (<6 mo ago) contact with infectious tuberculosis (, , , ) and 56 individuals from whom no recent history of contact with infectious tuberculosis could be elicited (, , ?, ). The median frequency of IFN- SFC in recent contacts was 65 by comparison with 25 in the remote group (p = 0.009). This relationship between T cell reactivity and recency of infectious contact held only for Acr2. B, Comparison of the T cell and B cell response to Acr2. The Ab response (as determined by ELISA) was compared with the frequency of IFN--secreting cells in 41 subjects. HS had lower Ab levels with higher frequencies of IFN--secreting cells, such that 9 of 20 had Ab OD values <0.3 and IFN--secreting cells >40 per 106 PBMC in comparison with just 2 of 21 patients (p = 0.0148).
In a subset of 41 subjects (20 HS and 21 TP), the availability of a serum sample allowed us to compare T and B cell Ab responses to Acr2. HS had lower Ab levels with higher frequencies of IFN--secreting cells, such that 9 of 20 HS had a response characterized by Ab OD values <0.3 and IFN- secreting cells >40 per 106 PBMC; only 2 of 21 TP shared this profile (p = 0.0148; Fig. 5B). However, responses were heterogeneous, and there was no overall inverse correlation between the Ab and T cell response, nor was there any correlation between Acr1- and Acr2-specific responses (data not shown). Because Acr2 shares 30% amino acid sequence identity with Acr1, we were also interested to investigate whether T cell recognition of the -crystallins was specific or cross-reactive. Of all the subjects in the study, 34% responded to both molecules, 14% to Acr1 alone, 30% to Acr2 alone, and 22% to neither. Thus, T cell recognition of one molecule was more common than recognition of both, implying specificity. This impression was reinforced by epitope mapping of both molecules in a subset of 24 responding donors (Fig. 6). Dominant epitopes in Acr1 were found between residues 81–124, consistent with our previous reports of two permissively recognized epitopes in this region (13, 14, 31). In contrast, no single peptide within Acr2 stimulated the T cells of >50% of the subjects, implying that Acr2 contains multiple epitopes restricted by specific MHC class II molecules. Recognition of peptides that contained >40% identical residues in both Acr1 and Acr2 was exceptional, occurring only for sequence 97–116 (10 identical residues: 50%) at a frequency of 7 of 11 for Acr1 and just 1 of 13 for Acr2. These data strongly imply specific recognition even of this, the most homologous peptide.
FIGURE 6. Epitope mapping of -crystallins in 24 responding donors. The T cell response to overlapping 20-mer peptides was assayed by the overnight IFN- ELISPOT assay. Only donors who responded to either or both -crystallins were included in this analysis. Dominant epitopes in Acr1 are found between residues 81 and 124, whereas no single peptide within Acr2 stimulated the T cells of >50% subjects. Sequence numbers in bold type indicate >40% identity between the Acr1 and Acr2 sequence. *, 125–144 for Acr1 sequence.
Discussion
We have investigated the gene expression, effect of deletion from the genome, and the immune response to Acr2, a novel -crystallin of MTB. MTB has two members of the -crystallin family of molecular chaperones. Although neither is absolutely required for growth in vivo or in vitro (32), Acr1 has been extensively characterized in the context of its role in the bacterial response to hypoxia/NO and as an immunodominant Ag. Our study demonstrates that Acr2 also makes a contribution to pathogen biology but more so to the host immune response. We found that acr2 gene expression was up-regulated shortly after entry into cells in response to exposure to host reactive oxygen. As a consequence, Acr2 is a prominent immune target, especially of T cells in individuals who are asymptomatic but recently exposed to infectious tuberculosis.
Expression of the acr2 gene is controlled by the heat shock regulator, HspR (5), and by the alternative factor, E, which is itself regulated by heat shock and by oxidative stress (33). It has recently been shown that acr2 is strongly induced following uptake of MTB by quiescent mouse macrophages, in contrast to acr1 that is induced only in activated macrophages (7). Consistent with findings in the murine system, we have observed strong induction of acr2 expression following uptake of MTB by human monocytes or MDM (Fig. 1). There was no induction of acr1 under these conditions. Preincubation of monocytes with NAC showed that acr2 induction was dependent on the generation of ROI, consistent with in vitro studies of acr2 regulation (7).
Building on our characterization of the role of acr2 in the pathogen response to infection, we reasoned that the early induction of acr2 expression could result in its availability as an attractive target for an early response on the part of the host immune system. Three lines of evidence suggest that this is indeed the case. First, the Acr2 Ag was strongly recognized by cattle infected by M. bovis, which contains an identical Acr2 gene (Fig. 3). Second, we had the opportunity to monitor the kinetics of T cell responses to different Ags in a single individual following accidental exposure to virulent M. bovis (Fig. 4). A strong response to Acr2 was observed within 1 wk of exposure, significantly in advance of responses to two other well-characterized immunodominant Ags, ESAT-6 and CFP-10 (34). Although the interpretation of data from a single donor has to be cautious, we take the data to indicate that infection with M. bovis did occur and that this infection rapidly re-expanded a population of previously undetectable Acr2-specific IFN--producing T cells. The kinetics of immune response to acr2 in this person, together with the transient response to ESAT-6, may indicate that the infection was cleared very rapidly. The third less direct evidence is derived from analysis of responses in groups of TP and HS who had clinical and immunological evidence of recent or past exposure to MTB. The frequency and number of IFN--producing T cells specific for Acr2 were higher among HS than among TP, with the highest responses seen in individuals with the most recent exposure to infection. These correlations were not seen in the case of responses to Acr1, ESAT-6, or CFP-10 (Fig. 5A).
Although BCG vaccination is of insufficient efficacy to control tuberculosis in human populations, only a few experimental approaches have improved its efficacy in animal models (3). Thus, attention has been drawn to the possibility of augmenting BCG by overexpression of its existing Ags or by combining it with Ags delivered either as DNA, protein subunit, or other live vector in a prime-boost strategy (35, 36, 37, 38). It is recognized that an early immune evasion strategy adopted by MTB is retardation of phagosomal maturation, crucially delaying its degradation and thus immune recognition (39). Nevertheless, our study indicates the early expression of acr2 gives away the presence of MTB to the immune response. The demonstration that Acr2 is an early immune target is encouraging, but insufficient to qualify it as a vaccine candidate. Our ongoing work is therefore directed toward determining whether Acr2 can be used to augment the vaccine efficacy of BCG in experimental models.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
Dr. Mark Nicol of the University of Cape Town is thanked for his critical reading of the manuscript. We thank the medical, microbiological, and nursing staff of Northwick Park Hospital for assistance in recruiting and characterizing subjects for the study, particularly Drs. Robert Davidson, Robert Wall, and Stuart Dickson, and Prof. Geoffrey Pasvol.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Wellcome Trust of Great Britain (Refs. 064261, 060079, 038997, 059141). H.M.V. is supported by Department for Environment Food and Rural Affairs.
2 K.A.W. and G.R.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Robert J. Wilkinson, Wellcome Trust Center for Research in Clinical Tropical Medicine, Faculty of Medicine, Imperial College London, Wright Fleming Institute, Norfolk Place, London W2 1PG, U.K. E-mail address: r.j.wilkinson@imperial.ac.uk
4 Abbreviations used in this paper: MTB, Mycobacterium tuberculosis; ROI, reactive oxygen intermediate; MDM, monocyte-derived macrophage; NAC, N-acetyl cysteine; RT, reverse transcription; CT, cycle threshold; TP, tuberculosis patient; HS, healthy sensitized subject; PPD, purified protein derivative; SFC, spot-forming cell; BCG, bacillus Calmette-Guérin; ESAT-6, early secreted antigenic target-6; CFP-10, culture filtrate protein-10.
Received for publication May 26, 2004. Accepted for publication December 29, 2004.
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